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The l(2)03605 P element induced lethality is not caused by disruption of the Drosophila melanogaster… James, Erick Robert 1999

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The 1(2)03605 P element induced lethality is not caused by disruption of the Drosophila melanogaster High Mobility Group genes D (HMG-D) or Z (HMG-Z) by Erick Robert James B.Sc, The University of British Columbia, 1995 A thesis submitted in partial fulfillment of the requirements for the degree Master of Science in The Faculty of Graduate Studies Department of Zoology We accept this thesis as conforming to the required standard The University of British Columbia 1999 © Erick Robert James, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. 1 further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract Chromatin is functionally defined as DNA, histones and non-histone chromosomal proteins (NHCP). The High Mobility Group (HMG) 1/2 proteins represent one well defined set of NHCP in eukaryotes . These proteins are thought to act as architectural components of chromatin and are well characterized biochemically but not biologically. H M G 1/2 proteins have been implicated in transcription, DNA replication, chromatin assembly and stability. The Drosophila HMG1 homologues are HMG-D and HMG-Z. The Drosphila HMGs are thought to play a role in establishing developmental specific regions of chromatin that are silent and easily replicatable, albeit at different developmental times and locations, but again their biological role is speculative. Unfortunately, no mutations exist in the H M G 1/2 class of proteins and mutants would be extremely useful in assigning a biological function to these proteins. However, the Drosophila H M G gene are cytologically mapped to a precise region, banded region 57F8-11 on chromosome 2. I attempted to identify P element insert mutations in HMG-D/Z using the Berkeley Drosophila Genome Project P element insertion lines. The Berkeley Drosophila Genome Project has undertaken the daunting task of P element tagging essential genes in Drosophila. Their P element inserts are mapped cytologically and the expression pattern of enhancer trap expression are known. One such recessive lethal line, l(2)03605/CyO has a P element inserted at 57F8-10, the same cytological location as HMG-D/Z. In addition, the insert shows fi-galactosidase expression in the nervous system, while HMG-D/Z mRNA is also found in embryonic neural tissues. This coincident expression pattern and the cytological location suggest that the 1(2)03605 P element insertion may have disrupted either or both genes. Therefore, I set out to determine whether the 1(2)03605 P element is either inserted into and/or disrupting the HMG-D/Z genes. This work shows the 1(2)03605 P element does not affect either HMG-D or HMG-Z and most likely disrupts another gene in the 57F8-10 region. ii Table of Contents Abstract ii Table of Contents iii List of Figures and Tables iv Abbreviations vi Acknowledgment vii Introduction 1 Materials and Methods 14 Results 22 Discussion 57 Conclusion..- '. 60 References 60 iii List of Figures and Tables Figure 1) The HMG-domain of HMG-D 5 Figure 2) Exon/intron organization of HMG-D and HMG-Z 9 Figure 3) Polytene in situ hybridization of DNA flanking 1(2)03605 P element 13 Figure 4) LacZ staining pattern of 1(2)03605 embryos 16 a) 12 hour b) 18 hour Figure 5) Plasmid Rescue 25 a) Map of pZ element b) Southern analysis of DNA from 1(2)03605 lethal flies, probed with kanamycin gene Figure 6) DNA sequence flanking the 1(2)03605 P element insertion site, including a primer designed for PCR (P1341B 28 Figure 7) PCR reaction using P1341B and Pout primers 30 Figure 8) Genomic southern analysis of wt vs P element (1(2)03605) containing fly DNA 32 a) Probe: HMG-Z cDNA b) Probe: Complete wt P element c) Probe: 263 bp DNA flanking P element Figure 9) Southern analysis on various genomic clones ( Plasmid and A,-clones) using 263 bp flanking DNA as a probe 36 iv Figure 10) Southern analysis on PI and XG1 clones using the 263 bp flanking fragment as a probe 38 Figure 11) Restriction map 40 a) HMG-D/HMG-Z genomic region contained in X-clones b) A,G1 clone Figure 12) Genomic Southern of wt vs 1(2)03605 lines 42 a) Probe: HMG-D ORF and flanking DNA (G2Sal2) b) Probe: 263 bp DNA flanking P element c) Probe: 7 kb Sal] fragment from X-clone d) Probe: HMG-Z exon 1 and 5'-UTR (G2Sall) Figure 13) Northern analysis of total RNA from adult female Df(2R)Pu-D17, 1(2)03605 and wt lines 45 Figure 14) Densitometric analysis of Northern blot 48 Figure 15) 1(2)03605 P element mobilization 50 Table 1) Table of lines generated from re-mobilization experiment 50 Table 2) Re-mobilization lines crossed to deficiency line (Df(2R)Pu-D17) 53 Figure 16) Genomic Southern analysis of the re-mobilization lines 56 v Abbreviations BDGP: Berkely Drosophila Genome Project CNS: Central Nervous Sytem HMG: High Mobily Group IPTG: Isoppropyl-l-thio-(3-D-galactoside LEF-1: Lymphoid Enhancer-binding Factor NHCP: Non-Histone Chromosomal Proteins ORE: Open Reading Frame PCA: perchloric acid PCR: Polymerase Chain Reaction PNS: Peripheral Nervous System SRY: Sex-determining factor TCA: trichloracetic acid UTR: Un-Translated Region wt: wild-type vi Acknowledgment I would like to thank Dr. Thomas A. Grigliatti for allowing me to pursue my scientific education in his laboratory. My Master's committee: Dr. Hugh W. Brock for all your constructive criticism and guidance, Dr. George Spiegelman. The "Back Lab": Michael O'Grady, James Whalen and Michael Harrington for all the technical and especially the moral support. The rest of the Grigliatti lab and finally my family and friends who put up with the "hermit" lifestyle science forces us into. vii Introduction High Mobility Group Chromatin Proteins The formation and organization of chromatin, functionally defined as a complex of DNA, histones, and non-histone proteins (Alberts et al., 1994) is an extremely important process in the life cycle of a eukaryotic cell. Not only must the DNA be condensed inside the small nucleus, but it must also remain a substrate for replication and transcription. Naked DNA is wrapped around a core of four basic proteins, collectively called histones: H2A, H2B, H3 and H4. This nucleosomal core is the basic structural unit of chromatin. Each nuclesome core consists of an octamer of histones associated with 146 nucleotide pairs of DNA. This octamer contains two each of histones H2A, H2B, H3 and H4. A fifth histone variant, HI is a linker histone binds to the DNA between nucleosome cores, further condensing the DNA into a chromatosome. This form of DNA is the 30-nm fibre. Higher order compaction is poorly understood; it arises by further coiling and looping of the chromatin and is maximally condensed in the metaphase chromosome (Alberts et ai, 1994). A significant component of chromatin are the non-histone chromosomal proteins (NHCP). These proteins include transcription factors, chromatin bound enzymes (replication, transcription and DNA repair machinery), scaffold proteins and the high mobility group proteins (Van Holde et al., 1995). The high mobility group (HMG) proteins are among the largest and best characterized group of non-histone chromosomal proteins (Bustin and Reeves, 1996). HMG proteins were originally identified in the early 1960s as acidic impurities in histone HI preparations (Johns, 1964). All HMG proteins bind DNA, are extractable from nuclei with 0.35 M NaCl, are soluble in 5% perchloric acid (PCA) or 2 -5% trichloroacetic acid 1 (TCA) and have a high electrophoretic mobility in polyacrylamide gels (Johns, 1982). Hence, the name High Mobility Group proteins. Typically HMG proteins have a molecular mass <30 kDa, and are highly charged. Despite early purification