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The cloning of polyhomeotic, a complex Drosophila locus required for segment determination and cuticular… Freeman, John Douglas 1987

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THE CLONING OF POLYHOMEOTIC, A COMPLEX DROSOPHILA LOCUS REQUIRED FOR SEGMENT DETERMINATION AND CUTICULAR DIFFERENTIATION By JOHN DOUGLAS FREEMAN B.Sc, The University of British Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA OCTOBER 1987 © Doug Freeman, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date Oc^.S , DE-6C3/81) ABSTRACT The polyhomeotic (ph) locus of Drosophila melanogaster has been characterized g e n e t i c a l l y . Early studies showed that gh i s a member of the Polycomb (Pc) group. These genes have s i m i l a r phenotypes and are required for normal segment determination. Recent analyses of amorphic gh mutations show that the gh locus i s complex, has a strong maternal e f f e c t and plays a role i n c u t i c u l a r development. To test the function of gh at the molecular l e v e l , the cloning of the gh locus was undertaken. One s t r a i n had been shown to contain a P element i n s e r t i o n near gh. A genomic l i b r a r y was prepared from this s t r a i n and a recombinant phage containing this P element i n s e r t i o n was i s o l a t e d by transposon tagging. The DNA flanking the i n s e r t i o n was used as a s t a r t i n g point for a chromosomal walk. A se r i e s of overlapping phage spanning 170 kilobases was i s o l a t e d . Southern blot analysis was used to determine the locations of important deficiency breakpoints within the region covered by the walk. A distance of approximately 35 kb was shown to separate the two deficiency breakpoints which include gh. This i n t e r v a l was found to contain rearrangements i n four of the seven gh a l l e l e s which were examined by Southern blot analysis. The i n t e r v a l also contains a repeated sequence. The r e l a t i o n s h i p between the genetic and molecular structure of gh i s discussed. - i i i -TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF FIGURES iv ACKNOWLEDGEMENT v ABBREVIATIONS vi GENERAL INTRODUCTION 1 CHAPTER 1 INTRODUCTION 8 MATERIALS AND METHODS 14 RESULTS 23 DISCUSSION 31 CHAPTER 2 INTRODUCTION 33 MATERIALS AND METHODS 38 RESULTS 42 DISCUSSION 62 CHAPTER 3 INTRODUCTION 65 MATERIALS AND METHODS 67 RESULTS 68 DISCUSSION 74 GENERAL DISCUSSION 77 REFERENCES 83 - i v -LIST OF FIGURES PAGE FIGURE 1. Partial digestion of ph.2 DNA. 25 FIGURE 2. Screening for phage containing P elements. 28 FIGURE 3. Localization of EMBL3-14P by in situ hybridization. 30 FIGURE 4. Deficiencies in the ph region. 35 FIGURE 5. Mapping of restriction enzyme sites 44 FIGURE 6. Screening with central and extreme fragments. 47 FIGURE 7. Comparison of positives from one step. 48 FIGURE 8. Determining the orientation of the walk. 50 FIGURE 9. Restriction map of the walk 52 FIGURE 10. Mapping deficiency breakpoints I. 55 FIGURE 11. Mapping deficiency breakpoints II. 57 FIGURE 12. Map of deficiency breakpoints and a repeated sequence. 60 FIGURE 13. Southern blot analysis of p_h mutants. 69 FIGURE 14. Map of ph rearrangements 73 - V -ACKNOWLEDGEMENT I would like to thank: My supervisor, Dr. H. Brock, for his enthusiasm and his support throughout this project. Dr. J. Berger, for helpful suggestions. Dr. D. Mager, Dr. N. Randsholt, Dr. R. Lansman and Margaret Smith for their invaluable assistance. Dan Nuttall and Lynn Freeman for their help in preparing the manuscript. ABBREVIATIONS Abbreviations used in this text are those accepted as standard by the Proceedings of the National Academy of Sciences (USA), Volume 84, pp. v i -v i i (1987). The components of important solutions are detailed on pages 14, 16 and 20. Media were as described in Maniatis e_t a l . (1982). Bacterial strains are described in Maniatis et a l . (1982) and Frischauf e_t a l . (1983). - 1 -GENERAL INTRODUCTION The fruit f l y , Drosophila melanogaster provides a useful model for the study of the genetics of development. Drosophila embryonic development is regulated by genes which f a l l into at least three categories. The f i r s t group of genes determine the dorsal-ventral, anterior-posterior and left-right axes of the embryo (Anderson and Nusslein-Volhard, 1984). Products of these genes are deposited in the egg before f e r t i l i z a t i o n and are therefore maternal-effect genes. The second group of genes is responsible for the division of the Drosophila embryo into segments. Segmentation occurs early in development and appears to be under the control of many zygotically-active genes (Nusslein-Volhard and Weischaus, 1980). Mutants in these genes show alterations in the normal segmentation pattern including a reduction in segment number or a change in polarity of individual segments. Mutational analysis has revealed a third group, the homeotic genes, which are required for determination of the segments. Mutants in these genes are characterized by a transformation of a l l or part of one segment into a homologous structure from another segment. Two major clusters of homeotic genes have been identified. The bithorax complex (BX-C) (Lewis 1978, reviewed by Lawrence and Morata 1983) contains genes which are required for the normal development of the thoracic and abdominal segments. The Antennapedia complex (ANT-C) (Kaufman et a l . 1980, Lewis et al- 1980, Struhl 1982) contains genes which are essential for development of the thorax and head. DNA from both complexes has been isolated (Bender e_t a l . 1983, Garber e_t a l . 1983, Kuroiwa et a l . 1984, Scott et a l . 1983) and the study of these regions continues at the molecular level. With results from genetic analysis, Lewis (1978) proposed a model to - 2 -explain the action of the bithorax complex (BX-C). According to the model, the developmental level of a segment is determined by the subset of BX-C genes expressed in that segment. The gene products of the complex would act by repressing or activating other genes which in turn determine the development of segmental structures. Lewis proposed that the mesothoracic segment represents a primitive level of development and that more posterior segments represent an elaboration on that primitive state via the action of the BX-C genes. His model predicts that each segment should have a unique set of activated BX-C genes and that the total number of BX-C genes expressed should increase from anterior to posterior, with a l l elements of the BX-C acting in the eighth abdominal segment. With the isolation of DNA from the BX-C, i t became possible to test some predictions of Lewis' model by molecular analysis. Although the exact distribution is not as predicted, the segment-specific expression of BX-C genes has been demonstrated using in situ hybridization to transcripts during embryogenesis. In general, the transcripts for a specific BX-C gene are concentrated in the segment most affected by mutations in that gene (Akam 1983, Akam and Martinez-Arias 1985, Harding et a l . 1985). Segment-specific localization of transcripts has also been demonstrated for elements of the Antennapedia complex (Hafen e_t a l . 1983, Levine e_t a l . 1983, McGinnis et a l . , 1984, Martinez-Arias 1986, Harding et a l . 1985). What regulatory processes restrict the expression of BX-C and ANT-C genes to certain regions? There appear to be at least three levels of regulation. First, the genes of the two complexes are controlled by a hierarchy of cross-regulatory interactions (Harding e_t a l . 1985, Hafen e_t a l . 1984 Struhl 1982, Morata and Kerridge 1982). The distribution of ANT-C transcripts in embryos lacking the BX-C is consistent with their being negatively regulated by elements of the BX-C. Removal of the BX-C results - 3 -in an accumulation of ANT-C transcripts in posterior regions. These transcripts are normally restricted to more anterior segments (Hafen e_t al . 1984, Harding et a l . 1985). This implies that one or more of the components of the BX-C negatively regulate the expression of ANT-C genes. Wedeen e_t a l . (1986) have also shown that one component of the BX-C, Ultrabithorax, may be negatively regulated by the other BX-C components abd-A and Abd-B. Insight into the possible mechanism of cross-regulatory interactions came with the discovery of regions of homology between the BX-C and ANT-P, termed 'homeo boxes' (McGinnis et a l . 1984, Scott and Weiner 1984). Harding et a l . (1985) have suggested that cross-regulatory interactions may be mediated by the homeo-domains of the ANT-C and BX-C genes. They propose a hierarchy of interactions between homeo box-encoded proteins and homeo box-containing transcription units. A second type of regulation is demonstrated by the segmentation genes fushi tarazu (ftz) and hunchback, (hb). Ftz is located within the ANT-C and was originally identified as a pair-rule segmentation mutant (Wakimoto and Kaufman, 1981). Hb is a member of the gap class of segmentation genes and is not associated with either of the two homeotic complexes. Like the homeotic members of the ANT-C, ftz contains a homeo box (Kuroiwa e_t a l . , 1984). The presence of a homeo box in a gene which had been implicated only in segmentation led to speculation that ftz might also be a homeotic gene. Recently, Ingham and Martinez-Arias (1986) and Duncan (1986) showed that ftz is required for the correct activation of BX-C and ANT-C genes. Examination of newly-discovered dominant alleles of ftz led Duncan (1986) to propose a model in which the ftz gene product functions f i r s t to direct segmentation and then to influence the expression of the genes of the BX-C. The dominant alleles which Duncan examined were found to have a homeotic phenotype; the f i r s t abdominal segment is transformed to resemble - 4 -the third abdominal segment. The same effect is seen with an increase in dosage of f t z + . In this model, f_tz+ f i r s t acts to create the proper segmental units (parasegments, according to the terminology of Martinez-Arias and Lawrence, 1985). Second, f t z + activates elements of the BX-C appropriate for each of the parasegments in which ftz is expressed. After analysis of ANT-C and BX-C transcript distribution in ft z ~ embryos, Ingham and Martinez-Arias also concluded that f t z + is required for the proper expression of the genes of the two complexes; absence of ftz results in an alteration in the early expression pattern of several BX-C and ANT-C transcripts. With the exception of hunchback. (White and Lehmann, 1986), which regulates the early distribution of Ubx, no other segmentation genes are known to alter early expression patterns of the BX-C and ANT-C. Third, i t has been shown that many genes outside the two complexes are important in the regulation of the BX-C and ANT-C. In his model for the expression of the BX-C, Lewis (1978) proposed that Polycomb (Pc) might be a negative regulator of the BX-C. However, Pc has been shown to be only one of a group of genes which may control the spatial expression of both BX-C and ANT-C genes (Jurgens 1985). Included in this group are Polycomblike (Pel) (Duncan 1982), extra sex combs (esc) (Struhl 1981), super sex combs (sxc) (Ingham 1984), Additional sex combs (Asx), Posterior sex combs (Psc), Sex comb on midleg (Scm) (Jurgens 1985) and polyhomeotic (ph) (Dura e_t a l . 1985). These Pc-group genes are typified by the appearance of sex combs on the second and third legs of mutant males. Sex combs are normally present only on the f i r s t legs of males. Extra sex combs are produced by certain gain of function alleles of the ANT-C gene Sex combs reduced (Scr). In addition to the sex comb phenotype, the members of the Pc-group show a variety of other homeotic transformations similar to those exhibited by dominant gain of function - 5 -mutations in the BX-C and ANT-C. Each member of the Pc-group has a unique spectrum of phenotypes, but general trends are evident. In heterozygous adults, segments are transformed to resemble more posterior segments. These transformations resemble those seen in f l i e s heterozygous for the dominant BX-C alleles Contrabithorax, (Cbx), Miscadestral pigmentation (Mcp) and intraabdominal (Uab) (Jurgens, 1985; Dura et a l . 