,uxw MM?" 3" .I u ‘4 515‘" ; nun-.45: ' w‘k‘ §.u‘3. n _i' - i - . ,_ ;-:: ~ :1: ‘ :-- 0-. (fl (Ill m. 1 ATS Alul lcdl ,. Wnn—Qs 2°” --------- m." '—f‘ 6 --------------- “or: (d I 2 I P u I I G I T + I! ‘ r”- 1’ ‘ _- I .. ~c I ~ "I. T r . =- I I .-___ I I .. t r ,— . _ . O I. c c “-— m s w r t __ 2" g "l a ("l ’- . - -4-" . lch I'htIIIrns' __‘2 l (InozuoulcDNAI'IIIIIIIIIuts' (O) 15 the exception of the 158 nucleotide band, were consistent with the transcription start site mapped with St and mung bean nuclease. The 158 nucleotide primer extension band could not be due to hybridization of P1 to a second site downstream on the U0 mRNA as the sequence of P1 has no similarity to any other site along the U0 mRNA, even allowing for 14 mismatches out of 30 nucleotides. The unexpected primer extension product of 158 nucleotides was not due to a second upstream promoter responsible for high levels of U0 mRNA. This conclusion was derived from an additional set of primer extension experiments with P2, a synthetic primer, just downstream of P1 (Figures 1. 2c and A3). Primer P2 was hybridized to poly(A)"' RNAs from Ore-R third instar larvae and ry2 12 hour adults, both sources having high levels of U0 mRNA. Only a single extension product of 126 nucleotides resulted (Figure 2d) which also maps the transcription start site of the U0 gene to position +1 (Figure 1). To confirm the location of the U0 transcription start site three different U0 cDNA clones from a third instar larval cDNA library were sequenced at the 5' end (Figures 1 and A1). These cDNA clones extend to nucleotide A at +1 followed by a G nucleotide not present at the corresponding position of either the Canton-S genomic sequence or the ecd1 U0 genomic sequence (Figure 29). No U0 cDNAs were isolated which had an additional 67 base pair 5' exon. A clue concerning the origin of the 158 nucleotide primer extension band was obtained when the Spel-EooRl fragment, containing the 5' transcribed region of the U0 gene including the sequence of P1 (Figure 1), was used as a probe to screen a third instar larval cDNA library. Two classes of cDNAs were obtained. One class of cDNAs hybridized to the EcoRl-Spel probe and also to cU02 (Figure A1). The second class of cDNAs hybridized to the EcoRl-Spel probe but not to cU02 and contained cDNAs of approximately 3800 base pairs in 16 length with restriction maps unlike that of the U0. This latter class of cDNAs may encode the message which is primed by P1 and gives rise to the 158 nucleotide extension product. No further work was done to characterize the 158 nucleotide extension product. 3. Spatial distribution of U0 mRNA by in situ hybridization. In situ hybridizations of U0 mRNA were performed in order to identify the population of cells of the Malpighian tubules containing U0 mRNA in the wild type 12 hour Ore-R adult and in the xanthine dehydrogenase deficient (ry2) 12 hour adult which has a five- to tenfold higher level of U0 mRNA (Friedman, 1973, Kral et al., 1986). At least two mechanisms could account for the five- to tenfold higher level of U0 mRNA in the Malpighian tubules of ry2 12 hour adults: (1) an increase in the amount of U0 mRNA within the same population of cells of Malpighian tubules or (2) recruitment of additional cells expressing U0 mRNA from those cells which comprise the Malpighian tubules. To examine these two possibilities, U0 sense and antisense RNA probes were hybridized in situ to sectioned and whole mount Malpighian tubules. An [ct- 35deUTP-labeled antisense RNA probe synthesized from the EcoRl-Aocl template of cU02 (Figure 1) hybridized exclusively to the Malpighian tubules in sections of ry2 12 hour adult abdomens (Figure 3a and b). No hybridization signal was detected using a sense U0 RNA probe hybridized to alternate sections (data not shown). Whole-mount Malpighian tubules from Ore-R adults and ry2 adults were hybridized with a U0 antisense RNA probe. There were no detectable U0 transcripts in the midgut, hindgut or the cells which comprise the transitional segment or the initial enlarged segment of the Malpighian tubules as defined by Wessing and Eichelberg (1978) (Figure 3c and d). There was a 17 Figure 3. Spatial distribution of U0 mRNA among the cells which comprise the D. melanogaster adult Malpighian tubules. Phase contrast (a) and darkfield illumination (b) of the autoradiographic image of a sectioned ry2 12 hour adult abdomen hybridized to a U0 antisense 358-labeled RNA probe synthesized from the EcoRl-Aocl restriction fragment of cU02 (Fig. 1c). Hybridization signals were detected exclusively within the Malpighian tubules (Mt). (c) Brightfield images of whole mount Malpighian tubules from a ry212 hour adult were hybridized to the antisense 35S-labeled RNA probes as described for panels (a) and (b). The autoradiographic image revealed hybridization mainly within the mid-segment (M) of the anterior pair of tubules (A) and along the entire posterior pair of tubules (P) with a small amount of hybridization within the cells of the ureter (U). Hybridization did not occur within the transitional segment (T) or the initial segment (I) of the Malpighian tubules. Dark areas along the gut (G) attached to the Malpighian tubules are not exposed silver grains but are opaque material within the preparations which also present in the control sense-strand in situ hybridizations (e). (d) Whole Malpighian tubules from an Ore-R adult 12 hour adult were hybridized to the antisense 35S-labeled U0 RNA probe in (a) and (b). (e) No detectable signal was present when ry212 hour adult Malpighian tubules were hybridized to a sense 35S-labeled U0 RNA probe. 18 Figure 3. 19 weak hybridization signal within the ureter and within the cells at the extreme distal end of the posterior tubule (Figure 3c and d). In both ry2 adults and 0re- R adults, U0 transcripts accumulate within the main segment cells of the anterior Malpighian tubules and along the length of the posterior tubules. There was a far greater number of silver grains over the whole-mount Malpighian tubules of the ry2 12 hour adult as compared to the same population of cells of the Ore-R 12 hour adult (compare Figure 3c and d), which is consistent with five- to tenfold higher level of U0 mRNA and U0 activity in the ry2 adult as compared to the Ore-R adult (Friedman, 1973; Kral et al., 1982; Kral et al., 1986). 4. Deduced amino acid sequence and protein sequence comparisons of U0. The D. melanogaster U0 transcription unit contains two in-frame methionine codons in the amino terminal region (Met-1 and Met-23, Figure 1). If Met-1 is utilized as the translation initiation codon, the deduced Mr for the U0 peptide would be 39,989 daltons which is not significantly different from the apparent Mr of 40,480 +1340 estimated for the purified U0 protein (Friedman and Barker, 1982). If Met-23 is used to initiate translation, the deduced Mr would be 37,701 daltons which is significantly less than the apparent molecular weight. The scanning model for translation initiation (Kozak, 1989) would predict that Met-1 is the translation start site for the U0 gene in D. melanogaster. Met-1 is in good sequence context for a Drosophila translation initiation codon (Cavener, 1987). The four nucleotides upstream of Met-1 of the U0 gene are identical to the four nucleotides preceding the start codon of other Drosophila genes (T 6r6k and Karch, 1980; 0'Tousa et al., 1985; Ito et al., 1988). Whether Met-1 or Met-23 is the translation start for the U0 protein could not be examined directly since the amino terminus of the purified U0 protein was blocked and could not be 20 sequenced (Friedman, unpublished results). Consequently, a molecular evolutionary comparison between urate oxidase of D. pseudoobscura and D. virilis (Figure 4) which diverged from D. melanogaster approximately 35 million and 60 million years ago, respectively (Beverley and Wilson, 1984) was used to determine which of the two in-frame methionine codons, Met-1 or Met-23, is the U0 translation start site. The sequences of the first seven amino acids of urate oxidase from D. melanogaster, D. pseudoobscura and D. virilis are identical (Figure 4). The D. pseudoobscura deduced U0 amino acid sequence has a methionine codon (Met-21) in the corresponding position to Met-23 of the D. meIanogaster deduced U0 protein sequence. However, the D. vin'lis deduced U0 amino acid sequence does not have a second methionine residue in the amino terminal region (Figure 4). The first eight codons of the deduced U0 protein in D. virilis contain four synonymous substitutions when compared to the first eight codons for the deduced amino acid sequence of D. melanogaster U0 protein, while immediately upstream of Met-1, in both species, the preceding 32 nucleotides show no DNA sequence similarity. Taken together, these data indicate that Met- 1 is the U0 translation start codon in all three species of Drosophila. The amino acid sequence comparison between urate oxidase of D. melanogaster, soybean (Nguyen et al., 1985), rat (Reddy et al., 1988), mouse, pig and baboon (Wu et al., 1989) is shown in Figure 4. There is 32% to 38% amino acid sequence identity between urate oxidase of Drosophila melanogaster and urate oxidase of the five other species. Though not indicated in Figure 4, many of the non-identities represent conservative evolutionary amino acid changes (Lipman and Pearson, 1985). Among the 22% of the amino acid residues identical in the deduced amino acid sequences of urate oxidase from soybean, rat, mouse, pig, baboon and Drosophila, there are four histidine residues (Dm: HIS-170, HIS-172, HIS-182 and HIS-308) which may be 21 HAOQEVVBGF 0°. ERR LL...- DP? 7...... III 9.9.... "H" 1.8111111 suntan?» TTTTTK .ka... 0000". VVVVT 535$ VTTTTV ' - - - - - - - - - - - - - - ~ - - - - - - - - - ‘ . - - - - - - - - - - - - - - - - - - - - - - - - I'IVBAYPNQRVCOIETRTNVNGKCB 22 3;? ~ 6 ran-w N15; uhxrt o A I 41! R.'~E,.0‘B!:~QZMQ7KmL;YGDIVLT' al.,.‘IItD it 225 P x o «.1:2,35."-‘8.,S{50‘K¢':_;,L:,;vio x o v I. s_-s o 1.3%: x I o P 225 P 2 pk e 3 arm yflo “ran-$0 1 o v x. 12,510 wt: 1 I o I 225 x ‘r z 3:518:33 {.LE’XD . v , say vii . 0.269 p OVm-valfi‘v il.