and well defined protein structure and biochemical characterization, their cellular function remains an enigma (Bustin and Reeves, 1996; Grosschedl et al, 1994). HMG proteins have been classified into three families based on their molecular mass, DNA-binding characteristics and amino acid sequence motifs. These families are the HMG-I(Y), HMG-14/-17 and the HMG-1/-2 class. The HMG-I(Y) family of proteins are known as "architectural transcription factors" due to their ability to function both as components of chromatin and as secondary transcription factors (Reeves and Wolffe, 1996). HMG-I(Y) is involved in both the induction and negative regulation of transcription (Bustin and Reeves, 1996). HMG-I(Y) also interacts specifically with known transcription factors (for example NF- KB and ATF-2) and induces DNA structure changes at promoter/enhancer sequences, aiding in transcription initiation (Falvo et ah, 1995; Thanos and Maniatis, 1995). How HMG-I(Y) specifically acts as an architectural transcription factor is unknown. Another class of HMG proteins is the HMG-14/-17 proteins, which are proposed to be transcriptional activators. By binding to nucleosome cores they change the rate of transcription elongation, but do not affect transcription initiation. HMG-14/17 are thought to be architectural components that help create an unfolded chromatin fibre that facilitates transcription by removing the repressive action of the histones (Bustin et al., 1995). The well studied HMG 1/2 group can be further subdivided according to the number of HMG domains present in the protein and their specificity of sequence recognition. The HMG domain is a DNA-binding motif of approximately 80 amino acids (Ner, 1992). In general proteins with multiple HMG domains show little or no sequence specificity, (for example HMG-1 and-2 with two HMG-domains). Alternatively those 2 proteins with a single HMG domain show interactions with specific DNA sequences, (for example the transcription factors: sex-determining factor (SRY) (Sinclair et al., 1990) and the lymphoid enhancer-binding factor (LEF1) (Travis et al., 1991)). The cellular function of HMG 1/2 proteins has yet to be clearly discovered. However, a role in transcription, replication, chromatin assembly and the stabilization of chromatin structure has been implicated (Landsman and Bustin, 1993). Recently, Xenopus HMG1 protein was found in early embryonic chromatin lacking HI (Nightingale et ai, 1996). With the stage set as HMG-proteins being architectural-chromatin proteins, a summary of our current understanding of two specific HMG 1/2 proteins, HMG-D and HMG-Z will ensue followed by the key question of this thesis as it relates to these two proteins. HMG-D and HMG-Z The Drosophila HMG-D protein is a highly abundant chromosomal protein that is closely related by amino acid sequence to the mammalian HMG1 and the yeast S. cerevisiae NHP6 proteins (Wagner et al., 1992). However, HMG-D only contains a single copy of the HMG-domain DNA binding motif (Landsman and Bustin, 1991, 1993). The HMG domain is followed by a basic region similar to the carboxyl terminal domain of histone HI and a short carboxyl terminal acidic stretch similar to those in HMG 1/2 (Churchill et al, 1995). The NMR structure of the HMG domain of HMG-D is similar to the B-domain of HMG-1, which is an L-shaped fold formed by three oc-helices (Jones et al., 1994) held together by two hydrophobic cores (Balaeff et al., 1998) (see Figure 1). Churchill et al. (1995) showed HMG-D preferentially binds to A/T rich DNA containing the dinucleotide sequence TG. This TG site is a readily deformable base combination and shows the preference of HMG-D for pre-bent DNA sites. Furthermore, HMG-D binding to DNA induces further bending in the DNA. HMG-D is encoded by a single copy gene located at region 57F8-11 on the right arm of chromosome 2 (Wagner et al., 1992). Northern analysis of HMG-D revealed a 1.3 3 Figure 1 The HMG-domain of HMG-D. The solution structure of HMG-D obtained by NMR. I, II and III represent oc-helices, while A and B refer to hydrophobic cores (Balaeff etal, 1998). 4 5 kb transcript and a 0.95 kb transcript. These transcripts are present at relatively equal and high levels in RNA prepared from 0-4 and 4-8 hr embryos with slightly lower amounts present in the 8-12 and 12-16 hr embryos. Trace amounts of HMG-D transcripts are found in later stages of development. Neither HMG-D mRNA is seen in the adult male, however both transcripts are present in the adult female (Ner et al., 1993; Wagner et al, 1992). Stroumbakis and Tolias (1994) examined the distribution of HMG-D mRNA in the female germline and early embryo. In stage 1-6 egg chambers, HMG-D mRNA is found throughout the germarium, in the germline nurse cells and in the somatic follicle cells. At stage 7, HMG-D transcripts stop accumulating in follicle cells, however transcripts continue to accumulate in the nurse cells until stage 10. During stage 11, the nurse cells begin to transfer HMG-D mRNA into the oocyte where these transcripts accumulate around the periphery of the oocyte until the end of oogenesis. Zygotic HMG-D rnRNA expression resumes in neuroblasts that are migrating from the neural ectoderm in the stage 9 embryo that is entering the slow phase of germband elongation. This zygotic expression continues throughout the remaining stages of embryogenesis, specifically in neuroblasts of the CNS. Most neuroblasts on both sides of the ventral cord and brain lobes and a subset of cells in the PNS express HMG-D mRNA. The suggestion that later zygotic HMG-D expression may play a significant role in the development of the Drosophila embryonic nervous system (Stroumbakis and Tolias, 1994) is not without precedent. Another vertebrate HMG1 homologue, HMG-X of Xenopus is also abundantly expressed in neuroectoderm-derived tissues during embryogenesis (Kinoshita et al, 1994). HMG-X is thought to participate in neural fate determination by regulating, through modulation of chromatin configuration, the ability of transcription factors to induce the expression of a specific set of genes. Interestingly there is a second HMG-1 related protein in Drosophila, HMG-Z (Ner et al, 1993). HMG-Z is 65% identical and 78% similar at the amino acid level to HMG-6 D. The genes encoding HMG-D and HMG-Z are adjacent on the chromosome and oriented head-to-head. HMG-D and HMG-Z each contain two introns, shared sequence and conserved intron/exon arrangements and their close proximity indicate a possible gene duplication (Ner et al., 1993). HMG-D's first intron named intervening sequence (IVS) one (IVS1) is 1.2 kb long and is within the 5' UTR, resulting in a short exon of 30 bp. The second exon, encoding the 39 N-terminal residues of the HMG-D protein is 215 bp long. IVS2 is only 65 bp and is followed by exon3 which contains the rest of the coding region and the 3' UTR. HMG-Z also contains three exons (exon 1-165 bp, exon2-214 bp and exon3-741 bp) which are separated by two introns (IVS1 >1 kb and IVS2 95 bp) (Figure 2) (Ner etal., 1993). Northern analysis of HMG-Z reveals a 0.65 kb and a 1.2 kb transcript. A small amount of the 1.2 kb transcript appears in 0-4 hr embryos suggesting either that the maternal contribution of HMG-Z is minor or it arises from early zygotic expression. Both transcripts are seen in 4-8 hr and 8-12 hr embryos, with the 1.2 kb transcript being more abundant. Both transcripts are present in trace amounts in later embryos. There is a high level of the 0.65 kb transcript in 1st instar larvae, suggesting a requirement for HMG-Z protein during larval development (Ner et al., 1993). Functionally, HMG-D and HMG-Z may play similar roles but at different times during development. HMG-D is present at a constant level in all stages of development as determined by Western analysis (Ner and Travers, 1994). In embryos HMG-D is confined to the nuclei from the start of embryogenesis to the blastoderm stage as shown by in situ localizations with anti-HMG-D antibodies. The most intense initial staining is observed in mitotic chromosomes and the polar bodies. The polyploid yolk nuclei and the pole cell nuclei also contain HMG-D. Interestingly, histone HI as detected by Western analysis first appears in the interphase and mitotic nuclei of some nuclear division cycle 7 embryos. During cycles 8 and 9, histone HI staining becomes more intense and increases dramatically due to zygotic transcription. Histone HI is an 7 Figure 2 Exon/intron organization of a) HMG-D and b) HMG-Z (Ner et al, 1993). 