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^ 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 / - » 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