1985). In embryos, recessive lethality is seen, together with an incomplete transformation of segments toward more posterior segments (Jurgens, 1985). The transformations exhibited by the Pc-group have been interpreted as resulting from the inappropriate expression of the BX-C and ANT-C, and not from the direct action of the Pc-group genes (Struhl, 1981; Duncan and Lewis, 1982; Duncan, 1982; Ingham, 1984; Dura et a l . , 1985). Jurgens (1985) suggested that the Pc-group genes may provide an integrated system for control of spatial expression of the homeotic complexes. Flies mutant for any pair of Pc-group genes show enhancement of homeotic transformations (Jurgens, 1985; Dura e_t a l . , 1985), suggesting that these genes work synergistically to regulate BX-C and ANT-C genes. Recent molecular evidence indicates that the Pc-group genes may not be responsible for the establishment of BX-C and ANT-C expression patterns (Struhl and Akam, 1985; Wedeen e_t a l . , 1986). Instead they may act to maintain the established pattern of expression. Struhl and Akam (1985) found that although the later distribution of Ubx transcripts is altered in esc - embryos, the early pattern of expression is the same as in wild type embryos. They speculate that esc is a component of the process which propagates segmental commitments made earlier. The other members of the Pc-group would also be components of this genetically.complex process. Whether a l l Pc-group genes act in the same manner as esc w i l l be determined when analyses of transcript distribution are compared for other - 6 -members of the group. I t i s p o s s i b l e that the Pc-group genes do not a c t d i r e c t l y to m a i n t a i n segment i d e n t i t y . I n s t e a d , these genes may have a more d i r e c t r o l e i n d e t e r m i n a t i o n and i n f l u e n c e the e x p r e s s i o n o f the BX-C and ANT-C i n d i r e c t l y . P o l yhomeotic, a member of the Pc-group, has r e c e n t l y been c h a r a c t e r i z e d (Dura et a l . , 1985; 1987). E a r l y s t u d i e s f o c u s e d on hypomorphic pji a l l e l e s . These a l l e l e s show homeotic t r a n s f o r m a t i o n s i n a d u l t s i n c l u d i n g the appearance of sex combs on the second and t h i r d l e g s and p o s t e r i o r t r a n s f o r m a t i o n o f t h o r a c i c and abdominal segments. I n t e r a c t i o n w i t h o t h e r Pc-group genes, l e a d i n g to an enhancement of homeotic phenotype, has a l s o been demonstrated (Dura e_t a l . , 1985). N u l l m u tations at t h i s l o c u s proved d i f f i c u l t to o b t a i n . Such mutants were i s o l a t e d o n l y a f t e r an e x i s t i n g gh mutation was s u b j e c t e d to a second round of mutagenesis (Dura e_t a l . , 1987). The need f o r two mutagenic events i n the c r e a t i o n of a n u l l mutant suggests that the ph l o c u s i s complex. F l i e s c a r r y i n g o n l y the n u l l a l l e l e ( p h - ) d i e i n mid-embryogenesis and f a i l to d e velop v e n t r a l c u t i c l e . Head segments and the extreme p o s t e r i o r appear u n a f f e c t e d . However death o c c u r s too e a r l y to a l l o w s c o r i n g f o r homeotic t r a n s f o r m a t i o n s i n the abdominal and t h o r a c i c segments. Lack of v e n t r a l c u t i c l e i s the embryonic phenotype of s e v e r a l D r o s o p h i l a mutations ( N u s s l e i n - V o l h a r d e_t a l . , 1984; Jurgens e_t a l . , 1984; Wieschaus ejt a l . , 1984). However, none of the o t h e r members of the Pc-group has been shown to be r e q u i r e d f o r c u t i c u l a r development. I t i s p o s s i b l e that ph p l a y s a r o l e i n the development of the e p i d e r m i s u n r e l a t e d to i t s r o l e i n segment d e t e r m i n a t i o n . S e v e r a l of the Pc-group genes show maternal e f f e c t s , i n d i c a t i n g that t h e i r e x p r e s s i o n i s r e q u i r e d i n the maternal g e r m l i n e ( S t r u h l , 1981; D e n e l l , 1982; Ingham, 1984) I n d u c t i o n of g e r m l i n e ph~/ph~ c l o n e s shows - 7 -that ph has a strong maternal effect. No progeny were obtained when mosaic females carrying ph~/ph~ germline clones were mated to £h + males (Dura e_t a l . , 1987). The maternal effect of ph is therefore much stronger than that observed for other Pc-group genes. Breen and Duncan (1986) showed that Asx, Pel, See and Scm have maternal effects which are partially or completely rescued by the paternal wildtype a l l e l e . The maternal effect of Pc (Lawrence et a l . , 1983), esc (Struhl, 1981, 1983) and sex (Ingham 1984) can also be rescued by the paternal contribution. The existence of two mutable regions, a requirement in cuticular development and a strong maternal effect make the ph locus unique among Drosophila Pc-group l o c i . To better understand the function of ph, the locus should be studied at the molecular level. Such studies require the isolation of DNA from the ph locus. In this thesis I report the cloning of ph and surrounding regions, plus the identification of genetic lesions associated with ph mutations. - 8 -CHAPTER 1 INTRODUCTION Genetic analysis permits the identification of genes which have interesting function, but whose gene products are unknown or not yet purified. Techniques have been developed whereby these genes, known only through genetic analysis, can be cloned. In prokaryotes and lower eukaryotes, genes can be recovered by transforming mutant strains with shotgun genomic libraries. The gene of interest is isolated by virtue of its a b i l i t y to correct the mutant phenotype. This approach has not been used for Drosophila because of the labour involved in the microinjection of large numbers of embryos. For eukaryotes with small genomes, i t is relatively simple to catalogue the entire genome as a series of overlapping clones. This approach is being undertaken for the nematode, Caenorhabditis elegans. Genes which are already cloned w i l l be mapped onto this ordered sequence. In the future i t w i l l be possible to clone genes knowing only their position on the genetic map. The systematic ordering of the entire Drosophila genome has not yet been undertaken, although portions have been characterized in this way. Three methods have been used successfully to clone Drosophila genes whose RNA or protein products are not known: microdissection from polytene chromosomes, chromosome walking and transposon tagging. If the cytological position of a gene is known, i t should be possible to clone the gene using microdissection (Scalenghe e_t a l . , 1981). The region containing the gene is cut out from lightly fixed chromosomes, DNA is purified and then cloned into phage vectors. These clones are then - 9 -used to isolate larger clones from the region using the technique of chromosome walking (see below). Due to d i f f i c u l t i e s inherent with this technique, microdissection has been used successfully by only a few laboratories, esc, a member of the polycomb-like group of genes (Frei e_t a l . , 1985) and Kruppel (Preiss e_t a l . , 1985) have been cloned in this way. Chromosome walking (Bender e_t a l . 1983) can be used to isolate a gene which is located at a distance from a previously cloned sequence. This sequence is used as a start point to isolate other clones which contain the same sequence but which extend further along the chromosome. The start point DNA is radioactively labeled and used as probe to isolate homologous sequences among other clones which have been immobilized on nitrocellulose f i l t e r s . Clones with homology are identified by autoradiography, purified and compared to the start point DNA by restriction mapping. The process is then repeated, using the extreme segments of the new clones as probes to isolate other DNAs which overlap partially, but which extend s t i l l further along the chromosome. Using this technique, genes have been isolated after walks of several hundred kilobase pairs (kb). Problems arise when repeated sequences are encountered in the walk. If a genomic library is screened with a probe containing a repeated sequence, clones w i l l be isolated which are non-contiguous but which have homology to the repeat. This problem can be overcome by selecting a probe which contains only unique sequence. If the stretch of repeated sequence is not too long, this probe can be used to isolate other clones which span the repeated sequence and extend beyond into unique sequence which can be used to begin the next step of the walk. If this approach f a i l s , the walk can be shifted to a library prepared from a Drosophila strain which has a different distribution of repeated sequences (Bender e_t a l . , 1983). - 10 -Chromosome walking requires a cloned starting sequence sufficiently close to the target gene, a way to determine the direction of the walk, and a means of identifying the destination. Many Drosophila sequences have been characterized and are available for use as starting points. In addition, many inversions and deletions are available which can be used to jump to other locations. The direction of the walk can be determined by in situ hybridization to polytene chromosomes. Clones that are two bands apart can be distinguished in this way (Bender e_t a l . , 1983). Recognition of the endpoint w i l l be discussed in Chapter 2. Chromosome walking is a reliable but time-consuming method of isolating genes from Drosophila. Isolation of transposable elements from Drosophila has made possible a third method for cloning a gene whose product is unknown; transposon tagging (Bingham e_t a l . 1981). The procedure is applicable to the cloning of any gene whose function has been impaired by the insertion of a transposable element that can be recognized molecularly. A gene library containing sequences representative of the entire genome is prepared from the mutant strain. This library is then screened to isolate a l l sequences which contain transposable element (TE) DNA. Each TE-containing clone is labelled and hybridized in situ to Drosophila polytene chromosomes to identify those clones that hybridize to the region of interest. These clones can then be used as probes to isolate the corresponding sequences in a library prepared from wild type DNA. Transposon tagging was f i r s t used to isolated sequences from the white locus into which the mobile element copia had inserted (Bingham e_t a l . 1981). Searles e_t a l . (1982) reported the f i r s t use of the Drosophila P-element and Carramolino e_t a l . (1982) reported the f i r s t use of gypsy to isolate a gene by tranposon tagging. - 11 -To clone ph, I decided to use a combination of two methods; transposon tagging and chromosome walking. The allele ph^ (Dura et a l . , 1985) had been isolated from a P-M hybrid dysgenic cross. In the germ line of P-M female progeny that result from crossing P males with M females, P elements are mobilized and mutations may arise as a result of insertions into new locations (Engels, 1983). Preliminary in situ hybridization data, using P-element DNA as a probe, showed that a P element was inserted in ph^ x chromosomes near the expected cytological location of gh. Although the genetic analysis of ph^ was not yet complete, I decided to isolate the P-containing sequence from that region. Even i f this P element was not responsible for the ph^ mutation, i t would s t i l l serve as a convenient start point for the cloning of ph^ by chromosome walking. For successful transposon tagging, the genomic library must contain representative sequences from the entire genome. Several factors must be considered in the construction of the library. First, the insert fragments must be produced randomly, so that a l l sequences have an equal chance of representation. Second, i f bacteriophage lambda derivatives are used as vectors, the insert fragments must be long enough to allow packaging of the recombinants into phage heads. Finally, enough recombinant phage must be produced to be reasonably certain that every sequence is represented. The general formula, to calculate the probability of representation for any sequence is N=ln (1-P) ln (1-f) where P = desired probability f = fractional proportion of the genome in one recombinant N = the number of recombinants needed to ensure representation - 12 -Thus For a Drosophila melanogaster library, where the desired probability is 0.99, the insert size is 20 kb and the haploid genome is 1.65 x 10^ kb (Maniatis et a l . 1978) N=ln (1-0.99) ln (20/1.65 x 105) =38,000 A library of 38,000 phage, each with 20 kb inserts, represents 7.6 x 10^ kb of insert DNA. Thus, in order to ensure, with a 99X probability, that any one sequence has been cloned, one must clone the equivalent of 4.6 genome lengths of DNA (7.6 x 10^ / 1.65 x 10^). in practice, this number is usually exceeded. Creation of random fragments is best done by mechanical shearing. However, this method requires many preparative steps before the fragments can be inserted into a vector (Lawn e_t a l . , 1978). A more convenient alternative is to digest the DNA with a restriction enzyme which recognizes many sites, under conditions which allow cutting of only a small number of these sites. By varying the conditions of the digest, one can maximize the number of fragments in any size range. Since the fragments w i l l have cohesive termini generated by the restriction enzyme, they can be ligated to vector arms with the same cohesive termini. The termini of the vector need not be generated by the same enzyme. For example, many vectors have been engineered so that the central region can be removed using Bam HI, an enzyme which produces cohesive termini with the same sequence as those generated by Mbo I. Vectors prepared in this - 13 -way can therefore be ligated to products of an Mbo partial digest (Frischauf et a l . , 1984). I prepared a library from jah^ DNA, using partial digestion with Mbo I to generate DNA fragments that were inserted into the replacement vector EMBL 3 (Frischauf at a l . , 1984). Using P-element DNA as a probe, I isolated many P-containing sequences from this library. These sequences were hybridized in situ to polytene chromosomes from a strain without P-elements. One recombinant phage was shown to originate from polytene band 2C1-2. - 14 -MATERIALS AND METHODS Recovery of ph^ embryos Embryos were collected from a population cage containing approximately 10,000 adult f l i e s of genotype ph^/ph^ or ph^/Y. Eggs were collected on trays containing 1.5% agar coated with a paste of brewer's yeast dissolved in 0.17% propionic acid. Using d i s t i l l e d water, the yeast/embryo mixture was rinsed through a strainer to separate adult f l i e s and other large debris and then rinsed in a #200 mesh strainer to remove yeast. Embryos were dechorionated in 50% chlorox, 1% Triton-X for two minutes, rinsed thoroughly with d i s t i l l e d water, and stored frozen at -70° or used immediately for DNA preparation. Isolation of DNA Dechorionated embryos were added to one volume of 0.2 M EDTA, 0.2 M Tris pH 8.0. After 1 hour at 50°, the solution was brought to 1.75% NaDodSO^, 100 ug/ml proteinase K and incubated overnight at 50°. The mixture was extracted gently four times with one volume of phenol/chloroform/isoamyl alcohol (25:24:1). The aqueous phase was dialysed against 1 1 of TE (lOmM Tris pH 8.0, 1 mM EDTA ) at 22° for 24-48 hr with four changes. The solution was brought to IM NaCl and 100 ug/ml RNase and incubated at 37° for 45 min. lOOug/ml pronase was added and the solution incubated at 37° for a further 45 min. This solution was gently extracted with an equal volume of chloroform/isoamyl alcohol (24:1). DNA was precipitated after addition of one tenth volume of 2.5M NaOAc and two - 15 -volumes of 95% ethanol. The precipitate was recovered by centifugation, washed with 70% ethanol and resuspended in a small volume of TE. The DNA prepared by this method was about 70 to 80 kb long, as shown by electrophoresis on 0.4% agarose gels in 1.0 x TBE. Electrophoresis DNA was separated electrophoretically on 0.4 - 1.0% agarose gels containing 1 x TBE (TBE is 0.089 M Tris-OH, 0.089 M boric acid and 2 mM EDTA) at 30 - 100 V in a submarine gel apparatus. Isolation of Sized Genomic DNA Restriction Fragments To determine optimal digestion conditions, genomic digests were performed as follows: 