8 HMG-D 1200 215 780/1030 165 > 1000 214 95 741 jmH$3A architectural protein that binds to the linker DNA flanking the nucleosome core, aiding in the assembly of higher-order chromatin structures (Wolffe, 1997). During the early stages of development, there is a dramatic shift in the relative amounts of HI and HMG-D, with HI eventually becoming the more abundant protein (Ner and Travers, 1994). Initially, during early embryogenesis HMG-D is associated with a less condensed chromatin structure that lacks histone HI. As histone HI increases it replaces HMG-D and the chromatin takes on a different, more compact chromatin structure. This is demonstrated by a decrease in the size of the mitotic chromatids as HI displaces HMG-D. The physiological significance of the differing states of chromatin during development is not known. However, there is the possibility that HMG-D containing chromatin is less compact, which would facilitate the rapid condensation and decondensation required during the very short early cleavage cycles (Ner and Travers, 1994). Also, there is a correlation of the appearance of histone HI and the initial transcriptional competence of the cell after the mid-blastula transition (Anderson and Lenhyel, 1980). This suggests that chromatin containing HMG-D is transcriptionally silent, and transcriptional competence is associated with replacement of HMG-D by histone HI. Thus, HMG-D and HMG-Z may function in organizing silent chromatin (Ner and Travers, 1994). The Xenopus HMG1 protein also causes a silent chromatin conformation in pre mid-blastula transition embryos. HMG1 represses in vitro transcription of a dinucleosomal template to the same level as histone HI. There is a high concentration of HMG1 in the Xenopus egg and embryo that may contribute to transcriptional silencing of the early embryonic chromatin, in the same unknown manner as HMG-D (Ura et ai, 1996). However, a 6-fold higher concentration of HMG1 over HI is required to repress transcription at the same level. In addition, histone HI has a 40-fold higher affinity for nucleosomal DNA than HMG1. Biochemical studies on HMG-D/Z protein structure (Jones et al., 1994) and their DNA binding properties (Balaeff et ai, 1998; Churchill et al., 1995) have shed little light 10 on these proteins biological significance. A recent study has discovered that there are two isoforms of an E. coli produced recombinant HMG-D. One isoform contains an oxidized methionine residue at position 13 of the protein while the other isoform is not oxidized at position 13. Furthermore, this oxidation of Met13 decreases the DNA binding affinity 3-fold over the non-oxidized isoform of HMG-D (Dow et al., 1997). Unfortunately, this result only exists in E.coli and appears to be a result of the recombinantly produced protein. Unless the Drosophila HMG-D protein is also shown to be oxidized, the results bear little biological significance. In the absence of biological data, we do not know exactly what roles HMG 1/2 proteins have during development and the cell cycle. The best way to get a handle on biological function is to perturb the system and find out what happens. Unfortunately, there are no known mutations in the HMG 1/2 proteins. The Berkeley Drosophila Genome Project (BDGP) has undertaken an extremely important and productive task of P element tagging essential genes within the fruitfly. A P element is a Drosophila transposable element that has been used extensively for mutagenesis and germline transformation (Griffiths et al., 1993). The P element Gene-disruption Project has produced many randomly induced P element lines, each containing a single P element insert that is recessive lethal. The cytological location of all inserts have been mapped by in situ hybridization to polytene chromosomes. In addition to cytological mapping, the project is also isolating and sequencing the DNA flanking the P element using either plasmid rescue or inverse PCR. With these two goals complete it will be possible to link the physical, cytological and genetic maps of the Drosophila genome (BDGP, 1996). This large pool of P element tagged lines allows for the recovery of insertional mutations in essential genes and the cloning of those genes, such as HMG-D/Z. The P 11 Figure 3 In situ hybridization of polytene chromosomes using DNA flanking 1(2)03605 P element as a probe. The figure indicates the P element is located on the right arm of chromosome 2 at cytological position 57F8-F10 (Spradling et al., 1995) 12 1(2)03605 / 57F8-57F10 2R element associated with the recessive lethal mutant 1(2)03605 was localized to the cytological location 57F8-F11 by in situ hybridization on polytene chromosomes (Figure 3), the same cytological location as the HMG-D and HMG-Z loci. The PZ-enhancer trap P element used to generate the recessive lethal lines contains the structural gene for /3-galactosidase. Upon insertion into the genome, the /5-galactosidase expression falls under the control of local promoters/enhancers (a classical enhancer trap experiment) (Mlodzik and Hiromi, 1992). The P-galactosidase expression pattern of the 1(2)03605 P element is confined to the nervous system (Figure 4). This expression pattern is coincident with the zygotic expression pattern of HMG-D and HMG-Z (Ner, personal communication; Stroumbakis and Tolias, 1994)] Therefore, the 1(2)03605 P element line is an excellent candidate for a mutant which disrupts HMG-D, HMG-Z or both to recessive lethality. This was the working hypothesis for this thesis. Unfortunately the 1(2)03605 P element was shown not to disrupt either HMG-D or HMG-Z. Therefore, it must be affecting some other gene nearby. Materials and Methods Fly strains The fly strains used in this study are described on Flybase ( and in Lindsley and Zimm (1992). All flies were obtained from the Bloomington stock centre. P element recessive lethal line: Stock #: PI341, Cytological location: 057F08-10 cn[l] P{ry+=PZ}l(2)03605°3605/CyO; ry506 Deficiency: Stock #: 2606, Breakpoints: 057B04;058B Df(2R)Pu-D17, cn bwx sp/SMl 14 Figure 4 LacZ expression pattern in 1(2)03605 late embryos is confined to the nervous system, (a) A lateral view of a 12 hour embryo with anterior to the left, posterior to the right, dorsal at the top and ventral at the bottom shows expression restricted to the CNS. (b) A dorsal view of a 18 hour embryo with anterior to the left and posterior to the right shows CNS expression but also expression in the PNS (Spradling et ah, 1995). 15 (a) Genomic DNA isolation Fifty flies were homogenized in 2 ml of a 0.2 M saccharose, 0.1 M Tris-HCl pH 9.2, 0.1 M NaCl, 0.05 M EDTA and 0.5% SDS solution at room temperature. The homegenate was then incubated at 65° C for 30 minutes, followed by the addition of 300 JJLI of 8M potassium acetate pH 10. This solution was then mixed on a vortex and placed on ice for 30 minutes, then centrifuged in a tabletop centrifuge. The supernatant was then treated with 5 ul of lflg/|ll RNase A for 30 minutes at room temperature, phenol/chloroform extracted with an equal volume of 1:1 phenol/chloroform , followed by ethanol precipitation with 95% ethanol, a wash with 70% ethanol and re-suspension in 50 | l l of IX TE. PI DNA isolation (PI phage clone # DS08966-94-38 from the Berkeley Genome Project contains the 1(2)03605 P element insertion site) A single PI containing colony was grown overnight in Luria-Bertani Medium (LB) with 25 |J.g/ml of kanamycin. 