‘..x.szltov ov - ov v ocvax'rzoo GLx---tlxsl: GLt---ilxes cnx---uxcc l: Figure 4. Alignment of the deduced amino acid sequences of U0 from Drosophila melanogaster (Dm), soybean (S), rat (R), mouse (M). pig (P) and baboon (B) and the first 39 and 43 amino acids from the amino termini of U0 from Drosophila virilis (Dv) and Dr080phila pseudoobscura (00). respectively. The single-letter amino acid code is used. Three deduced amino acid sequences for rat urate oxidase have been reported which differ from one . another at the amino and carboxy termini (Ito et al., 1988; Motojima et al., 1988; Reddy et al., 1988). In this figure the deduced rat urate oxidase sequence from Reddy et al. (1988) was used since it appears to be full length. To establish the comparison and accomodate the larger Dm U0 protein, a gap was introduced in the middle of all the other U0 amino acid sequences at a site which showed no sequence similarity between Dm and the other five urate oxidases. All other gaps were created by the FASTP program to optimize the alignments (Lipman and Pearson, 1985). The U0 amino acid sequence of D. melanogaster shows 32%, 38%, 37%, 36% and 35% identlty to the U0 amino acid sequence of soybean, rat, mouse, pig and baboon, respectively. Boxed areas indicate identical amino acids found in two or more of the U0 proteins. Amino acid residues of urate oxidase identical in all six species are shaded. The location of introns for D. melanogaster U0 and soybean uricase II are indicated by solid triang es. 23 involved in copper binding (Mahler, 1958; Wu et al., 1989). Urate oxidase is a peroxisomal enzyme (deDuve and Baudhuin, 1966; Lazarow and Fujiki, 1985; Hayashi et al., 1976) and the deduced amino acid sequence at the carboxy terminus of urate oxidase from Drosophila is Ser-His-Leu, soybean is Ser-Lys- Lou and rat, mouse, pig and baboon are Ser-Arg-Leu. These tripeptide sequences are also found at the carboxy termini of some, but not all, peroxisomal proteins (Miyazawa et al., 1989; Gould et al., 1990; Lewin et al., 1990). Any one of these three carboxy terminal tripeptides is sufficient for targeting a reporter protein to peroxisomes (Gould et al., 1990). On the basis of the similar carboxy termini of urate oxidase proteins compared here (Figure 4) and from the data on targeting of some peroxisomal proteins (Gould et al., 1988; Gould et al., 1989; Miyazawa et al., 1989), a serine residue followed by a positively charged amino acid and then a carboxy terminal leucine is likely to be involved in the peroxisomal targeting of U0 of Drosophila, plants and vertebrates. C. Discussion The U0 gene of D. melanogaster is structurally compact, comprised of two exons separated by a 69 base pair intron (Figure 1). The D. melanogaster U0 gene is transcribed from a single promoter yielding U0 mRNA of 1224, 1227 and 1244 nucleotides depending on which one of three 3' endonucleolytic cleavage sites is utilized (Figures 1 and 2). A few genes from both vertebrates and invertebrates have been shown to have multiple polyadenylation sites (Setzer et al., 1980; Mlodzik and Gehring, 1987; Dreesen et al., 1988; Garbe et al., 1989; Laird-Offringa et al., 1989) and in some cases; the polyadenylation sites are closely spaced (Johnson et al., 1987; Seeger et al., 1988; Quan and Forte, 1990). The biological significance of multiple adjacent polyadenylation signals and sites remains to be determined (Denome and Cole, 1988). 24 , The U0 gene of D. melanogaster has a complex developmental and tissue- specific pattern of expression. U0 mRNA is present within the main segment cells of the Malpighian tubules (Figure 3) of third instar larvae and adults (Friedman, 1873; Kral et al., 1986). it is assumed that the U0 gene is regulated at the level of transcription. The pattern of expression of a U0-lacZ fusion transgene supports this assumption (Figure 10, Chapter Three). However, nuclear run-on experiments are needed to address whether the dramatic decline in the steady state level of U0 mRNA at the end of the third instar stage is due to a decrease in U0 gene transcription and/or a decrease in the half-life of U0 mRNA. Putative regulatory sequences involved in tissue-specific expression, developmental timing and quantitative regulation of the U0 gene can be identified by several methods. Comparison of the DNA sequence of the flanking region of the homologous gene in different species is one approach for identifying putative cis-regulatory elements which are detected as conserved motifs highlighted amidst a background of dissimilar DNA sequence (Blackman and Meselson, 1986; Bray et al., 1988; Fenerjian et al., 1989; Kassis et al., 1989). The application of this method to examine the 5’ flanking DNA of the U0 gene is described in Chapter Three. Another method used for identifying putative cis-regulatory elements is to examine the flanking DNA of a particular gene for sequences which are well characterized cis-elements of known function. The sequence at position -31 to -37 of the D. melanogaster U0 gene was identified as a TATA box using this method. Bracketing the TATA box and the U0 transcription start site at +1 of the U0 gene is a perfect 13 base pair direct repeat (DR), AAGTGAGAGTGAT, beginning at positions -138 and +11. The sequence of the DR motif is similar to a proposed 20-hydroxyecdysone consensus sequence found upstream of six 20-hydroxyecdysone inducible genes of Dros0phila (Pongs, 1988). The possible role of the DR motif in 20- 25 hydroxyecdysone repression of the U0 gene is discussed in Chapter Three. A third method to identify cis-regulatory elements of a gene is to compare the flanking sequence with the regulatory regions of other genes that share some aspect of temporal and tissue-specific gene control. Urate oxidase is the only gene thus far reported in Drosophila which is expressed exclusively within the Malpighian tubules. The white gene (w) and the alcohol dehydrogenase gene (Adh) are expressed in the Malpighian tubules but also in other tissues (Fjose et al., 1984; Lockett and Ashburner, 1990). Nevertheless, there may be similar cis-acting regulatory elements in the flanking DNA of the w, Adh and U0 genes which may be involved in Malpighian tubule expression. An 864 bp region of the w gene of D. melanogaster was reported to be necessary for expression in the Malpighian tubules (Pirrotta et al., 1985). Tissue-specific expression of the Adh1 gene of D. muIIeri requires the presence of two regulatory elements, the “A box" and 'B box” (Fischer and Maniatis, 1988). A DNA sequence similarity search was performed using regions of the w and Adh-1 genes important for Malpighian tubule expression and the flanking DNA of the U0 gene. This search revealed a sequence in the 5’ flanking DNA of the U0 gene, AAAGTAAAGCG (-751, Figure 1), that was similar to the sequence AAAGTACAGTG in the Malpighian tubule-specific region of w (+4223, 0' Hare et al., 1984; Pirrotta et al., 1985) and to the sequence AAAGTAAAACG in the middle of the Adh-1 ‘3 box” (-227, Fischer and Maniatis, 1988) which is critical for transcription of Adh-f. (This sequence is not found in the upstream flanking DNA of the D. pseudoobscura U0 and the D. vin'lis U0 genes which are described in Chapter Two). The sequence AAAGTAAAGCG of the D. melanogaster U0 gene resides within the region deleted in the P-element construct P[(wA+)del(-808, -702)DmU0-lacZ] (Chapter Three) which will be returned to the germ line for functional testing. It will be of interest to examine 26 the tissue-specific expression of the U0-lacZ transgene in transformants carrying this construct in order to establish whether this sequence has a possible role in the Malpighian tubule-specific expression of the D. melanogaster U0 gene. In general, a fine structure map of cis-acting regulatory elements of a gene is the necessary first step to understanding the intriguing questions concerning gene regulation. The molecular analysis of D. melanogaster U0 determined that this gene is structurally compact and has a complex expression pattern and, therefore, is amendable to a variety of methodologies for studying gene regulation. The determination of the structure, sequence and pattern of expression of the D. melanogaster U0 gene was essential background information for the investigation of evolutionary changes in sequence and regulation of Drosophila U0. CHAPTER 1W0: Evolutionary changes in the sequence of the U0 gene of D. melanogaster, D. pseudoobscura and D. virilis. A. introduction Sequence comparisons of the homologous gene from different species can be used to identify important structural and regulatory features of a gene or protein (Lipman and Pearson, 1985; Doolittle, 1989). Evolutionary processes conserve sequence which has functional relevance and randomizes and sometimes eliminates sequence which is functionally unimportant. Sequence comparisons have been successful in the identification of important structural and regulatory features of many Drosophila genes (Bray et al., 1989; Treier et al. 1989; Seeger and Kaufman, 1990; Shea et al., 1990). An interspecific sequence comparison of the U0 gene was included as part of the molecular analysis reported here. Since conserved sequence is likely to be important, a molecular evolutionary analysis of the U0 gene was made in an attempt to (1) identify conserved regions of the U0 protein to establish structure-function relationships and (2) to identify cis-acting regulatory elements in the flanking DNA of the U0 gene that are potential candidates for cis-regulatory elements. The U0 genes from D. melanogaster; D. pseudoobscura and D. virilis were cloned (Kral et al., 1986; Friedman et al., 1991; Lootens et al., 1991) and compared. D. pseudoobscura and D. virilis are estimated to have diverged from D. melanogaster 35 million to 60 million years ago (Beverley and Wilson, 1984). These three species were chosen for the comparison since the time of divergence of these species has been shown to be long enough for randomization of nonessential DNA 27 28 sequence (Henikoff and Eghtedarzadeh, 1987; Riley, 1989). B. Results 1. Nucleotide and deduced amino acid comparisons of U0 of D. melanogaster, D. pseudoobscura and D. virilis. A restriction map of the DrOSOphiIa pseudoobscura genomic region showing the U0 transcription unit and the subclones used for sequencing 2.24 kb of DNA from the D. pseudoobscura U0 region are shown in Figure A3. Southern analyses demonstrated that the D. pseudoobscura U0 genomic clones were derived from D. pseudoobscura strain AH133 and that no obvious rearrangements had occurred during construction of the genomic library or of subclones used for sequencing. Southern analysis also confirmed that the U0 gene was single copy in D. pseudoobscura AH133 (Friedman et al., 1991). A restriction map of the Drosophila virilis genomic region and the subclones used for sequencing 1.8 kb of DNA from the D. virilis U0 region are shown in Figure A4. Extensive restriction mapping and Southern analyses were performed using the U0 genomic clones and DNA isolated from several strains of D. virilis (Lootens et al., 1991). These data clearly indicated that the U0 gene was tandemly duplicated in some strains of D. virilis and that the genomic clones were derived from a strain containing this tandem duplication of the U0 gene. The tandemly duplicated D. virilis U0 genes are designated Dv U01 and Dv U02 (Chapter Four). The Dv U01 sequence is presented here. Elucidation of the mechanism of recombination that resulted in this tandem duplication among some strains of the D. virilis is part of the Ph.D. thesis research project of Susan Lootens. The D. pseudoobscura and D. virilis genomic sequences are available in the EMBL GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X51940 and X57114, respectively. The transcription start 29 sites of the D. pseudoobscura and D. virilis U0 genes were inferred on the basis of the position and sequence of the experimentally determined D. melanogaster U0 transcription start site. Both the D. pseudoobscura and D. virilis inferred transcription start sites, GGCATCAGTCAGTCAT and GGCCTCATCGGAAT, respectively, (Figure A5) match the consensus transcription start site identified for several other Drosophila genes (ATCA(GI'l')T(C/'l'), Hultmark et al., 1986). In the D. pseudoobscura and D. virilis U0 transcribed region, the first ATG codon following the transcription start site is likely to be the translation initiation codon (Figure A5 and discussed in Chapter One). The sequence contexts of the U0 translation initiation codons (ATG) from D. pseudoobscura (TAG AATGTI'T) and D. virilis (CAGTAIGTI'T) are similar to those reported for other Drosophila genes (Cavener, 1987). Between the D. melanogaster and D. pseudoobscura deduced amino acid sequence, the first nine amino acid residues are identical with only two silent nucleotide substitutions. Between the D. melanogaster and D. virilis deduced amino acid sequence, the first eight amino acid residues are identical with eight silent nucleotide substitutions (Figure A5). The D. melanogaster, D. pseudoobscura and D. virilis U0 introns are 69 bp, 62 bp and 55 bp, respectively (Figure A5). The position of the D. pseudoobscura and D. virilis U0 introns was inferred from a comparison of the D. pseudoobscura and D. virilis sequence to the D. melanogaster U0 cDNA and genomic sequence. The intronic consensus donor (GT) and acceptor (AG) splice sites (Mount, 1982) present in the D. melanogaster U0 gene are conserved in the D. pseudoobscura U0 and D. virilis Dv U01 genes with the intronic positions bifurcating an aspartic acid codon between the first and second positions in all three species (Figures 5 and A5). The D. pseudoobscura and D. virilis U0 nucleotide sequences are 82.2% and 73.2% identical to the D. melanogaster U0 nucleotide sequence, 30 Alignment of the deduced amino acid sequence of U0 of from these three Drosophila species. Boxed areas indicate identical amino acid resides in two of the three U0 proteins. Shaded amino acid residues are D. melanogaster (Dm). D. pseudoobscura (Dp) and D. virilis (Dv). The single- letter amino acid code is used. There is 72% identity among the U0 protein Figure 5. . Dashes (-) represent gaps identified identical in all three Drosophila speci by FASTP (Lipman and Pearson, 1985). 