UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Molecular analysis of the Drosophila gene, Polyhomeotic Freeman, Sally Jean 1988

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1988_A6_7 F64_3.pdf [ 9.22MB ]
Metadata
JSON: 831-1.0097620.json
JSON-LD: 831-1.0097620-ld.json
RDF/XML (Pretty): 831-1.0097620-rdf.xml
RDF/JSON: 831-1.0097620-rdf.json
Turtle: 831-1.0097620-turtle.txt
N-Triples: 831-1.0097620-rdf-ntriples.txt
Original Record: 831-1.0097620-source.json
Full Text
831-1.0097620-fulltext.txt
Citation
831-1.0097620.ris

Full Text

MOLECULAR ANALYSIS OF THE DROSOPHILA GENE, POLYHOMEOTIC By SALLY JEAN FREEMAN B . S c , The U n i v e r s i t y o f A l b e r t a , 1968 A THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department o f Zoology) We accept t h i s t h e s i s as conforming t o the r e q u i r e d s t anda rd THE UNIVERSITY OF BRITISH COLUMBIA JUNE 1988 © S a l l y Freeman, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, 1 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. The University of British Columbia Vancouver, Canada Department of DE-6 (2/88) ABSTRACT Polyhomeotic (ph) i s a developmentally important gene i n Drosophila melanogaster which has been genetically characterized and recently cloned. p_h i s genetically and molecularly complex and has a strong maternal e f f e c t . Analysis of n u l l or amorphic a l l e l e s reveal phenotypic e f f e c t s that include embryonic l e t h a l i t y , c e l l death of the ventral epithelium, homeotic transformations, and a l t e r a t i o n i n the pattern of axon pathways. Two independent point mutations are required to produce a p_h n u l l a l l e l e . I have shown that the pji locus contains two, large, highly conserved, tandem repeats that are both transcribed. I have i d e n t i f i e d t r a n s c r i p t s that are altered i n p_h mutants and that are developmentally regulated. Fourteen cDNA's have been isolated, and mapped. Northern and Southern blot analysis, and comparisons between cDNA and genomic r e s t r i c t i o n maps shows that the cDNAs represent at least 4 d i f f e r e n t t r a n s c r i p t s that include d i s t i n c t products of both repeats as well as non-repeated sequence. Both the genetic behavior and molecular organization of the p_h locus are unique i n Drosophila. i i : TABLE OF CONTENTS PAGE ABSTRACT i i . TABLE OF CONTENTS '- " i i i LIST OF TABLES iv LIST OF FIGURES v. ACKNOWLEDGMENT v i ABBREVIATIONS v i i GENERAL INTRODUCTION 1 CHAPTER 1 : The p_h locus contains two, large tandem repeats as determined by cross-hybridization. INTRODUCTION 11 MATERIALS AND METHODS 15 RESULTS 21 DISCUSSION 33 CHAPTER 2 : Transcription of the p_h locus - Northern Analysis. INTRODUCTION 38 MATERIALS AND METHODS 42 RESULTS 4 6 DISCUSSION 65 CHAPTER 3 : Isolation and analysis of p_h cDNAs. INTRODUCTION 70 MATERIALS AND METHODS 74 RESULTS 78 DISCUSSION 97 GENERAL DISCUSSION 100 REFERENCES 103 i i i LIST OF TABLES TABLE I. Legend for Figure 4(a). TABLE II. Cross-hybridization of ph r e s t r i c t i o n fragments. TABLE I I I . Transcription of p_h and adjacent genes. TABLE IV. ph cDNAs. LIST OF FIGURES PAGE FIGURE 1.. Complementation and mutagenesis analysis. 8 FIGURE 2. Map of deletions and a l l e l e s . 13 FIGURE 3. Subcloned DNA. 23 FIGURE 4. Mapping of the repeat containing domains. 26 FIGURE 5. The repeat containing domains. 2 9 FIGURE 6. Northern analysis of wild-type embryos. 4 9 FIGURE 7. Transcription units of the ph, region. 51 FIGURE 8. Northern analysis of p_h mutants. 56 FIGURE 9. Developmental p r o f i l e of p_h t r a n s c r i p t i o n . 62 FIGURE 10. Screening the cDNA l i b r a r i e s . 80 FIGURE 11. Determining the genomic location of three cDNAs. 85 FIGURE 12. Fragments of genomic DNA to which cDNAs hybridize. 88 FIGURE 13. R e s t r i c t i o n maps of cDNAs. 92 Figure 14. Northern analysis of p_h cDNAs. 96 v ACKNOWLEDGMENT I would l i k e t o thank: My s u p e r v i s o r , Dr. H. Brock, f o r h i s e n t h u s i a s t i c encouragement, e x c e l l e n t p r o f e s s i o n a l a s s i s t a n c e , a d v i c e , and s u p p o r t . Dr. R. Lansman and Doug Freeman f o r a d v i c e and e x p e r t t e c h n i c a l a s s i s t a n c e . Dr. L. Kauvar and Dr. S. P o o l e f o r g i f t s o f cDNA l i b r a r i e s . v i ABBREVIATIONS Abbreviations used i n t h i s text are those accepted as standard by the Proceedings of the National Academy of Sciences (USA), pp. v i - v i i (1987). v i i GENERAL INTRODUCTION The objective of t h i s thesis to conduct a molecular analysis of the polyhomeotic (ph) locus of Drosophila melanooaster. Ph i s one of almost one hundred genes that have been analyzed and are known to be required i n the embryo for normal development (review: Akam, 1987; Jurgens, 1985). The central problem of developmental biology i s how the one dimensional information stored i n the DNA and RNA of an egg i s converted into the three dimensional structure of a developing embryo. This continuum of pattern formation must be the res u l t of a developmental program that consists of a precise s p a t i a l and temporal pattern of gene expression. The program ultimately results i n the correct expression of the thousands of genes that are required for the normal development of a complex m u l t i c e l l u l a r organism. The f r u i t f l y i s an excellent organism for investigating t h i s problem because of i t s well characterized developmental pattern, and the combination of genetic and molecular techniques that can be u t i l i z e d . In 198 6 H. Mienhardt proposed that, i n Drosophila, early embryogenesis i s controlled by a network of genes arranged i n a h i e r a r c h i c a l manner. The s p e c i f i c interactions between these genes ensures the proper timing of developmental events and generates the proper pattern. S o l i d support for t h i s model has been accumulating rapidly (Jackie et a l . , 1986; Frasch and Levine, 1987; Edgar and Odell, 1988; and Ingham et a l . , 1988). 1 In order to provide a temporal and morphological framework for the molecular analysis of gene expression discussed here I w i l l give a b r i e f introduction to Drosophila development. In Drosophila the zygote nucleus i n i t i a l l y undergoes 1 3 mitotic d i v i s i o n s . The daughter nuclei migrate to the periphery to form the s y n c i t i a l blastoderm at about 2 hours p o s t - f e r t i l i z a t i o n . At the fourteenth d i v i s i o n c e l l u r i z a t i o n occurs and results i n the c e l l u l a r blastoderm at about 2 . 5 hours. Gastrulation begins at 3 hours. At t h i s stage the mesoderm invaginates ventrally, and the anterior and posterior midgut invaginations form the endoderm. The germ band begins to move dorsally and then a n t e r i o r l y to form the f u l l y extended germ band at about 4 . 5 hours. The e a r l i e s t and most s t r i k i n g feature of embryonic pattern i s the establishment of repeating units or metameres that w i l l give r i s e to the segmented body plan. The f i r s t external sign of segmentation i s a series of ventral bulges that become v i s i b l e at about 3 . 5 hours. Germ band lengthening i s followed by the reverse movement, shortening, whereby the l a s t or eighth segment comes to l i e at the posterior end. Next, the gut i s formed and the head structures involute. These changes are complete by 12 hours and are followed by rapid c u t i c l e depostion over the entire external surface. The remaining 8 - 1 0 hours of embryonic development are devoted to the further d i f f e r e n t i a t i o n of the in t e r n a l organs and external c u t i c u l a r processes. Also, the delimitation of imaginal c e l l s from surrounding c e l l s takes place. Emergence of the f u l l y formed larva from the egg at approximately 2 2 hours marks the sta r t of postembryonic 2 development. Postembryonic development consists of 3 consecutive l a r v a l instars, followed by a prolonged pupal stage i n which the animal f i n a l l y transforms into the adult. More detailed descriptions of Drosophila embryogenesis and development are provided by Turner and Mahowald, (1976, 1977, 1979); Lohs-Schardin et al_., (1979); and Campos-Ortega and Harenstein, (1985). The study of the regulation of the events described above has been made possible by the i d e n t i f i c a t i o n of mutations i n approximately 50 genes that f a l l into three categories (review: Akam, 1987). The f i r s t group of genes determines the early p o l a r i t y of the embryo and sp e c i f i e s p o s i t i o n a l information i n a global manner. The products of these l o c i are deposited i n the egg before f e r t i l i z a t i o n and these genes are therefore maternal-effect genes (Nusslein-Volhard, 1979; Anderson and Nusslein-Volhard, 1984; and Schupbach and Wiechaus, 1986). The second group of genes i s responsible for the d i v i s i o n of the embryo into segments. These segmentation genes are zygotic-effect genes (Nusslein-Volhard and Wiechaus, 1980); and can be further divided into three classes based on the s p e c i f i c types of pattern defects observed i n mutant embryos: (1) the gap mutations cause deletions of contiguous groups of segments; (2) the p a i r - r u l e mutations cause periodic pattern deletions spaced at two segment i n t e r v a l s ; and (3) the segment p o l a r i t y mutations aff e c t the patterning within segments. The t h i r d group, the homeotic genes, are required for the 3 determination of segmental i d e n t i t y . Mutations i n 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 i d e n t i f i e d . The bithorax complex (BX-C) contains genes which are required for the normal development of the thoracic and abdominal segments (Lewis, 1 9 7 8 and reviewed by Peifer, 1 9 8 7 ) . The Antennapedia complex (ANT-C) contains genes which are essential for the development of the thorax and head. (Kaufman et a l . , 1 9 8 0 , Lewis, 1 9 8 0 and Schneuwly et a l . , 1 9 8 6 ) . The f i n a l i d e n t i t y of a segment i s ultimately under the control of at least one of the homeotic genes. In general, the tra n s c r i p t s and proteins for a s p e c i f i c gene of the BX-C or ANT-C are concentrated i n the segment most affected by mutations i n that gene (review: Akam, 1 9 8 7 ) . For t h i s reason a great deal of e f f o r t has been put into elucidating the regulatory processes that r e s t r i c t the expression of these genes. There appear to be at least three levels of regulation. F i r s t , the genes of the two complexes are controlled by a hierarchy of cross-regulatory interactions. Products of the BX-C appear to negatively regulate the expression of ANT-C genes (Hafen et a l . , 1 9 8 4 and Harding et a l . , 1 9 8 5 ) . Wedeen et a l . , ( 1 9 8 6 ) have shown that one gene of the BX-C, Ultrabithorax (Ubx) may be negatively regulated by the other BX-C genes, abd-A and abd-B. A second type of regulation i s demonstrated by the segmentation genes. Fushi tarazu ( f t z ) , a p a i r - r u l e gene, i s required either d i r e c t l y or i n d i r e c t l y for the correct 4 a c t i v a t i o n of the BX-C and ANT-C genes. This has been demonstrated genetically (Duncan, 1986) and by examining the d i s t r i b u t i o n of ANT-C and BX-C tr a n s c r i p t s i n f t z ~ embryos (Ingham and Martinez-Arias, 1986). Hunchback (hb), a gap gene, has been shown to affect the early d i s t r i b u t i o n of Ubx (White and Lehmann, 1986). F i n a l l y , i t has been shown that another group of genes that do not belong to any of the above categories are important in the regulation of the homeotic genes. Lewis, 1978, provided genetic evidence and 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, now known as the Pc-group genes, which may control the s p a t i a l expression of both BX-C and ANT-C genes (Jurgens, 1985). Included i n t h i s 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 mid lea (Scm) (Jurgens, 1985); 1(4)29 (Duncan, 1982); and polyhomeotic (ph), (Dura et a l . , 1985). A l l cause homeotic transformations i n embryos and adults similar to those described for certain gain of function a l l e l e s of the BX-C and ANT-C. These homeotic transformations occur because of inappropriate expression of BX-C and ANT-C, rather than being the d i r e c t e f f e c t of the Pc group mutations (Struhl, 1981; Duncan and Lewis, 1982; Duncan, 1982; Ingham, 1984; and Dura et a l . , 1985). The homeotic transformations of ind i v i d u a l Pc group genes are strongly enhanced i n individuals mutant for any 5 p a i r of Pc group genes (Hannah-Alava, 1958/ Jurgens, 1985; and Dura et al.,- 1985), suggesting that Pc group genes form a complex regulatory network. Molecular evidence indicates that the Pc-group genes may not be responsible for the establishment of BX-C and ANT-C expression patterns, but rather may act to maintain the established pattern of expression. The i n i t i a l d i s t r i b u t i o n of ANT-C and BX-C tr a n s c r i p t s i s unaffected i n esc" embryos, but la t e r i n development ANT-C and BX-C tr a n s c r i p t s accumulate inappropriately i n both esc" (Struhl and Akam, 1985), and Pc~ embryos (Wedeen et a l . , 1986). These results support the hypothesis that Pc-group genes cause homeotic transformations v i a inappropriate expression of homeotic genes, and suggest that they are required to ensure the s t a b i l i t y of segment-specific expression once i t i s established. Polyhomeotic i s a member of the Pc-group of genes. Early genetic studies i d e n t i f i e d hypomorphic a l l e l e s that show homeotic transformations i n adults including the appearance of sex combs on the second and t h i r d legs, and posterior transformation of the thoracic and abdominal segments. Also, these p_h a l l e l e s are sensitive to the wild type dose of BX-C and ANT-C genes and interact with Pc., Pel and esc to enhance the homeotic transformations. (Dura et al., 1985). To accurately deduce the function of a wild type gene i t i s necessary to study, the n u l l or amorphic (Muller, 1932) phenotype. The results of a screen for n u l l mutations are given i n Figure 1. It can be seen that r e l a t i v e to other complementation groups i n the region, ph. i s highly mutable and 6 Figure 1. Complementation and mutagenesis analysis of the ph region. The name of each complementation group and i t s position r e l a t i v e to to the def i c i e n c i e s i s indicated at the top of the figure. The order of complementation groups included i n brackets cannot be determined from the available data. Below each complementation group are the number of a l l e l e s induced with EMS and with X-rays, and the t o t a l number of a l l e l e s i s indicated, with the number of viable a l l e l e s given i n brackets. At the bottom right i s the number of mutant chromosomes analyzed with each mutagen. Over three times as many ph. a l l e l e s were recovered r e l a t i v e to any other complementation group. Two l i n e s are drawn below the p_h locus to suggest that two separate mutagenic events are required to obtain an amorphic or l e t h a l a l l e l e . (From Dura et a l . , 1987). 7 |(1)DF967 1(1)107 1(1)405 KDcsw 210 ph KDPgd l(1)C204 i i i i i i l l I ' 1 1 1 ' } j j ' Df(1)JA52 ] [ • j ' ' | n " — - • D f ( 1 ) p n " j i ! i ' I n : ** » Df(1)Pgd-kz J » ! i [ • ! i i it -•Df(1)64c1t 7 2 0 3 1 18 EMS 6100 0 1 1 3 0 6 RX 10200 7(1) 3(0) 1(0) 6(1) 1(1) 24(24) 16300 completely lacking n u l l mutations. Null mutations were not obtained u n t i l an e x i s t i n g p_h mutation was subjected to a second round of mutagenesis. Embryos that have only the n u l l a l l e l e (ph~) develop normally u n t i l about 10 hours post-f e r t i l i z a t i o n and then die at about 12 hours. The presence of V a r t l i k e ' sensory organs, that are normally seen only i n the eighth abdominal segment, i n a l l the segments suggest homeotic transformations. The embryos f a i l to develop ventral c u t i c l e . None of the other members of the Pc-group, except 1(4)29, has been shown to be required for c u t i c u l a r development. An extreme hypomorph was i s o l a t e d that exhibits posterior transformation of most segments towards the eighth abdominal segment. This i s si m i l a r to the phenotype of Pc~ (Dennell and Frederick, 1983) and esc" (Struhl, 1981) embryos. Smouse et al. (1988) have examined the developing central nervous system (CNS) of ph~ embryos and f i n d a dramatic a l t e r a t i o n i n the pattern of axon pathways. The wild type pattern of segmentally repeated commissures and connectives i s replaced by bundles of axons confined to the hemiganglia of o r i g i n . Interestingly, mutations at another Pc-group locus, Psc, produce a si m i l a r CNS phenotype. The Pc-group genes that have been c a r e f u l l y tested show a a maternal e f f e c t and are therefore required i n the maternal germline (Struhl, 1981; Denell, 1982; and Ingham, 1984). Homozygous ph~ clones were induced i n the germline of females who were then mated to ph"1" males. (J-M. Dura et a_l., i n press) . No progeny were obtained, in d i c a t i n g that the zygotic product was unable to rescue the maternal mutation. The maternal 9 e f f e c t s of Asx, Pel, See and Scm (Breen and Duncan, 1986)/ as well as Pc (Lawrence et a l . , 1983); esc (Struhl, 1981,1983) and sxc (Ingham, 1984) are a l l p a r t i a l l y or completely rescued by the paternal wild-type a l l e l e . The maternal e f f e c t of 1(4)29, l i k e that of ph., cannot be rescued by the paternal a l l e l e (Breen and Duncan, 198 6). In summary, p_h i s a complex locus that i s either d i r e c t l y or i n d i r e c t l y involved i n important developmental decisions that include the determination of segmental iden t i t y , the formation of c u t i c l e and the development of axon pathways. It shares some of these c h a r a c t e r i s t i c s with other members of the Pc-group of genes but the c u t i c u l a r e f f e c t and non-rescuable maternal e f f e c t are unique to p_h and 1(4)29 i n t h i s group. Understanding the structure and function of p_h gene has the po t e n t i a l to elucidate some aspects of some of the unsolved problems of developmental biology. Recently, ph and surrounding l o c i have been cloned by chromosomal walking (Dura et a l . , 1987). This work made i t possible to conduct the molecular analysis of rjh that I report in t h i s t h e s i s . The ultimate aim of the work described here i s to determine both the structure and function of the p_h gene. Towards t h i s end three d i f f e r e n t sets of experiments were conducted. F i r s t , cross-hybridization of p_h DNA was used to show the presence of repeated sequences at the locus. Second, t r a n s c r i p t i o n of the locus was examined by Northern analysis of RNA from wild-type and mutant embryos as well as from d i f f e r e n t stages of development. F i n a l l y complementary DNAs corresponding to the locus were i s o l a t e d . 