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The left telomere on chromosome iii in Saccharomyces cervisiae isolation and characterization Button, Linda Louise 1986

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THE LEFT TELOMERE ON CHROMOSOME III IN SACCHAROMYCESCEREVISIAE: ISOLATION AND CHARACTERIZATION By LINDA LOUISE BUTTON B.Sc. (hon.), University of New Brunswick, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1986 © Linda Louise Button, 1986 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department o r by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of Biochemistry The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date QfjAu% 1Q \ i i ABSTRACT Telomeres, the molecular ends of eukaryotic chromosomes, are essential for chromosome stability and complete replication of the termini. A yeast Saccharomvces cerevlslae telomerlc region was Isolated by chromosome walking from HMLff.. the most distal known gene on the left end of chromosome III. The terminal Sail restriction fragment on MIL (chromosome III left end) mapped 8.6 kb distal to HMLy. and was cloned in a circular vector to generate a probe for the MIL telomere.This Sail fragment on the end of chromosome MIL was heterodisperse In length , having an average size of 3.3 kb and a length distribution of +/- 0.2 kb. Southern blot and DNA sequence analyses Indicated that the IIIL end conforms to the T-X class, rather than the T-Y'-X class, of yeast telomeres. The terminal (T) region on IIIL consists of 0.6 kb (+/- 0.2 kb) of 5'-C i -3A-3' tandem repeat sequence, adjacent to the 1.2 kb type X AM region. There Is no homology to the type Y' AM region between HMLoc and the IIIL telomere. The telomerlc probe from IIIL hybridized to multiple genomic restriction fragments; both heterodisperse and defined length bands were observed in Southern hybridization analyses and these mapped to chromosome ends. Heterogeneity of the length of the terminal fragment on a given chromosome end was observed among various yeast strains and this length variation was localized to the T region. The cloned fragment from the IIIL telomere lacked the terminus due to the cloning method used to Isolate It, however this T-X fragment functioned as a telomere on linear plasmids in yeast. Plasmids constructed in vitro with IIIL ends were maintained as linear molecules in yeast, and were more stable mitotlcally than linear plasmids constructed with Tetrahymena rDNA termini. Plasmids constructed with IIIL end fragments that had the entire T region deleted were replicated as circular molecules In yeast. Addition of Y" regions did not occur on any of the linear plasmids constructed with fragments from the IIIL telomere during replication in yeast, but T region was presumably added to the equivalent extent as that observed on the natural chromosome IIIL end [0.6 kb (+/- 0.2 kb)]. A rationale is discussed for the existence of two telomerlc classes in yeast, T-X 8nd T-Y'-X, and for the addition of T region to chromosome ends 1n replication. i i i The region adjacent to H M L K , on I ML, which extends 8.6 kb toward the telomere, Is homologous to an alternate telomeric region In the genome of haplold yeast strains. Southern hybridization analyses with HMLc^ distal probes revealed that at least the terminal 6.5 kb of this region is retained In some circular chromosome III strains that were expected to have the MIL and I MR telomeric regions deleted. Northern hybridization indicated that an unidentified R N A transcript Is homologous with a fragment from the HMLfr distal region. i v Table of Contents Page I. Abstract i1 II. Table of Contents Iv III. List of Tables vii IV. List of Figures viii V. Acknowledgements x VI. List of Abbreviations xii VII. INTRODUCTION A. Yeast Chromosome Structure 1 B. The Function of Telomeres on Chromosomes 2 1. Telomeres and Chromosome Stability 2 2. Telomeres and DNA Replication 3 C. Cytology of Telomeres 6 D. Molecular Characterization of Telomeres 8 1. Extr8chromosomal Linear DNA 8 2. Yeast Chromosomes 9 3. Trypanosome Chromosomes 11 E. Artificial Chromosomes in Yeast 11 1. Heterologous Telomeres In Yeast 11 2. Artificial Chromosome Construction and Stability 12 F. Stabilization of Linear Plasmid Ends In Yeast 13 G. Genetic Control of Telomere Length in Yeast 13 H. Isolation and Characterization of the Telomeric Region from a Specific Yeast Chromosome 14 1. Chromosome Walking 14 2. Yeast Chromosome III 15 3. Characterization of the 11IL Telomeric Region 16 VIII. MATERIALS AND METHODS A. Materials 18 B. Strains and Media 18 1. Yeast 18 2. Bacteria 19 3. Phage and Plasm Ids 20 C. Basic Molecular Cloning Techniques 20 D. Yeast DNA Isolation 21 1. Large Scale DNA Preparation 21 2. Small Scale DNA Preparation 22 3. Yeast DNA Purification 23 E. Lambda Phage DNA Isolation 23 1. Large Scale DNA Preparation 23 2. Small Scale DNA Preparation 24 3. Phage DNA Purification 24 F. Plasmid DNA Isolation 24 V 1. Large Scale DNA Preparation 24 2. Small Scale DNA Preparation 25 3. Plasmid DNA Purification 25 G. Yeast RNA Isolation 26 H. Yeast Genomic DNA Library Preparation 27 I. Bacteriophage Lambda Techniques 28 J. Plasmid Dephosphorylatlon and Ligation 29 K. Ml3Cloning 29 1. Preparation of M13 Replicative Form DNA 29 2. Subcloning Fragments in Ml3 Vectors 30 3. M13 Template DNA Preparation 30 4. M13 Clone Complementation Test 31 L. Plasm1dandM13 DNA Transformations 31 M. DNA Probe Preparation 32 1. Nick Translation 32 2. M13 Primer Extension 33 N. Gel Hybridization Analyses 33 1. Agarose Gel Electrophoresis and Southern Hybridization 33 2. RNA Dot Blot and Northern Hybridization 34 0. Plaque and Colony Hybridization 35 1. Plaque Hybridization 35 2. Colony Hybridization 36 P. Recombination Screening 36 Q. SI and Bal31 Nuclease Digestion of Yeast DNA 37 R. DNA Sequence Determination 38 1. Preparation of Deletions with Exonuclease 111 and S1 Nuclease 38 2. DNA Sequence Determination 38 S. Construction of Plasmids to Assay for Telomere Function 39 1. Clones with Requisite Restriction Site Arrangements 39 2. Design of Linear Plasmids 41 T. Yeast Transformation 42 U. Mitotic Stability Determination 43 IX. RESULTS A. Chromosome Walking from HMLoy .44 1. Hybridization of Probe 1 * with Yeast Genomic DNA 44 2. Hybridization Screening of the Lambda Charon4A-Yeast DNA Library 46 3. Recombination Screening of the Lambda Charon4A-Yeast DNA Library 49 4. Hybridization Screening with other Yeast Genomic DNA Libraries 50 5. Southern hybridization of Yeast Genomic DNA with Probe 2* 53 B. The IIIL Distal Region is Retained in Some Circular III Strains 55 C. Transcription in the IIIL Distal Region 59 D. The IIIL Probe 2* is Adjacent to the IIIL Telomere 61 1. Bal31 Nuclease Sensitivity of the Probe 2* Region 61 2. The IIIL Alternate Region Is Telomere Proximal 63 3. The 11IL Telomere Associated Region Maps Distal to Probe 2* 63 E. Attempts to Clone the 111L Telomere by Marker Rescue 66 F. Attempt to Clone the 111L Telomere by Vector Addition at the Terminus 71 G. Cloning the I ML Terminal Fragment in a Circular Vector 73 H. The IIIL End Conforms to the T-X Class of Yeast Telomeres 75 1. The IIIL Terminal Probe is Homologous Exclusively with Telomeres 79 J . Length Heterogeneity of the 111L Telomere Among Yeast Strains 81 v i K. The MIL Telomere Is Retained In Some Ring III Yeast Strains 84 L. DNA Sequence Analysis of the Chromosome MIL Telomere 86 M. MIL Telomere Includes 1.8 kb at the Terminus 89 N. Construction of Clones to Study the Function of the I ML Telomere 91 0. The Cloned 1111_ Telomere Functions on Linear Plasmlds In Yeast 95 P. Deletion Fragments from the MIL Telomere are Not Stabilized with Y'Regions 103 Q. Structures of the Plasmlds with MIL End Fragments in Yeast 104 X. DISCUSSION A. Chromosome Walking 111 B. Chromosome 11IL End Is a T-X Class Telomere 113 C. Heterogeneity of the Length of the 111L Telomere Among Yeast Stral ns 114 D. Requirements for a Functional Telomere In Yeast 116 E. The 11 IL End is Stabilized with T Region but Without Y" Region Addition 118 F. Why are There Two Classes of Telomeres Maintained In Yeast? 120 G. How are Yeast Telomeres Repl icated? 122 H. I ML Distal versus MIL Alternate Region in the Yeast Genome 125 1. Future Prospects 129 XI. LITERATURE CITED 132 v i i List of Tables Eage. I. The IIIL Distal Region Is Retained In most Circular III Yeast Strains. 58 II. Stability and Structure of Plasmids with Telomerlc Fragments in Yeast. 97 v i i i Ltst of Figures Page 1. The 5'-End Problem In DNA Replication and Some Possible Mechanisms 4 Proposed as Solutions. 2. Organization of the Repeat Sequences at Yeast Telomeres. 10 3. Organization of Chromosomes 111 In the Yeast S. cerevlslae. 45.1 4. Southern Hybridization of Linear 111 and Ring 111 Yeast Strains 45.2 with the aula Probe 1*. 5. Chromosome Walking Steps from HMLfr on 11 IL. 48 6. Restriction Endonuclease Maps of Recombinant Phage AMGBO1-14. 52 7. Southern Hybridization with Probe 2* and Yeast Genomic DNA. 54 8. Southern Hybridization of Linear III and Ring 111 Yeast Strains 56 with Probes from the 11 IL Distal Region. 9. Hybridization Analysis of Yeast RNA with a 11 IL Distal Probe. 60 10. Length Heterogeneity and Bal31 Nuclease Sensitivity of the 11 IL 62 Distal Region. 11. The 11 IL Alternate Region is Bal31 Nuclease Sensitive. 64 12. Probe 2* Is not Homologous with Telomere Associated Regions in Yeast. 65 13. Strategy to Clone the 11 IL Telomere by Marker Insertion and Excision. 67 14. Characterization of the URA3 Integration Site in 11 IL. 69 15. Strategy for Cloning the 111L Telomere by Vector Addition. 72 16. Cloning the 11 IL Terminal Fragment In a Circular Vector. 74 17. The 11 IL Terminal Fragment Is Homologous with the X but not the Y' 76 Telomere Associated Region. 18. The IIIL Terminal Fragment Is Homologous with Yeast Repetitive DNA. 78 19. The IIIL Terminal Probe Hybridizes with other Chromosome Ends. 80 20. Southern Hybridization Indicates Length Heterogeneity of the 11 IL 82 Telomere Among Yeast Strains. IX 21. Southern Hybridization of Linear 111 and Ring 111 Yeast Strains with 85 the IIIL Terminal Probe. 22. DNA Sequence of the IIIL Terminal Fragment. 87 23. Extent of the X Region on the 11IL Telomere. 90 24. Construction of Linear Plasmid with Tetrahymena rDNA termini. 92 25. Construction of Plasmids to Study the Function of Fragments 94 from the IIIL Telomere. 26. Assay for Linear or Circular Plasmids In the Yeast Transformants. 100 27. Determination of the Restriction Maps for the Linear or Circular Plasmids 105 In the Yeast Transformants. 28. Restriction Maps of the Plasmids with Fragments from the IIIL Telomere. 106 following Replication In Yeast. 29. Model for the Retention ofthe IIIL End in a Ring III Yeast Strain. 127 x List of Abbreviations A absorbance ATP rlboadenosine 5'-tr1phosphate bp base pair (s) BSA bovine serum albumin cpm counts per minute DEP diethyl pyrocarbonate DNA deoxyribonucleic acid dATP deoxyrlboadenoslne 5-trlphosphate ddATP dldeoxyrlboadenoslne 5'-tr1phosphate dCTP deoxyrlbocytoslne 5'-tr1phosphate ddCTP dideoxyrlbocytoslne 5'-tr1phosphate dGTP deoxyrlboguanoslne 5-trlphosphate ddGTP . dldeoxyrlboguanoslne5-trlphosphate dTTP deoxyrlbothymldlne 5'-tr1phosphate ddTTP dldeoxyrlbothymldlne 5'-tr1phosphate dNTP deoxyrlbonucleotlde triphosphate ddNTP dldeoxyrlbonucleotlde triphosphate ds double-stranded DTT dlthlothreltol EDTA ethylene diamine tetraacetate EtBr ethldium bromide EtOH ethanol hr hour IIIL yeast chromosome III left arm IPTG Isopropylthlogalactoslde KAc potassium acetate kb kllobase pair (s) kd kilodalton (s) LB Lurla-Bertanl mA mllllamperes min minute (s) nt nucleotides PEG polyethylene glycol, carbowax pfu plaque-forming units RE restriction enzyme (s) RF repllcatfve form RNA ribonucleic acid rpm revolutions per minute RT room temperature (23°C) SDS sodium dodecyl sulfate sec second (s) ss single-stranded TEMED N.N.N' ,N'-tetramethylene aminomethane Tris tris (hydroxymethyl) amino methane u unit (s) UV ultraviolet V volts VRC vanadyl rlbonucleoslde complex W watts Xgal 5-dlbromo 4-chloro 3-lndolylgalactoslde To Mum and To Dave: If only you were here... 1 INTRODUCTION The replication and stability of eukaryotic chromosomes is dependent on at least three types of elements: centromeres, the attachment sites for mitotic and meiotic spindle fibers (25), ARS elements, the presumptive origins of replication (210), and telomeres, the molecular ends of the chromosomal DNA molecules (25). Such yeast chromosomal elements have been cloned and characterized using artificial yeast chromosomes (29,56, 140, H I ) . A. Yeast Chromosome Structure Yeast Saccharomvces cerevlsiae provides a choice system for eukaryotic chromosome structure and replication studies. Most details of genome organization and function are conserved in yeast and higher eukaryotic organisms (69) and the relatively simple yeast genome has been extensively characterized. Yeast chromosomes consist of linear, double-stranded DNA packaged with histones into nucleosomes (69, 155). Haploid yeast strains contain seventeen chromosomes or linkage groups which range in length from 150 kb to 2500 kb, as shown by genetic analyses (135, 136) and by electron microscopic observation of synaptonemal complexes in meiotic cells (35). The yeast genome consists of 9 x 10 9 daltons or 1.4 x 10 4 kb of DNA (118). About 80 - 85$ of yeast cellular DNA is contained in the nuclear chromosomes, and the other 15 - 20% is 2jxm plasmid and mitochondrial DNA (155). Only about 5% of the yeast genome Is repetitive DNA, as determined by renaturation kinetics (32, 118), and this 5 * consists largely of yeast rRNA and tRNA genes (69). Each yeast chromosome contains a single DNA molecule (102 -10^ kb), as determined by sucrose gradient sedimentation analysis (27, 152), electron microscopic analysis of DNA contour lengths (154), viscoelastic measurement (103, 118) and by karyotype analysis using orthogonal-field-alternation-gel electrophoresis, (OFAGE), (36,37, 173). The DNA molecule is continous through the centromere on the chromosome since circular derivatives of yeast chromosome III (109, 186), and yeast centromerlc regions (25, 51, 53) have been isolated. The ends of the chromosomal DNA molecules isolated from yeast behave as free ends as determined by their sensitivity to digestion with the double-stranded exonuclease Bal31 or the single-stranded S1 nuclease (175,193,206). 2 B. The Function of Telomeres on Chromosomes 1. Telomeres and Chromosome Stability The Importance of telomeres In providing stability to a chromosome was recognized through studying chromosomes that were broken by Irradiation (138) or by dicentric chromosome breakage during mitosis (120, 121,122). Whereas broken chromosome ends undergo fusion, degradation, and recombination events, telomeres provide stability to chromosome ends. In 1938 Muller coined the term "telomere" for any monopolar chromosome end In reference to the stable polarity of genes and chromosomes in the cell. DrosoDhila telomeres were regarded as Immutable structures due to the low frequency of healing events In Irradiated chromosomes (138, 165). Dicentric chromosomes are broken due to the force exerted by pulling the two centromeres In opposing directions on the mitotic spindle during the cell cycle. McCllntock described the breakage-fusion-bridge cycle in maize (120, 122), In which the unstable ends of the broken dicentric chromosome fuse, producing another dicentric chromosome which Is broken during the next mitotic cycle. However, broken maize chromosomes were sometimes stabilized or "healed" during the breakage-fusion-bridge cycle , presumably due to the addition of telomeres to the broken ends. Dicentric yeast chromosomes were constructed by selecting for recombination events between circular and linear chromosomes In yeast (82, 83), or by the addition of a second centromere to a monocentrlc yeast chromosome (191). Resolution of dicentric chromosomes In yeast results In a novel genome organization, Indicating the Instability of ends broken during mitosis or melosls, however the breakage-fusion-bridge cycle has yet to be demonstrated in yeast. The reactivity of broken ends, resulting In recombination, is evident in site specific substitutive yeast transformation. With the Introduction of linear DNA fragments lacking telomeric regions In yeast, the "broken" ends readily recomblne with genomic DNA at a region homologous to the ends of the Introduced DNA fragment (149, 150, 196). Alternatively plasmlds which contain telomeric DNA at the ends and a yeast origin of replication, ie. an autonomously replicating segment (AJiS region) (33,47,48, 49,70,184, 210), replicate autonomously as linear molecules or minichromosomes when Introduced into yeast (56,65,140,156,193). 3 2. Telomeres and DNA Replication The unique telomerlc structure at chromosome ends must provide a mechanism for the complete replication of the ends of a linear DNA molecule (208). All DNA polymerases that have been characterized require a polynucleotide primer with a 3' hydroxyl group (3" OH) to initiate DNA synthesis and the polymerases elongate exclusively in the 5' to 3* orientation, pj novo, synthesized RNA fragments usually provide the priming function (114), but these RNA fragments are later replaced by DNA which Is synthesized by DNA polymerase from a 5'-upstream primer. Mechanistic problems arise with linear DNA molecules because the removal of the 5'-terminal RNA primer leaves a 5'-gap, hence an incompletely replicated daughter strand on the progeny chromosomes (Fig. la). Circular DNA genomes do not require terminal gap filling mechanisms since primer removal, followed by strand elongation from the adjacent region around 8 circular template, inevitably juxtaposes the 5' and 3' ends in DNA replication. Some prokaryotlc viruses have linear DNA genomes with cohesive end regions or terminal redundancies, and consequently the termini of the DNA molecule are completely replicated through the temporary removal of the chromosome ends by circularIzatlon as in phage A or concatemer formation as In phage T4 or T7 (114, 208). An animal virus, the herpesvirus pseudorables, similarly replicates as circles or concatemers through the Intramolecular ligation of complementary terminal sequences on the linear double-stranded DNA genome (15, 96). The novel mechanism of protein priming for initiation of DNA synthesis described for bacteriophage $29 (158, 207) and adenovirus (44, 46, 81, 134, 161) obviates the requirement for a 5" terminal RNA primer. The 5' terminal proteins on the bacteriophage $29 or adenovirus genomes bind the initiating nucleotides from which DNA polymerase can elongate In DNA replication. In an in vitro system for Adenovirus replication, the 80 kd precursor protein for the 5'-term1ni reacts with dCTP to give an 80 kd-dCTP proteln-nucleotide primer. This interacts with the template DNA molecule at the origin of replication, located 9-18 bp from the terminus of the adenovirus genome, to form a 5' terminal complex on the daughter strand which is elongated by adenovirus DNA polymerase (45,46, 81, 197). 4 4a Figure 1. The 5"-end Problem in DNA Replication and Some Possible Mechanisms Proposed as Solutions. a. The 5' end problem In replication of a linear DNA molecule (208). The 5" ends of the daughter DNA strands remain unreplicated following removal of the terminal RNA primers. b. Cavalier-Smith model(41) for the role of terminal palindromic sequences in completing the chromosome ends after the DNA replication. The 3* overhang on the parental strand folds back on itself due to intrastrand complementarity. Ligation of the parental strand with the daughter strand, a specific endonucleolytic cleavage, and extension of the parental strand in the 5'->3' direction completes replication. c. Covalently closed loop model as proposed by Bateman (11). In this model, the termini are self-complementary and exist as covalently closed loops. DNA replication continues around the end and eliminates the requirement for a terminal primer. d. Crossed strand exchange model proposed by Heumann (87). Completion of the 5' end of the daughter strands occurs by recombination between the the terminal repeat sequences and a similar internal repeat region on the chromosome. e. Dancis and Holmquist fusion model (55,91) for termini completion. Transient fusion and fission of chromosome ends are due to telomeric associations and direct repeats on all termini. Formation and resolution of a cruciform structure In the fission process results in the reproduction of hairpin loop ends. f. Completion of chromosome ends by simple terminal repeat addition reviewed by Blackburn and Szostak (23, 25). Tandem, simple repeats containing specific single-strand gaps are present at chromosome ends. Further repeat unit addition during replication by recombination or a terminal transferase-like enzyme ensures complete replication of the ends. Replication is equivalent for both daughter DNA molecules, hence only the completion of a single DNA molecule is presented for models b, d, e, f. The parental DNA strands are represented by solid lines (—), newly replicated daughter strands are broken lines (----), the RNA primers are vertical lines (»•"•), and the 5" -> 3' polarity of a parental strand is represented by an arrowhead (•*•). Repeat sequences are represented by the boxes and complementary nucleotides are represented by open ( • ) and closed ( E S I ) boxes. Endonucleolytic cleavages In the replication completion mechanisms are indicated by triangles ( V ) . 5 Processing of the 80 kd protein to a 55 kd molecule results In the mature 5" terminal protein covalently linked to either 5'-end of the Adenovirus DNA molecule (43). Viruses provide relatively simple systems which are thought to reflect the replicative mechanisms utilized for completion of the host cell chromosomes. Consequently, models described for the complete replication of the DNA termini on eukaryotic chromosomes are based on terminal structures that have been characterized for eukaryotic viruses. All proposed schemes for the DNA replication of chromosome ends involve DNA strand exchange or temporary elimination of the extreme ends of the linear DNA strands and invoke specialized telomeric sequences or structures (Fig 1). The existence of palindromic DNA sequences that form hairpin structures at the chromosome ends (Fig. 1b) to complete DNA synthesis through strand exchange was proposed by Cavalier-Smith (41). Following gap-filling and ligation, a site-specific endonuclease cleavage, and hairpin transfer results in a 3' recessed end. Subsequently, the 5' to 3' elongation activity of DNA polymerase from the 3' OH completes the terminus. The rolling hairpin model for the replication of the linear single-stranded DNA of parvovirus was based on hairpin transfer (198). In this model a 5' terminal primer is not required because the priming function Is provided by the terminal hairpin structures (44, 134). Direct support for this model was obtained with the DNA sequence analyses of hairpin termini of both viral and replicative form DNA molecules (6, 8, 9, 119). Bateman simplified the Cavalier-Smith model by proposing covalently closed or cross-linked termini on chromosomes (Fig. 1 c) such that the chromosomal DNA molecule Is regarded as circular, self-complementary, and single-stranded. DNA replication then continues around the ends and hence eliminates the terminal gap problem (11). Daughter strands are resolved by an endonucleolytic cut which is specific for the telomeric region, resulting in unfolding of the ends, followed by refolding of the covalently closed ends on the progeny DNA molecules. The terminal structures determined for some linear DNA genomes support the Bateman model. One terminus of the linear mitochondrial DNA molecule of Paramecium contains a cross-link, at least transiently during replication (76). Terminal loops or crosslinks were observed near the ends of the vaccinia virus genome, since denatured viral DNA strands do not separate at the ends (73). DNA sequence analysis of the termini (10) indicated that tandem repeats exist near the 6 ends of the vaccinia virus genome and the terminal 104 nt region exists as a single-stranded loop which is not completely base paired. Alternately, the termini of linear DNA molecules may be completed in DNA replication by a crossed-strand exchange mechanism between an internal repeat sequence and a terminal repeat sequence (Fig. Id), (87) or a modified loopback mechanism described for Tetrahvmena mitochondrial DNA (77). In this model, the Internal repeat region is transferred to the incomplete terminus through recombination, and the resultant gap is completed by the 5' to 3' elongation activity of DNA Polymerase from the internal 3' OH. Cytological evidence of telomeric associations led Dancis and Holmquist (55, 91) to propose the completion of linear chromosome ends through the temporary removal of chromosome ends by a transient fusion followed by a fission process (Fig. 1 e). This model predicts similar DNA sequences, In the same orientation on all telomeres, since this would facilitate the recognition of fused ends during the fission process. Telomeric associations and DNA sequence analyses of telomeric regions in eukaryotic chromosomes or extrachromosomal fragments (described below) provides support for their model. Recently proposed models for completion of eukaryotic chromosome ends are based on the structures characterized for cloned chromosome ends (Fig. If). The addition of terminal repeat sequences to chromosome ends, and single-stranded gaps within the terminal repeats are explained by either recombinational mechanisms (40, 203, 205) or by invoking a novel terminal transferase-like activity for the completion of chromosome ends in DNA replication (175). C. Cytology of Telomeres Transient telomeric associations with one another and with the nuclear membrane are observed cytologically. During mitosis, meiosis, or interphase of the cell cycle, telomeric associations (Reviews 25,55, 91) may be essential for proper chromosome pairing and segregation as well as the non-random distribution of genetic material In interphase nuclei as shown in plants and insects (5, 74, 168, 214). Results are consistent with telomeres of both homologous and nonhomologous chromosomes becoming attached during interphase by chromatin connections. Possibly the termini of the DNA molecules associate by intermolecular ligation during DNA replication (55, 91). 7 Repetitive DNA sequences at telomeric regions In a given genomic complement, possibly resulting in heterochromatic structures at chromosome ends, may account for the transient telomerlc associations. The heterochromatic structure of telomeres in the maize genome was observed as a knob at the chromosome end which lengthens the chromosome end but does not add necessary genetic material (120). Heterochromatln has been observed in other species but It Is generally considered as a nonessential characteristic of telomeric structure (25). The primary structure of the repetitive DNA at the chromosome ends may be the necessary factor for telomerlc functions and associations. Telomeres in Secale species contain repeated sequences along with large blocks of heterochromatin, which display both intraspecies and interspecies heterozygosity (12, 14, 99). In situ hybridization of Drosophila polytene chromosomes with a telomerlc probe, has revealed repeated sequences at chromosome ends and homology of telomeres with heterochromatin in the centromeric region (168, 214). Similarly, repetitive DNA was localized to the heterochromatin of the centromeric regions and some telomeric regions In Rhvnchosciara polytene chromosomes through in situ hybridization with satellite RNA probes (66). Telomeric regions of Xenoous also contain repetive DNA (151). During replication, amplification of telomeric repeat blocks occurs at the ends of the germ line chromosomes in Ascaris which may explain the heterochromatic region observed at the termini (167). Conclusive evidence of repetitive sequences at the telomerlc regions of the chromosomes In a species was obtained using cloned telomeres from yeast (49, 175, 193), trypanosomes (24, 203), and the linear DNA fragments in the somatic macronuclei In dilates (20,22,23,113,147,212). Telomere specific binding proteins or alternate DNA secondary structures may also be responsible for the associative behavior of telomeres. The chromatin structures of the telomeric regions on macronuclear fragments in the single-celled eukaryotes Tetrahvmena. Physarum. and Oxytricha are non-nucleosomal (21,50,79). A deoxynucleoprotein complex exists at the termini of Oxytricha linear fragments (79) and the interaction between telomerlc complexes is proposed. Z-DNA or poly-dG regions may explain the aggregation of telomerlc regions since these structures will self- associate (4, 64,162). Z-DNA may exist at telomeric regions, as demonstrated by the binding 8 of anti-Z DNA antibodies (4) and poly-dG regions have been sequenced at the extreme ends of telomeric regions (Reviews 23,25). D. Molecular Characterization of Telomeres 1. Extrachromosomal Linear DNA In the ciliated protozoans, the germinal mlcronuclear DNA molecules are fragmented, the rDNA genes are amplified, and these linear rDNA gene fragments exist as short chromosome-like molecules in the somatic macronucleus. These naturally occurring amplified DNAs facilitated sequence and structural analyses of the termini on the linear DNA molecules (22). The ends of the amplified 21 kb rDNA palindrome in Tetrahvmena thermophila ( 1 c o p i e s per macronucleus), were the first to be extensively studied (101), and these contain twenty to seventy tandem repeats of the sequence 5'-CCCCAA-3' (20), also referred to 8S 5'-C4A2~3' repeats, with specific single-stranded gaps in the terminal 100 bp of the repeat units (22). The extreme ends of the linear rDNA molecules may be blocked by hairpin loops, since they are not accessible to end-labelling techniques, and do not appear to be covalently bound with a terminal protein. The S'-C^-Z' repeat region varies in chromatin structure from bulk macronuclear DNA which is arranged into typical nucleosomes, as determined by nuclease protection experiments (21). The terminal repeat sequence Is presumably required for telomeric function since other holotrlchous dilates such as Glaucoma (102) and Paramecium (212) have conserved the 5'-C4A2-3' repeat region which is added to the linear fragments during macronuclear development. The related hvpotrlchous dilates. Stvlonvchia (147). Oxvtricha (113), and Euolotes (113) have a similar terminal sequence on their macronuclear DNA fragments, which is po1y-5'-CCCCAAAA-3', or more simply referred to as 5'-C4A4-3' repeat units. Conservation of telomeric repeat sequences In macronuclear and mlcronuclear genomes was demonstrated for Oxytrlcha (59) since the mlcronuclear chromosomal DNA molecules also have variable lengths of terminal 5 ' r e p e a t sequences. Amplified, palindromic linear rDNA molecules In the slime molds Phvsarum (98) and Dictyostelium (67) contain the related terminal repeat sequences 5'-C3TA-3' and 5'-Ci-8^-3* respectively. In Phvsarum. the terminal restriction fragments display length and sequence 9 heterogeneity whereas the rDNA sequences are conserved (17). Single-stranded gaps are specifically located adjacent to the Inverted repeat sequences, 5'-CCCTA-3' or 5'-TAGGG-3' within the one kilobase terminal region in Physarum (17, 98). This region also contains more complex terminal repeat units that may form hairpin ends (17, 98). The extrachromosomal linear fragments in both Oxytricha (79) and Physarum (50) have non-nucleosomal termini and terminal protein complexes. 2. Yeast Chromosomes The molecular cloning of a yeast Saccharomyces cerevlsiae telomere (193) provided sufficient DNA for molecular characterization of telomeres on chromosomes that participate in mitotic and meiotic events during the cell cycle. Yeast telomeres were cloned using, a linear plasmid that had two Tetrahvmena termini and which replicates In yeast (193). Yeast fragments that replaced one of the Tetrahvmena ends were selected In yeast by assaying for the ability of the fragments to replicate the plasmid as a linear molecule (193). A yeast telomeric fragment derived from such a linear plasmid was extensively studied through Southern hybridization and DNA sequence analyses. The structure of yeast telomeres as they are presently understood is Illustrated In Figure 2. Like the ends of extrachromosomal DNA molecules in simple eukaryotes, yeast termini may be covalently closed by a hairpin-like structure (71, 72, 193). The termini on yeast chromosomes contain simple repeat sequences that are tandemly arranged (T region), adjacent to a more complex repeat region (Y' region 8nd X region) which is referred to as the telomere associated region (49, 175, 193). Variable amounts of T region sequence (about 100 bp) exist between the Y' and X regions (205). The 0.3 kb to 0.6 kb terminal T repeat sequence region, as well as the internal T region consists of 5'-C2-3A(CA) 1-4-3' units (175, 205, L.L.Button and C.R.Astell, In press), which are more simply described as 5'-C^ -3A-3' repeats. The T region at the terminus of the chromosome contains specific single-stranded gaps within the repeat units (49, 175). The telomere associated Y' and X regions represent a family of A M regions (47, 48,49), which are presumptive origins of DNA replication in ye8St (Review 210). TheY' regions are conserved 6.7 kb regions that consist of 131 and Y regions, and are present in a tandem array of 1-4 copies on more than 50$ of the chromosome ends in the yeast genome (49, 205, 206). Alternatively, X regions are heterogeneous in restriction map and 10; Figure 2. Organization of the Repeat Sequences at Yeast Telomeres. Yeast ARS elements of the Y* and X classes have been mapped to the telomere associated regions on yeast chromosomes. The arrangement of the repeat regions at yeast telomeres was described initially by Chan and Tye (49) and was modified by Walmsley et al. (205). The T region (WM ) represents 5'-Cj -3A-3' simple tandem repeat sequences located at the terminus of the chromosome as well as the Y'-X junction region. The first class of yeast telomeres described (a) has between 1 and 4 conserved 6.7 kb Y' elements, consisting of Y ( • ) and 131 ( I H ) regions, adjacent to a single, heterogeneous X (flTrnTi) element. A variable length of T region separates the Y" and X elements on these telomeres. The second class of telomeres (b) contains a single heterogeneous X element adjacent to the T region at the chromosome end. Unique DNA sequences on the chromosome ( ) map centromere proximal to the X element in both the T-Y'-X and the T-X types of telomeres. 10a, Y' T (1-4) [ . 3 - . 6 k b ] [ 6 . 7 k b ] -cen [ . 3 - 3 . 7 5 k b ] T X 11 length (0.3 - 3.75 kb), are present In single copy on all chromosome ends In the yeast genome, and have no homology with the Y' region ( 49). Consequently, there are at least two classes for yeast chromosome ends: (1) T-Y'-X telomeres, (Fig. 2a) and (2) T-X telomeres, (Fig. 2b). The telomere isolated on the linear yeast plasmid by Szostak and Blackburn (193) belongs to the T-Y'-X class and the T-X yeast telomere has been observed in Southern hybridization analyses with yeast telomeric probes (93,205, 206). 3. Trypanosome Chromosomes Telomeres of the hemoflagellate Trypanosoma brucei have been isolated by exploiting the known properties of chromosome ends: simple terminal repeats, single-stranded gaps near the ends, and Bal31 nuclease sensitivity of the termini (24, 203). The termini of T. brucei chromosomes consist of multiple tandem repeats of the sequence 5'-CCCTAA-3', also referred to as 5'-C3TA2-3" repeats, adjacent to a region of more complex repeats (24, 203). Other flagellate species apparently have retained the terminal simple repeat region (24). Progressive growth of telomerlc restriction fragments (7-10 bp per generation) has been observed in T. brucei (18) through Southern hybridization analyses with probes from the 3' ends of telomerically located variant surface-antigen genes (YSG). Prosposals for the terminal addition of 5 -C3TA2-3 ' repeats during replication explain telomerlc growth properties (24, 203). E. Artificial Chromosomes in Yeast 1. Heterologous Telomeres in Yeast The cloning of yeast telomeres was preceded by the demonstration that termini from Tetrahymena rDNA molecules provided replicative stability to a linear plasmid in yeast (193). Following the propagation of the linear plasmids In yeast, the Tetrahymena rDNA ends had been transformed into yeast-like telomeres in that yeast terminal repeat 5'-C ] -3A-3" regions (100-300 bp), with yeast specific terminal gaps, were added to the ends of the Tetrahvmena 5 -C4A2 -3 ' repeats (175,193,204). Similarly, linear plasmids were stabilized In yeast with Oxvtrlcha fallax termini 12 (5'-C4A4-3* repeats), and these were capped by yeast terminal repeat addition (300-1000 bp of 5'-Ci_3A-3' repeats), (156). The ability of termini from dilate macronuclear DNA to function In the ohvlooenetlcallv distinct yeast S. cerevlslae may reflect the conservation of a mechanism for DNA replication of chromosomal termini (56, 65, 140, 193, 195). Presumably terminal repeat structure, rather than recognition of a specific sequence, Is essential for telomere replication and stability. Consistent with this Idea, the Phvsarum polvcephalum rDNA molecule Is maintained in yeast in a linear form, In the absence of selection (117). 