20 yg of DNA plus 5 ul of Bethesda Research Laboratories (BRL) core buffer was brought to 50 ul with d i s t i l l e d water and then one unit of BRL Mbo I was added and the reaction incubated at 37°. Aliquots of 5 ul were removed at 2.5 min intervals, brought to 20 mM EDTA and placed on ice. The aliquots were heated to 70° for 10 min and electrophoresed on a 0.4% agarose gel in 1.0 x TBE overnight. To determine the digestion time producing maximum fluorescence in the 20-40 kb range, gels were stained with ethidium bromide at 10 ug/ml. A f u l l -scale digest was carried out using the optimal reaction time in a reaction containing 100 yg of DNA in a total volume of 250 ul. The DNA was precipitated with one tenth volume of 2.5 M NaOAc and two volumes 95% - 16 -ethanol, washed with 70% ethanol, dried and resuspended in 50 y l TE. The DNA was layered on a salt gradient prepared as follows: ice-cold solutions of 24%, 20%, 15%, 10% and 5% NaCl in 0.25 M EDTA were layered in 1 ml steps in 5 ml SW50.1 centrifuge tubes. The gradients were spun for three hours at 37,000 rpm at 2°. Fractions of 125ul were collected by dripping and DNA was precipitated with 1 volume of water and two volumes of 95% ethanol. Each fraction was washed with 70% ethanol, dried and resuspended in 30yl 0.1X TE. 3 y l of each of the last fifteen fractions was electrophoresed on a 0.4% agarose gel to determine which fractions showed peak fluorescence in the 20 kb range. These fractions were used in subsequent steps. Preparation of EMBL3 Bacteriophage EMBL3 bacteriophage (Frischauf e_t a l . , 1983) were recovered from plate lysates on 15 X 150 mm petri plates containing 1.5% agarose in NZCYM. For each plate 5 X 105 pfu were mixed with 200yl K802 plating cells prepared by precipitating bacteria from 5 ml of an overnight culture and resuspending in 2.5 ml 10 mM MgSO^ . The phage/bacteria mixture was incubated at 37° for 15 minutes and plated with 10 ml of NZCYM top agarose onto 150 mm plates containing NZCYM AND 1.5% agarose. The plates were incubated at 37° for 12 hours and placed at 4°. Each plate was overlaid with 15 ml cold SM (SM is 100 mM NaCl, 8 mM MgS04, 50 mM Tris pH 8.0 and 0.01% gelatin ) and placed at 4° for a minimum of 6 hours. The eluate was collected and spun at 7500 x g for 10 min. to remove debris. Phage were pelleted at 25,000 rpm for 2 hours in an SW27 rotor. Pellets were resuspended in 500yl SM and spun in an micro-centrifuge for 30 seconds to - 17 -remove contaminating agarose. Each 500 u l suspension was used in a 5 ml CsCl equilibrium gradient. The suspension was brought to 2.33 ml with SM and mixed with 2.67 ml 7.2 M CsCl in SM. The mixture was spun at 30,000 rpm in an SW50.1 rotor at 22° for 16 hr. Phage bands were removed by side puncture with a 22G needle and removed to a 5 ml ultracentrifuge tube. The volume was brought to 1 ml with 7.2 M CsCl in SM. 3 ml of 5 M CsCl in SM was layered onto this solution, followed by 1 ml of 3 M CsCl in SM. These step gradients were spun for 1 hour, 30,000 rpm in an SW50.1 rotor. Phage were removed by side puncture as described above. Vector Preparation Phage in CsCl were dialysed twice for 1 hr against 1 1 of 10 mM Nacl, 50 mM Tris pH 8.0 and lOmM MgS0A for. EDTA was added to 20mM, pronase to 0.5 mg/ml and NaDodS04 to 0.5%. The mixture was incubated at 37° for 1 hr, then extracted twice with phenol/chloroform and once with chloroform alone. The DNA was then dialysed against 11 of TE with three changes. EMBL3 DNA was digested to completion with Bam HI and EcoRI as follows: 20 ug of EMBL3 DNA was cut with 30 U of Bam HI in a total reaction volume of 250 u l with 50 mM NaCl, 10 mM Tris pH 7.5, 10 mM MgCl2, and 1 mM DTT. After two hours at 37° the reaction was brought to 100 mM NaCl and 30 U of Eco RI added for a further two hours. The digest was brought to 20 mM EDTA and extracted with phenol/chloroform and chloroform alone. The digest was precipitated with 1/2 volume of NH^ OAc and two volumes of 95% ethanol. The precipitate was washed twice with 70% ethanol, dried and resuspended in 20 yl of 0.1X TE. - 18 -Ligation of Vector and Insert Vector and insert were ligated at a molar ratio of 2:1 vector:insert. 1.0 yg of prepared EMBL3 arms were ligated to 200 ng ph^ Mbo I fragments using 3U of BRL T4 ligase in a 5 y l volume for 16 hours at 12°. Preparation of Packaging Extracts Recombinant phage were packaged using packaging extracts prepared from SMR10, generously donated by Susan Rosenberg, University of Oregon. SMR10 is a lysogen lacking cos sites. Extracts were prepared as follows: 0.5 ml of a fresh overnight culture of SMR10 was used to inoculate 450 ml LBK (lOg Bactotryptone, lOg NaCl, 5g yeast extract and 4 ml IN NaOH in 11 dH20) prewarmed to 34°. Incubation was at 34° until the OD55Q was 0.60. The culture was then warmed to 44° in a water bath and shaken vigorously for 15 min. The flask was shaken for 90 min at 37° and subsequently chilled at 0° for 5 min with continuous swirling. After centrifugation at 6000 rpm for 6 min, 4° in prechilled bottles, the supernatant was removed and the pellet resuspended in 0.35 ml TSP (0.1 M Spermidine, 0.1 M Putrescine and 0.04 M Tris pH 7.9). 20 yl of this suspension was added to Eppendorf tubes containing 5 yl of a solution of 50% DMSO and 7.5 mM ATP on ice. Each tube was vortexed and quick-frozen in liquid nitrogen. - 19 -Packaging of Recombinant Phage 4 y l of the ligation mixture was mixed with 10 y l of CH buffer (40 mM Tris pH 8.0, 1 mM spermidine, 1 mM putrescine, 0.1% 6-mercaptoethanol and 1% DMSO) on ice. 1 y l of 0.1 mM ATP was added and the volume was brought to 20 y l with 66mM Tris pH 8.0. This mixture was added to a freshly thawed packaging mix and placed at 37° for 1 hour. A second packaging extract, preincubated with 5 y l of DNase (1000 yg/ml) and 2.5 y l of 0.5 M MgCl2 for 10 minutes, was added and the incubation continued at 37° for a further 30 minutes with frequent mixing. The volume was then brought to 1 ml with SM and 5 y l of chloroform added. The library was titred on K802 to estimate total bacteriophage and on NM528 to estimate total recombinants. Amplification was carried out as follows: The entire library, representing 70,000 recombinants, was plated on NM528 and the plates overlain with SM, which was collected and stored at 4° with 5 yl/ml chloroform. Screening The Library 75,000 bacteriophage were plated on 15 X 150 mm petri plates at a density of 15,000 per plate. Two sets of nitrocellulose f i l t e r s were l i f t e d ; the f i l t e r s were placed on the plates for 3 minutes, marked with needle and ink, and placed on a surface saturated with 1.5 M NaCl, 0.5 N NaOH for three minutes. After a second three minute denaturation in this buffer, the f i l t e r s were blotted dry and then twice neutralized in 1.5 M NaCl 0.5 M Tris pH 8.0. The f i l t e r s were then baked at 80° for 2 hours. - 20 -Filters were pre-hybridized in 50% formamide, 5X Denhardt's solution (Denhardt, 1966), 5X SSPE, 0.1% NaDodS04 and 100 ug/ml denatured salmon sperm DNA in heat-sealed bags. Prehybridization was at 42° for 2 hours in a volume of 6 ml per 150mm f i l t e r . Heat-denatured 32p_i a5 ei e (j probe DNA was added and hybridization continued for 16 hours at 42°. The f i l t e r s were twice washed at room temperature with 2X SSC (lx SSC is 0.15 M NaCl and 15 mM NaCitrate), 0.1% NaDodS04 for 10 min. A second wash with 0.2X SSC, 0.1% NaDodSO^ was carried out at 65° for thirty minutes. The f i l t e r s were placed against a backing sheet, covered with plastic wrap and placed with X-ray film and one intensifying screen at -70°. Filters were aligned with the autoradiogram and the location of a l l f i l t e r markings indicated on the autoradiogram with ink. The library plates were aligned with the autoradiogram and areas which aligned with positive signals were picked and transferred to 1 ml of SM. Purification of Positives and DNA Preparation Purification of positives was as follows: 5 y l of a 10-3 dilution of isolated plaques in SM was incubated with 10 yl of K802 plating bacteria at 37° for 15 min., mixed with 1 ml of NZCYM top agarose and plated on 50 mm petri plates containing 3 ml NZCYM bottom agar. Nitrocellulose f i l t e r s were l i f t e d and treated as described above for library screening. Single isolated plaques were identified and transferred to 1 ml of SM using a capillary pipette. 500 yl of the suspension was used to infect 100 y l of K802 plating bacteria at 37° for 15 min. This mixture was added to 4 ml of NZCYM and incubated overnight at 37°. 400 yl of the lysate was brought to 1 yg/ml RNase and lyg/ml DNase and incubated for a further 30 minutes - 21 -at 37°. 1 y l of diethylpyrocarbonate, 10 y l of 10% sodium dodecyl sulphate (NaDodSO^ and 50 y l of a 2M Tris 0.2M EDTA pH 8.0 solution were added and the mixture was incubated at 70° for 5 min. The solution was then extracted with equal volumes of phenol, phenol/chloroform and chloroform alone. DNA was precipitated with 40 y l 2.5M NaOAc pH 5.2 and two volumes of 95% ethanol. The pellet was washed with 70% ethanol, dried and resuspended in 50 y l TE. Comparison of Positives and Southern Blotting Positives were compared using restriction enzyme digestion and Southern blot analysis. Each DNA was digested with two enzymes under conditions specified by the manufacturer. Either Eco Rl and Xho I or Hind III and Pst I were used. The digested DNA, together with size standards was separated electrophoretically on 0.8 % agarose gels in 1 x TBE. The gel containing the electrophoresed fragments was soaked in 0.5M NaOH and 1.5M NaCl for 30 minutes. The gel was then neutralized with 0.5M Tris pH 8.0, 1.5M NaCl for 30 min and placed on a bed of 3MM chromatography paper with wicks extending into a reservoir of 10 x SSC. The gel was covered with nitrocellulose, two layers of 3MM paper, paper towels and weighted. After blotting overnight, the nitrocellulose was removed and baked 2 hours at 80° (Southern, 1975). Hybridization was essentially as described in Maniatis et a l . (1982): Filters were floated on 6 x SSC and transferred to heat-sealable plastic bags. Prehybridization fluid (50% formamide, 6 x SSC, 0.5% NaDodSO^ 5 x Denhardt's solution and 100 yg/ml denatured salmon sperm DNA) was added to a volume of 200 yl/cm 2 of nitrocellulose. Incubation was at 42° for 2 to 4 hours. The prehybridization fluid was removed and replaced with a - 22 -smaller volume (50 y l per cm^ ) of hybridization fluid (prehybridization fluid with 0.01M EDTA). Approximately 100 ng of probe DNA with a specific activity of 10? to 10^ cpm/ug was added. Hybridizations were for 4 to 16 hours at 42°. Filters were washed twice for 15 min. in 2.0 x SSC, 0.1% NaDodSO^ at room temperature and twice for 30 min. in 0.2 x SSC, 0.1% NaDodS04 at 65°, before exposure of X-ray film overnight. Nick Translation Probes for use in library screening or for Southern blot analysis were prepared using a32p_dCTP whereas probes for in situ hybridizations were prepared using ^H-dTTP. Reactions were as follows: 500 ng- 1 ug of DNA was mixed with 5.0 y l 10 x nick translation buffer (0.5M Tris pH 7.2, 0.1M MgSO^ , ImM dithiothreitol and 500 ug/ml bovine serum albumin), 100 yCi of 32P-dCTP or 50 yCi of 3H-dTTP, 1 nmole of each of the three cold dNTPs and 5 units of DNA polymerase I in a total reaction volume of 50 y l . 0.5 y l of DNase (0.1 yg/ml) was added and the mixture was incubated at 15° for 1 hour. The reaction was stopped by addition of EDTA to 20mM. Labelled DNA was separated from unincorporated nucleotides by precipitation twice with NH^ OAc and ethanol or by passage through a 1 ml minicolumn of Sephadex G50®. In Situ Hybridization In situ hybridization was carried out by Dr. H. W. Brock using the procedure of Brock and Roberts (1983). - 23 -RESULTS The ph 2 a l l e l e of polyhomeotic was isolated after a dysgenic cross between P males and M females, and was shown to be a l l e l i c to other p_h mutants (Dura et a l . 1985). Brock showed by in situ hybridization that a P element was present at band 2C1-2 in the polytene chromosomes of this strain (unpublished results). When this thesis project was begun, the analysis of ph 2 was not complete. It had not been demonstrated that the P element at band 2C1-2 was responsible for the p_h mutation in this strain. However, the cytological location of the P element was within the interval known to contain gh; at that time localized only to the interval 2C-D. It was decided to attempt to clone the P element and flanking DNA from 2C1-2 concurrent with the genetic analysis. If the P element at 2C1-2 proved to be responsible for the phenotype of ph 2, then the cloned DNA would contain sequences in or adjacent to the ph locus i t s e l f . If the P element proved not to be associated with p_h, its flanking DNA would s t i l l provide a useful starting point for a chromosome walk. Construction of a ph 2 genomic library. ph 2 embryos were collected, dechorionated and stored as described in Materials and Methods. When compared to adult tissue, embryos consistently gave better yields of high molecular weight DNA suitable for library construction (unpublished observations). Embryos were solubilized with detergent and protease, extracted with phenol to remove protein, and the DNA precipitated with ethanol after treatment with RNase. The yield from 2 grams of embryos varied between 200 and 300 ug of DNA. Typical - 24 -preparations yielded DNA that was between 60 and 90 kb long as estimated after electrophoresis in low percentage agarose gels. The DNA was of a size sufficient to allow the generation of a good yield of partially digested fragments for library construction. Lambda replacement vectors have been engineered to accept inserts of up to 23 kb in length (Frischauf e_t a l . 