500 mis of LB with 25 flg/ml of kanamycin and 5% sucrose were inoculated with 500 (il of the overnight culture and shaken at ~ 250 rpm at 37° C for about 5 hours (OD 5 5 0 = 0.15). Five mis of filter-sterilized 0.1 M IPTG were added to the culture and further grown with shaking at 37° C for another 5 hours (OD 5 5 0 = 1.3 to 1.5). The cells were then harvested by centrifugation and the DNA isolated with a Qiagen maxi-prep kit according to the manufacturer's instructions. The resulting DNA was then re-suspended in 100 (il of TE. LB medium 17 T e n g r a m s o f b a c t o - t r y p t o n e , 5 g b a c t o - y e a s t ex trac t , 10 g N a C l w e r e a d d e d to 950 m i s d e i o n i z e d H 2 0 a n d s h a k e n u n t i l the so lu tes h a d d i s s o l v e d . T h e p H w a s a d j u s t e d to 7.0 w i t h the a d d i t i o n o f 5 N N a O H . T h e v o l u m e w a s a d j u s t e d to 1 l i ter w i t h d e i o n i z e d H 2 0 a n d the m e d i u m w a s then s t e r i l i z e d b y a u t o c l a v i n g f o r 20 m i n u t e s at 15 l b / s q . i n . o n the l i q u i d c y c l e ( S a m b r o o k et al., 1989). Total RNA isolation F i f t y f l ies w e r e h o m o g e n i z e d i n 1 m l o f T R I z o l ® R e a g e n t ( G I B C O B R L ) . T h e i n s o l u b l e m a t e r i a l w a s r e m o v e d b y c e n t r i f u g a t i o n at 12,000 x g f o r 10 m i n u t e s at 4° C . T h e c l e a r e d h o m o g e n a t e s o l u t i o n w a s t h e n t r a n s f e r r e d to a f r e s h t u b e f o r p h a s e s e p a r a t i o n . T h i s h o m o g e n a t e s o l u t i o n w a s then i n c u b a t e d at r o o m t e m p e r a t u r e f o r 5 m i n u t e s . 200 ( l l o f c h l o r o f o r m w a s a d d e d , f o l l o w e d b y i n c u b a t i o n at r o o m t e m p e r a t u r e f o r 2 to 3 m i n u t e s . T h e s a m p l e s w e r e then c e n t r i f u g e d at 12,000 x g f o r 15 m i n u t e s at 4° C . A f t e r c e n t r i f u g a t i o n the a q u e o u s p h a s e w a s t r a n s f e r r e d to a f r e s h tube , a n d the R N A w a s p r e c i p i t a t e d b y the a d d i t i o n o f 500 u l o f i s o p r o p y l a l c o h o l . T h e s a m p l e s w e r e t h e n i n c u b a t e d at r o o m t e m p e r a t u r e f o r 10 m i n u t e s a n d c e n t r i f u g e d at 12,000 x g f o r 10 m i n u t e s at 4° C . T h e supernatant w a s s u b s e q u e n t l y r e m o v e d a n d the R N A p e l l e t w a s h e d w i t h 1 m l o f 75% e t h a n o l . T h e R N A p e l l e t w a s then b r i e f l y a i r - d r i e d a n d r e d i s s o l v e d i n R N a s e - f r e e d i s t i l l e d water . Plasmid rescue T w o to 5 g e n o m i c e q u i v a l e n t s o f g e n o m i c D N A w e r e d i g e s t e d w i t h Xba I, Xba \INhe I, a n d Xba llSpe I f o l l o w e d b y e i ther heat i n a c t i v a t i o n o f the r e s t r i c t i o n e n z y m e o r p h e n o l / c h l o r o f o r m e x t r a c t i o n . D i g e s t e d D N A w a s r e s u s p e n d e d i n 20 - 50 u.1 o f T E . H a l f 18 of the DNA was used for the subsequent ligation reaction. The ligation reaction was performed in a 200 (il volume to reduce intermolecular ligation events. Therefore 20 jil of 10X ligation buffer and 2 u,l of 1 U/|il ligase was used and the remaining volume made up with distilled water. The ligation reaction was incubated at 16° C overnight. The DNA in the ligation mix was then precipitated by the addition of 20 u.1 of 3 M sodium acetate and 2 volumes of ethanol. The DNA pellet was then resuspended in 10 to 20 (il of TE. The resuspended DNA was then transformed into either Subcloning Efficiency DH5a™ Competent Cells (GEBCOBRL) following the manufacturer's instructions or lab made electro-competent E.coli (DH5oc™ ) using the BTX Transporator™ Plus (see below for protocol). Preparation of electro-competent cells Five hundred mis of LB were innoculated with 1-5 mis of an overnight bacterial; culture and incubated at 37° C until OD6oo = 0.6. The culture was then chilled on ice for 30 minutes and the cells harvested by centrifugation at 4000 g for 15 minutes at 4° C. The resulting pellet of cells was then washed twice with 250 mis of ice-cold 10% glycerol (autoclaved), harvested by centrifugation at 4000 g for 15 minutes at 4° C and resuspended in 1 ml ice-cold GYT medium (10% glycerol, 0.125% yeast extract, 0.25% tryptone, autoclaved to sterilize). The competent cells were aliquoted into 1.5 ml ice-cold eppendorf tubes, flash frozen in liquid nitrogen and stored at -80° C. Electroporation BTX Disposable Cuvettes (P/N 610 (1 mm gap)) were used with a set charging voltage of 1.4 kV, field strength of 13.0 - 15.0 kV/cm and a pulse length of 5 - 6 msec. Forty \\\ of 19 electro-competent cells were placed in an ice-cold cuvette along with 1 ng/ul of DNA, mixed by 'flicking' and placed on ice for 1 minute. The electrical charge was applied and 960 (il of LB was added immediately, followed by gentle mixing with a pipette. The cuvette was then transferred to 37° C for 1 hour, followed by transfer to the appropriate agar plate. Calculation of theoretical number of rescue plasmids One can determine how many plasmids should be recovered for a set of rescue experiments based on the ratio of rescuable plasmid DNA to total diploid genomic DNA. The minimum size of the rescue plasmid would be 7 kb (Figure 5a) and the total diploid genome size of Drosophila melanogaster is 330 Mb (Rubin, 1998). Thus, the ratio of rescuable plasmid DNA to total genomic DNA is 7 kb:330 Mb or 2.12 x 10"5. In the transformation portion of the rescue experiment 100 ng of DNA was transformed 100 independent times. Therefore 10,000 ng of DNA was transformed. To determine how much of this total DNA is plasmid DNA, we simply multiply 10,000 ng of DNA to the 2.12 x 10"5 ratio, coming up with 0.212 ng of plasmid DNA. The transformation efficiency (CFU) of the competent E.coli is 25 ng DNA giving 1 x 104 colonies. Consequently 2.5 x 10"3 ng of DNA gives rise to a single colony. Simply dividing the total amount of plasmid DNA 0.212 ng by 2.5 x 10"3 ng yields 84.8 plasmids. Therefore, approximately 85 rescue plasmid should have been recovered. DNA purification for labeled probes DNA from a restriction enzyme digest or from PCR was subjected to electrophoresis and the desired band cut out of the gel and placed in a eppendorf tube at 20 -80° C for 15 minutes. The gel slice was allowed to thaw completely and centrifuged in a tabletop centrifuge at 14, 000 rpm for 10 minutes. The supernatant was transferred to a new tube with the addition of 1/10 volume of sodium acetate and 2 volumes of 95% ethanol. The precipitated DNA was again spun and washed with 70% ethanol and then air dried. The resulting DNA pellet was resuspended in 10 ul of TE. lul of the sample was then subjected to electrophoresis for quantification. Southern and Northern analysis Radioactively labeled (Amersham a 3 2 PadATP) DNA probes were made with the Boehringer-ManNheim random prime DNA labeling kit according to manufacturer's instructions and the probe purified with Pharmacia Biotoech MicroSpin™ S-300 HR columns. Transfer and hybridization procedures were followed according to standard protocols (Sambrook et al., 1989). Pre-hybridization and hybridization was done in 25 mM KP0 4 pH 7.4, 5X SSC, 5X Denhardt's solution, 50 Ug/ml sheared salmon sperm DNA, 50% formamide and 10% dextran sulfate at 42° C. Washes were as follows: once at room temperature to 60° C (room temperature wash was added and