2. Artificial Chromosome Construction and Stability The construction of artificial chromosomes In yeast was facilitated by the following observation. The terminus of the Tetrahymena rDNA molecule Is not essential for a functional telomere In yeast since a circular clone containing an inverted repeat of Tetrahvmena rDNA ends (1e. lacking the terminus region but containing a portion of the S ' - C ^ ^ ' repeat region) Is resolved Into a linear molecule with two functional telomeres In yeast (195). Cloned Tetrahymena rDNA ends were readily available and could be Inserted as an Inverted repeat in a circular plasmid construct, then resolved in yeast to render linear mlnlchromosomes (65,140,194,195). The four known elements required for chromosome stability and replication are genes, ARS elements, centromeric region, and telomeres, and these were recomblned In vitro to render artificial yeast chromosomes (56, 140, Reviews 26, 141). Natural yeast chromosomes are maintained in single copy in haplold cells and the mitotic stability (chromosome loss per generation) ranges from 10~ 4 to 10~ 5 (139). Centromeric mlnlchromosomes, containing the LEU2 gene, CEN3 and ARS1 regions, with Tetrahymena rDNA termini are maintained In single copy in yeast cells If the length approaches the range of chromosomal DNAs ( 10 2 to 10°* kb), (140) although the mitotic stabilities of long artificial chromosomes remain at least two orders of magnitude less than natural chromosomes. Possibly an unidentified element or specific spacing of elements may be required for stability. Artificial yeast chromosomes have not been constructed with natural yeast telomeres but these may be required for the proper mitotic segregation of artificial chromosomes due to requisite telomerlc associations In the cell (56,140, Review 25). 13 F. Stabilization of Linear Plasmlds In Yeast Yeast telomere associated Y' regions enhance chromosome end stability and provide healing functions to broken chromosome ends (65). A linear plasmid with a partial Y' region (ie. broken artificial chromosome end) acquires additional Y' regions through a RAD52 dependent recombination mechanism and the mitotic stability of the linear plasmid Increases with further Y' region addition (65). The yeast RAD52 gene product is required in recombinational events that involve double-strand break Initiation (Review 196). Linear plasmlds with Tetrahvmena termini are stabilized or "healed" in yeast with the addition of T region repeats (5'-Ci_3A-3') by a RAD52 Independent mechanism (65, 215), followed by BAT25_2_ dependent Y' region addition as observed with broken yeast chromosome ends (65). Addition of Y' ARS regions to Tetrahymena termini reflects the requirement for strong ARS activity at yeast chromosome ends (65). Increased ARS activity refers to a more efficient and regulated replicator which is defined by increased transformation frequency and mitotic stability on plasmids in yeast (33, 42, 104). Internal to the S ' - C ^ - 0 " ^ P 6 9 * r 8 9 ^ o n o n * n e Tetrahymena rDNA end there is a region which contains the ARS consensus sequence (33), and which functions as a yeast origin of replication (1,105,139,140). However, the yeast Y' and X telomeric ARS regions have higher ARS activity than the region on the Tetrahvmena rDNA end (65). The role of the telomere associated X region in telomere healing or chromosome stability has not yet been defined. Y" region recombination occurs among chromosome ends within a given yeast strain. Although the telomere associated Y' region is highly conserved, there are restriction enzyme site polymorphisms among different yeast strains, and among meiotic segregants within a given strain (93). The extensive homology of the yeast chromosome ends (49) and the DNA repeat sequence units within the Y region (92) may result in the rearrangement of Y regions, along with the stabilization of broken chromosome ends, through gene conversion or other recomblnational events. 6. Genetic Control of Telomere Length In Yeast Different yeast strains display variation in the terminal restriction fragment lengths due to strain specific lengths of the T region (5 ' -Ci -3A-3 ' repeat units), (40, 93, 206). All chromosome ends within a given yeast strain have similar amounts of telomeric T region repeats and apparently the 14 length Is genetically controlled by a set of genes that are co-dominant In a diploid yeast strain (206). The cell division cycle CDC17 gene is at least partially responsible for the control of T region length at the chromosome end (40). Strains with temperature sensitive mutations In CDC 17 demonstrate progressive growth In telomere length in a BAI>5_2 Independent manner, due to deficiency In functional CJ)CJ2g8ne product (40). H. Isolation and Characterization of the Telomerlc Region from a Specific Yeast Chromosome 1. Chromosome Walking Limited information was known about the structure of eukaryotic chromosome ends when this research project, Involving the Isolation a specific yeast telomere, was proposed. Tetrahvmena rDNA macronuclear fragments were shown to terminate In variable lengths of 5'-C4A2-3' repeats and possibly with hairpin protected ends (20). Yeast S. cerevlsiae chromosome ends were thought to exist as covalently closed loops (71, 72), but other terminal structures such as protein primers, palindromes, and repeat sequences (discussed earlier) were also plausible. Consequently, we decided to Isolate a S. cerevlsiae telomere by chromosome walking from a distal locus that was characterized on the chromosome end, since this cloning method does not select for a specific terminal structure or function. The technique of chromosome walking (13,51) involves the use of a cloned chromosomal fragment as a hybridization probe to screen collections of genomic DNA clones for the Isolation of novel fragments that overlap the original sequence. Hence through progressive steps, contiguous stretches of a particular chromosomal region can be analyzed. Chromosome walking Is facilitated In yeast compared to higher eukarotes due to the relatively small amount of repetitive DNA, (about 5%), In the yeast genome (32, 69). Chlnault and Carbon (51) were able to clone the centromere from yeast chromosome III (CEN3 region) by chromosome walking from LEU2 on the left end (IIIL) to CDC 10 on the right end of chromsome III (MIR). Identification of CEN3 in the Isolated region was confirmed by assaying for the centromeric stabilization of autonomous plasmids in yeast (53). 15 A similar approach was adopted In this study to Isolate the telomeric region on chromosome 11 IL by chromosome walking from the most distal known gene on 11 IL toward the telomere. The average length of the terminal regions on yeast chromosomes was estimated, given several assumptions. Firstly, the yeast genetic linkage map, which consists of seventeen linkage groups (136), is complete between the distal markers on any given yeast chromosome. If so, then any DNA that Is not accounted for In the linkage map must exist at the termini of the chromosomes. Secondly, the physical size and genetic linkage map proportionality of 2.7 kb/cM, determined for chromosome III by Strathern et al. (186), represents an average value for other regions of the yeast genome. Thirdly, equivalent lengths of terminal regions exist on all chromosome ends. The average terminal length was calculated as follows. The haplold yeast genome contains 1.4 x 10 4 kb of DNA which Is equivalent to 5185 cM. The genetic linkage map encompasses about 3500 cM (136), hence 1685 cM Is unaccounted for and may be distributed equally among the thirty-four chromosome ends. If these assumptions are valid, about 50 cM or 135 kb separates the distal genetic marker from the terminus on each yeast chromosome. Based on this estimated length, chromosome walking would appear to be a feasible approach to telomere Isolation since at least 15-20 kb could be covered per step with clones prepared with lambda vectors (125). Since telomeres provide stability and complete replication to chromosomes, telomeric DNA must have distinctive characteristics recognizable through DNA sequence analyses, Southern hybridization and by assays for a functional telomere on linear plasmlds In yeast. In this manner a yeast telomeric region could be Isolated without prior knowledge of its structure, and the entire region between the telomere and the unique DNA on the chromosome end could be characterized. 2. Yeast Chromosome III Yeast chromosome III was selected for the isolation of a telomere by chromosome walking for several reasons. The most distal known genetic marker on 11 IL is HMLpc. one of the mating type genes. Mating type genes HMLpy. MAT, and HMRa have been cloned (142), sequenced (7), and their orientation on chromosome III Is known (187). Yeast strains containing circular derivatives of chromosome III were available (109) and were Invaluable controls for progression towards the 16 telomere during chromosome walking experiments. Such circular III strains resulted from recombination between the mating type loci, as occurs during yeast mating type interconversion or switching events (143, 109). Information at the MAI locus situated on the right arm of III determines mating type. HML and HMR are unexpressed storage cassettes for <x and a Information respectively, and are repressed by the concerted action of unlinked loci known as SJR, MAR. or CML genes. Mating type interconversion is a gene conversion event, and is initiated in homothallic (HO) yeast strains by the HO endonuclease double-stranded break (115, 116) within the recipient MAI locus (111, 188). In standard yeast strains (MAR+), only MAI switches mating types while the HML and HJjJi loci remain unchanged (106). However, mjr ior macZ strains switch efficiently at HH loci as well as MAI (108). Circular chromosome III strains were derived from a J i a i i parental strain by recombination events between HML and HMR (109). Presumably the telomeric regions of chromosome III are deleted in the circular III strains. Haploid circular III yeast strains are viable (109), indicating there are no essential genes distal to HMLo, and that the distance between the IIIL telomere and HML<x Is relatively short. Electrophoretic karyotype analyses (36, 37, 173) indicate that chromosome III is 370 - 390 kb in length. Since 350 kb separate the chromosome III distal markers HMLy, and MAL2 (107), the total amount of DNA distal to HMLtt, and MAL2 is probably 20 -40 kb. 3, Characterization of the Telomeric Region on 111L In this study it was found that the IIIL terminus mapped 12 kb distal toHMLo, as determined by Bal 31 nuclease sensitivity and the presence of a T region consisting of 5'-[C2-3A(CA) j -4 ] -3 ' repeat units. Characterization of the cloned telomeric region from IIIL by Southern hybridization and DNA sequencing analyses revealed that the 11IL end Is a T-X class telomere which extends distal to the IIIL unique region with an average length of 1.8 kb and a size distribution of + /_ 0.2 kb for the terminal length. Telomeric properties described using probes from T-Y'-X telomeres (49, 65, 92, 93, 175, 193, 205, 206) were assayed with the novel T-X telomeric probe which hybridized exclusively to chromosome ends. The ability to transform yeast with cloned DNA (89) facilitated the 17 studies for telomeric function that were conducted by cloning the IIIL end on linear plasmlds In yeast (193, 194, 195). The required Information for a functional IIIL telomere was 50 bp (or possibly less) of the T region and the adjacent X A£S region. Linear plasmids containing deletions in the telomeric fragments from IIIL had T region addition but were never healed with Y' regions. Hence the T-X class of the IIIL telomere was maintained on linear plasmlds in yeast and a rationale for the existence of two classes of yeast telomeres, T-X and T-Y'-X, is discussed. Hybridization analyses of linear 111 and ring Ml yeast strains with HMLft distal probes indicated that the 9 kb terminal region on MIL Is retained in most circular Ml yeast strains through replacement recombination with an alternate homologous region In the yeast genome. HMLqi distal probes hybridized with an undefined yeast RNA species which led to the novel suggestion that there may be a unit of transcription in the HMLoi distal region which is functionally duplicated at the alternate region. 18 MATERIALS AND METHODS A. Materials All chemicals were analytical or reagent grade. Acrylamide, blsacrylamlde and TEMED were purchased from B1o-Rad Laboratories; agarose (Type I) was from Sigma Chemical Company, and Low Melting Point (LMP) Agarose was from Bethesda Research Laboratories. Nitrocellulose filters (BA85) were from Schleicher and Schuell; Gene Screen or Gene Screen Plus was purchased from New England Nuclear. [<x 3 2 P ] dNTPs (2000-3000 Ci/mmol) were either from New England Nuclear or Amersham Corp., deoxy NTPs and dldeoxy NTPs were obtained from P.L. BiochemIcals Inc. M13 oligonucleotide primers were synthesized on an Applied Blosystems oligonucleotide synthesizer. Restriction enzymes were purchased from New England Blolabs, Bethesda Research Laboratories, or Boehringer Mannheim and used as specified by the supplier. L fflli DNA Polymerase I (Klenow fragment) was supplied by Bethesda Research Laboratories, Boehringer Mannheim, or Promega Blotec; LcpJ i DNA Polymerase I (Kornberg enzyme) was supplied by Boehringer Mannheim or Bethesda Research Laboratories. Nuclease Ba131 and Exonuclease III were supplied by New England Blolabs; SI nuclease and T4 DNA Llgase were from Bethesda Research Laboratories. DNase I and Bacterial Alkaline Phosphatase (BAP) were purchased from Worthlngton, Calf Intestinal Alkaline Phosphatase (CIP) was from Boehringer Mannheim. Zymolase W8S from Kirin Breweries Co. Ltd. and Glusulase was from DuPont Pharmaceuticals. Bovine Serum Albumin (BSA) and Lysozyme were purchased from Sigma Chemical Co. BSA (DNase free) was supplied by Bethesda Research Laboratories. B. Strains and Media 1. Yeast Yeast strain AB20a XP8-10B (MA fa hohis61eutmetltrp5- ]gal2cant), described by Nasmyth and Tatchell (142), was used for cloning the IIIL telomere. Strain K45 ( H M U & MAM HMfa mar1hotrptthr1drg4lys1ade8his2ura1mar) and the HML -///Wfusion derivatives, K191 and K192, as well as other circular III strains K193 [ MA fa (HML -HMRfu$ion)manade8- 10lyst mar], K195 [MAfc (HML -HMR fusion) mar I ura/ /ys\, and K196 \MAfa (HML-HMR 19 fusion)mar Itrp)'- f/ysh were gifts from A. Klar, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (109). AB972 {MAfctrp tochri), a S288c related strain, was provided by M. Olson, Washington University, St. Louis, Mo. (37, 169). Strains SR25-1A ( MA fa his4-9!2ira3-52 and SR26-12C ( M A f c h J s 4 - 9 t 2 > r a 3 - 5 2 e u 2 - 3 , / /jPwere gifts from S. Roeder, Yale University, New Haven, Conn. Strains T388 [ ( M A M leu2-3,112ii$3-11,13ura3lrp Item 1) + pSZ216 ( L£U2his3)\, (193), and A281 (formerly LL20-11-2), (63), a d r ° derivative of A2 {MAM leu3,112hi$3-11, IScanh were gifts from J . Szostak, Harvard Medical School, Boston, Mass. (195). Yeast strains were grown at 30 °C with aeration in complete medium, YEPD (\% yeast extract, 2% peptone, 2% glucose), or in synthetic complete medium ( 0.67* yeast nitrogen base without amino acids, 2% glucose with all amino acids added except the selected marker amino acid), (176). Amino acids were prepared as a 100 x mixture containing 2 mg/ml of each of L-trp, L-ala, L-cys, L-met, L-pro, 3 mg/ml for each of L-11e, L-lys, L-tyr, L-leu, 5 mg/ml L-asp, L-glu, L-phe, and 10 mg/ml for L-ser, L-thr, L-val. The 100 x stock also contained 1 mg/ml uracil, and 2 mg/ml adenine sulfate, (166). Yeast Regeneration Agar was equivalent to selection medium, with the addition of 1M Sorbitol, and 2 * Agar (177). 2. Bacteria Escherichia coll strains DP50 [f, tonA53,dapD8,lacY1 ,glnV44XsupE4¥, Mga/-uvrH47,\-,tyrT5#>supF5%gyrA29, A ( thyAST, hsd$3\ or LE392 [F,hsdR5f4rfmf), supE44$upFS8JacY1or A ( lacl2y6ga1K2galT22/netB 1 JtrpR55, A - ] (126) were hosts for ACharon4A clones. Growth was In LBMgT medium which consists of LB medium [ 10 g/1 bactotryptone, 5 g/1 yeast extract, 5 g/1 NaCl, (pH 7.2)], (57), supplemented with 10 mM MgCl2 and 50 mg/1 thymidine. LBMgT plates contained 1.5 % agar, while top agar contained 0.7 % agar or 0.7* agarose. L e s l i e \r,hsdS2(Krg,m£r),ara- t4,proA2,lacYt,ga)K2,rpsL2(KSm r), xyl-5mtl-1, ^ W A ] , ( 1 2 6 ) , D H 1 [ F-,recA1,endA1,gyrA96,thi,hsdRlAr£ mf),supE44,relA1?, A - ] or MM294 [ F-,endA l.hsdRfK rf ) ?upE44, thi- / ^ " ], (130) were host strains for plasmid clones. Growth of RR1, DH1, or MM294 was In LB medium for mlnipreparations (5-10 ml), and In M9-m1nimal salts medium (6g/1 Na2HP04, 3 g/1 KH2PO4, 1g/l NH4CI, 0.5 g/1 NaCl, ImM 2 0 MgS04,0.1mMCaCl2, 1 mM thiamine HC1, and 0.2$ glucose), (57), for 0.5 -1 liter cultures. For selective growth, amplclllln (AMP) was added to 50 mg/1, tetracycline (TET) was added to 12.5 mg/1, or chloramphenicol (CAM) was added to 20 mg/1. Amplification of plasmid DNA in cultures was with 170 mg/1 chloramphenicol, or 10 mg/1 for amplification on agar plates. L Qili JM 101 ( supE , th i , -ldcproF'traD36proABjaclQZ-Ml$), (132) was propagated In M9-m1n1mal salts or YT media, [8 g/1 Tryptone, 5 g/1 yeast extract, and 5 g/1 NaCl, (pH 7.2-7.4 )], (132). All strains were incubated at 37 °C, with aeration. 3. Phaoe and Plasmids Lambda Charon4A vector DNA and phage packaging extracts were gifts from T. Snutch and I. Kovesdl, respectively, Simon Frassr University, Burnaby, B.C. The recombination screening system, I1AN7 vector and host strain MC1061 (p3), (H. Huang, Washington University, St. Louis, Mo.), was a gift from R. MacOlllivray, University of British Columbia, Vancouver, B.C. The AMG14-Yeast (AB972) genomic library was kindly supplied by M. Olson, Washington University, St. Louis, Mo. The yeast genomic library in plasmid vector YEp 13, prepared by K. Nasmyth (142), was a gift from M. Zoller, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Plasmids pSZ220 (193), pSZ93, pSZ221, pSZ222, (195, 150), and pSZ218 (194) were kindly provided by J.W. Szostak, Harvard Medical School, Boston, Mass. Plasmids YRp131A and YRp131B (48, 49) were gifts from C.Chan and B.-K. Tye, Cornell University, Ithaca, N.Y. C. Basic Molecular Cloning Techniques Most basic molecular cloning techniques were conducted as described by Manlatls et al. (126). Those routine methods Include agarose and acrylamide gel preparation, restriction enzyme site mapping, Isolation of DNA fragments from low melting point (LMP) agarose, phenol/chloroform (CHCI3) extractions, ethanol precipitations, and quantitation of DNA using EtBr/agarose plates. 21 D. Yeast DNA Isolation 1. Large Scale DNA Preparation High molecular weight yeast DNA for genomic library construction was isolated from S^ . cerevlsiae AB20a XP8-10B cells by the spheroplast lysis - sucrose density gradient and EtBr cesium chloride equilibrium gradient purification method of Olson et al., (148). Cells from a 500 ml culture grown in YPD medium, at 30 °C, to late log or stationary phase (1 x 10 8 cells/ml), were pelleted at 4000 g, 10 min, 4 °C. After washing the cells with 30 ml distilled H2O, resuspenslon was with 2 ml spheroplastlng mix per gram wet weight of cells, [ 1 M Sorbitol, 0.1 ti NaCltrate, 0.06 M EDTA (pH 7.0), 1 $ 2-mercaptoethanol]. Zymolase 5000 (5 mg per gram of cells) was added and the suspension was Incubated at 37 °C for 2 hr. Cells were lysed by slowly adding 3.5 ml lysis buffer per gram of cells, [3$ sarkosyl, 0.5 M Tris, 0.2 M EDTA (pH 9.0)] and Incubating at 65°C, 15 min. After quick chilling the lysed cell mixture on ice until the temperature was reduced to about 23°C, the mixture was loaded on sucrose step gradients (3 ml 50$ sucrose cushion, 26 ml 10$ - 30$ w/v sucrose) prepared In Beckman polyallomer SW27 tubes. Gradients were centrifuged at 11000 rpm, 16 hr, 9 °C in a SW27 rotor. After removing the top layer, the viscous fraction at the 15 - 20$ sucrose Interface was collected with a wide bore plpet. The DNA solution was dlalyzed against TE [ 10 mM Tris, 1 mM EDTA, (pH 7.5)] for about 12 hr at 4 °C, with at least one buffer change. Since there are no DNA pelleting and resuspension steps in this protocol, the probability of shearing DNA during the preparation Is minimal. However, the preparation of sucrose gradients, subsequent fractionation, and dialysis of DNA fractions makes this method more tedious than later more efficient methods. The preferred method for isolation of yeast high molecular weight DNA from 50 ml to 1 liter cultures was a modification of the Stiles protocol (183). For each 50 ml stationary phase culture (2x10 8 cells/ml), the cells were pelleted at 4000g for 5 min, and washed with 3.5 ml TE [50 mM Tris, 50 mM EDTA, (pH7.5)]. Resuspenslon was In 3.5 ml spheroplast solution [ 1 M Sorbitol, 0.1 M EDTA (pH8.0), 14 mM 2-Mercaptoethanol], and Zymolase 60,000 (2.5 mg) was added. After Incubation at 37 °C for 1 hr, spheroplasts were pelleted at 4000 g, 5 min, 4 °C. The spheroplasts 22 were resuspended in 3.2 ml TE [ 10 mM Tris, 1 mM EDTA, (pH 7.5)] followed by the addition of 0.32 ml 0.5 M EDTA(pH8.0), 0.16 ml 2 M Tris base, 0.16 ml 10 * SDS. Cell lysis was completed by Incubation at 65 °C for 30 min. Cell debris was removed by precipitation with 0.8 ml 5 M KAc (pH 4.8), Incubation on ice for 1 hr, and centrlfugatlon at 20,000 g for 15 min at 0°C. The supernatant was transferred to a fresh tube and DNA was precipitated with 2.5 volumes of EtOH (95*) at 23 °C. DNA was pelleted by 12,000 g centrifugation at 23 °C for 15 min, and resuspended In 5 ml TE [ 10 mM Tris, 1 mM EDTA, (pH 7.5)]. DNA was purified by centrifugation in EtBr-ceslum chloride equilibrium density gradients. Alternatively, yeast DNA was isolated by a similar spheroplast lysis protocol (177), however It was more time consuming and produced DNA that frequently gave partial digestion with restriction enzymes. 2. Small Scale DNA Preparation Minlpreparatlon yeast DNA (58) was used for the preliminary analysis of yeast transformants. Each transformant was grown in a 5 ml culture of selection medium if the selected amino acid marker was on an autonomously replicating (ARS) plasmid. If the selected marker was Integrated Into the genomic DNA of the yeast transformant, growth was in complete medium (YPD). Cells were pelleted for 5 min in a table top centrifuge (2500 g), then washed with 1 ml 1 M Sorbitol and transferred to eppendorf tubes. The cell pellet was resuspended in 0.5 ml of spheroplast solution (1 M Sorbitol, 50 mM KH2PO4, 14 mM 2-mercaptoethanol) and 0.2 mg Zymolase 5000 was added. Following Incubation at 30 °C for 30 min, spheroplasts were pelleted, resuspended In 0.5 ml lysis solution [50 mM EDTA (pH8), 0.2* SDS], and Incubated at 70 °C for 15 min. To remove cell debris and SDS, 50 Jul 5 M KAc (pH 4.8) was added, tubes were left on Ice 30 min, then centrifuged at 12,000 g at 4 °C for 15 min. Supernatants were 8dded to 2 volumes EtOH (95*) at 23 °C, then centrifuged at 12,000 g at 23 °C for 15 sec. DNA pellets wer8 dried In air, and resuspended in 50 JJLI TE [ 10 mM Tris, 1 mM EDTA, (pH7.5)] with 10 jjig/ml RNaseA. If DNA was to be used for restriction enzyme digestion, it was first purified by Phenol/CHCl3 (1:1) extraction and EtOH precipitation. 23 3. Yeast DNA Purification Yeast DNA was purified by EtBr-cesium chloride equilibrium density gradient centrffugation. DNA isolated from a 500 ml culture was made to 9.4 ml with TE [ 10 mM Tris, 1 mM EDTA, (pH 7.5)], then 0.5 ml lOOx TE was added, along with 10.02 g CsCl. After transfer to a Beckman polyallomer 16 x 76 mm quick seal tube, 0.15 ml ethldlum bromide (10 mg/ml) was added. Centrifugation was at 60,000 rpm in a Ti70.1 rotor for 16 - 20 hr at 15 °C. The DNA band was visualized with long wave (366 nm) UV light, recovered with a syringe and 20G needle. EtBr was removed with n-butanol extractions, and the DNA solution was dialyzed against TE for 12 hr at 4 °C, with 1 or 2 buffer changes. DNA concentration was determined by A 2 6 0 measurement, and by comparison with DNA standards using agarose/EtBr gel electrophoresis and UV fluorescence (126). E. Lambda Phage DNA Isolation 1. Laroe Scale DNA Preparation DNA from phage lambda clones was Isolated using a modification of the reported procedures (126). Host LE392 bacteria (1.25 ml fresh stationary phase culture) were diluted with 1.25 ml of 10 mM CaCl2,10 mM M0CI2, then Infected with about 1.2 x 10 8 pfu of a given phage clone. Following adsorption at 37 °C for 10 min, cells and phage were used to Inoculate 250 ml LBMgT medium that was preincubated at 37°C. The culture was shaken vigorously (250 rpm) in a New Brunswick Scientific incubator until cells were lysed ( 6 - 8 hours). Complete lysis was ensured by adding 3 ml CHCI3 and shaking (100 rpm) for an additional 3 min. Cells were pelleted at 8000 g at 4 °C for 15 min. The supernatant was added to 0.15 x volume 5 M NaCl, and 0.3 x volume 50 % PEG, mixed by inversion, and incubated overnight (16hr) at 4 °C. The precipitated phage were collected by centrifugation at 8000 g, at 4°C for 15 min. The phage pellet was resuspended In 5 ml DNase I buffer [50 mM Tris (pH 7.5), 5 mM MgCfc, 0.5 mM CaC^], and 50 JAI DNase I (1 mg/ml) plus 100 )i1 RNase A (5 mg/ml) were added. After incubation at 37 °C for 30 min, the phage mixture was centrifuged at 14,000 g for 5 m1n. Phage supernatant was treated with 0.5 ml 10$ SDS, 50 yl 0.5 M EDTA (pH7.5),and 150 }*1 proteinase K (5 mg/ml), and Incubated at 68 °C for 1 hr. DNA was purified by 24 phenol / CHCI3 extractions and EtOH (95*) precipitation. DNA (about 0.5 mg) that was Isolated from a 250 ml culture, was resuspended with 0.5 ml TE [ 10 mM Tris, 1 mM EDTA (pH7.5)]. 2. Small Scale DNA Preparation The procedure described above was also used to isolate phage DNA from 10 ml cultures. A single plaque was used to infect 50 jil of host LE392 cells. Prior to removal of the phage coat with SDS, EDTA, and proteinase K, an aliquot (10*) was reserved at 4 °C for inoculation of large scale preparation. Phage DNA prepared in this manner was restriction enzyme digested in the presence of Bovine Serum Albumin, or BSA (50 jig/ml) and RNase A (50 jig/ml). 5. Phaoe DNA Purification On occasslon, phage DNA preparations were not readily digested with restriction enzymes. Such DNAs were purified by one of the following methods, (a) DNA was ethanol precipitated in the presence of ammonium acetate, with 0.5 volume 7.5 M NH4OAC and 2 volumes ethanol (95*). For DNA concentrations greater than 50 jig/ml , Incubation at -70 °C for 30 min was sufficient to precipitate DNA. DNA concentrations less than 50 jig/ml required -20 °C incubation for 12 hr. (b) Impurities were efficiently removed by CsCl density gradient centrifugation. Phage DNA (100 jxg) was brought to 3.8 ml volume in TE [50 mM Tris, 10 mM EDTA, (pH 8.0)], then CsCl (3.9 g) and EtBr (0.3 ml of 10 mg/ml solution) were added. Centrifugation was in Beckman 1 3 x 5 1 mm polyallomer tubes in a VT165 rotor at 50,000 rpm at 20 °C for 16 hr. The phage DNA band was recovered, n-butanol extracted to remove EtBr, adjusted to 3 ml with TE [ 10 mM Tris, 1 mM EDTA, (pH 7.5)], and DNA was precipitated with 3 volumes ethanol (95*) at -20 °C for 12 hr. F. Plasmid DNA Isolation All solutions used for large or small scale plasmid DNA preparations were chilled on ice prior to use. 1. Large Scale DNA Preparation Preparative scale plasmid DNA was Isolated by the detergent lysis protocol (57) from 500 ml cultures of M9-minimal medium that were amplified with CAM. Cells were pelleted at 4000 g at 4 OC for 10 min, and washed with 25 ml TE [35 mM Tris, 100 mM EDTA, (pH 8.0)]. Cells were 25 resuspended In 10 ml STE [18$ sucrose, 35 mM Tris, 100 mM EDTA, (pH 8.0)], and 2 ml fresh lysozyme solution (10 mg/ml In STE) was added. Following Incubation on ice for 20 min, 2 ml 0.5 M EDTA (pH 8.0) and 0.5 ml RNaseA (2 mg/ml) were added. Following RT incubation for 5 min, cells were treated with 20 ml of lysis solution [ 1 $ Triton X, 15 mM EDTA, 50 mM Tris, (pH 8.5)] at 0 °C for 10 min. Cell debris was pelleted by centrifugation In a T145 rotor at 30,000 rpm at 4 °C for 1 hr. The supernatant was phenol extracted, then 2 or 3 volumes of ethanol (95$) were added, and left at -20 °C, overnight. Precipitated nucleic acid was pelleted at 2500 g at 4 °C for 10 min, and resuspended In 1 mlTE[10mMTr1s,0.1 mM EDTA (pH 8.0)]. 2. Small Scale DNA Preparation Analytical preparations of plasmid DNA were Isolated by the alkaline lysis procedure (19) from 5 ml stationary phase cultures. Cells from 1.5 ml aliquots were pelleted by centrifugation in an Eppendorf microcentrifuge, at 12,000 g at RT for 1 min. Cells were resuspended in 0.1 ml fresh lysozyme buffer [50 mM glucose, 25 mM Tris (pH 8.0), 4 mg/ml lysozyme]. After 5 min at RT, cells were lysed by adding 0.2 ml fresh lysis solution (0.2 N NaOH, 1$ SDS). The tubes were Inverted several times, then left 5 min at 0 °C. Cell debris and SDS were precipitated with 0.15 ml Ice cold KAc (pH 4.8). Tubes were vortexed for 10 sec, left 5 min at 0 °C, then centrifuged at 12,000 g at 4 °C for 5 min. The supernatant was extracted with phenol/CHCl3. Nucleic acid was precipitated by addition of 2 volumes RT ethanol (95$) with incubation at RT for 2 min and was pelleted with centrifugation at RT for 5 min. The precipate was washed with 1 ml ethanol (70$), dried under vacuum, and resuspended In 50 j*l TE [ 10 mM Tris, 1 mM EDTA (pH 7.5)]. RNaseA was added to 20 jug/ml and plasmid DNA was stored at -20 °C. 3. Plasmid DNA Purification Preparative scale plasmid DNA was purified on EtBr cesium chloride equilibrium density gradients (1 or 2 gradients per DNA preparation from a 500 ml cell culture). For each gradient, the DNA solution was adjusted to 9.2 ml with TE [ 10 mM Tris, 1 mM EDTA( pH 7.5)], and 0.5 ml 100 x TE and 9.7 g CsCl were added. After transfer to a polyallomer 16 x 76 mm quick seal tube, about 0.8 ml EtBr solution (10 mg/ml) was added in the neck of the tube using a syringe and needle. The 26 tube was heat sealed, then shaken to mix the EtBr and CsCl solutions. Centrifugation was at 60,000 rpm In a T170.1 rotor for 16 hr at 15 °C. The closed circular band of plasmid DNA was recovered from the gradients as described for yeast DNA purification on CsCl gradients. 6. Yeast RNA Isolation Total yeast RNA was isolated according A. Spence (manuscript in preparation), and RNA from strain GM3C2 or BM-CYC+ was provided by A. Spence and B. McNeil, U.B.C., Vancouver, B.C. Yeast cells from a 50 ml log phase culture (2 x 10 7 cells/ml) were treated with 0.25 ml cyclohexlmlde solution (20 mg/ml In 9 5 * ethanol), and cells were pelleted at 3000 g at 4 °C for 5 min. Cells were washed in 10ml ice cold extraction buffer [0.1 M Tris (pH 7.5), 0.15 M NaCl, 0.1* DEP], pelleted, and resuspended In 0.25 - 0.5 ml extraction buffer at 0 °C. Cells were broken after adding 0.05 volume VRC (0.2 M) and acid treated glass beads (0.45 - 0.50 mm diameter) to below the surface level of the liquid in the tube. Vortexing at maximum speed for 5 times in 15 sec pulses, with 45 sec intervals on Ice between each pulse, served to break the cells open as determined by phase contrast microscope analysis of an aliquot t8ken from the supernatant. The supernatant was transferred to a sterile Eppendorf tube along with 3 x 100 >il rinse solution from beads. The combined supernatant was centrifuged at 12,000 g In an Eppendorf microcentrifuge for 5 min at 4 °C. Following transfer of the supernatant to a fresh tube, SDS was added to make the solution 0.5* SDS. VRC was added to 0.05 x volume and proteinase K was added to 0.6 mg/ml. After incubation at 37 °C for 60 min, the mixture was extracted with phenol/CHCl3 (1:1 mix), and ethanol (95*) precipitated. The pellet was rinsed with ethanol (70*) , and resuspended with 0.5 ml 10mM EDTA (pH 8.0), then 0.5 ml 4 M L1C1 was added. RNA was precipitated at 0°C for 16 hr, and pelleted with centrifugation at 12,000 g at 4 °C for 15 min. Following resuspension of RNA in 10mM EDTA (pH 8.0), undissolved material was removed with a second centrifugation at 12,000 g. The RNA In the supernatant was ethanol precipitated and resuspended in 0.15 - 0.25 ml H2O. The RNA yield was estimated by A26O measurement (126). 27 H. Yeast Genomic DNA Library Preparation Lambda Charon4A DNA was Isolated by the formamlde extraction method from phage prepared by the PDS procedure and purified on CsCl gradients (28, 126). Following EcoRI digestion of ACh4A phage DNA, the cohesive ends of the 20 kb and 10 kb EcoRI vector arms were annealed by incubation at 42°C for 2 hr. The resultant 30 kb vector DNA fragment was separated from the 6.6 kb and 7.8 kb phage DNA stuffer fragments by electrophoresis in a 0.5$ Low Melting Point (LMP) agarose gel at 1.5 V/cm for 40 hr. Isolation of the 30 kb band was by electrophoresis onto a Whatman 3MM filter paper strip, backed by a piece of dialysis membrane, (75) placed in a slit in the gel that was cut directly below the 30 kb band. DNA was recovered from the filter paper and membrane with several washes of elutlon buffer [ 10 mM Tris (pH 7.6), 10 mM NaCl, 1 mM EDTA, 0.2$ SDS]. The isolated vector DNA was ethanol precipitated and quantitated by agarose gel electrophoresis. A second preparative 0.5$ LMP agarose gel purification of the 30 kb fragment which encompasses the 20 kb and 10 kb vector arms was performed to ensure the complete removal of stuffer fragments and a 1:1 ratio of vector 20 kb (left) and 10 kb (right) arms. Yeast DNA 16-20 kb partial EcoRI digest fragments were prepared by digesting high molecular weight yeast DNA with a range of EcoRI concentrations (0.25 - 1.0 u/jug), with aliquots taken at 15 min, 30 min, and 60 min Intervals from each reaction. The aliquots were combined, and were fractionated alongside lambda DNA size markers on a 0.5$ LMP agarose gel at 2 V/cm for 16 hr. Fragments In the 17 kb range were isolated by the filter paper strip method (75). Fragments larger than 21 kb were prevented from contaminating the 16-20 kb fraction by Inserting an additional filter paper-dialysis membrane strip at the 21 kb region to collect higher molecular weight fragments. Concentrations of ACh4A vector and yeast DNA fragments were determined by the EtBr agarose plate method (126). Ligation mixes required a 2:1 molar ratio of ACh 4A vector arms to yeast EcoRI fragments, and a minimum DNA concentration of 0.25 jig/jil in the ligation mixture. (125). The 10 jil ligation mix contained 2 jig of the vector arm fragment (30 kb) and 0.5 jjig yeast EcoRI fragments (15 kb). The vector arm fragment was Incubated at 42°C to ensure that all 20 kb and 10 kb arm fragments were joined at the cohesive ends. The yeast DNA fragments and ligation buffer were added 28 along with 2 u T4 DNA Ligase. Incubation was at 4 °C for 20 hr. The extent of ligation was assayed by analysis of 5 * of the ligation mix on a 0.3* agarose mlnlgel alongside undigested ACh 4A vector (0.2 jig) and EcoRI digested vector (0.2 jig). In vitro packaging was carried out with ligated vector plusyeast insert (68,90). One packaging extract (50 jjtl, stored at -70 °C) was thawed on ice for 3 min and the ligation mix ( 9.5 jil) was added along with 1.5 JJLI ATP (0.1 M), and 20 jil CH buffer [40 mM Tris(pH 8.0), 10 mM spermidine, 0.1* 2-mercaptoethanol, 7 * DMSO, 1.5 mM ATP], The suspension was mixed with a glass rod at 15 min Intervals during incubation in a 37 °C H2O bath for 1 hr. A second packaging extract aliquot was thawed 3 min on Ice, and 10 jil DNasel (1 mg/ml) plus 2.5 Jul MgCl2 (1 M) were added. Half of the extract (25 jil) was added to the ACh4A/yeast DNA packaging reaction, and 37 °C incubation was continued for 30 min, with stirring at 10 min intervals. Finally, 0.9 ml SM [0.1 M NaCl, 0.01 M Tris (pH 7.5), 0.01 M MgCl 2, 0.02* gelatin] was added along with a few drops CHCI3. The genomic library was stored at 4 °C. I. Bacteriophage Lambda Techniques Host bacteria cells ( DP50 SupF or LE392) were grown to stationary phase ( 1 0 9 cells/ml) in a 10 ml culture and pelleted in a table top centrifuge at 2000 rpm for 10 min. Cells were resuspended in 0.5 x volume of A dll [10 mM Tris (pH 7.5), 10 mM MgSO^. Plating bacteria (0.15 ml) were mixed with the appropriate number of phage and Incubated for 15 min at 37°C to allow phage adsorption. Cells and phage were added to 3 ml of 0.7* top agar or agarose at 42 °C, and the mixture was poured onto a 37 °C agar plate. The top agar layer was hardened at RT for 10 min, then plates were Inverted and incubated at 37 °C for 10-14 hr. Phage stocks were prepared by plating about 10^ phage per 85 mm plate. After 37 °C incubation to obtain confluent lysis, 5 ml of A dll were added to each plate, and plates were incubated (right side up) for 12 hr at 4 °C. The overlay solution was removed with a pasteur pipet and 0.1 ml CHCI3 was added. Bacterial and agar debris were removed with a brief spin in the table top centrifuge. The supernatant was made to 0.3* CHCI3, and stored at 4 °C. 29 J . Plasmid Dephosphorylatlon and Ligation Reaction conditions with bacterial alkaline phosphatase (BAP), (30), for plasmid DNA dephosphorylation consisted of restriction enzyme digested vector (0.01 iig/u.1), and BAP (0.05 u/jig) In 50 mM Tris (pH 8.0). After 30 min at 37 °C, the reaction was stopped with the addition of 1 ill 0.25 M EDTA (pH 8.0), followed by phenol extraction and ethanol precipitation. Conditions used for calf intestinal alkaline phosphatase (CIP) were restriction enzyme digested DNA (0.05 |ig/|i1) andCIP (0.05 u/jig) in 1 xCIP buffer [50 mM Tris (pH 9.0), 1 mM MgCl2,0.1 mM ZnS04, 1 mM spermidine]. Incubation was at 60 °C for 30 min and at 68 °C for 1 hr. Dephosphorylated DNA was ethanol precipitated before the subsequent ligation reactions. Inter molecular ligations with plasmid DNA typically contained 0.1 - 0.2 jig of linearized plasmid DNA and 2-5 x molar excess of Insert fragment in 20-25 ill reaction volumes with 1 -2 u T4 DNA Ligase, and incubation at 16°C for a minimum of 12 hr. Intramolecular ligations were carried out in large volumes (100-1000 ill) under otherwise similar conditions. Ligation buffer used for initial cloning reactions contained 66 mM Tris (pH 7.6), 6.6 mM MgCl2, 10 mM DTT, 1 mM ATP. Later work Indicated better ligation efficiencies were obtained with T4 DNA Ligase using ligation buffer containing 50 mM Tris (pH 7.5), 10 mM MgCl2, 10 mM DTT, 1 mM spermidine, 0.1 mg/ml BSA, and 1 mM ATP. DC. Ml3 Cloning 1. Preparation of M13 Replicative Form DNA M13 Replicative Form DNA (RF) was prepared by either the in yjyjo or the ia vitro methods (132), however in vitro preparations were preferred due to the simplicity and rapidity of the method. In vitro M13 RF DNA was prepared by annealing 2-4 iig M13 template clone DNA with 4 pmole M13 universal primer in a 20 ill reaction mixture containing 10 mM Tris (pH8.0) and 5 mM MgCl2 at 55 °C for 10 min. The primer W8S elongated with DNA Polymerase I, Klenow fragment (2u), and 0.25 mM dNTPs for 20 min at RT. The reaction was stopped by heat inactivation of the DNA Polymerase I, Klenow fragment at 70 °C for 10 min. inyjyo M13 RF DNA (132, 213) was prepared from a 500 ml culture of JMl01 cells Infected with the M13 clone of interest. Stationary phase 30 JM 101 cells grown in M9 medium (about 10^ cells) and a M13 clone plaque picked from a fresh plate were used to Inoculate 5 ml of 2 x YT medium. Growth at 37°C for 4 hr with vigorous shaking produced sufficient phage for infection of a 500 ml culture. The 5 ml phage supernatant and 5 ml of stationary phase JM101 cells were added to 500 ml of 2x YT medium and Incubated at 37 °C for 4-5 hr with high aeration. Cells were harvested by centrifugation at 4000 g at 4 °C for 10 min. M13 RF DNA was Isolated by the alkaline lysis procedure described for plasmid DNA Isolation (126), and purified by EtBr-ceslum chloride equilibrium density gradient centrifugation. 