31 respectively (Figure 5 and Table A3). The calculated peptide molecular weights for the D. pseudoobscura and D. virilis U0 protein are 39,266 and 38,680 daltons, reSDectively, as compared to the D. melanogaster value of 39,989 daltons. This difference in size of the U0 protein among these three Drosophila species has been confirmed by Western blot analysis (Figure 6). The first exon of the D. pseudoobscura and D. virilis U0 gene encodes 185 and 181 amino acids, respectively, while the first exon of D. melanogaster U0 encodes 191 amino acids. The second exon of the D. melanogaster, D. pseudoobscura and D. virilis U0 genes each encodes 161 amino acids (Table A3). Between D. pseudoobscura and D. melanogaster, the U0 protein-coding region has accumulated 185 nucleotide substitutions from a total of 1038 nucleotides (17.8%) resulting in 42 amino acid replacements, 79% having occurred in exon 1 (Figure A5 and Table A2). Between D. virilis and D. melanogaster, the U0 protein-coding region has accumulated 275 nucleotide substitutions from a total of 1026 nucleotides (26.8%) resulting in 86 amino acid replacements, with 68% occurring in exon 1 (Figure A5 and Table A2 ). The D. melanogaster, D. pseudoobscura and D. virilis U0 exon 1 are 607, 590 and 585 nucleotides, respectively. Between D. pseudoobscura and D. melanogaster there are 106 nucleotide substitutions in U0 exon 1 resulting in 48 synonymous changes and 33 amino acid replacements, of which 29 are evolutionarily conserved as defined by Lipman and Pearson (1985). Between D. virilis and D. melanogaster there are 162 nucleotide substitutions in U0 exon 1 resulting in 53 synonymous changes and 59 amino acid replacements, of which 39 are evolutionary conserved changes. The protein coding region of exon 2 of the U0 gene from both D. pseudoobscura and D. melanogaster has 482 bp with 79 nucleotide substitutions of which 56 (70.9%) are in the third codon position. When compared with exon 2 of D. melanogaster U0, exon 2 of D. pseudoobscura U0 32 94,000— 67,000- 43,000— 30,000— .Flgure 6. Western analysis of U0 protein of D. meIanogaster, D. pseudoobscura and D. virilis. Whole cell homogenate from two Malpighian tubules of D. melanogaster third instar larvae (lane 1), one half of a Malpighian tubule from D. pseudoobscura (lane 2) and one half of a Malpighian tubule from D. virilis (lane 3). U0 protein was detected by Western analysis using rabbit polyclonal antibodies made against apparently homogeneous pure D. melanogaster U0 protein (Friedman and Barker, 1982; Kral et al., 1986). The positions of protein molecular weight standards are given in the left margin. The U0 M, calculated from the deduced amino acid sequence of U0 of D. melanogaster, D. pseudoobscura and D. virilis is 39,989, 39,266 and 38.680 daltions, respectively. 33 has only 9 amino acid replacements of which 8 are conserved replacements. The protein coding regions of exon 2, for both D. virilis and D. melanogaster U0 genes, have 482 bp with 113 nucleotide substitutions of which 80 (70.8%) are in the third codon position. When compared to exon 2 of the D. melanogaster U0 gene, the exon 2 of D. virilis U0 has 27 amino acid replacements with 26 being conserved replacements. 2. D. melanogaster, D. pseudoobscura and D. virilis U0 codon usage. Third-codon-positions evolve at a rate approximating selective neutrality and have been used as a measure of the degree of divergence among species (Henikoff and Eghtedarzadeh, 1987; Riley, 1989). In Drosophila, the third- codon-position averages 71.4% G+C (Stramer and Sullivan, 1989). The third position of D. melanogaster, D. pseudoobscura and D. virilis U0 are 71.0%, 76.3% and 63.7% G+C, respectively. The codon usage for U0 genes of D. melanogaster, D. pseudoobscura and D virilis (T able A3) is similar to other Drosophila proteins (Shields et al., 1988). To examine the degree of divergence among the U0 genes of D. melanogaster, D. pseudoobscura and D. virilis, third-codon-position differences, adjusted for codon bias, were compared for threonine, proline, alanine, glycine and valine, each having four codons. The deduced amino acid sequences of U0 of D. melanogaster and D. pseudoobscura contain 90 conserved residues for these five amino acids. With random codon usage, 68 (75%) of the third-codon-positions of these amino acids would be expected to have changed to synonymous codons. When corrected for codon usage bias as described in Materials and Methods (Appendix A), 47 different third-codon- positions would be expected to have changed while 37 (79%) were observed to have changed since the divergence of D. melanogaster and D. pseudoobscura (Table A4). 34 The deduced D. melanogaster and D. virilis U0 amino acid sequences contain 79 conserved residues for the five amino acids listed above. With random codon usage, 60 of the third-codon-positions of these amino acids would be expected to have changed to synonymous codons. When corrected for codon usage bias, 43 different third-codon-positions would be expected to have changed and in fact, 43 were observed to have changed since the divergence of D. melanogaster and D. virilis (Table A4). These results indicate that the additional time of divergence between D. virilis and D. melanogaster; as compared to D. melanogaster and D. maudoobscura, has allowed for most of the unconstrained sequence of the U0 gene to have changed. 3. DNA sequence comparisons in the 5’ flanking DNA of the D. melanogaster, D. pseudoobscura and D. virilis U0 genes. When comparing the flanking DNA of a homologous gene among sufficiently diverged species, only sequence with important coding and regulatory function is conserved. The D. melanogaster, D. pseudoobscura and D. virilis U0 flanking sequences were compared using dot matrix analyses (Pustell and Kafatos, 1982; Pustell and Kafatos, 1984). Conserved stretches of nine or more nucleotides, allowing for one nucleotide mismatch, were used as the search parameters. Eight conserved sequence elements 9 to 16 bp in length (E1-E6 and E8) were identified after a comparison of the 5' flanking DNA of the D. melanogaster U0 gene to the 5’ flanking DNA of the D. pseudoobscura U0 gene. Four conserved sequence elements (E1, E2, E7 and E8) were identified when comparing the 5' flanking DNA of the D. melanogaster U0 gene to that of the D. virilis U0 gene, with three of these elements (E1, E2 and E8) also shared between the 5' flanking DNA of the D. virilis and D. pseudoobscura 5' U0 genes. The position and sequence of each conserved element is shown in 35 Drosophila pseudoobscura AH 133 2 rr’ MET M '347 ~343 -288 -197 -lO7 -34 +35 ' Drosophila melanogaster Canton 5 a 7 c s 4 or: 2”?» NET 5 §::!§§:3| -531-527 “509 -386 -242 -l38 -101 '31 +1 +34 Drosophila virilis 105l.0 8 7 2 11 NET -532 -339 +43 WW position W WW maniac 1 GGCATCATCAGTAT -a pGCATCAg'rCAorcAT -s GGCCTCATCGGMT -s 2 'rArAAAAcs -31 TATAAAAAGA -34 TATAAAAIG .24 3 CTTTCTACGAAAT 401 cmcrACIAMT 407 NP 4 TGGAGATAGAAA .242 TGGAGATAGAAA -2ss NP 5 ' rcrcrAAAAATrA ~3se TCTGTAGAAAITTA .197 NP 6 TTGTGAAATA -509 1769er .347 NP 7 TAATGTTAT .527 NP TAATGTTAT 4339 a GAAATAATGT .531 GAMTAGTGT ~343 MMTAQTGT ~532 Figure 7. The relative positions and the sequences of the conserved elements in the 5' flanking DNA of the U0 genes of D. melanogaster, D. pseudoobscura and D. virilis. E1 matches the consensus transcription initiation site for Dros0phila genes (Hultmark et al., 1986). E1 in the D. melanogaster U0 5' flanking DNA has been experimentally determined to be the U0 transcription start site (Figure 2, Chapter One). E2 corresponds to a consensus TATA box sequence (Corden et al., 1980). The DR element is present only in the D. melanogaster U0 gene and is similar in sequence to a proposed 20- hydroxyecdysone receptor binding element (Pongs, 1988). 36 Figures 7 and A5. Two of the elements, E1 and E2, are in similar positions relative to the transcription start sites of the D. melanogaster, D. pseudoobscura and D. virilis U0 genes. E1 matches the Drosophila transcription start consensus sequence (Hultmark et al., 1986) and contains the nucleotide that has been identified for D. melanogaster (Figure 1) and inferred for D. pseudoobscura and D. virilis (Figure A5) to be the U0 transcription initiation site. E2 matches the TATA box consensus sequence (Corden et al., 1980) and is at position -31, -34 and -24 with respect to the transcription start site of the D. melanogaster, D. pseudoobscura and D. virilis U0 genes. Other than E1 and E2, the sequences of the evolutionary conserved elements, do not correspond to any cis-regulatory element reported to date. 0. Discussion An interspecific DNA sequence comparison of the U0 gene from different Drosophila species was chosen as the starting point to identify Drosophila U0 protein structure/function relationships and Drosophila UO cis-regulatory elements. Due to the short length of the U0 gene, interspecific comparisons of U0 were tractable. A sufficient period of time has transpired since the divergence of D. melanogaster, D. pseudoobscura and D. virilis that only sequence with functional importance should be conserved. Approximately 78% of the expected third-codon-position changes for alanine, glycine, proline, threonine and valine, when corrected for codon bias, have changed between D. melanogaster and D. pseudoobscura and 100% between D. meIanogaster and D. virilis U0 genes. This amount of third-codon-position change found in the Drosophila UO genes is consistent with observations from comparisons of the Adh (Schaeffer and Aquadro, 1987), Garf (Henikoff and Eghtedarzadeh, 1987), hsp82 (Blackman and Meselson, 1986) and th (Riley, 1989) genes of 37 D. melanogaster and D. pseudoobscura and the hsp82 (Henikoff and Eghtedarzadeh, 1987) gene of D. melanogaster and D. virilis. 