the solution brought to 60° C) in IX SSC, 0.1% SDS, followed by a 15 minute wash at 60° C with the same wash solution. The blots were then washed twice with either 0.5X SSC, 0.1% SDS (low stringency) or 0.1X SSC, 0.1% SDS (high stringency) at 60° C for 15 minutes. Blots were exposed to Kodak XOMAT film at -70°C for 1-3 days. Densitometic analysis Analysis was carried out using NIH Image according to Chow and Evans ( 21 Polymerase Chain Reactions (PCR) Reactions were completed in 50 p.1 reaction volumes. The final concentrations of the various components of the PCR are as follows: IX of the 10X reaction buffer; 2.5 mM MgCl 2; 1.0 mM dNTPs; 50 ng DNA; 25 pmoles of primers; 1 U Taq DNA polymerase. The cycles were as follows: 1) 94° C for 1 minute; 2) 92° C for 1 minute; 3) 58° C for 1 minute; 4) 72° C for 2.5 minutes and 5) steps 2 through 4 were repeated 32 times. Primers: Poul: 5' CGG GAC CAC CTT ATG TTA TTT CAT C 3' PI 34 IB: 5' GGA ATT CCG AC A GAA GAA TAC CGC A A A CTC 3' Fly crosses All crosses were carried out at 25° C on standard cornmeal sucrose media containing 0.04 % Tegosept as a mold inhibitor. In the reversion experiment 50 vials, each containing 1 (1(2)03605) male and 5 (A2-3) females were set up and allowed to lay eggs for 3 days, the adults were then transferred to fresh vials and allowed to lay for 3 more days. Dysgenic males were crossed to 1(2)03605 females and ry males collected. These males were crossed to CyO females for balanced stock generation. In the re-mobilization lines crossed to the deficiency experiment 5 re-mobilized males/females were crossed to 5 deficiency containing females/males. Offspring were then scored for straight vs curly wings. Results Failed plasmid rescue indicates the 1(2)03605 P-element may be deleted 22 Plasmid rescue of the DNA flanking the P element insertion in strain 1(2)03605 was attempted in order to obtain DNA to fine localize the P element insertion site. Unfortunately the plasmid rescue experiment failed to yield any rescue plasmids. Genomic l(2)03605/CyO DNA was cut with enzymes suitable for rescuing the DNA flanking the P element, DNA was cut singly with Xba I and doubly with Xba I/Nhe I and Xba II Spe I. Cutting with Xba I/Nhe I or Xba 1/ Spe I still allow for re-circularization of the plasmid, as the enzyme combinations produce compatible ends. Approximately 85 rescue plasmids should have been recovered (see Materials and Methods for calculation of this number), but a rescue plasmid was never recovered. This suggested that the P element had undergone some rearrangement or deletion that no longer allows for plasmid rescue. The expected size of rescue plasmid should be > 7 kb as the unique Xba I site is ~ 7 kb from the 5' end of the pZ element and the next restriction site is upstream of the P element within the genomic DNA (Figure 5a). To determine the actual size of the rescue plasmid, genomic Southern analysis was undertaken (Figure 5b). 1(2)03605 genomic DNA was cut with the same enzymes designed for the rescue: Xba I, Xba I/Nhe I and Xba I/Spe I, blotted and subsequently probed with a fragment of the kanamycin gene (343 bp Pvu II Nru I restriction fragment from Novagen's pET-28a(+)). The resulting bands in each digest are all less than 3 kb. Also, the Xba I/Nhe I digest shows two bands indicating a new Nhe I site within the pZ element. These results support the notion the 1(2)03605 P element has undergone a rearrangement or deletion that disrupts plasmid rescue. 23 Figure 5 Determination of the size of rescue plasmid from 1(2)03605 flies, (a) Shows a map of the pZ plasmid with the unique Xba I restriction site ~ 7 kb from the 5' end of the vector, (b) 1(2)03605 genomic Southern probed with a 343 bp Pvu II Nru I kanamycin gene fragment from Novagen's pET-28a(+). DNA was cut with enzymes designed for plasmid rescue: Xba I, Xba UNhe I and Xba l/Spe I. Southern shows bands less than the expected >7 kb, indicating the P element is deleted. 24 (a) pZ M Xi Pout p lacZ hsp| 170 Kmr ORI rosy + 1 kb (b) Z C/3 cd cd cd X i X> X X X 2.5 Polymerase chain reaction indicates that the 1(2)03605 P element has not moved since its initial characterization Since the plasmid rescue experiment was unsuccessful and the Genome Project managed to obtain the sequence of the DNA flanking the 1(2)03605 P element, the incorrect fly line may have been used in my plasmid rescue experiments. To test this, a PCR experiment was undertaken to ensure the corrrect line had been obtained from the stock centre. A primer (P1342B) was designed from the Genome Project's sequence for use in a PCR experiment (Figure 6). Using this primer and a primer within the pZ element (Pout) should yield a product of 263 bp if the P element fly line is indeed the original 1(2)03605 line. Also, the experiment will confirm if the P element is in the original location determined by the Genome Project. The PCR experiment (Figure 7) yields the expected single 263 bp band (see lanes 2 and 3). The single primer controls failed to yield any products. This 263 bp product from a genomic primer and a P element specific primer indicates the P element has not moved and is in the Genome Project's suggested location and is therefore the correct 1(2)03605 Berkeley Genome Project characterized P element line. The 1(2)03605 P element induced recessive lethality is not caused by a disruption of the HMG-Z gene. With both the enhancer trap R-galactosidase expression from the P element and the HMG-Z mRNA being confined to the nervous system the possibility the P element is disrupting the HMG-Z gene exists. A genomic Southern analysis of l(2)03605/CyO and Oregon-R (a wild-type strain) DNA using an HMG-Z cDNA as a probe was undertaken to 26 Figure 6 Sequence of DNA flanking the P element, including a primer (P1341B) designed for PCR. An Eco RI restriction cut site was included in the primer for ease of future molecular manipulation of any PCR products. The 5' end (nucleotide 1) is directly next to the P element. The sequencing was done by the Berkeley Drosophila Genome project from a successful plasmid rescue. 27 1 C T C C T G C C T T T N G T T A T G T G T G T G A G G A G A T T G A T A T A G A A T G T G C T A A A 51 T A A T A C C A G G A A T G A C A G N C A C T C T A C T T C C A C T C T A C A G A T C C G G C A C T 101 C T A G C T C C T T N G T A G C C A A A T T A A T T A T C T A C C T C A A T A T C A G N A G A A A A 151 C T A G G T A T T T A A G A A G T T A T T T C A G T T G T T T G G A N T C A T T T T A T T A A T A T 201 GTTTNCGTTT C T T G A A G A G T T T G C G G T A T T CTTCTGTCTT G A A C A A A T A N 251 T T G A C A A C T C G A T T T A A G C G A C A A C T T N G G T C T T T A T A A C C T A C A T A T G T P1341B: 5' > G G A A T T C C G A C A G A A G A A T A C C G C A A A C T C < 3 ' 28 Figure 7 Polymerase chain reaction on 1(2)03605 genomic DNA using Pout and PI34IB as primers. Lane 1 is a molecular weight marker, A. DNA cut with Hind III. Lanes 1,2,3,4 and 5 are the products of the PCR. Lanes 1 and 2 are identical and contain all the components required by the reaction. Lanes 3 and 4 are missing a single primer. An expected size band of 263 bp is shown in lanes 2 and 3. 