10 CHAPTER ONE: The ph. locus contains two, large, tandem repeats as determined by cross-hybridization. INTRODUCTION Genetic analysis has shown that the p_h locus i s highly mutable, and that two independent mutagenic events are required to produce an amorphic a l l e l e . Furthermore, Df(1)Pgd-kz i s like, a p_h viable a l l e l e phenotypically and i n i t s complementation behavior. It i s viable when heterozygous with viable p_h a l l e l e s , but l e t h a l over either Df (1) JA52 or l e t h a l ph a l l e l e s . A l l of these observations suggest that the p_h locus contains two mutable regions that are functionally interchangeable. According to t h i s model, hypomorphic (viable) mutations, including Df(1)Pgd-kz, a l t e r one mutable region whereas amorphic (lethal) mutations a l t e r both mutable regions. Molecular analysis has shown that 4 p_h a l l e l e s map d i s t a l to the Df(1)Pgd-kz breakpoint while 4 map proximal to the same breakpoint. F i n a l l y , there appears to be some duplication of r e s t r i c t i o n s i t e s on either side of the breakpoint. These analyses have been published by Dura et al.. (1987) and are summarized i n Figure 2. Together, the molecular and genetic data suggest that the p_h locus may actually contain two regions, with similar functions. The presence of duplicated sequences i n the region defined Figure 2. Map of deletions and a l l e l e s i n the ph. region. The thick black l i n e represents the wild type DNA, and the r e s t r i c t i o n s i t e s are indicated B(Bam HI), E (Eco RI), H(Hind III, P (Pst I), S (Sal I), X(Xho I ) . The coordinates are given i n kilobases. The map i s oriented with the telomere to the l e f t and the centromere to the r i g h t . Below are shown deletions, indicated with thinner black l i n e s ; insertions, represented as tri a n g l e s ; and inversions, indicated with t h i n l i n e s , for the DNA rearrangements and ph. mutations mapped. The uncertainty i n the location of the breakpoints i s indicated by open boxes and the i d e n t i t y of each chromosomal rearrangement i s shown on the right or l e f t . JA52, p n 3 8 , and Pgd-kz refer to Df(l)JA52, D f ( l ) p n 3 8 and Df(l)Pgd-kz respectively^ T h e superscripts of the d i f f e r e n t ph a l l e l e s : p_hA ph3-, ph-S ph-^-, p h ^ s ph4-^-, and p h — , are shown as figures to the right or l e f t of each rearrangement. phMLz anci pfrijul a r e v i s i b l e inversions but t h e i r precise nature has not been determined. It i s l i k e l y that they are complex rearrangements and not just simple inversions (see Chapter 2). (From Dura et a l . , 1987). 12 by the p_h lesions and Df (1) Pgd-kz i s strongly suggested by a l l of these observations. I used Southern b l o t t i n g to demonstrate the presence of duplicated sequences and define t h e i r p o s i t i o n on the r e s t r i c t i o n map of the p_h region. Subcloned DNA from the ph. region was digested with r e s t r i c t i o n enzymes, separated by electrophoresis, and then blotted to n i t r o c e l l u l o s e . Individual r a d i o l a b e l l e d r e s t r i c t i o n fragments from the p_h region were then used to probe the b l o t . I was able to determine the number of times a p a r t i c u l a r sequence was present i n the p_h region, determine i t s location within a s p e c i f i c r e s t r i c t i o n subfragment(s) and locate the proximal and d i s t a l l i m i t s of the duplication. 14 MATERIALS AND METHODS Subcloning Genomic DNA cloned into EMBL 4 and Charon 4A phage was subcloned into the plasmid vector pUC12 (Vierra and Messing, 1982), as described by Maniatis et a l . (1982) with some modifications. Two approaches were taken. 1. Shotgun cloning One to 3 of phage DNA was digested with 5 units of appropriate r e s t r i c t i o n enzyme in 20 /fl of high s a l t buffer (150 mM NaCl, 50 mM T r i s - C l pH 7.5, 10 mM MgCl 2 and 1 mM d i t h i o t h r e i t o l ) for 1 h at 37°C. The DNA was p r e c i p i t a t e d by addition of 1/2 volume of 7.5 M ammonium acetate and 2 volumes of 95% ethanol, rinsed i n 70% ethanol, dried, and resuspended in 20 j»l of TE (10 mM Tris-HCl pH 8.0 and 1 mM EDTA pH 8.0) . One /4q of pUC12 vector was digested as above and then phosphorylated af t e r resuspension i n 25 ^1 of c a l f i n t e s t i n a l a l k a l i n e phosphatase buffer (0.5 M T r i s - C l pH 9.0, 10 mM MgCl 2, 1 mM ZnCl 2 and 10 mM spermidine) containing 1 unit of c a l f i n t e s t i n a l a l k aline phosphatase. The reaction proceeded for 15 min. at 55°C and was stopped by adding 5 / t l of 10 mM t r i n i t r i l o a c e t i c acid and 70 1 of H 20. DNA was then p r e c i p i t a t e d with ethanol as above and resuspended i n 50^1 of TE. The digested phage and vector preparations were separated e l e c t r o p h o r e t i c a l l y on a 1% agarose gel containing Tris-borate 15 buffer (89 mM T r i s - C l pH 7.5, 89 mM boric acid, 2 mM EDTA) and 1 /tg/ml of ethidium bromide. The concentration of phage and vector digests were estimated by comparison of band int e n s i t y to a known lambda phage standard under UV illumination. The l i g a t i o n reaction contained a 3:1 r a t i o of vector to insert i n a t o t a l volume to 20 /*! of ligase buffer (66 mM T r i s - C l , 5 mM MgCl, 5 mM d i t h i o t h r e i t o l , 1 mM ATP) and 1 unit of T4 DNA l i g a s e . The reaction was allowed to proceed overnight at 14"C. Competent JM83 Ej_ c o l i were prepared as follows: C e l l s were grown to an o p t i c a l density (ODgQg) °f 0*6 a n c * c h i l l e d on ice for 10 min. The c e l l s were centrifuged for 10 min at 5000 rpm and the supernatant was removed. The c e l l s were resuspended i n one half volume of ice cold 50 mM CaC^/ c h i l l e d on ice for 20 min, and then recentrifuged as above and resuspended i n a 1/10 volume of 50 mM CaCl2 • 10jnl of the l i g a t i o n mix was added to 200^1 of the competent c e l l s and kept on ice for 40 min. The c e l l s were heatshocked for 45 sec at 42°C. 1 ml of L-broth (10 g Bacto-tryptone, 5 g Bacto-yeast extract, 10 g NaCl per l i t r e pH 7.5) was added and the c e l l s were incubated for 1 hour at 37°C to allow expression of a m p i c i l l i n resistance. F i f t y #1 of the transformed c e l l s were plated on L-broth plates (1.5% agarose) containing 50^<g/ml of a m p i c i l l i n and that had been treated with 50^1 X-gal (20 g/ml in N-N' dimethylformamide) and 20 /Ul isopropylthiogalactoside (20 ^g/ml i n d i s t i l l e d water) . Subclones were obtained by selecting white colonies. 16 2. Unique fragment cloning The insert was prepared by digesting the phage with the appropriate r e s t r i c t i o n enzyme(s) and separating the res u l t i n g fragments on an agarose g e l . The desired fragment was extracted from the gel by crushing the excised gel s l i c e i n 200/ul of phenol, vortexing for 10 sec and freezing at -70°C for 15 min. After centrifugation for 15 min at 12,000xg the supernatant (containing DNA) was extracted twice more with phenol and once with CIA (chloroform:isoamyl alcohol [24:1]). The DNA was then p r e c i p i t a t e d by adding 1/2 volume of 7.5 M ammonium acetate and 2 volumes of 95% ethanol. After washing in 70% ethanol, drying and resuspending, the DNA was used i n l i g a t i o n reactions as described above. Mini plasmid preparations Colonies which contained recombinant plasmids were grown overnight i n 4 ml of L-broth containing 50 g/ml a m p i c i l l i n . Plasmid DNA was prepared according to a modification of the alka l i n e l y s i s method (Birnboim and Doly, 1979). Three ml of culture was centrifuged for 5 min to pr e c i p i t a t e the c e l l s and the supernatant was discarded. The c e l l s were resuspended i n 200 ^1 of ice. cold 50 mM glucose, 10 mM EDTA and 25 mM T r i s - C l (pH8) . Four hundred /ul of 0.2N NaOH, 1% SDS was added and the tubes were inverted to mix and l e f t at room temperature for 10 min. Three hundred/il of ice cold potassium acetate (pH4.8) was added, and after gentle vortexing the lysate was c h i l l e d for 15 17 min on i c e . Debris, including denatured protein and genomic DNA, was removed by centrifugation for 15 min at 12,000xg. The supernatant was removed and the nucleic acids p r e c i p i t a t e d with 0.6 volumes of isopropyl alcohol. The p e l l e t was washed in 70% ethanol and resuspended i n TE containing 50 /<g/ml of RNase A. The subclones were i d e n t i f i e d by digesting the plasmids and the parent phage with the appropriate r e s t r i c t i o n enzymes, separating the fragments on an agarose gel, and comparing the band patterns between the plasmid and parent phage digests. Subclone i d e n t i t y was assumed i f the subcloned fragments had the same mobility as fragments i n the parent phage. • Preparation of Southern f i l t e r s Plasmids containing subcloned DNA spanning 30 kb of the putative ph region were digested with r e s t r i c t i o n enzymes to produce fragments that were 2 kb or less i n length. These fragments were separated by electrophoresis on an agarose gel and blotted to n i t r o c e l l u l o s e as described by Southern(1975). The gel was soaked i n 1.5 M NaCl and 0.5 M NaOH for 1 hour, and then neutralized by soaking i n IM T r i s - C l and 1.5 M NaCl for 1 hour. Gels were blotted overnight i n lOx SSC (lx SSC i s 0.15 M NaCl, 0.015 M sodium c i t r a t e ) . The n i t r o c e l l u l o s e f i l t e r was baked at 80°C for 2 hours to f i x the DNA to the f i l t e r . 18 Preparation of hybridization probes Subcloned fragments i s o l a t e d from agarose gels a f t e r digestion and electrophoretic separation were rad i o l a b e l l e d by nic k - t r a n s l a t i o n . One hundred to 500 ng of DNA was suspended i n a t o t a l volume of 25^1 containing 5 / t l of 5x nick-t r a n s l a t i o n buffer (0.5 M T r i s - C l , 0.1 M MgS04 , 1 mM dithiothrei.tol, 500 ^g/ml bovine serum albumin) , 20 uM each of dGTP, dCTP and dTTP, 0.5^1 of lx 10~ 5 d i l u t i o n of 1 /jg/ml DNase I, 5 units of DNA polymerase I and 5/^1 of ( o t 3 2P)dATP (3000 mCi/mM). The reaction was allowed to proceed for 1 hour at 15°C. 72 yul of TE and 3^1 of 0.5 M EDTA was added to stop the reaction. Nick-translated DNA was separated from unincorporated nucleotides by centrifugation at 2000 RPM i n a bench centrifuge through a 1 ml column of Sephadex G-50. The probes had a s p e c i f i c a c t i v i t y of -10^ DPM//<g. Hybridization of Southern f i l t e r s Southern f i l t e r s were prehybridized i n hybridization buffer (50% formamide, 6x SSC, 0.01 M EDTA, 5x Denhardt's solution(0.1% F i c o l l , 0.1% polyvinylpyrrolidone, 0.1% BSA), 0.5% SDS, 100 g/ml denatured herring sperm DNA) for 5-20 hours at 42°C. Ten to 100 ng of r a d i o l a b e l l e d probe was added to the hybridization buffer and allowed to hybridize to the f i l t e r s for 5 to 12 hours. 19 The f i l t e r s were washed 3 times under the following conditions: low stringency (2x SSC and 0.1% SDS at room temperature) for 15 min; medium stringency (0.2x SSC and 0.1% SDS at 50°C) for 1 hour; high stringency (0.1% SSC and 0.1% SDS at 65 °C) for 1 hour. The f i l t e r s were p a r t i a l l y dried, wrapped i n Saran wrap and exposed to X-ray f i l m . 20 RESULTS To f a c i l i t a t e the experiments described here, I subcloned the p_h DNA from phage lambda into plasmid vectors as described i n Materials and Methods. The subcloned DNA spanned the region from map positions 115 to 156 and the s p e c i f i c subclones obtained are shown i n Figure 3. This made i t possible to produce Southern blots of r e s t r i c t i o n fragments that represented the cloned DNA from the entire region. A photograph of the ethidium bromide stained gel that was used to make one of these blots i s shown i n Figure 4a. The subclones run i n each lane, the enzymes they were digested with and the fragments generated from each subclone are shown i n Table I. To determine i f DNA from t h i s region was present i n more than one copy s p e c i f i c r e s t r i c t i o n fragments were ra d i o l a b e l l e d and hybridized to the Southern b l o t . The fragments that were used as probes and the fragments to which they hybridized are l i s t e d i n Table I I . This information i s depicted diagramatically i n Figure 5. I w i l l discuss one of these hybridization steps i n d e t a i l . Other results were obtained and interpreted s i m i l a r l y except where noted below. The 1.8 Sal I/Hind III r e s t r i c t i o n fragment from map pos i t i o n 127.8-129.6 was hybridized to a blot of the gel shown i n Figure 4a. The autoradiograph i s shown i n Figure 4b. In lane-6, the probe hybridizes to i t s e l f (two fragments comigrating at 0.9 kb). The 1.8 fragment hybridizes strongly at two other positions as shown i n lane 9. These bands F i g u r e 3. Subc loned DNA of the ph. r e g i o n . The r e s t r i c t i o n map from F i g u r e 2. i s r e p r o d u c e d . The fragments s u b c l o n e d are shown as t h i n n e r l i n e s below the a p p r o p r i a t e r e s t r i c t i o n f ragments . The r e s t r i c t i o n enzymes used t o generate each subc lone are r e p r e s e n t e d by l e t t e r s : E , Eco R I ; B , Bam H I ; S, S a l I ; and X, Xho I 22 E E XE BSX SS BS SXBX SS X X B XBEBXBXB XE ESB SEX | ' U . UJ 1_1 ^ _ J J II II I I l I i 1 | || 1 1 1 1 II i n 11° 120 130 140 150 160 9.0 B 2.8 B i t 2.1 B 4.3 E .2.2E . 3.6 E , . 2.5SE , 4.8E 1.0 E 3.7E 0 8 S 7.0 E S 2.3S " 4.0 S 1.5S 3.9S 2.0SE * 8.7X i 2.0X. 2.2 XB 3.4X CO CM LANE SUBCLONE DIGESTED WITH FRAGMENTS GENERATED 1 2.2 E E 1.6 E/X, 0.6 X/E 2 3.6 E E P 1.3 E/P, 0.75 P/E, 0.7 P, 0.5 P, 0.4 P 3 8.7 X E P A 5.0 P, 1.3 E/P, 1.1 P/X, 0.7 P, 0.5 P, 0.4 P 4 2.3 S S X P 0.2 S/X, 2.1 X/S 5 0.8 S S P 0.45 S/P,0.35 P/S 6 4.0 S B H P 1.2 H/B, 1.0 S/P, ' 1.0. B/S, 0.8 P/H 7 1.5 S S P 1.5 S ' 8 3.9 S H P S X 1.4 X/S, 1.1 H, 0.55 X/H, 0.45 H/X 9 2.0 E S E P X 0.5 P/E, 0.45 P/S, 0.35 S/P, 0.4 P, 0.2 S/P 10 1.95 X P A 1.5 X/A, 0.45 A/X 11 2.2 X B undigested 4.9 S 12 9.0 B B S P 4.8 P/B, 1.9 B/P, 0.6 P/S, 0.5 S/P, 0.4 P, 0.4 P/S,0.2 S/P 13 2.8 B B X 2.5 X/B, 0.3 B/X 14 3.4 X B P E 1.3 B/E, 1.1 B/E, 0.4 E/B, 0.3 X/P, 0.3 P/X 15 4.8 E B E X 1.5 X, 1.1 B/X, 1.0 X/B, 0.45 B, 0.4 E/B 16 1.0 E E 1.0 E 17 3.7 E E P S 1.4 S/E, 0.7 S/P, 0.6 P/B, 0.5 B/S, 0.4 E/S TABLE I. The numbers i n the f i r s t column refer to the numbered lanes i n Figure 3 and i d e n t i f y subclones from the ph. region. The numerical order corresponds to the order on the chromosome from d i s t a l to proximal. For exact positions see Figure 2. These subclones were digested with r e s t r i c t i o n enzymes i d e n t i f i e d by l e t t e r s : E, Eco RI; B, Bam HI; P, Pst I, S, Sal I; A, Xba I, and X, Xho I. The fragments generated are i d e n t i f i e d by size and the r e s t r i c t i o n s i t e s at which they were cut. They are l i s t e d i n order of size and thus po s i t i o n on the gel from top to bottom. 24 Figure 4. Mapping of the repeat containing domains of the ph region. (a) Ethidium bromide stained gel of e l e c t r o p h o r e t i c a l l y separated fragments of subcloned DNA. To i d e n t i f y the subclones and fragments i n each numbered lane see Table I. The same DNA was run i n another gel with lambda digested with Hind I I I . The size and p o s i t i o n of these marker fragments are indicated i n kb on the r i g h t . (b) Autoradiograph of the above Southern blot probed with 1.8 S/H(127.8-129.6). Again size and position of marker fragments are given on the r i g h t . The size of the fragments to which the probe hybridized are given on the l e f t i n kb. 25 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 _0 . 2 6 PROBE MAP POSITION SELF HYB. (lane) ADDITIONAL HYB. (lane) MAP POSITION DOMAIN 0.8 S 137.1 -137. 9 0.45 S/P 0.35 P/S (5) 0.8 S 127-127.8 b 1.8 S/H 127.8 -129.6 1.0 S/P 0.8 P/H (6) 0.2 S/P 0.4 P (9,12) 137.9-138. 