2. Subclonlnp Fragments In M13 Vectors Fragments to be subcloned In the Smal site of the M13RF vector were made blunt-ended using DNA Polymerase I, Klenow Fragment (126). After digesting the DNA with restriction enzymes, it was treated with Klenow fragment (2u), In a 50 ul reaction mixture [50 mM Tris (pH 7.4), 10 mM M0SO4,0.01 mM DTT, 50 ug/ml BSA], containing 8 uM dNTPs. After Incubation at RT for 20 min, the DNA Polymerase I Klenow fragment was heat Inactivated at 70 °C for 10 min. The resultant blunt ended fragments were purified following electrophoresis In 0.7* LMP agarose by extraction with phenol/CHCl3 (126). M13 cloning ligation mixes usually had 20-30 ul volumes and routinely contained 0.8 pmoles/ml Ml3 vector DNA, 5x molar excess of Insert fragment, ligation buffer and 1 -2 u T4 DNA ligase. Reactions were incubated at 16 °C for 16-20 hr (132). 3. M13 Template DNA Preparation The single-stranded M13 template DNA was Isolated after infecting 1.2 ml JM101 cells (early log phase) with a selected M13 plaque, and growing for 4 hr at 37 °C, with vigorous shaking. Cells were pelleted In an Eppendorf microcentrifuge for 3 min. The ph8ge supernatant (800 ul) was added to 200 ul PEG solution (15* PEG 4000, 2.5 M NaCl) and Incubated for 15 min at 23 °C. Precipitated phage were pelleted by centrifugation at 12,000 g at RT for 10 min in an Eppendorf microcentrifuge and the supernatant was removed by aspiration. After resuspenslon of phage with 100 ul TE [10 mM Tris, 1 mM EDTA (pH 7.5)], phage DNA was Isolated with phenol/CHCl3 extractions and EtOH precipitation. DNA W8S resuspended In 40 ulTE and stored at -20 °C. 31 4. h 15 Clone Complementation Test To establish the orientation of the Insert In a given M13 clone, hybridization screening (132) using a clone of defined Insert orientation as reference was performed on selected Ml3 clone templates. Hybridization buffer consisted of 10 mM Tris (pH7.5), 0.1 M NaCl, 0.1$ SDS and 1 mM MgCl2- The number of clones tested for insert orientation (x) determined the volumes of premix (+) and premix (-) reagents. The volume of the premix ( + ) or (-) reagent was 8(x+2) j i l , and each contained (x+2) ill of 10x hybridization buffer. The premix ( + ) reagent also had 2(x+2) ill of template DNA from a homologous M13 clone of known insert orientation. For each clone to be tested, 2 ill of template DNA was added to 8 til of each premix reagent. Hybridization was for 1 hr at 65 °C. The reactions were terminated with the addition of 5 ill of 60$ sucrose, 0.02$ Bromophenol Blue, 0.02$ Xylene Cyanol, and 0.025 M EDTA (pH8.0). For each tested clone, premix ( + ) and (-) reaction mixes were run in adjacent lanes on a 0.7$ agarose gel at 150 V for 3 hr. Tested M13 clones having the opposite Insert orientation to the standard M13 clone migrate as a single band In the premix (+ ) lane, with slower mobility than the premix (-) sample due to the double stranded region of the hybridized Insert region. M13 clones having the same insert orientation as the standard clone have i equivalent mobilities In premix (+) and (-) lanes. L. Plasmid and Ml3 Transformations Plasmid or M13 DNA was Introduced into bacteria cells that were made competent by incubation in CaCl2 (54, 57, 126, 132). An overnight culture of bacteria was diluted 100-fold in culture medium (0.5 ml in 50 ml) 8nd incubated at 37 °C with shaking, until mid-log phase or about 2 hr. Cells were pelleted in sterile 30 ml tubes at 4000 g at 4 °C for 5 min. Cells were resuspended in 0.5 x volume of cold CaCl 2 (50 or 100 mM) and kept on ice 15 min. After pelleting the cells by centrifugation as done previously , the cells were resuspended In either 0.5 ml 100 mM CaCl2 or 4 ml 50 mM CaCl2 . For RR1, DH1, or MM294 cells, plasmid DNA transformation efficiencies were improved by incubating the resuspended cells In the CaCl2 solution at 4 °C for 12-16 hr prior to use. Freshly prepared JM 101 competent cells were used for M13 transformations. 32 For plasmid DNA transformation, 0.1 ml of 100 mM CaCl2 cells or 0.2 ml of 50 mM CaCl2 cells were used per reaction. After adding DNA In TE [ 10 mM Tris, 1 mM EDTA, (pH 7.5)] or ligation buffer, cells were incubated on Ice for 30 min. Cells were heat shocked at 37 °C for 5 min, and 1 ml of LB or YT medium was added per tube. Cell wall recovery was at 37°C for 1 hr without shaking. Aliquots of the transformation mix were plated on selection plates and colonies were visible after 12-16 hr at 37 °C. For transformations with M13 DNA (ss template of RF DNA), 0.3 ml 50 mM CaCl2 treated JMl01 cells and Ml3 DNA (2 ng), or ligation mix aliquots, were incubated on ice for 40 min. Following heat shocking at 45 °C for 2 min, cells were added to 3 ml YT top 8gar that contained 50 jil Xgal (2%), 10 ill IPTG (100 mM), and 0.2 ml exponential JM 101 cells. Cells and top agar mixtures were plated on YT agar plates and plaques were visible after incubation at 37 °C for 10 - 14 hr. M. DNA Probe Preparation 1. Nick Translation Nick translated probes (126, 163) were used predominantly for isolating clones by plaque hybridization or colony hybridization. A typical nick translation reaction contained 0.5 jig DNA, 5 jul 10 x nick translation buffer [0.5 M Tris (pH 7.4), 0.1 MMgCl2. 1 mM DTT, 0.5 mg/ml BSA], 5 ill 2 mM CaCl2,5 til each of 0.1 mM dGTP and 0.1 mM dCTP, 0.2 ill each of 0.1 mM dATP and 0.1 mM dTTP, 2.5 ill each of [<x 32 p] dATp a n ( j [ a 32 p] unp, 0.5 jil DNase I (25 pg/jil, freshly diluted from 1 iig/ul stock), 0.5 ill DNA polymerase I, Kornberg enzyme (2.5u) and H2O to 50 jxl total volume. Incubation was at 16 °C for 2.5 hr. The reaction was terminated with the addition of 2 JJLI 0.5 M EDTA (pH 8.0) and incubation at 68 °C for 10 min. After chilling the reaction mix on Ice, the unincorporated radioactivity was removed by gel filtration chromatography using AcA 54 (5000 -7000) matrix, and a BioRad dispocolumn (17 x 1 cm). The column buffer was 0.2 M NaCl, 10 mM Tris, 0.25 mM EDTA (pH 7.8). Fractions (500 - 700 jil) were collected in Eppendorf tubes. The probe eluted 1n the void volume (fractions 3-4) and had an average specific activity of 10 7 cpm/jig, (Cerenkov counts). The probe was denatured by adding 0.04 volume of 5 M NaOH and heating at 90-33 100 °C in a H2O bath for 2 min. and quick chilling at 0 °C. The probe solution was neutralized with 0.04 volume of 5 M HC1 and 0.1 volume of 1 M Tris (pH 7.4). 2. M13 Primer Extension Most Southern hybridization probes were prepared from fragments cloned in M13 vectors using a modification of the M13 sequencing protocol (132). The M13 universal sequencing primer was annealed to the template clone DNA in a 10 ul reaction mixture containing 0.5 - 1 ug single-stranded template DNA and 2 pmole primer in reaction buffer [ 10 mM Tris (pH 8.0), 5 mM MgCl2]. The annealing reaction was incubated at 55 °C for 15 min and cooled at room temperature for 10 min. After adding 5 uldNTP mixture (100 uMdGTP, 100 uM dCTP, 15uMdATP, 15 uM dTTP), 20 uCI each of [ ot 3 2 P] dATP and [ot 3 2P] dTTP, plus 2 u DNA polymerase I Klenow fragment, incubation was at 23 °C for 20 min. The primer extension reaction was stopped with incubation at 68 °C for 10 min. The unincorporated radioactivity was removed from M13 probe preparations by gel filtration chromatography on AcA54 columns then probes were denatured with alkali and heat, as described for nick translated probes. Specific activities of 1 0 7 - 1 0 8 cpm/ug (Cerenkov counts) were routinely obtained for M13 primer extended probes. N. Gel Hybridization Analyses 1. Agarose Gel Electrophoresis and Southern Hybridization Agarose gel electrophoresis of DNA samples (126) was conducted using between 0.5* to 0.75* agarose gels, containing 1 ug/ml EtBr, and Tris borate buffer [TBE: 50 mM Tris, 50 mM boric acid, 1 mM EDTA, (pH 8.3). Sucrose-dye stop solution [ 50 * sucrose, 25 mM EDTA (pH 7.4), 0.02* Bromophenol Blue and 0.02* Xylene Cyanol] was added to samples prior to gel loading such that the final sucrose concentration was between 5 * and 10* (w/v). Yeast genomic DNA or high molecular weight DNA fragments were subjected to electrophoresis at low voltage gradients (1-2.5 V/cm) for 13 - 17 hr. Visualization of DNA samples by UV fluorescence was conducted with either short wave UV (254 nm) or long wave UV (300 -360 nm). 34 Southern hybridizations were conducted on DNA samples in situ on dried agarose gels (201) or DNA was transferred, either bidlrectlonally (179) or unldlrectlonally (129), to nitrocellulose, NEN Gene Screen, or NEN Gene Screen Plus filters. NEN Gene Screen Plus was the preferred membrane for Southern analyses, due to Its ease of handling and Us efficient binding capacity. Prehybrldlzatlon and hybridization conditions were routinely 6 x SSC [1 x SSC: 0.15 M NaCl, 0.015 M NaCltrate (pH 8.3)]; 10 x Denhardt's mix (0.2* Ficoll, 0.2* polyvinyl pyrrolldone, 0.2* BSA); 0.5* SDS, and 0.1 mg/ml carrier salmon sperm DNA (sheared and denatured). Filters were Incubated in sealed bags at 68 °C, in a H2O bath for 12 - 24 hr. Following hybridization, the Initial washes were In 2 x SSC, 0.5* SDS at RT for 2 x 15 min. Stringent washes were conducted in 1 x SSC, 0.5* SDS at 68 °C, with slow shaking in a H2O bath for 2 x 1 hr. Filters were exposed to Kodak XAR-5 film at -20 °C or -70 °C, with intensifying screens. For secondary hybridization, initial probes were removed from the transfer filters as follows: (1) Nitrocellulose filters were washed In 0.01 x SSC, 0.1 * SDS, with 5 - 10 min Incubation at 100 °C; (2) Gene Screen was washed for 1 to 3 hr with constant agitation at 68 °C, In 250 -300 ml of manufacturer's suggested buffer [5 mM Tris, 0.2 mM EDTA (pH 8.0], 0.05* sodium pyrophosphate, 0.1 x Denhardt's mix]; (3) Gene Screen Plus membrane was not completely dried following hybridization, if subsequent probe removal was required. The probe was efficiently removed with incubation in a 68°C shaking H2O bath, for 1 hr using 100-200 ml of 0.4 N NaOH followed by 100-200 ml of 0.1 x SSC, 0.1 * SDS, 0.2 M Tris, (pH 7.5). Filters were exposed to X-ray film to assay for the removal of the Initial probe prior to hybridization with a second probe. 2. RNA Dot Blot and Northern Hybridization Yeast RNA was subjected to dot blot analysis (199, 200) using a BRL Hybri-Dot Manifold to spot RNA (1-20 jig) on Gene Screen Plus filter that was wetted with H2O and 20 x SSC. Prehybrldlzatlon and hybridization conditions were those used for Southern analyses. Northern analysis was performed on RNA samples (199, 200) fractionated in 1 * agarose - formaldehyde gels with 1 x MOPS buffer [5 x MOPS: 0.2 M morpholinopropanesulfonlc acid (MOPS, pH 7.0), 50 mM sodium acetate, 1 mM EDTA (pH 8.0)], (126). Following electrophoresis at 30 V for 20 hr, a portion 35 of the gel was stained with 33 jig/ml acridine orange in 10 mM Na2HP04 (pH 6.5) for 10 min, then destained with 3 x 20 min washes in 10 mM N82HP04 (pH 6.5) (129). RNA was visable in the stained gel by UV fluorescense at 300 nm. RNA was transferred to NEN gene screen plus (126) and baked In vacuo at 80 °C for 2 hr. Filters were prehybrldized at 60 °C for a minimum of 6 hr in prehybridization mix containing 5 x Denhardt's mix, 1 M NaCl, 0.5$ SDS, 10$ dextran sulfate. Denatured, sheared salmon sperm DNA (0.1 mg/ml) and denatured, nick-translated probe were added. Hybridization was at 60 °C, in a H2O bath for 15 - 20 hr, with constant agitation. Washes were conducted with contlnous agitation of filters for 2 x 5 min at RT, In 300 ml 2 x SSC, followed by 2 x 45 min washes at 60 °C In 300 ml 2 x SSC, 1 $ SDS. Finally, filters were washed in 300 ml of 0.2 x SSC at RT. Membranes were dried at RT and exposed to CIUREX film, with an intensifying screen, for 1 week. 0. Plaque and Colony Hybridization 1. Plaoue Hybridization Bacteriophage lambda recombinant libraries were screened by plaque hybridization (16, 126). A maximum 10 4 plaques per plate, in LBMgT top agarose, were transferred to nitrocellulose filter discs, by laying a sterile filter on a plate for 5 min. India Ink spots, positioned asymmetrically on the periphery of the agarose surface, served to orient the filter on the plate. Phage were denatured and fixed to filters by submerging the filters in petri plates filled with 1.5 M NaCl and 0.5 M NaOH, for 5 min. Filters were transferred to plates containing 3 M NaCl, 1 M Tris (pH 7.0) for 5 min and this wash was repeated. After filters were rinsed In 2 x SSC for 5 min, they were blotted on Whatman 3MM paper and dried at 68 °C for 1 hr. Bacteriophage M13 were similarly transferred to nitrocellulose filters for screening. Denaturation and neutralization steps were carried out in duplicate. Rather than submerging filters in petri plates, cells and phage were denatured on Whatman 3MM paper soaked with 0.5 M NaOH, 1.5 M NaCl and neutralized on filters soaked with 1 M Tris (pH 8.0), 1.5 M NaCl. Nitrocellulose filters were washed in 6 x SSC for 2 min, and filters were dried on Whatman 3MM paper and baked in a 68 °C oven for 1 hr. 36 2. Colony Hybridization Bacterial colonies which contained recombinant plasmid DNA were screened by the high colony density method (84, 126). Colonies were plated on sterile nitrocellulose filters (maximum lO^per filter) on LB-AMP plates. Following growth at 37 °C for 12 hr, colonies were transferred to replica filters and grown on LB-AMP at 37 °C for 8 hr. Colonies were amplified on LB-AMP/CAM plates at 37 °C for 12 hr. Alternatively, colonies were transferred to LB-AMP grid pattern plates, grown at 37 °C until colonies were 0.2 - 0.5 cm diameter, and transferred to sterile nitrocellulose filters as for plaque hybridization. Colonies were lysed by laying filters on buffer saturated Whatman 3MM paper as described for M13 plaque hybridization. Filters were transferred to Whatman 3MM wetted with 2 x SSPE [ 0.36 M NaCl, 20 mM NaH 2P0 4 (pH 7.4), 2 mM EDTA ] for 5 min, then filters were dried briefly on Whatman 3MM paper at RT and baked for 2 hr at 68 °C. Bacterial debris was removed from the filters by rinsing with 100 ml of prewash solution [50 mM Tris (pH 8.0), 1 M NaCl, 1 mM EDTA, 0.1 % SDS], at 42 °C, for 1 to 2 hr followed by a brief rinse In 6 x SSC prior to prehybrldlzatlon. Filter hybridization and wash conditions were equivalent to those described for Southern hybridization analyses, using 2 to 3 ml of hybridization mix per filter. Filters were dried at RT on Whatman 3MM and exposed to XAR-5 film with intensifying screen at -70 °C. P. Recombination Screening ]n v M recombination selection (126, 174) was used to screen the ACh4A/yeast genomic library. In this screening method the probe fragment is cloned in a microplasmid vector (I1AN7), which contains an amber suppressor tRNA gene. The genomic library, which has amber mutations in the phage vector, is grown in a recombination proficient bacterial strain that carries the probe clone. Genomic clones which have insert fragments homologous to the probe may acquire the amber supressor tRNA gene by reciprocal recombination with the microplasmid clone. The subsequent growth of the genomic library in a supressor deficient bacterial strain selects for genomic clones which have an amber supressor tRNA gene Integrated in the probe fragment region. The riAN7 ( 885 bp microplasmid which contains the ColEI repllcon, supF gene, and polylinker region) was Isolated from LcoJi MCI 061 (p3) (IIAN7) by alkaline lysis (126). The 37 microplasmid was separated from L ffili genomic DNA by electrophoresis in 0.7$ LMP agarose at 30 V for 16 hr and was purified by phenol/CHCl3 extraction of the Isolated gel band (126). The probe fragment was cloned In the polylinker region of ITAN7, using 0.3 jig microplasmid vector and 1 or 3 x molar excess of Insert fragment. Aliquots from the ligation mixture were assayed via mlnlagarose gel electrophoresis before and after the addition of 2 u T4 DNA Ligase and 16 hr Incubation at 15 °C. Ligation mixes were transformed in MC1061(p3) cells made competent with 100 mM CaCl2 (54) and transformants were plated on LB-AMP (12.5 jig/ml) -TET (7.5 jig/ml) plates. DNA was Isolated from selected transformants by the small scale alkaline lysis preparation (19) and analyzed for IIAN7 microplasmid containing the Insert probe fragment by agarose gel electrophoresis. Bacteria containing the desired recombinant microplasmid were infected with either the amplified or nonampllfied ACh4A-yeast library using 1 0 5 - 1 0 6 phage per 2.5 ml overnight cell culture (2 x 10 8 cells/ml). Phage were plated In LB-AMP (12.5 jig/ml) - TET (7.5 iig/ml) top agar and grown at 37 °C for 12-16 hr. Phage stocks were prepared and plated on Su + host LE392 (5 x 10 4 dilution) or Su~ host MCI061 (nondiluted) to determine the Su + phage resulting from microplasmid and phage recombination. TrueSu + recombinant phage were distinguished from amber revertants by plaque hybridization of phage grown on MC1061. The hybridization probe was either the fragment that was used for the recombination screening or a fragment that mapped centromere proximal to that region on the chromosome. Q. SI and Bal31 Nuclease Digestion of Yeast DNA Yeast high molecular weight DNA (10 jig) was digested with SI nuclease (1 u) In 100 Jul reaction volume, containing 1 x SI buffer [30 mM NaOAc (pH 4.6), 150 mM NaCl, 1 mM ZnS04, 5$ glycerol]. Following incubation at RT for 30 min , reactions were stopped by phenol/CHCl3 extraction. Yeast high molecular weight DNA (10 jug) was treated with the ds exonuclease Bal31 (1 u) in 100 Jul reaction volume containing Bal31 buffer [600 mM NaCl, 12 mM CaCl2, 12 mM MgCl2, 20 mM Tris (pH 8.0), 1 mM EDTA] with incubation was at 30 °C. Aliquots were removed at periodic Intervals between 0 and 30 min and added to 0.1 volume of 0.2 M EGTA (pH 8.0) to terminate the 38 reaction. Prior to restriction enzyme digestion, aliquots were phenol/CHCl3 extracted and ethanol precipitated. R. DNA Sequence Determination 1. Preparation of Deletions with Exonuclease 111 and S1 Nuclease The terminal IIIL EcoRI fragment was isolated from MTLB6411 RF, made blunt ended with DNA Polymerase I Klenow fragment, then ligated to Smal digested M13mp18 RF (213). Sets of deletion clones were prepared with Exonuclease III (Exolll) and SI nuclease for both orientations of the insert present in the subclones MTLB6S12 and MTLB6S21, as described by Henikoff (86). For each clone, 10 ug DNA was treated with 800 u Exolll in 100 ul buffer [66 mM Tris (pH 8.0), 0.66 mM MgCy, at 37 °C. Aliquots (7.5 ul) were taken at 30 sec intervals for 5 min, added to 22.5 ul Exolll stop mix [0.2 M NaCl, 5 mM EDTA (pH 8.0)] per time point, and incubated at 70°C for 10 min. DNA was precipitated with 3 volumes EtOH (95*) per aliquot, resuspended in 50 ul SI nuclease buffer [0.25 M NaCl, 30 mM KAc (pH 4.8), 1 mM ZnS04,5* glycerol] and treated with 1 u SI nuclease per aliquot for 30 min at 23 °C. The reaction was terminated with 6 ul SI stop solution [0.5 M Tris, 0.125 M EDTA, (pH 8.0)], followed by phenol / CHCI3 extraction and EtOH precipitation. The termini were repaired by treatment with 0.1 u DNA Polymerase I Klenow fragment per aliquot in 10 ul Klenow repair buffer [20 mM Tris (pH 8.0), 7 mM MgCl2], at 37 °C for 2 min, followed by the addition of 1 ul dNTP mix (0.125 mM) and Incubation at 37 °C for an additional 2 min. The deletion clones were rellgated in 40 ul ligation buffer per time point aliquot containing spermidine, BSA, and 1 u T4 DNA Ligase and Incubated at 23 °C for 4 hr. Ligation mix aliquots (10 ul) were transformed in CaCl2 treated JM101 cells. The extent of deletion was determined by agarose gel electrophoresis of a sample of template DNA clones for each Exol 11 time point aliquot and comparison of the clone length with the parental clone and the M13mp 18 vector. 2. DNA Sequence Determination Deletion clone sets were sequenced by the dideoxy chain terminator method with single-stranded DNA templates (132, 170, 171). Template DNA (5u1 or 1-2 ug) was annealed with 2 pmoles M13 unversal sequencing primer (or insert sequence specific primer) in 10 ul annealing 39 buffer [ 10 mM Tris (pH 8.0), 5 mM MgCl2] at 55 °C for 5-10 min. After 10 min at 23 °C, 1 yl [<x32P] dATP (10 iiCi) and 1 ill 12.5 JIM dATP were added to the annealed mixture. In sequencing poly dC-dA repeat region, ie. poly dG-dT template strand, the concentration of dATP was doubled by adding 25 jiM dATP rather than 12.5 iiM dATP. Four tubes were labelled C.T.A, or 6, and 2 jil of DNA / primer mix plus 2 ill of each respective dNTP/ ddNTP mix were added. The following nucleotide mixes were used: dC/ddCTP: 0.014 mM dCTP, 0.25 mM ddCTP, 0.11 mM dGTP, 0.11mM dTTP. dT/ddTTP: 0.0055mM dTTP, 0.50 mM ddTTP, 0.11 mM dCTP, 0.11 mM dGTP. dA/ddATP: 0.05 mM ddATP, 0.075 mM dCTP, 0.075 mM dTTP, 0.075 mM dGTP. dG/ddGTP: 0.055 mM dGTP, 0.30 mM ddGTP, 0.11 mM dCTP, 0.11 mM dTTP. Tubes were prelncubated in a 30 °C H2O bath for 5 min. DNA Polymerase I Klenow fragment was diluted to 0.25 u/jil in 1 x Kpn buffer [6 mM NaCl, 6 mM MgC12, 6 mM Tris (pH 7.5), 6 mM 2-mercaptoethanol]. At time 0 min, 2 ill of the diluted Klenow fragment (0.5 u) was added to each CTAG tube. Following incubation at 30 °C for 15 min, 2 ill of the diluted Klenow fragment (0.5 u) plus 2 ill dNTP (0.5 mM) chase mix were added per tube. Following a second 15 min, 30 °C incubation, 5 ill formamide dye stop mix (95$ deionized formamide, 10 mM EDTA, 0.1 % Xylene Cyanol, 0.1 % Bromophenol Blue) was added. Samples were heated to 90-100°C for 2-3 min in a H2O bath to denature DNA and were immediately quick chilled on ice. Electrophoresis conditions were 6$ acrylamide, 7 M urea gels, 30 W (constant power), 30 mA maximum current. Samples (2 ill) were loaded at times 0, 1.5, and 3.5 hr, and electrophoresis was stopped at 4.5 hr. Gels were dried on Whatman 3MM paper using a vacuum gel dryer. Exposure of XRP-1 film was at RT for 12-16 hr. S. Construction of Plasmids to Assay for Telomere Function 1. Clones with Requisite Restriction Site Arrangements DNA sequence and Southern hybridization results indicated clone MTLB6S12 (Fig. 16) contained the IIIL telomere, however it was necessary to determine whether it functioned as a yeast telomere. Plasmids replicate as linear molecules in yeast if the ends are stabilized by telomeric fragments (193) and circular plasmids which contain Inverted telomerlc repeats are resolved into 40 linear plasmlds In yeast (194,195). The IIIL telomere In MTLB was tested for telomere function by subclonlng It as an Inverted repeat In the yeast plasmid pSZ218 (194), introducing the plasmid into yeast by transformation, and assaying for presence of linear plasmlds. In the same manner, deletion derivatives of the IIIL telomeric fragment were assayed for the stabilization of linear plasmids in yeast. Initially, the telomeric fragments in MTLB6S12 and the derivative Exolll/Sl nuclease deletion clones (MTLB6SD12 clones) were subcloned into the Smal site of M13mp19 to provide the requisite flanking restriction sites. For example, the pSZ218 vector has a single Bglll site for cloning BamHI fragments as inverted repeats (194), hence It was necessary that the telomeric fragments have a BamHI site on the centromere proximal end and an EcoRI site on the distal end (Fig. 25). Telomeric fragments In MTLB6S12 and MTLB6SD12 clones were excised from the M13mp 18 vector by digestion of the M13RF clone DNAs with EcoRI/BamHI for MTLB6S12 or EcoRI/Hlndlll for the Exolll/Sl deletion MTLB6SD12 clones. Following treatment with DNA Poll Klenow fragment, blunt ended fragments were Isolated from 0.7* LMP agarose gels. These fragments were llgated In the Smal site of M13mp19RF (213) and clones were selected by M13 clone complementation tests with the MTLB6S12 clone as the reference orientation clone. The selected clones had the telomeric fragments oriented with the BamHI /Sail sites from the M13 vector on the proximal end, adjacent to the X region and the Hlndlll/EcoRI sites from the Ml3 vector on the distal end, adjacent to the T region. All subclones were sequenced to ensure that the correct orientations and deletions of the IIIL telomeric region had been obtained. The M13mp 19 subclone which contained the full length telomeric fragment from MTLB6S12 was named TF1. The subclones TF2 to TF7 contained the set of MTLB6SD12 fragments with progressive deletions of the telomere In M13mp19 (Fig. 25). A deletion clone constructed for DNA sequence analysis of the opposite orientation clone (MTLB6S21) was used to construct a plasmid to assay for the requirement of an A M region on the telomere. Clone MTLB6SD21-66 lacks the A M region of the IIIL telomere, but retains the 5'-Ci-3A-3" repeat region. It did not require subclonlng Into M13mp 19 because the telomeric fragment h8S the necessary 41 flanking RE sites In the M13mp18 vector but MTLB6SD21 -66 was referred to as TF8 for the linear plasmid constructions (Fig. 25). 2. Deslon of Linear Plasmids For each telomerlc subclone (TF1 - TF8), double-stranded M13 clone DNA was prepared by the in vitro RF method. For cloning in pSZ218, BamHI/EcoRI telomeric fragments were isolated from telomeric clones TF1 - TF7. The LEU2 gene was isolated from pSZ218 as a Sall/Xhol fragment (2.2 kb) or a Sall/Hlndlll fragment (2.5 kb) for preparing a set of linear yeast plasmids which lacked the pBR322 region of pSZ218 (65). Telomerlc fragments were Isolated from clones TF1 - TF8 as Sall/EcoRI or Hindlll/EcoRI fragments for the LEU2 plasmid constructions. As an experimental control for testing telomerlc function, the telomere of the Tetrahvmena rDNA linear molecule was cloned as an Inverted repeat in the yeast plasmid pSZ93, (195) since the Tetrahvmena rDNA end is known to function In yeast (56,65,139, 140, 193, 194, 195,215). The Tetrahvmena rDNA end fragment (0.7 kb) which contains 0.33 kb of 5'-CCCCAA-3' terminal repeat units and 0.36 kb of rDNA unique DNA, was Isolated from pSZ222 (195) by Xhol/Hhal digestion (Fig. 24). All fragments were Isolated by 0.7$ LMP agarose gel electrophoresis and purified by pheno1/CHCl3 extraction. Ligation mixes were similar to those described for previous linear plasmid constructions In yeast (194, 195). Yeast telomerlc fragments were llgated in 10x molar excess to yeast plasmid vector pSZ218/Bglll (0.2 jig) or to the LEU2 fragment (0.1 jig) In 30 ul reaction volumes. The Tetrahvmena rDNA end fragment was llgated In 10x molar excess to yeast plasmid vector pSZ93 (0.2 ug) In a 30 ul reaction volume. Ligation buffer contained 50 mM NaCl, 25 mM Tris (pH 7.4), 10 mM MgCl2,10 mM DTT, 1 mM spermidine, 0.1 mg/ml BSA, and 1 mM ATP. When ligation Involved joining fragments with isochlzomer restriction site ends, the corresponding restriction enzymes (RE) were Included In the ligation mixture. This reduced the percentage of unwanted ligation events in the reaction since the joining of ends produced by the same enzyme is followed by RE cleavage, while joints from Isochlzomer ends are not recognized by either enzyme. For example, in ligations of pSZ218/Bglll andBamHI/EcoRI telomerlc fragments, BamHI (2 u) and Bglll (2 u) were included in ligation mixes to enhance the proportion of BamHI/Bglll ligations. Ligations were 42 Incubated for 20 hr, at 16 °C after the addition of 1.5 u T4 DNA Llgase, and 2 u of each required restriction enzyme. Enzymes were heat Inactivated by Incubation at 70 °C for 10 m1n. For ligation mixes which contained restriction enzymes, further RE aliquots (2 u) were added and reactions were Incububated for an additional 2 hr at 37 °C to ensure the complete digestion of undesirable ligation products, and reduce the frequency of background transformants. Ligation mixes were stored at -70°C and used In yeast transformations. T. Yeast Transformation Yeast spheroplasts were transformed with plasmids as described by Sherman et al. (177) and modified by Orr-Weaver et al. (150). Cells were pelleted at mid log phase (10 7 cells/ml) in a table top centrifuge at 2000 g. For each 50 ml culture, cells were washed twice with 10 ml 1 M Sorbitol, then resuspended in 5 ml 1 M Sorbitol. Spheroplasts were generated with 150 ul glusulase and 5 ul 2-Mercaptoethanol, at 30 °C for 1 hr. Gentle washing was critical since spheroplasts were easily disrupted. Spheroplasts were washed twice with 10 ml 1 M Sorbitol, then once in Sorbitol buffer [0.9 M Sorbitol, 10 mM Tris (pH 7.4), 10 mM CaC^]. The final resuspension was in 1.5 ml STC [1 M Sorbitol, 10mMTris(pH7.4), 10 mM CaC^l and0.3 ml were used per transformation. For each transformation, the 25 ul ligation mix and 5 ul shearedLooJicarrier DNA (8 ug) were added to the spheroplast aliquots and 1ncub8tion was at 23 °C for 15 min. A 3 ml solution of 45 * PEG 4000, 10 mM Tris (pH 7.4), 10 mMCaCl2 was added to each transformation mix, followed by incubation at 23 °C for 15 min. The PEG was removed after pelleting the cells in a table top centrifuge. Resuspension was in 0.5 ml STC. Cells (0.3 ml) were plated In 30 ml regeneration agar with selection for LEU+ prototrophs and incubation at 30 °C for about 4 days. Transformants were purified by streaking individual colonies onto selective medium plates. After removal of the PEG solution from the pelleted sheroplasts, resuspension of the spheroplasts was difficult, and this may reduce the yeast transformation frequency. To test this, an alternate set of transformations was performed In which the final spheroplast pelleting step was omitted and the PEG/spheroplast mixture was plated directly In regeneration agar. The volumes of the transformation mixtures were reduced such that the PEG concentration in the spheroplast / regeneration agar mixture was low. In this alternate protocol, 43 spheroplasts were resuspended In 0.5 ml buffer per 50 ml cell culture and 60 ul were used per transformation with the addition of 10 ul of DNA. Following Incubation at 23 °C for 15 min, 0.6 ml PEG solution was added, and Incubation was continued at 23 °C for 15 min. Cells (0.3 ml) were plated directly In 30 ml regeneration agar. Fewer spheroplast washing steps made this modified protocol more efficient, however the transformation frequencies obtained were equivalent using either version of the procedure (average 25 transformants per ligation mixture). U. Mitotic Stability Determination Mitotic stabilities were reported as the fraction of cells containing a plasmid after growth on nonselective medium (65, 140, 195). Following growth of transformants to log phase In selection medium ( SC-leu), aliquots were diluted and plated on complete medium (YEPD) to render about 100 colonies per plate. Plates were Incubated for 2 days at 30 °C, then colonies were replica plated to selective medium plates using a transfer block and sterile Whatman No. 1 filter paper. The percentage of colonies growing on selection versus complete medium was recorded as the mitotic stability for a given transformant. 44 RESULTS A. Chromosome Walking from HMLa 1. Hybridization of Probe 1 * with Yeast Genomic DNA The strains of veast S. cerevlsiae that contain a linear chromosome III (Fig. 3a) oraclrcular chromosome III, also referred to as ring 111, (Fig. 3b) differ in the HMLoc and the HMRa distal regions as these regions are deleted with the HMR-HMLy, fusion event In ring 111 strains (109). The initial probes used for chromosome walking were Hindi Il/Xhol and Hindlll/Xbal fragments (probe 1*) isolated from the cloned region (142), and were distal to HMLft on chromosome IIIL (Fig. 3c). Southern hybridization characteristics of probe 1* with restriction enzyme digested and electrophoretlcally fractionated DNA from linear chromosome III and circular III yeast strains are shown In Figure 4. Two EcoRI fragments hybridized with equal intensity to the HMLfr distal probe (1*) in linear III strains AB 20a, XP8-10B (Fig. 4a, lane 1), AB972 (Fig. 4a, lane 2), and K45 (Fig. 4a, lane 3 and Fig. 4b, lane 1). The lack of restriction enzyme site polymorphisms In these two regions Is evidenced by the conserved 8.5 kb and 4.9 kb EcoRI fragments homologous to probe 1* In the three linear III yeast strain genomic DNAs. Similarly, there were two Hindi 11 and two Sail fragments that were homologous with probe 1* in the linear III strain K45 (Fig. 4b, lane 1). The hybridization of a H J I L a distal probe with two regions In the haploid yeast genome has been reported (109), but the map position of the alternate region Is unknown. For each RE digest, one fragment In the linear III strain K45 that hybridized with probe 1* was missing In the circular III strains , 1e. the 4.9 kb EcoRI, 6.5 kb Hindi 11 and >15 kb Sail fragment (Fig. 4b). Presumably each fragment that was missing In the ring 111 strains maps distal to HML% and consequently these fragments were referred to as "IIIL distal fragments". Alternatively, the fragments that were homologous to probe 1* and that were retained In the circular III strains, 1e. the 8.5 EcoRI, 5 kb Hindi 11, and 0.4 kb Sail fragments (Fig. 4b) must map to the alternate region In the haploid yeast genome and will be referred to as "IIIL alternate fragments". One IIIL alternate fragment, the 0.4 kb Sail fragment, had a slower mobility In the ring III strains K191 and K196, and Is missing in K193, a ring III strain. The explanation for this heterogeneous hybridization pattern with different circular III strains was not obvious since 45.1 Figure 3. Organization of Chromosome III In the Yeast S. cerevisiae . a. The relative map positions for the distinguishing markers on chromosome III are indicated. The most distal known genetic markers on the left and right ends of chromosome III, HMLoi and MAL2 respectively, are ca. 350 kb apart, if physical and genetic map distances are equated at 2.7 kb/cM (107,186). About 12 kb separate HULa and the left telomere (underlined), but the MAL2 to right end distance remains to be determined. Electrophoretic karyotype analysis of yeast chromosomes (37) indicates that chromosome III is approximately 370 kb in length. Since the region between the IIIL terminus and MAL2 region accounts for about 360 kb, the distance between MAL2 and the I MR telomere must be in the range of 10-20 kb. It is unknown if the IIIL and MIR telomeres are identical, so they are distinguished by different symbols. The solid dot on the chromosome represents the centromere. b. Yeast strains which contain this circular version of chromosome III result from recombination between the homologous regions In the mating type cassettes, HML<x and HMRa (109). The fusion cassette HML-HMRK . is the product of this recombination event, and the regions which are distal to HMLft and HMRa , Ie. the IIIL and IIIR telomeres, are apparently deleted during the fusion event. c. Initial probes used for chromosome walking. Probe 1* is the 2.2 kb Hindlll/Xbal fragment or the 1.4 kb Hindlll/Xhol fragment which maps distal to HML%. Probe 1* was isolated from the 6.47 kb Hindlll fragment in the pHMLot, clone (7, 142). The 1.4 kb Xbal and 2.3 kb Hindlll/Xbal fragments map within the HMLoi cassette region and were used as probes in restriction mapping experiments. The W, X, Y<x, Z1, and Z2 regions of the HMLp, cassette, and the orientation of HMLoc on IIIL are indicated. Restriction sites: H, Hindlll; X, Xbal; Xh, Xhol. 4 5 . 