29 PQ CQ 2.3 kb 2.0 kb PH £ + J + J +j O O O PH OH OH PH 263 bp 30 Figure 8 Replicative genomic Southern of 1(2)03605 vs wt D N A cut with various restriction enzymes, (a) Blot probed with HMG-Z cDNA shows there is no difference between 1(2)03605 and wt D N A (b) Same blot probed with complete P element probe indicates the 1(2)03605 line contains P elements while the wt (Oregon-R) line does not. (c) Same blot probed with 263 bp flanking D N A indicates a polymorphism between the 1(2)03605 line and wt. 31 E 06 o o UJ T3 C f oo i— O 0 ' 1 o >> u u — IT) O o °r ro 00 l-i CN CN >i—' O pel oo " O 1 r o o Pi o o W es pes 00 "O 1 r O IT) o o m CN o - o -c f 00 u O "CP u o T 3 Cf 00 1 -O "CT1 r o IT) o r o PQ o o UJ C N Cf 00 1 -O "cy r >> u m o o CN P f oo r> Ii CN O -T3 o "o1 r u uo O o r o O o r o o — • * • • • • • • IMBW^^B? ^^^^ ip^jp (a) (b) (c) H M G - Z P-element 263 bp f l ank ing D N A 32-test this hypothesis (Figure 8a). Genomic DNA was cut with three restriction enzymes: Bam HI, Eco RI and Hind III, separated by electrophoresis, blotted and probed with a 3 2 P labeled HMG-Z cDNA. The autoradiograph indicates there is no polymorphism detected between the two fly lines. Therefore, it is unlikely the induced recessive lethality was caused by an insertion of a P element within the HMG-Z gene. To confirm that the 1(2)03605 line contained P element sequences the same blot was stripped and re-probed with a probe made of the complete 2.9 kb P element (Figure 8b). The autoradiograph indicates as expected there are no P.elements in the Oregon-R strain and P elements are present within the 1(2)03605 line. As a positive control to validate the 263 bp PCR product being composed of DNA flanking the P element, it was used as a probe on the previously probed and stripped blot (Figure 8c). This probe should show a polymorphism between the two fly lines. The blot indeed indicates a polymorphism is detected between the 1(2)03605 and wt lines. In both the Bam HI and Hind III digest the probe detected two bands in the 1(2)03605 line and only a single band in the wt (Oregon-R) fly. In both of these digests the upper band present in both the wt and l(2)03605/CyO line comes from the non-P element mutagenized chromosome. That is, in the wt case both chromosomes are "normal", but in the l(2)03605/CyO line the CyO chromosome is "normal" and the 1(2)03605 chromosome is mutant. Hence, the CyO and wt restriction cut patterns are the same. The novel band in the l(2)03605/CyO line is from the mutagenized chromosome. This polymorphism demonstrates that the 263 bp PCR product is next to the 1(2)03605 P element, and that the 263 bp PCR product can detect a genomic polymorphism between wt and 1(2)03605 DNA. 33 The P element insertion site is contained within both a lambda clone for the HMG-D/HMG-Z genomic region and a PI clone for the region. To determine if the 263 bp flanking DNA and therefore the P element insertion site is contained within genomic clones for the region, Southern analysis was undertaken. A lambda clone (A.G1) (Ner et al., 1993) and various plasmid clones (Ner, unpublished) of the region were digested with Eco RI, blotted and probed with the 263 bp flanking DNA (Figure 9). The above clones for the 57F8-F11 genomic region were isolated in the initial cloning experiments of HMG-D and HMG-Z (Ner et al, 1993; Ner and Travers, 1994). The autoradiograph indicates the 263 bp flanking DNA is not located within any of the plasmid clones, but is located within the lambda clone (?iGl). To further elucidate where the P element is inserted within the A.G1 clone, a restriction digest analysis was completed. The XG1 clone was digested singly with BamU I and Sal I, blotted and probed with the 263 bp flanking DNA. In conjunction a PI phage clone (clone # DS086966-94-38) of the 57F8-11 region known to contain the 1(2)03605 P element insertion site (mapped to this PI clone by the Berkeley Genome Project) was digested with Bam HI and included on the blot (Figure 10). The autoradiograph indicates the P element insertion site is contained within a ~ 10 kb Bam HI fragment of the PI clone, a ~ 9 kb Bam HI and a ~ 7-8 kb Sal I fragment. This places the 1(2)03605 P element downstream of the HMG-D gene (Figure 11). To confirm this location of the P element insertion site, genomic Southern analysis comparing 1(2)03605 and wt DNA cut with Sal I and hybridized with various probes was completed (Figure 12). The genomic blot was first probed with the 263 bp 34 Figure 9 Southern analysis of various X and plasmid clones of the HMG-D/HMG-Z genomic region (Ner et al., 1993) cut with Eco RI using the 263 bp flanking DNA as a probe. The blot indicates the flanking DNA and thus the P element insertion site is contained within the XGl clone. 35 a r n a 5 ' ' Bt 111 «J g" <_ * «3 5 / 3 ^ O M N N £ pt$ O O O a a a a & 2 3 -9.4-6.5> 3 . 0 -2 . 0 -3b Figure 1 0 Southern analysis of a PI clone (DS086966-94-38) for the region cut with BamH I and the \G\ (Ner et al, 1993) clone cut with either BamH I or Sal I using the 263 bp flanking DNA as a probe. The blot indicates the flanking DNA and thus the P element insertion site is contained within a ~ 10 kb BamH I fragment of the PI clone and a ~ 9 kb BamH I and a ~ 7-8 kb Sal I fragment of the A.G1 clone. 37 X E CQ X e x cd cC CQ W 38 Figure 1 1 (a) A schematic diagram of the HMG-D/HMG-Z genomic region indicating the location of the 1(2)03605 P element (Triangle), as well as some of the plasmid clones for the region from (Ner et al., 1993). (b) A schematic diagram of the ?iGl clone indicating the location of the 1(2)03605 P element insertion site (Triangle), the HMG-D gene as well as the location of the plasmid clones used in the previous P element localization studies (Ner, unpublished). The map indicates the 1(2)03605 P element insertion site is downstream of the HMG-D gene. 39 (a) HMG-D W PG2Sal2 U ?BgaSall f T U_i f K hfG2 pG2SaB ' ' U i XG4 Gl HMG-Z (b) pRG3.8 pWhapR!G3.2 pG2Sal3 pG2Sall/pWhp-BHl Sal I Sal I Sail 1.5 3.6 Sail Sail HMG-D 1 '1(2)03605 7-8 10 BamHl BamHl MO Figure 12 Genomic southern analysis of 1(2)03605 vs wt DNA cut with Sal I using (a) 263 bp flanking DNA, (b) G2Sall (HMG-D), (c) 7-8 kb XGl-Sal I fragment and (d) G2Sal2 (HMG-Z) as probes. The blot indicates a polymorphism is uncovered between the 1(2)03605 and the wt line when using either the (a) 263 bp flanking DNA or (b) the G2Sall (HMG-D) clone as a probe. The blot also shows that when using the (c) 7-8 kb XGl-Sal I fragment as a probe the same bands light up as with the 263 bp flanking DNA and the G2Sall (HMG-D) clone probes. Thus, the 7-8 kb XGl-Sal I fragment spans both the 263 bp flanking DNA and the G2Sall (HMG-D) clone. Panel (d) indicates there is no difference between the 1(2)03605 and wt lines when using G2Sal2 (HMG-Z) as a probe. 41 H2_ flanking DNA and this shows an expected polymorphism between the two fly lines (Figure 12a). The blot was stripped and re-probed with a plasmid clone of the region, G2Sall (see Figure 11). This clone contains the complete HMG-D ORF and also some downstream flanking DNA. The G2Sall clone likewise detects a polymorphism between the 1(2)03605 and wt lines. Since, the P element was localized to a 7-8 kb Sal I fragment of the A.G1 clones (see Figure 1 lb) it was also used as a probe on the same blot (Figure 12c). This probe detects a polymorphism between the two lines and in fact detects the same bands as the 263 bp flanking DNA probe and the G2Sall probe. Therefore, this probe must span both of these DNA fragments. As a negative control the blot was re-probed with a plasmid clone that contains part of the HMG-Z gene (G2Sal2) (Figure 12d -see Figure 11 for location of plasmid clone). As expected this probe fails to detect a polymorphism between the two lines. This genomic Southern analysis confirms the P element insertion site is downstream of HMG-D. The 1(2)03605 P element does not affect HMG-D transcripts from adult females. With the P element insertion site close to the HMG-D locus it might be disrupting expression of HMG-D. To test if the recessive lethality is associated with a reduction/elimination of HMG-D transcripts, total RNA from adult 1(2)03605/CyO, Oregon-R and Df(2R)Pu-D17/SMl females was isolated and used in a Northern analysis with a 3 2 P labeled HMG-D cDNA as a probe (Figure 13). This same Northern blot was also probed with RP49 as a quantitative control for equal loading. The autoradiograph with the RP49 probe indicates the wt and 1(2)03605 RNA levels are approximately equal, 43 Figure 13 Northern analysis of total RNA isolated from adult females of Df(2R)Pu-D17, Oregon-R and 1(2)03605 using both a HMG-D cDNA and RP49 as probes. The RP49 signal indicates that equal amounts of RNA were loaded from the Oregon-R and 1(2)03605 flies, however the Df(2R)Pu-D17/SMl lane is overloaded. The blot also indicates there is no difference in the two 1.3 kb and 0.95 kb HMG-D transcripts in RNA from Oregon-R and the 1(2)03605 line. Therefore, the 1(2)03605 P element is not affecting the expression HMG-D transcripts. 