138.1-138. 1 5 c 2.2 H/S 129.6 -131.8 1.2 H/B 1.0 B/S (6) 0.3 P/B 2.2 B/X (13) 143.3-143. 143.6-145. 6 8 d 1.5 S 131.8 -133.3 1.5 S (7) 2.2 B/X (13) 143.6-145. 8 e 0.7 S/H 133.3 -134 0.45 H/X 0.55 X/H (8) unique 1.3 S/B 133.3 -134.6 0.45 H/X 0.55 X/H 1.1 H (8) unique 1.1 H 134 -135.1 1.1 H (8) 1.2 P/S (3) 123.5-124. 7 a 2.0 H/S 135.1 -137.1 1.4 X/S 0.45 S/X (8) 2.3 S (4) 1.2 P/S (3) 124.7-127 a 3.4 X 145.8 -149.2 3.4 X ( a l l 4.8 E (1.1 0.4 bands) B/X, E/B) uniqui (14 and 15) TABLE I I . Cross-hybridization of ph. r e s t r i c t i o n fragments. The r e s t r i c t i o n fragments used to probe Southern blots are l i s t e d i n the f i r s t column. They are designated by t h e i r length i n kilobases and the symbols for the r e s t r i c t i o n s i t e s that were cut to produce them: B(Bam HI), E(Eco RI), H(Hind I I I ) , P(Pst I), S(Sal I) and X(Xho I ) . The fragments they hybridized to ( s e l f hyb. and additional hyb) are named i n the same way and are l i s t e d i n the t h i r d and fourth columns. The numbers i n brackets indicate the lanes i n Table I. 27 Figure 5. The repeat containing domains of the p_h region. The r e s t r i c t i o n map from Figure 2 i s reproduced. The open boxes above the l i n e show the locations of domains that contain duplicated sequences. The l e t t e r s that label the domains show which fragments on one side were sim i l a r to the correspondingly labeled fragment on the other side. DNA without an open box above i t i s unique. The location of the deletions and a l l e l e s of the region are given below the r e s t r i c t i o n map. 28 29 correspond to the 0.4 Pst 1(138.1-138.5) and 0.2 Sal I/Pst 1(137.9-138.1) fragments. Note that these fragments are proximal (nearest centromere) to the Df(1)Pgd-kz breakpoint whereas the probing fragment i s d i s t a l (nearest telomere). The i d e n t i c a l pattern i n lane 12 i s due to hybridization to the same d i s t a l fragments represented i n a d i f f e r e n t subcloned fragment. The minor bands i n lane 9 are due to incomplete digestion of the subcloned fragment and are absent i n lane 12. The data shows that there i s sequence s i m i l a r i t y between a d i s t a l fragment and two proximal fragments. This domain of sequence s i m i l a r i t y was labeled xc' and i s shown in Figure 5. A number of conclusions can be made from t h i s r e s u l t . F i r s t , a sequence i n the d i s t a l region i s s i m i l a r to a sequence in the proximal region. Second, the length of t h i s sequence must be less than 0.6 kb (the length of the proximal 'c' domain) and therefore there must be at least 1.2 kb of unique sequence i n the probe fragment. Third, the degree of s i m i l a r i t y i s high, because the in t e n s i t y of hybridization to proximal and d i s t a l fragments i s similar even though less DNA i s present i n lanes 9 and 12. The s i m i l a r i t y i n t h i s hybridization signal was not affected by high stringency washing (O.lxSSC and 0.1% SDS at 65 C for l-2hours). In a s i m i l a r manner, hybridization data was obtained for three other domains of sequence s i m i l a r i t y . These were named *a', xd' and ye'. This data i s summarized i n Table II and was used to determine the locations shown in Figure 4c. Three probes were required to delineate the 'a' domain. The 2.0 Hind I l l / S a l 1(135.1-137.1) fragment hybridizes to the 2.3 Sal 1(124.7-127) 30 and weakly to the 1.2 Pst I/Sal 1(123.5-124.7). The 1.1 Hind III (134-135.1) fragment also hybridizes to the 1.2 Pst I/Sal 1(123.5-124.7) but more strongly. This s i m i l a r i t y i s lim i t e d in the proximal region to the Bam HI s i t e (134.6) because the 1 . 3 Sal I/Bam HI(13 3.3-134.6) hybridizes only to i t s e l f . The M' domain was mapped with the 2.2 Hind I l l / S a l I fragment(129.6-131.8). It hybridizes to the 0.3 Pst I/Bam HI(143.3-143.6) and the 2.2 Bam Hl/Xho 1(143.6-145.8) fragments. The 1.5 Sal 1(130.8-132.3) fragment or xe' domain also hybridizes to the 2.2 Bam Hl/Xho I fragment. Therefore, the M' and ye' domains can be separated i n the d i s t a l region but not i n the proximal region. A modified method was used to map the xb' region. No d i s t a l counterpart of the 0.8 Sal 1(137.5-138.3) fragment has been subcloned. However, EMBL 4 phage E33.1 which had been i s o l a t e d i n the chromosomal walk (Dura et a l . . 1987) contains an insert which includes the DNA from map positions 116 to 137. The subcloned 0.8 Sal I fragment hybridizes to the 0.8 Sal I fragment i n E33.1 as well as to i t s counterpart i n the 9.0 Bam HI(134.6-143.6) proximal fragment (data not shown). These results allow a l i m i t e d characterization of the d i s t a l and proximal repeated regions. The d i s t a l region i s approximately 9 kb i n length. Its d i s t a l l i m i t i s the Pst I s i t e at map position 124, and i t s proximal l i m i t i s the Sal I s i t e at map p o s i t i o n 132.3. It includes some unique sequence, but i t s extent cannot be determined by t h i s analysis. The proximal region spans about 10 kb. Its d i s t a l l i m i t i s the Bam 31 HI s i t e at map p o s i t i o n 134.3 and i t s proximal l i m i t i s the Xho I s i t e at map p o s i t i o n 145.8. This region has 4.5 kb of unique sequence between the Ac' and M' domains. F i n a l l y , the degree of s i m i l a r i t y between proximal and d i s t a l counterparts of domains xa', xb' M' and *e' i s high because as for domain Ac' high stringency washing could not di s t i n g u i s h between hybridization of the probe to i t s e l f or to the corresponding repeated sequence. The melting temperature (Tm) of two p e r f e c t l y matched DNA strands, i n a 0.1% SSC solution, can be determined as follows: Tm = 69.3 + 0.41 x (G+C)% - 650/L where L = the average length of the probe i n nucleotides. (Maniatis, 1982) In these experiments G+C content i s unknown so i s assumed to be 50% and the average probe length i s approximately 500 base p a i r s . Therefore the Tm i s approximately 88°C. The Tm of a duplex DNA decreases by 1°C with every increase of 1% i n number of mismatched pairs, (Maniatis, 1982), so the wash conditions used here allowed divergence of approximately 23%. Greater divergence would resu l t i n melting and a less intense signal as compared to s e l f hybridization. No difference i n i n t e n s i t y was observed so the s i m i l a r i t y must be at least 77%. 32 DISCUSSION The ph. locus contains two large regions of duplicated sequence. There are at least 4 sequences that are repeated i n the same li n e a r order i n the two regions. There i s at least one sequence i n each of domains xa', xb' and yc' and at least one sequence i n the xd-e'domain. If there i s only one duplicated sequence i n t h i s l a t t e r domain i t would necessarily include the Sal I s i t e at 131.8. It i s possible that each domain contains more than one duplicated sequence. It would be possible to improve t h i s analysis i f smaller r e s t r i c t i o n fragments were used as probes, but the most e f f i c i e n t way to determine t h e i r exact size and location w i l l be to sequence both regions. The boundaries of each domain indicate i t s outer l i m i t . In each case there could be unique sequence both within and between domains. Indeed, t h i s analysis demonstrated the presence of 4.5 kilobases of unique sequence between the xc' and \d' domains i n the proximal region. If there i s an analogous unique sequence i n the d i s t a l region i t would necessarily be smaller since together the yc' and M' domains here span only 4 kb. It i s in t e r e s t i n g to re-examine the molecular and genetic data i n l i g h t of t h i s new information. It has been proposed that there are two si m i l a r regions at the p_h locus with similar functions (Dura e_t a_l. 1987) . According to t h i s model the viable p_h a l l e l e s altered the function of one mutable region, 33 whereas l e t h a l a l l e l e s affected both mutable regions. Complementation analysis predicted that one p_h mutable region should be proximal and the other should be d i s t a l to the Df(1)Pgd-kz breakpoint, located between coordinates 133.7 and 134.6 (Dura et a l . 1987) (Figure 5). The d i s t a l repeat therefore, l i e s completely outside t h i s deficiency, whereas the proximal repeat l i e s completely within i t . DF(1)Pgd-kz behaves l i k e a viable hypomorphic p_h a l l e l e . These observations suggest that when the deficiency removes only the proximal repeat, the d i s t a l repeat provides some residual p_h function. Thus the tandem repeats appear to be coincident with the mutable regions suggested by the i n i t i a l genetic and molecular analysis of the locus. According to the above model, viable a l l e l e s would result from a,lesion i n either of the two regions. This would occur frequently compared to other smaller l o c i and genetic analysis has shown that r e l a t i v e to other complementation groups i n the region p_h viable a l l e l e s were recovered about three times more often than l e t h a l a l l e l e s at neighboring l o c i (Dura et al.,1987). Lethal p_h a l l e l e s , on the other hand, would only be produced i f a double le s i o n mutated both repeats. A second p o s s i b i l i t y i s that there i s a small sequence necessary for the correct function of both repeats, and only a lesion i n t h i s small sequence would produce an amorph. The genetic analysis strongly supports the f i r s t p o s s i b i l i t y because two independent mutagenic events were required to produce an amorph (Dura et a l . , 1987) . The location of various p_h rearrangements that give r i s e to hypomorphic phenotypes can be correlated with the location of the repeats and i s consistent with the model. Eight such -5 7 mutations have been mapped (Figure 5). Four of these; ph—, ph—, pft4_0_9 a n ( ^ pfriJLO. a r e either insertions or inversions and affect only the proximal region. According to the model, these mutations are viable because the d i s t a l repeat remains intact, and i s able to provide some p_h a c t i v i t y . The reciprocal argument can be made for ph^ -Q-^ , a deletion of part of the ? 4 d i s t a l region. The si t u a t i o n i s d i f f e r e n t for ph—, ph— and p h — . These lesions remove most of the d i s t a l region, the unique region between them, and probably some of the proximal duplicated sequence from domain ya'. The model holds, however, because one whole set of domains i s s t i l l present. In these mutants the xa' domain from the d i s t a l repeat and the xb', *c', yd' and Ae' from the proximal repeat are intact and provide a f u l l complement of domains. Thus far, a l l the genetic and molecular data predicts that the p_h locus i s organized i n such a way that two regions in the locus can substitute for each other to produce a viable f l y . The simplest interpretation i s that there i s a gene duplication at the p_h locus. This s i t u a t i o n i s not without precedent i n Drosophila. The p_h duplication d i f f e r s from the example of the duplicated heat shock 70 protein that has no phenotype even when a l l copies are mutant (Grausz e_t a l . , 1981) probably owing to the presence of the homologous heat shock cognate gene, ph also d i f f e r s from another gene duplication, the l a r v a l serum 35 proteins of Drosophila, that do not have a detectable phenotype when only one or two of the three l o c i are deleted. Recently, however, a number of Drosophila l o c i have been i d e n t i f i e d that have sequence s i m i l a r i t y to clo s e l y linked t r a n s c r i p t i o n units and may prove to have some s i m i l a r i t i e s to ph. Near the transformer locus of there i s a tandem duplication of approximately 8 kb (McKeon et a l . , 1987) and as with ph the two copies have sim i l a r but not i d e n t i c a l r e s t r i c t i o n maps. The repeat i s transcribed but i t s function i s unknown. There i s evidence for two genes at t h i s locus so t h i s s i t u a t i o n may prove to be analogous to ph. The achaete-scute complex d i f f e r s from the previously mentioned examples including ph. i n that here the s i m i l a r i t y between the two t r a n s c r i p t i o n units i s due to domains of amino acid s i m i l a r i t y and i s not r e f l e c t e d by a comparable l e v e l of nucleic acid s i m i l a r i t y due to codon degeneracy ( V i l l a r e s and Cabrera, 1987). In t h i s complex there may be up to 6 t r a n s c r i p t i o n units that encode similar polypeptide domains. Interestingly these products are involved i n neurogenic development and the d i f f e r e n t i a t i o n of chaetae. Other examples of adjacent t r a n s c r i p t i o n units with sequence s i m i l a r i t y are Enhancer of  s p l i t (Knust et a l . , submitted), gooseberry (Baumgartner et a l . , 1987) and engrailed (Coleman et a l . , 1987). If ph i s indeed two genes, one would consist of the proximal repeat plus flanking regions, and the other would consist of the d i s t a l repeat plus flanking regions. However, the precise boundaries of the p_h gene or genes cannot be determined from t h i s analysis. The model given above predicts that any viable p_h a l l e l e should affect only one of the two mutable regions that contribute to joh function. As stated above t h i s i s not the case for ph—, ph—, and ph-3J^. These lesions remove most of the d i s t a l repeat, the unique region between the repeats, and may remove some proximal repeated sequences. Therefore the unique sequence between the repeats cannot be es s e n t i a l for complete function of both ph. mutable regions, and i s probably part of the d i s t a l repeat. The results presented above do not demonstrate unequivocally that there are two separate genes at the p_h locus with similar functions, rather that one complex gene with two domains having sim i l a r functions. It i s possible a single gene with variable s p l i c i n g that includes each duplicated region in alternative t r a n s c r i p t s could account for the data. A l t e r n a t i v e l y i t i s possible that there i s one gene that produces a large protein with two functional domains. This i s the case for the qlycinamide r i b o t i d e transformylase gene of Drosophila that has a 260 amino acid motif repeated twice (Henikoff et al.. 1983) . For p_h the protein would presumably have residual function i f only one domain was present. It became obvious that t r a n s c r i p t i o n a l analysis of both wild type and mutant f l i e s at d i f f e r e n t stages of development might answer some of the questions raised here. This analysis i s the subject of the next chapter. 37 CHAPTER TWO: Transcription of the ph locus - Northern analysis. INTRODUCTION Complementation analysis and deletion mapping suggest a minimum of three t r a n s c r i p t i o n units within and immediately adjacent to the ph. locus (Figure 1) . The Df (1) Pgd-kz breakpoint f a l l s within the ph. unit or, al t e r n a t i v e l y , between two p_h t r a n s c r i p t i o n u n i t s . D i s t a l to p_h but proximal to the DfJiLpn breakpoint i s the csw locus and i t s corresponding t r a n s c r i p t i o n unit (s) plus any others between t h i s locus and p_h that were undetected i n the genetic analysis. Proximal to the Df (1) JA52 breakpoint and p_h i s the Pgd-kz locus. The tr a n s c r i p t i o n unit for t h i s locus and any genetically u n i d e n t i f i e d l o c i are i n t h i s region. Transcriptional analysis using genomic DNA from the walk through t h i s region should i d e n t i f y a minimum of three t r a n s c r i p t i o n units, at least one for each of the three genetically i d e n t i f i e d l o c i . This analysis i s complicated by two related problems. F i r s t , as indicated above, i f there are genetically u n i d e n t i f i e d l o c i i n the region these w i l l show up as additional t r a n s c r i p t i o n units. Second, some l o c i i n Drosophila are complex and produce a number of t r a n s c r i p t s . Ph i s large, highly mutable and has complex complementation behavior. Other complex l o c i i n Drosophila, with si m i l a r c h a r a c t e r i s t i c s , include the bithorax complex (BX-C) (Lewis, 3 8 1978 and Bender, 1983) and the achaete-scute complex (AS-C) (Garcia-Bellido, 1979). Both of these complex l o c i have been shown to have a number of t r a n s c r i p t s . (0'Conner, 1988 and V i l l a r e s , 1987). Given the p o s s i b i l i t i e s , i t i s d i f f i c u l t to id e n t i f y a p a r t i c u l a r t r a n s c r i p t as either a product of an unid e n t i f i e d locus or that of an i d e n t i f i e d but complex locus. Nevertheless, t r a n s c r i p t i o n a l analysis i s necessary to determine the t o t a l number of tran s c r i p t s i n the region, and to determine the possible l i m i t s of the p_h gene. I made small probes that represented the genomic DNA from map positions 114-157. These probes were hybridized i n d i v i d u a l l y to Northern blots of embryo, l a r v a l , pupal and adult p o l y ( A ) + RNA. These experiments revealed the t o t a l number and size of unique t r a n s c r i p t s i n the region. It also made clear which probes I should use i n subsequent experiments, because s p e c i f i c probes hybridized to subsets of the t o t a l number of