1 * . ® H M L Q HIS4 L E U 2 M A T Q THR4 H M R a M A L 2 i 1 • 1 1 ^ir»| L I N E A R III 6 ' ' ' 50 kb R I N G III .1 1 1 1 1 45.2, Figure 4. Southern Hybridization of Linear III and Ring III Yeast Strains with the HMLa Distal Probe 1*. a. Yeast genomic DNA from the linear chromosome 111 strains AB20a XP8-10B, AB972, K45, and the circular III strain K191, was digested with EcoRI, fractionated on a 0.7* agarose gel, transferred to nitrocellulose, and probed with a nick-translated 2.2 kb Hindlll/Xbal pHMLtx fragment (probe 1*). The 4.9 kb EcoRI fragment that was absent In ring III strain K191, but present in the linear III strains, must map distal to HMLft on IIIL. The 8.5 kb EcoRI fragment that hybridized with probe 1* maps to the IIIL alternate region. b. Identification of fragments that map distal to HMLtx. on IIIL by using various circular III yeast strains. DNA from linear III strain K45, (lane 1) and circular III strains K191, (lane 2), K192, (lane 3), K193, (lane 4), K195, (lane 5), and K196, (lane 6) were digested with EcoRI, Hindlll, or Sail as indicated. After fractionation on a 0.65* agarose gel, the DNA was transferred bidirectionally to gene screen plus filters. One filter was hybridized with an Ml3 probe that contained the 2.6 kb EcoRI/Sall fragment which maps to the probe 1* region, (MTeLB2-2.6, or probe E in Fig. 8). The HMiffi distal fragments were those absent in circular III strains but present in linear 111 strain K45. Size markers are A/Hindi 11/EcoRI fragment positions. 45.2 a 46 restriction fragment length polymorphisms were not detected with other restriction enzymes. Later hybridization analyses suggested that a recombination event occurred in the HMLtt. distal region during formation of the ring III strains, likely in the vicinity of the 0.4 kb Sail fragment (see discussion). 2. Hybridization Screening of the Lambda Charon4A-Yeast DNA Library Genomic DNA libraries are constructed with lambda or cosmld vectors for chromosome walking studies, since the average length of the Insert fragments is greater than that In libraries made with plasmid vectors. A lambda Charon4A (hereafter referred to as ACh4A) rather than a cosmld vector-yeast genomic DNA library was prepared for Isolating the IIIL telomere by chromosome walking for two reasons: (1) The probability of cloning noncontiguous chromosomal DNA In a given clone Is higher for cosmld vectors since the average length of the Insert fragments (30-45 kb) Is double that for lambda vectors (15-20 kb). Consequently, the possibility of "chromosome hopping" rather than "chromosome walking" is enhanced with a cosmld vector library. (2) Restriction mapping the yeast DNA Inserts would be simpler for the lambda vector versus the cosmld vector clones due to the shorter Insert lengths. The genomic DNA library of yeast strain AB20a XP8-10B was constructed by ligating 16 -20 kb EcoRI partial digest fragments with the EcoRI arm fragments of the ACh4A vector (28) and packaging the resulting DNA concatamers with lambda phage extracts in vitro (68, 90). A titer of 10 7 pfu/ml was obtained when the yeast library was plated on LE392 cells. An aliquot from the library was grown In culture and the phage DNA was digested with EcoRI and fractionated by agarose gel electrophoresis. Prominent ACh4A stuffer fragments, (6.6 kb and 7.8 kb EcoRI fragments), Indicated that nonrecomblnant ACh4A phage DNA was present in the yeast genomic DNA library. The proportion of ACh4A nonrecomblnant phage to ACh4A phage with yeast Inserts could not be estimated since the nonrecomblnant ACh4A phage may have a growth advantage over recombinant phage in liquid culture. The number of recombinant phage having an average Insert length of 17 kb that should be screened for a 99$ probability of recovering a unique yeast genomic sequence was calculated as 4 x 10 3 phage (52). Plaque hybridization of 5 x 10 3 phage using the Initial nick-translated probe (1*) 47 Identified three recombinant phage, named A4ALB1-4, A4ALB2-4, and A4ALB3-4 Fig. 5a). The recombinant phage named A4ALB4-4 was isolated from the yeast genomic library in a subsequent hybridization screening experiment using probe 1 *. These phage clones were purified through a series of four plaque hybridizations since the background ACh4A nonrecomblnant phage present in the library outgrew the selected recombinant phage in liquid culture. The DNA isolated from the phage clones was analyzed by restriction enzyme digestion and agarose gel electrophoresis. The Insert fragments In the phage clones differed only in the presence or absence of the 0.5 kb EcoRI fragments that flank the 16.1 kb region containing the4.6kb,4.9 kb,5.7kb,and0.9 kb EcoRI fragments (Fig. 5a). Phage clones A4ALB1 -4 and A4ALB3-4 contained the 16.1 kb region while A4ALB2-4 and A4ALB4-4 contained the 16.1 kb region flanked by the two 0.5 kb EcoRI fragments. The restriction map of the HliLot distal region In the phage clones (Fig. 5b) was derived by restriction enzyme analysis using single and double enzyme digestions and Southern hybridization using the probe 1* or the adjacent 2.3 kb Xbal /Hindi 11 fragment from the pHML clone (Fig. 3c). The fragments that were mapped distal to the HML<x region In the Southern hybridization experiment with yeast genomic DNA and probe 1 * (Fig. 4b) were present in the phage clones, ie. the 4.9 kb EcoRI, 6.5 kb Hindi 11, and > 15 kb Sail fragment. None of the clones Isolated from the ACh4A/yeast library had Insert fragments that mapped to the IIIL alternate region. The lack or absence of the IIIL alternate region In the ACh4A/yeast library may be explained by an unfavourable arrangement of EcoRI sites In that region for cloning 16-20 kb partial digest fragments (125). The EcoRI fragments in A4ALB4-4 were shotgun cloned into pBR325 which h8d been EcoRI digested and dephosphorylated with Bacterial Alkaline Phosphatase (BAP), (29, 30). The subclones with the yeast EcoRI fragments that mapped to the region distal to HMLft.. were referred to as pTel-1 (4.6 kb Insert) and pTel-2 (4.9 kb Insert). The subclones pTel-3 (5.7 kb Insert), pTel-4 (0.9 kb Insert), and pTel-5 (0.5 kb Insert) mapped to the centromere proximal side of HMLct (Fig. 5c). The 0.5 kb EcoRI fragment that mapped distal to pTel-1 In the phage clone A4ALB4-4 was not cloned, due to the problem of distinguishing It from the upstream 0.5 kb fragment. The restriction map established for the region distal to HliLa, In the phage 48 Figure 5. Chromosome Walking Steps From HMLa on IIIL. a. Genomic clones Isolated from the ACh4A/ yeast library with a nick-translated pHMLot probe 1* (2.2 kb Hindlll/Xb8l). The extent of the yeast DNA inserts in the phage are indicated, and the region homologous with probe 1* Is depicted by the bar above the insert DNA. Phage A4ALB1-4 and A4ALB3-4 were Identical (16.1 kb partial EcoRI digest fragment), while phage A4ALB2-4 andA4ALB4-4 h8d identical 17.1 kb Inserts of EcoRI fragments. b. Restriction map of the region isolated on chromosome IIIL. Restriction sites were mapped using multiple restriction enzyme digestions and Southern hybridization analysis. Restriction sites: R, EcoRI; H, Hindi 11; B, BamHI; S, Sail; Xh, Xhol; and X, Xbal. The orientation of the map, with respect to the centromere on chromosome III, is indicated as distal at the telomeric end or proximal at the centromerlc end. c. EcoRI fragments from phage A4ALB2-4 were subcloned into the EcoRI site of pBR325. The position of the Insert fragments for the subclones pTel-1, pTel-2, pTel-3, and pTel-4 on the restriction map of the IIIL region is indicated. Further restriction sites were mapped in the pTel-1 and pTel-2 subclones that were distal to HMLtx : T, BstEII; P2, Pvull; N, Ncol; K, Kpnl; and P, Pstl. The distal 1 kbEcoRI/Sall fragment or the 0.5 kb and 1.2 kb EcoRI/Pvull fragments in pTel-1 were referred to as probe 2* and used for further chromsome walking experiments. d. Genomic phage clones A4ALB12-5 and A4ALB102-3 were isolated from the ACh4A/yeast library using probe 2*. The Insert fragments In both recombinant phage extended no further toward the telomere on IIIL than probe 2*. Phage A4ALB102-3 contained an additional centromere proximal 3.8 kb EcoRI fragment. e. The phage clone, A4ARLB301, was Isolated by in viva recombination selection from the ACh4A/yeast genomic library, using the recombinant microplasmid npT-T. Other phage clones Isolated by this screening method, A4ARLB302-A4ARLB312 were identical to A4ARLB301. The 1.9 kb EcoRI fragment that mapped distal to probe 2* was present in all recombination selection clones, and it resulted from the integration of the ITpT-T microplasmid into the phage and the duplication of the target 1 kb EcoRI/Sall fragment (probe 2*) upon integration. 48 OL St) A4ALB1-4 A4ALB3-4 I-A4ALB2-4 A4ALB4-4, (g) (D ® MIL TEL DISTAL — H —I— I -XhS B R 1 * HMLQ -m H — i —i i i BHS XhB X R X X HXh pTsl-2 2* pT«l-1 i I I It I H H — I R P2 S ' J_T / L B R K P2 Xh S -t-H H—H 1 BHS XhB PX R A4ALB12-5. A4ALB103-I-12 14 pT«l-3 A4ARLB301 # 1 1— 16 kb pTel-4 CEN3 PROXIMAL R R npT-T 49 clones was confirmed through Southern hybridization of restriction enzyme digested DNA from the A4ALB phage clones using the pTel subclones as probes. The most distal probe, pTel-1, extended 9.6 kb distal to HMLo; on IIIL. The second set of chromosome walking probes, referred to as probe 2* , were isolated from the distal end of pTel-1. The probe 2* fragments extended 7.3 kb from probe 1* toward the left end of chromosome III and included the 1 kb EcoRI/Sall, 3.8 kb EcoRI/BamHI, and 0.5 kb EcoRI/Pvull fragments (Fig. 5c). The ACh4A-yeast DNA library was screened by plaque hybridization with probe 2* using at least ten times the required number of recombinant phage to encompass the yeast genome (5 x 10 4 pfu). The phage clones A4ALB12-5 and A4A102-3 were Isolated using probe 2* (Fig. 5d), however probe 2* was the most distal yeast fragment in both of these clones. 5. Recombination Screening of the Lambda Charon4A-Yeast DNA Library To determine whether phage clones that contained inserts from the IIIL region that was distal to probe 2* were present but 8t very low frequency In the yeast library, the ACh4A-ye8st DNA library was screened with probe 2* using In YJYQ recombination selection (126, 174). The sensitivity of this screening method allows the selection of a single recombinant phage from a population of 10 6 pfu. A probe 2* fragment (1 kb EcoRI/Sall) was cloned into the EcoRI/Sall sites In the polyllnker region of the IIAN7 microplasmid which contains the amber supressor gene supF, to produce the recombinant ripT-T. The ACh4A-yeast DNA library was amplified and both the unamplified (10 5 pfu) and amplified (10 6 pfu) phage populations were passed through the E. coli strain MC1061(p3) (Su - cells) that h8d been transformed with npT-T. The resultant phage titers were determined on the E. coli strain LE392 (Su + cells), then 10 6 - 1 0 7 phage were plated on MC1061(p3) to select for phage containing the supF gene by a prior recombination event with npT-T. The expected recombination frequency for the probe 2* and the phage library Is 10~6 (126, 174) but the recombination frequency of the unamplified library was 4 x 10~5 and the amplified library was 4 x 10~3 . The elevated recombination frequencies obtained for the ACh4A library presumably reflect the reversion of the amber mutations in the ACh4A phage since amber 50 revertants can also grow on MC1061(p3). The proportion of amber revertants In the ACh4A-yeast DNA library was determined by plating the amplified and the unampllfled ACh4A/yeast library (10 4 pfu) on the MC1061(p3) Si r strain without prior passage through the microplasmid bearing strains. There was no phage growth for the unampllfled ph8ge library, but a IO - 2 reversion frequency W8S obtained for the amplified library. Apparently the amber revertants had a growth advantage during the amplification of the ACh4A-yeast DNA library. To distinguish between revertant Su + phage and true recombinant Su + phage, probes from the region that was distal to HMLK. on IIIL were used for secondary screening of the Su + phage by plaque hybridization. The SupF recombinant phage Isolated from the unampllfled ACh4A-yeast DNA library were selected by growth on the Su _ host MC1061(p3), and screened by plaque hybridization with either probe 2* or the 1.2 kb EcoRI/Sall fragment that mapped upstream from probe 2* In pTel-1 (Fig. 5c). About 75$ of the Su + phage hybridized with both probes. A set of the positive phage clones named A4ARLB and numbered 301 to 312 were purified and characterized by restriction enzyme digestion of the Isolated DNA. All of the recombinant Su + clones had Identical restriction maps to the phage clone A4ALB1-4 except for an additional 1.9 kb EcoRI fragment that mapped adjacent to pTEL-1, (F1g. 5e). However, this extra fragment resulted from the recombination event between the IIIL distal 1 kb EcoRI/Sal I fragment and the homologous probe 2* fragment In IIpT-T which results In the duplication of the target site upon integration of the IIpT-T microplasmid. 4. Hybridization Screening with other Yeast Genomic DNA Libraries The probe 2* distal region was apparently not represented In the yeast genomic DNA library that was constructed with yeast EcoRI fragments and the EcoRI arm fragments of the phage ACh4A. Presumably a genomic DNA library prepared using a restriction enzyme that has a four base pair recognition site would have a more random distribution of Insert fragments and may contain clones with further distal fragments from the IIIL telomerlc region than the ACh4A library that was constructed using EcoRI which has a six base pair recognition site. A yeast genomic DNA library had been prepared by partially digesting the DNA of the strain AB972 (169) with Sau3A to obtain 51 fragments that were 15 kb average length. The phage vector that was used for cloning the 5au3A fragments was AMG14 (fi. Olson, personal communication) which consisted of the left arm of A1059 (100) and the right arm of ACh30 (164). Southern hybridization of restriction enzyme digested DNA from yeast strain AB972 with a HMLtx distal fragment (probe 1*) did not reveal any restriction fragment length polymorphisms between AB20* XP8-10B (ACh4A-yeast DNA library ) and AB972 (AMG14-yeast DNA library), (Fig. 4a). The AMG14-yeast DNA library Initially had 100* recombinants but had been amplified prior to plaque hybridization with the IIIL distal fragments. Hybridization screening of aboutl-2 x 10 4 pfu in total, (10 3 pfu/85 mm plate), resulted in the Identification of multiple recombinant phage (designated AMGBO 1 to AMGB014) that were homologous with probe 1*. Mapping studies indicated that the inserts In the AMGBO phage clones had different restriction fragments than those contained In the recombinant phage Isolated from the ACh4A-yeast DNA library with probe 1*. The restriction maps that were derived for the AMGB01 to AMGB014 clones (Fig. 6) show that all phage have Insert regions about 17 kb long, which differ only in the presence or absence of the distal Hindlll or EcoRI sites. The 2.2 kb Hindlll/Xbal/ pTel-2 fragment, (probe 1*), hybridized with the 5 kb Hindlll fragment and the 1.0 kb EcoRI/Sall pTel-1 fragment (probe 2*) did not hybridize with any of the AMGBO clones. Southern hybridization analysis of yeast DNA from linear and circular III strains (Fig. 4b) revealed that a 5 kb Hindlll fragment was homologous with probe 1 * but It mapped to the 111L alternate region. Presumably the AMGBO clones that were Isolated contain fragments from the IIIL alternate region, not the IIIL distal region. Plaque hybridization of the AMG14-yeast DNA library with probe 2* did not detect homologous recombinant phage, even when 50 genomic equivalents of phage (5 x 10 4 pfu) were screened. Possibly the IIIL distal region was poorly represented In the AMG14-yeast library and consequently It was eliminated during the amplification of the library. 52 Figure 6. Restriction Endonuclease Maps of Recombinant Phage AM6B01 -14. Restriction maps the yeast DNA inserts in the recombinant phage isolated from the AMG14/yeast library, using nick-translated probe 1* fragments ( pHMLot, 2.2 kb Hindlll/Xbal or 1.4 kb Hindi U/Xhol illustrated in Fig. 3c). All clones contained similar partial Sau3A fragment inserts (about 17 kb), with Hindi 11, (H) and EcoRI, (R) sites indicated. The four identified clone sets differed only in the presence or absence of flanking Hindi 11 and EcoRI sites. Probe 1* hybridized to a 5 kb Hindi 11 fragment, and a >5 kb EcoRI fragment in all clones. 1* H R H H R R H R H 4 MGB01 ^ 4 M6PQ2,3,5 • 4 MGB04, 8, 13. 14 ¥ « M G B 0 6 . 7 . 9 . 10. 11. 12 , 0 2 4 6 8 10 12 14 16 IS kb 53 As an alternate approach, a yeast genomic library that was prepared with 15 kb Sau3A fragments of yeast DNA and the yeast plasmid vector Yep 13 (142), was screened with pTel-1 fragments (probe 2*) by high-density screening of at least 2 x 10 4 clones (84,85) and by grid plate screening using sufficient clones to Include the entire yeast genome (5000 colonies). As a positive control for the hybridization, the HMLfr 1.4 kb Xbal/Hlndlll fragment (Fig. 3c) hybridized with clones on duplicate filters, with the expected frequency. Probe 2* did not hybridize to any colonies, Indicating the IIIL distal region was not represented In the Yep13-yeast DNA library which has been used successfully to Isolate many yeast genomic segments Including the yeast mating type genes (142). 5. Southern Hybridization of Yeast Genomic DNA with Probe 2* The absence of probe 2* distal fragments In yeast genomic DNA libraries suggested that probe 2* was In the proximity of the IIIL telomere and the structure of the telomere may have precluded the cloning of the probe 2* distal region In the libraries. To establish whether probe 2* which mapped 8.6 kb distal to HHI& was near the chromosome IIIL end, Its hybridization characteristics with yeast genomic DNA were studied. Probe 2* hybridized exclusively to the IIIL distal region, as evidenced by the single fragment homologous with probe 2* for most restriction enzyme digestions (Fig. 7a). Two fragments (3.0 kb and 1.2 kb) hybridized with probe 2* In Pvull digestion (Fig. 7a, lane 4), due to the Pvull restriction site within the probe 2* (1 kb EcoRI/Sall) fragment (Fig. 7b). The restriction fragments that were homologous with probe 2* In the yeast genomic DNA digestions (Fig. 7a) agreed with the fragment lengths that were mapped on the IIIL clones Isolated from the ACh4A-yeast genomic DNA library (Fig. 5b). It was possible to predict the distance between probe 2* and the distal restriction enzyme sites on IIIL since the sites on the proximal side of probe 2* had been mapped (Fig. 5b). For example, the 10 - 12 kb Pstl fragment (Fig. 7a, lane 3) must extend 1.7 to 3.7 kb on the telomeric side of probe 2* . since the upstream Pstl site mapped 8.3 kb centromere proximal from the EcoRI site on the distal end of probe 2* (Fig. 7b). In the same manner, the distal Pvull site was estimated to be 2.5 kb from probe 2* . (Fig. 7b), since the 3.0 kb Pvull fragment (Fig. 7a, lane 4) encompasses the distal 0.5 kb of probe 2*. None of the distal restriction enzyme sites that were estimated in this manner extended further than 3.5 kb from probe 2* toward the IIIL telomere 54 Figure 7. Southern Hybridization with Probe 2* and Yeast Genomic DNA. a. Aliquots of DNA from yeast AB20<x XP8-10B (3.3 ug) were digested with restriction enzymes, fractionated on a 0.65$ agarose gel, and transferred to gene screen plus filter. Probe 2* was a 1 kb EcoRI/Sall fragment Isolated from pTel-1 and cloned In M13mp8. This probe 2* clone was called MTeLB 1 - 1 , and it mapped 8.6 kb distal to HrlUx. Restriction enzyme digestions: lane 1, EcoRI; 2, H1ndlll;3,Pstl;4,Pvull;5 IBstEII;6,Kpnl;7 1BamHI;8,Xbal;9,Sall;and 10, Xhol. Size markers are A/Hindi 11/EcoRI fragment positions. b. Estimated positions for the restriction enzyme sites in the region that Is distal to probe 2*. The RE map in the region between HMLK. and probe 2* was determined from the genomic phage clones and the plasmid subclones (Fig. 5b,c). The length of the fragments homologous with probe 2* were determined from Southern hybridization (Fig. 7a). For each restriction enzyme, this fragment length was the distance between the centromere proximal site and the centromere distal site on either side of probe 2*. Fragments 1 -10 In the restriction maps for the probe 2* region correspond to lanes 1-10 in the Southern blot. 1 2 3 4 5 6 7 8 9 10 kb -2t5 44 36 b = 8 - 1 7 • - - 13 - 1P - Q9 0.6 HML MIL EMU .CEN3 1 EcoRI Hindlll I 1 Pstl " I 1 1 P » " " | | BslEII 6 | | Kpnl BamHI Sai l 1 o 15kb 55 (Ftg. 7b). In the Southern hybridization with probe 2* , heterodisperse bands were observed in the Pvull (Fig. 7a, lane 4) and Sail (Fig. 7a, lane 9) digestions . Studies have shown that telomerlc fragments In yeast are heterogeneous in length due to variable amounts of simple 5'-C 1-3A-3' repeat units at chromosome ends (49, 175, 206). This suggested that the Sail and Pvull fragments homologous with probe 2* contained the variable repeat lengths of the IIIL telomerlc region and hence probe 2* was proximal to telomerlc DNA. However, probe 2* hybridized to a unique region of the yeast genome and therefore It was apparently not Included In the telomerlc repeat region that contains the complex X or Y' A M regions and the simple T region repeats (48,49). B. The IIIL Distal Region Is Retained In Some Circular III Strains The region between probes 1* and 2* that was Isolated from the yeast genomic library was hybridized to the genomic DNA from yeast strains that were similar except for the presence of a linear chromosome III or circular chromosome III In the haploid genome. The rationale was that the differences In the Southern hybridization patterns for the circular III and linear III strains would show that the IIIL distal region rather than the IIIL alternate region had been Isolated in the phage clones since It was expected that the entire HMLK, distal region was deleted In ring III strains (109). However, results from the Southern hybridization analysis Indicated that the region distal to probe 1 * is retained In most of the circular 111 strains examined. A single fragment was homologous to the distal 1 kb EcoRI/Sall fragment (probe A) In the linear III strain K45 and all circular 111 strains except K192 (Fig. 8A, lane 3). The lengths of the EcoRI, Hindi 11, and Sail fragments were consistent for K45, a linear III strain, (Fig. 8A, lane 1) and for those fragments retained in the K191, K193, K195, and K196 ring III strains (Fig. 8A, lanes 2,4,5,6) . The 2.4 kb Sail fragment (probe B) hybridized with two regions In the linear III strain K45, and to a single fragment In all of the circular III strains (Fig. 8B). For most circular III strains, the hybridization pattern for probe B was contrary to the expected result. The IIIL distal fragments that were homologous with probe B were retained In the circular III strains K191, K193, K195, and K196, while the IIIL alternate fragments that were homologous with probe B wereajjsjoi (Fig. 8B, lanes 2, 4, 5, 6). Only circular III strain K192 had the expected hybridization pattern since the IIIL distal fragments were absent and the IIIL alternate 56 Figure 8. Southern hybridization of Linear III and Ring III Yeast Strains with Probes from the IIIL Distal Region. Fragments that mapped to the region distal to HMLft, between probe 1 * and probe 2*, were cloned as Sail or Sall/EcoRI inserts in M13mp8. Probes A to E refer to the following Ml 3 clones: A, MTeLBI-1.0; B, MTeLB1-2.4; C, MTeLB 1-1.2; D, MTeLB2-2.3; and E, MTeLB2-2.6; and the number following the hyphen in each name indicates the length of the insert fragment. Genomic DNA from the linear 111 yeast strain K45 and the circular 111 yeast strains K191, K192, K193, K195, and K196 was digested with EcoRI .Hindll I, or Sail, (3.3 ug DNA/digest), fractionated on a 0.65* agarose gel, and transferred bidirectionally to gene screen plus filters. Southern hybridization with probes A,B ,C, and D resulted in the blots labelled A to D respectively. The Southern blot with probe E was included with the probe 1* hybridization results (Fig. 4b). Lanes: 1, K45; 2, K191; 3, K192; 4, K193; 5, K195; and 6, K196; and EcoRI, Hindlll, or Sail enzyme digestions are indicated. The restriction fragments which map to the IIIL distal region are Indicated by the symbols on the right side of the K45 lane (lane 1) for EcoRI ( • ) , Hindlll ( • ) , and Sail (A) digestions. Size standards were A/Hlndlll/EcoRI fragment positions which are indicated by the horizontal bars at the left, and the positions of A/Hindi 11 fragments at the right side of the southern blots for A-D. 56 a. MIL END HML i—h 1 — h CEN 3 A +• B C <*• D EcoRI -HindJII Sall_ B -EfifiBl Hindlll Sail 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 kb 213-kb -|237 -95 -67 -43 -J20 -Sal] U ECORI Hindlll 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 1 2 3 4 5 6 57 region fragments were retained and hybridized with probe B for each of the EcoRI, Hindi 11, and Sail digestions (Fig. 8B, lane 3). These hybridization characteristics for the various ring 111 strains were also observed with the adjacent 1.2 kb Sall/EcoRI (probe C) fragment. The IIIL distal region was deleted In circular 111 strain K192. but retained in all of the other circular III strains, K191, K193, K195, and K196 (Fig. 8C). The 11IL alternate region was retained in K192, and was deleted in K191, K193, K195, and K196 (Fig. 8C). Since both the 111L distal and the 111L alternate region had a 3.6 kb Sail fragment homologous with probe C, no difference In hybridization was detected between K192 and the other ring ill strains for Sail digested DNA (Fig. 8C-Sa1l, lanes 2-6). The pTel-2, 2.3 kb EcoRI/Sall fragment (probe D) had a mixed hybridization pattern for the IIIL distal and the IIIL alternate region fragments that were retained in the circular III strains (Fig 8D). With EcoRI digestion, the IIIL alternate region 8.5 kb fragment was retained, and the IIIL distal 4.9 kb fragment was deleted in all circular III strains (Fig 8D-EcoRI, lanes 2-6). With Hindi 11 digestion, K192 was the only circular 111 strain that had deleted the 11 IL distal 7 kb Hindi 11 fragment and retained the 11 IL alternate 6 kb Hindi II fragment (Fig. 8D-Hindlll, lane 3 ). The other ring III strains had the reverse Hindi 11 fragment pattern which was similar to that observed with probes A, B, and C. The IIIL distal fragment (7 kb Hindi 11) was retained in the circular 111 strains K191, K193, K195, and K196, while the IIIL alternate fragment (6 kb Hlndlll) was deleted (Fig. 8D-Hindlll, lanes 2, 4, 5, 6). The pTel-2,2.6 kb Sall/EcoRI (probe E) hybridized exclusively to IIIL alternate region fragments In all circular III strains examined (Fig. 4b). Possibly, that portion of the IIIL distal region was deleted during the HMLp, - HMRa fusion event (109) that produced the circular 111 strains. These Southern hybridization results are summarized In Table I. The presence of the IIIL distal fragments in a given yeast strain Is Indicated by closed symbols and the IIIL alternate region fragments are Indicated by open symbols. Since K45 has a linear chromosome III, both the IIIL distal and alternate region fragments were present for probes B to E. Probe A was unique In the yeast genome and hybridized to only the IIIL distal region. Strains with a circular chromosome III (K191, K192, K193, K195, K196) have deleted either the IIIL distal or IIIL alternate fragments that hybridized with probes B to E. Circular III strain K192 was unique In having the entire IIIL distal region deleted while the 58 Table I. The IIIL Distal Region Is Retained In most Circular III Strains. The Southern hybridization data from Figures 4 and 8 are presented in this table to demonstrate the pattern of hybridization for the probes from the IIIL distal region. Fragments homologous with probes A to E are represented by the symbols, where the closed symbols are fragments which mapped to the IIIL distal region, and the open symbols are fragments that mapped to the IIIL alternate region. Both IIIL distal and IIIL alternate regions are present in the linear III strain K45 for all the probes except for A which maps exclusively to the IIIL distal region. The presence of the fragments from the IIIL distal or 11 IL alternate regions in the circular 111 strains (K191, K192, K193, K195, and K196) is indicated by closed or open symbols. A hybrid pattern of open and closed symbols is evident in most circular III strains and this may reflect the position of a recombination or conversion event. The fragments from the IIIL distal region that were retained in the genomes of the ring III strains K191, K193, K195, and K196 and the complete deletion of the IIIL distal region in the strain K192, is sketched on the map below the table. Probe Enzyme K45 K191 A EcoRI • 4.6 • Hindlll • 7.0 • Soil • 3.3 A B EcoRI • 4.6D3.8 • Hindlll • 7.0O6.0 • Soil • 2.4A 2.5 A C EcoRI • 4.6 • 3.8 • Hindlll • 7.0 O 6.0 • Soil A 3.6 A 3.6 A D EcoRI • 4.9 • 8.5 • Hindlll •7 .0 O 6.0 • Sail A3,6 A 3.6 A E EcoRI • 4.9 • 8.5 • Hindlll • 6 . 5 O 5.0 O Sail A>20A 0.4 A K192 K193 K195 K196 — • — • - A • • o • A A • • O • A A • • O • A A • • O O A • • • • A A • • • • A A • • • • A A • • • • A A • • O O A A IIIL End** " 4 " K191 K193 K195 K196 A B C A -4-D HML CEN3 R E T A I N M D E L E T E ^ 59 IIIL alternate region was conserved. Circular 111 stralns K191, K193, K195, and K196, had a break point In the hybridization pattern for the IIIL distal versus IIIL alternate fragments In the probe D region. These circular III strains retained IIIL distal fragments In the probe A to D region, presumably at the IIIL alternate region since the IIIL alternate fragments were absent for the probe A to D region. Possibly gene conversion events occurred concomlnantly with the HHL& - HMRa fusion events, replacing a portion of the IIIL alternate region with the IIIL distal region (Table I). C. Transcription In the IIIL Distal Region To Investigate the reason for the strong homology between the IIIL distal and IIIL alternate regions and for the retention of the IIIL distal region in most circular III strains, I asked whether there is a transcription unit In the IIIL distal region that Is repeated at the IIIL alternate region in the genome of haploid yeast strains. An essential, but functionally duplicated gene In the HMLq. distal region could answer these problems and also explain why circular III strains that contain only one of these loci are viable as haplolds (109). Preliminary dot blot analysis of total yeast RNA Isolated from linear III or circular chromosome III strains hybridized with the HMLtt, distal probe pTel-1, which includes the probes A, B, and C used in the previous Southern analysis (Fig. 8), indicated that there are RNA transcripts homologous with this region (Fig. 9a). Northern analysis of the total RNA from linear III strain K45 and circular III strain K191 (Fig. 9b) indicated that the pTel-1 probe hybridized to a single transcript sized at approximately 1.4 kb. Since pTel-1 maps 5 kb distal to HMLtt,and It is homologous to a 1.4 kb transcript, a transcription unit In the IIIL distal region must map at least 4-5 kb distal to HMLtt, and this corresponds to the 11 IL distal region that Is retained at the IIIL alternate region In four of the five circular III strains studied. Considering that such a transcript Is separated from HMLq. by more than 4 kb, it cannot be a run-off transcript from HMLtt, in the marl strains K45 and K191 and It maps to the middle of the fragment that was retained In the circular 111 strains. Since a RNA transcript Is homologous with the pTel-1 probe in the circular III strain K192 which lacks the IIIL distal region (Fig. 8, Table I), there must also be a transcription unit at the IIIL 60 Figure 9. Hybridization Analysis of Yeast RNA with a IIIL Distal Probe, a. RNA Dot Blot Analysis. Total yeast RNA was spotted on a Gene screen plus filter using a BRL Hybrldot Manifold. Yeast strains AB20a XP80-10B, K45, K191, K192, and GM3C2 (ACYC) had 1, 3, 5, 10, and 20 ug RNA as Indicated, but strain BM-CYC+ had only the 1, 3, 5, and 10 ug RNA samples. Probe pTel-1, the distal 4.6 kb EcoRI fragment on IIIL which Includes probe 2* In the vector pBR325, was nick-translated to a specific activity of 3.5 x 10 7 cpm/ug (Cerenkov counts). A pBR322 probe that was used as a hybridization control did not show homology with any of the RNA samples except for strain BM-CYC+. b. RNA Northern Analysis. Total yeast RNA from the yeast strains K45 and K191 was fractionated on a 1* agarose - formaldehyde gel, transferred to a gene screen plus filter, and was hybridized with pTel-1, that was nick-translated to a specific activity of 4 x 10 7 cpm/ug (Cerenkov counts). Lanes: 1,K45,(7.5ug); 2, K45, (15 ug); 3, K191, (7.5 ug); 4, K191, (15 ug). Size standards were the positions for denatured A/Hlndlll/EcoRI fragments. 60a. 61 alternate region. If the IIIL distal region and the IIIL alternate region have equivalent transcription units, then the IIIL distal region was retained In some of the circular III strains as a consequence of the recombination event with a broken chromosome end rather than as a functional selection. D. Probe 2* Is Adjacent to the IIIL Telomere 1. Ba131 Nuclease Sensitivity of the Probe 2* Region Chromosome ends In yeast (175, 206) and trypanosomes (18, 24, 60, 203, 209) are sensitive to digestion with the double-stranded exonuclease Bal31 (80). The rationale behind the following set of experiments was that If probe 2* from the IIIL distal region mapped to within a few kllobase pairs of the IIIL terminus, some restriction fragments would contain both the IIIL end and probe 2*. Such fragments would be sensitive to Bal31 nuclease digestion and would hybridize with probe 2* In Southern hybridization analysis. Determination of the Bal31 nuclease sensitivity of yeast genomic DNA and the length of time required to shorten the restriction fragments that are homologous with probe 2* would reflect the distance between the IIIL terminus and probe 2*. Yeast DNA was digested with Bal31 nuclease for Increasing periods of time (Fig. 10). Aliquots of DNA digested with Bal31 nuclease were subsequently digested with the restriction enzymes Sail or Pvull, and analyzed by Southern hybridization with probe 2*. Since variable lengths of chromosome ends had been demonstrated in yeast genomic DNA (40, 49, 93, 175, 206) and since probe 2* hybridized to heterodisperse Sail and Pvull fragments (Fig. 7b), I asked whether the IIIL terminus was Included In these fragments. The 3.3 kb Sail and the 3.0 kb Pvul I fragments were readily digested with Bal31 nuclease and the region homologous with probe 2* was completely deleted after 15 min of Ba131 nuclease treatment. With the digestion conditions used for Bal31 nuclease, about 150 bp/mln were deleted from the IIIL end, hence probe 2* was a minimum of 2.3 kb from the IIIL end. The sensitivity of the 3.0 kb Pvull fragment to Bal31 nuclease digestion and the resistance of the upstream 1.2 kb Pvull fragment that was homologous with probe 2*, provided an Internal control for the specificity of Bal31 nuclease digestion at the termini of chromosomes. The length heterogeneity and the Bal31 sensitivity of the 3.3 kb Sail and 3.0 kb Pvull fragments suggested that these fragments contain the IIIL chromosome end, and that the terminus maps 2.3 kb distal to probe 2*. 