1983). To minimize the number of phage required for a representative library, i t is best to generate fragments close to, but not exceeding the maximum cloning capacity. To avoid competition by smaller molecules, and to avoid the ligation of non-contiguous genomic DNA fragments, fragments of less than 12 kb should not be included. A portion of the genomic DNA was digested with the restriction endonuclease Mbo I for varying lengths of time to determine conditions which maximized the yield of fragments in the 15 to 23 kb range. Aliquots of DNA that had been digested for different times were compared after electrophoresis on agarose gels (figure la). The time required for the optimal degree of digestion was determined and the reaction was scaled up to digest 100 yg of DNA with Mbo I. The partially digested DNA was fractionated on NaCl gradients as described by Hadfield (1983). Gradient fractions were collected and precipitated with ethanol and the size range of the DNA from each fraction was determined using gel electrophoresis (figure lb). Fractions containing fragments 15 to 23 kb long were pooled. The partially digested genomic DNA was cloned into the Bam HI site of the vector EMBL3 (the enzymes Mbo I and Bam HI produce termini which can be ligated together). EMBL3 was chosen as the vector because i t has several features which simplify the cloning procedure. Phage arms do not need to be separated from the stuffer fragment as is the case for many lambda vectors. There are Eco RI sites within the stuffer fragment Figure 1. Partial digestion of ph 2 DNA DNA was isolated from ph 2 f l i e s and digested with Mbo I. Aliquots were removed at intervals, treated to inactivate the enzyme and analysed on 0.4% agarose gels. Reaction conditions which gave a maximum fluorescence in the 20 to 40 kilobase range were used to digest 100 pg £h 2 DNA. The DNA was centrifuged on 5 ml salt gradients. Fractions were collected, precipitated, and a portion of each was analysed on an agarose gel. (a) Photograph of an agarose gel comparing digestion timepoints. Marker= X Hind III digest plus undigested X DNA. Uncut= undigested ph 2 DNA. 0.5 through 30 = digest time in minutes. Maximum fluorescence in desired range occurs at 1 minute, (b) Photograph of an agarose gel showing gradient fractions 7 through 22. Marker as in (a). Fractions 14 and 15 show maximum fluorescence in the 15 to 23 kb range. - 26 -adjacent to the Bam HI sites at each end. Complete digestion with Eco Rl makes i t impossible for the stuffer fragment to ligate back to the phage arms. The very small Eco RI/Bam HI fragments are lost in the precipitation which follows the Eco Rl digestion (Frischauf e_t a l . 1983). EMBL3 DNA was prepared by digestion with both Bam HI and Eco Rl. The DNA was phenol extracted, precipitated and ligated to Mbo I genomic fragments at a ratio of 1 ug vector to 200 ng insert. This corresponds to a molar ratio of approximately 2:1. A small aliquot was compared by electrophoresis with non-ligated arms and insert in order to monitor ligation efficiency. 500 ng of ligated DNA was packaged in vitro as described in Materials and Methods. EMBL3 contains sequences in the stuffer fragment which prohibit growth in strains carrying a P2 lysogen (Zissler et a l . , 1971). However, recombinant phage lacking the stuffer fragment are able to grow in such strains. Therefore, equal aliqouts of the packaged DNA were plated on both NM528, a P2 lysogen which wi l l allow only recombinants to grow, and on K802, a strain which allows the growth of a l l phage. The number of phage obtained on either strain was virtually identical, indicating only a low background of non-recombinant phage. The packaging efficiencies of this ligation ranged from 1 x 10^ to 2 x 10^ pfu/ug. A total of 77,000 recombinant phage were recovered from the pji 2 library ligation after packaging and plating on NM528. The plated phage were recovered by diffusion into phage buffer and were stored for use in subsequent steps. Screening the library for P-element-Containing clones. To identify recombinants containing a P element, the library was - 27 -plated and transferred to replica nitrocellulose f i l t e r s . The f i l t e r s were then hybridized to ^ 2P-labelled plasmid DNA. Two P element-containing plasmids were used. One set of f i l t e r s was hybridized to pn25.1 (O'Hare and Rubin, 1983) which contains an intact P factor as well as flanking DNA from the white locus. The other set of f i l t e r s was hybridized to pn.6.1 which contains a P element plus flanking DNA from polytene band 17C (Spradling and Rubin, 1982). Recombinant phage containing P element sequences should hybridize, to both probes. Phage with homology to white or 17C-specific sequences should hybridize only to pn.25.1 or pn.6.1 respectively. A typical pair of f i l t e r s is shown in figure 2. A total of 53 positive signals were obtained. Seven phage showed hybridization to pn25.1, six showed hybridization to pn6.1 and forty hybridized to both probes. The forty putative positive clones were then purified as described in Materials and Methods. DNA was prepared from each of the forty positives, digested with restriction enzymes and separated by electrophoresis on agarose gels. The DNA was blotted onto nitrocellulose (Southern, 1975) and pn25.1 DNA was used as a probe. Phage with identical patterns of hybridization were presumed to represent isolates of the same sequence. In this way, duplications were eliminated and the number of positives to be screened by in situ hybridization was reduced to twenty-five. Localization of phage by in situ hybridization. To identify which of the 25 phage hybridized to the 2C1-2 region, phage DNA was labelled with ^H-dCTP and hybridized in situ to polytene chromosomes. To minimize the number of in situ hybridizations required, the phage DNA was pooled in groups of five. Equal amounts of DNA from -28-p*6.1 p*25.1 Figure 2. Screening for phage containing P elements. Replica nitrocellulose f i l t e r s were l i f t e d from the ph 2 library and treated to denature and bind bacteriophage DNA. Duplicate f i l t e r s were hybridized to radiolabelled P element-containing plasmids pn6.1 or pit25 .1. Shown is a pair of f i l t e r s hybridized to pn6.1 (left) or pn25.1 (right). The position of phage which hybridized to both probes is indicated by open circles. The positions of phage which hybridized to only pit25.1 are indicated by arrows. - 29 -each phage was mixed, labelled by nick-translation and hybridized in situ to polytene chromosomes from an M strain. Since M strains lack P elements, hybridization w i l l result only from DNA flanking the P elements. One pool of five phage contained a recombinant which hybridized to 2C1-2. DNA from each phage in this pool was nick-translated and hybridized separately to polytene chromosomes. One phage, designated EMBL3-14P, was shown to originate from 2C1-2 (figure 3). Localization of EMBL3-14P relative to neighbouring deficiencies. Attempts to revert the ph 2 mutant by subjecting this strain to further rounds of dysgenesis were unsuccessful, suggesting that the P element in 2C1-2 was not inserted at the nh locus (Dura and Brock, unpublished observations). With the cloning of DNA from 2C1-2 i t became possible to test the location of this DNA in relation to deficiencies known to include ph. EMBL3-14P was radio-labelled and hybridized to polytene chromosomes from Df(l)pn38/+ females. Complementation analysis has shown that gh is included in Df(l)pn^8 (Dura et a l . , 1985). In those nuclei in which the pn38 a n ( j w i i d type chromosomes were asynapsed, hybridization was observed on both homologues. EMBL3-14P was therefore not included in Df(l)pn^8 (Brock, unpublished observation). This result was confirmed later, when cytological examination of a new small deletion, Df(l)JA52 (Perrimon ejt a l . , 1985) showed that gh had to be between 2D2 and 2D4. Therefore the DNA cloned from 2C1-2 became the start point for a walk, as described in Chapter 2. -30-Figure 3. Localization of EMBL3-14P by in situ hybridization. Top: A drawing of the tip of the X chromosome (based on the map by Bridges (1938)). Bottom: In situ hybridization of EMBL3-14P to an Oregon R polytene X chromosome, aligned with the line drawing to cla r i f y band positions. The arrow indicates the position of silver grains which obscure band 2C1-2. - 31 -DISCUSSION I cloned a DNA segment originating from band 2C1-2 of the Drosophila  melanogaster X chromosome. Using transposon tagging, the sequence was isolated from a genomic library prepared from the mutant strain, ph 2. For transposon tagging to be successful, the library screened must be representative of the entire genome. In addition to my success in cloning the desired fragment from 2C1-2, other lines of evidence suggest that the ph 2 library is representative: The cloning strategy involved the use of two P element-containing probes. Each of these probes also contains unique sequence from either 17C or white. The number of phage hybridizing only to one probe but not the other gives an indication of how many recombinants contain sequence from these l o c i . If one assumes an average insert size of 17 kb for the 70,000 recombinants, then a total of 1.2 x 10 6 kb were screened. Since the haploid genome length for Drosophila is 1.65 x 10-", the amount of DNA screened represents approximately seven genome-lengths. On average therefore, one would expect any given sequence to be present in the library approximately seven times. The number of phage isolated by 17C (6) and white (7) is close to expected values for unique sequences. The total number of P element-containing phage isolated also supports the premise that the £h 2 library is representative of the genome. Bingham et. a l (1982) report that there are typically 30-50 P-element copies per haploid genome in a P strain. I have reported the isolation of 25 different P elements from the ph 2 library. The true number of P-containing phage in the library could actually have been higher than the number isolated. Since an amplified copy of the library was screened, i t - 32 -is possible that not a l l of the original clones were represented in the fin a l plating. Also, i t is possible that phage which contained only a small piece of a P element were not detected under the hybridization conditions described. The phage EMBL3-14P does not contain ph, as shown by i t s cytological position with respect to the deletion breakpoint of Df(l)pn-^^. However, since i t originates not far from the region presumed to contain gh, i t could serve as a convenient start point for a walk towards gh. An alternate strategy would have been to isolate other gh mutants from hybrid dysgenic crosses, in hope of isolating a mutant in which a P element was clearly inserted near gh i t s e l f . Reversion studies would be required in order to demonstrate that the mutation was due to the insertion of a P element. This approach was not followed for two reasons. First, the amount of time required to isolate further mutants, to perform the necessary reversion and outcrossing, to create and screen a library, and to identify positives by in situ hybridization is comparable to the amount of time required to complete a walk (see chapter 2). Second, several other gh mutants had been isolated from dysgenic crosses, yet none had been shown to contain a P element at 2D3 (Dura, unpublished results) Thus i t appeared possible that insertion of a P element at gh is a rare occurence and that attempts to isolate a P-containing mutant might f a i l . I decided to use EMBL3-14P as the start point for a walk towards gh. The walk towards gh is the topic of the next chapter. - 33 -CHAPTER 2 INTRODUCTION With the development of genomic libraries, i t was immediately apparent that i t would be possible to isolate overlapping clones and thus isolate linked genes (Maniatis et a l . , 1978). An early application of this procedure was the cloning of the human 6-like globin gene cluster (Fritsch et a l . , 1980). In Drosophila, walks containing a series of overlapping clones have been used to isolate several interesting l o c i , including the BX-C (Bender at a l . , 1983) the ANT-C (Scott et a l . , 1983) rosy-ace (Bender et a l . , 1983), achaete-scute (Campuzano et a l . , 1985; Carramolino e_t a l . 1982), Notch (Artavanis-Tsakonas et a l . , 1983), yellow (Biessman, 1985), paired (Kilchherr et a l . , 1986), glucose dehydrogenase (Cavener et a l . , 1986) , engrailed (Kuner et a l . , 1985) and transformer (McKeown et a l . , 1987) . Walking in Drosophila is made easier by the many well-characterized chromosomal rearragements available. These deletions and inversions are extremely useful for the investigator attempting to conduct a chromosomal walk in Drosophila; they can be used as markers to monitor the progress of a walk by Southern blot analysis and by in situ hybridization to polytene chromosomes. The fusion of DNA strands from different chromosomal regions creates restriction fragments of different length in the region of the breakpoint. The fragments can be readily distinguished from wild type fragments i f radiolabeled DNA from the breakpoint region is hybridized to Southern genomic blots of mutant DNA. A rearrangement breakpoint near the beginning of a walk can be extremely useful since the the orientation of a walk can be easily determined using - 34 -only Southern blotting. Rearrangements can also be used as bridges to jump into chromosomal regions distant from the start point; once the location of a deficiency or inversion breakpoint is identified, a library derived from the rearranged stock can be screened with wild type DNA from the breakpoint region to isolate clones which contain a breakpoint fusion fragment. This fragment can then be used to screen for wild type sequence from the other side of the rearrangement (Bender et a l . , 1983). Often no useful breakpoints are located near a start point. In this case there is no way to determine immediately in which direction the walk should proceed. It is therefore necessary to walk in both directions until the two ends of the walk can be distinguished when hybridized in situ to polytene chromosomes. According to Bender e_t a l . (1983) a distance corresponding to one or two polytene bands must f i r s t be spanned. A knowledge of the rearrangements available in the region 2C-E is needed for an attempt to clone ph by chromosomal walking. The ph locus was mapped by complementation analysis against previously characterized deficiencies which extend through 2C-E (Dura e_t a l . , 1985). A portion of those results is reproduced in figure 4. This figure shows the extent of the deficiencies shown to have breakpoints in the region of ph. Dura et a l . (1985) showed that the deficiency Df(l)Pgd-kz complements the hypomorphic alleles of ph. However, this deficiency does not complement the recently isolated null alleles of ph (Dura e_t a l . , 1987) and the embryonic phenotype of Df(1)Pgd-kz/ph~ shows that the deficiency behaves as a viable a l l e l e of gh. In contrast, another deficiency in the region, Df(l)pn38, f a i l s to complement a l l gh alleles examined. The region containing gh has also been well characterized genetically by Perrimon et a l . (1985). Their analysis covered the 2C-D region and included an analysis of the position of gh in relation to the same deficiencies used in the analysis of Dura et al.