t r a n s c r i p t s . To i d e n t i f y ph-specific t r a n s c r i p t s , Northern analysis of RNA from p_h mutants that contain DNA rearrangements was undertaken, because these mutants should also have altered t r a n s c r i p t size and or number (see Figure 2). These rearrangements must not affect the tra n s c r i p t s of flanking genes so any altered t r a n s c r i p t s must be ph products. Some of the DNA rearrangements aff e c t only the d i s t a l or only the proximal repeat of the ph locus and these are viable a l l e l e s . A model proposed by Dura et a l . (1987), states that the ph locus has two mutable regions and each produces 39 functionally s i m i l a r products. At least 2 wild type copies of the p_h products are required for v i a b i l i t y . This model predicts that f l i e s homozygous for ph. viable lesions w i l l have at least one wild type t r a n s c r i p t . Therefore, t h i s analysis w i l l test the model and could provide some information about the organization of the locus. I analyzed poly (A) + RNA prepared from mutants pfr2,401,410, a n c j .40_9_> These mutants were chosen s p e c i f i c a l l y because Southern analysis shows ph— and ph-4-^- aff e c t the d i s t a l repeat while ph-4-^ 9- a n c ; pfr4_10 a f f e c t the proximal repeat (see Figure 2). A possible molecular interpretation of the above model i s that each repeat i s transcribed separately from i t s own promoter and DNA rearrangements a f f e c t i n g only one repeat should a l t e r the migration of only one t r a n s c r i p t . If the ph locus has only one promoter, DNA rearrangements a f f e c t i n g only one repeat might aff e c t both t r a n s c r i p t s . Nevertheless, more complex models are possible, and the data may not exclude any model. Developmental genetic studies show that p_h has a strong maternal e f f e c t , a homeotic ef f e c t , and i s required for the formation of ventral c u t i c l e early i n development. Clonal analysis shows that the p_h product i s required for development of imaginal disk c e l l s during the f i r s t three l a r v a l instars (Dura, 1985 and 1987). It i s possible that additional requirements for ph do not have a phenotype and have been missed i n the genetic analysis. Independent studies by Smouse et a l . (1987) have shown that p_h i s required for the embryonic development of axon pathways. These observations suggest that 40 ph has important roles i n normal development and undergoes complex developmental regulation, ph. should be expressed i n females, embryos, and i n imaginal discs, and perhaps at other developmental stages. I examined the developmental p r o f i l e of ph expression. Poly(A) + RNA was prepared from 0-3, 3-12 and 12-24 hours embryos, pooled larvae, pupae and adults and probed with fragments believed to i d e n t i f y a l l major ph t r a n s c r i p t s . The information presented i n t h i s chapter shows that there are at least 4 ph t r a n s c r i p t s , suggest that there are 2 t r a n s c r i p t i o n units at the ph. locus, and shows that ph undergoes complex developmental regulation. 41 MATERIALS AND METHODS Preparation of RNA RNA was prepared from fresh or frozen Canton S embryos, larvae, pupae and adults. One ml of tissue was homogenized i n 5 ml of 65°C l y s i s buffer (0.3 M sodium acetate pH 5.0, 50 mM EDTA, 1% SDS) and 5 ml of 65°C phenol (made pH 5.0 with 0.1 M sodium acetate) i n a Dounce homogenizer with a B pestle. The mixture was incubated at 65°C for 10 min with 3 or 4 vigorous mixings. The phases were separated by centrifugation at 4000 RPM for 10 min. The aqueous layer was transferred to a new tube, 5 ml of phenol was added, and incubation and centrifugation were repeated as above. Phenol extraction was repeated 2 or 3 times u n t i l both phases were transparent. The sample was extracted with 5 ml of phenol at 65°C for 10 min. After c h i l l i n g on ice, 5 ml of CIA was added, and the phases were again separated by centrifugation. The f i n a l organic extraction used CIA alone. The RNA was pre c i p i t a t e d with 2 volumes of 95% ethanol afte r the addition of 1/10 volume of sodium acetate pH 5.0. The suspension was centrifuged for 10 min at 10,000 RPM. The p e l l e t was rinsed with 70% ethanol, dried, and resuspended i n water. The i n t e g r i t y of the RNA was checked i n a 50% formamide, 1% agarose gel made with 0.02 M sodium phosphate buffer and run in the same buffer at 80 volts for 1.5 hours. 42 Selection of poly(A ) RNA. The technique used i s as described by Maniatis(1982) with some modifications. An oligo(dT)-cellulose column was prepared by e q u i l i b r a t i n g 150 mg of oligo(dT)-cellulose i n loading buffer (20 mM T r i s - C l pH 7.5, 0.5 M NaCl, 1 mM EDTA, .01% SDS) and poured into a 5 ml disposable pipette. The column was washed with 3 bed volumes each of water, 0.1 M NaOH and 5mM EDTA, and water again and then with 5 volumes of loading buffer. Two to 3 mg of t o t a l RNA was heated to 65°C for 5 min i n an equal volume of 2x loading buffer, cooled, then loaded onto the column. The column was washed with 5 volumes of loading buffer and subsequently with 3 volumes of elution buffer(10 mM T r i s - C l , IMm EDTA, 0.05% SDS). The eluted poly(A +) f r a c t i o n was pr e c i p i t a t e d with 95% ethanol after the addition of 1/10 volume of sodium acetate (pH 5.0). Y i e l d was t y p i c a l l y about 2% of the t o t a l RNA loaded. The RNA was rinsed i n 70% ethanol, dried and resuspended i n water to give a concentration of approximately 2 mg/ml. Preparation of Northern f i l t e r s . P o l y ( A ) + RNA was separated by electrophoresis through a formaldehyde g e l . Each sample was prepared by mixing 10 g of poly (A) + RNA i n 2.5^1 of water with 4.0,41 of 5x gel running buffer (0.2 M morpholinopropanesulfonic acid pH 7.0, 50 mM 43 sodium acetate, 5 mM EDTA pH 8.0), 3.5 yOl of formaldehyde, 10 jul of formamide and 0.5 /u.g of ethidium bromide, and then incubating at 70°C for 10 min. One /ug of lambda, predigested with Hind III was treated i n the same way and included i n each set of samples as a s i z i n g marker. The samples were loaded onto a 1% agarose gel (2.2 M formaldehyde and lx gel running buffer) and separated by electrophoresis at 35 volts for 12 hours. Pr i o r to b l o t t i n g to n i t r o c e l l u l o s e , the gel was rinsed with water then soaked i n 50 mM NaOH and 10 mM NaCl for 45 min, neutralized with 0.1 M Tris-HCl pH 8.0 for 45 min. and soaked i n 20x SSC for 1 hour. The gel was blotted 10 to 15 hours i n 2Ox SSC. The f i l t e r was then removed and baked for 2 hours at 80°C. Preparation of hybridization probes Probes were radi o l a b e l l e d by nick- t r a n s l a t i o n as described i n chapter one or by random priming (Feinberg and Vogelstein, 1982) . A 13^1 mixture was prepared containing 5^1 1 M Hepes pH 6.6, 5^1 dNTP buffer (100 uM each dCTP, dGTP and dTTP, 25 mM MgCl2/ 50 mM 2-mercaptoethanol, pH 8), 1.4^1 oligomer buffer (1 mM Tris-HCl pH 7.5, 1 mM EDTA, 4.5 mg/ml oligomer), 1^1 BSA (1 mg/ml), and 2.5 units of Klenow. 100 ng of DNA was heated for 2 minutes at 100°C i n 12 >1 of water, cooled and added to the above mixture. 5^1 of (* 32P)dATP (3000 mCi/mMole) was added and the reaction was allowed to proceed 10 to 20 hours at room temperature. Unincorporated dNTP's were removed by 44 centrifugation at 2000 RPM i n a bench centrifuge through a 1 ml column of Sephadex G-50. The probes had a s p e c i f i c a c t i v i t y of approximately 5x10 DPM//ig. Hybridization of Northern f i l t e r s Northern f i l t e r s were prehybridized, hybridized, and washed as described for Southern f i l t e r s i n Chapter 1. 45 RESULTS I d e n t i f i c a t i o n o f ph. and a d j a c e n t t r a n s c r i p t i o n u n i t s . RNA was p r e p a r e d from Canton S embryos, l a r v a e , pupae and a d u l t s as d e s c r i b e d i n M a t e r i a l s and Methods. Twenty ,/fg o f t o t a l embryo RNA was probed w i t h the 2.3 S a l I r e s t r i c t i o n fragment from c o o r d i n a t e s 124 .7-127 . No h y b r i d i z a t i o n t o the t o t a l RNA was d e t e c t e d so 10 /<g o f p o l y ( A ) + was probed w i t h the same fragment . I t h y b r i d i z e d to two RNA of 6.4 and 6.1 kb ( F i g u r e 6a, l ane 1 ) . T h e r e f o r e i n subsequent exper iment s , each lane c o n t a i n e d 10 yug o f p o l y (A) + RNA. To determine the number o f d i f f e r e n t t r a n s c r i p t i o n u n i t s between the Df (1) pn^^2- d i s t a l b r e a k p o i n t and the phosphoqluconate dehydrogenase (Pgd) gene, known t o be l o c a t e d between c o o r d i n a t e s 154 and 158 ( L u c c h e s i , p e r s o n a l communicat ion) , I h y b r i d i z e d genomic fragments from c o o r d i n a t e s 114-157 to f i l t e r s t h a t c o n t a i n e d p o l y ( A ) + RNA. The i n f o r m a t i o n o b t a i n e d from these exper iments i s g i v e n i n T a b l e I I I and r e p r e s e n t a t i v e da ta are shown i n F i g u r e 6. In most cases da ta were o b t a i n e d f o r deve lopmenta l s tages o t h e r t h a n embryo and where a v a i l a b l e t h i s i n f o r m a t i o n i s g i v e n . The t r a n s c r i p t i o n u n i t s i d e n t i f i e d by t h i s a n a l y s i s are diagrammed i n F i g u r e 7. The most d i s t a l fragment used i n t h i s a n a l y s i s was the 3.6 Eco RI fragment from c o o r d i n a t e s 1 1 6 . 5 - 1 2 0 . 1 . T h i s fragment TRANSCRIPTIONAL ANALYSIS OF PH AND ADJACENT GENES PROBE COORDINATES EMBRYO LARVAL PUPAL ADULT 3 . 6 E 116.3-125 6 . 6 5.7 4.95 3.5 6.6 5.7 4.95 3.5 6.6 5.7 4.95 3.5 4.95 2.3 S 124.7-127 6.4 6.1 6.1* 6.6 6.1 smear* 0.8 S 127-127.8 6.4 6.1 6.1* 6.6* 6.1 4.0 S 127.8-131.8 6.4 6.1 6.6 6.1 1.5 S 131.8-133.3 ND 6.6 6.1 1.3 S/B 133.3-134.6 6.4* 6.1 6.6* 6.1 2.0 X 139.4-141.4 6.4 6.1 6.6 6.1 6.1* 2.2 X/B 141.4-143.6 6.4 6.1 6.6 6.1 6.1 9.0 B 134.6-143.6 6.4* 6.1 smear smear* smear* smear* 2.8 B 143.6-146.4 3.4 X 145.8-149.2 2.0 2.0 2.0 2.0 1.0 E 152.4-153.4 3.7 E 153.4-157.1 2.5 ND ND ND 1.6 Table I I I . The r e s t r i c t i o n fragments used to probe Northern blots are l i s t e d i n the f i r s t column and are designated by t h e i r length and end r e s t r i c t i o n s i t e s : B(Bam HI), E (Eco RI), X(Xho I) and S(Sal I ) . The map coordinates of these fragments are given i n the second column. In the l a s t 4 columns the length i n kb of each RNA species to which the probe hybridized i s given for embryos, larvae, pupae and adults. A blank indicates that the experiment was done but no tra n s c r i p t s were revealed; ND indicates that the experiment was not done and an asterisk indicates that data i s not shown i n Figure 6. Figure 6. Northern analysis of tra n s c r i p t s from the p_h region in wild-type embryos. (a) Autoradiographs of Northern blots of p o l y ( A ) + embryo RNA probed with r e s t r i c t i o n fragments from the p_h region. The r e s t r i c t i o n fragments (with coordinates) used i n each lane are as follows: 1 - 3.6 Eco RI (118.9-122.5) 2 and 3 - 2.3 Sal I (124.7-127) 4 - 0.8 Sal I (137.1-137.8) 5 - 4.0 S a l I (127.8-131.8) 6 - 2.0 Xho I (139.4-141.4) 7 - 2.2 Xho 1/ Bam HI, (141.4-143.6) 8 - 3.4 Xho I (145.8-149.2) 9 - 3.7 Eco RI (148.7-152.4) The data shown i n lane 2 was obtained from RNA that was separated by electrophoresis at 25 volts for 24 hours. For a l l other experiments separation was at 25 volts for 12 hours. (b) As for (a) except l a r v a l , pupal and adult poly (A) + RNA i s probed. The stage used i n each lane i s indicated below each lane; l a r v a l - L, pupal - P, and adult. The r e s t r i c t i o n fragments used i n each lane are as follows: 1,2 and 3 - 3.6 Eco RI (118.9-122.5) 4 - 4.0 Sal I (127.8-131.8) 5 - 2.0 Xho I (139.4-141.4) 6 - 1.5 Sal I (131.8-133.3) 7,8 and 9 - 2.2 Xho 1/ Bam HI (141.4-143.6) 10,11 and 12 - 3.4 Xho I (145.8-149.2) 48 49 Figure 7. Transcription units of the p_h region. The t r a n s c r i p t i o n units i d e n t i f i e d by Northern analysis are indicated with s o l i d l i n e s below the r e s t r i c t i o n map reproduced from Figure 2. They are labeled A(csw), B(p_h), C (unidentified) and D (Pdo) . 50 51 hybridizes to t r a n s c r i p t s 6.6, 5.7, 5.0 and 3.5 kb (Figure 6a, lane 1). The 8.7 Xho I fragment, coordinates 116-125, overlaps t h i s fragment and approximately 1.5 kb of the 'a' domain of the d i s t a l repeat. It hybridizes to the four t r a n s c r i p t s mentioned above, plus the 6.4 and 6.1 kb t r a n s c r i p t s (data not shown). No other fragments from coordinates 116.3 to 152.4 hybridize to the 4 t r a n s c r i p t s i d e n t i f i e d by the 3.6 Eco RI fragment. The simplest interpretation of t h i s result i s that at least one of these 4 t r a n s c r i p t s correspond to the csw gene t r a n s c r i p t . It may be that csw i s a complex locus because only one complementation group was detected genetically, i n which case a l l 4 t r a n s c r i p t s might belong to the csw locus. Nevertheless, i t cannot be ruled out that these tr a n s c r i p t s are products of more than one t r a n s c r i p t i o n unit. This alternative could be eliminated by demonstrating that the putative csw t r a n s c r i p t s are altered i n csw mutants. For convenience, these tr a n s c r i p t s w i l l referred to as the A t r a n s c r i p t i o n unit (Figure 7). A l l are expressed in embryos, larvae and pupae but only the 5.0 t r a n s c r i p t was detected i n adults (Figure 6b, lanes 1, 2, and 3). A l l fragments from coordinates 116 to 143.6 hybridize to two major tra n s c r i p t s of 6.4 and 6.1 kb i n embryos. The d i s t a l boundary of t h i s t r a n s c r i p t i o n unit (B i n Figure 7) could not be resolved from the proximal boundary of the A unit between coordinates 117 and 124.7 because no fragment used hybridized to one or the other set of t r a n s c r i p t s . The proximal l i m i t of the DNA responsible for producing the 6.4 and 6.1 kb t r a n s c r i p t s i s map position 143.6. The 2.3 Sal I and 9.0 Bam HI 52 fragments often produced intense smears of s i l v e r grains that almost obscured the 6.4 and 6.1 kb bands (data not shown). This smear may be due to the presence of the M repeat (Wharton et a l . , 1985) . This repeat i s present i n the *a' domain (Dura et a l . , 1985). The B unit i s large, i s located i n the region genetically determined to be the p_h locus and i t l i k e l y expresses the p_h products. Fragments from unit B were also hybridized to l a r v a l , pupal and adult poly (A) + RNA. (Table III and Figure 6 ) . Faint tr a n s c r i p t s , approximately 6.1 kb are seen i n larvae and adults (Figure 6b, lanes 7 and 9 respectively). In pupae 2 tra n s c r i p t s 6.6 kb and 6.1 kb are e a s i l y detected and resolved (Figure 6, lanes 4, 5, 6 and 8 ). The 2.8 Bam Hi fragment, coordinates 143.6-146.4, does not hybridize to any t r a n s c r i p t s . The 3.4 Xho I and 4.8 Eco RI fragments proximal to t h i s fragment and extending to map po s i t i o n 152.4 hybridize to a 2.0 kb t r a n s c r i p t i n embryos, larvae, pupae and adults. (Figure 6a, lane 8 and 6b lanes 10, 11 and 12). These fragments include the entire t r a n s c r i p t i o n unit (C i n Figure 7) for t h i s RNA. This unit i s proximal to the repeats but d i s t a l to the Pod locus. It i s unclear i f i t i s part of the p_h locus or i s the product of some as yet genetically u n i d e n t i f i e d locus. Proximal to t h i s t r a n s c r i p t i o n unit the 1.0 Eco RI fragment, coordinates 152.4-153.4, hybridizes to no RNA. The neighboring 3.7 Eco RI fragment, coordinates 153.4-157.1, on the other hand hybridizes to two novel tr a n s c r i p t s 2.5 and 1.6 53 kb (Figure 6a, lane 9). These RNA probably correspond to the (Pgd) t r a n s c r i p t i o n unit (D i n Figure 7) because an independent investigation has determined the location of t h i s gene between these coordinates (Lucchesi, personal communication). The approximate location of the p_h locus had been determined by genetic analysis i n combination with the molecular mapping of deficiency breakpoints. In t h i s region I have i d e n t i f i e d at least.four t r a n s c r i p t i o n units, at least one of these i s ph. Transcription i n ph mutants Further evidence was required to v e r i f y that t r a n s c r i p t i o n units B and possibly C were ph, and that ph function was not coded for by any sequence beyond these two units. This evidence was obtained by analyzing t r a n s c r i p t i o n i n ph mutants. Approximately 10 g of poly (A) + for each of ph ±'-2- '-ilil/ a n G : were used to prepare duplicate Northern b l o t s . These were probed with fragments s p e c i f i c for each t r a n s c r i p t i o n unit and a c t i n . The data are shown i n Figure 8. DNA from the regions flanking the B t r a n s c r i p t i o n unit (s) does not hybridize to any RNA's that are altered i n ph. mutants. The 3.6 Eco RI (114-117.6) fragment, as described e a r l i e r , hybridizes to four RNAs, and t h i s region i s referred to as the A unit for convenience. As expected, these t r a n s c r i p t s are unaltered i n ph mutants (Figure 8a). The 3.7 Eco RI fragment (153.4-157.1) hybridizes to t r a n s c r i p t s of 2.5 and 1.6 kb that Figure 8. Northern analysis of t r a n s c r i p t s from p_h mutants. (a - e) Autoradiographs of a single Northern blot of embryo poly (A) + RNA prepared from wild type (lane 1), ph— (lane 2), pfrituii ( i a n e 3) and ph-4-^-. The blot was probed sequentially with: a - 3.6 Eco RI b - 4.0.Sal I and actin c - 3.4 Xho I d - 3.7 Eco RI a (lanes 1 - 4 ) - 4.0 Sal I and actin (lanes 5 and 6) - 2.2 Xho I/Bam HI and ac t i n . (f) Autoradiograph of a Northern blot of pupal POiy (A) RNA prepared from wild type (lane 1), ph— (lane 2), ph-4-^- (lane 3), phitOJi ( i a n e 4) and ph4-^- (lane 5) . The blot was hybridized to the 4.0 Sal I fragment and a c t i n . The approximate sizes of the tran s c r i p t s are shown i n kb on the l e f t and r i g h t . Lambds Hind III fragments were used as markers. 