62 Figure 10. Length Heterogeneity and Bal31 Nuclease Sensitivity of the IIIL Distal Region. Yeast genomic DNA (AB20a XP8-10B) was treated with exonuclease Ba131 (0.1 u/ug) for up to 30 min. DNA was digested with Salt or Pvull for each time point aliquot, fractionated on a 0.65* agarose gel (3.3 ug/lane), and transferred to gene screen plus filters. Hybridization was with probe 2* (MTeLB 1-1.0). Size markers are A/Hindi 11/EcoRI fragment positions. SaiJ . Pvuii 6 9 15 30 0 3 6 9 15 30 63 2. The IIIL Alternate Region Is Telomere Proximal Since the IIIL distal region consisted of 12 kb between HMLfl, and the IIIL telomere, I asked whether the homologous IIIL alternate region was also near a chromosome end. Bal31 nuclease digestion with yeast genomic DNA from the linear III strain K45 and the circular III strains K191 or K192 provided an answer to this question. The 1.2 kb Sall/EcoRI fragment from the pTel-1 clone (Fig. 5) was used as probe for Southern hybridization analysis since it hybridized to both the IIIL distal and the IIIL alternate region (Fig. 8C). The 7 kb Hindlll fragment from the IIIL distal region and the 6 kb Hindlll fragment from the IIIL alternate region were sensitive to Bal31 nuclease digestion (Fig. 11), suggesting that both IIIL distal and IIIL alternate regions are telomeric. The hybridization signal was lost after 10 mfn of Bal31 nuclease digestion for the IIIL alternate region, while the IIIL distal region remained for at least 20 mfn of digestion with Bal31 nuclease. Assuming the probe mapped 5.7 kb from the end of the 11 IL distal region and the rate of Bal31 digestion was equivalent for all chromosome ends in the genome of K45, the IIIL alternate region had the homologous region only 2.8 kb from a chromosome end. For circular III strain K191 which had retained a large portion of the IIIL distal region, the kinetics of digestion of the 7 kb Hindlll fragment with Bal31 nuclease was equivalent for K45 and K191 Indicating the distance from the probe to the chromosome end was equivalent for each (5.7 kb). If the IIIL distal region replaced the IIIL alternate region in strain K191 by a recombination or conversion event, then either the IIIL distal telomere replaced the IIIL alternate telomere or the distance between the probe 8nd the telomere at the 111L alternate region was Increased. Alternatively, strain K192 which did not retain the fragments from the IIIL distal region and which contained the 6 kb Hindlll fragment from the IIIL alternate region, had the same kinetics of Bal31 nuclease digestion as the IIIL alternate region In K45. 3. The 11 IL Telomere Associated Region Maps Distal to Probe 2* The length of telomeres on yeast chromosomes Is genetically controlled (40), hence the length of the terminal DNA restriction fragment on a chromosome Is strain specific (93, 206). A 3.3 kb (+/- 0.2 kb) Sail fragment hybridized with probe 2* In the yeast strain AB20ot, XP8-10B (Fig. 12c, lane 2), but a 3.0 kb CV- 0.2 kb) Sail fragment hybridized with probe 2* In strain K45 64 Figure 11. The IIIL Alternate Region Is Bal31 Nuclease Sensitive. DNA from the linear III yeast strain K45, and the circular III strains K191 or K192 was digested with Bal31 nuclease (0.32 u/jig) for up to 20 min. Following HindifI digestion, fractionation on a 0.6$ agarose gel, and transfer to gene screen filters, hybridization was with MTeLB 1 -1.2 (probe C, Fig. 8C) since it shares homology with both the IIIL distal and IIIL alternate regions in the yeast genome. For each yeast strain the DNA samples were U, Bal31 nuclease untreated, followed by 0 min, 5 min, 10 min, 15 min, and 20 min Bal31 nuclease digestions as indicated. Lane M contains the X/Hindll I fragments that were used as size markers. 64cu 65 Figure 12. Probe 2* is not Homologous with Telomere Associated Regions in Yeast. a. Restriction maps of the yeast telomere associated region in clones: pSZ220, (193); YRp 131A or YRp 131B, (48,49). The extent of the X region, Y region, and 131 region in each clone 1s indicated. Restriction sites: N, Ncol; PI, Pvul; S, Sail; Sc, Sacl. b. DNA from yeast, phage A clones, and plasmlds was digested with restriction enzymes and fractionated on a 0.65* agarose gel which was stained with EtBr. The banding pattern for the DNAs was observed by UV fluorescence. Lanes 1-12: 1, A/Hindi 11/EcoRI; 2, AB20* XP8-10B/Sall, (4 ug);3,K45/Sall,(4ug);4,K191/Sall,(4ug); 5, K192/Sall, (4ug); 6, pSZ220/Pvul/Sacl, (0.5ug); 7, pTel-1/EcoRI/Sall, (0.25 ug); 8, A4ALB4-3/ EcoRI, (0.25 ug); 9, A4ALB102-3/EcoRI, (0.25 ug); 10, YRp131A/Sall/Ncol, (0.25 ug); 11, YRp131B/ Sall/Ncol, (0.25 ug); 12, A/Hindlll, (0.5 ug). c. DNA was transferred to gene screen plus filter, and hybridized with the IIIL distal probe 2* (MTeLB1-1.0). PSZ220 Yrp 131A Yrp131B PI Sc Y 0.8 kb S HIM I1IMI 1 I Mil I I mil ll UM I I Mil ri 11 1111 ll I I S N 131 10 kb N N N S 6.7 kb 65 a. b. c. 1 2 3 49810 12 J ? kb <237 « a s « 6.7 « 43 4 23 -A 20 66 (Fig. 12c lane 3). The length heterogeneity observed for different yeast strains supports the Idea that the Sail fragment homologous with probe 2* Includes the IIIL terminus. Probe 2* was unique to the IIIL distal region and did not hybridize with the ring III strain K192 (Fig. 12c, lane 5) which has deleted the entire IIIL distal region. However, the ring III strain K191 has a Sail fragment that Is homologous with probe 2* and it has an equivalent length to that in the parental linear 111 strain K45 (Fig. 12c, lanes 3&4). If this 3.0 kb ( V - 0.2 kb) Sail fragment does contain the IIIL telomere In K191, then the conversion event that replaced the IIIL alternate region with the IIIL distal region (Table I) also converted the telomere at the IIIL alternate region to the telomere from the IIIL distal region. Since probe 2* hybridized to yeast fragments which Included the IIIL terminus, I asked whether probe 2* also contained sequences that are homologous to the telomere associated region which consists of X and Y' repeats (49). The clones pSZ220 (193), YRp131A, and YRp131B, (47, 48, 49), (Fig. 12a) Isolated from the telomere associated regions In yeast were restriction enzyme digested, fractionated by agarose gel electrophoresis, and analyzed by Southern hybridization with probe 2* (Fig. 12b, 12c, lanes 6,10,11) alongside control clones that are homologous with probe 2* (Fig. 12b, 12c, lanes 7,8,9). However, probe 2* was not homologous with the type X AJiS. clone, YRp131A (Fig. 12c, lane 10) or the type Y _A£S_ clones, YRp131B and pSZ220, (Fig. 12c, lanes 6&11). Furthermore, Southern hybridization analyses using type X or Y probes to screen the cloned IIIL distal region did not reveal any telomere associated sequences In the 9.6 kb HMLtt; distal region (data not shown). The telomere associated region on IIIL must be distal to probe 2* and must be within the terminal 2.3 kb on 111L. E. Attempts to Clone the IIIL Telomere by Marker Rescue One strategy that I used in attempting to Isolate the 111L end involved the introduction of a yeast selectable marker in the proximity of the IIIL telomere. Cloning of the closely linked IIIL telomere would conceivably be possible on a linear or circular plasmid by recovery of the plasmid through selection for the marker on the vector, as outlined schematically (Fig. 13). This strategy was analogous to the marker insertion and eviction method that was developed to clone several yeast genes 67 Figure 13. Strategy to Clone the IIIL Telomere by Marker Insertion and Excision. a. Integrative plasmid pLB21 -17 contained the 3.8 kb EcoRI/BamHI fragment from pTel-1 in the Ylp5 vector (189). pLB21 -17 was digested with Xhol, which cuts 0.75 kb from the BamHI end of the insert fragment, to target pLB21 -17 for insertion in the IIIL distal region. b. Transformation of yeast strain SR25-1A (uracil auxotroph) with pLB21 -17/Xhol using the lithium acetate, alkali cation method (94) produced mitotically stable transformant YeLB21-17-26. The expected map for Integation at the IIIL distal region is indicated. c. Excision of URA3 from the IIIL distal region and co-isolation of the IIIL telomere. Transformant DNA is partially digested with Hindi 11 or Xhol under conditions such that the 11 IL end, probe 2* region, and the AMP to URA3 region on the vector are contained on the same restriction fragment. d. Recovery of the fragment as a linear plasmid could be accomplished by adding Tetrahymena rDNA termini to the Hindi 11 or Xhol end, as 0.7 kb Hindlll/Hhal or Xhol/Hhal fragments from pSZ221 and pSZ222 respectively (195). The linear plasmids would then be introduced into yeast strain SR25-1A and uracil prototrophic transformants would be characterized for linear plasmids containing the IIIL end. e. Recovery of the AMP marker along with part of the IIIL end is possible on a circular plasmid. YeLB21 -17-26 DNA is partially digested with Hindi 11 or Xhol, and treated with S1 nuclease to render blunt ends on the fragment, followed by Intramolecular ligation and selection for AMP in L coli. Colony hybridization with a IIIL distal probe would identify clones with a fragment from the IIIL telomeric region. Since the yeast telomerlc single-stranded breaks are sensitive to SI nuclease digestion (175), some of the terminal region will be deleted in cloning the IIIL telomere in a circular vector. Restriction sites: B, BamHI; H, Hindi 11; P, Pstl; R, EcoRI; S, Sail; X, Xhol. 67a. A M P / ^ \ X U R A 3 PLB 2 1 - 1 7 I l i t IIIL END 3 £ T T T "EH Ik 1 R CEN3 L2i_i URA3 AMP I2» I X B sis—XB 1 >? • 2*r i URA3 AMP • 2 * I (J IIIL END ' 2 * ' . U.1A3 AMP l 2 f I T«t rDNA END IIIL END AMP URA3 68 (183, 211). Plasmid pLB21-17 (Fig. 13a) was constructed by cloning the 3.8 kb EcoRI/BamHI fragment from pTel-1 (Includes probe 2* region) In Ylp5, which Is a yeast vector that contains the URA3_ marker and lacks a yeast origin of replication (189). Insertion of the URA3 marker In the IIIL distal region was achieved through the genetic transformation of yeast strain SR25-1A (which contains the non-reverting ura3-52 mutation) with pLB21 -17 that was digested with Xhol to target the Insertion (149, 150) and selection for uracil prototrophic transformants. Since Ylp5 cannot replicate autonomously In yeast, URA3 transformants presumably had pLB21 -17 Integrated by site directed, homologous recombination into yeast genomic DNA (149, 150, 196), (Fig. 13b). Transformants were obtained at a low frequency (5 transformants per 2.5 ug DNA), and these transformants had a mltotlcally stable URA3 marker which Is Indicative of vector Integration. Transformant YeLB21-17-26 appeared to have the plasmid pLB21-17 integrated in the IIIL distal region, as determined by restriction enzyme mapping and Southern hybridization analysis (Fig. 14). Sizes of the fragments that were homologous with probe 2* In transformant YeLB21-17-26, (Fig. 16c) differed by the predicted lengths from fragments on the IIIL distal end In yeast strain AB20o, XP8-10B (used for cloning and mapping the IIIL distal region) or host strain SR25-1A (Fig. 14a, 14b). An exception to the predicted fragment pattern was the extra 7 kb Hindi 11 fragment In YeLB21 -17-26 where only the > 15 kb fragment was expected to hybridize with probe 2* (Fig. 14b). Also the low molecular weight fragments that hybridized with reduced Intensity to probe 2* in the Pstl, Hindlll, and EcoRI digests of AB20ct XP8-10B and SR25-1A were not previously detected. Two additional transformants Isolated In the same experiment had the same Southern hybridization pattern as YeLB21-17-26. To establish whether the extra Hindi 11 fragment In transformant YeLB21 -17-26 was a result of vector integration at the IIIL alternate region in the yeast genome, the probe 2* was removed from the filter (Fig. 14c), and It was used for Southern hybridization with the 1.2 kb Sall/EcoRI fragment (probe C*) that mapped proximal to probe 2* on IIIL, since this fragment shares homology with both 69 Figure 14. Characterization of the URA3 Integration Site in IIIL. a. Restriction map of the integration site in the probe 2* region for the yeast strain AB20ot XP8-10B, and the lengths of the fragments which are homologous with the probe 2* are indicated. Control probe, C*. Isa 1.2 kb Sall/EcoRI (ProbeC, Fig. 8C) fragment which hybridizes to both IIIL distal and alternate regions In AB20<xXP8-10B. b. Expected restriction map following Integration of pLB21-17, which results In the duplication of the target 3.8 kb EcoRI/BamHI fragment in the IIIL distal region. Positions of homology for probe 2* and probe C* and the fragment lengths expected in the URA3 transformant YeLB21-17-26 are Indicated. Restriction fragment lengths are indicated in kb and restriction enzyme sites are B, BamHI; H, Hindlll; P, Pstl; R, EcoRI. c. Southern hybridization of probe 2*. (MTeLB 1 -1.0), with DNA from AB20ot XP8-10B, (lane 1), SR25-1A, (lane 2), and YeLB21-17-26 (lane 3) digested with Pstl, BamHI, Hindlll, and EcoRI as indicated. Following hybridization with probe 2* , and development of the probe 2* autoradiogram, probe 2* was removed, and secondary hybridization of probe C* was conducted. Size standards are A/Hindi 11/EcoRI fragment positions. 69 o. MIL E N D l 2 * 1 T^ 1 C * l l_ R B H ~ 1 • I 12 kb TT i TIT C E N 3 4.8 IIIL E N D 4 _2*i flHBR~ "67T q U,BA3_ _ A'V'p B P P R "t 1" "TS" H I B R B H -]—r-P R C E N 3 " 4 ^ B a m H I "'"<"" EcoRI Pstl BamHI Hindlll EcoRI 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 ~ 2 ~ 3 i T S TTS — 215 — I - 4.4 -- 3 6 — (- 17—I 13—, I Probe 2 Probe C 70 the IIIL distal and IIIL alternate regions In the genome of other yeast strains (probe C*. Fig. 80 . Results from this Southern blot were difficult to interpret since the IIIL alternate region fragments that were observed In strain AB20a XP8-10B were absent In the host strain SR25-1A that was used In the transformation. The faint bands that hybridized with probe C* in the genomic DNA digests of SR25-1A and AB20ot XP8-10B were also present In the probe 2* Southern blot (Fig. 14c), and were not previously detected In Southern hybridization. The reduced or lack of homology between probe C* and a IIIL alternate region In the host strain SR25-1A suggested that Integration did occur at the IIIL distal region in the SR25-1A transformants, the region homologous to the target site in pLB21-17 (180). In addition, the fragments that were homologous with probe C* In YeLB21-17-26 (Fig. 14c) agreed with those predicted for Integration of pLB21 -17 In the IIIL distal region (Fig. 14b) except for Hlndll I digestion where both 15 kb and 7 kb fragments hybridized with probe C*. Genetic linkage analysis (176,177) would conclusively show whether URA3 was linked with the IIIL distal region In YeLB21 -17-26, however I decided to first test the feasibility of recovering the marker along with the chromosome end In Ye21-17-26. Since both the IIIL distal and IIIL alternate regions are proximal to telomeres as shown In the previous section by their sensitivity to Ba131 nuclease digestion, the URA3 marker along with the flanking chromosome end should be recovered for Insertions at either region. Genomic DNA from transformant YeLB21-17-26 was digested partially with Hindlll or Xhol such that the yeast URA3 marker, the bacterial AMP marker, the probe 2* region, and the chromosome IIIL terminus would be contained on the same restriction fragment (Fig. 13c). Tetrahymena rDNA termini were Isolated as Hlndlll/Hhal or Xhol/Hhal fragments from pSZ221 or pSZ222 respectively (195). Ligation mixes containing the partially digested DNA fragments from YeLB21-17-26 and the Tetrahymena rDNA end fragments (Fig. 13d) were Introduced by genetic transformation into yeast strain SR25-1A, but no URA3 prototrophic transformants were produced. The second approach to marker recovery involved the circularfzation of the Hindlll or Xhol partial digest DNA fragments from YeLB21-17-26 and selection for the AMP marker In bacteria. Blunt chromosome ends were generated on the partial RE digest fragments using S1 nuclease (20,24,175). These were circularized by Intramolecular ligation, Introduced In E. coli 71 DH 1 cells by transformation, and AMP resistant colonies were selected (Fig. 13e). No transformants were obtained with either Hindlll or Xhol digested and Hgated DNA (5 ug) from YeLB21-17-26, however the control digestion and intramolecular ligation with DNA from the host strain SR25-1A produced 60 colonies. Southern hybridization analysis demonstrated that pBR322 homology exists In the genomic DNA Isolated from strain SR25-1A and possibly that region was recovered In the selection for AMP resistant colonies. The question remains as to why no AMP resistant colonies were produced with YeLB21-17-26 DNA, since the genome of this URA3 yeast transformant contained the host pBR322 sequences of SR25-1A plus pLB21-17 vector sequences. Problems with Identifying the marker Insertion region In the transformants Isolated from host strain SR25-1A along with difficulties encountered with marker recovery suggested that this strategy was not an efficient method to use for the isolation of the IIIL telomere. F. Attempt to Clone the IIIL Telomere by Vector Addition at the Terminus A variation of the marker Insertion strategy for IIIL telomere cloning was the addition of the vector fragment that contains a selectable marker at the chromosome ends, analogous to the method developed by VanderPloeg et al. (203) for cloning Trypanosoma telomeric regions. This method involves the ligation of a plasmid vector to the ends of double-stranded DNA in high molecular weight genomic DNA which presumably are chromosome ends, followed by intramolecular ligation, and selection In E. coll for the antibiotic resistance marker on the plasmid vector (Fig. 15). AB20* XP8-10B DNA was treated with Bal31 nuclease for Increasing periods of time (20 ug aliquots at 1, 3, 5, 7, and 9 min digestion) to ensure that chromosomal DNA was blunt ended. The pBR322 vector (3 ug) was digested with Pvul I, dephosphorylated to prevent self-llgation of the plasmid, and Hgated to Bal31 digested AB20<x XP8-10B DNA (7 ug), and the ligation mixture was partially digested with EcoRI (lu/ug for 10, 20, and 30 min digestion). Intramolecular ligation of the products followed by transformation in DH1 cells resulted In about 1200 AMP resistant colonies of which 5 hybridized to probe 2*. The plasmlds In these transformants consisted of a 2 kb and a 4 kb Hindlll fragment that both hybridized with probe 2*. However a single Hindlll fragment that Is >5 kb Is homologous with probe 2* on chromosome IIIL Indicating that deletion or rearrangement events occurred in the clones. 72 Figure 15. Strategy for Cloning the IIIL Telomere by Vector Addition. High molecular weight DNA from yeast strain AB20a XP8-10B was treated with Bal31 nuclease, to produce blunt ended chromosomes. pBR322 was Pvull digested, CIP treated, and Hgated to Bal31 treated genomic DNA. Following partial digestion with EcoRI, fragments were intramolecularly Hgated, and transformed in L cgJi DH1 cells. Selection was initially for AMP resistance clones, followed by colony hybridization with the IIIL distal probe 2*. Restriction sites: B, BamHI; H, Hindlll; P2, Pvull; R, EcoRI; andS, Sail. Chromosomal DNA * • PBR3221IIIL END i_2*| i_2*i 1 AMH | « « — r n — r R ,JHR S -l 1 n-S R HS Bal31 Nuclease Digest ion PBR322 Ligation 1 1 S R EcoRI Partial Digest ion Intramolecular L igat ion AMP S e l e c t i o n Probe 2 S c r e e n 73 G. Cloning the IIIL Terminal Fragment In a Circular Vector Southern hybridization results indicated that probe 2* hybridized with a 3.3 kb ( V - 0.2 kb) Sail fragment that contained the IIIL terminus (Fig. 10). The method chosen to clone the IIIL terminus was to prepare blunt-ended chromosomes with Bal31 nuclease, such that the terminal restriction fragment on IIIL could be cloned In a circular vector (24, 175,193, 203), (Fig. 16a). Some of the telomeric region Is deleted in generating blunt-ended chromosomes with Bal31 nuclease, however reports indicate that the terminal 5'-Ci_3A-3' repeat sequence extends for 0.3 -0.5 kb on yeast chromosomal termini (49,175, 204,206). High molecular weight DNA from yeast strain AB20ct XP8-10B was treated briefly with Bal31 nuclease (2 and 4 min), and repaired with DNA Polymerase I, Klenow fragment such that the IIIL end could be Hgated with a blunt-ended vector. DNA was digested to completion with Sail, and Ba131 nuclease/Sall digested fragments in the 3-4 kb range (including the 3.3 kb IIIL terminal Sail fragment) were isolated by preparative 0.7* LMP agarose gel electrophoresis. Fragments were dlrectlonally cloned Into Sall/Smal digested M13mp9RF, hence telomeric fragments were selected since only they should contain the necessary Ba131 blunt-end for cloning in the Smal site. Following hybridization with probe 2* . clone MTLB6411 was purified (Fig. 16b). The 3 kb Insert had not undergone any significant recombination events in cloning since Its restriction map was Identical to that established through mapping of the region distal to probe 2* on IIIL (Fig. 7b). Apparently 0.3 kb ( + / - 0.2 kb) were removed In the Ba131 nuclease treatment during cloning of the 3.3 kb ( + / - 0.2 kb) Sail fragment on the Intact IIIL terminus. MTLB6411 contained the 0.5 kb EcoRI fragment distal to probe 2* . as well as a 1.5 kb EcoRI fragment, presumably the IIIL terminal fragment. Preliminary sequencing analysis confirmed that the 1.5 kb EcoRI fragment contained the 5'-Ci_3A-3' repeat sequence adjacent to the Smal site in MTLB6411, which is Indicative of yeast telomeric structure. The terminal 1.5 kb EcoRI/Ba131 fragment was subcloned to remove probe 2* sequences that might confuse the characterization of the IIIL terminus (probe 3*). Also Ii13mp9 was not an appropriate vector to use for hybridization probes, since It contains a short region that Is homologous 74 Figure 16. Cloning the IIIL Terminal Fragment in a Circular Vector. a. High molecular weight yeast AB20ot XP8-10B DNA was treated with Bal31 nuclease (0.1 u/ug, 2 min and 4 min aliquots) and repaired with DNA Polymerase I, Klenow fragment to yield blunt chromosome ends which can be cloned. Following complete digestion with Sail, fragments which Include the 3.3 kb Sal I /chromosome IIIL end were isolated by preparative 0.7% LMP agarose gel electrophoresis and electroelution. b. Purified Bal31 /Sail fragments were cloned in the M13mp9RF vector which was digested with Sail and Smal. JMl01 transformants were screened with a nick-translated probe 2* fragment and this resulted in the Identification and purification of the IIIL telomeric clone MTLB6411. The 3 kb Sall/END fragment in MTLB6411 corresponds to the 3.3 kb (V-0.2 kb )IIIL end with 0.3 kb ( + / -0.2 kb) excised by the prior Bal31 nuclease treatment. c. The IIIL terminal fragment, probe 3* , wassubcloned for DNA sequence determination and analysis by Southern hybridization. The EcoRI ends on probe 3*. one derived from yeast DNA IIIL end and the other from the polylinker region of M13mp9, were repaired with DNA Polymerase I, Klenow fragment. Probe 3* was cloned into the Smal site of M13mp18RF to result in MTLB6S12, which has the same orientation of the insert as MTLB6411. Symbols: telomeres ( M , • ); blunt-ended chromosome IIIL ( B E ) ; M13 universal sequencing primer ); and the M13mp9 or M13mp18 vectors ( i ). Restriction sites: B, BamHI; H, Hindi 11; P, Pstl; S, Sail; R, EcoRI; Z, Smal. 74 a. 75 with the pBR322 plasmid (131) and hence hybridizes with the vector regions of clones which could lead to problems In Interpreting Southern hybridization results. The IIIL terminal fragment (probe 3*), (Fig. 16b), was released from MTLB6411 by EcoRI digestion which made use of both the M13mp9 polyllnker site and the EcoRI site on the yeast IIIL terminal fragment. Probe 3* was subcloned in the Smal site of M13mp18RF, after DNA Polymerase I, Klenow fragment repair of the EcoRI ends (Fig. 16c). Consequently, probe 3* was flanked by several restriction sites In the polyllnker region of M13mp18, such that deletion clones could easily be generated with Exonuclease III (also referred to as Exolll) for DNA sequence determination (86). MTLB6S12 had the same Insert orientation In the Ml3 vector as MTLB6411, which contained S ' - G ^ T - S ' repeat units adjacent to the binding site In Ml3 of the universal sequencing primer. The IIIL terminal fragment was also cloned In the opposite orientation, as an EcoRI fragment in the EcoRI site of M13mp 18, (referred to as MTLB6S21) such that deletion clones could be generated with Exolll and SI nucleases from the opposite end of the 11 IL telomerlc fragment and the sequence of both DNA strands could be established. H. The IIIL End Conforms to the T-X Class of Yeast Telomeres The arrangement of yeast telomerlc regions is conserved. Southern hybridization analysis Indicates that yeast telomeres contain a telomere associated region that consists of complex repeat units referred to as type X and Y', and these repeat units contain A M regions (49, 93, 193, 206) which are thought to be origins of replication in yeast (210). As expected for a telomerlc fragment, the IIIL terminal EcoRI fragment (probe 3*) hybridized with multiple fragments In the yeast genome, of either discreet or heterodisperse lengths (Fig. 17, lanes 1 -3). Southern hybridization was used to determine whether the IIIL telomerlc region (probe 3*) was homologous with clones YRp131A, YRp131B, and pSZ220 from theyeast telomere associated region (Fig. 17a, 17b). The IIIL telomeric probe was homologous with the type X ARS region of YRp131A(48,49), hybridizing with the 0.9 and 3.3 kb Sall/Ncol fragments (lane 5) and the 3.8 kb Sall/Hlndlll fragment (lane 6). The IIIL telomerlc probe did not hybridize with the type Y' A M region In either YRp 131B (lane 4) or pSZ220 (lane 7), (48,49, 193). Since probe 3* . but not the adjacent probe 2* (Fig. 12), was homologous with a telomere associated region clone, the telomerlc region must be contained within the 76 Figure 17. The IIIL Terminal Fragment Is Homologous with the X but not the Y' Telomere Associated Region. a. Southern hybridization using MTLB6S12 as the probe for the IIIL telomere (probe 3*) with the following DNA samples: 1, AB20a XP8-10B/BamHl, (4 ug); 2, AB20aXP8-10B/Pvul 1,(4 ug); 3, AB20ot XP8-10B/Hindt 11, (4 ug); 4, YRp 131 B/Sall/Ncol, (0.12 ug); 5, YRp131A/Sall /Ncol, (0.12ug);6,YRp131A/Sall/H1ndlll,(0.12ug); 7, pSZ220/Alul, (0.25 ug). Size markers on the right of the autoradlogram Indicate positions of A/Hindlll/EcoRI fragments. Chromosome IIIL terminal fragments are distinguished from the other bands by the open triangles (O). b. Restriction maps of the yeast telomere associated regions in clones pSZ220 (92, 193), YRp131A, and YRp 131B (48,49). Restriction sites: A, Alul; H, HindiII; N, Ncol; P1, Pvul; S, Sail; Sc, Sacl. 1 2 3 4 5 6 7 Yeast Y * V' b. 0 . 8 k b PSZ220 ^ - ^ c r # # « Y Y r p 1 3 , A - I I 1 2— 1 — I — I I - 1 0 k b S N N N N N N S 1 * 3 * 1 X Yrp131B H H ' 1 C » 6 . 7 k b S N S 131 Y 77 terminal 2.3 kb on IIIL and It Is reasonable that the type Y'_AMrepeat region which Is usually 6.7 kb In length, Is not present within the short telomeric region on the MIL end. The IIIL telomere contains only the simple repeat T region distal to the X A M region and was therefore classified as a type T-X telomere, as opposed to the previously Isolated and characterized type T-Y'-X telomere (48,49, 175, 193). Restriction enzyme digestions of yeast genomic DNA were probed with the IIIL telomeric fragment, probe 3* . (Fig. 18) to determine whether both class T-X and T-Y'-X telomeres are homologous with the IIIL telomeric fragment. The estimated lengths for the components of yeast telomeres (49, 205) are: (1) simple repeat T region, 0.3-0.5 kb, (2) conserved Y' A M region, 6.7 kb, (3) Internal simple repeat region, 0.1-0.2 kb, and (4) the heterogeneous X A M region, 0.3-3.75 kb, (Fig. 2). Hence yeast telomeres conforming to the T-Y'-X class are at least 7.5 kb in length and the distance between the X region and the terminus of the chromosome Is about 7 kb on the T-Y'-X telomeres. Consequently, the heterodlsperse, low molecular weight (<7 kb), and intensely hybridizing bands in the Southern hybridization analysis with the IIIL terminal probe (Fig. 18) must represent telomeres that lack a Y' A M region, and therefore can be categorized as type T-X telomeres ( 49,93,175,205,206). Such T-X telomeres represent about 30 * of the yeast chromosome ends that hybridized with probe 3*. The heterodisperse, low molecular weight (<7 kb), and weakly hybridizing bands, evident In Pstl digestion (1.0 kb), Kpnl digestion (1.1 kb) and Xhol digestion (1.4 kb), (Fig. 18, lanes 3, 6, 10), probably shared homology with only the simple repeat region (T) of probe 3* and therefore represent type T-Y'-X telomeres. The sizes of these fragments correspond to the Pstl, Kpnl, and Xhol restriction sites within the conserved Y' A M region on most yeast telomeres (175, 193, 204). The low molecular weight fragments with defined fragments lengths must represent chromosome ends which contain the specific restriction enzyme site between theX region and the terminal heterodisperse (T) region (Fig. 18, lanes 1, 3, 4, 6, 7, 9). It was not possible to categorize those fragments Into T-Y'-X or T- X class telomeres, since the proximities of the X regions to the respective chromosomal termini were unknown. The telomeres from the seventeen chromosomes In the haploid yeast genome were not distinguishable by different fragment lengths in 78, Figure 18. The IIIL Terminal Fragment is Homologous with Yeast Repetitive DNA. Southern hybridization of yeast AB20aXP8- 10B DNA with MTLB6S12, the terminal probe from IIIL. DNA (3.3 ug) was digested with each of the following restriction enzymes: Lane 1, EcoRI; 2, Hindi 11; 3, Pstl; 4, Pvull; 5, BstEII; 6, Kpnl; 7, BamHI; 8, Xbal; 9, Sail; and 10, Xhol. Open triangles on the autoradlogram distinguish the IIIL end fragments from the other telomeric bands. Size markers are A/Hindl 11/EcoRI fragments on the left and A/Hlndlll fragment positions on the right edge of the autoradiogram, as indicated by the horizontal bars. 79 any of the restriction enzyme digestions (Fig. 18). The maximum number of fragments that hybridized with IIIL T-X probe (12-16 fragments) was observed for Pvull (lane 4) or BstEII (lane 5) digestions. Similar restriction site arrangements In the type X A M flanking regions must preclude the generation of different RE fragment lengths for each telomere. I. The IIIL Terminal Probe Is Homologous Exclusively with Telomeres Bal31 nuclease digestion was used to estimate the distance from the X region (probe 3*) to the respective chromosome ends. AB20<x XP8-10B genomic DNA was digested with Bal31 nuclease for up to 30 minutes, digested with Sail or Pvull, and analyzed by Southern hybridization with probe 3* (Fig. 19). A subset of Sail fragments that hybridized with the T-X probe was sensitive to digestion with Bal31 nuclease whereas all of the Pvull fragments that hybridized with the T-X probe were digested progressively with Bal31 nuclease. Assuming that Bal31 nuclease Initiates digestion at chromosome ends In high molecular weight DNA ( 18, 24, 60, 175, 203, 206, 209), all Pvull fragments detected by the probe 3* must Include the terminus of a yeast chromosome. The difference In sensitivity to deletion with Bal31 nuclease observed for Sail and Pvull fragments is a consequence of the restriction site differences in the type X and Y' A M regions. The representative X and Y" clone maps (48, 49) have no Pvull sites distal to the A M region in any of the heterogeneous type X clones isolated, and the conserved Y* region is void of Pvull sites. A Sail recognition site separates the type X and Y" regions In all of the clones, hence all type T-Y'-X telomeres are digested with Sail at the Y'-X junction. As a result, all Pvull fragments homologous with the X region probe contain the Ba131 nuclease sensitive chromosome end. Alternatively, Sail fragments that contain an X region and which map to T-X telomeres belong to the subset of Sail fragments that contains the Bal31 nuclease sensitive region. The other Sail fragments that hybridize to the X region probe are from T-Y'-X telomeres and these fragments display Bal31 nuclease sensitivity only after the T-Y' region (>7 kb) Is completely deleted by Ba131 nuclease. These assumptions are valid only If all X regions are void of Sail and Pvull recognition sites in the region distal to A M . which is the case for all X regions that have been Isolated 80 Figure 19. The IIIL Terminal Probe Hybridizes with other Chromosome Ends. Yeast AB20* XP8-10B DNA was treated with Bal31 nuclease (0.1 u/ug) for up to 30 min, (deletes 125-150 bp per minute), and was digested with Sail or Pvull as Indicated. After fractionation on a 0.65* agarose gel, the DNA fragments were transferred to gene screen plus filters, and hybridized with MTLB6S12 (probe 3*). The Sail or Pvull fragments on the IIIL end are Indicated by open triangles. Size markers are A/Hindi 11/EcoRI fragment positions on the left side and A/Hindi 11 fragments on the right edge of the autoradiogram. 1.0 0.9 81 (48, 49). If T-Y'-X telomeres have a minimum length of 7.5 kb, then all Pvull fragments that are homologous with the T-X region from IIIL and shorter than 7.5 kb (Fig. 19) must represent telomeres of the alternate T-X category. Based on this reasoning, together with the proportion of Sail fragments that display Bal31 nuclease sensitivity, I estimate that at least 30$ of chromosome ends In the haploid yeast genome belong to the T-X class of telomeres. J . Length Heterogeneity of the IIIL Telomere Among Yeast Strains Restriction endonuclease site polymorphisms and DNA rearrangements among telomeric regions in various yeast strains have been examined using a Y' probe, pSZ220, in Southern hybridization analysis (93, 193). Extensive differences In hybridization patterns, both In the size and relative abundance of restriction fragments have been observed and It was concluded that the number of Y' homologous regions varies considerably among different yeast strains (93). The T-X probe from the IIIL telomere hybridized to both yeast telomere classes, and consequently was used to examine strain differences In the T-X region of yeast telomeres. The unique fragment on the distal end of chromosome IIIL (probe 2*) was homologous with fragments that had conserved lengths among different yeast strains, if the fragments did not include the IIIL terminus (Fig. 20a, lanes 1-6, Pvull, EcoRI). The 1.2 kb Pvull fragment and the 4.6 kb EcoRI fragment extend In the centromere proximal direction from probe 2* , and both fragments were conserved In the various strains. The Identity of the extra bands In SR25-1A (lane 5, Pvull, EcoRI, Sail) Is unknown. DNA from this strain hybridized with pBR322 vector sequences, hence the extra bands may be due this foreign DNA of unknown origin that was Introduced into this strain. Fragments homologous with probe 2* that contained the IIIL terminus in strain AB20a XP8-10B varied In their average length with a distribution V - 200 bp compared to other yeast strains (lanes 1-6, Pvull, Sail). The average length of the Pvull 3.0 kb ( V - 0.2 kb) fragment and Sail 3.3 kb ( V - 0.2 kb) fragment In AB20ot XP8-10B (lane 1) was different In other strains, however these telomerlc fragments remained equally heterodisperse ( V - 0.2 kb) in all strains. Since heterodisperse Sail and Pvull fragments hybridized with probe 2* in each of the various strains, the length heterogeneity In 82 Figure 20. Southern Hybridization Indicates Length Heterogeneity of the IIIL Telomere Among Yeast Strains. a. Yeast DNA (4 ug/lane) was digested with either Pvull, EcoRI, or Sail, and fractionated on a 0.65* agarose gel. Lanes: 1 ,AB20aXP8-10B; 2, K45; 3, T388; 4, AB972; 5, SR25-1A; 6, SR26-12C. Following unidirectional transfer of DNA to gene screen plus, the filter was hybridized to probe 2*, (MTeLB 1-1.0). b. Secondary hybridization of the filter was with probe 3* . (MTLB6S12), after removing probe 2*. Fragments that are expected to map to the IIIL end are distinguished by open triangles, while open circles indicate restriction fragment length polymorphisms observed in the X or Y' regions among the various yeast strains. Size markers are positions of A/Hindi 11/EcoRI fragments and are indicated in the center section separating Southern blots a and b. 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 k b f 2 1 5 ••'•M •••HI •4k - 4 4 , 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 83 the IIIL terminal region among the strains Is not due to differences in Sail or Pvull restriction sites in the T-X region of the IIIL telomere. Presumably the length differences are due to different average amounts of simple repeat T sequences (5'-Ci_3A-3' units) at the chromosome IIIL end In the various strains (40,175,206). Southern hybridization of the various yeast DNAs that were restriction enzyme digested, and probed with the T-X region from IIIL (probe 3*) suggested that the variation in the length of the T repeat region was a characteristic of most T-X telomeres (Fig. 20b). For yeast DNA digested with Pvull, EcoRI, and Sail, the low molecular weight (<4 kb) heterodisperse bands varied In average length by about 0.2 kb among the different strains. Such relatively small length differences were not detectable for the large Pvull fragments (>7 kb), which presumably represent T-Y'-X telomeres, due to the limitations of the assay system. Variations In terminal repeat length at both T-X and T-Y'-X chromosome ends, either within a given yeast strain or among different strains, have been described recently (40,93, 206). There were some restriction enzyme site polymorphisms or DNA rearrangements in the telomeric regions of the various yeast strains that were detected with the the T-X probe from IIIL (probe 3*). The Pvull fragments that are polymorphic among the various strains are larger than 7 kb, the EcoRI fragments are larger than 4.5 kb, and the Sail fragments are larger than 4 kb (Fig. 20b). As described In a previous section, Pvull fragments shorter than 7.5 kb were categorized as T-X telomeres, and those 7.5 kb or longer were classified as T-Y'-X telomeres. Apparently, the polymorphisms that were detected with the T-X probe, (probe 3*), map to T-Y'-X telomeres. It Is also conceivable that the polymorphisms detected, exist In the Y' region of these telomeres, and the X region Is void of strain specific variations. The hybridization pattern of the T-X region probe 3* with the genomic DNA from various yeast strains is similar, and the proportion of high and low molecular weight fragments that hybridized with the T-X probe is conserved among the yeast strains (Fig. 20b). This suggests conserved ratio of T-X class to T-Y'-X class telomeres In the genomes of the various haploid yeast strains. 