1. Deduced amino acid sequence comparisons of D. melanogaster, D. pseudoobscura and D. virilis U0. There is 72% identity among the deduced amino sequences of D. melanogaster, D. pseudoobscura and D. virilis U0 (Figures 5 and Table A2). The amino terminal extension of DroSOphiIa melanogaster U0, not found in vertebrate or plant U0 (Figure 4), is also present in D. pseudoobscura and D. virilis U0. Perhaps this amino extension has a functional role specific for Drosophila U0. The histidine residues conserved among D. melanogaster, vertebrate and plant U0, may be involved in copper binding and were discussed in Chapter One. The carboxy terminal tripeptide sequence (SHL) of U0 is found in all three species of Drosophila and may be involved in peroxisomal targeting of U0. This issue was discussed in Chapter One. The variable region of Drosophila UO, from D. melanogaster U0 amino acid positions 138 to 169 (Figure 5), is coincident with a region not present in the vertebrate and plant U0 (Figure 4). This variable region may represent evolutionarily unconstrained sequence. 2. Conserved sequence elements in the 5’ flanking DNA of the D. meIanogaster, D. pseudoobscura and D. virilis U0 genes. In addition to tentatively identifying structure/function relationships of U0 by molecular evolutionary comparisons, a second goal of the interspecific sequence comparisons was to determine the location and sequence of putative D. melanogaster U0 cis-acting regulatory elements which may be responsible for the timing, tissue-specific expression and level of U0 gene expression during development. Depending on the particular gene, cis-acting regulatory 38 elements may reside close to (Raghavan et al., 1986; Martin et al., 1989; Cereghini et al., 1987) or far from (Giangrande et al., 1987; Johnson et al., 1989; Bergson and McGinnis, 1990) the coding region as well as within an intron (Bingham et al., 1988; Bruhat et al., 1990; Kassis, 1990) and may be as small as 8 to 14 bp (Berg and von Hippel, 1988; Karim et al., 1990). Well characterized consensus cis-regulatory elements have been compiled for several viral, mammalian, Drosophila and plant genes (Jones et al., 1988; Wingender, 1988; Biggin and Tjian, 1989). Since there are no obvious sequence landmarks or rules for predicting the locations of uncharacterized cis- regulatory elements, the location and assignment of function to cis-regulatory elements and identification of the transcription factors interacting with a particular gene can be a difficult task. An interspecific sequence comparison is a high resolution method facilitating the location and sequence of cis-acting regulatory elements. Using computer based homology matrix analyses (Pustell and Kafatos, 1984; Martinez-Cruzado et al., 1988), comparisons are made between the DNA sequence flanking the coding region of homologous genes from different species. Naturally occurring mutations will eliminate ancient homologies where nucleotide sequences are not under tight functional constraints, while the nucleotide sequences of some cis-regulatory elements are conserved. Interspecific sequence comparisons have been made for several Drosophila genes to identify potential functional regions of proteins and putative cis—acting regulatory elements (Table A5). For some of these Drosophila genes, candidate cis-acting regulatory elements were identified as evolutionarily conserved sequence. Subsequently, experimental evidence demonstrated that many of the small evolutionarily conserved sequence motifs in the flanking DNA are functional regulatory cis-elements that bound particular trans-acting factors (Table A5). For example, a 16 bp element conserved within the 5’ flanking DNA 39 of the Drosophila dopa decarboxylase (Ddc) gene of D. meIanogaster and D. virilis has been shown to be essential for Ddc transcription in the central nervous system and binds to a factor present in embryonic nuclear extracts (Bray et al., 1988; Bray et al., 1989). ‘ For several reasons a molecular evolutionary comparison probably does not identify all cis-acting regulatory elements of a particular gene. Cis—regulatory elements smaller than 7 bp are not easily discovered in sequence comparisons. It is also possible that a particular trans-acting factor may bind to cis-elements of strikingly different sequence and still bring about the same regulatory effect. There are examples in which regulatory factors bind dissimilar sequence elements (Pfeifer et al., 1987; Baumruker, 1988). A particular cis-acting sequence and trans-acting factor of a given species could have experienced co-evolution such that their counterparts in the other species no longer share sequence similarity. For these reasons, a molecular evolutionary comparison should be coupled with complementary methods, such as deletion analyses and fine structure mapping with point mutations, when identifying the regulatory elements of a gene. As for the Drosophila U0 gene, molecular evolutionary sequence analyses revealed several potential candidate cis-regulatory elements (Figures 7 and A5). The elements E1 and E2 represent the transcription start site and the TATA box of the U0 genes, respectively. The sequence of the conserved elements E3 to E8 do not match the sequence of any characterized cis- regulatory element. The functions of these elements, if any, cannot currently be discerned. From P-element mediated germ line transformation of a UO-lacZ fusion gene (Chapter 3), the DNA sequence 826 bp upstream and 350 bp downstream of the D. melanogaster U0 transcription start site has been shown to be sufficient for the appropriate D. melanogaster U0 temporal and spatial pattern of 4O expression. The elements in the 5' flanking DNA conserved between D. melanogaster, D. pseudoobscura and D. virilis reside within this DNA sequence and are likely to be cis-acting regulatory elements of the D. melanogaster UO gene. The location of these conserved elements has been used as one guide for selecting regions to be deleted from the the 5’ flanking DNA of a UO-lacZ fusion gene (Chapter Three). lll. CHAPTER THREE: Identification of the location of regulatory regions required for D. melanogaster U0 gene expression. A. Introduction Appropriate expression of most eukaryotic genes depends on the interaction of trans-acting regulatory factors and cis-acting DNA sequences. With the ability to clone a particular gene from an organism, make alterations in the regulatory region of that gene, introduce the altered gene into the genome of the organism and assay the effects of the alterations on transcription of the gene, many of the mysteries concerning gene regulation have been revealed. This approach has been used to understand the regulation of several genes of mice, Drosophila and plants (Garbadebian et al., 1986; Chung and Keller, 1990; Ohshima et al., 1990; Todo et al., 1990; Vassar et al., 1991). Studies using in vivo functional analysis of gene regulation have revealed generalities and continue to uncover novelties with regard to the location and sequence of cis-regulatory elements. With the exception of house-keeping genes, cis-acting regulatory sequences are frequently comprised of general promoter elements, the transcription start site and TATA box, as well as other factor-binding sites that may be relatively close or at great distances from the transcription start site (Reghavan, 1986; Giangrande et al., 1987; Bergson and McGinnis, 1990). This chapter concentrates on the localization of cis-regulatory elements for the D. melanogaster UO gene and includes the application of a newly formulated method of deletion analysis to the 5’ region of the U0 gene. The method involves the site-specific removal of stretches of DNA as large as 235 bp in length. This technique is expedient, reliable and generally applicable to 41 42 the study of any region of DNA for which the sequence is known. B. Results Determination of the location of the regulatory region of the D. melanogaster U0 gene was possible because the D. melanogaster U0 gene is structurally ' compact with a transcribed region of approximately 1.4 kb. Moreover, there is an efficient germ line transformation system available for Drosophila (Rubin and Spradling, 1982). When reintroduced into the genome of D. melanogaster, most D. melanogaster genes with a sufficient amount of flanking DNA, are appropriately regulated (Goldberg et al., 1983; Richards, 1983; Scholnick et al., 1983; Spradling and Rubin, 1983). Transformed stocks are stable for many years and analyses of gene expression can be performed throughout development and on large numbers of isogenic animals. For these reasons, different P-element plasmids containing the U0 gene were constructed and transformed into D. melanogaster using P-element transformation (Rubin and Spradling, 1982). 1. U0 flanking DNA sequence sufficient for appropriate U0 gene expression. P[(w+A)DmU0PstI], a P-element construct containing the 3.2 kb Psfl fragment which includes the U0 gene from the sod1 strain (Figure A1), was inserted into the P-element vector CaSpeR (Pirrotta, 1988) and integrated into the genome of D. melanogaster using P-element mediated transformation described by Rubin and Spradling (1982) with some modifications. A detailed description of the transformation procedure used here is found in Materials and Methods (Appendix A). This 3.2 kb Pstl fragment contains the U0 gene with 826 bp of 5’ flanking DNA and approximately 1200 bp of 3’ flanking DNA. By performing the appropriate genetic crosses of the P-element transformants (see 43 Materials and Methods, Appendix A), five independent lines were made homozygous for a single P-element insertion. An example of a genomic Southern analysis performed to determine the number of P-element integrations is shown in Figure A6. In vivo transcription of the U0 transgene of construct P[(w'*A)DmUOPstl] was examined by Northern analysis. An oligonucleotide specific for message transcribed from the U0 transgene of construct P[(w“'A)DmUOPstI] (Chapter One, Materials and Methods and Table A6) detected UO mRNA from transformed third instar larvae and adults but not from pupae on Northern blots. ln wild type DrOSOphiIa melanogaster, UO expression is confined to the Malpighian tubules. Northern analysis of RNA from hand-dissected Malpighian tubules of third instar larvae and adult transformants compared to RNA extracted from the remaining tissues demonstrated that U0 mRNA from the U0 transgene is only expressed within the Malpighian tubules. Shown in Figure 8 is the Northern analysis of a transformed line, designated 1M1iD, possessing the U0 transgene of construct P[(w"'A)DmUOPstI]. Four additional independent transformed lines were analyzed in an identical fashion with the same results as in Figure 8. A list of all the U0 P-element constructs used in this study, the different transformed lines and the results of functional analysis of the U0 transgenes is shown in Table A7. 2. U0 flanking DNA insufficient for appropriate UO gene expression. In order to determine if 174 bp of 5' flanking DNA and approximately 300 bp of 3' flanking DNA would provide appropriate UO transgene expression, a second P-element plasmid designated P[(hspw+)DmUOSpelStuI] was constructed. The SpeI-Stul restriction fragment containing the U0 gene _i 55"5-5' -l -I Em'b ('0 3': kb $23o<33<0"... 