55 56 are also unaltered i n ph. mutants (Figure 8d) . These RNAs probably correspond to the Pgd locus. The 2.0 kb RNA that i s produced by t r a n s c r i p t i o n unit C i s unaltered i n the ph mutants tested, and i t i s unclear i f t h i s i s a ph tr a n s c r i p t that i s unaltered i n these mutants or i f i t i s expressed by an unide n t i f i e d locus situated between p_h and Pgd (Figure 8c). The 2.0 kb tr a n s c r i p t cannot be eliminated as a pote n t i a l ph product for 2 reasons. F i r s t , none of the ph mutants examined thus far either eliminate or have breakpoints within t h i s unit, so i t i s not reasonable to expect any change in t r a n s c r i p t s i z e . Second, a ph. cDNA, C26, has been i s o l a t e d that contains sequence represented i n both the B and C tr a n s c r i p t i o n units (see Chapter 3). The B t r a n s c r i p t i o n unit, that produces the 6.4 and 6.1 kb RNA, includes the 4.0 Sal I fragment (127.8-131.8). This fragment was used as a probe to determine the t r a n s c r i p t i o n pattern of these RNAs i n ph mutants because i t hybridizes well to both species (Figure 6a, lane 5). The same blot was also probed with the 2.2 Xho I/Bam HI (141.4-143.6) fragment because i t i s mostly unique to the proximal repeat. Transcription of the 6.4 and 6.1 embryo RNA i s c l e a r l y altered i n the mutants (Figure 8b and e). p In ph— two novel t r a n s c r i p t s , both of which are larger than the wild-type species are expressed (Figure 8d, lanes 2 and 5). This mutation removes portions of both repeats and the unique region between (Figure 5). It was argued i n Chapter 1 that because one complete set of domains was l e f t intact the 57 mutation was viable. The results here show that p_h i s expressed, a l b e i t abnormally. The nature of these products cannot be deduced from t h i s analysis but one or both must provide enough ph function for v i a b i l i t y . The 4.0 Sal I fragment l i k e l y hybridizes to only one tra n s c r i p t i n ph-4-^-, although t h i s i s not c l e a r l y apparent i n the photograph because the two wild type bands were not well resolved (Figure 8e, lane 4). This conclusion i s strengthened by the fact that the 2.2 Xho I/Bam HI, hybridizes to a single smaller RNA, 3.5 kb, i n the same mutant (Figure 8e,lane 6). This fragment hybridizes to 6.4 - 6.1 kb species i n wild-type embryos (Figure 6a, lane 7). Transcription i s d e f i n i t e l y altered i n ph 4 1^' and t h i s datum has further si g n i f i c a n c e . A fragment of d i s t a l o r i g i n (4.0 Sal I) hybridizes to one unaltered t r a n s c r i p t while a proximal s p e c i f i c fragment (2.2 Xho I/Bam HI) hybridizes to a smaller tra n s c r i p t not present i n wild-type organisms. The DNA rearrangement i n ph4-^- affects only the proximal repeat (see Figure 5). Therefore the simplest interpretation of these results i s that both repeats produce in d i v i d u a l t r a n s c r i p t s from separate promoters. In t h i s mutant the proximal but not the d i s t a l product has been altered. The interpretation that there are both d i s t a l and proximal s p e c i f i c products was not obvious when ph fragments were used to probe wild-type RNA because the s i m i l a r i t y of repeated sequence results i n hybridization of most probes to both t r a n s c r i p t s . Also, i n most experiments i t was not possible to d i f f e r e n t i a t e between the 6.4 and 6.1 species or even to determine i f one or both were present. 58 The data obtained for ph^-^- are more d i f f i c u l t to interpret. This mutant i s also due to a DNA rearrangement i n the proximal repeat (see Figure 5). No a l t e r a t i o n i n tr a n s c r i p t size i s detected when either probe i s used (Figure 8e, lanes 3 and 5), but there i s an overabundance of one of the pn40_9. species (compare Figure 8e lanes 3,4 and 5 to lane 1) . The ph4-^- product i s present at wild type le v e l s , while that of ph ^0-2- i s expressed at levels approximately 4 f o l d higher. When the same blot was hybridized to an actin probe, the amount of RNA i n each lane i s shown to be approximately equal (Figure 8e). This overexpression i s not seen when the 2.2 Xho I/Bam HI probe i s used (compare the difference between lanes 3 and 4 to that between lanes 5 and 6, Figure 8d). It may be that the ph4_0_9 m u t a t i o n causes a change i n the l e v e l of expression of one of the t r a n s c r i p t s but not of the other. It i s puzzling that both of the probes hybridize to f u l l length species i n pn4QJ. k u t t 0 a fun i e n g t h and a smaller species i n ph4-^-. These rearrangements are s i m i l a r . It i s possible that i n p h — , 3.5 kb of the proximal species has been separated from the remainder of the message but i s s t i l l transcribed. In pn4_Q_9 o n ffoe other hand none or only small portion of the proximal species i s transcribed. Thus, only the d i s t a l f u l l length species i s transcribed and i t has some s i m i l a r i t y to the 2.2 Xho I/Bam HI fragment. These data provide i n d i r e c t evidence that the t r a n s c r i p t i o n of the two repeats are under the control of separate promoters and that unit B may i n fact be two t r a n s c r i p t i o n units. 59 Our collaborators have shown that only the 6.4 kb message i s produced i n p h — (N. Randsholt, personal communication) . This i s consistent with my results and suggests 6.4 and 6.1 kb species are the d i s t a l and proximal products, respectively. Interesting results were obtained when pupal poly (A) + RNA of ph mutants was analyzed. P o l y ( A ) + RNA prepared from wild-type, ph—, ph 4-^, p h — and p h — pupae was probed with 4.0 Sal I fragment used i n the previously described experiment (Figure 8e, lanes 1 - 5). Note that for the wild type RNA the 6.6 and 6.1 kb species have not been well resolved (lane 1). The data for ph— (lane 2) are d i f f i c u l t to interpret because of underloading. In ph-4-^- the wild type t r a n s c r i p t i o n pattern i s altered. There are 2 d i s t i n c t species, one approximately 6.6 kb and another about 2.0 kb (lane 3) . In ph4-0-^- and ph •4-^°- there appears to be only one 6.1 kb t r a n s c r i p t (lanes 4 and 5) . Ph4-^-i s a small deletion that removes at least part of the xc' and xd' domains of the d i s t a l repeat. The simplest interpretation of these data i s that the ph 4-^ deletion removes part of the d i s t a l 6.1 kb species but leaves the proximal 6.6 kb species i n t a c t . In ph4-^0- and ph-4-^- only the unaltered d i s t a l 6.1 kb species i s detected because t h i s rearrangement affects the proximal repeat. Note that the s i t u a t i o n here i s d i f f e r e n t than in embryos, since the larger species i s produced by the proximal and the smaller by the d i s t a l repeat. It seems that at least 4 d i s t i n c t RNAs are produced by the B unit; a 6.4 and 6.1 kb i n embryos, and a d i f f e r e n t 6.1 and a 6.4 kb i n pupae. In summary, the analysis of t r a n s c r i p t i o n i n ph. mutants has shown that t r a n s c r i p t i o n units A and D flank the ph locus. 60 It provides i n d i r e c t evidence that both the repeats of unit B may be independently transcribed from separate promoters so that B would then consist of 2 t r a n s c r i p t i o n units. It does not rule out the p o s s i b i l i t y of an additional p_h product produced by t r a n s c r i p t i o n unit C, a region that i s unique and proximal to both repeats. Developmental p r o f i l e of p_h t r a n s c r i p t i o n . It has been established that ph has an important and p l e i o t r o p i c role i n development (Dura et al., 1985 and 1987), and i t i s l i k e l y that the expression of t h i s locus i s d i f f e r e n t i a l l y regulated throughout development. Regulation at the l e v e l of t r a n s c r i p t i o n i s e a s i l y detected by Northern analysis of RNA prepared from d i f f e r e n t stages of development. Most important developmental decisions are made during embryogenesis so i t was necessary to use staged preparations of RNA for the early stages of development. Embryos were co l l e c t e d at 0-3, 3-12, and 12-24 hours p o s t - f e r t i l i z a t i o n . P o l y ( A ) + RNA from these preparations was probed with representative ph. genomic fragments and cDNA's. Similar results were obtained for the various probes and the result obtained for a cDNA, C3, i s shown i n Figure 9, lanes 3, 12, and 24. A blot that included RNA from the 3 embryonic stages as well as pupae and adults was prepared and probed separately with the 2.3 Sal I fragment and the C3 cDNA in order to get a Figure 9. Developmental p r o f i l e of pJi t r a n s c r i p t i o n Autoradiographs of Northern blots of poly (A) + RNA prepared from 0-3 hour embryos (3), 3-12 hour embryos (12), 12-24 hour embryos (24), larvae (L), pupae (P) and adults (A) and hybridized to cDNA C3. Lanes 3, 12 and 24 were also hybridized to a c t i n . The approximate sizes of the t r a n s c r i p t s are shown on the l e f t . Lambda Hind III fragments were used as size markers. 62 t o t a l developmental p r o f i l e . S i m i l a r r e s u l t s were obtained f o r each probe and the data f o r C3 pupae and a d u l t s are shown i n Figure 9 lanes P and A. No l a r v a l RNA was i n c l u d e d on e i t h e r b l o t so l a r v a l data from a separate experiment i s i n c l u d e d i n t h i s Figure 9, lane L. The 6.1 kb t r a n s c r i p t i s e a s i l y detected at 0-3 and 3-12 hours but becomes more abundant l a t e r at 12-24 hours. The 6.4 t r a n s c r i p t on the other hand i s b a r e l y detectable at 0-3 hours and i t s l e v e l of expression peaks at 3-12 hours and i s maintained at t h i s l e v e l at 12-24 hours so that at t h i s stage the abundance of both t r a n s c r i p t s i s approximately equal. This f i n a l l e v e l of expression i s what was seen i n the t o t a l embryonic preparations described e a r l i e r (Figure 6). At l e a s t 1 of the l a r g e species i s expressed i n l a r v a e but i t i s not p o s s i b l e t o determine the r e l a t i v e l e v e l of t r a n s c r i p t i o n from these data. P r e l i m i n a r y r e s u l t s show that the 6.4 and 6.1 kb species are expressed i n f i r s t i n s t a r l a rvae but that the l e v e l f a l l s o f f i n second and t h i r d i n s t a r l a rvae (data not shown). On Northerns prepared from pupal RNA, fragments from the ph region h y b r i d i z e d t o two RNA of 6.6 and 6.1 kb. The l a t t e r i s i n most cases approximately 5 times more abundant than the former. These two species are e a s i l y seen i n Figure 6b, lane 8, but i n Figure 9, lane P, two d i s t i n c t species are not so apparent. I t may be t h a t i n the l a t t e r f i g u r e they are not resolved, or that the 6.6 kb species i s not present i n great enough concentration to be detected. In adult RNA there appears to be only one t r a n s c r i p t , 63 approximately 6.1 kb that i s present at lower lev e l s than i n pupae or embryos. The 2.0 kb RNA was detected i n a l l stages at approximately equal levels (Figure 6a, lane 8 and Figure 6b, lanes 10, 11, and 12. The data discussed above and shown i n Figure 9 reveals that the p_h locus i s d i f f e r e n t i a l l y expressed throughout development. The developmental p r o f i l e of p_h i s probably as follows. In embryos and f i r s t i nstar larvae there i s complex, d i f f e r e n t i a l expression of 6.4 and 6.1 kb t r a n s c r i p t s followed by an abrupt decline of both during the late l a r v a l stages. During pupal development a novel 6.1 RNA i s transcribed at late embryonic levels and a 6.6 kb t r a n s c r i p t i s t r a n s i e n t l y expressed at low l e v e l s . F i n a l l y i n the adult, a 6.1 kb species i s expressed at low l e v e l s . The 2.0 kb RNA i s not d i f f e r e n t i a l l y expressed i n development. 64 DISCUSSION The analysis of the t r a n s c r i p t i o n of the ph. region i n wild type embryos has i d e n t i f i e d at least 4 t r a n s c r i p t i o n units. The analysis of the t r a n s c r i p t i o n of t h i s region i n ph mutants confirms that ph had been cloned because every mutation examined a l t e r s at least one of the RNA products of the region. The l i m i t s of the ph t r a n s c r i p t i o n units have been determined and preliminary i n d i r e c t evidence that the two repeats are separately transcribed has been obtained. F i n a l l y , d i f f e r e n t i a l developmental expression of the locus has been demonstrated. The ph locus contains two regions of repeated sequence, and the DNA i n t h i s region produces two t r a n s c r i p t s i n embryos and two i n pupae. Transcripts of each p a i r are distinguishable but of s i m i l a r size and contain high amounts of the repeated sequence. These facts, combined with the observation that two mutagenic events are required to eliminate ph. function (see discussion Chapter 1), suggest that t h i s region consists of two separate mutable regions that are functionally autonomous, but that produce messages of similar sequence and function. Another p o s s i b i l i t y i s that there i s one t r a n s c r i p t i o n unit that produces two messages by alternative s p l i c i n g of a primary tr a n s c r i p t , or one t r a n s c r i p t i o n unit with multiple promoters or termination s i t e s or both. These p o s s i b i l i t i e s cannot be d i f f e r e n t i a t e d between on the basis of Northern hybridization of genomic probes to wild type RNA because of the high 65 sequence s i m i l a r i t y between the two repeats. However, analyzing t r a n s c r i p t i o n i n the ph. mutants supports the hypothesis that there are two t r a n s c r i p t i o n units. If there i s one p_h tr a n s c r i p t i o n unit with alternative s p l i c i n g or multiple promoters, a p_h mutation a l t e r i n g either the proximal or d i s t a l repeat could affect both or one of the t r a n s c r i p t s . However, i f there are two p_h tr a n s c r i p t i o n units corresponding to the proximal and d i s t a l repeats, then a p_h mutation a f f e c t i n g only the proximal or d i s t a l repeat should affect one but not both mRNA's. The preliminary data presented here favor the l a t t e r interpretation. In embryos, ph-4-^- and p h — affect only the tran s c r i p t from the proximal repeat, and i n pupae ph-4-^- a l t e r s the t r a n s c r i p t from the d i s t a l repeat. More di r e c t evidence i s required to determine i f the B tr a n s c r i p t i o n unit can be separated into two units and i f the sit u a t i o n i s the same i n a l l developmental stages. The experiments described here should be repeated for embryos and pupae of the same mutants and any others that af f e c t only one repeat. Good candidates would be ph-2^ -'-S-*- and—. Df (1)Pgd-kz would be es p e c i a l l y valuable because t h i s deficiency behaves l i k e a p_h viable a l l e l e and i t s d i s t a l breakpoint maps i n the unique region between the repeats. The prediction i s that t h i s deficiency would completely eliminate the 6.1 kb species but leave the 6.4 kb i n t a c t . However, i t would be d i f f i c u l t to c o l l e c t s u f f i c i e n t embryos to prepare RNA for t h i s experiment because Df(1)Pgd-kz i s homozygous l e t h a l . These experiments should be conducted so that the two species can be e a s i l y resolved. 