44 3 f i n cs Q O o >» u o r o C N 1.3 0.95 kb. kb-R P 4 9 -M5 and thus there are no differences in the levels of either the 1.3 kb or 0.95 kb HMG-D transcripts. The deficiency lane is overloaded and therefore is not informative. Unfortunately, the Northern analysis indicates the 1(2)03605 P element induced lethality is not caused by a reduction in HMG-D transcript levels. Densitometric analysis was completed on the above Northern blot to ensure visual analysis of the band intensity was accurate (Figure 14). The numbers under the curves represent the area under that curve and is a measure of band intensity and thus RNA abundance. The densitometric analysis indicates the wt and 1(2)03605 HMG-D RNA levels are equal with respect to both the 1.3 and 0.95 kb transcripts (Figure 14a,b). 4787 for wt vs 5185 for 1(2)03605 of the 1.3 kb transcript and 8458 for wt vs 8189 for 1(2)03605 of the 0.95 kb transcript. The RP49 quantitative control indicates there are equal levels of RNA in the wt and 1(2)03605 lanes, and that the deficiency lane is overloaded (24391 for Df(2R)Pu-Dl 7 vs 17169 for wt vs 15444 for 1(2)03605 of the RP49 transcript). Therefore, as stated previously there is no difference in the HMG-D RNA levels between wt and 1(2)03605 flies. The 1(2)03605 recessive lethality is reversible and the newly generated re-mobilized lines fall into 3 categories: "precise" excisions, "imprecise" excisions and "local hops". The close proximity of the 1(2)03605 P element to the HMG-D gene, based on the mapping of the P element insertion site, may allow for the generation of new HMG-D alleles in a P element re-mobilization experiment. A mutant HMG-D allele could be generated if upon excision the 1(2)03605 P element deleted flanking DNA into the HMG-46 Figure 1 4 Densitometry analysis of Northern blot. Area plots are aligned as in the Northern from Figure 12. (a - b) Show the relationship between fly lines for the 1.3 kb and the 0.95 kb transcripts. The deficiency is twice the level of the wt and 1(2)03605 lines due to unequal loading (see RP49). The wt and 1(2)03605 lanes are essentially equal, indicating no difference between the mutant and wild-type, (c) Shows the RP49 control with the overloaded deficiency lane and the equally loaded wt and 1(2)03605 lanes. 47 48 Figure 15 Re-mobilization cross of 1(2)03605 line. 1(2)03605 males were crossed to a transposase source (A2-3) and dysgenic males were collected and then crossed to 1(2)03605 females. Male progeny were collected based on the loss of the rosy gene supplied by the pZ P element, ry' males were then balanced over CyO. Table 1 The re-mobilized lines were checked for recessive lethality. 4 lines are homozygous viable and therefore classified as "precise" excisions. 17 lines are still recessive lethal and have been classified as "imprecise" excisions that may have removed part of the flanking DNA surrounding the P element. * Homozygous flies were made of lines R15, R22 and R42. R46/R46 males are sterile and therefore a homozygous stock was not generated. 49 C? 1(2)03605 ry+ : rv 5 0 6 x Sp_ ; 5M2-3 CyO ry 506 22°C CyO TM6Ubx <S l(2)03605(ry); SM2-3 x T J L Q L ; D T 9 CyO ry CyO ry 506 Cf l(2)03605*(rv) ; ry CyO ry" x 7 M ; £v5 0 6 9 506 CyO ry" l(2)03605R fry0 ; rv 5 0 6 CyO ry" C?,9 Homozygous viable lines Homozygous lethal lines R15,R22,R42, R46 RI, R2, R8, R9, RIO, RI 1, RI6, RI9, R20, R21, R28, R31, R33, R37, R38, R43, R47 50 D locus. This re-mobilization experiment began with the mobilization of the 1(2)03605 P element and the generation of new lines. As shown is Figure 15, the 1(2)03605 P element was re-mobilized as follows: 1(2)03605 males were crossed to female flies carrying a transposase source, P[A2-3ry+](99B) abbreviated as A2-3 and single dysgenic male progeny were then crossed back to 1(2)03605 females. Females that had lost the rosy+ phenotype supplied by pZ were collected and balanced over CyO. Twenty-one independently generated re-mobilization lines were established. Each line was then examined for straight (1(2)03605*11(2)03605*) and curly wing (1(2)03605* I CyO) phenotypes. Four homozygous viable lines were generated and 17 recessive lethal lines were recovered (Table 1). Those lines that contain straight winged flies are presumably revertants of the 1(2)03605 P element induced lethality. To determine which ry lines still had a disruption of the 1(2)03605 locus or were simply "local-hops" out of the 1(2)03605 locus and into a nearby essential gene, the lines were crossed to the deficiency Df(2R)Pu-D17, which is deleted for bands 57B4 - 58B (Table 2). The original 1(2)03605 line was crossed to the deficiency and as expected is lethal over Df(2R)Pu-D 17 (Table 2a). The 4 homozygous viable lines are all viable over the deficiency indicating they are "precise" excisions (Table 2b). Interestingly, the R46 line is only partially viable over the deficiency, depending on the direction of the cross. R46IDf(2R)Pu-D17 flies are only recovered when the male parent contributed the deletion and the female contributes the re-mobilized P element. This line may be a partial revertant as homozygous R46 flies are rare and no progeny are recovered when homozygous R46 males are crossed to wt virgin females, indicating the homozygous R46 males are sterile. The re-mobilized lines that 51 Table 2 Re-mobilization lines were crossed to a deficiency for the region (Df(2R)Pu-DJ7). (a) Parental 1(2)03605 line crossed to the deficiency. As expected the transheterozygotes are not viable, (b) 4 of the lines are homozygous viable and viable over the deficiency and these are classed as "precise" excisions, (c) 5 lines that are recessive lethal but lethal over the deficiency were recovered, these lines are classed as "imprecise" excisions and may have deleted DNA flanking the P element, (d) 7 lines that are recessive lethal but viable over the deficiency are classed as "local-hops" and probably reflect the P element excising precisely and jumping into another essential gene not uncovered by the deficiency. 52 Cf RXX xDf(2R)Pu-D17Q & C? Df(2R)Pu-D17 xRXX 9 CyO SMI SMI CyO (d) Curly Straight Curly Straight Line C? 9 c? 9 Cf 9 Cf 9 1(2)03605 22 26 0 0 21 18 0 0 R15 9 7 18 22 8 8 2 3 R22 7 28 10 12 - - - -R42 20 22 9 6 14 10 2 1 R46* 13 14 0 0 22 20 1 3 R16 24 25 0 0 17 28 0 0 R20 20 26 0 0 20 24 0 0 R21 21 13 0 0 - - - -R42 9 7 0 0 - - - -R43 24 19 0 0 9 13 0 0 R8 20 20 8 10 17 21 14 15 RIO 26 24 20 7 6 5 3 0 R28 29 24 15 10 8 12 2 5 R31 21 31 11 22 16 8 4 4 R33 20 19 26 22 20 15 3 7 R37 13 24 19 13 17 15 3 4 R47 19 18 4 8 12 19 15 14 53 are still recessive lethal fall into two classes when crossed to the deficiency (Table 2c,d). Five of the lines are lethal over the deficiency and most likely represent an "imprecise" excision from the 1(2)03605 locus or simply a disruption of the ry+ gene. The other 7 lines are all viable over the deficiency and probably represent a "local-hop" restoring the 1(2)03605 locus and disrupting another essential gene outside the boundaries of the deficiency. These "local-hops" are ry", showing that the mobilized element has suffered a partial deletion of the ry+ gene. The re-mobilized P-element fails to generate any new HMG-D alleles. To determine if any of the re-mobilized lines deleted any of the HMG-D open