3.58 w P¢<9n P2<8 P1407 Psi—GB 1‘.3 nu.4 433-7 405 2 40m 1 kb U0 mRNA . $1.4 kb 2.37— 1 35'- -1 .8 kb ras mRNA H. 0‘...“ Figure 16. 77 the developmental expression of the D. virilis U02 transgene. A low level of D. virilis U02 transgene expression was detected in the pupae, third instar larva and adult. Furthermore, the majority of D. virilis U02 mRNA was present in the tissue remaining after dissection and removal of the Malpighian tubules. A representative Northern analysis of one transformed line, 4F130, is shown in Figure 16C. Detection of U0 mRNA from the D. virilis U02 transgene by Northern analysis required an eight-fold longer autoradiographic exposure time than for detection of U0 mRNA from the D. virilis U01 transgene. Data presented elsewhere indicates that as a result of the site of unequal recombination giving rise to the U0 duplication event, the D. virilis U02 gene lacks upstream sequences containing UO cis-acting regulatory elements. As a consequence, the D. virilis U02 gene has an altered temporal and tissue- specific pattern of expression. We attribute the low level of U0 mRNA detected in the adult of D. virilis strain 1051.0 (Figure 14) to transcription from the D. virilis U02 gene. 0. Discussion The genetic bases for interspecific differences in the regulation of several Dros0phila genes have been investigated by creating interspecific hybrids or P- element transformants. Bray and Hirsh (1986) have identified differences in the expression of the D. melanogaster and D. virilis dope decarboxylase (Ddc) genes. The D. virilis Ddc enzyme activity profile shows a 3-4 fold increase between the early third instar larval and late third instar larval stages while the D. melanogaster Ddc enzyme activity profile has a 15-30 fold increase during this same developmental period. In addition, the D. virilis Ddc gene is expressed at higher levels than the D. melanogaster enzyme in tissues other than the cuticle and central nervous system. This tissue-specific pattern and 78 quantitative level of expression of the D. virilis Ddc gene persisted when integrated into the D. melanogaster genome. There are other examples of species differences in gene expression attributed to cis-acting changes. The alcohol dehydrogenase (Adh) gene of D. melanogaster is expressed in the fat body, hindgut, rectum, Malpighian tubules and male genital disc derivatives while the Adh-2 gene of D. mulleri is only expressed in the adult fat body, hindgut and rectum. Expression of the D. mulleri Adh-2 transgene in the D. melanogaster genome coincides with the tissue-specific pattern of the D. mulleri Adh-2 gene (Fischer and Maniatis, 1986). Brennan and Dickenson (1988), Brennan et al. (1988) and Wu et al. (1990) have shown that there are tissue-specific differences in the expression of the Adh gene of D. hawaiiensis, D. affinidisiuncta and D. grimshawi compared to expression of the Adh gene of D. melanogaster. The majority of these differences also behave in a cis-dominant fashion when the Adh genes of D. hawaiiensis, D. affinidisiuncta and D. grimshawi are separately introduced into the D. melanogaster genome. The esterase-5 gene of D. pseudoobscura is expressed in the eyes and the hemolymph, whereas, the D. melanogaster homologue, esterase-6, is expressed in the ejaculatory duct. The D. pseudoobscura esterase-5 gene retains its sex- and tissue-specific expression when transformed into a D. melanogaster genome (Brady and Richmond, 1990). For the examples summarized here, interspecific regulatory variation is clearly cis-dominant since the species-specific patterns of expression of the transgenes are maintained in the presence of D. melanogastertrans-acting factors. There are reports of species-specific quantitative differences in gene products that are due to evolutionary changes of trans-acting regulators. Csink and McDonald (1990) discovered differences in the amount of copia mRNA among 37 populations world wide of D. melanogaster, D. simulans and 79 D. mauritiana. There were also intraspecific differences in the level of copia mRNA within D. melanogaster populations that showed no correlation with the copy number of copia elements. Using interpopulation hybrids, variation in the amount of copia mRNA among natural populations of D. melanogaster, D. simulans and D. mauritiana was attributed to differences in trans-acting controlling factors. In addition, quantitative differences in the amount of Adh activity and cross-reacting material in D. melanogaster and D. simulans have been suggested to be a consequence of trans-acting modifiers that alter the rate of translation of Adh mRNA or the stability of Adh protein (Laurie et al., 1990). There are also a few examples in which species-specific expression of a gene may be the result of both cis-acting and trans-acting genetic differences (Dickenson, 1980; Brennan and Dickenson, 1988; Cavener et al., 1989; Krasney et al., 1990). One of the most clearcut examples is that concerning the regulation of the aldehyde oxidase gene of D. grimshawi and D. formella, two Hawaiian picture-winged Drosophila (Dickenson, 1980). Aldehyde oxidase (AD-1) is present in D. grimshawi third instar larvae at moderate levels in the fat body and barely detectable levels in the salivary gland and carcass. In contrast, A0-1 is present at high levels in D. formella third instar larval salivary glands but at a barely detectable levels in the fat body and carcass. When hybrids were made between D. grimshawi and D. formella, the hybrids had barely detectable levels of A0-1 in the salivary gland and fat body, implying negative regulation due to trans-acting controlling factors supplied by D. grimshawi. However, the hybrids had high levels of A0-1 in the carcass. This AO-1 found in the carcass of the hybrids migrated electrophoretically as the form distinctive of D. formella. This implies that the expression of A01 in the carcass is a cis- acting property of the D. formella aldehyde oxidase gene. As I have shown, the D. melanogaster, D. pseudoobscura and D. virilis UO genes have different temporal expression patterns (Figures 8, 13, 14 and A7). 80 Two non-mutually exclusive hypotheses to account for the different temporal patterns of U0 gene expression among these three species are: (1) the differences in the temporal pattern of expression of the U0 gene are due to changes in the cis-acting elements of the U0 gene and (2) the differences in the pattern of expression of the U0 gene are due to changes in the temporal profile of one or more trans-regulators required for U0 gene transcription in the third instar larval and adult stages. The first hypothesis would predict that the species-specific expression of D. pseudoobscura or D. virilis UO transgenes would persist when integrated into a D. melanogaster genome. The second hypothesis would predict that the D. pseudoobscura and the D. virilis UO transgenes would be expressed in a temporal pattern identical to the D. melanogaster UO gene, provided that the D. melanogaster UO trans-acting factors recognize the cis-regulatory sequences of the D. pseudoobscura and D. melanogaster UO genes. When the D. pseudoobscura UO gene and the D. virilis U01 gene were transformed into a D. melanogaster genome, a temporal pattern identical to that of the D. melanogaster UO gene was observed (Figures 153 and 16B), consistent with the second hypothesis. There is an alternative, but more complex explanation for the D. melanogaster UO-like temporal expression pattern displayed by the D. pseudoobscura UO transgene and the D. virilis U01 transgene in a D. melanogaster genome. This alternative explanation requires the presence of distant negative regulatory elements involved in UO gene repression. It is possible that a cis-element required for repression of the D. pseudoobscura UO gene during the third instar larval stage resides outside the boundaries of the D. pseudoobscura UO flanking DNA included in the P-element construct P[(w"’A)DpUORl] (Figure 15A). Similarly, an adult cis-acting regulatory element involved in repression of the D. virilis U01 gene would have to reside at a 81 distance greater than 3200 bp, the 5’ limit of the DNA present in the P-element construct P[(w+A)DvUO1Pstlj (Figure 16A). There are examples of Drosophila genes with distant cis-acting regulatory elements (Giangrande et al., 1987; Johnson et al., 1989; Bergson and McGinnis, 1990). However, on the basis of expression of a UO-lacZ fusion gene, 826 bp of 5' U0 flanking DNA and the first 350 bp of the D. melanogaster U0 transcribed region are sufficient for appropriate regulation of the D. melanogaster UO gene when reintegrated into the D. melanogaster genome (Figure 10). If the D. pseudoobscura and the . D. virilis UO cis-regulatory elements are also within approximately 800 bp of 5' flanking DNA they would have been included in the P-element constructs employed here. Additional P-elements could be constructed that contain the D. pseudoobscura and D. virilis U0 genes with additional 5’ and 3’ flanking DNA, transformed into D. melanogaster and analyzed for U0 transgene expression. The problem with such an approach is that there are limitations to the amount of DNA capable of being efficiently transformed into D. melanogaster via P-element transposition, with an upper limit of approximately 50 kb (Spradling, 1986). Additional evidence is needed to unequivocally demonstrate that the species-specific temporal patterns of U0 expression are due to changes in trans-acting regulatory genes and not to the exclusion of U0 cis-regulatory elements form the P-element constructs. A test, when feasible, would be to perform reciprocal P-element transformations of the U0 genes with D. melanogaster, D. pseudoobscura and D. vin'lis. The D. melanogaster U0, D. pseudoobscura U0 and D. virilis U01 transgenes in the D. virilis genome would be predicted to be expressed only during the third instar larval stage, identical to the developmental pattern of the endogenous D. virilis U01 gene. Similarly, the D. melanogaster U0, D. pseudoobscura U0 and D. virilis U01 82 transgenes in the D. pseudoobscura genome would be expected to be expressed only in the adult. Reciprocal P-element transformations of D. pseudoobscura and D. virilis are currently not feasible for several reasons. Although successful transformations of a few Drosophila species other than D. melanogaster have been reported, none have involved D. pseudoobscura and D. virilis (Brennan et al., 1984; Scavarda and Hartl, 1984; Daniels et al., 1985; Daniels et al., 1989; Laurie et al., 1990). It is not known whether the promoters of the D. melanogaster reporter genes used for screening transformants are recognized by D. virilis transcription factors and whether any of the D. melanogaster reporter genes would be expressed in D. virilis at levels sufficient for screening. Once the genes for the U0 transcription factors are cloned, evolutionary changes in their expression patterns can be examined in D. melanogaster, D. pseudoobscura and D. virilis. A change in the temporal pattern of expression of a trans-acting regulatory gene might be expected to influence the expression of more than one gene since many transcription factors regulate more than one target gene (Costa et al., 1988; Hardon et al., 1988; Hoey et al., 1988; Rupperl et al., 1990). If the species differences in the