(1985) In addition, Perrimon et a l . reported -35-l(1)DF967 1(1)107 1(1)405 l(1)csw ph l(1)Pgd l(1)C204 i i Df(1)JA52 Df(1)pn38 m**m*> Df(1)Pgd-kZ •^••» Df(1)64c18 Figure 4. Deficiencies in the nh region. Deficiencies are represented by solid horizontal lines. The position of loci in the region of ph as determined by their complementation behaviour with the deficiencies is indicated above. The order of bracketed loci is unknown. Two vertical dotted lines are used for ph to indicate that two mutagenic events are required to produce a null a l l e l e . Adapted from Dura et a l . (1987). - 36 -the discovery of a small deficiency, Df(1)JA52, which f a i l s to complement a l l gh alleles and is lethal in combination with Df(l)Pgd-kz. This complementation behaviour is also consistent with the designation of Df(l)Pgd-kz as a hypomorphic allele of ph. Unfortunately, no deficiencies existed that would f a c i l i t a t e a jump to 2D3 from my start point in 2C1. However, the deficiencies shown in figure 4 are useful in that they can be used to monitor the progress of a walk. Since Df(l)pn^^ and Df(1)JA52 do not complement gh, the locus must l i e within the interval covered by both Df(l)pn38 and Df(1)JA52. Since Df(l)Pgd-kz appears to interfere with gh function, the breakpoint of that deficiency should l i e in or near the locus i t s e l f . It is therefore possible to predict the order in which the deficiency breakpoints would be encountered and the location of gh in relation to those breakpoints. The distal breakpoint of Df(1)JA52 should be discovered f i r s t , followed by the distal breakpoint of Df(l)pn^8. These two breakpoints should be distal to ph. The distal breakpoint of Df(l)Pgd-kz should be found next, and lastly the proximal breakpoint of Df(1)JA52 should be encountered. Previously published walks provide a means of estimating the distance from band 2C1-2 to 2D3. Spierer et a l . , (1983) analysed the cytological origin of clones from a walk of 315 kb (Bender et a l . , 1983), using fragments from their walk as probes for in situ hybridization. With this data they also were able to correlate distances in kb with chromomeric (band-interband) distances seen cytologically. They classified bands into four categories; very faint, faint, readily visible and large. In the interval which they examined, they were able to assign a mean length of 17+5 kb and 20+8 kb to the readily visible bands. The mean length for faint and very faint bands was 5.6+1.1 kb. A large band in the region (a doublet according to Bridges (1938)) was assigned a length of 160 kb. If - 37 -one assumes that chromomeric units of similar size in the interval 2C1-2 to 2D3 can be compared with those studied by Spierer e_t a l . , then i t is possible to estimate the distance which must be covered in a walk through the region; the interval contains 4 bands which can be classified as readily visible, as well as 6 faint bands and 4 very faint bands. Using the maximum from the range of values estimated by Spierer et a l . , for faint and readily visible bands, the maximum distance from 2C1-2 to 2D3 can be estimated as 170 kb. Since the longest walks reported exceeded this distance by twofold, the cloning of ph by walking from 2C1-2 seemed feasible. This chapter describes this walk and the identification of the rearrangement breakpoints in the vicinity of ph. - 38 -MATERIALS AND METHODS Drosophila stocks Flies were reared at 20-28° in half pint milk bottles containing standard sucrose-cornmeal-agar medium. The strains Df(l)Pgd-kz/FM6, Df(l)JA52/FM6 and Df(l)pn 3 8/FM7c were maintained as balanced stocks. Preparation of Drosophila DNA DNA was prepared from females using the following procedure. 100 f l i e s were homogenized in an Eppendorf tube containing 200 ul of homogenization buffer (50 mM Tris pH 8.0, 100 mM EDTA and 200ug/ml proteinase k) on ice. More buffer was added until the total volume reached 500 y l . The mixture was left 10 min. on ice. NaDodSO^ was added to a fi n a l concentration of 1% and the mixture was incubated at 65° for 30 min. The solution was cooled to 37° and proteinase K was again added to a fin a l concentration of 400 yg/ml, followed by incubation at 37° for 3 hr. The total volume was then brought to 7 ml with TE (10 mM Tris pH 8.0, 1 mM EDTA) and 7 g of CsCl was added and mixed gently. 0.8 ml of ethidium bromide (10 mg/ml in H20) was added and the mixture was spun for 10 min at 10 K in an SS34 rotor to sediment proteins. After centrifugation for 36 hr, 45K. rpm, 15° in a 50Ti rotor, the DNA band was made visible using a UV light source and the band was collected by side puncture. Ethidium bromide was removed by successive extractions with an equal volume of NaCl-saturated isopropanol until no ethidium bromide could be detected in - 39 -either phase. Three volumes of TE were then added and the DNA was precipitated with 1/10 volume of 3 M NaOAc and two volumes 95% ethanol, followed by centrifugation in Eppendorf tubes for 10 min. The pellets were washed with 70% ethanol, air dried and resuspended overnight at room temperature in 200 y l TE. Digestion of genomic DNA and Southern blotting DNA isolated from the deficiency strains was digested to completion with restriction enzymes and electrophoresed on 0.6 or 0.8% agarose gels. For each lane, 2 yg of genomic DNA was digested with Bam HI, Eco RI, Sal I or Xho I in a total volume of 200 y l for 16 hrs. The DNA was then precipitated with ethanol and resuspended in a small volume of TE. DNA was electrophoresed at 25-30 V for 16-20 hr in a submarine gel apparatus as described in chapter 1. Southern blotting was as described in chapter 1. Isolation of DNA Fragments from Agarose Gels Restriction fragments were cut out of agarose gels with a st e r i l e razor blade. Gel slices were sealed in dialysis tubing together with 0.5 ml 0.5 x TBE. The dialysis tubing was placed in an electrophoresis tank f i l l e d with 0.5 x TBE. After 2-3 hr at 100 V, the current was reversed for 30 seconds. The gel slice was removed from the tubing and the solution was transferred to an Eppendorf tube. The eluted DNA was then extracted once with phenol/chloroform and once with chloroform alone. The - 40 -DNA fragment was then precipitated with NaOAc and ethanol, washed with 70% ethanol and resuspended in 10 y l TE. Yields were typically 40-50%. Fragments were 3 2P-labelled as described in chapter 1. Library Screening The EMBL4 Oregon R library was a gif t from the laboratory of V. Pirotta. The Charon 4a Canton S library was obtained from the laboratory of T. Maniatis. Plating of phage was as described in chapter 1 except that phage were plated on the E. coli strain C600. Plaque density was 6000 per 85 mm petri plate. A total of 12 plates were used for each screen. Three nitrocellulose replicas were l i f t e d from each plate. Treatment of f i l t e r s and hybridization conditions were as described in chapter 1. Two sets of f i l t e r s were hybridized to the extreme probe and one set was hybridized to the more central probe. Identification of positives was as in Chapter 1. The region of a plate containing a positive was picked and transferred to 1 ml SM. Positives were purified as follows: 100 y l of an overnight culture of C600 was mixed with 3 ml of NZCYM top agar and plated on 85 mm petri plates containing 20 ml bottom agar. When the top agar had solidified a sterile loop was inoculated with the phage suspension and streaked over the plate surface. The plate was incubated overnight at 37°. Single plaques were identified as in chapter 1 and transferred to 1 ml SM. - 41 -Isolation of Phage DNA Phage were recovered from plate lysates as outlined for the preparation of EMBL3 in chapter 1. 75ul of the phage suspension was used to infect 100 ul of C600 for each 85 mm petri plate. Ten plates were used for each preparation. Treatment of plate lysates was as in chapter 1 except that only 5 ml of SM was added per plate. CsCl gradients and collection of phage bands were as described in chapter 1. Extraction of DNA was by dialysis against 100ml of 50% formamide, 0.2 M Tris (pH 8.5) and 20 mM EDTA for 2-6 hrs at room temperature. Three changes of TE were used to purify the DNA which could then be used directly in restriction digests or nick translations. Yields were typically 20-50 ug. Mapping of Phage Inserts Phage DNA was digested in a l l possible combinations with the enzymes Bam HI, Eco Rl, Sal I and Xho I under conditions specified by the supplier. The digests were electrophoresed and Southern blotting was performed as described in chapter 1. - 42 -RESULTS The Walk The recombinant phage EMBL3-14P, whose recovery was detailed in chapter 1, was used as the start point for a chromosomal walk which eventually spanned 175 kb of the Drosophila melanogaster X chromosome. I used two libraries for the walk. The library used for most of the walk was from the laboratory of V. Pirotta. This library is constructed in EMBL4, and contains Mbo I fragments from the Drosophila  melanogaster wild type strain Oregon R. The second library was from the laboratory of T. Maniatis. This library consists of Drosophila  melanogaster DNA fragmented by shearing and inserted into the vector Charon 4 using Eco Rl linkers. The genomic DNA originated from the strain Canton S (Maniatis et a l . , 1978). The EMBL4 library was used preferentially, since i t has a higher average insert size than the Charon 4 library. Longer inserts would reduce the number of steps required to reach ph. However, the Charon 4 library was used i f a previous step using the EMBL4 library had been unsuccessful. The following is a description of only one step in the walk, beginning with the characterization of a newly isolated phage and ending with the purification of the next phage in the walk. The same procedure was followed in a l l of the many steps of the walk. To determine which restriction fragments originated from the ends of the insert, a newly isolated phage f i r s t had to be mapped with restriction enzymes. The enzymes Bam HI, Eco Rl, Sal I and Xho I were used in a l l combinations of single, double, triple and quadruple digests. These enzymes were chosen - 43 -because they do not cut the EMBL4 vector and so analysis of the restriction digests was not complicated by the presence of vector fragments. In addition, the insert DNA in EMBL4 is flanked by Eco RI sites, and so one can determine the length of the insert by totalling the lengths of the Eco RI fragments. The digests were run on 0.8% or 1.0% agarose gels together with size markers. Length of fragment versus distance of migration was plotted for the marker DNA fragments and this graph was used to determine the length of the phage restriction fragments, figure 5a shows a typical restriction digest from the walk and the size of the fragments observed. The gel was then blotted onto nitrocellulose and the blot was hybridized to labelled phage DNA from the previous step in the walk. After autoradiography, fragments which overlapped with the previous phage were easily identified. Figure 5b is an autoradiogram prepared from a blot of the gel in figure 5a. Using the information from the mapping gel and the autoradiogram (comparison of fragment sizes in single, double, triple and quadruple digests and extent of overlap with the previous phage), I was able to generate a restriction map showing the location of sites for the four restriction enzymes along the insert (figure 5c,d). Once I had determined the restriction pattern, I could then choose a restriction fragment furthest from the previous clone to use in screening the library for the next step. I also chose a more central fragment for screening; phage which hybridize to an extreme fragment but not to a central fragment should extend further from the previous phage than those which hybridize to both. The appropriate fragments were separated on agarose gels, the band cut out and the DNA isolated by electroelution into dialysis tubing. The DNA was cleaned by phenol and chloroform extraction followed by ethanol precipitation. DNA fragments were labelled with 3 2P -44-Figure 5. Mapping of restriction enzyme sites. The phage A36 was digested with the enzymes Bam HI, Eco Rl, Sal I and Xho I in a l l combinations, (a) An agarose gel showing fragments produced upon digestion of A36 with the above enzymes. Ml: X DNA cut with Hind III. M2: X DNA cut with Hind III and Eco Rl. Representative fragment sizes in kb are indicated beside the photograph. J35 EX: The previous phage in the walk, J35, digested with Eco Rl and Xho I. B = Bam HI, E = Eco Rl, S = Sal I, X = Xho I. (b) Autoradiograph of a nitrocellulose f i l t e r prepared from the gel in (a) and hybridized to radiolabelled J35. Labels are as in (a), (c) Size in kb of A36 restriction fragments as determined from the gel in (a). Fragments which hybridize to J35, as determined in (b), are underlined. Fragments smaller than 0.4 kb are not detected, (d) The map of A36 generated using the information from (b) and (c). The region of overlap with J35 is shown by the hatched box below the map. -vs-te) X LU ( b ) ; 2 " a w co x <o x x x eo l i l to X CO X X UJ LU CO CO a i ; a a a i u u > a a a a i u a 2 23.1 a x x x a *~ co luexcoxxujujeocouj'-2 2 "» i n i c ^ l i i i i i i i i R i a i U B l 2 3 . 1 1 . 6 -0.8-3 ftr«M 0 . 8 -( c ) A36 RESTRICTION FRAGMENTS B: 6.9, 3.4, 2,0, 1.75, 0.4 E: 4.7, 4.45, 3.6, 1.5. 1.05 S: 4.0, 2.4, 1.2 X: 6.2, 1^ 5, 1.5 BE: 3.65, 3.4, 2.2, 2_0, BS: 3.9, 3.3, 2.25, 2_0, BX: 4.3, 2.65, 1.75, 1.7, ES: 4 45, 2.5, 2.4, 1 5. EX: 3.75, 3.55. 1.5, 1.5, 1.15, 1 35, 15, 12, 1J_. 15, 1.05, 0.95, 0.65, 0.8, 0.4 IU , 0_95 1.2. 1.05. 