84 K. The IIIL Telomere is Retained In Some Ring III Yeast Strains Southern hybridization of genomic DNA from linear and circular chromosome III strains with the IIIL telomerlc probe served to Investigate the fate of the IIIL end In the HMLtt,-HMRa fusion event that formed the circular chromosome III (Fig. 21). The average length of the IIIL terminal fragment In yeast strain K45 was about 0.3 kb shorter than that In strain AB20a XP8-10B, as described In the previous section (Fig. 20a). In K45,the 1.5 kb (+/- 0.2 kb) EcoRI, 1.6 kb (+/- 0.2 kb) Hindi 11, and 3.0 kb (+/- 0.2 kb) Sail fragments (Fig. 21, lane 1) mapped to the IIIL end and these fragments were absent in strain K192 In which the IIIL distal region Is deleted, (lane 3). The telomerlc fragments from chromosome IIIL were retained In the ring III strain K191 (lane 2), which supports the proposal that the 11 IL distal region was retained In K191 by replacing the 11 IL alternate region In a telomere conversion or recombination event. Both K191 and K192 were derived from K45, hence the telomerlc fragments, other than those on chromosome III In K45, should be conserved In these ring III strains. Other than the differences In the telomerlc fragments from IIIL between the linear III and ring III strains, the fragments from the right end of chromosome III (MIR) should hybridize with the T-X probe In K45 and these should be absent In the ring III strains. For each restriction enzyme digestion of the genomic DNA from linear III and ring III strains, one fragment was Identified that may map to the IIIR end: a >10 kb EcoRI fragment, a 8 kb Hindi 11 fragment, and a 4.5 kb Sail fragment ( lane 1), but these have not been characterized. A 3.6 kb EcoRI fragment that was conserved In K45 and K192, but absent In K191 may correspond to the telomere of the IIIL alternate region that was replaced by the IIIL telomere In K191. Although strains K193, K195, K196 are circular III strains which were derived from an alternate host strain than K45, the majority of fragments that were homologous with the 11 IL telomerlc probe In the K191 and K192 strains were maintained In the K193, K195, and K196 strains. Similar to ring III strain K191, the restriction fragments which contain the IIIL telomere were retained In the ring III strains K193, K195, and K196 (lanes 4,5,6). 85 Figure 21. Southern hybridization of Linear lit and Ring III Yeast Strains with the IIIL Terminal Probe. Yeast DNA was digested with EcoRI, Hindi 11, or Sail as indicated, then fractionated on a 0.65$ agarose gel and transferred to gene screen plus filters. Lanes: 1, K45; 2, K191; 3, K192; 4, K193; 5, K195; and 6, Kl96. This Southern hybridization filter was used for the IIIL distal probes in Figure 8). After removing the other IIIL distal probes, hybridization was with the IIIL terminal probe 3* (MTLB6S12). The length of the IIIL terminal fragment in linear III strain K45 is indicated for EcoRI ( • ) , Hindi 11 ( • ) , and Sail ) digestions by closed symbols. The estimated positions for the IIIR terminal fragments in the EcoRI, Hindi 11, and Sail digestions are Indicated by the equivalent open symbols (d.O.O). Size Marker positions are indicated by horizontal bars at the left for A/Hindlll/EcoRI fragments and positions at the right arefor theA/Hindlll fragments (23.7 kb, 9.5 kb, 6.7 kb, 4.3 kb, 2.3 kb, 2.0kb,and0.6kb). 85 ECQBJ Hindlll Sail 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 8 6 L. DNA Sequence Analysis of the Chromosome IIIL Telomere To establish the extent of the terminal repeat region on 11 IL, to compare the DNA sequence with that reported for a Y" containing telomere, to locate the position of the A M consensus sequence within the X region on IIIL, and to search for tandem units of repeat sequences In the X region on IIIL, the complete DNA sequence of the cloned fragment from the T-X telomere on 11 IL was determined. Sets of deletion clones were prepared with Exonuclease III (referred to Exolll) and SI nuclease according to Henlkoff (86) from the subclones MTLB6S12 and MTLB6S21(both insert orientations) with processlve Exolll digestion from the BamHI site In each clone. Dideoxynucleotlde chain terminator sequence determination (170,171) of both sets of deletion clones (Fig. 22a) gave the complete DNA sequence for the IIIL telomeric fragment (Fig. 22b). The set of deletion clones for MTLB6S21 was Incomplete In the T region, possibly due to problems with Exolll digestion In the G-rlch region of the template strand of MTLB6S2I. To resolve this problem, a sequence specific primer was used to complete the sequence of the T simple repeat region of MTLB6S21. Repeat DNA sequences from yeast telomeres (175) had been previously determined using the Maxam and Gilbert chemical sequencing method (128), however simple terminal repeat sequence for the IIIL end was completely determined with the enzymatic method for DNA sequence determination (170, 171), (Fig. 22c). The repeat sequence S ' - G ^ T - S ' was obtained using a MTLB6S21 deletion clone and the synthetic primer that was specific for the Insert in the clone, while the alternate strand 5'-Ci_3A-3' repeat sequence was determined from a MTLB6S12 deletion clone using the M13 universal sequencing primer. The 356 nt sequence of the T region In MTLB6S12 was more accurately defined as 5'-[C2-3A(CA) i_4]-3' and the distal end of the cloned 1539 nt IIIL telomeric fragment contained fifty-three repeat units. An 80 nt repeat region from a Y' containing telomere was sequenced by Shampay et al. (175) and It has the same simple repeat unit (5'-Ci_3A-3') as the IIIL telomere. However, the defined formula for this telomere Is 5'-[C2-3A(CA) ^-3]-3* since It has a maximum of three tandem CA dlnucleotldes whereas the repeat units at the IIIL end are defined by 5'-[C2-3A(CA) 1_4]-3' since a repeat unit in the fragment from the IIIL telomere had four tandem CA repeats. The simple repeat region on a telomere Is thought to extend to the terminus of that chromosome (22, 23, 24, 25, 175, 203, 206). This 87 Figure 22. DNA Sequence of the IIIL Terminal Fragment. a. Extent and orientation of DNA sequence determination for the cloned 1539 nt EcoRI/Ba131 nuclease treated fragment from the IIIL terminus. Position 0 refers to the Initial nucleotide in the sequence of the cloned telomere. The 0.3 kb (V-0 .2 kb) which extends distal to this 1539 bp fragment on the end of chromosome IIIL were deleted during cloning of the IIIL telomere . Dotted lines represent the M13mp 18 vector sequences which flank the insert fragment. Arrows below represent the start point and extent of sequence obtained from each Exolll/SI nuclease deletion clone. The sequence of the MTLB6S12 clones extend toward the 5' end of the insert fragment, while the MTLB6S21 clones extend toward the 3' end, and hence the entire insert is covered in both directions. The arrow with an open circle at its base In the MTLB6S21 deletion set distinguishes the sequence obtained using a site-specific, synthetic oligonucleotide primer. b. Nucleotide sequence of the IIIL telomerlc region, that extends 1539 bp from the IIIL terminal EcoRI site, as determined by dideoxynucleotide chain terminator sequencing (170,171). The 5' to 3' DNA strand on the chromosome contains the 5'-C \ - 3 A - 3 ' repeat sequences. A perfect A£S_ concensus sequence (33, 42, 104) Is underlined at positions 1036-1048 nt. The simple repeat region differs from the reported (175) formula 5'-C2-3A[CA] 1 - 3 - 3 ' at nt 152-163 by having a [CA]4 block. The only 6 bp recognition restriction enzyme site in the terminal IIIL cloned fragment Is BstEII at nt 1175-1181. c. IIIL telomerlc repeat sequence determined by dideoxynucleotide chain terminator method (170, 171). Repeat sequences 3"-Gj_3T-5' and 5'-Ci_3A-3" were determined from the reverse orientation clones, MTLB6S21 and MTLB6S12 respectively, from the IIIL telomere. The MTLB6S21 sequence was determined with the site-specific synthetic primer, and the sequence prior to the Z'-G] - 3 T - 5 ' repeat region in MTLB6S21 stems from the MIL X region which flanks the T region. The MTLB6S12 sequence was determined using the M13 universal forward primer, and the sequence prior to the 5'-C)-3A-3' repeat region In MTLB6S12 is the M13mp18 polycloning site. The only C3A(CA)4 repeat unit sequenced in the IIIL T region is indicated at the top of the MTLB6S12 sequence. a 87 _*_B_S_ I I I L E N D ['•*• o 14SS I4'0 C ro*&i»c* i»*rc i* i r f» i t r«occcc**c*. (UCICTTSTilUaiTtOtUIUICGCGeiTGT M T L B 6 S 2 1 M T L B 6 S 1 2 I 1 I 1 C TAG C T AG 88 proposal Is supported by the finding that a section of the 5'-Ci_3A-3' repeat region adjacent to a telomere associated A£S_region 1s sufficient to provide telomere function 1n yeast (65,175,195, L.L. Button and CR. Astell, manuscript In preparation). It 1s assumed that the 0.3 kb (+ /-0.2 kb) of DNA removed from the IIIL end with Bal31 nuclease in cloning the IIIL telomere consisted of additional S ' - C ^ - 3 " repeat sequences. Adjacent to the T region on IIIL was the type X A£S_ region (Fig. 22b). Distal portions of X regions that were derived from T-Y'-X telomeres were sequenced by YYalmsley et al. (205) and the distal 260 bp of such X regions are 80-90* homologous with the X region which flanks the T region on IIIL. The consensus sequence for yeast presumptive origins of replication, or A M regions, Is 5'-A/T-T-T-T-A-T-Pu-T-T-T-A/T-3' (34, 42, 104, 182). A perfect A M consensus sequence, 5-T-T-T-T-A-T-G-T-T-T-T-3' , is located at nt 1036-1047 on the cloned IIIL end or 1.2 kb (+/-0.2 kb) from the terminus on the intact chromosome IIIL end. The ARS_consensus in the Y" region of a T-Y'-X telomere that was partially sequenced (175) Is 700 bp from the chromosome end, and in the opposite orientation with respect to the 5'-Ci_3A-3" containing strand compared with the ARS region on the 111L end. The effect of the orientation of the A M consensus sequence on A M activity has not been reported. The flanking regions are known to participate In ARS function and the sequences adjacent to the consensus sequence vary to a large extent In the A M regions that have been characterized (34,42, 104,182). Consequently, the strength of the ARS regions In X and Y' cannot be estimated simply by DNA sequence analyses. Instead, comparison of the X and Y' A M regions must Involve assays for ARS function. Nucleotide sequence studies with the Y' containing clone pSZ220 (92, 193) revealed a region of tandem direct repeats which were proposed to have a role in telomeric replication, maintenance, and homogenlzation of telomeric sequences. In contrast to the Y' region, no direct repeat region was apparent in the X region on IIIL and the lack of G residues in the 5 - 0 ^ - 3 ' containing strand was its only distinguishing feature (Fig. 22b). 89 M. The IIIL Telomere Includes 1.8 kb at the Termtnus The extent of the X region on the end of IIIL, and the effect of the T region on the hybridization pattern obtained with the T-X probe, were measured by Southern hybridization analysis with probes that contained progressive deletions In the IIIL telomeric region (Fig. 23). The full length clone, MTLB6S12, (probe A, 1539 nt Insert) was homologous with multiple fragments In restriction enzyme digestions of yeast genomic DNA and with the X region fragments In YRp 131A (Fig. 23A). The removal of the entire 5'-Cj-3A-3' repeat region in probe B did not significantly alter the hybridization pattern or the relative band Intensities (Fig. 23B) from those detected with probe A. The overall reduction In hybridization Intensity with probe B (Fig. 23B) compared with probe A (Fig. 23A) was the result of the asymmetric transfer of DNA In bidirectional Southern analysis. In transferring DNA from an agarose gel to two filters, I have consistently found that the top filter (Fig. 23, A,C) contained a greater percentage of DNA than the bottom filter (Fig. 23, B,D). All of the T region and a portion of the X region was deleted In probe C, which had a total deletion of 1051 nt compared with probe A (MTLB6S12). This deletion completely eliminates the A M consensus sequence in MTLB6S12 (Fig. 22b) and probe C did not hybridize with the 0.9 kb Sall/Ncol fragment In YRp131A (Fig. 23C, lane 5) which maps adjacent to the A M region In YRp131A (Fig. 17b). However, the 500 nt fragment from IIIL In probe C contained further X region sequence since probe C hybridized with the 3.3 kb Sall/Ncol and 3.8 kb Sall/Hlndlll fragments (Fig. 23C, lanes 5&6) which Include the region upstream of the A M consensus sequence In YRp131A (Fig. 17b). A subset of genomic DNA fragments (high molecular weight) that were homologous with probes A and B, did not hybridize with the X region In probe C. Possibly these fragments represent yeast telomeres that are homologous with the region between the A M consensus sequence and the T region on the IIIL telomere, since they hybridize with probes A and B but not with probe C. The hybridization intensity of the fragments from the IIIL telomere has Increased relative to the other telomerlc fragments that are homologous with probe C (Fig. 23C, lanes 1 -4), due to greater heterogeneity on the centromeric side of the A M consensus sequence than on the telomerlc side for the X regions. In support of this idea, the restriction maps of the heterogenous X region clones (48, 49) display more conservation In 90 Figure 23. Extent of the X Region on the IIIL Telomere. Bidirectional Southern hybridization analysis, with deletion fragments from the IIIL telomere as probes, was conducted on restriction enzyme digested yeast and plasmid DNA samples that were fractionated on duplicate 0.65$ agarose gels. Lane: 1, AB20ot XP8-10B/Sall, (4 ug); 2, AB20a XP8-10B/Hlndl 11, (4 ug); 3, AB20* XP8-10B/BamHI, (4 ug); 4, AB20* XP8-10B/Xhol, (4 ug); 5, YRp131A/Sall/Ncol, (0.1 ug); 6, YRp131A/Sall /Hlndlll, (0.06 ug). Probes A, B, C, and D were M13 Exol 11 /S1 nuclease deletion clones that were prepared for DNA sequence analysis. For probes A, B, C, and D, approximately 300 bp, 650 bp, 1350 bp, and 1550 bp respectively were removed from the intact 1.8 kb (+/-0.2 kb) EcoRI fragment at the chromosome IIIL terminus. The map below the Southern blots Indicates the positions of probes A, B, C, and D on the 11 IL end. The heavy arrow on the IIIL end represents the 600 (+/- 200)bp of 5'-C i -3A-3' simple repeat region. Open triangles on the autoradlogram Indicate the framents on the IIIL end and the closed triangles at the right Indicate the positions A/Hindlll/EcoRI fragments. 90CL T E L 4> 11 P S 91 restriction sites on the telomerlc side of A£S compared to the centromeric side. Finally, probe D had a 1239 nt total deletion compared with probe A (MTLB6S12). The 300 nt insert In probe D had no significant homology with the X region in YRp131A (Fig. 23D, lanes 5&6). For each restriction enzyme digestion, there were only two or three genomic fragments that were homologous with probe D (Fig. 23D, lanes 1-4). Since X regions are homologous with one another by definition (48, 49), there must be minimal X region sequence remaining In probe D. The boundary between DNA that is unique to chromosome IIIL and the DNA that Is repeated on all yeast telomeres must be near the terminal EcoRI site on chromosome IIIL, about 1.8 kb from the IIIL terminus. The fragments that are homologous with probe D, which do not map to the IIIL terminal region (Fig. 23D, lanes 1-4), presumably represent other chromosome ends that contain X regions which are highly homologous to that on 11 IL. It Is unknown whether these chromosome ends are also T-X telomeres. N. Construction of Clones to Study the Function of the IIIL Telomere I determined whether the cloned IIIL telomere, which had the terminal 0.3 kb (V-0 .2 kb) DNA deleted, contained sufficient information to maintain a linear plasmid in yeast (193). The required amount of telomerlc DNA for recognition as a chromosome end by the cell's replication machinery was also analyzed, through using progressive deletions of the IIIL end fragment. The assay system W8S analogous to that developed by Szostak et al. (65,140,194,195) to study replication and resolution of Tetrahvmena thermophlla macronuclear rDNA ends on linear plasmids in yeast S_. cerevlsiae. Since the cloned Tetrahvmena termini function as telomeres on linear plasmids in yeast, I prepared a yeast plasmid with Tetrahvmena rDNA end fragments to use as a control In yeast for the function of clones constructed with IIIL ends (Fig. 24). The Tetrahymena rDNA end in pSZ222 Is a 0.7 kb Xhol / Hhal fragment, which has 330 bp of Tetrahymena 5'-C4A2-3' terminal repeat sequence adjacent to 360 bp of rDNA which contains a yeast AFiS_ consensus sequence and displays low ARS activity In yeast (1,65,105,139,195). The vector used to prepare the linear plasmid was pSZ93 (Fig. 24a), (150). Its salient features Include the LEU2 gene (160) for selection in yeast, and the 92 Figure 24. Construction of a Linear Plasmid with Tetrahymena rDNA Termini. a. Th8 700 bp Xhol/Hhal fragment from the end of Tetrahymena rDNA was isolated from pSZ222, (195). The 330 bp C4A2 region is oriented toward the Hhal end of the fragment as indicated by the arrow, and the adjacent 360 bp region of Tetrahymena rDNA has weak A M activity in yeast (1, 65, 105). There are 9 bp of pBR322 DNA separating the Hhal site and the C4A2 region (195). The pSZ93 vector (150, 195) is a 7.4 kb plasmid that contains the ARS1 region for autonomous replication (184) along with the LEU2 gene (2) for selection in yeast. The ligation mix of pSZ93/Sall and p5Z222/Xhol/Hhal Included the restriction enzymes Sail and Xhol, to digest rellgated pSZ93 or telomerlc fragment concatemers produced during ligation, and thereby enhance the proportion of vector llgated with two Tetrahvmena rDNA end fragments. b. Proposed structure of the linear plasmid following resolution in yeast of circular or linear plasmids present in the ligation mixture. An inverted repeat of the end fragments was not prepared prior to ligation of the end fragments in pSZ93. Presumably one Xhol/Hhal end fragment would ligate to each Sail end of pSZ93 by inter molecular ligation, then circular ization would occur by intramolecular ligation of the Hhal sites. In yeast, recognition of and inverted repeat structure of Tetrahymena end fragments results in conversion of the ligation products to monomeric linear molecules (194, 195). Restriction sites: B, BamHI; H, Hindi 11; Hh, Hhal; K, Kpnl; R, EcoRI; S, Sail; X, Xhol. 92 GL 93 ARS1 region (184) which functions as a strong origin of replication and hence provides high mitotic stability to linear plasmlds In yeast (56, 65, 140). The restriction enzymes Sail and Xhol were included In the ligation mixture since this increases the percentage of ligation products with inverted Tetrahvmena ends in the circular pSZ93 vector (195). Presumably, other ligation products include linear plasmid monomers and concatamers. Upon transformation of the ligation mixture Into yeast, the Tetrahvmena Inverted repeat region Is recognized and resolved to give predominantly linear plasmid molecules (Fig. 24b), (65, 194, 195). The 9 bp of pBR322 sequence which separates the 5 - 0 ^ 2 - 3 ' repeat sequence region from the Hhal site on the Tetrahymena end fragment does not interfere with the resolution process in yeast (195). The terminal 1.8 kb (+/_ 0.2 kb) EcoRI fragment on IIIL (Fig. 25a) contains a 1.2 kb type X A M region upstream from the 0.6 kb (+/_ 0.2 kb) 5 ' -C 1 _ 3 A-3' repeat region. The cloned telomere from IIIL, referred to here as TF1, Is a 1.54 kb EcoRI fragment which has 356 bp of 5 ' -C 1 _ 3 A-3' repeat sequence adjacent to a 1.2 kb type X A M region (Fig. 25a). Telomeric fragments which have progressive deletions at the IIIL end were prepared by Exolll / SI nuclease digestion of MTLB6S12 to produce clones TF2 to TF7. The TF8 deletion clone was prepared by Exolll/Sl nuclease digestion of the reverse orientation clone MTLB6S21. The telomeric fragments were transferred initially from the MTLB6S12 deletion clones in the M13mp 18 vector to M13mp 19 (213). The resulting TF deletion clones had the IIIL fragments flanked by the required restriction sites for subclonlng In the yeast vector pSZ218 (Fig. 25b) to produce the linear plasmlds. Vector pSZ218 (194) rather than pSZ93 W8S used for preparing plasmlds with IIIL termini, since pSZ218does not contain a yeast A M region. Consequently, the A M activity of the X region on the IIIL telomeric fragments is a determining factor in the mitotic stability of the linear plasmlds In yeast (65). The BamHI / EcoRI fragments were isolated for each of TF1 to TF7 for cloning into the single Bglll site of pSZ218. The restriction enzymes Bglll and BamHI were Included to select for the desired ligation products which were either circular plasmlds containing Inverted telomeric repeats or linear plasmlds with IIIL telomeric fragments at the ends (194, 195). The assumption was that upon transformation In yeast, plasmlds with sufficient telomeric DNA Information should be recognized, resolved, and then be maintained as 94, Figure 25. Construction of Plasmids to Study the Function of Fragments from the IIIL Telomere. a. Maps of the telomeric region isolated from chromosome IIIL, the extent of the cloned IIIL telomere (TF1), and the Exolll / SI nuclease derivatives of TF1 (TF2 - TF7) or the reverse orientation deletion clone (TF8). The IIIL telomere contains a 0.6 kb (V-0 .2 kb) region of 5'-Ci_3A-3' repeat sequence adjacent to a 1.2 kb type X A M region. The A£§_ consensus sequence (33, 42, 104) maps 0.52 kb distal to the IIIL terminal EcoRI site. Short flanking sequences from the M13 polycloning site are terminated by BamHI, Sail, Hindiii, or EcoRI and this region exists on the centromere proximal and distal ends of the telomeric TF1 to TF7 fragments as indicated for TF1. TF8 is flanked by Hindi 11 on the centromere proximal end and EcoRI on the distal end. The lengths of the deletion fragments are: TF1, 1.54 kb; TF2, 1.23 kb; TF3, 1.18 kb; TF4, 0.92 kb; TF5, 0.72 kb; TF6, 0.47 kb; TF7, 0.29 kb; TF8, 0.71 kb. b. Cloning the fragments from the IIIL telomere in pSZ218. Vector pSZ218, (194), was digested with Bglll and used for cloning BamHI/EcoRI fragments from the IIIL telomeric region. TF1 to TF7 fragments were llgated to pSZ218 in the presence of BamHI and Bglll. The linear monomer is the predicted plasmid structure following transformation of the ligation mixture in yeast, if the circular and dimeric molecules are resolved in ye8st (65, 194, 195). The estimated length of resultant plasmid is the combined length of the pSZ218 vector (5 kb) plus two end fragments. Transformants were named according to the telomeric fragments used in the particular plasmid construction, ie. YeTF 1 -1 to YeTF7-1 contained plasmids with TF 1 to TF7 telomeric fragments respectively. c. Cloning IIIL telomeric fragments on a LEU2 fragment. The LEU2 fragment was Isolated as a 2.2 kb Sall/Xhol fragment from pSZ218. It was ligated to TF1/Sall/EcoRI (IIIL X and T regions), or to TF3/Sall/EcoRI ( entire T region deleted), in the presence of Xhol. Sail was not included since Sail ligation was required to add a telomeric fragment at one end of the LEU2 fragment. d. The effect of different telomerlc fragments on either end of the LEU2. fragment was determined. The LEU2 Sail/Hindi 11 fragment (2.5 kb) was isolated from pSZ218 and ligated with a combination of telomeric fragments, either TF3/Sall/EcoRI and TF2/Hindlll/EcoRI or TF3/Sall/EcoRI and TF8/Hindlll/EcoRI. The monomeric linear plasmid expected to result following transformation in yeast is sketched for each construction. Symbols: X region ( ), AfiS consensus sequence ( • ) , T region ( • • ) , IIIL terminus ( • ) . Restriction sites: B, BamHI; Bg, Bglll; H, Hindi 11; R, EcoRI; S, Sal I; X, Xhol. 94 <x HSTT i— • - - - X Region ARS T Region - - - | ( c w * ) 1,2kb . . - r . -0.5 5kb- IIIL End I TF 1 TF2 TF3 TF4 TF 5 TF6 TF7 TF8 ,. pSZ2 18 5 kb-LEU2 BgHR riuiri'ftH'If/mr^L TF 1 HR S R =r3 BgJB Y e T F 1-1 (*YoTF7-l) TF 1 i2kb 1 S R TF3 YeTF 1-3,1-4 5.3 kb XS YeTF3-4 4.6 kb . 2J5kb-TF2 TF3 S R X, H 1 / TF8 TF3 YeTF3-6 4.9 kb — ^ YeTF»6 4.4 kb 95 linear plasmlds In the yeast cell. Due to the numerous subclonlng steps Involved In preparing the BamHI / EcoRI fragments from the cloned IIIL telomere, there were about 30 nucleotides of M13 vector DNA sequence separating the IIIL distal end (the 5'-C 1 _ 3 A-3" repeat end) and the EcoRI site on each fragment. Studies conducted with Tetrahymena ends cloned on linear plasmlds In yeast Indicate that up to 54 bp of foreign DNA sequence, adjacent to the 5 - 0 ^ 2 - 3 ' repeat region, do not significantly affect the resolution of Inverted telomeric repeats since the majority of transformants had linear plasmlds while the rest had a mixture of linear and circular configurations ( 195). Further plasmlds were designed to determine whether a yeast LEU2 fragment, without the adjacent plasmid sequences, can be stabilized and subsequently be maintained In a linear form In yeast (65) with IIIL telomeres. TheLIIJ2.fragment, (2.2 kb Sail / Xhol), was Isolated from pSZ218 (2, 194) and Hgated to TF1 or TF3, to determine whether the 5'-C 1 _ 3 A-3 ' repeat sequences are required at the ends of the plasmlds to result In linear plasmid resolution and maintenance In yeast (Fig. 25c). The requirement for 5'-Ci_3A-3' repeat sequence and the A£S_ region at one or both ends of a linear plasmid was addressed by Hgatlng different IIIL fragments to either end of the LEU2 fragment (Fig. 25d). In one case, the TF2 and TF3 fragments were used as ends since both have A£S_ regions, but TF3 lacks the 5'-Ci_3A-3' repeat region, whereas TF2 contains 49 bp of the T region repeat sequence. Secondly, the TF3 and TF8 fragments were used as ends on the LEU2 fragment since TF8 lacks the A£S_ region, but contains 356 bp of the 5 - 0 ^ - 3 ' repeat region whereas TF3 has the type X A M region but lacks the T region . The estimated length Is given for each plasmid construction but it does not account for any addition reactions that are known to occur at the ends of linear plasmlds In yeast (65, 175, 195). Instead, It represents the minimum linear plasmid length for the various classes of plasmid constructions. 0. The Cloned IIIL Telomere Functions on Linear Plasmlds In Yeast Ligation mixes for each of the plasmid constructions were transformed Into the yeast strain A281, which has a nonrevertlng ]ejr genotype, (65, 195) and prototrophic LEU+ transformants were selected. The TF 1 to TF7 fragments are numbered according to the extent of the deletion from the 111L telomere. Telomeric fragments TF6 and TF7 (Fig. 25a) contained only 0.47 kb and 0.29 kb 96 respectively of the X region and did not produce transformants when cloned in pSZ218. The TF5 fragments contained 0.72 kb of the X region and gave very few transformants when cloned In pSZ218. The TF4 to TF 1 fragments contained between 0.92 kb to 1.54 kb from the 1.8 kb ( V - 0.2 kb) IIIL telomere. The transformation frequencies for each of these fragments cloned on pSZ218 was equivalent (between 10 and 25 transformants per ligation mixture). The molar ratio of IIIL insert to pSZ218 vector was equivalent for all of the plasmid constructions (10 x excess). Apparently the extent of the deletions from the IIIL telomere, which averaged between 1.1 kb and 1.5 kb In TF5, TF6, andTF7, precluded the cloning of these fragments on pSZ218 in yeast. The ARS consensus sequence (34, 42, 104, 182) was deleted In the TF6 and TF7 fragments (Fig. 25a), hence there was no functional origin of replication on the pSZ218 plasmlds Hgated with TF6 or TF7. The TF5 fragment extended 200 bp distal to the A M consensus sequence (Fig. 25a). The low transformation frequency observed with the TF5 fragments ligated with pSZ218 may reflect an A M domain that Is required for optimal A M activity and that extends beyond the TF5 deletion (42, 182). Transformants were selected to assay the mitotic stability and structure of the resident LEU2 plasmid for each of the plasmlds constructed with deletion fragments that contained thetype X- A M region (TF5 - TF 1). Mitotic stability, as represented by the percentage of plasmid bearing cells In the population (195) was determined by growing the transformants in selection medium that lacked leucine until log phase, plating an aliquot on complete medium (YPD), Incubating at 30 °C for 1-2 days, then replica plating on selection medium ( SC-leu). A summary of the plasmid constructions and the mitotic stabilities determined for representative transformants are presented in Table II. Transformants with Tetrahymena rDNA ends on pSZ93 (Fig. 24b), had average stabilities of 40* (eg. YeTFSZ-1), which Is slightly lower than previously reported values for linear ARS1 plasmlds with Tetrahvmena rDNA ends In yeast (56,65,139, 140). Alternatively, LEU* transformants containing the circular PSZ93 plasmid (eg. YeSZ93-1> had mitotic stabilities of 5-10*. Transformants that contained the LEJJ2. marker on pSZ218 ligated with the IIIL telomeric fragments displayed a wide range of stabilities (15-100*) and the LEU+ marker stability was Inversely proportional to the extent of the telomeric deletion. Transformants that contained plasmlds with TF 1 fragments (average 0.3 kb deleted from the 97 Table II. Stability and Structure of Plasmlds with Telomeric Fragments In Yeast. AH plasmids were transformed Into the yeast strain A281. The plasmid constructions used for each set of genetic transformations are given In the left column. The estimated length of each plasmid is the sum of vector length plus the length of two telomeric fragments. Transformants were named according to the telomeric fragments and the vector used in the plasmid construction (first and second numbers in the name) and to the number of the particular transformant that was isolated and characterized (third number). For example, transformant YeTF 1-1-1 had TF 1 ends, on the vector pSZ218, and it was the first transformant Isolated that contained this type of plasmid construction. The percentage of plasmid bearing cells represents the mitotic stability of the plasmid in the transformant which was determined by measuring the fraction of colonies that grow on leucine selection medium, after replica plating colonies from nonselective medium. The linear or circular nature of the plasmid in each transformant was data derived from agarose gel electrophoresis and Southern hybridization analysis of DNA from the transformants (Fig. 26 and 27). 97 cx Construction Estimated Transformant Mitotic Linear Circular Length Stability Plasmid PSZ93 • IgJ rDNA 8.8 kb YeTFSZ-1 455i YeTFSZ-2 35 PSZ93 74 kb YeSZ93-l 10 **** YeSZ93-2 7.5 **** PSZ218 + TF1 8.1 kb YeTFl-1-1 75 **** YeTFl-1-2 75 **** YeTFl-1-10 70 **** YeTFl-1-11 75 ||u|f||tj|( LEU2 + TF1 5.3 kb YeTFl-3-1 85 ** -i- integrated YeTFl-3-4 80 YeTFl-4-1 75 +*** YeTF 1-4-2 70 *+** YeTFl-4-4 75 **** DSZ218 + TF2 7.5 kb YeTF2-1-1 75 YeTF2-1-26 55 **** YeTF2-1-27 75 **** PSZ218 + TF3 74 kb YeTF3-1-l 20 i k i k i k i l i YeTF3-1-4 65 **** YeTF3-1-33 25 **** YeTF3-1-37 15 **** LEU2 + TF3 4.6 kb YeTF3-4-2 15 **** YeTF3-4-3 30 YeTF3-4-4 40 **** LEU2 + TF3 4.7 kb YeTF3-5-1 100 integrated TF2 YeTF3-5-3 75 **** YeTF3-5-5 65 LEU2 + TF3 4.2 kb YeTF3-6-1 75 TF8 YeTF3-6-4 75 **** YeTF3-6-5 70 s|cifcs|u|t PSZ218 + TF4 6.9 kb YeTF4-1-l 15 **** YeTF4-l-2 25 **** YeTF4-1-3 25 PSZ218 + TF5 6.5 kb YeTFS-1-1 100 integrated + <*) 98 IIIL telomere in strain AB20a XP8-1 OB), ligated with either pSZ218 (YeTF 1 -1) or the LEU2 fragment (YeTF 1-3 or YeTF 1-4), had the highest average plasmid stabilities (70-80*). In transformants with mitotic stabilities approaching 100* (YeTF 1 - 3 - 1 , YeTF3-5-1, YeTF5-1 -1) , the LEU2 marker may have Integrated Into the yeast genomic DNA. The homology of the Sal I /Xhol ends of the LEJi2. fragment with the ]ejr gene In genomic DNA provides the preferred conditions for marker integration through homologous recombination with genomic DNA (149, 150, 196). The YeTF2-1 transformants had pSZ218 plasmids with only 49 bp remaining of the T region (5'-Ci-3A-3' repeat sequence) compared with 356 bp of T region on the plasmids in the YeTF 1 -1 transformants. However, the mitotic stability of the LEU2 plasmids In the YeTF2-1 transformants was 55 -75* , and equivalent to that observed for the TF 1 -1 transformants. The deletions in the TF3 and TF4 fragments extended past the T region of the IIIL telomere (Fig. 25a) and this resulted in marked reductions in the mitotic stabilities of the LEU2 marker 1n most of the YeTF3-1 and YeTF4-1 transformants. For example, pSZ218 with TF3 or TF4 fragments had average stabilities of 20*. An exception was transformant YeTF3-1 -4 which had a mitotic stability equivalent to that observed for transformants that contained pSZ218 plasmids with TF1 or TF2 fragments. Transformants such as YeTF3-4-3 that contained the LIU2_fragment llgated with TF3 ends had mitotic stabilities between 15 and 40 * , and equivalent to the stabilities for TF3 fragments cloned on pSZ218 plasmids (15-25*). However, plasmids constructed with the T region or the T-X region at one end of LEU2 and the X region (TF3) at the other such as In the transformants YeTF3-5-3 and YeTF3-6-1, had mitotic stabilities of 75*. Such high mitotic stabilities were equivalent to those determined for transformants that contain plasmids with a type X ARS region and a T region at both ends of the LEU2 plasmid (eg. YeTF 1 - 4-1). The ability of the IIIL end fragments to function as yeast telomeres was determined by analyzing the structure of the plasmids In the genomic yeast DNA prepared from selected transformants. A IIIL fragment was defined as a functional telomere If it maintained the LEU2 marker 8S a linear molecule in yeast. The estimated length for the linear plasmid expected to result from a given plasmid construction was the combined length for the vector fragment and two telomerlc fragments (Table 11). The transformants obtained for a given plasmid construction were screened for 99 the presence of linear plasmids In two ways . First, If a linear plasmid Is present In the transformant, an extrachromosomal band Is observed in the DNA of the transformant which is absent In the DNA from the host strain A281. The extrachromosomal band Is detected by fractionation of the genomic DNA that has not been digested with restriction enzymes by agarose gel electrophoresis and observation of the EtBr stained gel by UV-fluorescense (Fig. 26A). Since the genomic DNA from the host strain A281 Is void of extrachromosomal bands such as the yeast endogenous 2urn plasmid DNA, an extra band In transformant DNA which results from multiple copies of a linear plasmid can be identified (63, 65,139,195). Secondly, linear plasmids can be detected by Southern hybridization using probes from the vectors pBR322 or pSZ218 which hybridize with a single band In transformants that contain linear plasmids. However, if the transformant contains a circular plasmid, the probes hybridize with multiple bands which correspond to supercoiled, monomer, dimer and concatamerlc plasmids (Fig. 26B), (195). The presence or absence of linear plasmids In the selected yeast transformants (Fig. 26, A&B) is given In Table II for the various plasmid constructions. The mitotic stability of the LEI marker In a given transformant correlates with the linear or circular nature of the plasmid in the transformant such that a high mitotic stability Is indicative of a linear plasmid in the transformant. The total genomic DNA, that was not digested with restriction enzymes, contained only the chromosomal DNA smear In the host strain A281. Extrachromosomal bands that had lower molecular weight than the genomic DNA band were evident In the total DNA isolated from selected transformants (Fig. 26A). An additional band was evident In transformants that contained plasmids constructed with Tetrahvmena rDNA ends at the expected 9 kb region (Fig. 26A, lanes 18,21, 22). Plasmid pSZ218 with TF1 or TF2 ends (Fig. 26A, lanes 4 - 11), or theiEy_2 fragment with TF1 ends (Fig. 26A, lanes 24-28) were linear In some transformants since extrachromosomal bands were observed with the predicted lengths. Southern hybridization analyses with pBR322 or pSZ218 probes (F1g. 26B) demonstrated that these extrachromosomal bands corresponded to the linear plasmids. The pSZ218 probe, which contains the LEU2 fragment was used to detect plasmids constructed with the LEU2 fragment. The leu. region In the yeast chromosomal DNA was also homologous with the LEU2 region of pSZ218, and this 100; Figure 26. Assay for Linear or Circular Plasmids in the Yeast Transformants. A. Yeast DNA was isolated from selected yeast transformants, and undigested DNA was fractionated by electrophoresis on 0.65* agarose gels, alongside size markers. Ethidlum bromide stained gels were observed by UV fluorescence. Samples lanes 1 -18 and 19- 38 represent two separate agarose gels. Positions of extra low MW bands in some transformants are indicated by at left of gels in 6 kb -10 kb region. Sample lanes: (1) A/H/R, (2) A/H, (3) A281, (4) YeTF 1 -1 - 1 , (5) YeTF1-1-2, (6) YeTFl-1-10, (7) YeTF 1-1-11. (8) YeTF2-1-1, (9) YeTF2-1-2, (10) YeTF2-1-26, (11) YeTF2-1-27, (12) YeTF3-1-1, (13) YeTF3-1-33, (14) YeTF3-1-37, (15) YeTF4-1-1, (16) YeTF4-1-2,(17) YeTF5-1-1,(18) YeTFSZ-1. (19) A/H/R, (20) A281, (21) YeTFSZ-1, (22) YeTFSZ-2, (23) YepSZ93-2, (24) YeTF1-3-1, (25)YeTF1-3-4, (26) YeTF1-4-1, (27) YeTF1-4-2, (28) YeTF1-4-4, (29) YeTF3-4-2, (30) YeTF3-4-3, (31) YeTF3-4-4, (32) YeTF3-5-1, (33) YeTF3-5-3, (34)YeTF3-5-5, (35) YeTF3-6-1, (36) YeTF3-6-4, (37) YeTF3-6-5, (38) A/H/R. B. DNA was transferred bidirectionally from the agarose gels to gene screen plus filters for Southern hybridization analysis. The bottom filters (contain less DNA than top filters) were hybridized with nick-translated pBR322 for samples 1-18 and with nick-translated pSZ218 for samples 19-38. Linear plasmids are identified in the transformant DNA as single or double bands that are a minimum length of 6 kb. Circular plasmids exist as multiple bands in the transformants, ranging in length from <4 kb (covalently closed circles) to >24 kb (multimeric circles). Transformants which contain linear plasmid bands (6-10 kb region) are indicated by arrows at the bottom of the lanes. Size standards are A/Hindi 11/EcoRI fragment positions. C. The filters taken from the top of the agarose gels during bidirectional transfer of the DNA samples were hybridized with a nick-translated Y" region probe, the 1.7 kb Bglll fragment from Yrp131B. There are no obvious differences between linear plasmid and circular plasmid containing transformants. i 1 , T • T i V • T i 1. 