developmental pattern of the U0 gene are a consequence of evolutionary changes in the temporal expression or availability of particular trans-acting factors essential for U0 gene expression, it is possible that, in addition to U0, other genes are targets of the same transcription factor(s) and have also experienced a change in regulation since the divergence of D. virilis, D. pseudoobscura and D. melanogaster. Although the temporal expression pattern of the D. melanogaster UO, D. pseudoobscura U0 and D. virilis UO1 genes have diverged, the Malpighian tubule-specific expression has been conserved. D. pseudoobscura and D. virilis diverged from D. melanogaster approximately 35 million and 60 million years ago, respectively (Beverley and Wilson, 1984). The D. pseudoobscura 83 U0 and the D. virilis U01 genes transformed into the genome of D. melanogaster are expressed only in the Malpighian tubules. This result implies that the cis-acting elements and the transcription factor(s) required for restricting UO gene expression to the Malpighian tubules have been conserved among these three Drosophila species. it is interesting to note that although the D. pseudoobscura and D. virilis U01 transgenes were expressed in the third instar larva and adult Malpighian tubules of D. melanogaster, there is very little sequence similarity among the 5' flanking DNA of the D. melanogaster, D. pseudoobscura and D. virilis U01 genes (Figures 7 and A5). Given these results, it seems likely that the U0 gene is under the control of cis-acting elements that have small core binding sites or that are poorly conserved at the nucleotide level. Only by performing a thorough mutagenesis analysis of the regulatory region of the U0 gene as described in Chapter Three, will the sequences of such elements be revealed. CONCLUSION There is evidence that evolutionary changes in cis-acting regulatory elements of a particular gene and evolutionary changes in the trans-acting factors have brought about species differences in gene regulation (Bray and Hirsh, 1986; Fischer and Maniatis, 1986; Brady and Richmond, 1990; Csink and McDonald, 1990; Laurie et al., 1990). However, such differences are often subtle variations of spatial patterns or quantity of mRNA or protein product (Bray and Hirsh, 1986; Fischer and Maniatis, 1986; Laurie et al., 1990). Minor variations in gene regulation make data concerning gene expression of interspecific hybrids and P-element transformants difficult to decipher. In contrast, the differences in the temporal pattern of U0 expression are overt with easily discernable results of interspecific P-element transformations. The Drosophila UO gene has proven to be an excellent and unique model system for studying evolutionary changes in gene regulation. The data collected thus far support the idea that the pattern of U0 regulation among D. melanogaster, D. pseudoobscura and D. virilis is different as a consequence of a change in the temporal pattern of one or more trans-acting regulatory factors. Additional tests are needed to unequivocally demonstrate that an evolutionary change in a U0 trans-acting regulator has occurred. An obvious first step to understand the differences in U0 regulation of these three Dros0phila species at the molecular level would be, for example, to identify UO trans-acting factors which are expressed in D. melanogaster third instar larvae and adults but only in D. pseudoobscura adults and only in D. virilis third instar larvae. The identification of the U0 trans-acting factors could be accomplished once a thorough analysis of the U0 cis-acting regulatory elements has been 84 85 completed. This analysis would include the identification of the base pairs within UO cis-acting regulatory elements which are essential for the binding of U0 transcription factors. The small size of the U0 gene makes mutation analysis coupled with in vivo functional testing possible and expedient. The D. melanogaster UO gene has a transcribed region of approximately 1.2 kb and a regulatory region which extends no more than 826 bp 5’ of the transcription start site. The D. pseudoobscura U0 and D. vin'lis U01 genes are also structurally compact and could be subjected to a similar type of analysis to identify their UO cis- acting regulatory sequences. In an attempt to obtain clues to the location and sequence of the U0 cis- acting regulatory elements, an interspecific sequence comparison of the flanking DNA of the U0 gene from D. melanogaster, D. pseudoobscura and D. virilis was performed. Several elements were conserved between D. melanogaster U0 and D. pseudoobscura U0 and between D. melanogaster U0 and D. vin'lis U0, but only one conserved element, discounting basal promoter elements (T ATA box and transcription start site), was conserved among all three species. It was surprising to discover that the D. melanogaster U0, D. pseudoobscura U0 and D. virilis U01 genes did not have several conserved elements in 5' regulatory regions of all three genes since the U0 genes from these three Drosophila species are all expressed exclusively in the Malpighian tubules. Moreover, the D. pseudoobscura U0 and D. vin'lis U01 transgenes were expressed in a pattern identical to the D. melanogaster U0 gene. There are several explanations that could account for so few conserved putative UO cis-acting regulatory elements among these three species: (1) the cis-acting elements of the U0 gene may consist of small core of binding sites which were missed in the similarity search, (2) the important sites for binding 86 transcription factors within a U0 cis-acting element may be noncontiguous, (3) a particular UO transcription factor may bind a functionally equivalent U0 cis- acting element with a dramatically different sequence among these three species, and (4) only a single cis-acting control region of the U0 gene, in addition to basal promoter elements, is required for the complex temporal and tissue-specific pattern of expression of the U0 gene. The analysis of the U0 gene presented in this thesis is the most clearcut case demonstrated to date of species differences in regulation attributable to changes in trans-acting regulatory factors. The U0 gene of Drosophila will continue to be a valuable paradigm for studying the evolution of gene regulation. APPENDIX A Materials and Methods APPENDIX A: Materials and Methods 1. Drosophila stocks. The Drosophila stocks and their phenotypes used in this study are listed in Table A1. Drosophila virilis strains 1051.48 and 1051.1 were obtained from J.S. Yoon of the the National Drosophila Species Resource Center, Bowling Green, Ohio. The Drosophila pseudoobscura strain AH133 and the Drosophila melanogaster white deficient strain Df(1)w,y57023(2) were gifts of W. Anderson (University of Georgia, Athens) and V. Pirrotta (Baylor University, Houston) respectively. All stocks were kept at 25°C or at room temperature with the exception of the temperature sensitive mutant ecd1(Lepesant, 1978) which was reared at 19°C. Drosophila stocks were maintained on standard media (188.64 9 sucrose, 29.1 g brewers yeast, 185.6 g cornmeal, 24 g carageenan per 2230 ml water and 15.2 ml of propionic acid as a preservative) supplemented with active yeast . 2. DNA isolation. a. Large scale plasmid DNA preparation. This protocol is a modification of the plasmid purification procedure described by Birnboim and Doly (1979). 1. Grow 10 ml overnight culture of the bacterial strain harboring the plasmid of interest in LB media (10 g bactotryptone, 5 g yeast extract and 10 g NaCI 87 88 per liter) supplemented with the appropriate antibiotic. 2. Add 10 ml overnight culture to one liter of M9CA media and periodically monitor 00. 3. When A500 = 0.4 to 0.5 add 5 ml of chloramphenicol (34mglml in EtOH). 4. Amplify plasmid overnight (12-16 hours) at 37°C with constant shaking. 5. Chill cells 5 minutes on ice and centrifuge in an lEC Centra-7R at 2,800 rpm for 40 minutes at 4°C. Collect pellet. 6. Pool cells in 40 ml of wash buffer. 7. Centrifuge at 8,000 RPM for 10 minutes in JA21 at 4°C. 8. Resuspend in 3 ml of fresh lysozyme buffer, then add 1 ml of fresh lysozyme buffer containing 8 mg of lysozyme. 9. Incubate 0°C for 30 minutes. 10. Add 8 ml of alkaline SDS. Incubate 0°C for 10 minutes. 11. Add 6 ml of 3 M NaOAc, pH 4.8. Mix by inversion. Incubate 0°C 1 hour. 12. Centrifuge at 17,000 RPM in JA21 for 20 minutes at 4°C. 13. Remove supernatant and place in 40 ml centrifuge tube. Add 1 volume of isopropanol to supernatant and precipitate at -20°C overnight or at -70°C 1 houn 14. Centrifuge at 10,000 RPM for 15 minutes at 4°C. 15. Pour off supernatant, dry pellet in dissector, resuspend in 4 ml of TE and add1 ml of1 M NaCl. 16. Add 10 ml of EtOH. Precipitate overnight at -20°C or 1 hour at -70°C. 17. Centrifuge at 10,000 RPM for 15 minutes at 4°C, pour off supernatant and dry pellet under vacuum. 89 18. Resuspend pellet in 15 ml of TE (10 mM Tris, 1mM EDTA, pH 7.5). 19. Add 15.9 g CsCl and 0.75 ml of ethidium bromide (10mglml). 20. Transfer to 25 ml Oakridge tube. Centrifuge using Beckman Tl50.2 at 33K for 36 to 48 hours at room temperature. 21. Collect plasmid band. 22. Extract with H20 saturated sec-butanol until pink color is lost. 23. Dialyze against one liter of TE for 3 days with several changes of buffer. Solutions for Plasmid Prep: 84188115 6 grams Na2HPO4 3 grams KH2PO4 0.5 grams NaCl 1 gram NH4CI dH20 to 1 liter pH to 7.4 We 1000 ml M9 Salts 25 ml 20% glucose 25 ml 20% Casamino acids 2 ml 1% thiamine 1 ml 1 M MgCl2 0.1 ml1 M CaCl2 4 ml 25 mglmi stock Ampicillin (or appropriate amount of a different antibiotic) 90 WUOmM Tris, pH 8, 1 mM EDTA) 10 ml 0.1 M Tris pH 8 0.4 ml 0.25 M EDTA pH 7 18 grams sucrose dH20 to 100 ml Mimmfepafe fr92:7") 225 ml 20% glucose 200 ml 0.25 M EDTA 125 ml1 M Tris pH 8 01120 10 5 ml Alkaline SQS (prepare fresh) 2 ml 1.0 N NaOH 1.0 ml 10% see dH20 to 10 ml b. Small scale plasmid DNA isolation. This procedure is used to rapidly obtain approximately one to two micrograms of plasmid DNA starting from a 1.5 ml saturated bacterial culture. 1. WM: pick a single plaque on a plate with a sterile toothpick and place in 1.5 ml of 2XTY (2XTY is 16 g bactotryptone, 10 g yeast extract and Sg NaCI per liter of water) media containing a 1:100 dilution of a saturated culture of an E. coli strain capable of being infected by M13 and grow for 12 to 16 hours at 37°C with shaking. 91 E. coli MV1193 was used as the M13 host. Egr plasmid isglatign other than M13: inoculate 1.5 ml of LB media (10 g bactotryptone, 5 g yeast extract and 10 9 NaCl per liter) supplemented with the appropriate antibiotic with a single colony of bacteria harboring a plasmid and grow for 12 to 16 hours at 37°C with shaking. 2. Transfer culture to a 1.5 ml microtube and centrifuge for 10 seconds. 3. Discard supernatant (retain supernatant for M13 phage DNA isolation, see below) and wash pellet with 200 pl of TE pH 8.5. 4. Centrifuge for 10 seconds and discard the supernatant. 5. Resuspend the pellet in 25 pl of Solution I (8% sucrose, 50 mM Tris, 75 mM EDTA, pH 8.5). 6. Add 5 pl of 10 mglml lysozyme in 10 mM TE, pH 8.5. 7. Incubate 3 minutes at room temperature. 8. Add 25 pl of Solution H (8% sucrose, 50 mM Tris-HCI, 75 mMEDTA, 1% triton, pH 8.5) and vortex. 9. Place the tube in a boiling water bath for exactly 45 seconds and quickly chill on wet ice. 10. Add 250 pl of Solution III (0.5 M NaCl, 10 mM Tris, pH 8.0) and mix by inversion. 