66 Ultimately, understanding the organization of the locus w i l l require the sequencing of cDNAs from d i f f e r e n t stages, and of genomic DNA. If there are two separate t r a n s c r i p t i o n units in the B region i t should be possible to i s o l a t e side s p e c i f i c cDNAs with separate promoters. Developmental P r o f i l e of ph Transcripts The developmental p r o f i l e of ph expression i s complex. At least 4 species of RNA are d i f f e r e n t i a l l y expressed throughout development. I n i t i a l l y , at 0-3 hours, a 6.1 kb t r a n s c r i p t i s present i n r e l a t i v e l y low amounts. This species p e r s i s t s and i t s l e v e l increases approximately 5 f o l d i n the 12 hours p r i o r to hatching. It continues to be expressed during the f i r s t i n s t a r larva, after which there i s an abrupt decline (data not shown). Transcripts of s i m i l a r size are apparent i n pupae at equivalent lev e l s (Figure 9, lane P) and i n adults at lower l e v e l s . It may be that the species detected i n the adult i s actually only present i n female ovaries. This hypothesis i s consistent with the strong maternal e f f e c t that has been demonstrated for ph but remains speculation u n t i l separate preparations of male and female RNA are probed. The case for the 6.4 kb species i s somewhat d i f f e r e n t . It i s i n i t i a l l y present at 3-12 hours, and i t s abundance becomes sim i l a r to the 6.1 kb species i n the next 12 hours after which i t s l e v e l becomes undectable. Two RNA's, 6.6 and 6.1 kb are present i n pupae, and although they have sequence s i m i l a r i t y to the 67 embryonic species they are probably pupal s p e c i f i c . The 6.6 kb RNA i s larger than either embryonic species and the mutant data suggest that 6.1 kb pupal RNA species i s a d i s t a l product while i t s embryonic counterpart i s a proximal product. F i n a l l y a f i f t h 2.0 kb RNA i s present throughout development but i t i s unclear i f t h i s i s a ph product or that of a genetically u n i d e n t i f i e d gene. It i s i n t e r e s t i n g to re-examine the genetic data (Dura et a l . , 1987 and Brock and Weddell, unpublished data) i n l i g h t of t h i s developmental analysis of t r a n s c r i p t i o n . The ph locus has both a maternal and a zygotic e f f e c t . The requirement for a ph, product i n the maternal germ l i n e was demonstrated by generating germline clones that were homozygous for ph n u l l (ph—) a l l e l e s . Embryos derived from these clones show almost complete lack of d i f f e r e n t i a t i o n and have a ventral hole i n the c u t i c l e . The zygotic phenotype i s somewhat d i f f e r e n t . Embryos that are homozygous or hemizygous ph— develop normally for 10 hours but development arrests at 12 hours. Mutant embryos possess no ventral c u t i c l e , but head segments and the posterior region are unaffected and embryonic defects are confined to the thoracic and abdominal segments. Another a l l e l e , ph - ^ L , i s an embryonic l e t h a l that i s not amorphic, since the phenotype i s c l e a r l y d i f f e r e n t from that of ph— a l l e l e s . The embryo develops u n t i l about 24 hours when i t dies p r i o r to hatching. Thoracic and abdominal segments i n these embryos resemble the eighth abdominal segment. The three phenotypes described here r e f l e c t temporal and functional differences i n the embryonic requirements for the ph 68 products. This i s consistent with developmental p r o f i l e described above. Only the 2.0 and 6.1 kb species are available in the f i r s t hour of development. One or both of these must be responsible for the strong maternal e f f e c t . The absolute zygotic requirement i s between 10 and 12 hours, and t h i s coincides with the detection of the 6.4 kb RNA. The abundance of both large t r a n s c r i p t s peaks between 12 and 24 hours, and i t i s at the end of t h i s period that the extreme hypomorph, ph^^, dies. This analysis shows that there are at least 4 p_h t r a n s c r i p t s . It has, however, raised questions as to the nature of the p_h t r a n s c r i p t i o n units. Mutant analysis favors the interpretation that there are 2 t r a n s c r i p t i o n units that correspond to the two repeats. However, confirmation of t h i s hypothesis w i l l require sequence data. A comparison of the genomic sequence to that of the 6.4 and 6.1 mRNA's should reveal the true structure of the locus. One way i n which t h i s could be done i s by i s o l a t i n g cDNA's that correspond to the 6.4 and 6.1 t r a n s c r i p t s and then sequencing both the cDNA's and genomic clones. Even before sequence data i s available an analysis of the r e s t r i c t i o n maps of the cDNA's compared to that of the genomic clones and an analysis of cross hybridization between the cDNA's and genomic clones may allow either acceptance or r e j e c t i o n of the above hypotheses. 69 CHAPTER THREE: Isolation and analysis of ph cDNAs INTRODUCTION Complementary DNA (cDNA) i s a f a i t h f u l DNA copy of an mRNA synthesized i n v i t r o using reverse transcriptase. A cDNA l i b r a r y i s a c o l l e c t i o n of cDNAs synthesized from a p a r t i c u l a r population of mRNAs. Ideally, the synthesized f i r s t strand cDNA w i l l be representative of the t o t a l population of mRNAs. Following p a r t i a l hydrolysis of RNA templates a second DNA strand i s synthesized with DNA polymerase to make a double-stranded cDNA. These molecules are then cloned into bacteriophage or plasmids, thus forming a l i b r a r y of DNA molecules complementary to the o r i g i n a l mRNAs. Such l i b r a r i e s are useful for i s o l a t i n g the processed version of a gene when the genomic version has already been cloned or when a homologue from another species i s available. The molecular characterization of a gene i s greatly f a c i l i t a t e d once cDNAs are available. F i r s t , the r e s t r i c t i o n maps of cDNA clones can be compared to genomic clones to determine i f the gene contains introns. When a gene possesses one or more introns the genomic clones contain sequences that are not present within the corresponding cDNA, and thus there i s a discrepancy between the maps. It may be d i f f i c u l t to i s o l a t e large genes i n the form of single cDNAs, i n which case clones representing parts of the gene must be characterized i n d i v i d u a l l y . In such cases i t i s necessary to clone cDNAs whose sequences overlap, to ensure that a l l parts of the gene 70 are characterized. Ultimately, a comparison of the nucleotide sequences of the genomic and cDNA clones shows exactly where and how large the introns are. Second, a cDNA i s an ideal probe for Northerns and Southerns because i t lacks introns and flanking sequence outside the tr a n s c r i p t i o n unit that may be present i n a genomic clone. Therefore, the length of homologous sequence i n a cDNA clone for a s p e c i f i c mRNA w i l l be higher than that of a genomic clone. If genomic DNA of another species i s analyzed, a cDNA i s a superior probe because only exons, which show a high rate of evolutionary conservation, are analysed. Third, determining the sequence of a gene i s usually more e f f i c i e n t i f cDNAs can be used. This i s espe c i a l l y true i f the introns are large. F i n a l l y , the sequence of a cDNA can be used to determine the open reading frame and then appropriate fragments can be used to construct expression vectors i n order to generate protein products. Many factors complicate the search for cDNAs. F i r s t , rare t r a n s c r i p t s may not be represented i n the l i b r a r y . Since, the frequency of a given mRNA i n a population cannot e a s i l y be measured accurately, large numbers of recombinants should be screened to ensure that even the rarest members of a l i b r a r y are l i k e l y to be recovered. Second, i t i s more d i f f i c u l t to make f u l l length cDNAs of large mRNAs because the i n t e g r i t y of the complex formed between reverse transcriptase and the mRNA being copied must be maintained longer. Third, s p e c i f i c sequences or secondary structure of some mRNAs may preclude copying by reverse transcriptase. F i n a l l y , amplification of 71 the l i b r a r i e s w i l l result i n sele c t i v e loss of cDNAs that reduce the v i a b i l i t y of lambda phage. These l a s t three problems a l l make i t d i f f i c u l t to i s o l a t e large, rare, cDNAs. Northern analysis has shown that the major ph tra n s c r i p t s are r e l a t i v e l y long, at 6.1 and 6.4 kb, and r e l a t i v e l y rare so problems were anticipated. However, as further molecular analysis of ph. required the i s o l a t i o n of cDNAs, a screen for ph cDNAs was undertaken. Even i f only small ph cDNAs were obtained they can s t i l l be used as primers to produce extended ph cDNAs. I used subcloned fragments from the ph locus to screen lambda gtlO cDNA l i b r a r i e s from 6 d i f f e r e n t stages of development: 1.5-5 hour embryos (M. Goldshmidt-Clermont i n prep); 0-3, 3-12 and 12-24 hour embryos, and early and late stage pupae (Poole et al., 1985) . These l i b r a r i e s have been used to successfully i s o l a t e cDNAs for many Drosophila genes, including Antennapedia (Schneuwly, 1986), transformer (Boggs,1987) and T o l l (Hashimoto,1988). These stages were chosen because Northern analysis had shown that ph messages are more abundant at these stages of development (see Chapter 2). No obviously d i f f e r e n t ph tr a n s c r i p t s are expressed i n either larvae or adults so these stages were not screened. I hoped to i s o l a t e cDNAs complementary to the 6.4 and 6.1 kb t r a n s c r i p t s i n the embryo l i b r a r i e s , cDNAs complementary to 6.6 and 6.1 tr a n s c r i p t s i n the pupal l i b r a r i e s , and representatives of the ubiquitous 2 kb t r a n s c r i p t i n at least some of the stages. The Goldschmidt-Clermont embryo l i b r a r y contained 25 x 10 4 phage i n 5 subpools of 5 x 10 4 phage each. To determine 72 the number of phage that must be screened to obtain any single cDNA from a subpool, the following formula was used: N = ln_d(l-P) In (1-F) where P = desired p r o b a b i l i t y F = f r a c t i o n a l proportion of the t o t a l cRNA population that a single cDNA represents. N = the number of recombinants to be screened to ensure representation. The desired p r o b a b i l i t y was 0.99 and each subpool contained 5 x 10 4 phage so N = ln_(l-0.99) ln (1/50,000) = 283,241 Therefore, approximately 3 x 10^ phage of each subpool were screened. Each Poole l i b r a r y contained 3 x 10^ phage and 3 x 10 of each of these l i b r a r i e s were screened. In t o t a l , 3.0 x 10^ phage were screened and positives were obtained for the 1.5-5 h embryo, 0-3 h embryo, 3-12 h embryo and early pupal stages. The approximate genomic location of each cDNA was determined by hybridization to genomic DNA and r e s t r i c t i o n mapping. Also, representative cDNAs were used to probe Northerns and Southerns of genomic DNA to test i f s p e c i f i c cDNAs could be assigned to a p a r t i c u l a r t r a n s c r i p t . MATERIALS AND METHODS Screening the cDNA l i b r a r i e s CDNA clones were i s o l a t e d from two lambda gtlO l i b r a r i e s constructed and kindly donated by M. Goldschmidt-Clermont (M. Goldschmidt-Clermont et a l . , i n prep) and L. Kauvar (Poole et a l . 1985). Phage were plated at a density of 30,000 pfu/150 mm p e t r i dish on 1.5% NZCYM agar i n a top layer of 7.5 ml of NZCYM agar containing 0.7% agar and 300 /<1 of an overnight culture of Ej. c o l i C600 (Appleyard, 1954). NZCYM i s 10 g NZ amine, 5 g NaCl, 1 g casamino acids, 5 g Bacto-yeast extract, 2 g MgS04 per l i t e r , pH 7.5. The plates were incubated at 37°C u n t i l near confluent l y s i s , then c h i l l e d . A c i r c u l a r n i t r o c e l l u l o s e f i l t e r was placed on each plate for 1 min then l i f t e d and placed DNA side up i n 15 ml denaturing solution (1.5 M NaCl, 0.5 M NaOH) for 5 min, and then transferred to 15 ml of ne u t r a l i z i n g solution(1.5 M NaCl, 0.5 M Tris.CI pH8.0) for 5 min. Duplicate f i l t e r s were l i f t e d i n the same manner. DNA was fix e d to the f i l t e r by baking for 2 hours at 80°C. Hybridization probes were radi o l a b e l l e d by nick t r a n s l a t i o n or random priming as described i n Chapters 1 and 2 respectively. The f i l t e r s were prehybridized, hybridized, and washed as described for Southern f i l t e r s i n Chapter 1. Duplicated p o s i t i v e plaques were picked and suspended i n 1 ml of SM (5.8 g NaCl, 2 g MgS04 - 7H20, 50 ml 1 M T r i s - C l (pH 7.5), 5 ml 2% ge l a t i n per l i t e r of H20) to elute phage. 74 The phage were plated at a density of 1000 pfu per 90 mm p e t r i dish, l i f t e d and hybridized to the same probe as described above. This step was repeated u n t i l i n d i v i d u a l plaques could be is o l a t e d and plates were 100% p o s i t i v e . Preparation of phage DNA Small-scale preparations of phage DNA of a l l positives were used to i d e n t i f y cDNA clones. Large-scale preparations of these clones were used as sources of DNA to be mapped, subcloned and stored. 1. Small-scale phage and phage DNA preparations Phage were eluted from a single p u r i f i e d plaque i n SM. 50 ^1 of t h i s suspension, 50^1 of a C600 overnight culture and 3 ml of NZCYM were incubated together at 37°C i n a shaking incubator u n t i l l y s i s was apparent, approximately 10 hours. The c e l l s were p e l l e t e d by centrifugation for 10 min. 1.2 ml of the lysate were mixed with 300 /ul of a 20% polyethylene g l y c o l 8000, 2.5 M NaCl solution and c h i l l e d for 15 min. The phage were pr e c i p i t a t e d by centrifugation i n a bench top centrifuge for 10 min. The supernatant was removed and the phage p e l l e t resuspended i n 0.8 ml SM. The phage were lysed by adding 20 /<1 of 10% SDS, 100 1 of a 2M T r i s - C l pH8.0, 0.2 M EDTA solution and heating at 70°C for 5 min. 100^1 of 5 M sodium acetate was added and after 15 min on ice the DNA was pre c i p i t a t e d with 0.6 ml of isopropyl alcohol, pelleted, rinsed 75 i n 70% ethanol, dried and resuspended i n TE. 2. Large-scale phage and phage DNA preparation. 10 8 phage were incubated with 1 0 1 0 C600 c e l l s for 15 min at 37°C and then added to 500 ml of prewarmed NZCYM. The cultures lysed after approximately 8 hours i n a shaking incubator at 37°C. 5 ml of CIA was added and shaking was continued for 30 min. After the addition of 29.2 g of NaCl, the c e l l s were p e l l e t e d by centrifugation at 8000 rpm for 10 min i n a Sorvall GSA rotor at 4°C. 50 g of polyethylene gl y c o l 8000 was added to the decanted supernatant, which was then c h i l l e d on ice for one hour. The suspension was centrifuged as above and the supernatant discarded. The phage containing p e l l e t was resuspended i n 4 ml of SM with 40^g of deoxyribonuclease and then extracted with 2.5 ml of CIA. The aqueous phase was removed and adjusted to a density of 1.46 g/ml with cesium chloride. The phage were i s o l a t e d by two 16-20 hour centrifugations at 36,000 rpm at 4°C i n a SW50.1 rotor. Cesium chloride was removed from the phage preparation by d i a l y s i s for 1 hour against a 1000 f o l d volume of 10 mM NaCl, 50 mM T r i s - C l pH 8.0 and 10 mM MgCl 2. The buffer was changed once and the d i a l y s i s repeated for an additional hour. The phage suspension was adjusted to 0.025 M EDTA, extracted with an equal volume of phenol and spun at 4000 rpm for 5 min i n an HS-4 Sorvall rotor to separate the phases. The aqueous phase was removed and extracted as above with an equal volume of phenol:CIA (1:1). A f i n a l extraction was performed with an CIA 76 alone. The aqueous phase was removed and adjusted to 0.3 M sodium acetate, p r e c i p i t a t e d with 2 volumes of 95% ethanol and centrifuged to p e l l e t the DNA. The p e l l e t was rinsed i n 70% ethanol, dried and resuspended i n TE. Subcloning and Southern and Northern hybridizations of cDNAs. The procedures used for subcloning, Southern f i l t e r preparation and Southern hybridization were performed as described i n Chapter 1. Northern analysis was performed using methods described i n Chapter 2. 77 RESULTS Screening the cDNA l i b r a r i e s . To recover recombinants containing p_h mRNA sequence, six separate l i b r a r i e s from d i f f e r e n t stages of development were screened. Each l i b r a r y was i n d i v i d u a l l y plated and transferred to r e p l i c a n i t r o c e l l u l o s e f i l t e r s . The f i l t e r s were then hybridized to J P-labeled phage or plasmid DNA from the p_h region. A t y p i c a l pair of f i l t e r s i s shown i n Figure 10a. F i f t y thousand phage were present on the plate from which these f i l t e r s were made and only one contained sequence highly similar to a p_h probe, the 4.0 Sal I fragment (see large arrows Figure 10a). The phage that produced t h i s signal contains the cDNA named Pla. Approximately 10 additional f a i n t p o s i tives are present (see small arrows, Figure 10a) but representatives of t h i s class of positives were shown to contain pUC 13 sequence and were subsequently ignored. The fa i n t signal i s probably due to small amounts of pUC sequence present i n the probe. Each p o s i t i v e was p u r i f i e d to homogeneity as described i n Materials and Methods. To ensure that representatives from each t r a n s c r i p t i o n unit were obtained, subcloned DNA representing the entire p_h region from coordinates 124.7 to 149.2 was used to probe the the 1.5 - 5.0 hour and 0-3.0 hour and 3 -12 hour embryo l i b r a r i e s . Northern analysis, however, had shown that a subset 78 Figure 10. Screening the cDNA l i b r a r i e s . (a) Autoradiographs of two r e p l i c a f i l t e r s are shown. The large arrows indicate a duplicated p_h p o s i t i v e clone. The small arrows indicate duplicated signals that were shown to contain pUC 13 sequence.(b) R e s t r i c t i o n digests of DNA prepared from p u r i f i e d phage. M i s lambda phage cut with Hind III and the sizes of the fragments are shown i n kb on the l e f t . The follow-ing cDNAs are present i n the numbered lanes: lane 1, Pla; lane 2, E5; lane 3, E3; lane 4 and 5, fals e positives and lane 6, D13. 