4 i T i 1P I I I V ••••• 2a7 * 95 * — ( 57 • 4.3 ' I A A A A A A A • •237 • 9.5 • 6 7 • 43 A A A * A * 1 nfjtyftfmi j kb •237 • 05 • 6.7 • 43 101 resulted In bands with relatively weak hybridization Intensities In the Southern blots (Fig. 26B, lanes 20 - 37). The hybridization intensity of the single copy ley. gene in the yeast genome provided a useful Internal control, In Southern blots with a JL£U_ probe, for estimating the copy number of the LEU plasmids In the transformants. In Southern analysis with the pBR322 or pSZ218 probes, a 9.0-9.5 kb band hybridized In transformants YeTFSZ-1 and YeTFSZ-2 that contained the pSZ93 plasmid with Tetrahvmena rDNA ends, (Fig. 26B, lanes 18, 21, 22). The estimated length for a linear plasmid In the YeTFSZ transformants was 8.8 kb but the addition of 5'-C 1 _ 3 A-3 ' repeat sequence may account for the extra 0.7 kb In plasmid length observed In the transformants (40, 65, 175, 206). Plasmids constructed with TF 1 or TF2 ends were present In transformants as linear molecules and in most cases, the length of the plasmids agreed with that estimated for linear monomers (Fig. 26B, lanes 4 - 11, and 24 -28). In Southern analysis, the pBR322 or pSZ218 probes hybridized with other bands than the monomeric linear plasmids for some transformants, and the length of these bands suggested that they were dlmerlc linear plasmids (Fig. 26B, lanes 10, 11, 24 - 28). Transformant YeTF 1-3-1 (Fig. 26B, lane 24) was an exception since It had predominantly Integrated or multimeric plasmid, and a minor DNA band which migrated ahead of the position expected for linear monomeric plasmids (5.5 kb). In all transformants, the lengths of the linear plasmids exceeded the estimated lengths calculated from the combined lengths of the vector and telomerlc fragments. Possibly addition of 5'-C]_3A-3' repeat units occurred at the IIIL ends on the linear plasmids, as described for linear plasmids constructed with Tetrahvmena ends (65,175) or for yeast ends (40,206). The transformants that contained the plasmids constructed with pSZ218 and TF3 or TF4 ends, YeTF3-1 and YeTF4-1 transformants respectively (Fig. 26B, lanes 12-16), had multiple plasmid bands that hybridized with the pBR322 probe. This reflected the circular structure of the plasmids, and the multiple bands were the closed circular, open circular, and concatamerlc forms of the plasmid DNA. Similarly, the plasmids constructed with the LEU2 fragment from pSZ218 and the TF3 end fragments were maintained as circular plasmids in the YeTF3-4 transformants (Fig. 26B, lanes 29 -31). However, If the plasmid was constructed with the 5'-Ci_3A-3' repeat region at only one end of 102 the LEU2 fragment and the ARjj region from the IIIL telomere (TF3) at the opposite end, some transformants maintained the plasmids as linear molecules (F1g. 26B, lanes 33,35,37). Linear plasmids were observed in some of the YeTF3-5 and YeTF3-6 transformants but these linear molecules were longer than the structures predicted for the plasmids constructions (Table II). The increase in length was presumably due to terminal T region addition at the TF3 end which lacked the terminal 5'-Ci_3A-3' repeat region. There were also plasmid bands in the YeTF3-5-3, YeTF3-6-1 and YeTF3-6-5 transformants that were longer than the 9.5 kb linear monomer fragments, and these may be due to inefficient resolution of the partial IIIL telomeres. The single transformant that was Isolated for the plasmid constructed with pSZ218 and TF5 telomerlc fragments had the vector Integrated In the yeast chromosomal DNA. This was concluded from the high mitotic stability of the LEU In YeTF5-1 -1 (Table II), and the Southern hybridization with pBR322 which showed the vector sequences were present in low copy number and the vector had the same mobility as yeast genomic DNA in YeTF5-1-1(F1g. 26B, lane 17). In Southern hybridization, the DNA from transformants with circular plasmids such as pSZ93 had weak hybridization signals compared to the DNA from transformants with linear plasmids, reflecting the relatively low copy number of circular plasmids (Fig. 26B, lane 23). In previous reports, the linear ARS1 plasmid copy number was estimated as 25 to 50 copies per plasmid bearing cell (HO). Differences In the intensity of hybridization signals between the genomic leu2 and the linear plasmid LEU2 region (Fig. 26B, lanes 20 - 37) reflect a similar copy number (25 - 50 copies) for linear plasmids having IIIL ends. For all of the transformants examined, high mitotic stabilities correspond to linear plasmids (Table II); linear plasmids with IIIL ends displayed high mitotic stabilities (average 75*) while transformants with circular plasmids containing the IIIL end region had moderate to low mitotic stabilities (average 20*). 103 P. Linear Plasmids With Partial IIIL Telomeres are Not Stabilized with Y' Regions Short linear plasmids that lack an ARS region and have Tetrahvmena rDNA ends, or similar linear plasmids that contain a portion of the Y' A M region at one end, are stabilized by the addition of Y" A M elements to one end of the plasmid In RAD+ yeast strains (65). I asked whether Y' addition events occurred on the linear plasmids that were constructed with either a complete or partial X A M region from the IIIL telomere in the RAD+ yeast strain A281. Duplicate filters of those hybridized with pBR322 or pSZ218 (Fig. 26B), were probed with a Y' A M region probe, the 1.7 kb Bglll fragment from YRp131B (48,49), (Fig. 26C). This Y' fragment hybridizes with between 20 and 30 regions in the haploid yeast genome (49, 93). In the yeast transformants that contained plasmids constructed with the deletion fragments from the IIIL telomere, linear plasmids existed in multiple copies and migrated as extrachromosomal bands In undigested DNA samples (Table II, Fig. 26B). Therefore, healing events that Involved Y' A M region addition on the ends of the linear plasmids should be obvious In Southern hybridization analysis with a Y' probe (Fig. 26C). If the linear plasmids were stabilized with Y' regions at the ends, the Y' probe should hybridize Intensely with the linear extrachromosomal plasmids, but should not hybridize with the circular extrachromosomal plasmids, that were detected with the pBR322 or pSZ218 probes (Fig. 26B). In Southern analysis, the Y' probe hybridized with only the high molecular weight chromosomal DNA band in the transformants, and this hybridization pattern was similar for the transformants with circular or linear plasmids (Fig. 26C). Variation in hybridization Intensities with the Y' probe for the transformants reflects variation In the amount of DNA In each sample lane rather than varying amounts of Y' regions in the transformants, since the intensity of UV fluorescence is not uniform for the EtBr stained DNA samples (Fig. 26A). As with the structure of the T-X telomere on chromosome 111L, the telomerlc fragments Isolated from 111L did not require the presence of the conserved Y' regions for maintaining stable linear plasmids In the ye8St transformants. 104 Q. Structures of the Plasmids with IIIL End Fragments In Yeast To elucidate the structure of plasmids containing the TF fragments from the IIIL telomere following their replication In yeast, genomic DNA was Isolated from selected transformants, DNAs were digested with restriction enzymes and subjected to Southern analysis using pSZ218 as hybridization probe (Fig. 27). The restriction maps for the linear or circular plasmids were deduced from the Southern blots and are represented schematically (Fig. 28, A-0). The plasmids constructed with the vector pSZ218 and the TF 1 or TF2 fragments which contain the A£S_ and 5'-C 1 _ 3 A-3' repeat regions from the IIIL end, were maintained as linear monomers or dlmers in yeast (Fig. 27A, lanes 3-18, Fig. 28, A&B). The pSZ218 probe hybridized with a single linear plasmid with the expected length for a linear monomer in transformants YeTF 1 -1 -1 and YeTF2-1 - 1 , however YeTF 1 -1 -10 and YeTF2-1 -26 contained linear plasmids with the expected lengths for both monomers and dlmers. The plasmid constructed with pSZ218 and TF1 or TF2 ends was predicted to exist as a linear monomer, about 8 kb In length (Fig. 25b) which corresponds to the plasmid band at about 8-9 kb In the undigested DNA samples (Fig. 27, lanes 3, 7, 11, 15). For the linear plasmid that exists as a monomer, Xhol digestion results In the removal of one end and Sail digestion cuts the linear plasmid In the central region (Fig. 25b). The 7 kb Xhol fragment and the 4.3 kb to 4.7 kb Sail fragments (Fig. 27) agreed with the predicted structure for a linear monomer, If the length of the TF1 and TF2 fragments was about 1.7 kb (Fig. 28A). Since the TF1 fragment was Initially 1.54 kb and the TF2 fragment was 1.23 kb, different extents of T region addition must have occurred at these ends. Such addition reactions would result in equivalent lengths of T region on the linear plasmids and on the end of chromosome IIIL in the yeast genome. This agrees with recent reports showing that the length of the yeast telomerlc repeat (T region) Is under genetic control (40) and It is similar for all chromosome ends In a given yeast strain (206). The undigested DNA from transformants YeTF 1 -1 -10 and YeTF2-1 -26 had plasmid bands that were about twice the length of the linear monomeric plasmids (Fig. 27, lanes 7, 15). This together with the extra Sail fragment that hybridized with pSZ218 fn these transformants, Indicated that YeTF 1-1-10 and YeTF2-1 -26 contained dimeric as well as the monomeric linear plasmids. The extra Sail fragment was about 7 kb In length (Fig. 27, lanes 9 & 17) 105 Figure 27. Determination of the Restriction Maps for the Linear or Circular Plasmids in the Yeast Transformants. Yeast transformant DNA (2 ug) was digested with restriction enzymes and fractionated on 0.65* agarose gels. After unidirectional transfer to gene screen plus filters, Southern hybridization was a with nick translated pSZ218 probe (10 7 cpm/filter). Sample lanes: (1) pSZ218/SalI (O.lug), (2) pSZ218/EcoRI (0.1 ug), (3) YeTF 1 -1 -1/undigested, (4) YeTF 1-1-1 /Xhol,(5) YeTF 1 -1 -1 /Sai l , (6) YeTF 1-1-1 /EcoRI,(7) YeTF 1 -1 -10/undigested, (8)YeTF 1-1-10/Xhol,(9)YeTF 1-1-10/Sall,(10)YeTF 1-1-10/EcoRI,(11 )YeTF2-1 -1 /undigested, (12)YeTF2-1 -1 /Xhol,(13)YeTF2-1 -1 /Sail,(14)YeTF2-1 -1 /EcoRI,(15)YeTF2-1 -26/undigested, (16) YeTF2-1 -26/Xhol ,(17) YeTF2-1 -26/Sall,(18) YeTF2-1 -26/EcoRI. (19)YeTF3-1-1 /undigested/20)YeTF3-1 -1 /Xhol ,(21 )YeTF3-1 -1 /Sail ,(22) YeTF3-1 -1 / EcoRI, (23)YeTF3-1 -4/undigested,(24)YeTF3-1 -4/Xhol,(25)YeTF3-1 -4/Sall,(26) YeTF3-1 -4/EcoRI, (27)YeTF4-1 -1 /undigested/28)YeTF4-1 -1 /Xhol,(29)YeTF4-1 -1 /Sail,(30) YeTF4-1 -1 /EcoRI, (31 )YeTF4-1 -3/und1gested,(32)YeTF4-1 -3/Xhol ,(33)YeTF4-1 -3/Sall,(34) YeTF4-1-3/ EcoRI. (35)YeTF 1-4-1 /undigested/ 36)YeTF 1-4-1 /Sail,(37)YeTF 1-4-1 /EcoRI,(38)YeTF3-4-1 / undigested ,(39) YeTF3-4-1/Sall,(40)YeTF3-4-1/EcoRI. (41)pSZ218/Hindlll/Sall(0.1 ug),(42)YeTF3-5-1/undigested/43) YeTF3-5-3/ undigested/44) YeTF3-5-3/SalI,(45)YeTF3-5-3/H1ndl 11,(46)YeTF3-6-1 /undigested, (47)YeTF3-6-5/uncut, (48) YeTF3-6-5/Sall, (49) YeTF3-6-5/Hindlll. Sample lanes 1-18, 19-34,35-40, and 41-49 are separate agarose gels and Southern hybridization analyses. The positions of A/Hlndlll/EcoRI fragment size markers are indicated for each. 105a, ? i 1 . y i T i T i T i ¥ , T , V kb 2X5 • kb 4215 44 • 3.6 • 20 • 13 • 1.7 • W • - t - 44 4 3JB 4 1.7 4 13 ¥ i ¥ i 4P 106 Figure 28. Restriction Maps of the Plasmids with Fragments from the IIIL Telomere following Replication In Yeast. A. Linear monomers were resolved in the transformants that contained plasmids constructed from pSZ218/Bglll and the IIIL telomeric fragments, TF1, TF2, or TF3 (BamHI/EcoRI fragments). Transformants YeTF 1 -1 - 1 , YeTF2-l-1, and YeTF3-1-4 contain exclusively linear monomeric plasmids. B. Linear dimers were resolved from the constructions with pSZ218/Bglll and the TF1 or TF2 fragments from the IIIL telomere. The 1.5 - 2.0 kb region at the center of symmetry in the linear dimers must be a partially deleted inverted telomeric dimer as judged from the restriction digestion patterns in Southern analyses (Fig. 27, lanes 7-10, 15-18). Plasmids in transformants YeTF 1 -1 -10 and YeTF2-1-26 exist as both linear monomers and dimers. C. Circular pSZ218 with inverted telomeric repeat fragments inserted at Bgll I was maintained in the transformants Indicated, that resulted from plasmid constructions with TF3 or TF4 fragments. Dimerlc circles must also be present as determined from the doublet pattern observed with restriction enzyme digestion in the Southern analyses (Fig. 27, lanes 19-22,27-34). D. Ligation of TF1 ends on the LEU2 fragment resulted in monomers and dimers of linear plasmids following replication in yeast. E. Plasmids constructed with TF3 ends on the LEU2 fragment were not resolved into linear molecules in the yeast transformants, as in YeTF3-4-1. The TF3 fragments were maintained as an inverted dimer in a circular plasmid. F. Resolution from a circular to a linear plasmid occurred for the plasmid constructed with a TF2 fragment on one end of the TF3-LEU2 plasmid in the transformant YeTF3-5-3. G. Linear plasmid resulted from resolution of TF8 fragment on one end of the TF3-LEU2 fragment in transformant YeTF3-6-5. Restriction sites. B, BamHI; Bg, Bglll; H, Hindi 11; R, EcoRI; S, Sail; X, Xhol. Symbols: T region repeat sequence/ ); type X A M region/ ); ARS consensus sequence ( • ); pSZ218, ( t ^^ ) ;LEU2 i ( IV Lengths determined for the X and T regions are indicated for each type of plasmid construction in the yeast transformants. Telomeric fragments on linear plasmids were elongated to approximately 1.7 kb except for TF8 which was 0.9 kb, presumably by T region addition to render equivalent T region length on all linear plasmids. Y e T F 3 - 6 - 5 107 and It must map to the central region of the dlmerlc linear plasmids In YeTF 1 -1 -10 and YeTF2-1 -26 (Fig. 28B). Presumably two linear monomers were llgated together in a head to tall arrangement, and the Inverted repeats of the TF1 or TF2 fragments In the central region of the molecule were not resolved to yield two linear monomers upon replication In yeast. Instead, part of the Inverted repeat region was deleted, leaving about 2 kb for the 3 kb TF 1 Inverted repeats In YeTF 1 -1 -10 or for the 2.4 kb TF2 Inverted repeats In YeTF2-1 -26. The other 5 kb In the 7 kb Sail fragment Is the pSZ218 vector (Fig. 2813). A deletion In the Inverted repeats of the telomerlc fragments would explain the Incomplete resolution of the plasmids In transformants YeTF 1 -1 -10 and YeTF2-1 -26 that resulted in both linear monomers and dlmers. The TF3 fragments had the entire A£S_ region from the IIIL end but none of the S ' -C^sA-S ' repeat region (Fig. 25a), and when ligated to pSZ218 resulted In plasmids that were replicated as linear as well as circular molecules in yeast (Fig. 27, lanes 19-26 and Fig. 28, A & C). In transformant YeTF3-1 -1 (Fig. 27, l8nes 19-22 ), circular monomers were present as Indicated by the 7.5 kb Xhol fragment and the 7.5 kb Sail fragment which are the expected size for a plasmid containing pSZ218 (5 kb) and two TF3 fragments (2.36 kb), (Fig. 25a,b). Apparently, there was also a population of circular plasmids that contained a 1 kb deletion, likely in the Inverted repeat region of the TF3 fragments, to result In the 6.5 kb Xhol fragment and the 6.5 kb Sail fragment in YeTF3-1 -1 . The 4.4 kb Sail fragment that Is heterodisperse may result from the generation of a Sail site during the proposed deletion event In the TF3 region or alternatively the 4.4 kb Sail fragment may reflect the resolution from circular to linear plasmids in some YeTF3-1 -1 transformants (Fig. 27, lane 21). Transformant YeTF3-1 - 4 also contained plasmids constructed with pSZ218 and TF3 end fragments, however only plasmids maintained as linear monomers were observed In YeTF3-1 -4 (F1g. 27, lanes 23-26). As described for transformants YeTF 1-1-1 and YeTF2-1 - 1 , a single 8-9 kb plasmid band was present in the undigested sample and a 7 kb Xhol fragment and the 4.3-4.7 kb Sail fragments agreed with the predicted map for the linear plasmids (Fig. 25b, 28A). The lengths of the Xhol and Sail fragments Indicate that the TF3 fragments were increased from 1.18 kb to the 1.7 kb ends on the linear plasmid, similar to the addition events described for the TF1 and TF2 fragments. 108 Presumably, the extra 0.5 kb on the TF3 fragments Is T repeat sequence, which resulted In the stabilization of the linear plasmid In YeTF3-1 -4. This speculation, however, has not been tested by Southern hybridization with a T region probe. If the addition is T region, it may be required for the efficient resolution of circular plasmlds and maintenance of linear plasmlds in yeast since the telomeric fragments which lacked the T region (TF3) existed as circular molecules In YeTF3-1-1 but as linear molecules with additions to the ends In YeTF3-1 -4. Similarly, the telomeric fragments that contained a portion of the T region, such as TF 1 (356 bp of T region) or TF2 (49 bp of T region) were present exclusively on linear plasmlds. Plasmlds constructed with pSZ218 and the TF4 fragments, which had only 919 nt of the X region and no T region (Fig. 25a), were maintained exclusively as circular molecules In the transformants (Fig. 27, lanes 27-34, Fig. 28C). The 7 kb Sail and Xhol fragments are expected for circular plasmlds constructed with pSZ218 (5 kb) and inverted TF4 repeats (1.84 kb), (Fig. 280. The 6 kb plasmid fragments In Sail or Xhol digestions may be circular plasmlds that contain a deletion In the TF4 Inverted repeat region (Fig. 27, lanes 28,29,32, 33). For each of the constructions with pSZ218 and the TF fragments, a deletion event in the inverted repeat region of the telomeric fragments has been proposed to explain the extra bands observed In the Southern blot. Presumably, an Inverted repeat structure Is unstable for telomeric repeats in yeast and a deletion event rather than a resolution event may occur In this region especially if there is no T repeat region in the telomeric fragments. Plasmlds were constructed with only the yeast L1U2g e n e fragment from pSZ218 and the IIIL end fragments (Fig. 25c, d) and these were replicated as linear molecules in yeast if at least one end of the plasmid contained the 5'-C^_3A-3' repeat region and one had an A M region. The LEU2 fragment with TF 1 end fragments was maintained as linear monomers and dimers In transformant YeTF 1 -4-1 (Fig. 27, lanes 35-37 and Fig. 28D), and the TF 1 ends were lengthened from 1.54 kb to 1.7 kb on the linear plasmlds, as observed for pSZ218 with TF 1 end fragments In YeTF 1-1-10. The length of the TF 1 end was heterodisperse following replication In yeast, as evidenced by the smeared 5-6 kb band in the undigested DNA which was the linear plasmid monomer( Fig. 27, lane 35). Sail digestion removes one TF1 end from the linear monomer and bisects the linear dimer (Fig. 28D), resulting In a 109 heterodisperse 3.6-4.1 kb fragment (Fig. 27, lane 36) that Includes the 2.2 kb LEU2 fragment plus a TF1 fragment. Presumably heterogeneity of the TF 1 fragment length 1.7 kb (+/-0.2 kb), equivalent to that on the end of chromosome MIL, Is the explanation for the heterodisperse lengths observed for the plasmid In YeTF 1 -4 -1 . The plasmid constructed with the LEU2 fragment and the TF3 telomerlc fragment was maintained as a circular monomer which contained the TF3 Inverted repeat In transformant YeTF3-4-1 (Fig. 27, lanes 38-40, and Fig. 28E). The low copy number for circular plasmids versus linear plasmids in yeast is evident In the reduced Intensity of pSZ218 hybridization forYeTF3-4-1 compared to YeTF 1-4-1 (Fig. 27, lanes 35-40). If the plasmid was constructed with a 5 '-C 1 _ 3 A-3' repeat region at only one end of the LEU2 plasmid, such as with the TF2 or TF8 fragments (Fig. 25a, d), plasmids were maintained as linear monomers and dlmers in transformants YeTF3-5-3 and YeTF3-6-5 (Fig. 27, lanes 43-49 and Fig. 28, F&G) however circular plasmids remained in transformants YeTF3-5-1 and YeTF3-6-1(F1g. 27, lane 42). To establish whether addition to ends of the linear plasmids had occurred in YeTF3-5-3 and YeTF3-6-5, the lengths of the terminal fragments on the linear plasmids were estimated through restriction enzyme digestions which remove one of the two ends. The plasmid In YeTF3-5-3 (Fig. 28F) was constructed using the LEU2 fragment (2.5 kb), a TF3 end (1.18 kb) and a TF2 end (1.23 kb). Sail digestion removes both ends from the 5.5-6.0 kb linear plasmid leaving the 2.5 kb vector fragment (Fig. 27, lane 44) and digestion with Hindi 11 removes only the TF2 end, leaving a 4.2-4.4 kb fragment containing the LEU2 fragment plus a TF3 fragment (Fig. 27, lane 45). Consequently, both TF3 and TF2 ends must have been increased to an average length of 1.7 kb, and addition reactions at the ends presumably resulted In the heterodisperslty observed for the length of the linear monomeric plasmid (Fig. 27, lanes 43). Similar characterization of YeTF3-6-5, which contained the LEU2 fragment with TF3 and TF8 end fragments, indicated that addition had occurred at the ends of the linear plasmid resulting in a 1.7 kb TF3 endanda 0.85 kb TF8 end (Fig. 27, lanes 47-49, Fig. 280). It Is interesting that the TF1 and TF8 fragments which initially had equivalent T region lengths (0.36 kb) were both lengthened by about 0.15 kb on the linear plasmids in yeast, regardless of the X region differences for the TF 1 and TF8 fragments (Fig. 25A). The similar addition of DNA to fragments that initially had equal T region 1 1 0 but contrasting X region extents, provides evidence for the assumption that additions to the TF 1 and TF8 end were T region and not X A M region sequences. The length of T repeat region was maintained at 0.5 kb ( + / - 0.2 kb) at both ends of the various linear plasmids In the transformants that were characterized. Therefore, the addition of T region to the linear plasmids was regulated to maintain the same average length on all ends which supports the idea that the telomerlc repeat length at chromosome ends Is regulated genetically (40, 206). It was not determined whether the 30 bp of M13 sequence present Initially on the end of each telomerlc fragment had been removed during this process of telomere addition. Its presence did not appear to hinder the resolution of circular to linear plasmids, since plasmids with TF1 or TF2 ends were maintained exclusively as linear molecules, The extra 60 bp at the center of the inverted repeat may have been one reason for the Inappropriate resolution of linear dimers to monomers in some transformants(YeTFI-1-10, YeTF2-1-26,YeTF 1-4-1 ). I l l DISCUSSION A. Chromosome Walking In this study the telomeric region from the left end of yeast chromosome III was isolated by chromosome walking from the distal genetic marker, H M L K . to the chromosome IIIL end. This telomeric region was functional in replicating a plasmid as a linear minichromosome in yeast. This approach for Isolating the 11 IL telomere Is analogous to that used In cloning the first yeast centromeric region, which was Isolated from chromosome III (51, 53). The first yeast telomere that was molecular 1y characterized was Isolated by the alternate approach, in which selection was for the function of the telomere in stabilizing a linear plasmid in yeast (193). The fragments that exhibited the functional characteristics of telomeres, were mapped to the ends of chromosomes by Southern hybridization, however the chromosomal origin of each telomerlc fragment remains unknown. Chromosome walking offered the advantage of identifying a given telomeric region with a specific chromosome arm. In addition, the Isolation of a telomere by this method allowed the molecular characterization of a specific end structure and comparative studies with other telomeric regions in the yeast genome. The problem with this method was the unknown distance separating the telomere from HMLot, on chromosome IIIL. The genetic map distance on chromosome III is about 130 cM as estimated from the yeast genetic linkage map (107, 136, 146). Equating physical and genetic map distances on chromosome III at 2.7 kb/cM (186), the physical length of chromosome III was calculated as 350 kb between the distal genetic markers HMLoc and MAL2. on IIIL and I MR respectively. Yeast karyotype analysis, which could estimate the physical length of chromosome III, was necessary to establish the amount of DNA on chromosome III that is unaccounted for In the genetic map which presumably consists of the regions distal to HMLa and MAL2. Due to the lack of condensation of yeast chromosomes, (69,78) a useful karyotype was not available until recently when an electrophoretlc karyotype for yeast was established with the physical lengths of yeast chromosomes being estimated using orthogonal-field-alternation gel electrophoresis, (0FA6E), (36, 37, 173). Chromosome III was estimated to be about 370 kb in length on OFAGE gels (36, 37). Since the genetic map accounts for about 350 kb, about 20 kb must include both the distance from HMLoy to the 11 IL terminus and the 112 distance from MAL2 to the IIIR terminus. Had the relatively small length between HMLoi and the telomeric region on IIIL been known at the outset, the negative results obtained for chromosome walking experiments could have been interpreted. The first problem was that the screening of many yeast genome equivalents in the phage DNA library failed to yield clones from the chromosome IIIL region that were more than 10 kb distal to HMLot,. The lack of clones from the 11 IL distal region in yeast genomic libraries could be attributed to a nonrandom distribution of insert fragments In the ACh4A-yeast or AMG14-yeast DNA libraries, or to the proximity of a region on chromosome IIIL, possibly the telomere, that cannot be cloned in phage or circular plasmid vectors. The latter of these two possibilities was shown to be correct. The distance from the IIIL telomere to the most distal probe on IIIL that was isolated from the phage clones was only 2.3 kb ( + /-0.2 kb) and was determined through Bal31 nuclease digestion of chromosomal DNA. Bal31 nuclease is specific for the ends of chromosomal DNA molecules in the genome, hence telomeres yield shorter restriction fragments when obtained from chromosomes that have been digested progressively with Bal31 nuclease while fragments from the internal regions on these chromosomes are of constant length (18, 24, 60, 175, 203, 206, 209). The 1 kb EcoRI/Sall fragment on IIIL which was 8.6 kb distal to HMLot, (probe 2*) was unique in the yeast genome and hybridized to Bal31 nuclease sensitive IIIL fragments. Further evidence that this IIIL distal probe (probe 2*) was identifying restriction fragments which contained the IIIL telomere was the heterodisperse length of fragments that hybridized with probe 2* for some of the restriction enzyme digestions. Previous studies using non-unique telomeric probes from yeast chromosomes demonstrated the length heterogeneity of telomeric fragments, attributed to variable amounts of simple repeat sequences that are present at chromosome ends (18,24,40,49,60,93, 175, 193, 203,206,209). However, length variability in individual chromosome ends could not be distinguished in those reports since the telomeric probes were not chromosome specific. In defining the role of telomere elongation in replicating yeast chromosomes, it is essential to determine events at a defined telomere, as in the case of trypanosome telomeres (18, 60, 209). The unique probe on chromosome IIIL (probe 2*) that hybridizes with the IIIL telomere demonstrated that this chromosome end varies in length in an 113 asynchronous cell population, since it hybridized to MIL terminal fragments that had a size distribution o f + / - 0.2 kb. Telomere addition and elimination reactions with the terminal 5'-C).3A-3' repeat sequences during DNA replication are presumably the cause of the length variability on chromosome ends In yeast (40, 65, 175, 193, 195, 205, 206). The IIIL telomeric region that was Isolated In the MTLB6S12 clone contained 356 bp of 5'-C 1 _ 3 A-3' simple repeat sequence. The length of this T region on the end of chromosome IIIL in the AB20«, XP8-10B genome was estimated by Southern hybridization with restriction enzyme digests of chromsomes to be 0.6 kb (V-0 .2 kb). Therefore between 50 bp to 400 bp of repeat sequence was deleted from the IIIL end with Bal31 nuclease during cloning. If the length of the T region at yeast telomeres Is in a constant state of flux with addition and elimination reactions during DNA replication, the deletion of 50 to 400 bp from the T region on IIIL leaving 356 bp of T region, should not have a detrimental effect on telomere function. This shown to be the case since the telomerlc region from IIIL in MTLB6S12 provided a functional telomere on linear plasmids In yeast. This was also Indirect proof that the deleted 50-400 bp region from the IIIL end consisted of additional T region sequence (5'-Ci_3A-3'), which is predicted to extend to the terminus of yeast chromsomes (40,49,65, 175,206). B. Chromosome IIIL End Is a T-X Class Telomere Yeast chromosome ends are distinguished by the complex repeat regions referred to as X and Y* that are proximal to the T region which consists of simple 5'-Cj_3A-3' repeat units at the terminus of the chromosome. The telomere associated X and Y' regions represent a family of ARS regions that are thought to be origins of replication in yeast (47, 48, 49). The majority of yeast telomeres have a type X and between one to four type Y' AB£ elements and are referred to as the T-Y'-X class. The second class has a single type X ARS region and is referred to as T-X telomeres. In T-Y'-X class, the T region separates the X and Y' regions as well as terminates the chromosome (49, 93, 205, 206). Yeast telomeres that were previously Isolated represent the T-Y'-X class (49, 193). The T-X class has been postulated to exist because of the results obtained from Southern hybridization with restriction enzyme digestions of yeast chromosomes (93, 175, 205, 206), but no example has been Isolated until this report. 114 The proximity of the IIIL terminus with the unique region on the end of chromosome IIIL (probe 2*) was the first indication that the IIIL telomere represented the T-X class. Telomeric regions of the T-Y'-X class are apparently longer than 7.5kb(49) whereas the IIIL telomeric region was confined a region that was less than 2.3 kb. Further characterization by Southern hybridization with previously Isolated X and Y' region clones (48,49) showed that the cloned telomeric region from chromosome IIIL contained homology with the X ARS region, but not the Y' ARS region. The X region on IIIL was localized to the 1.2 kb region adjacent to the telomeric S ' - C ^ - S ' repeats, with an ARS consensus sequence In the center of the X region. Although the type X AJiS regions are heterogeneous in restriction enzyme sites and in length, they are defined as X regions by their homology with one another (48,49). It is assumed that all yeast telomeres contain an X region (49, 205), hence the X region from the end of chromsome IIIL should hybridize to each of the 34 chromosome ends in the haploid yeast genome. In Southern analysis of chromosomal DNA, numerous fragments were homologous with the X region probe and no additional fragments were detected with the T-X region probe from chromosome IIIL. Thus, all telomeres that contain a T region must also contain an X region. It was not possible to distinguish X region fragments corresponding to each chromosome end in the haploid yeast genome. Perhaps the telomeric fragments from some of the chromosomes had identical lengths or at least sizes that were too close to be separated by the conditions used for fractionation by electrophoresis. Ba131 nuclease sensitivity assays confirm that all fragments homologous to the X region on chromosome IIIL 8re telomere proximal, and at least 30 * of the chromosome ends in the yeast genome are of the T-X class (49,205, 206). C. Heterogeneity of the Length of the IIIL Telomere Among Yeast Strains Restriction enzyme site and length heterogeneity of the telomere associated Y' region and length heterogeneity of the terminal T region has been observed in Southern hybridization analysis of the telomeres in various yeast strains (40,92,93,206). The length of the terminal fragments from T-X class telomeres was heterogeneous among yeast strains, as observed by Southern hybridization with the IIIL telomeric probe. The differences observed In the fragment lengths for the T-X telomeres were attributed to variation in the length of the T region and not in the type X ARS. region. For the T-X 115 telomeres that were identified using the prooe from the MIL end, heterogeneity among strains was obvious In the length of the terminal restriction fragment. In any given yeast strain, the length heterodlsperslty of the terminal fragment on a T-X telomere was limited in size range ( V - 0.2 kb), but the average length of this terminal fragment also varied by ( + / -0.2 kb) among the strains examined. Recently, the length of the terminal T region was shown to be genetically controlled and it appears that regulation is by more than one genetic locus (40,206). Possibly the variation observed for the average length of the terminal fragment on a given chromosome end in different yeast strains is a consequence of the genetic cloning of a particular T region length for a given chromosome end in one strain. This T region length then determines the average length for the telomere, with addition and elimination reactions at this end resulting In variation for the T region length within the particular strain. Genetic regulation of the addition and elimination reactions would explain the conservation of the average length, and the limited variability ( V - 0.2 kb) of the average length. Among different yeast strains, there are some examples of restriction-fragment-length-polymorphisms on chromosomes that are homologous with the X region probe from the IIIL end. However, these variations occurred with fragments that mapped to the T-Y'-X class of telomeres. Alternatively, the fragments that mapped to T-X telomeres had variation in the length of the T region and conservation of the length of the X region. This may reflect the requirement for a conserved X region at T-X telomeres, since it provides the ARS function, while the X region at T-Y'-X telomeres may vary since the A M function may be from the Y' region. Furthermore, Southern hybridization with the T-X probe from the 11 IL telomere indicated that the ratio of T-Y'-X to T-X class telomeres is maintained In the genomes of the different yeast strains. Perhaps this ratio Is essential for proper telomeric associations and chromosome segregation. The reported changes In the number of Y' regions in different strains and in the meiotic segregants of a given strain (93) possibly reflect variations at the T-Y'-X class of telomeres rather than conversion events between T-X and T-Y'-X telomere classes. Recently, Surosky and Tye constructed a telocentric chromosome III In a diploid yeast strain by deleting 80-100 kb from the left end of chromosome III (190). The deletion on chromosome IIIL was thought to result from a double reciprocal recombination event between chromosome III and a 116 short fragment, containing both a portion of the Y* ARS region and a LEU2 or CEN3 region, that was Introduced Into the yeast strain 3285 by transformation. It was assumed that deletion of the region between LEU2 or CEN3 and end of IIIL occurred by replacement recombination with the short fragment. However, there is no Y' region on the end of chromosome IIIL If yeast strain 3285 is like those examined by Southern hybridization analyses with the telomere proximal fragment (probe 2*) that is unique to IIIL . Instead, the alternate explanation for the production of the telocentric chromosome III was more probable (190); deletions were generated by a single recombination event between LEU2 or CEN5 on the Introduced fragment and LEU2 or CEN3 on chromosome III. This event would result In the replacement of the region distal to LEU2 or CEN3 on IIIL with the partial Y' region on the Introduced fragment. Presumably, this partial Y" region on IIIL was functionally healed with a Y' region from a T-Y'-X chromosome end. This explanation also agrees with the proposed function of Y' regions in providing stability to broken chromosome ends (65). The alteration of the telomere associated region from X to Y' on IIIL In the telocentric chromosome III strain does not significantly affect the mitotic stability of chromosome 111 but It does result In slight defects In melotlc pairing and segregation (190). This suggests that the telomere associated regions, X and Y', are at least partly responsible for telomeric associations and the proper segregation of chromosomes In meiosis (49). D. Requirements for a Functional Telomere In Yeast The termini of Tetrahymena rDNA function as telomeres in yeast as shown by cloning the rDNA ends on linear plasmlds, then selecting for the plasmid marker in yeast (193). A yeast telomere was Isolated by the replacement of one Tetrahvmena end on the linear plasmid with a yeast fragment that displays telomeric function (193). Characterization of replication intermediates for yeast chromosomes (195), studies on yeast chromosome mechanics and stability (56, 88, 139, 140), as well as the healing events that occur at chromosome ends in yeast (65, 215) were conducted in yeast using linear plasmlds which had termini from Tetrahvmena. Oxvtricha or S_. cerevisiae. Similarly, to establish that the cloned chromosome IIIL telomere contained the essential Information for telomere function, the IIIL end fragment was cloned on linear plasmlds in yeast. 