11 . Centrifuge for 10 minutes. 12. Remove pellet with toothpick and discard pellet. 13. Add 250 pl of cold isopropyl alcohol and precipitate at -20°C for 1 hour. 14. Centrifuge for 10 minutes at 4°C. 15. Wash pellet with 200 pl of 70% EtOH. 16. Centrifuge for 10 minutes at 4°C. 17. Vacuum dry pellet and resuspend in 20 pl of TE. 92 c. Small scale M13 phage DNA isolation. M13 phage DNA for sequencing and site-directed mutagenesis was prepared according to the following protocol. 1. Obtain supernatant form step 3 of small scale plasmid DNA isolation procedure (above). 2. Centrifuge for 10 minutes and transfer supernatant to a new tube. 3. Centrifuge for 10 minutes and transfer supernatant to a new tube. 4. Add 300 pl of 20% PEG, 2.5 M NaCI to supernatant and incubate at room temperature for 15 to 30 minutes. 5. Centrifuge for 10 minutes and discard supernatant. 6. Centrifuge pellet again for 10 minutes and discard supernatant. 7. Resuspend the pellet in 160 pl of TES (20 mM TrisHCl, 10 mM NaCI, 0.1 M EDTA, pH 7.5). 8. Add 160 pl of phenol/chloroform (1:1) and vortex for 5 minutes. 9. Centrifuge for 5 minutes and transfer 140 pl of the top layer to a new tube. 10. Add 140 pl of chloroform and vortex for 5 minutes. 11. Centrifuge for 5 minutes and transfer 120 pl of the top layer to a new tube. 12. Add 15 ul of 2.5 M sodium acetate and 250 pl of EtOH. 13. Precipitate at -20°C for at least 1 hour. 14. Centrifuge 10 minutes at 4°C. 15. Wash pellet with 200 pl of 70% EtOH. 16. Centrifuge for 10 minutes at 4°C. 17. Vacuum dry pellet and resuspend in 10 pl of TES. 93 3. Genomic DNA extraction from a small number of files. This protocol is a modification of the DNA extraction procedure of Bender et al. (1983). 1. Add 187 pl of grinding buffer (0.1 M NaCL, 0.2 M sucrose, 0.1 M Tris-HCL, 0.05M EDTA, pH 9.1), 3 pl DEPC (diethylpyrocarbonate) and 10 pl of 10% SDS to a 1.5 ml microcentrifuge tube. 2. Add 1 to 12 files and grind at room temperature with a glass pestle that fits snugly inside the tube. 3. Incubate at 65°C for 30 minutes. 4. Add 30 pl of 8 M potassium acetate and incubate at 0°C for 30 minutes. 5. Centrifuge for 10 minutes at 4°C. Transfer the supernatant to a new centrifuge tube and repeat the centrifugation. 6. Mix the supernatant with 250 pl of ethanol. Allow to stand at room temperature for 5 minutes. 7. Centrifuge for 10 minutes at 4°C. 8. Wash pellet 3 times with 70% ethanol, dry pellet under vacuum and resuspend DNA in 10 pl of TE QB wash pellet once with 70% ethanol, dry under vacuum, resuspend in 10 pl of TE and perform dot dialysis for 30 minutes. W: Float a Millipore dot dialysis membrane (#VMWP 01300) with the shiny side facing up in a petri dish or small container filled with TE. Carefully load the DNA sample onto the center of the membrane. Allow dialysis to occur for 30 minutes. Remove the sample from the membrane using a pipet. There may be a small loss or gain of sample volume during the dialysis. 9. Digest the entire DNA sample with 10 units of restriction enzyme (less may also work) for 3 hours and load the entire sample into a single lane of an agarose gel. 4. Southern analysis. DNA samples were electophoresed and blotted onto Hybond-N (Amersham) or Immobilon (Millipore) membrane according to Maniatis et al. (1982). Transferred DNA was crosslinked to the nylon membrane by ultravilolet irradiation. Southern blots was prehybridized at 42°C overnight in 50% formamide, 50 mglml sheared salmon sperm DNA, 0.1 M PIPES, pH 7.04, 0.1 M NaCl, 0.1% Sarkosyl, 0.1% Ficoll, 0.1% PVP-40 and 0.1% BSA. Modified prehybridization solution with 40% formamide and 10% dextran sulfate served as the hybridization mixture. Restriction fragments used as probes for Southern blots were size separated on agarose gels, isolated by eiectroelution (Maniatis et al., 1982) and further purified using an Elutip-D column (Schleicher and Schuell). DNA fragments were labeled with [a-32deATP to a specific activity of 1.3 x109 to 8.2 X108 cpmlpg by the method of Feinberg and Vogelstein (1984). Approximately 1X107 to 2 X107 cpm were added to 30 ml of hybridization solution and incubated with the blot at 42°C overnight. After hybridization, the blots were rinsed in 2XSSC, 0.05 % N-laurylsarkosine, 0.02% sodium pyrophosphate and washed at 50°C for two hours in two to four washes of 0.1XSSC, 0.05% N-laurylsarkosine, 0.02% sodium pyrophosphate. Blots were autoradiographed at -70°C with one screen. 95 5. Small scale total RNA isolation. This RNA isolation procedure was taken form Richards et al. (1983) with modifications of the volumes used. 1. 30 larvae, pupae or adults or 30 hand dissected Malpighian tubules and the remaining tissue are placed into 450 pl of extraction buffer prepared with DEPC treated water (3 M LiCl, 6 M urea, 10 mM NaOAc pH 5.0, 0.2 mglml heparin, 0.1% SDS) in a microcentrifuge and ground with a glass pestle. 2. Incubate on ice for one hour to overnight. 3. Centrifuge for 15 minutes at 4°C. 4. Wash the pellet with 100pl of wash buffer (4 M LiCl, 8 M urea). 5. Centrifuge for 15 minutes at 4°C. 6. Resuspend pellet in 100 pl of 0.1 M NaOAc, 0.1% SDS, pH 5.0 prepared with DEPC treated water. 7. Add 100 pl of phenol/chloroform (prepared according to Maniatis et al., 1982) and vortex for 20 minutes and centrifuge for 10 minutes. 8. Remove aqueous layer transfer to a new tube, add 100 pl of chloroform, vortex and centrifuge for 10 minutes. 9. Remove aqueous layer and transfer to a new tube. Adjust to 0.2 M NaOAc, pH 5.0 and add 2 volumes of EtOH. 10. Place at 20°C for 4 hours or longer, centrifuge for 15 minutes at 4°C, wash pellet with 80% EtOH and vacuum dry pellet. 11. Bring up in 10 pl of denaturing solution (250 pl formamide, 89 pl 37% formaldehyde solution) and load onto Northern gel as described below. 96 6. Northern analysis. RNA was recovered from ethanol precipitation (see above) by centrifugation for 15 minutes, washed with 70% ethanol, vacuum dried and dissolved in 74% formamide and 9.7% formaldehyde, denatured for 5 minutes at 65°C and loaded onto 1.2 % agarose, 6.66% formaldehyde gel in 1X MOPS (10X MOPS is 0.5 M MOPS, 0.01 M EDTA, pH 7.0). RNA was size-fractionated in agarose- formaldehyde gels by electrophoresis in 1X MOPS. After electrophoresis, the portion of a gel containing samples to be transferred to a nylon membrane was incubated for 30 minutes at room temperature in 0.15 M NaCI, 0.05 M NaOH with shaking and then in 0.15 M NaCI, 0.1 M Tris pH 8.0 for 30 minutes with shaking. RNA was transferred onto nylon membrane (Hybond-N, Amersham or Immobilon, Millipore) with 10XSSC and crosslinked to the membrane using ultravilolet irradiation. The portion of the gel containing RNA standards was incubated in DEPC treated water for 15 minutes at room temperature and then stained for 15 minutes in 200 ml of DEPC water with 10 pl of EtBr stock solution (10mglml). The gel was destained by soaking in DEPC treated water for 5 hours to overnight. DEPC treated water is prepared by adding 250 pl of DEPC to one liter of distilled water, incubating at room temperature overnight and then autoclaving. The Northern blots in Figures 8 and 9 were prehybridized and hybridized in solutions identical to those of Wood et al. (1985). The oligonucleotide 4 (T able A6), which detects UO mRNA transcribed from the U0 gene of the D. melanogaster and1 strain, but not from the U0 gene of the D. melanogaster Canton-S strain, was end-labeled to a specific activity of 1.3x108 cpm/mg with [y-3ZP] dATP using T4 polynucleotide kinase (Maniatis et al., 1982). After six hours of prehybridization at 42°C, the Northern blots were hybridized for 3 days 97 at 50°C in 20 ml of hybridization solution with 1.6x107 cpm of the end-labeled oligonucleotide. Since the oligonucleotide probe is 66% A+T, the Northern blots were washed at 50°C according to Wood et al. (1985) in the presence of tetramethylammonium chloride which raises the melting temperature of AT base pairs to that of GO base pairs. DNA restriction fragments, when used as probes for Northern analyses, were prepared according to those described for Southern analysis above and labeled to a specific activity of 3.7xio8 to 9.3x108 cpm/pg using [a-32deATP (Feinberg and Vogelstein, 1984). Northern blots were prehybridized, hybridized and washed according to procedures for Southern analyses described above. For the D. pseudoobscura and D. virilis U0 probes, Northern blots shown in Figures 15 and 16 were washed at 60°C rather than 50°C. By increasing the temperature of the wash solutions, D. pseudoobscura U0 and D. virilis U0 probes only hybridized to mRNA from the D. pseudoobscura and D. virilis UO transgenes, respectively, and not to the U0 mRNA transcribed from the endogenous D. melanogaster U0 gene. To verify the presence of RNA in lanes showing no autoradiographic signal after hybridization with a U0 probe, all Northern blots were stripped of the U0 probe by boiling for ten minutes in 0.1% SDS and reprobed with a Hindlll-Pstl fragment of the D. melanogaster ras gene (Mozer et al., 1985). The ras gene is expressed uniformly throughout development of D. melanogaster. Northern blots were autoradiographed at -70°C with one screen. 98 7. Sequencing. Restriction fragments containing the U0 transcription unit of D. melanogaster, D. pseudoobscura and D. virilis were cloned into the single stranded bacteriophage vectors M13mp18 and M13mp19 (Messing and Vieira, 1982) phagemid vectors puc119 (Vieira and Messing, 1987) or pBluescript (Stratagene). The sequencing reactions were performed according to Sanger et al. (1977) using [oi-35S]dATP and DNA polymerase I, Klenow (Bethesda Research Laboratories, BRL) and the universal primer (BRL; United State Biochemical, USB ) or a synthetic oligonucleotide primer complementary to the sequence internal to a cloned UO sequence (Table A6). After performing sequencing reactions with Klenow purchased from various sources, Klenow obtained from Bethesda Research Laboratories was found to be superior to the other sources under the conditions used here. Sequence ambiguities due to secondary structure of the cloned DNA were resolved by sequencing with AMV reverse transcriptase (BioRad), Taq polymerase (Strategene) or Sequenase (USB) or substituting 7-deaza-2'- deoxyguanosine-5'-triphosphate for deoxyguanosine-5'-triphosphate (Boehringer Mannheim, BM). When sequencing with Taq polymerase, Sequenase and reverse transcriptase the manufacturers’ protocols were used. The majority of the sequencing was performed with Klenow using the protocol found in BRL’s M13 Cloning/Dideoxy Sequence instruction Manual with modifications of the concentrations of the dideoxynucleotides. Each M13 subclone containing a U0 sequence greater than 100 bp was sequenced using high concentrations (final concentrations: ddATP, 1.5 X 10'5M; ddGTP, 1.5 x 10'4 M; ddCTP, 4.5 x 10 '5; ddTTP, 4 x 10'4 M) and low concentrations (final concentrations: ddATP, 6.6 X 10'6 M; ddGTP, 2 X 10"5 M; ddCTP, 99 3 x 10-5 M; ddTTP, 6.6 x 105) of dideoxynucleotides and the DNA was separated on 8% and 6% polyacrylamide sequencing gels, respectively. Many subclones were sequenced twice. Figures 1A, 3A and 4A show the sequencing strategies for the D. melanogaster, D. pseudoobscura and D. virilis subclones, respectively. All restriction sites used in constructing the subclones were verified by sequencing across the same restriction site present within other subclones. I thank Tom Friedman, Jean Burnett, Janice Moskowitz and Susan Lootens for making some of the M13 subclones used for sequencing. 