79 of fragments hybridized to a l l of the tr a n s c r i p t s from both the B and C units so i n some cases only one or two fragments were used as probes. The s p e c i f i c fragments chosen for each screening are l i s t e d i n Table 4. For the l i b r a r i e s from which positives were i s o l a t e d the mean number of po s i t i v e s was 5.7 for every 3 x 10 recombinants screened (see Table IV). Based on these numbers the abundance of ph cDNAs i s approximately 1/50,000. It should be noted, however, that according to Northern analysis ph messages are most abundant late i n embryogenesis and no cDNAs were obtained for the 12-24 hour embryo l i b r a r y . It i s l i k e l y that the number of cDNAs i s o l a t e d i s a r e f l e c t i o n of the quality of the l i b r a r y rather than the actual abundance of ph messages. Nevertheless, t h i s result i s consistent with Northern analysis which shows that ph i s rare. Mapping of cDNAs Small-scale phage and DNA preparations were made of each p u r i f i e d p o s i t i v e (see Materials and Methods). The size of the cDNA was determined by digesting the prepared DNA with Eco RI to separate the insert from the phage. A representative photograph of these digests i s shown i n Figure 10b. The cDNAs present are Pla (lane 1), E5 (lane 2), E3 (lane 3) and D13, (lane 6). It i s probable that a l l of the ph sequences represented i n these l i b r a r i e s were characterized. A t o t a l of 14 d i f f e r e n t cDNAs were i s o l a t e d from d i f f e r e n t stages and 81 LIBRARY PROBE # POSITIVES UNIQUE SIZE(KB) POSITIVES 1 . 5 - 5 embryo ef a 2 C 2 6 6 . 0 ab,cd,gh,ij b 12 C l 3 . 6 C 3 1 . 7 C 5 2 . 3 C6 0 . 8 0 - 3 c 1 3 D 1 3 1 . 8 embryo 3 - 1 2 d 8 E l 3 . 6 embryo E 6 0 . 8 E 7 1 . 6 c 9 E3 1.0 E5 0.6 12-24 e embryo 5.5-9 day d 7 PI 1.9 pupal Pla 2.2 P6 2.1 7-9 day pupal TABLE IV. The results of cDNA l i b r a r y screens are given. The 1.5-5 hour l i b r a r y was divided into 5 subpools designated ab/ cd, ef, gh and i j . The ef subpool was screened separately and the remaining 4 were pooled and screened together. A l l of the probes used were combinations of subcloned r e s t r i c t i o n fragments and are l i s t e d below. Their position on the r e s t r i c t i o n map can be seen i n Figure 2 except for the 1.9 Eco RI fragment that was subcloned from cDNA C2 6. combination probes a - 1.5 Sal I, 2.2 Xho I/BamH I b - 2.3 Sal I, 4.0 Sal I, 1.5 Sal I, 9.0 Bam HI, 1.9 Eco RI(cDNA) c - 2.3 Sal I, 1.5 Sal I, 2.8 Bam HI 9.0 Bam HI, 1.9 Eco RI(cDNA) d - 4.0 Sal I, 1.9 Eco RI e - 4.0 Sal I 82 these are designated with a l e t t e r and number and are l i s t e d i n Table IV. Of the 11 embryo cDNAs, there were 5 cDNAs (C2 6, C l , C3, C5, C6) i s o l a t e d from the 1.5-5 hour l i b r a r y , 5 cDNAs (El, E3, E5, E6, E7) from the 3-12 hour l i b r a r y and one cDNA (D13) from the 0-3 hour l i b r a r y . Three cDNAs labeled PI, Pla and P6 were i s o l a t e d from the early pupal l i b r a r y . Table 4 also gives the probes that were used, the number of positives that were obtained for each stage and the size of each cDNA. "SO Each of the 14 cDNAs were i n d i v i d u a l l y labeled with J P and hybridized to Southern blots of r e s t r i c t i o n fragments which represented the cloned DNA from the entire ph region. A photograph of the ethidium bromide stained gel used to make one of these blots i s shown i n Figure 11a. The autoradiographs that were produced when E7, D13 and Cl were separately hybridized to t h i s blot are shown i n Figure 11 b,c and d. Similar data was obtained for a l l cDNAs. I w i l l discuss how t h i s method was used to map the posi t i o n of cDNA E7. This cDNA hybridizes strongly to 3 fragments i n the ph region (Figure l i d - lanes 5 and 8). These are the 1.8 Sal I/Hind III and adjacent 1.2 Hind III/Bam HI fragments (127.8-130.8) and the 1.2 Sal I/Xho I (137.9-139.1) fragment. See Figure 12 for the map pos i t i o n that was determined from t h i s data. E7 i s 1.6 kb and hybridizes to fragments that t o t a l 4.2 kb. It i s possible that E7 i s transcribed from sequences i n a l l 3 fragments and i s a product of parts of both repeats. However, the simplest explanation i s that E7 i s from one or the other repeat and that the hybridization to one repeat i s due to sequence s i m i l a r i t y to the xc' domain. If t h i s i s the case, E7 83 Figure 11. Determining the genomic location of 3 cDNAs (a) Ethidium bromide stained gel of digested and e l e c t r o p h o r e t i c a l l y separated fragments of subcloned DNA. The gel was blotted to n i t r o c e l l u l o s e , probed separately with d i f f e r e n t labeled cDNAs and exposed to X-ray f i l m . b),c) and d) The autoradiographs produced when cDNAs D13, CI and E7 respectively were used as probes. M i s lambda phage' cut with Hind III and the sizes of the fragments are shown i n kb on the l e f t and r i g h t . The subclones present i n each lane are l i s t e d below. The r e s t r i c t i o n s i t e s and enzymes are indicated B(Bam HI), E(Eco RI), H(Hind III, P(Pst I), S(Sal I ) , X(Xho I ) . lane subclone digested 1 3.6 E EP 2 7.0 ES EP 3 2.3 S SX 4 0.8 S PS 5 4.0 S BHS 6 1.5 S S 7 3.9 S HPSX 8 2.0 SE EPS 9 2.0 X EP 10 2.2 XB BX 11 ? 12 2.8 XB BX 13 3.4 X BPE 14 4.8 E BEX 15 1.0 E E 84 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 c. d. cannot be transcribed from the proximal repeat because i t i s 1.6 kb long and the fragment to which i t hybridizes i n t h i s repeat i s only 1.2 kb. These data indicate that E7 i s complementary to a d i s t a l s p e c i f i c message, and that i t contains sequence unique to the d i s t a l repeat. E6 hybridized to the same fragments but i s smaller than E7 so may be a truncated version of the the same cDNA. The data i n Figure l i b are for D13. D13 hybridizes to 4 fragments between 149.2 and 152 (Figure l i b , lanes 13, 14 and Figure 12). PI hybridized to the same fragments plus a 0.5 kb fragment between 147 and 148.5. The coordinates for D13 and PI f a l l within the region i d e n t i f i e d as t r a n s c r i p t i o n unit C i n the Northern analysis. The data i n Figure 11c are for E l . Identical data were obtained for CI. These cDNAs hybridize to two fragments between 139.5 and 143.7 (Figure 11c, lanes 9 and 10, and Figure 12) . This sequence i s unique to the proximal region except for a very small portion of the M' domain and there i s a some sl i g h t cross hybridization to a fragment i n the d i s t a l M' domain (Figure 11c lane 5). E l and CI are unique to the proximal region. The remaining 11 cDNAs; C26, CI, C3, C5, C6, E l , E3, E5, E6, Pla and P6 were mapped i n the same way and the information on map position obtained from these experiments i s summarized in Figure 12. C5 and P6 map to the same positions, the xa' domains of both repeats, and are the same size but C5 i s from embryos whereas P6 i s from pupae. C3 and E3 map to the same positions, also the ^a' domains of both repeats, but are not 86 Figure 12. Fragments of genonmic DNA to which cDNAs hybridize. The r e s t r i c t i o n map and the map of repeated domains i s reproduced from Figure 5 and extended to coordinate 155. The cDNAs that were used as probes are l i s t e d on the l e f t and the fragments to which they hybridized are shown as dark l i n e s immediately to the ri g h t . The dark l i n e s indicate a strong signal with high stringency wash conditions whereas the dotted l i n e s indicate a weak signal with high stringency wash conditions. Examples of strong and weak signals can be seen i n Figure 11. 87 - r j - r j m m m m o o O O O CT> - » Ol VI W - ^ - ^ 0 > Ul W N ft! go O) -OD •(/) -x • v> D •I - OS - V) x •I CO • X X (ft •o tft •"0 ID cr - X "0 -00 • X CO a 00 TJ tn e co x 00 m Cft OD 88 the same size and and were i s o l a t e d from d i f f e r e n t embryo l i b r a r i e s . Pla maps to the same pos i t i o n as E3 except for additional hybridization to *b' d i s t a l domain. This suggests that Pla i s d i s t a l s p e c i f i c . E5 on the other hand hybridizes to the Aa' and *b' domains of both repeats and i s 0.8 kb. It may be transcribed from either or both repeats. C6 hybridizes yc' domains of both repeats and also may be transcribed from either or both. The hybridization data obtained for these 13 cDNAs can be summarized as follows: D13 and PI are products of the C t r a n s c r i p t i o n unit and the rest are products of either or both of the repeated regions of the B t r a n s c r i p t i o n unit. It i s in t e r e s t i n g to note that the majority of the B s p e c i f i c cDNAs map to the \a' domain. Since both of the l i b r a r i e s were made using oligo d(T) as primer, and the i n i t i a l products of reverse transcriptase predominate, i t i s probable that one or both of the Aa' domains contain the 3 prime end(s) of the ph products. F i n a l l y , i n some cases there i s evidence that s p e c i f i c cDNAs can be t e n t a t i v e l y assigned to either the d i s t a l or proximal repeated region; E7, E6 and Pla to the d i s t a l repeat and E l and Cl to the proximal repeat. C2 6 i s 6.0 kb and therefore i s the only cDNA is o l a t e d that i s near the size of the major ph 6.0 kb t r a n s c r i p t s . However, i t maps to the B and C t r a n s c r i p t i o n units, and Northern analysis shows that the large ph t r a n s c r i p t s are products of the B t r a n s c r i p t i o n unit alone. Also, i t i s unusual i n that i t i s cut out of lambda at an Eco RI and a Hind III s i t e . This 89 l i b r a r y was made by c l o n i n g cDNAs i n t o Eco RI s i t e s o n l y and t h i s s u g g e s t s t h a t C26 has l o s t an Eco RI s i t e . F i n a l l y , p r e l i m i n a r y sequence d a t a r e v e a l s t h a t C26 has b o t h exons and i n t r o n s (H. Brock and M. D e C a m i l l i s , u n p u b l i s h e d d a t a ) . Taken t o g e t h e r t h e s e d a t a suggest t h a t C2 6 does not r e p r e s e n t an a u t h e n t i c cDNA. T h i s s i t u a t i o n i s not w i t h o u t p r e c e d e n t . Large numbers o f *junk' cDNAs have been i s o l a t e d f o r t h e Ubx gene from t h e same l i b r a r y (M. O'Connor, p e r s o n a l communication). Each cDNA, ex c e p t f o r E6 and P6, was s u b c l o n e d i n t o pUC 13. The map o f t h e r e s t r i c t i o n s i t e s f o r t h e enzymes Eco R I , Bam HI, H i n d I I I , P s t I , S a l I and Xho I was d e t e r m i n e d f o r each cDNA and t h e s e maps a r e g i v e n i n F i g u r e 13. These d a t a might have a l l o w e d more p r e c i s e mapping o f t h e cDNAs onto t h e genomic DNA and shown i f any o f them were o v e r l a p p i n g . These e x p e c t a t i o n s were o n l y p a r t i a l l y r e a l i z e d . The C l , E l p a i r and E7 have r e s t r i c t i o n maps t h a t c o i n c i d e w i t h t h e genomic r e s t r i c t i o n map. T h i s s t r e n g t h e n s t h e argument t h a t t h e s e a r i s e from t h e d i s t a l and p r o x i m a l r e g i o n s r e s p e c t i v e l y . None of t h e o t h e r cDNAs t h a t were mapped can be a s s i g n e d t o a genomic r e g i o n based on t h e i r r e s t r i c t i o n maps. T h i s i s not s u r p r i s i n g because cDNAs a r e u s u a l l y e x t e n s i v e l y p r o c e s s e d , depending on t h e s i z e and number -of i n t r o n s , and r e s t r i c t i o n s i t e s may be removed and new ones may be added. T h i s i s p o s s i b l y t h e case f o r most o f t h e s e cDNAs and o n l y a comparison o f t h e i r sequences t o t h e genomic sequence w i l l i d e n t i f y t h e i r e x a c t o r i g i n . 90 Figure 13 Re s t r i c t i o n maps of cDNAs. The r e s t r i c t i o n maps are drawn for the cDNAs l i s t e d on the l e f t . The s i t e s mapped are as follows: B(Bam HI), E(Eco RI), H(Hind III, P (Pst I), S(Sal I), X(Xho I ) . The size of each cDNA i s given i n kb on the ri g h t . 91 C26 C5 C3 C6 C1.E1 E3 E5 E7 013 PI Pla H U B H X S I 1 I B H X EH X E S X PSE P E B B I I I B X I I I E B I • I B X I III EX P S 6.0 2.3 1.7 0.8 3.6 1.0 0.6 1.6 1.8 1.9 2.1 Some of the r e s t r i c t i o n maps do appear to overlap. This i s the case for C5 and C3. Because t h e i r hybridization maps also overlap, i t i s l i k e l y that together they span approximately 2.6 kb of a processed message. It i s possible that E3, that has none of the 6 r e s t r i c t i o n s i t e s , i s contained within the 1.3 kb region of t h i s overlap that also has none of these r e s t r i c t i o n s i t e s . In the C tr a n s c r i p t i o n unit PI and D13 also overlap both i n respect to t h e i r r e s t r i c t i o n maps and hybridization data. Together they are approximately 2.0 kb and t h i s i s the size of the major t r a n s c r i p t of the C tr a n s c r i p t i o n unit. Overlap between E3 and E5 i s suggested by the hybridization data and t h i s i s not ruled out by the r e s t r i c t i o n maps. Northern analysis of ph cDNAs CDNAs were hybridized to Northerns of p o l y ( A ) + embryonic RNA to determine, i f possible, the tr a n s c r i p t i o n unit or repeated region to which they belonged and to determine i f they represented p_h tran s c r i p t s not i d e n t i f i e d i n the e a r l i e r t r a n s c r i p t i o n analysis. The cDNA insert, separated from the phage, or the entire plasmid containing the subcloned cDNA was T O labeled with ~^P by random priming (see Materials and Methods) then hybridized to 0-24 hour or staged embryonic p o l y ( A ) + RNA. Representative data from these experiments are given i n Figure 14. 93 In general, cDNAs that hybridize to some portion of the B tr a n s c r i p t i o n unit also hybridize to the 6.4 and 6.1 kb tran s c r i p t s with the same embryonic p r o f i l e described i n Chapter 2. Data are shown for E5, C3 and Pla (Figure 12 -b,c,d,and e). Pla was also used to probe a 0-24 hour RNA preparation and hybridized to both species (Figure 14 (e). From these data i t cannot be determined i f the cDNAs tested belong to one or the other repeat. This i s probably also true for the other cDNAs that map to the B t r a n s c r i p t i o n unit. The only exception i s E l . Other investigators have determined that i t hybridizes to a single t r a n s c r i p t approximately 6.1 kb (N. Randsholt, personal communication). This result i s i n agreement with the hybridization data which suggests that E l and Cl are derived from the proximal s p e c i f i c sequence. It should be noted, however, that the genomic fragments to which these cDNAs hybridize, themselves hybridize to both the 6.4 and 6.1 kb messages (see Chapter 2). One possible explanation i s that sequence similar to the 6.4 kb species i n the genomic sequence has been s p l i c e d out of the cDNA sequence. Complementary DNA's that hybridize to fragments of the C tr a n s c r i p t i o n unit hybridize to the 2.0 kb t r a n s c r i p t . These include PI, D13 and the 2.0 Eco Rl fragment of C26. Data are shown for D13 and 2.0 kb C26 fragment (Figure 14 - f and g). It i s not possible to assign cDNAs that map to the repeated domains to one or the other repeat on the basis of t h i s Northern analysis. It did, however, show that none of the cDNAs i s o l a t e d represented u n i d e n t i f i e d t r a n s c r i p t s . 94 Figure 14. Northern analysis of p_h cDNAs. Autoradiographs of Northern blots.of poly (A) + RNA prepared from 0-3 hours embryos (3), 3-12 hours embryos (12) and 12-24 hours embryos (24) are shown i n a, b, and c; and from 0-24 hours embryos i n d and e. The cDNA to which each was hybridized i s shown at the bottom. The approximate sizes of the tran s c r i p t s are shown on the l e f t and r i g h t . 95 96 DISCUSSION I have isolated, p u r i f i e d and characterized 14 cDNAs that are products of the p_h region. Taken together they span the region from map positions 134 to 152 excluding the 1.2 kb gap between the repeats. They hybridize to the major t r a n s c r i p t s of the region, and are most l i k e l y truncated cDNAs that correspond to the 6.4, 6.1 and 2.0 kb t r a n s c r i p t s . The most disappointing aspect of t h i s analysis was the f a i l u r e to i s o l a t e a f u l l length cDNA of the 6.6, 6.4 and two 6.1 kb t r a n s c r i p t s . The Northern analysis described i n Chapter 2 had shown these to be the major products of the locus. C26 approaches t h i s size, but for reasons l i s t e d e a r l i e r i s not a true complement of either of these messages. The d i f f i c u l t i e s encountered here are the result of either problems with the l i b r a r i e s screened or problems created by p_h locus i t s e l f . The conditions under which the l i b r a r i e s were made may not have been conducive to the production of long cDNAs. This i s probably the case, because published results of successful screens of these l i b r a r i e s have been for smaller genes or have recovered overlapping cDNAs of the larger genes. (Boggs, 1987 and Hashimoto, 1988). Also, these l i b r a r i e s have been amplified an unknown number of times, and t h i s may have resulted i n the selec t i v e loss of some phage. The majority of the small cDNAs isolated, that i s , C3, C5, C6, E3, E5, Pla and P6 map primarily to the xa' domain (see Figure 12). This may be because the xc', xd' or xe' domains contain secondary structure or s p e c i f i c sequences that act as a strong *stop' signal for reverse transcriptase. The problems encountered i n t h i s screen indicate that a considerable amount of work w i l l need to be done to obtain f u l l length ph cDNAs. The following experiments should be undertaken i n the order given u n t i l true f u l l length cDNAs for a l l of the ph products have been is o l a t e d . F i r s t , screen high quality, unamplified l i b r a r i e s that have recently become available (M. O'Connor, personal communication). Second, make a new l i b r a r y using a random sequence rather than oligo d(T) as a primer and taking precautions to prevent the formation of secondary structure. In t h i s way, i t may be possible to get cDNA fragments on the 5 prime side of a strong stop. The gap in the region of the stop would have to be f i l l e d with genomic sequence. F i n a l l y , sequence cDNAs already i s o l a t e d and use t h i s sequence to make oligomers. These w i l l serve as primers to make a l i b r a r y that i s highly enriched for p_h cDNAs. Depending on the oligomer used, the l i b r a r y w i l l be enriched for cDNAs from a s p e c i f i c p o sition within the ph. region. In t h i s way a c o l l e c t i o n of cDNA fragments w i l l be accumulated that can be pieced together to give f u l l length cDNAs for a l l the the ph products. The screen described i n t h i s chapter was not successful i n i s o l a t i n g cDNAs for a l l the products of the ph locus, but was the simplest, most dire c t experiment to do. These studies show that there i s no point i n further screening of the same l i b r a r i e s and new approaches such as those described above must 98 be taken. The cDNAs is o l a t e d can be sequenced to as s i s t further screening attempts, to confirm the d i r e c t i o n of tr a n s c r i p t i o n and to f i n d at least part of the coding region. The l a t t e r information can be used to compare ph amino acid sequence to other known proteins and to b u i l d expression vectors for the expression of at least part of the ph protein. These proteins can be used to raise antibodies to the ph. product so that the d i s t r i b u t i o n of the protein can be determined. This analysis has provided a necessary l i n k between the cloning of the ph gene and i t s further molecular characterization. 