0.75 1.05, 0.5 1.5, 1.2 0 4 SX: 2.4, 2.18, 1.8, 1.55, _ BES: 2.5, 2.2, 2.15, 2J), 1.15, 1.05, 0.95, 0.8, 0.75, 0.65, 0.4 BEX: 3.4, 2.65, 1.5, 1.15, V05, 1.05, 0JJ5, 0.95, 0.65, 0.5 BSX: 2.18, 2.15, 1.75, 1.55, 1.5, 1.35, It, 0.95, 0.8, 0.4 ESX: 2.4, 1.75, 1.5, 15, 15, 1.2, 1.15, U)5, 1.05, 0.75, 0.5 BESX: 2.18, 1.75, 1.5, 1 15, 1_05. 1.05, 0.95, 0.8, 0.75, 0.65, 0.5, 0.4 ( d ) A36 B BX J_L F S B B S I I ,1 kb . - 46 -dCTP by nick translation, denatured, and added to bags containing nitrocellulose f i l t e r replicas of the genomic library and hybridization buffer. To distinguish false positives, three sets of f i l t e r s were always l i f t e d ; one set was hybridized to the central probe and the other two were hybridized to the extreme fragment. In a successful step, signals would be obtained from the same location on both of the duplicate f i l t e r s hybridized to the extreme fragment, but not at the corresponding position of the f i l t e r hybridized to the central fragment. A typical autoradiogram is shown in figure 6. The number of positives varied from step to step. If more than one was isolated, the clones were compared to see which extended further along the chromosome. After aligning the original plates with the autoradiogram, I picked an area of the plate containing each positive. Since the library was densely plated, i t was not possible to pick only the positive and so a purification step was necessary. Dilutions were streaked onto bacterial lawns to generate single isolated plaques. Filters were l i f t e d from these plates and hybridized to the extreme fragment probe. Autoradiography revealed the location of single positive plaques. To isolate DNA for subsequent manipulations, the plaques were used to infect bacteria as outlined in materials and methods. After purification, DNA from each positive was cut with Eco RI and electrophoresed on an agarose gel to determine insert size. The previous phage from the walk, was also digested and run in parallel. By comparing the restriction pattern and total insert size of the phage, I could determine which of the positives extended furthest from the phage of the previous step (figure 7). This phage was then mapped with restriction enzymes to begin another step of the walk. -47-I II III Figure 6. Screening the the genomic library. The 1.5 kb Eco Rl fragment of A36 (see figure 5) was purified for use as an extreme probe. The 4.0 kb Sal I fragment of the same phage was purified for use as a central probe. The fragments were radiolabelled and hybridized to nitrocellulose f i l t e r s which had been l i f t e d from the EMBL 4 Oregon R library. The 4.0 kb Sal I fragment was hybridized to f i l t e r set I and the 1.5 kb Eco Rl fragment was hybridized to f i l t e r sets I and II. The autoradiograph prepared from the three replica f i l t e r s is shown. Two phage are identified which hybridize to both probes (circles) and one phage hybridized to only the extreme fragment (arrows). -48-(a) . o o g o _ co co co co co O I 2 O 2 (b) _ ru cn B 9 2 8 eg n to 2 n 2 2 O X 5 O 21.2-8.1-4.3-2.0-1 .6-1.4-(c) RESTRICTION FRAGMENT SIZES J29: SK>, 3,7, 2^ 55 C30: 8J), 37, 32, 1.7, 1.55 H30: 12J), 37, 3J. M30: 7.1, 3.4, 3^ 2, 1.7, V45 030: 7.1, 4.4, 3^ 2, 2.0, 1.7 (d) J29 9 0 3.7 2 S5 • C30 « 0 3 7 3.2 i r ,i 55. 1 2 3 7 3 2 M30 ,»•«•, 3 2 . 1 7 . 7.1 . 3.4 030- 1 1 . 1.7 . 7.1 4.4 Figure 7. Comparison of positives from one step, (a) Restriction digests of DNA from the phage J29 and from the four phage isolated from the library screen in which the extreme and central Eco RI fragments of J29 were used as probes. A l l of the newly isolated phage hybridized to both the extreme and the central fragment of J29. DNA was digested with Eco RI and electrophoresed on an agarose gel together with three markers (Ml, M2, M3). (b) Autoradiogram of a Southern blot prepared from the agarose gel in (a) and hybridized to radiolabeled J29 DNA. (c) Sizes of the Eco RI fragments identified in (a) in kb. Fragments shown to hybridize to J29 in (b) are underlined, (d) Eco RI restriction map of J29 and the four newly isolated phage derived from the information in (c). - 49 -Determining the Orientation of the Walk. When a chromosomal walk is begun, i t is impossible to determine the orientation of the f i r s t clone with respect to the chromosome. If one intends to walk in one direction only, i t is f i r s t necessary to walk in both directions until a known rearrangement breakpoint is reached or unt i l the extremes of the walk can be distinguished when hybridized in situ to polytene chromosomes. Since no rearrangements were available immediate to the start point in 2C1-2, the direction of the walk was determined using in situ hybridizations. The relevant portion of the walk is shown in figure 8. A l l attempts to walk in one direction from EMBL3-14P (to the le f t in the figure) had failed. None of the clones isolated extended further than the clone C123. However, the walk was extended successfully in the other direction. When a total of approximately 80 kb had been spanned,the extreme clones were compared by in si tu hybridization. figure 8 shows the hybridization patterns observed. The phage EMBL4 A26 clearly shows a position of hybridization proximal to that of the start point clone. Thus the walk had been successfully extended almost 80 kb in the proper direction and no further effort was made to walk in the other direction. Eventually, overlapping clones were isolated which spanned a total of 172 kb. The extent of the walk is shown in figure 9. A total of sixteen successful steps were required to extend from EMBL3-14P to a point beyond the region known to contain ph (see below). Thirteen of the phage originated from the EMBL4 Oregon R library, and the Charon 4a Canton S library was used to isolate three phage. -50-I Figure 8. Determining the orientation of the walk. In s i tu hybridizat ion of (a) C123 and (b) A26 DNA to polytene chromosomes. Arrows denote the position of s i l v e r grains. (c) A map of the walk to A26. The bottom l i n e shows the scale i n kb. The extent of each phage insert i s indicated by a s o l i d l i n e above. -52-Figure 9. Restriction map of the walk. The central line shows the scale in kb. The extent of each phage insert is indicated by a solid line above. The restriction sites for the enzymes Eco RI, Bam HI, Sal I and Xho I are indicated below. The Sal I restriction polymorphism at position 52 is indicated by a bracket. J 4 P , G25 ,L28 • C123 , F16 E23 .F27 ,J21 , A26 0 10 20 30 40 50 60 70 8 0 FcoRI i n I i 11 i • n i I I I I i i i • i i • i i i Ram HI i i • • • i i i i i 1 i i . . i S a l 1 i i • i (i) i i i i • i i Y h A 1 i I i i " i • i i • .J31 • F37 L28 . 030 E33 ,A36 .J29 , J35 t 90 100 110 120 130 140 150 160 170 EcoRI i i i I i i I I J—I 1 1 1 1 BamHI i i i i i i I 1—i u 1 1 Sal I I ' • i i I u i 1 i_i u 1— Xho I • • i i u I i I i—i 1 i 1 1—I—I 1 1 - 54 -Localization of Deficiency Breakpoints To determine the location of ph in the walk, i t was necessary to locate the position of the breakpoints of deficiencies known to be in the region: the proximal and distal breakpoints of Df(1)JA52, the distal breakpoint of Df(l)pn38 and the distal breakpoint of Df(l)Pgd-kz. As the walk was extended, phage were tested against Southern blots of DNA isolated from deficiency strain females and cut with each of the four enzymes used when mapping the phage (Bam HI, Xho I, Eco RI and Sal I). DNA from a wild type strain was included as a control. If a new band was detected in two or more of the Southern blots, then the process was repeated using restriction fragments from the phage as probes. Since a wild type chromosome was always present, no wild type bands were seen to disappear. However, a reduction in intensity of hybridization could often be seen for some bands. This reduction in intensity could be used to determine which fragments were to be used to localize the breakpoints more precisely. With this approach i t was possible to determine, within a few kb, the location of each of the four breakpoints. The strategy used to determine the location of each of the breakpoints is outlined below and in figures 10 and 11. The Df(1)JA52 distal breakpoint The phage A26 and F27 detected an anomolous fragment when hybridized to blots of Df(1)JA52 DNA cut with Eco RI and Xho I . The phage L28 did not detect the new fragment. The pattern observed using F27 as a probe is shown in figure 10. Two fragments from F27, the 3.6 and 7.5 kb Eco RI -55-EcoRI BamHI S a l I Xho I , A26 .FIT , 60 70 8 0 • i i i | 3.6 i 7.5 | i i • l tt I i l l I i i i i 6 .7 ll 4.0 12.01 4.2 . 4 . 0 . 5.6 + CN < eg in < Eco Rl Xho I Figure 10. Mapping deficiency breakpoints I. The relevant portion of the walk from f i g . 9 is reproduced. The sizes of fragments important to the interpretation of the Southern blots below are indicated. Fragments used as probes are indicated by dotted lines. DNA was prepared from wild type (+) and Df(1)JA52 (JA52) f l i e s , digested with the indicated enzymes, electrophoresed on agarose gels and blotted onto nitrocellulose. The autoradiogam on the left shows the pattern observed when the 7 . 5 kb Eco Rl fragment of F27 was hybridized to genomic DNA digested with Eco Rl. The phage F27 produced the pattern seen on the right when hybridized to genomic DNA digested with Xho I. - 56 -fragments, were isolated and used as probes. The 7.5 kb fragment, but not the 3.6 kb fragment, showed hybridization to a new band in blots of Df(1)JA52 DNA cut with Eco RI (figure 10b). Therefore the distal breakpoint of Df(1)JA52 is within the interval covered by the 7.5 kb Eco RI fragment. The D f ( l ) p n 3 8 distal breakpoint The D f ( l ) p n 3 8 breakpoint was f i r s t detected using the phage 030 as a probe. The 6.4 kb Xho I fragment was able to detect the anomolous fragments seen with 030 (figure l i b ) . The small size of the new Xho I fragment indicates that the breakpoint of Df(l)pn 3 8 must be towards the distal end of the 6.4 kb Xho I fragment. The Df(l)Pgd-kz distal breakpoint New bands were detected on genomic Southern blots when the phage J35 was used as a probe. The 1.5 Sal I fragment was shown to hybridize to these bands. The Df(l)Pgd-kz breakpoint should therefore l i e within the interval covered by the 1.5 kb Sal I fragment (figure 11c). The Df(1)JA52 proximal breakpoint The phage J35 also detected new bands in Southern blots of Df(1)JA52 DNA. The Df(1)JA52 breakpoint was localized to the 2.8 kb Bam HI fragment: This fragment was used as a probe in the blots shown in figure l i d , which identifies anomolous patterns of hybridization for Xho I and Sal I digests. The 1.6 Bam HI fragment immediately proximal to the 2.8 kb -57-Figure 11. Mapping deficiency breakpoints II. (a) The relevant portion of the walk from f i g . 9. The sizes of important restriction fragments are indicated. The restriction enzymes used to digest the genomic DNA is indicated below each lane. (b) Southern blots of wild type (+) and Df ( l ) p n 3 8 DNA with the 6.4 kb Xho I fragment used as a probe, (c) Southern blots of wild type and Df(l)Pgd-kz DNA with the 1.5 kb Sal I fragment of J35 used as a probe, (d) Southern blots of wild type and Df(1)JA52 DNA probed with the 2.8 kb Bam HI fragment of J35. (e) Southern blots of wild type and Df(l)pn 3 8 DNA probed with J35 DNA. (a) ,J31 030 E33 , A36 , J35 I ' 1 • • ' ' 110 120 130 140 150 E c o R I _ _ l I I 30,8 l L BamHI i LJ. • 3.s • s j I.JS^ 8.J_J u S a l I 22 | LJ ,1 .8 3.9 , i Xho I _ _ J . . . . . « . v 4 . I l a.7 , i • i 4 . 5 i 3 . 4 i i I Xho I Bam HI Sal I - 59 -Bam HI fragment did not reveal the anomolous fragments when used as a probe. It should be noted that hybridization to a fragment of 8.7 kb occurs in both the control and mutant Xho I genomic blots in figure l i d . This band is not predicted from the restriction map and is due to the presence of a repeated sequence which is discussed below. A Repeated Sequence shared by E33 and J35 When the phage E33 was hybridized to a blot of a gel used to map the next phage in the walk (J35), a hybridization pattern was observed which was inconsistent with the restriction map derived for J35. In addition to the hybridization predicted for the region of overlap, homology between E33 and the proximal portions of J35 was observed. This pattern can be explained i f there is a sequence in E33 which is repeated in the non-overlapping portion of J35. The location of the repeated sequence is reflected in the pattern of restriction sites in the region. The detailed characterization of this repeat w i l l be reported elsewhere (Dura et a l . , 1987). The extent of this repeat, as revealed by these studies, is shown in figure 12. Restriction Site Polymorphism Only one restriction site polymorphism was encountered, at the region of overlap of the clones G25 and E23. G25, derived from an Oregon R stock., contained a Sal I site in this interval while E23, derived from Canton S, did not. The location of this polymorphic site is indicated in figure 9. - 6 0 -T • t> c ; d ! e a :b:c: :d e: E E X E BSX SS B S S X B X S S X X B X B E B X B | 1 1 1 t 111 1 1 " " " ' ' ' , ' ' ' ' " ' | 110 120 130 140 150 38 - - Pn JA52 - -t— . _ P g d - k z Figure 12. Map of deficiency breakpoints and a repeated sequence. The s o l i d l i n e shows the coordinates i n kb and the r e s t r i c t i o n map of the region. E = Eco Rl , B = Bam HI, S = Sal I and X = Xho I. The p o s i t i o n of the 2 kb i n s e r t i o n at position 140 i n FM7c DNA i s indicated by the tri a n g l e below. The extent of d e f i c i e n c i e s i n this region i s shown by the s o l i d black l i n e s below. JA52, p n 3 8 and Pgd-kz refer to Df(1)JA52, D f ( l ) p n 3 8 and Df(l)Pgd-kz. Open boxes are used to show uncertainty i n the loc a t i o n of breakpoints. The boxes above the r e s t r i c t i o n map indicate the extent of the repeated sequence as determined by Dura e_t a l . (1987). Fragments with the same l e t t e r designation share c l o s e l y related sequences as revealed by cross-hybridization studies; the fragments marked by black arrows were r a d i o l a b e l e d and shown by Southern blot analysis to hybridize to fragments with the same l e t t e r designation. - 61 -Insertional Polymorphism A l l strains carrying the balancer chromosome FM7c but not those carrying FM6, showed an altered pattern of bands on Southern blots when the phage J35 was used as a probe. The pattern can best be explained as resulting from an insertion of approximately 2 kb at position 140 of the walk. The location of this insertion is indicated in figure 12. The assignment of this insertion to the balancer chromosome is supported by the pattern observed for Df(l)pn 3 8/FM7c. On the deficiency chromosome, this region is deleted. The restriction fragments must be derived only from the balancer chromosome, FM7c. See figure l i e . - 62 -DISCUSSION Overlapping clones spanning 172 kb were isolated in a Drosophila  melanogaster chromosomal walk. After a total of sixteen successful steps, the walk extended past the region of the chromosome known to contain the locus polyhomeotic; the breakpoints of deficiencies bracketing gh were revealed by Southern blot analysis. The order in which these deficiency breakpoints were encountered is the same as the order expected from genetic analyses (Dura et a l . 