117 Plasmids constructed with the 1.2 kD type x ARS region from IIIL along with 4 9 bp or 356 bp of T region (5'-C^_3A-3' repeats) at the termini of the plasmid, were replicated exclusively as linear molecules in yeast. The ligation mixture of circular and linear plasmids was Introduced into yeast by genetic transformation, and the plasmids were resolved Into linear molecules In the yeast, whether the vector portion of the plasmid was the entire pSZ218 plasmid (194) or a LEU2 fragment from pSZ218 (2, 65). The mitotic stability of linear plasmids with IIIL termini was about 75* plasmid bearing cells while the stability for linear plasmids which contained the ARS1 region, which is a strong origin of replication in yeast (184), and termini from Tetrahvmena rDNA (194, 195) was only 40* plasmid bearing cells. The doubling of the mitotic stability for linear plasmids with yeast telomeres rather than Tetrahvmena ends may reflect the cellular recognition of yeast ends, the preferred association with yeast telomeres for the linear plasmids with yeast chromosome ends, or to the efficient ARS activity in the X region of the yeast IIIL ends. In addition, the ARS regions at yeast ends ( X or Y regions) may stabilize these mlnlchromosomes due to their interaction with a cellular factor (124). Since the isolated T-X region from chromosome IIIL allowed the resolution and replication of linear plasmids In yeast that had high mitotic stabilities for the LEU plasmid marker, the terminal region on the IIIL end that was deleted with Bal31 nuclease for cloning in a circular vector, was not essential for a functional telomere. Presumably this 50 bp to 400 bp terminal region on IIIL consisted of additional T region sequences (5-0} -3A-3' repeats) that extended the 356 bp of T region that was present on the isolated IIIL end. At least a portion of the T region is required for the recogition of telomeric fragments in circular plasmids and the resolution of circular plasmids to linear molecules In yeast. Deletion of the entire 5'-Cj_3A-3' repeat region from the T-X fragment, leaving the 1.2 kb X region from IIIL on the telomeric fragments, resulted in the replication of circular plasmids in yeast for most transformants. However, one yeast transformant did contain linear plasmid molecules for the plasmid that was constructed with X region termini. Presumably, the stabilization of this plasmid was the result of a recombination event in yeast between X regions on the plasmid and on a chromosome that resulted in T region addition at the termini of the plasmid. Deletion of the T region plus the adjacent 200 bp from 118 the X region on the IIIL end resulted in a fragment that was not recognized as telomeric and hence only circular plasmlds containing this deletion were replicated in yeast. The distal portion of the X region (ie. X-T border region) appeared largely conserved among X regions as determined by Southern hybridization analysis with deletion probes from the X region or by comparison of the restriction maps for different X regions (48, 49). Perhaps the elimination of the distal portion of the X region removed a region that was essential for homologous recombination with chromosomal X regions and prevented the subsequent addition of the T region required to stabilize linear plasmlds. The transformation frequency of circular plasmlds that were constructed with X regions that had the distal 0.2 kb portion deleted was equivalent to that observed for linear plasmlds with the full length X region from the IIIL end. Consequently, It was estimated that the distal domain of the ARS region on the IIIL end (42,182) was In the distal 0.2 - 0.4 kb of the X region. Deletion of more than 400 bp from the distal end of the X region produced few or no transformants, presumably since the deletion interferred with the ARS function. Plasmlds constructed with the T region on just one end of the plasmid and the IIIL ARS. region on either one or both ends were replicated and stably maintained as linear molecules In yeast, for some transformants. These linear plasmlds were constructed with the T region at only one end of the plasmid, however T region addition apparently occurred at both ends of the linear molecule during replication. The plasmlds constructed with an X region at only one end of the plasmid did not acquire additional X or Y' regions during replication In yeast. On these short linear plasmlds that are replicated in high copy number, there Is no requirement for proper segregation like there is for natural chromosomes in the yeast cell. Therefore, a telomere associated region may not be needed at both ends of the linear plasmlds, whereas all evidence to date indicates that all chromosome ends In yeast have telomere associated regions. The T region was present at the ends of all linear plasmlds, which reflects Its essential role in replication and In stabilization of the ends of linear molecules. E. The IIIL End Is Stabilized with T Region but without Y' Region Addition Linear plasmlds that had IIIL telomeric fragments at the termini had the T regions lengthened in yeast by a yet uncharacterlzed addition reaction (40, 49, 65, 175, 206) such that the average length of the T region on the linear plasmlds was equivalent to that on the natural IIIL end. This may 119 reflect the genetic control of the length of terminal fragments on chromosome ends which agrees with previous reports showing the genetic variation of T region lengths between parental strains and their meiotic segregants or among various yeast strains (40, 206). A linear plasmid that was constructed with a defined length of the IIIL telomeric fragment had a heterodisperse length for the terminal fragment following its propagation in yeast which provides further evidence for telomere addition and elimination events at yeast telomeres (18,175). Telomeres are stabilized or "healed" through the addition of Y' ARS regions and T regions at the ends of linear plasmids in yeast if the plasmid contains only weak A£S_ regions, such as those on Tetrahvmena termini ( 1, 65, 105, 195, 215) or if the plasmid has only a partial Y' region (65, 190). There was no evidence for the addition of Y' regions on the ends of the linear plasmids constructed with IIIL fragments, using either complete or partial X ARS. regions as telomeres. If interaction or recombination did occur between the X region from the IIIL telomere that was on the linear plasmids and the telomeres on yeast chromosomes, ft must have been with the T-X class and not the T-Y'-X class of yeast telomeres. The A M region on the end of chromosome IIIL must be a highly active replication origin on linear plasmids, and consequently Y' ARS regions were not added to the 11 IL termini since this would not enhance the stability of the linear plasmids (65). The IIIL end fragments which had deletions extending into the X A M region resulted in few viable transformants and these were not healed with Y' or X A M regions. This lack of recombination events at the partial IIIL ends is reasonable since recombination with genomic DNA In yeast is enhanced only when broken ends, highly homologous with chromosomal DNA, are Introduced into yeast on a linear fragment (149, 150, 196). However, the plasmids with X region deletions did not conform to this arrangement since: (1) The majority of the plasmid DNA was circular In the ligation mixes used for the genetic transformations, and these remained as circular plasmids in yeast since the absence of a T region precluded the resolution of circular to linear molecules; (2) Sequence heterogeneity exists among X regions, especially on the centromere proximal side of the A M region (48, 49); (3) X and Y' regions share relatively no homologous regions for recombination events to occur between them (48, 49). 120 F. Why are There Two Classes of Telomeres Maintained in Yeast? If an X ARS. region provides the requirement for a telomere associated region without the Y' region, then for the simplest arrangement of chromosome ends in yeast, one would expect all telomeres to belong to the T-X class. The existence of two classes of telomeres in yeast may be due to the requirement for a strong origin of replication at the telomere to initiate replication at the end of the chromosome (49). The type of telomeric arrangement would therefore depend on the ARS activity in the telomere associated region. If the ARS In the X region provides the necessary function, then no additional Y* region sequences are required. However, should the ARS in the X region suffer mutations, deletions, rearrangements, or other damaging events, the Y' ARS regions would provide the ARS activity at the telomere , as observed by their role in healing telomeric regions (65). Addition of Y" regions to inactivated T-X class telomeres would possibly occur by recombination between the distal T region on the T-X telomere and the Internal T region (between Y" and X regions) on the T-Y'-X class telomere (65, 205). The telomere healing experiments conducted by Dunn et al. (65) support these ideas. In these studies, chromosome ends were healed by recombination with Y' regions if a partial Y' region (no X region) was at the end of a linear plasmid or If the Tetrahymena rDNA ends (weak ARS regions) were used as termini. Addition of Y' regions to Tetrahymena rDNA termini occurred by recombination with the T region that separates the X-Y' regions on T-X-Y' telomeres, similar to the mechanism proposed for the healing of T-X telomeres with Y' regions. Alternatively, a strong origin of replication (ARS1) at the plasmid end precluded Y" region addition. In the same manner, the T-X telomeres may have sufficiently strong A M activity in the X region such that further Y' region activity Is not essential. The ability of yeast A M regions to mediate high frequency transformation In yeast is affected by domains surrounding the A M consensus sequence (42, 104, 182). Different sequences surround the various yeast ARS regions that have been Isolated (34,42,104, 184, 202), hence these domains cannot be defined In a general manner with respect to DNA sequence requirements. Because point mutations within the A M consensus sequence destroy A M activity (42, 104, 182), this sequence must be conserved In Y' or X regions for a functional origin of replication. The X regions isolated 121 previously (48, 49) belong to the T-Y'-X telomere class since all clones contain the 131 region which forms part of the Y' region (49). Conversely, the X region on the MIL end, the only T-X telomere Isolated to date, had no homology with the 131 region. Comparison of the restriction enzyme maps for the X regions Indicates they are heterogeneous in length, restriction map, and extent of the ARS region. Perhaps the ARS activity in these X regions also displays such variability. Variation in the stability of the A£S for X regions from T-Y'-X telomeres was demonstrated In mutant strains defective in minichromosome maintenance, whereas the stability of the Y' A£S region was conserved for different Y' regions on the mlnichromosomes In these mutant strains (124). Different yeast strains were apparently polymorphic at X regions belonging to T-Y'-X telomeres and conserved at T-X telomere associated regions. This may reflect the requirement for ARS function In the X region of T-X telomeres. The Y' region may provide AJRS_ activity at T-Y'-X telomeres and hence there is less selection pressure for X region maintenance at such telomeres. The repetition of Y' regions on some telomeres (maximum of 4), (49) may be the result of gene conversion or other recombination events between the homologous Y' regions of different telomeres. The complex direct repeat sequences that are conserved in the Y' region (92) are thought to be Involved in the interaction of different Y' regions. Perhaps the variable number of Y' regions on yeast telomeres has a regulatory role in replication, such as control of replication initiation points in the cell cycle (49, 65, 70), or In the telomerlc associations Important in meiotic pairing and segregation events (25,35,49, 55, 91). Alternatively, the multiple nature of Y' regions may be a consequence of rather than a cause for telomeric associations. Possibly conversion events between Y' regions resulting In the addition of Y' regions at the ends of chromosomes by a RAD52+ dependent mechanism, (65) maintains the DMA sequence of the Y' ARS region which ensures that a functional ARS region is present at the end of the chromosome. The finding that Y' regions vary substantially in number among yeast strains for T-Y'-X telomeres (93) suggests that there Is no control mechanism for the number of Y' regions at a given telomere, and the only requirement may be the presence of at least one functional ARS region. Further support for the idea that a functional origin of replication is essential at the telomeric region is obtained from the studies conducted on heterologous telomeres 122 which also stabilize linear plasmlds in yeast. These heterologous telomeres may also contain ARS regions that are functional in yeast, as described for Tetrahvmena rDNA termini (1, 25, 65, 105, 156, 193). To establish that a region with strong ARS activity is required at chromosome ends, linear plasmids could be constructed with T-X termini, using the X region derived from a T-X telomere for one plasmid and the X region from a T-Y'-X telomere for a second plasmid construction. After replication of these plasmids in yeast, transformants would be screened by Southern hybridization to discern whether Y' regions are used to repair the T-X telomeres that were constructed with X regions from T-Y'-X telomeres. The linear plasmids constructed with T-X termini from T-X telomeres should not have Y' addition at the ends, If these are like the linear plasmids constructed with the T-X telomere from chromosome IIIL. Differences in the mitotic stability of linear plasmids with different X ARS regions could be detected using the colony color sector assay test (88) which can distinguish chromosome nondisjunction and chromosome loss events. Similarly, the effect that additional Y' regions on the ends of the linear plasmlds has on plasmid stabilities could be assayed in this manner. Cloning an X region from either a T-X or T-Y'-X telomere on a linear plasmid in yeast, followed by comparison of the replication patterns, mitotic stabilities, and telomere modifications of the T-X termini would demonstrate any differences in the ARS activities for X regions of the two telomere classes. These results would Indicate whether conversion between The T-X and T-Y'-X telomere classes can occur on specific chromosome ends in yeast. G. How are Yeast Telomeres Replicated? Simple repeat sequences exist at all eukaryotic chromosome ends that have been sequenced (20, 24, 67, 98, 102, 113, 147, 175, 203, 212) and these 8dhere to the general formula 5'-[Ci_e(A/T )i_4]-3\ (23). Although the sequence of the repeat units is divergent In heterologous systems, the function of this T region is maintained. Both Tetrahymena ( 5 - ^ 2 - 3 ' ) repeat units, (56, 65, 139, 140, 193, 194, 195) and Oxvtrlcha (5'-C4A 4-3') repeat units, (156) are functional as telomeres on linear plasmid vectors in yeast, although yeast chromosomes have 5'-Ci_3A-3' repeat units at the T region of the telomeres (175). In forming the macronuclear genome from mlcronuclear fragments in dilates such as Tetrahymena or Oxvtrlcha. the repeat 123 sequence must be added to the ends of the linear fragments (22), but the addition mechanism Is not yet defined. Similarly, addition of T region sequences occurs on chromosome ends in yeast. The yeast 5 ' -C 1 _ 3 A-3' repeat units are added to both Tetrahymena and Oxytricha termini (156,175) and to the deleted IIIL telomerlc fragments on linear plasmids In yeast. Further evidence for terminal addition and elimination events at telomeres In yeast is the heterogeneous length of the terminal fragment from chromosome IIIL observed on the chromsome or on linear plasmids in yeast. Presumably, simple repeat sequences are routinely added to or deleted from telomeres In replication (18, 65, 175, 203). Heterogeneity of the length of the terminal restriction fragment on yeast chromosomes suggests that a solution for the problem of completing chromosome ends in DNA replication (208) must only provide a mechanism for maintaining an average length for the telomere rather than the specific length of the parental DNA strand. Consequently, the gap resulting from removal of the 5' terminal primer in DNA replication may remain Incomplete and produce a shorter telomeric repeat length on the progeny chromosome. Simple telomerlc repeat sequences presumably conserve the average length through an undefined addition reaction. Models proposed for the addition of repeat region sequences at chromosome ends In DNA replication Involve either a recombination mechanism (17, 77, 87, 91, 203, 205) or a novel terminal transferase-llke enzyme activity (80a, 175, 195). However, neither alternative fully explains the addition events at chromosome ends since the model must accomodate the following: (1) heterodlspersity of terminal fragment length, (2) addition of T region repeats but malntalnance of the telomerlc length within a given range, (3) terminal single-stranded breaks, (4) inability to label or ligate the terminus, (5) addition of the T region to heterologous telomeric repeats, (6) a RAD52 Independent mechanism is responsible for addition of the 5 - 0 ^ - 3 ' repeat sequences to the linear plasmid ends (40,65, 215). If the average length of the T region at the chromosomal end Is maintained via recombination between the simple repeat units, then recombination must occur by Intrachromosomal or interchromosomal events that are BAJ252. Independent (65, 215). Both an Intrachromosomal loop-back model (203) and a model that Involves recombination between the T region on all chromosomes (205) have been proposed. In addition, unequal sister chromatid exchange which occurs by a RAD52 124 independent mechanism (157, 192, 196, 203, 216) may contribute to the observed length heterogeneity of telomeres. Deletion and elongation events that occur at the terminal simple repeat units are equivalent to the sister chromatid exchange events that occur within the repeat units of yeast ribosomal DNA (192). The length variation of the terminal fragment on the yeast chromosomes is within a given range ( V - 0.2 kb) which could indicate that chromatid pairing occurs in an ordered manner, and the exchange processes are regulated to maintain an average terminal length. Cross-over events In the loop-back model (203), interchromosomal recombination model (205), or unequal sister chromatid exchange model may be initiated at the single-stranded gaps near the terminus of the chromosome. Possibly, the regulation of a telomere specific endonuclease may simultaneously regulate the extent of cross-over events between chromatids. Activity of the CDC 17 gene product may fulfill this role since it prevents the telomeric region from continually elongating (40). Reduced amounts of CDC 17 gene product results in the length of the average telomere being increased every generation. Perhaps fewer single-stranded gaps are present at the termini with reduced amounts of CDC 17 protein, and recombination at more internal sites would produce longer extensions on some chromosomes (40). If unequal sister chromatid exchange Is responsible for telomere elongation, then longer telomeric regions may have a selective advantage for chromosomal stability and hence longer average lengths for the telomeric repeats are maintained in the CDC 17 mutants (40). According to this mechanism, the X and Y' regions at yeast telomeres play an Integral role in the replication of chromosomes by mediating the telomeric associations that are required for the interactions and recombination between T regions. Recombination events occur among telomeric regions during the healing of Incomplete telomeres In yeast, resulting In the addition of Y' regions to chromosome ends by a RAD52 dependent mechanism (65). In a similar manner, telomeric associations may result in recombination at the T regions to maintain the average length of the T region In a given yeast strain (206). This model for completion of chromosome ends by recomblnatlonal mechanisms can accomodate protected chromosomal termini, either by DNA structure or protein blockage. Addition of the yeast T region to heterologous telomeres may result from interaction between the terminal repeat regions on the yeast chromosomes and the linear plasmids with Tetrahymena or 125 oxvtricha ends, resulting from a similar secondary structure of the terminal repeat units in the cell followed by recombination at the single-stranded nicks in the repeat sequences. Completion of chromosome ends by recombinatlonal mechanisms does not require any novel molecular mechanisms unlike those that Invoke a terminal transferase-like activity for the addition of terminal repeats (80a, 175). The terminal transferase mechanism is based on the addition reactions that have been observed for most telomeres (Reviews 22,25) and the addition of yeast repeat units to heterologous telomeres In yeast (156,175). The transferase Is thought to add specific repeat units to the incomplete termini in DNA replication, and these eventually loop back to prime the synthesis of the opposite strand (80a, 175). While terminal transferase enzymes have been characterized (31, 159), none of these add specific repeat units with the possible exception of the tRNA nucleotidyltransferase that adds a single CCA unit to the 3' end of tRNA (62). However, support for this model was recently described; a terminal transferase activity In Tetrahymena whole cell extracts has been Identified that adds specific 5 ' -T 2 G 4 -3 ' repeat units to telomerlc DNA sequencesJn vitro (80a). Perhaps a terminal transferase-like enzyme in yeast synthesizes 5 ,-[C2_3A(CA)1_4 ]-3' repeat units at chromosome ends and regulates the extent of terminal addition as observed for the IIIL termini on linear plasmids fn yeast. One must also consider that both mechanisms may be Involved in replicating the yeast telomeres. A terminal transferase-like enzyme could result in T region addition at the chromosome ends, and the recombination of the T regions on the chromosome ends with the linear plasmid ends could explain the equivalent T region sequence and length on the natural chromosomes and on linear plasmids in yeast. H. IIIL Distal versus IIIL Alternate Region In the Yeast Genome The 8.6 kb region distal to HMLtt, on chromosome IIIL shared homology with an alternate and yet unidentified region in the haploid yeast genome referred to as the IIIL alternate region. Dual homology of a HML& distal fragment in the linear III yeast strain K45 and the deletion of the IIIL distal fragments In the ring III yeast strains K191 and K192, was detected by Southern hybridization (109). Comparative Southern hybridization studies using probes from the HMLtt distal region with linear III or circular III yeast strains distinguished IIIL distal and IIIL alternate region fragments that 126 were maintained in the circular III strains. However, only ring III strain K192 had the IIIL distal region completely deleted, the expected consequence of the HMLtx. - HMRa fusion event that produced ring III strains (109). Other ring III strains studied (K191, K193, K195, and K196) had retained most of the IIIL distal region fragments and these replaced the IIIL alternate region fragments presumably by a recombination or gene conversion-like event, perhaps analogous to mating- type switching in yeast (110,143). As Illustrated In Figure 29, the YZ endonuclease produces a double-stranded cleavage at HMlcs, in the mai l parental strains (106, 108, 109, 111, 115, 116, 188) which results in two broken ends that are recombinogenic (82, 83,123,149,150, 196). Homology between HMLft and HMRa resulted 1n recombination and fusion to form the ring 111 chromosome, while the HMLKf distal region invaded the IIIL alternate region and replaced It. This scenario was considered by Klar et al. (109), although they proposed that gene conversion occurred at HMLpc with the HMRa region by recombination of flanking markers to produce a ring chromosome III plus an acentric fragment containing the chromosome ends which was subsequently lost in mitosis. However, my Southern hybridization results indicate gene conversion-like events without exchange of flanking markers as seen for other Intrachromosomal gene conversion events such as M A I switching (109, 110, 112), or like the healing events observed for broken ends on yeast chromosomes (82, 83, 123). The IIIL distal region was retained in most ring III strains, indicating it was not lost on a fragment formed from the telomeric ends from the H M L J X and HMRa distal regions. Instead the IIIL distal region was attached to another chromosome, but It was exonucleolytlcally shortened at the broken end prior to this because about 2.5 kb of the region adjacent to HMLft was absent from all ring III strains examined. The remaining IIIL distal region, Including the telomere, was introduced to and replaced a homologous region on a different chromosome In most ring 111 strains (K191, K193, K195, K196) presumably by an interchromosomal gene conversion event (97,110, 172). Ring III strain K192 was the exception where the entire IIIL distal region was deleted during the fusion event. Perhaps the mechanism proposed by Klar et al. (109) was operative in this case; the broken IIIL and 127 Figure 29. Model for the Retention of the IIIL End In a Ring III Yeast Strain. a. Mating type interconversion at HMRa in K45 occurs due to the marl mutation (108), and is initiated by the YZ endonuclease (115,116). b. Regions flanking the YZ cut site are subjected to exonuclease digestion at the unstable ends as indicated by the arrows. Exposure of the HJjBa cassette X region results in homologous recombination at HMLtx,, with strand invasion of the broken HMRa end. c. Recombination results in a circular chromosome III and the terminal acentric IIIL fragment. The unstable IIIL broken is exonuclease digested In K191. d. The fragment with the recombinogenlc broken end from the HMLot, distal region bears strong homology with an alternate region, which Is also near a telomere, elsewhere in the genome. This results in the IIIL distal fragment replacing the alternate region by a gene conversion-like or a "telomere conversion" event. 127 HML 4 [] mism-III H V 2 IIIL K45 MAT [1 • [| B - H H-UIKVZ HMR H VZ - f t I l l ' " t IIIR VZ endonuclease b. c. MAT K191 HML •wzm 7 HML-HMR MAT X -as •-HMR ••fi-IIIR Ewonuclease 11 IL Distal K -tr-c IIIL Rlternate 128 MIR ends fused in this strain to form an acentric fragment with stable telomerlc ends, that was subsequently lost in mitosis. This fusion event for the broken end from IIIL has precedence in the breakage fusion bridge cycle described by McCHntock for broken chromosome ends In maize (120, 121, 122). The HMRa distal region shares homolgy with regions elsewhere in the yeast genome (109). It was not determined If portions of the MR distal region were retained in ring III strains, due to the lack of appropriate probes. The IIIL alternate region remains unmapped In the haploid yeast genome. Bal31 nuclease studies indicated that it is telomere proximal, like the IIIL distal region. The rate of Bal31 nuclease digestion Indicated that the fragment from the IIIL distal region hybridized to a region that was closer to the terminus of the chromosome terminus at the IIIL alternate region compared with the IIIL distal region. This indicates that length of the telomere at the IIIL alternate region Is less than 2.3 kb, and that It belongs to the T-X class of yeast telomeres similar to the IIIL telomere. The chromosomal origin of the IIIL alternate region could conceivably be determined through Southern hybridization analysis of the chromosomes from linear III and ring III strains using IIIL distal probes and OFAGE gels such as those used in yeast electrophoretlc karyotyping (36, 37,173). Since ring III strains are viable as haplolds, Klar et al. (109) proposed that there are no essential genes In the regions distal to HMLot, or HMRa. However, the possibility was also considered that there are essential but functionally duplicate loci since IIIL and I MR distal probes are homologous to other regions 1n the yeast genome. An essential gene in the HML& distal region that was present but was less transcriptionally active at the IIIL alternate region would provide an explanation for the retention of the IIIL distal region In most ring III yeast strains. It would also explain the sequence conservation between the IIIL distal and IIIL alternate regions within a given yeast strain and among the different strains that were used for Southern hybridization studies in this report (AB20& XP8-10B,AB972,andK45). The idea that the telomere proximal MIL distal or IIIL alternate regions may be transcribed Is supported by the finding that some gene families, such as yeast invertase (SUC) genes, and maltose fermentation (MAD genes, map to the ends of different chromosomes (38, 39, 133,136). In all yeast strains examined In this study, RNA hybridization analyses Indicated that a 129 fragment from the IIIL distal region was homologous with a RNA transcript. Since the RNA transcript was observed in ring III strain K192 which has the entire IIIL distal region deleted, It appears that the IIIL alternate region was transcribed. Preliminary searches for transcription units in the IIIL region with RNA Northern analysis Indicated that there Is transcript with an approximate length of 1.4 kb that Is.homologous with the region at least 4 kb distal to HMLtt,. Further transcript mapping studies have yet to be conducted to accurately determine the transcript length, to determine whether both the IIIL distal and IIIL alternate regions are transcribed, and to define the region of trancrlption. Since the entire IIIL distal region has been cloned, the proposed gene could easily be sequenced and compared to other yeast characterized genes as a first step in establishing the function of the gene product. I. Future Prospects Our understanding of the structure of yeast telomeric regions has increased considerably over the past five years. Most progress has been made in characterizing the elements at yeast telomeres that may function In chromosome replication and stability. Isolation and characterization of the telomeric region from chromosome IIIL region provided answers for questions concerning the structure of a specific chromosome end that could not be determined from the characterization of the general structure for a yeast telomere. The mechanism whereby telomeres are replicated remains to be determined. Is there a terminal transferase-like enzyme, or is a recombination mechanism responsible for completion of the termini of chromosomes in DNA replication? Is there a particular DNA secondary structure or chromatin conformation at chromosome ends resulting from the simple repeat sequences? Will yeast telomeres function In heterologous systems or higher eukaryotic systems? Why are there two classes of yeast telomeres? What are the regulatory factors involved in maintaining the average length of a telomere? The Isolation of defined chromosome ends, such as the IIIL region, may be useful In answering some of these questions. The Isolation and characterization of the IIIL telomere may be useful in the construction of artificial chromosomes in yeast. Chromosomes constructed previously had the ends of Tetrahvmena rDNA as the telomeric regions (26, 56, 88, 139, 140, 141). In all constructions, these minichromosomes were less stable than natural yeast chromosomes, although the mitotic stability does 130 Increase with longer artificial chromosomes. If interactions between telomere associated regions and strong ABS_activity are required at chromosome ends, then an authentic yeast telomere may improve the mitotic stability of artificial yeast chromosomes. The acentric linear plasmids that were constructed with the telomeric region from chromosome IIIL are more mitotically stable than similar plasmids containing Tetrahvmena ends and the ARS1 region. Perhaps homology with yeast chromosomes Is required at the ends of the linear plasmlds to stabilize them. Interaction and recombination between artificial chromosome ends and yeast chromosome ends does occur when yeast sequences are present on the ends of the linear plasmlds, as shown by the addition of Y' regions to linear plasmids in yeast (65). Artificial yeast chromosomes that behave like authentic yeast chromosomes in their stabilities could be invaluable for a variety of investigations (194): (1) Introduction of the artificial chromosomes Into other organisms to study the function of yeast elements in higher eukaryotic systems and the functional similarity of eukaryotic chromosomal components. (2) Artificial minlchromosomes In yeast may provide a simple system to study chromosome mechanics and recombination. (3) Cloning of long regions on artificial chromosomes may be possible since length Is not a confining factor for linear plasmlds while it is for circular plasmlds. Perhaps regions that are unstable in circular plasmlds due to DNA secondary structure may be cloned In linear minlchromosome vectors. Cloning of an extensive chromosomal region would allow the study of copy number effect, polarity effects, and regulatory domains around a given gene. (4) Determination of the potential to form Z-DNAwith the 5'-Ci_3A-3' repeats in the T region may be feasible with the IIIL telomeric clones. The remarkable stability afforded to yeast chromosome ends by the S'-Cj-sA-S' may be due to the secondary structure it adopts (23). Left-handed Z-DNA has an Inherent tendency to self-aggregate to form networks referred to as Z*-DNA (4) and Z* DNA may form the basis for a stable telomeric structure. Moreover, Z-DNA has a high potential for recombination (145, 162) which may explain the associative nature of yeast telomeres, and the 131 lengthening of T regions with 5'-Ci_3A-3" repeats to chromosome ends. The presence of Z-DNA at telomerlc regions, detected through binding of anti-Z-DNA antibodies, has been described for diptera Chironomus thummi and Drosophila melanoaaster (4) and for the Secale species (99). The alternating purine-pyrimidlne sequence poly d(C-A) * poly d(G-T) adopts Z-DNA conformation under physiological conditions (145,162). 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