reading frame, genomic Southern analysis of the original 1(2)03605, wt and all the re-mobilization lines was undertaken. Genomic DNA was doubly digested with Bgl II and Sal I, which in wildtype flies produces a 1.5 kb fragment containing most of the HMG-D ORF, blotted and probed with a 3 2 P labeled cDNA for HMG-D. Probing with the HMG-D cDNA should reveal any polymorphisms between the parental 1(2)03605 line and the re-mobilized lines, thus allowing detection of any deletions/re-arrangements created by mobilizing the 1(2)03605 P element that disrupt the HMG-D coding region (Figure 16). The ethidium bromide stained gel is presented (Figure 16a) to show the wavy gel front which leads to a slight difference in mobility for a DNA of given size, which is also reflected in the autoradiograph (Figure 16b). The autoradiograph shows no polymorphisms between the original 1(2)03605, wt and re-mobilized lines. However, re-mobilization line R43 was not digested completely and shows a higher band. This band 54 Figure 16 Genomic southern analysis of re-mobilization line DNA cut with Bgl II and Sal I which cuts out the HMG-D ORF. The blot is probed with an HMG-D cDNA. (a) Ethidium bromide stained gel prior to blotting indicates lines R15, R42 are under loaded and line R43 failed to cut. Note the gel front is wavy which leads to a slight difference in mobility for a DNA of given size, this is reflected in the autoradiogram. (b) The autoradiogram indicates there are no polymorphisms detected between the wt, 1(2)03605 and re-mobilization lines. Therefore, no new HMG-D alleles were created upon mobilization of the 1(2)03605 P element. (Molecular weight values indicate migration of the molecular weight size markers and are thus not entirely accurate across the gel) 55 represents uncut DNA. Therefore no new HMG-D alleles were created in the P element re-mobilization experiment. Discussion Summary A study was undertaken using a P element induced recessive lethal fly line obtained from the Berkeley Drosophila Genome Project to determine if this lethality was caused by disruption of either of the HMG-D or HMG-Z genes. In order to address this hypothesis the 1(2)03605 P element insertion site had to be defined. The location of the P element insertion site, downstream of the HMG-D gene, was determined by mapping it to various genomic clones of the region. With the P elements close proximity to HMG-D, both (i) Northern analysis to determine the P element's affect on HMG-D transcript levels and (ii) re-mobilization of the P element in an attempt to generate novel mutant HMG-D alleles was undertaken. Unfortunately, the 1(2)03605 P element does not disrupt either HMG-D or HMG-Z and no new HMG-D alleles were created. The 1(2)03605 line is the same line initially characterized by the Genome Project A plasmid rescue experiment using a Berkeley Drosophila Genome Project single P insert in the region where HMG-D and HMG-Z map was performed to obtain the genomic DNA next to the P element insertion. The failure to recover any rescue plasmids suggested that the P element may be deleted or re-arranged. Genomic Southern analysis (Figure 5b - Kanamycin gene probe) of the 1(2)03605 P element fly line confirmed this. In contrast, the BDGP had originally rescued the DNA flanking the 1(2)03605 P element. The sequence was obtained in 1993 and the 1(2)03605 P element stock was 57 maintained for a number of years until this project was started in 1997. Since Drosophila stocks must be maintained by replica/transfer rather than freezing, it is quite possible that a deletion or re-arrangement of the 1(2)03605 P element occurred during that time interval. The 1(2)03605 P element does not disrupt either HMG-D or HMG-Z The failure to recover P "rescue" plasmids from the 1(2)03605 line not withstanding the goal of testing whether the 1(2)03605 P element caused a knockout of HMG-D or HMG-D still remained. Evidence that led to this initial hypothesis was the coincident cytological location (57F8-11) of HMG-D/Z and the P element insertion site and the similar mRNA expression pattern of both in the developing nervous system. The hypothesis was a gamble, but one that was worth accepting. Unfortunately, the 1(2)03605 P element does not disrupt either HMG-D or HMG-Z . What makes the primary premise of this hypothesis (a gene of interest mutated to lethality by a transposable element) such a gamble are outlined below: (i) the number of essential genes in Drosophila (ii) the susceptibility of these gene to disruption by insertional mutagenesis and finally (iii) the extent to which these essential genes have been mutated to saturation. It is estimated that there are between 8000 to 15 000 genes in the Drosophila genome (Rubin, 1998). Approximately 3600 of these are essential loci based on the Miklos and Rubin (1996) estimate of 12 000 genes. The BDGP strain library has so far disrupted about one-third of the essential loci located on the autosomes. Specifically, 1182 P element tagged lines exist representing approximately 31 % of the 3200 autosomal genes that can mutate to a distinguishable phenotype (Rubin, 1998). Thus, the probability that a particular gene of interest is disrupted by a P element is about 33%. However, 58 more and more lines are being generated and this will increase the feasibility of tagging susceptible loci. The 1(2)03605 P element is likely disrupting an unidentifed gene Even though the 1(2)03605 P element did not insert within HMG-D/Z, it did insert less than 10 kb downstream of HMG-D (see Figure 11). Since the P insert mutation has a recessive lethal phenotype, the 1(2)03605 P element is likely affecting another gene downstream of HMG-D. There are two candidate genes within the 57F8-11 region, however neither of these appear to have expression confined to the embryonic nervous system. These genes are: TATA Binding Protein (TBP) at cytological map position 57F8-10 (Hoey et al., 1990) and Enhancer of eye roughness (ER2-5) at cytological map position 57F-58A (Karim et al, 1996). Therefore the 1(2)03605 P element induced lethality is probably caused by disruption of a novel gene. Re-mobilization of the 1(2)03605 P element failed to generate new HMG-D alleles The 1(2)03605 P elements insertion site downstream of HMG-D may have allowed for the generation of new HMG-D alleles by P element imprecise excision. Unfortunately, no new HMG-D alleles were created as the re-mobilization screen was initially carried out as a reversion analysis and not enough flies were screened for imprecise excisions. The reversion screen was successful and four homozygous lines were generated (Figure 15, Table 1). If a more extensive screen is carried out, in the order of 7000 screened chromosomes (Salz and Schedl, 1987; Tsubota and Schedl, 1986), deleted HMG-D alleles should be recovered. 59 Conclusion Since the lethality associated with the 1(2)03605 P element insert does not result from a disruption of either HMG-D or HMG-Z, the question remains "what gene is the P insert affecting?". Using a PI clone of the region as a probe on a comparative developmental Northern of 1(2)03605 vs wild type flies is a simple way to determine (i) if there is one or more transcribed genes in the region and (ii) if the P element is causing lethality by changing the expression pattern of some transcripts. Hopefully, with an observed difference it should be a simple matter of using fragments of the PI clone as probes on the Northern to narrow down the location of the gene of interest. With the fragment identified, the DNA can be sequenced and compared with known genes to determine if the gene has been previously identified or is a novel one. Also, the PI fragment can be used to screen a cDNA library to identify cDNA clones of the affected gene. 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