1985;1987). The total length of the walk is close to the value of 170 kb estimated previously (see introduction), indicating that the size of the chromomeric units in the interval 2C1-2 to 2D3 can be estimated using the values of Spierer et a l . (1983). It is interesting to compare the gh walk with other Drosophila walks. The walk is of average length in comparison to other reported Drosophila walks, which range in size from 80 kb for the cloning of Notch (Artavanis-Tsakonas et a l . , 1983) to 315 kb for the walk through the BX-C (Bender et a l . , 1983). Since the total haploid length of the Drosophila genome is estimated to be 1.65 x 10-> kb, the ordered clones which I have isolated represent approximately 0.1% of the genome. The speed at which other walks progressed is d i f f i c u l t to ascertain. The time required to complete one step in a walk varies depending on the set of procedures followed and the manpower available. Using the procedures outlined in Materials and Methods, I completed the walk in eleven months. A successful step was typically completed in fourteen days. The minimum time required to complete a step was nine days. Pirotta (1986), using a different set of procedures, produced a typical timetable for one cycle of a chromosomal walk; under optimal conditions, the time - 63 -required for one step is nine days. If DNA fragments containing repeated sequences are used as probes in a chromosomal walk, phage w i l l be isolated which have homology to the repeated sequence but which originate from elsewhere in the genome. The walk may be delayed until a suitable unique sequence can be selected for use as a probe in the next step. Hadfield (1983) and Pirotta (1986) recommend the routine screening of a l l isolated DNA for repeated sequences; phage DNA is digested with restriction enzymes, separated by electrophoresis, blotted onto nitrocellulose and hybridized to radiolabelled whole genomic DNA. Fragments which contain repeated sequence are detected by autoradiography, since repeated sequences are represented more frequently in the radiolabeled genomic DNA. I did not use this procedure routinely, since repeated sequences did not hinder the progress of the walk. Only two restriction fragment length polymorphisms were encountered in the 172 kb of DNA comprising the walk. Bender et a l . (1983) reported numerous polymorphisms in their walk through the BX-C; many of the polymorphisms were detected as differences between Oregon R and Canton S strains. Their walk was conducted using both Canton S and Oregon R libraries for the entire length of the walk. In contrast, the walk described here consists mainly of DNA from Oregon R with only a few clones isolated from a Canton S library. One of the polymorphisms was detected as a difference between the two strains in a region of overlap. It is possible that more polymorphisms would have been revealed i f both libraries were used throughout the walk. The second polymorphism observed was due to the presence of an insertion in the balancer chromosome FM7c. The origin of this insertion is unknown. If the segment containing the insertion were cloned, i t would be possible to compare i t by cross-- 64 -hybridization to previously characterized transposable elements. The region spanned by the walk contains numerous loci identified by Perrimon et a l . (1985) including corkscrew and ultraspiracle. The locus Phosphogluconate dehydrogenase should also be included in the region proximal to the Df(l)Pgd-kz breakpoint. A comparison of the restriction map of a clone containing Pgd with the restriction map from the walk shows that Pgd is contained within the phage A36 (Gutierrez, personal communication). A portion of the walk, the region between the distal D f ( l ) p n 3 8 breakpoint and the proximal Df(1)JA52 breakpoint, must contain polyhomeotic. The detailed search of this region for genetic lesions associated with ph alleles is the subject of the next chapter. - 65 -CHAPTER 3 INTRODUCTION Genetic analysis of gh_ has shown that the gene is complex. Null mutations were produced only when a hypomorphic al l e l e was subjected to a second round of mutagenesis (Dura et a l . , 1987). One possible interpretation is that there are two separately mutable regions within the ph locus. Only i f both regions are mutated is a gh null produced. The gh locus may actually contain duplicate genes, each with similar or identical function. This possibility is supported by the discovery of a sequence that is repeated within the interval known to contain ph. The genetic evidence also suggests that the gh locus may be large. The locus was found to be highly mutable in comparison to other loci contained in the interval deleted by Df(1)JA52. There are a total of five complementation groups, including gh, in this region (figure 4). In mutagenic screens designed to isolate mutants in the Df(1)JA52 interval, a total of twenty-five gh mutants were produced. In contrast, the number of mutants for each of the other four complementation groups ranged from one to seven (Dura et a l . , 1987). The gh locus therefore appears to be more susceptible to the action of the mutagens than the other loci in the region. This susceptibility suggests that ph occupies more of the Df(1)JA52 interval than the other loci and therefore offers a larger target for mutagenesis. Alternatively, some other physical property of the locus may make i t more available for the mutagenic activity of these mutagens. The disparity between the number of gh mutants and the numbers obtained for other loci becomes more pronounced when one looks only at the - 66 -interval between the proximal breakpoint of Df(1)JA52 and the dis t a l breakpoint of Df(l)pn 3 8. In chapter 2, this interval was shown to measure approximately 35 kb. This region contains only ph and csw. Only six csw mutants were produced in the screens which produced twenty-five gh mutants. If the large number of gh mutants is an indication of the size of the locus, then ph should occupy much of the 35 kb between the two deficiency breakpoints. In the same way as i t is employed to locate deficiency breakpoints, Southern blotting can be used to identify mutations which have altered the arrangement of restriction sites within a locus. One must be careful to choose alleles which are likely to contain rearrangements large enough for detection by this method; point mutations are rarely detected. The best mutants for this purpose are those which have been induced by mutagens which usually cause larger aberrations such as insertions or deletions. I examined ph alleles which had been induced by X-rays or by P-M mutagenesis, since these mutagens are more likely to effect gross changes in DNA ( G r i g l i a t t i , 1986; Kidwell, 1986). - 67 -MATERIALS AND METHODS A l l methods were as described in Chapter 2. Drosophila stocks Flies were reared as described in Chapter 2. ph 2 and pJy^Ol were maintained as homozygous stocks, ph 3 and ph? were maintained over FM6. pnA00> ph^03 a n c j were maintained over FM7c. Males carried the translocation Yw+. A l l DNA was prepared from females. - 68 -RESULTS Localization of polyhomeotic Mutants To determine the location of DNA arrangements associated with gh mutations, DNA was extracted from f l i e s homozygous (gh 2, gh 4 0 1) or heterozygous (gh 3, gh 7, gh 1 0, g h 4 0 0 , g h 4 0 3 , gh 40 4) for different alleles. The DNA was digested with the restriction enzymes Bam HI, Eco Rl Sal I or Xho I, electrophoresed on agarose gels and transferred to nitrocellulose. The phage J31 and J35, which together contain most of the DNA in the interval known to contain gh, were used to screen these genomic Southern blots. For many alleles, anomalous bands were detected with the phage J35 but not with J31. For two alleles (ph 2 and ph401), anomalies were detected with both phage. Restriction fragments from J35 were used as probes to further delimit the location of most rearrangements. If a probe detected anomolous fragments in Southern blots prepared with several different enzymes, then that probe was assumed to originate from a region of rearrangement. The results obtained from Southern blot analysis are summarized below and in figures 12 and 13. ph 2: This a l l e l e was induced by hybrid dysgenesis. Southern blot analysis shows that there is a deletion of approximately 10 kb in ph 2 f l i e s . Since DNA was prepared from homozygotes, the loss of certain restriction fragments is obvious in the blots for which J35 was used as a probe (figure 13a). No new bands were observed in any of the blots examined. Much of the lef t hand repeat appears to have been deleted (see discussion). -69-Figure 13. Southern blot analysis of gh mutants. Fragment sizes are indicated to the left of each blot. The restriction enzyme used is indicated below each lane. (a) A reproduction of the relevant portion of figure 9. Fragments used as probes are indicated by dotted lines. The sizes of important fragments are indicated, (b) Southern blot analysis of wild type, gh 2, ph^ OO and gh^ul (abbreviated as + , 2, 400 and 401) probed with J35. The pattern observed for gh^O differs from wild type due to the presence of the 2 kb insertion at position 140 in both the gh^OO and FM7c chromosomes. The gh^Ol chromosome also carries the insertion at this position, (c) Southern blot analysis of wild type, ph 3 and ph? (abbreviated as +, 3 and 7) probed with the 2.2 kb Bam Hl/Xho I fragment, (d) Southern blot analysis of wlid type, ph 3 and gh? probed with the 2.8 kb Bam HI fragment. - T O -E33 , A36 LJ35 E c o Rl BamHI S a l I Xho I 120 130 140 30.S 1S0 1 1 l 6.7 i 3.5 i 8.0 1 2.8 in.51 II i 1 1 3.9 i1 .8 3.8 i i 15.6 1 8.7 ,1.81 4.3 ,2.0, 4.5 i 3.4 l l 1 Bam HI Sal I Xho I + 3 7 + 3 7 3 305-7.4-1 5 . 5 -1 3 . 0 -10.5-'-8 7 - • 6 0 -3.4-3 . 2 -Eco Rl Sal I Xho I - 71 -ph401. This a l l e l e was induced by ionizing radiation. Like ph 2, ph^Ol is homozygous viable, pJi^Ol also contains a deletion in the same region as ph 2. However, fewer bands were missing when J35 was used as a probe. The deletion spans approximately 7 kb. An insertion of 2 kb at position 140, f i r s t noticed in FM7c (see chapter 2), is also present in ph^Ol. p h 7 : This allele was induced by hybrid dysgenesis. New bands became visible when J35 was used as a probe. When the 8.9 kb Bam HI fragment of J35 was used as a probe, both a 10.5 kb and a 8.9 kb fragment were detected, indicating that there is an insertion of approximately 1.5 kb in this region. J35 also revealed the presence of two new Sal I fragments of 11.0 and 6.0 kb. A 2.2 kb Bam Hl/Xho I f ragment isolated from the proximal end of the 8.9 kb Bam HI fragment was found to hybridize to the 6.0 kb Sal I fragment. The 2.8 kb Bam HI fragment of J35 hybridized to the 11.0 kb Sal I fragment. The wild type 15.5 kb Sal I fragment in this region therefore contains an insertion of approximately 1.5 kb in the right hand repeat of the ph 7 chromosome. This insertion carries a Sal I site, resulting in the replacement of the 15.5 kb fragment by the 11 and 6.0 kb Sal I fragments. The insertion must be toward the proximal end of the 8.9 kb Bam HI fragment since the 2.2 Bam Hl/Xho I fragment derived from i t did not hybridize appreciably to the new 11.0 kb Sal I fragment. ph 3: This a l l e l e was also induced by hybrid dysgenesis. Hybridization to J35 revealed two new bands from Eco Rl, Sal I and Xho I Southern blots, while one new band was revealed for Bam HI blots. The 2.2 kb Bam Hl/Xho I fragment hybridized to the new 12 kb Bam HI band, indicating the presence of an insertion of approximately 3 kb into the wild type 8.9 kb Bam HI - 72 -fragment. The 2.2 kb Bam Hl/Xho I fragment also hybridized to one of each of the two new Eco RI, Sal I and Xho I fragments. The 2.8 kb Bam HI fragment of J35 hybridized to the other new Eco RI, Sal I and Xho I fragments. Taken together, these results suggest the presence of an insertion of approximately 3 kb, located like pji? near the proximal end of the 8.9 kb Bam HI fragment, and containing sites for the enzymes Eco RI, Sal I and Xho I. p}1400> ph403> £h404. Using J31, J35 or A36 as probes, no deviation from wild type could be detected for these X-ray induced alleles, with the exception of the 2 kb insertion at position 140. The pattern observed for pjj400 i s included in figure 13. -73-a ib? c • d ! e a itxc. : d e: E x E Etsx s s BS 11 SXBX - I U L S S X X . X B EB X B I I II M 110 i 120 130 1 140 150 38 JA52 - -i i - - Pgd-kz 401 , -25- 3 2 m -zr- 7 Figure 14. Map of ph rearrangements. The map in figure 12 is reproduced with the addition of lines below to indicate the position of rearrangements associated with ph 2 , D h 3 , ph7 a n d £11401, Triangles represent insertions and black lines represent deletions. The open boxes indicate uncertainty in the location of breakpoints. - 74 -DISCUSSION Four of the seven gh mutants examined contained DNA rearrangements which could be detected by Southern blot analysis. A l l rearrangements l i e within the interval predicted by genetic studies. The rearrangements map to two regions within this 35 kb interval. These two regions correspond to each of the two repeated sequences identified in chapter 2. Although conclusive evidence is s t i l l required (see general discussion), the results from Southern blot analysis suggest that the repeated