99 GENERAL DISCUSSION The molecular analysis described here lays the groundwork and begins the extensive investigations that w i l l be required to determine the structure and function of the ph locus. Genetic analysis showed that p_h was a complex locus and 2 separate mutagenic events were required to produce a n u l l a l l e l e . On the basis of these results i t was suggested that there are two mutable regions that function as independent genetic units at the ph locus. Complementation analysis predicted that one ph mutable region should be proximal and the other should be d i s t a l to the Df(1)Pgd-kz breakpoint. The cross-hybridization data that was given i n Chapter one i s consistent with t h i s model. Two tandem repeats of highly s i m i l a r sequence flank the Df(1)Pgd-kz breakpoint. The ph hypomorphic mutations map to one or the other repeat. This combination of genetic behavior and molecular structure has not been previously described. Transcriptional analysis has i d e n t i f i e d two embryonic and two pupal tr a n s c r i p t s that are altered i n the ph viable a l l e l e s . These, therefore, are ph s p e c i f i c products. For the most part each le s i o n affects only one of the stage s p e c i f i c t r a n s c r i p t s . The simplest interpretation of these results i s that there are two separate t r a n s c r i p t i o n units at the ph locus. The results however are not unequivocal and i t i s 100 possible that there i s a single t r a n s c r i p t i o n unit corresponding to the two repeats that constitutes a complex gene with two domains having similar functions. A single unit with variable s p l i c i n g that includes each duplicated region i n alternative t r a n s c r i p t s could account for the data. Fourteen cDNAs corresponding to the f i v e t r a n s c r i p t s have been is o l a t e d . There i s preliminary evidence that some of these are s p e c i f i c products of either the d i s t a l or proximal repeat. None of these, however, are f u l l length copies and t h i s work predicts that obtaining them w i l l not be straightforward. The f a i l u r e to obtain true f u l l length cDNAs that represent authentic t r a n s c r i p t s , combined with the facts that i t i s d i f f i c u l t to di s t i n g u i s h between products of the two repeats and that there i s d i f f e r e n t i a l expression of the products throughout development indicates that the molecular analysis of t h i s locus w i l l require more work than was f i r s t anticipated. The developmental p r o f i l e of p_h expression consists of i n t r i c a t e d i f f e r e n t i a l expression of at least 4 p_h products, 6.6, 6.4 and two 6.1 kb. There i s homogeneous expression of a 2.0 kb t r a n s c r i p t that may also be a p_h product. This analysis i d e n t i f i e s a complex t r a n s c r i p t i o n pattern for ph.. Elucidating the exact nature of a l l the t r a n s c r i p t i o n products, as well as the time and place of t h e i r expression w i l l be a formidable task. The Ubx gene has presented a similar challenge. As part of the BX-C, i t was the f i r s t gene to be cloned by the technique of chromosomal walking (Bender, 1983) 101 and i s , at present, one of the most intensely studied Drosophila l o c i (for review see Peifer, 1987). Recent reports indicate that t r a n s c r i p t i o n a l and cDNA analysis i s s t i l l incomplete (Peifer, 1987; Saari and Bienz, 1987; O'Conner, 1988 and M. O'Conner, personal communication). Thus, the fact that i t appears that the molecular analysis of ph i s going to be d i f f i c u l t i s not without precedent. Ultimately i t w i l l be necessary to d i f f e r e n t i a t e between a l l of the RNA and protein products. This w i l l require the i d e n t i f i c a t i o n of coding sequence and possibly regulatory sequence that i s unique to each product. Product s p e c i f i c sequences can be used as probes for i n s i t u hybridization to tiss u e sections of staged embryos and larvae. They can also be cloned into protein expression vectors so that ph peptides and subsequently antibodies can be prepared. This would allow the detection of s p e c i f i c ph protein d i s t r i b u t i o n i n staged wild-type and mutant embryos. These analyses w i l l reveal the s p a t i a l and temporal s p e c i f i c i t y of expression and possibly the significance of the repeated domains. Ph w i l l be an i n t e r e s t i n g example of complexity of gene organization and regulation i n eukaryotes. Also, i t i s either d i r e c t l y or i n d i r e c t l y involved i n the i n t r i c a t e program that establishes the metameric pattern of Drosophila melanooaster. The work described here begins the analyses that w i l l determine the structure and function of ph, including i t s role i n development. 102 REFERENCES Akam, M., (1987). The molecular basis for metameric pattern i n the Drosophila embryo. Development 101, 1-22. Anderson, K.V., and Nusslein-Volhard, C. (1984). Information for the dorsal-ventral axis i s stored as maternal RNA. Nature 311. 223-227. Appleyard, R.K., (1954). Segregation of new lysogenic types during growth of a doubly lysogenic s t r a i n derived from Escherichia c o l i K12. Genetics 39., 440. Baumgartner, S., Bopp, D., Burri, M., and Nol l , M. (1987). Structure of two genes at the gooseberry locus related to the paired gene and t h e i r s p a t i a l expression during drosophila embryogenesis. Genes Dev. 1, 1247-1267. Bender, W., Akam, M., Karch, F., Beachy, P.A., Peifer, M., Spierer, P., Lewis, E.B. and Hogness, D.S. (1983). Molecular genetics of the Bithorax complex in Drosophila melanoqaster. Birnboim, H.C. and Doly, J. (1979), A rapid extraction procedure for screening recombinant plasmid DNA. Nuc. Acids Res. 1 Boggs, R.T., Gregor, P., Idr i s s , S.,.Belote, J.M. and McKeown, M. (1987) . Regulation of sexual d i f f e r e n t i a t i o n i n Drosophila  melanogaster v i a alternate s p l i c i n g of the transformer gene. C e l l 50/ 739-747. Breen, T.R. and Duncan, I.M. (1986). Maternal expression of genes that regulate the bithorax complex of Drsosohila  melanoqaster. Dev. B i o l . 118, 442-456. Campos-Ortega, J.A. and Hartenstein, V., (1985). The Embryonic  Development of Drosophila melanoqaster. Springer-Verlag, New York. Coleman, K.G., Poole, S.J., Weir, M.P., Soeller, W.C., and Kornberg, T. (1987). The invected gene of Drosophila: sequence analysis and expression studies reveal a close kinship to the engrailed gene.. Genes Dev. 1, 19-28. Denell, R.E., and Frederick, R.D. (1983). Homeosis i n Drosophila: a description of the Polycomb l e t h a l syndrome. Dev. B i o l . 17, 34-47. Denell, R.E. (1982) . Homeosis i n Drosophila: evidence for a maternal e f f e c t of the Polycomb locus. Dev. Genetics. 97, 103-113. 103 Dura, J-M., Brock, H.W. and Santamaria, P. (1985). Polyhomeotic: A gene of Drosophila melanoqaster required for correct expression of segmental i d e n t i t y . Mol. Gen. Genet. 198, 231-220. Dura, J-M., Randsholt, N.B., Deatrick, J., Erk, I., Santamaria, P., Freeman, J.D., Freeman, S.J., Weddell, D., and Brock, H.W. (1987). Maternal and zygotic requirement for polyhomeotic, a genetically complex locus required for normal expression of segmental i d e n t i t y and cu t i c u l a r development. C e l l 5_1, 687-877. Duncan, I. (1986). Control of bithorax complex functions by the segmentation gene Fushi-tarazu of Drosophila melanoqaster. C e l l 47, 297-309. Duncan, I. and Lewis, E.B. (1982). Genetic control of body segment d i f f e r n t i a t i o n i n Drosophila i n Developmental Order:  i t s Origin and Regulation (ed. S. Subtelny). New York: L i s s . Symp. Soc. Devi. 40./ 533-554. Edgar, B. and Odell, G. (1988) Modeling segmentation gene interactions, (submitted). Frasch, M. and Levine, M. (1987). Complementary patterns of even-shipped and f u s h i t a r a z u expression i n v o l v e t h e i r d i f f e r e n t i a l regulation by a common set of segmentation genes in Drosophila. Genes and Devel. JL, 981-995. Garcia-Bellido, A. (1979) . Genetic analysis of the achaete- scute system of Drosophila melanogaster. Grausz, J., Gyurkovics, H., Bencze, G., Awad, A.A.M., Holden, J.J. and Ish-Horowicz, D. (1981). Genetic characterization of the region between 8 6F1-2 and 87B15 on chromosome 3 of Drosophila melanogaster. Genetics .98., 775-789. Hafen, E., Levine, M., Gehring, 1984. Refulation of Antennapedia t r a n s c r i p t d i s t r i b u t i o n by the bithorax complex i n Drosophila. Nature, 307./ 287-289. Hannah-Alava, A. (1958). Developmental genetics of the posterior legs i n Drosophila melanogaster. Genetics 43., 878-905. Harding, K., Wedee, C , McGinnis, W. and Levine, M. (1985). S p a t i a l l y regulated expression of homeotic genes i n Drosophila. Science, 229, 1236-1242. Hashimoto, C , Hudson, K.L., and Anderson K.V. (1988). The T o l l gene of Drosophila. required for dorsal-ventral embryonic p o l a r i t y , appears to encode a transmembrane protein. C e l l , 52, 269-279. 104 Henikoff, S., Sloan, J.A., Kelly, J.D. (1983). A Drosophila metabolic gene t r a n s c r i p t i s a l t e r n a t i v e l y processed. C e l l 34., 405-414. Ingham, P.W., Baker, N.E., and Martinez-Arias, A. (1988). Regulation of segment p o l a r i t y genes i n the Drosophila blastoderm by fushi tarazu and even-skipped. Nature 331, 73-75. Ingham, P.W. and Martinez-Arias, A., (1986). The correct act i v a t i o n of Antennapedia and bithorax complex genes requires the fushi-tarazu gene. Nature, 324, 592-597. Ingham, P. (1984). A gene that regulates the bithoraz complex d i f f e r e n t i a l l y i n l a r v a l and adult cells- of Drosophila. C e l l 37, 815-823. Jackie, H. Tautz, D., Schu, R., S e i f e r t , E. and Lehmann, R. (1986). Cross-regulatory interactions among the gap genes of Drosophila. Nature 324, 668-670. Jurgens, G. (1985). A group pf genes c o n t r o l l i n g the expression of the bithorax complex i n Drosophila. Nature 316, 153-155. Kaufman, T.C, Lewis, R. and Wakimoto, B., 1980. Cytogenetic analysis of chromosome 3 i n Drosophila melanoqaster: The Homeotic gene complex i n polytene i n t e r v a l 84A-B. Genetics 94: 115-133. Lawrence, P.A, Johnston, P. and Struhl, G. (1983). Different requirements for homeotic genes i n the soma and the germ l i n e of Drosophila. C e l l 35, 27-34. Lewis, E.B. (1978). A gene complex c o n t r o l l i n g segmentation i n Drosophila. Nature 276, 565-570. Lewis, R.A., Kaufman, T.C, Dennell, R.E., T a l l e r i c o , P. (1980). Genetic analysis of the Antennnapedia gene complex (ANT-C) and adjacent chromosomal regions of Drosophila  melanoqaster I. Polytene chromosome segements 84B-D. Genetics 95, 367-381. Lewis, R.A., Wakimoto, B., Dennell, R.E., Kaufman, T.C. (1980). Genetic analysis of the Antennnapedia gene complex (ANT-C) and adjacent chromosomal regions of Drosophila melanoqaster I I . Polytene chromosome segements 84A-B1,2. Genetics 95., 367-381. Lohs-Schardin, M., Cremer, C. and Nusslien-Volhard, C. (1979). A fate map for the l a r v a l epidermis of Drosophila melanoqaster: l o c a l i z e d c u t i c l e defects following i r r a d i a t i o n of the blastoderm with an u l t r a v i o l e t laser microbeam. Devi. B i o l . , 73 239-255. Maniatis, T., F r i t s c h , E.F., and Sambrook, J. (1982). Molecular  cloning: a laboratory manual. Cold Spring Harbor Laboratory, 105 Cold Spring Harbor, N.Y. Meinhardt, H. (1986). Hierarchical inductions of c e l l states; A model for segmentation i n Drosophila. J. C e l l S c i . Suppl. 4., 357-381. McKeon, M., Belote, J.M. and Baker, B.S. (1987). A molecular analysis of transformer, a gene i n Drosophila melanoqaster that controls female sexual d i f f e r e n t i a t i o n . C e l l 4.8, 489-499. Muller, H.J. (1932). Further studies on the nature and causes of gene mutation. Proc. Sixth Intern. Cong. Genet. JL, 231-235. Nusslein-Volhard, C , (1979). Maternal e f f e c t mutations that a l t e r the s p a t i a l coordinates of the embryo of LJK. melanoqaster. In Determinants of s p a t i a l organization (ed. I.R. Konisberg and S. Subtelny), 185-211. Academic Press, New York. Nusslein-Volhard, C , and Wiechaus E., (1980). Mutations a f f e c t i n g segment number and p o l a r i t y i n Drosophila. Nature 287, 795-801. O'Connor, M.B., Bin a r i , R., Perkins, L.A. and Bender, W. (1988). Alternative RNA products from the Ultrabithorax domain of the bithorax complex. EMBO 1_, 435-445. Peifer, M., Karch, F., and Bender, W., (1987). The bithorax complex: control of segmental i d e n t i t y . Genes and Dev. JL, 891-898 . Poole, S.J., Kauvar, L.M., Drees, B., and Romberg, T. (1985) The engrailed locus of Drosophila: s t r u c t u r a l analysis of an embryonic t r a n s c r i p t . C e l l 4.0, 37-43. Saari, G. and Bienz, M. (1987), The structure of the Ultrabithorax promoter of Drosophila melanogaster. EMBO 6. 1775-1780. Schneuwly, S., Kuroiwa, A., Baumgartner,P., and Ghering, W.J. (1986) . Structural organization and sequence of the homeotic gene Antennapedia, of Drosophila melanogaster. EMBO J. 5., 733-739. Schupbach, T., and Wieschaus, E. (1986). Maternal-effect mutations a l t e r i n g the antero-posterior pattern of the Drosophila embryo. Wilhelm Roux' Arch. Dev. B i o l . 195, 302-317. Smouse, D., Goodman, C , Mahowald, A., and Perrimon, N. (1988) . Polyhomeotic: A gene required for the embryonic development of ason pathways i n the central nervous system of Drosophila• submitted. Southern, E.M. (1975). Detection of s p e c i f i c sequences among DNA fragments separated by gel electrophoresis. J. Mol. B i o l . 98, 503-517. 106 Struhl, G. and Akam, M. (1985). Altered d i s t r i b u t i o n s of Ultrabithorax t r a n s c r i p t s i n extra sex combs mutant embryos of Drosophila. EMBO 4, 3259-3264. Struhl, G. (1983) . Role of the esc— gene product i n ensureing the sele c t i v e expression of segment s p e c i f i c homeotic genes i n Drosophila. J. Embryol. Exp. Morphol. 76./ 297-331. Struhl, G. (1981) A gene product required for the correct i n i t i a t i o n of segment determination i n Drosophila. Nature 293. 36-41. Turner, R.F., and Mahowald, A.P. (1976). Scanning electron microscopy of Drosophila embryogenisis. 1. The structure of the egg envelopes and the formation of the c e l l u l a r blastoderm. Dev. B i o l . 50, 95-108. Turner, R.F., and Mahowald, A.P. (1977). Scanning electron microscopy of Drosophila embryogenisis. I I . Gastrulation and segmentation. Dev. B i o l . 57, 403-416. Turner, R.F., and Mahowald, A.P. (1979). Scanning electron microscopy of Drosophila embryogenisis. I I I . Formation of the head and caudal segments. Dev. B i o l . .68, 96-109. V i e i r a , J. and Messing, J. (1982). The pUC plasmids, an M13mp7-derived system for in s e r t i o n mutagenesis and sequencing with synthetic universal primers. Gene JL9., 259-268. V i l l a r e s , R. and Cabrera, C , (1987) . The achaete-scute gene complex of Drosophila melanoqaster: conserved domains i n a subset of genes required for neurogenesis and t h e i r homology to mvc. C e l l 50 315-424. Wedeen, C , Harding, K. and Levine, M. (1986). Spatial regulation of Antennapedia and bithorax gene gene expression by the polvcomb locus. C e l l 44./ 739-748. White, R. and Lehmann, R. (1986)/ A gap gene, hunchback, regulates the s p a t i a l expression of Ultrabithorax. C e l l , 47., 311-321. 107 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0097620/manifest

Comment

Related Items