sequences found in this region result from the duplication of a locus, and that the locus is gh. The presence of two genes, each with the same or similar function, would explain the need for two mutational events in the creation of a null mutant. Other predictions were confirmed with the localization of these rearrangements. The rearrangements are found over an interval of 20 kb, indicating that ph occupies most of the region between the distal breakpoint of D f ( l ) p n 3 8 and the proximal breakpoint of Df(1)JA52. The location of the Df(l)Pgd-kz distal breakpoint with respect to the gh rearrangements is also as predicted. Df(l)Pgd-kz overlaps those mutations which affect only the right hand (proximal) repeat. The position of this breakpoint is consistent with the designation of Df(l)Pgd-kz as a hypomorphic allele of ph. Two alleles have deletions in the left hand (distal) repeat. In ph 2, approximately 10 kb are deleted, while the deletion associated with pjv401 removes approximately 7 kb. The deletion of DNA in gh^Ol is consistent with its origin from X-ray mutagenesis. However, ph 2 was isolated from a hybrid dysgenic screen. The P element-containing phage 14P was isolated - 75 -from a library constructed using this strain (chapter 1). The molecular evidence confirms that no P elements are inserted in this strain in the vicinity of gh. The failure to obtain revertants is also explained. The amount of DNA removed by the gh 2 deletion corresponds to the distance between the two repeated sequences. The deletion appears to have fused the lef t and right hand repeats. It is not possible from Southern blot analysis to determine the exact fusion point of the repeats. However, the data are consistent with the existence of a deletion with breakpoints in or near the 0.8 kb Sal I fragments present in each of the repeats (figure 10). Since ph 2 is viable, this rearrangement must produce a functional a l l e l e , even though both repeats are affected. It is possible that the ph 2 a l l e l e originated from a recombination event. Hybrid dysgenesis has been shown to result in deletions and chromosomal rearrangements (reviewed by Kidwell, 1986). Two other alleles isolated from dysgenic screens, ph3 and gh?, contain insertions. Each of these insertions occurs in the same region of the right hand repeat. For the ph 3 insert, the presence of sites for the restriction enzymes Sal I, Eco RI and Xho I (as revealed by Southern blot analysis) matches the pattern of sites determined for the P element (O'Hare and Rubin, 1983). However, positive identification of the origin of the gh 3 and ph? insertions would require the cloning of the region from each strain and the hybridization of these sequences to characterized mobile elements. Three other X-ray induced alleles, gh^O, gh^ 0 3 and gh^O^, showed no rearrangements detectable by Southern blot analysis. Molecular studies of X-ray induced alleles at the rosy (Cote e_t a l . , 1986) and Alcohol  dehydrogenase (Kelley e_t a l . , 1985) loci have shown that such alleles often do not have detectable rearrangements. - 76 -The results from Southern blot analysis therefore support the hypothesis that the gh locus consists of two separately mutable regions. One region is proximal to the Df(l)Pgd-kz breakpoint and the second is dist a l to the breakpoint. With the exception of gh 2, each of the hypomorphic alleles examined showed rearrangements confined to one of the two regions. If two mutagenic events are required to produce a null mutation, then Southern blot analysis of such mutants should reveal rearrangements in both regions. Unfortunately, no null alleles were available at the time this study was undertaken. It is interesting to note that the 2.0 kb insertion at position 140, discovered in FM7c (see chapter 2), does not produce a gh phenotype. This insertion is very close to the insertions found in ph 3 and gh 7. It is possible that this insertion occurs in an intron and does not affect the function of the gene. Alternate explanations for the structure of the gh locus exist. Possibly there is a single gene with a complex pattern of post-transcriptional splicing. The repeated regions may represent exons which are present in alternative transcripts. The repeated regions may represent alternate controlling regions for one gene. The procedures required to determine the structure of the gh locus are outlined in the general discussion. - 77 -GENERAL DISCUSSION As outlined in the previous chapters, I isolated DNA from the Drosophila melanogaster X chromosome containing the locus polyhomeotic. I demonstrated that hypomorphic ph alleles show altered restriction patterns which map to one of two regions. These two regions are related in sequence, as shown by similarities in restriction maps and by cross-hybridization studies. Genetic evidence shows that the £h locus is complex, since two mutagenic events are required to produce a null mutation. These observations can be explained i f ph consists of two mutable regions each with the same or similar function. The loss of one region results in the hypomorphic homeotic phenotype, whereas i f both copies are mutated, a null mutant is produced. Other duplicate loci have been reported in Drosophila species. The three larval serum proteins (LSP) are produced by duplicate genes at separate chromosomal locations in Drosophila melanogaster (Roberts et a l . , 1985). This contrasts with p_h, in which both putative copies are closely linked. Another important difference is revealed by the deletion of LSP genes. Deletion of one or two of the three LSP loci produces no change in phenotype (Roberts e_t a l . , 1985), in contrast to ph. Closely linked duplicate genes have also been reported. Included in this group are the larval serum protein genes of most Drosophila species (Brock and Roberts, 1983) and Alcohol dehydrogenase in Drosophila mulleri (Fischer and Maniatis, 1986). Recently, a duplicated sequence near the transformer locus of Drosophila melanogaster was described (McKeown e_t a l . , 1987). A tandemly duplicated sequence of approximately 8 kb was detected in the - 78 -course of the chromosomal walk towards transformer. As is the case with ph, the two copies of the repeat have similar, but not identical restriction maps. The repeat is transcribed, but its function is unknown. Analysis of this sequence in other species showed that i t is duplicated in Drosophila mauritania but is present as only a single copy in Drosophila simulans (McKeown et a l . , 1987). It would be of interest to determine whether the repeats present in the region of ph in Drosophila melanogaster are also present in related species. Future Work Future experiments can be divided into two groups: those designed to determine the extent and structure of the gh locus and those designed to determine the function of ph. For the second group of experiments to proceed, the boundaries of the gene must f i r s t be determined. Southern blot analysis of the DNA rearrangements of mutant alleles indicates the general position of a locus. The identification of transcripts by northern blot analysis (Maniatis et a l . , 1982) is the next step in determining the extent of a locus. RNA isolated at different stages of development and from different mutant strains should be electrophoresed on agarose gels, transferred to a membrane and probed with DNA from the region of interest. In this way, transcripts which are expressed at correct developmental stages (correlating to the expression of the gene as determined by genetic studies) and which are altered in mutant strains, can be identified. Such transcripts are most likely to originate from the locus in question. Northern blot analysis of the region containing gh has recently been completed (Dura et a l . , 1987). Subcloned DNA fragments from the phage - 79 -J31, J35 and A36 were used to probe northern blots prepared from embryos, larvae and adults. RNA was extracted from wild type f l i e s and from gh mutants. Several transcripts were identified from this region. However, only the region of the walk which contains the repeated sequence was found to hybridize to transcripts whose mobility was altered in gh mutants. Each of the repeats is transcribed, as shown by the presence of transcripts in mutants lacking either the le f t or the right hand repeat (Randsholt et a l . in preparation). Several sizes of transcript are found, indicating that a family of transcripts is produced by each of the repeats. Positive identification of the ph transcription unit(s) would be possible using P element-mediated transformation (Spradling and Rubin, 1982). Up to 40 kb of genomic DNA can be introduced into Drosophila embryos using vectors containing P element sequences required for integration. After injection, the DNA integrates by transposition into cells which w i l l form the germ line. Additional factor(s) necessary for • integration are provided in trans by other coinjected P elements. A helper P element has been engineered which provides these factors but which cannot i t s e l f integrate (Karess and Rubin, 1984) thus avoiding the continuing transposition which would result from integration of an intact P element. The chromosomal position of integrated sequences can be determined by in situ hybridization to polytene chromosomes. It is possible to demonstrate the presence of a required function within a specific sequence i f that sequence can be integrated into Drosophila mutants and shown to rescue the mutant phenotype. Wild type DNA from the ph region would be inserted into an appropriate P element vector and used to obtain germ line transformants of ph mutants. Progeny of the injected f l i e s would be tested by genetic crosses, in situ hybridization and - 80 -Southern blotting to determine the location of inserted sequences. The inserts would also be tested for their a b i l i t y to rescue the gh phenotypes. In this way, i t would be possible to demonstrate conclusively the location of gh within the interval characterized in this thesis. The structure of a locus can also be studied using SI nuclease mapping, to determine the location and extent of exons within the transcribed sequence (Maniatis ejt a l . , 1982). Labelled fragments from the ph region would be hybridized to RNA prepared from wild type f l i e s and then digested with SI, a nuclease which digests single stranded nucleic acids. Only exon sequences, which hybridize to the message, would be protected. The sizes of these fragments can be determined after electrophoresis on acrylamide gels and autoradiography. For gh, the study of transcripts by this method would be complicated by the presence of transcripts from each of the two repeats. It may be more efficient to perform SI analysis using gh mutants in which one of the two repeats is deleted. An alternate strategy for determining the location of exons is to study complementary DNA (cDNA) clones of gh transcripts. Using DNA fragments from the gh region as probes, cDNA libraries prepared from different developmental stages would be screened to isolate clones corresponding to a l l gh transcripts. Since the two repeats do not have identical restriction enzyme sites, the restriction sites of the DNA clones from each repeat should also differ. Extensive restriction mapping of the the genomic sequence and of the cDNA clones should allow the assignment of each cDNA to one of the two transcription units. DNA sequence analysis of both genomic and cDNA clones could then be performed to precisely determine which regions are represented in mature transcripts. - 81 -Sequence analysis is the f i r s t step in the investigation into the function of nh. The amino acid sequence of any ph protein, as determined from the DNA sequence, would be compared to other amino acid sequences for regions of homology. Discovery of regions of nh with homology to sequences with known enzymatic or structural function would imply that these regions have similar structural or enzymatic roles. Other techniques are available to investigate the function of the gh transcription unit(s). The temporal and spatial distribution of most transcripts can be determined by in situ hybridization of labelled probes to tissue sections of Drosophila embryos and larvae (Hafen et a l . , 1983). in situ hybridization has been used to determine the transcript distribution of numerous homeotic and segmentation genes, including ftz (Hafen et a l . , 1984), scr (Kuroiwa et a l . , 1985), Antp (Levine et a l . , 1983) and Ubx (Akam, 1983). Using this technique, i t should be possible to determine the distribution of ph transcripts in wild type and gh mutant embryos. These experiments should reveal the location(s) and the stage(s) of maximal gh transcript accumulation. It would be interesting to determine the pattern of expression revealed by in situ hybridizations within the regions most affected by the gh phenotype. The characterization of cDNA clones should reveal which fragments could be used as transcript-specific probes. Using these fragments as probes, the distribution would be determined for each member of the gh family of transcripts. Once the distribution of gh transcripts in wild type embryos is determined i t would be important to determine the distribution of these transcripts in other Drosophila mutants. In particular, the distribution of gh transcripts in mutants of the BX-C, the ANTP-C and the other members of the Pc-group should be investigated. The reciprocal experiments, in - 82 -which gh mutants are tested for distribution of transcripts from the above mentioned genes, should also be performed in those cases where suitable probes are available. The distribution of the ph protein product(s) at different l i f e stages should also be determined. Antibodies to the protein(s) would be prepared, allowing the detection of ph protein distribution by indirect immunofluorescence. Using this technique, i t may also be possible to determine the intracellular location of the gh protein(s) at certain stages of development. Genetic analysis has shown that polyhomeotic locus of Drosophila  melanogaster plays an important role in segment determination and cuticular development. As described above, experiments to test the function of gh are now possible. 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