8. S1 and mung bean nuclease mapping. To determine the transcription start site for the U0 gene, S1 and mung bean nuclease mapping were performed by end-labeling 1 pg of the genomic restriction fragment, HhaI-EcoRl (Fig. 2) with T4 polynucleotide kinase and [y- 32P1dATP (30000ilmmol) (Maniatis, 1982). The two strands of the 17s nucleotide end-labeled fragment were separated on a 6% sequencing gel and the strand complementary to UO mRNA, designated probe S (Figure 3c), was isolated. Total RNA was extracted with guanidine HCl from whole third instar larvae and adults (Krawetz and Anwar, 1985). Poly(A)+ RNA was isolated on an oligo(d‘l')-cellulose column (Pharmacia). Approximately 1X105 cpm of probe 8 and 20 pg of poly(A)+ RNA from Ore-R third instar larvae, 10 pg poly(A)+ RNA from Ore-R adults (data not shown) or 10 pg of poly(A)"' RNA from ry212 hour adults were dissolved in 40 mi of 80% formamide, 40 mM PIPES pH 6.4, 400 mM-NaCI and 1 mM EDTA, boiled for 10 minutes and allowed to hybridize for approximately 12 hours at 42°C. Following the annealing step, 350 pl of S1 100 reaction buffer (Davis et al., 1986) were added to each sample along with 50 units of S1 nuclease (Boehringer Mannheim) or 150 units of mung bean nuclease (Pharmacia) and the samples were incubated at 37°C for one hour. The concentration of S1 and mung bean nuclease was optimized to achieve nearly complete digestion of the single stranded DNA while minimizing the slower rate of digestion of the DNAzRNA duplex. Mung bean nuclease is reported to create fewer digestion artifacts than some preparations of S1 nuclease (Murray, 1986). After S1 or mung bean nuclease treatment, the reaction was phenol/chloroform extracted, ethanol precipitated, dissolved in 6 pl of standard DNA sequencing loading dye, denatured for 3 minutes at 90°C and loaded into single lanes of a 6% sequencing gel. As a control, the same amount of labeled probe was hybridized to 10 pg of calf thymus tRNA (BM) and treated in an identical fashion as described above. 9. Primer extension analyses. Primer extension was performed by digesting 1 pg of the Spel-EcoRl restriction fragment (Figure 2) with Alul endonuclease and end-labeling the resulting restriction fragments with T4 polynucleotide kinase and [y-32deATP (Maniatis et al., 1982). The mixture of end-labeled fragments was separated on an 8% sequencing gel and the 30 nucleotide strand complementary to U0 mRNA, designated P1 (Figure 3c), was purified. Approximately 4x104 cpm of the labeled P1 were added to either 5 pg or 6 pg of poly(A)"’ RNA from Ore-R third instar larvae, from ry 2 12 hour adults or from Ore-R 12 hour adults. Poly(A)"' RNA was isolated as described above. Probe P1 and the poly(A)+ RNA were dried and then dissolved in 20 pl of hybridization solution (Hirsh et 101 al., 1986), placed at 65°C for 1 minute and incubated at 37°C for 1 hour. The hybridization products were ethanol precipitated and dissolved in reaction buffer (Hirsh et al., 1986) containing fresh ultrapure dNTPs (Pharmacia) and 10 units of AMV reverse transcriptase (Pharmacia) and incubated at 42°C for 1 hour. The extension products were phenol/chloroform extracted, ethanol precipitated, vacuum dried, dissolved in 6 pl of standard DNA sequencing loading dye, denatured for 3 minutes at 90°C and then loaded into single lanes of an 8% sequencing gel. Additional primer extension reactions were performed by end-labeling 400 ng of a synthetic 30 mer, P2, just downstream of P1 (Figures 2 and 3c). Hybridization and extension reactions were performed as described above using 4x104 cpm of the labeled P2 and 10 pg of poly(A)+ RNA from Ore-R third instar larvae or 10 pg of poly(A)+ RNA from ryz 12 hour adults. The same results for the primer extension reactions were obtained from several experiments using RNA from two independent isolations of each developmental stage and from each Drosophila strain. 10. Tissue in situ hybridizations. Abdomens from ry2 12 hour adults were frozen, sectioned and hybridized to sense and antisense UO RNA probes according to Raikhel et al. (1988). The RNA hybridization probes were generated by transcription in the presence of [at-35$]dUTP from the T7 and T3 promoters of pBluescript KS(+) (Strategene) containing the EcoRI-Accl restriction fragment of cU02 (Figure A1 c). Full length transcripts, confirmed by Northern analyses, were hydrolyzed for 30 minutes in the presence of 0.1M NaHCO3 at 60°C yielding an array of RNA fragments of approximately 200 nucleotides capable of permeating sectioned and whole- 102 mount tissue. Hybridization of the partially hydrolyzed UO sense and antisense RNA probes to the sectioned abdomens and autoradiography were performed according to Raikhel et al. (1988). Malpighian tubules were hand dissected in Drosophila Ringer solution (Ursprung, 1967), permeablized and fixed according to a procedure developed for DrOSOphila embryos (Edgar and O’Farrell, 1989). I am greatful to B. Edgar for making this procedure available to me prior to publication. The U0 sense and antisense RNA probes were prepared as described for the sectioned tissue in situ hybridizations. After hybridization and final washes, the Malpighian tubules were placed between a poly-D-lysine coated slide and a glass coverslip treated with Sigmacote (Sigma, SL-2) and flattened by pressing firmly on the coverslip. The coverslip was then removed by rinsing in 95% ethanol. Autoradiography was performed according to Raikhel et al. (1988). The preparations were photographed under phase contrast and dark-field illumination using an Olympus Vanox-S phase contrast microscope and a 10X objective. 11. Sequence comparisons. a. Deduced amino acid sequence comparisons. The deduced amino acid sequences comparisons in Figures 4 and 5 were made by dividing each deduced amino acid sequence into approximately three equal parts and making all pairwise comparisons. This was done to determine the similarity throughout the entire deduced amino acid sequence since the FASTP algorithm will only detect and optimize an alignment with the insertion of gaps in a single region established by the initial alignment (Lipman and Pearson, 1985, footnote 1 5). 103 b. Flanking DNA sequence comparisons. Comparisons of the flanking DNA of the U0 gene of D. melanogaster, D. pseudoobscura and D. virilis were performed using dot matrix homology analyses (Pustell and Kafatos, 1982; Pustell and Kafatos, 1984) using DNA Inspector Ile (Textco). All pair-wise combinations of D. melanogaster, D. pseudoobscura and D. virilis UO flanking DNA were made using search parameters which identified nucleotide stretches of nine or greater matching nucleotides, allowing for one nucleotide mismatch. c. Calculating the expected number of silent site substitutions. The number of the expected silent third-position-codon differences between D. melanogaster and D. pseudoobscura and D. melanogaster and D. virilis were calculated according to Henikoff and Eghtedarzadeh (1987). The codon bias percentages for Drosophila for the amino acids threonine, proline, alanine, glycine and valine were taken from Shields et al. (1988) and are listed in Table A4. The expected number of third-codon-position differences for each of the five animo acids was calculated using the formula: (n) X {1 -£[0.01 X bias %)2}], where n is the number of matching residues between D. melanogaster and D. pseudoobscura UO or D. melanogaster and D. virilis UO for one of the five amino acids. 12. P-element Plasmid Construction. a. P[(w"’A)DmU0Pstl]. The 3.2 kb genomic Pstl restriction fragment encoding the U0 gene from the ecd1 strain was cloned into the Pstl site of the P-element vector CaSpeR containing a modified D. melanogaster white (w) reporter gene (Pirrotta, 1988). The Pstl sites are at position 826 bp upstream and approximately 1.2 kb 104 downstream of the U0 transcribed region. b. P[(hspw"')DmUOSpel-Stul]. The Spel-Stul restriction fragment encoding the U0 gene from the sod1 strain was cloned into the Xhal and Stul sites of the P-element vector pW8 which has the promoter of the hsp70 gene linked to the body of the white gene as a reporter (Klemenz et al., 1988). The Spel site is 171 bp from the transcription start site of the D. melanogaster UO gene and the Stul site is approximately 300 bp downstream of the U0 transcribed region. Heat shock is not required for screening transformants since the hsp70 promoter is transcribed at low levels in the absence of heat shock, providing enough white product to produce pigmentation within the eyes of transformed adults. c. P[(w"'A)DpUORI]. The EcoRl restriction fragment containing the D. pseudoobscura UO gene (Figure 15A) was cloned into the EcoRI site of the P-element vector CaSpeR. This construct contained the D. pseudoobscura UO gene with approximately 700 bp of 5' flanking DNA and approximately 200 bp of 3' flanking DNA . d. P[(w"'A)DvU01Pstl]. The 4900 bp Pstl fragment containing the D. virilis U01 gene (Figure 16A) with approximately 3200 bp of 5' flanking DNA and 400 bp of 3' flanking DNA was cloned into the Pstl site of the P-element vector CaSpeR. 105 e. P[(w"'A)DvUO2Pstl]. The 5500 bp Pstl restriction fragment that includes the D. vin'lis U02 gene (Figure 16A) with approximately 4000 bp of 5' flanking DNA and 300 bp of 3' flanking DNA was cloned into the Pstl site of CaSpeR. f. P[(w+A)pmuo-laczl. The Bglll fragment containing the Canton-S UO gene (Figure A1) was isolated from a lambda genomic clone possessing the U0 gene. The Bglll fragment was subcloned into the Bglll site of pKC7 (Maniatis et al., 1982) and the resulting plasmid is designated pKC7IB (Figure M1). The subsequent steps performed to make the P-element construct P[(w+A)DmUO-lacZ] are diagramed in Figure M1. The Bglll fragment was isolated from pKC7IB and then digested with Aval. The Bglll-Aval fragment containing 826 bp of U0 5’ flanking DNA and the first 346 bp of transcribed UO sequence was isolated and the 3’ recessed ends were filled in by Klenow in the presence of dNTPs (Maniatis et al., 1982). BamHl linkers (NEB) were ligated to the filled in ends. The products of the ligation were digested with BamHI. Excess BamHl linkers and their cleavage products were removed by fractionating the mixture through a 25 cm x 1.7 cm BioGel P-60 column with TE (BioRad). The fraction containing the BamHI linkered fragment containing the U0 gene was digested a second time with BamHI and the digest was loaded onto a 1% agarose gel. The band containing the BamHI linkered fragment was excised from the gel, electroeluted and ethanol precipitated. This Bglll-Aval restriction fragment, now possessing terminal BamHl sites was cloned into the BamHI site of puc119 (Vieira and Messing, 1987). I would like to thank Robin Steinman for ligating the BamHI linkers onto the Bglll-Aval UO fragment. This UO-containing BamHI fragment was isolated from puc119 and cloned 106 Figure M1. Construction of the UO-IacZ fusion gene. pKC7lB is a subclone of the Canton-S Bglll restriction fragment containing the U0 gene (Figure A1). A portion of the U0 coding region is represented by the open rectangle, the IacZ coding sequences are represented by a cross-hatched rectangle, the white gene coding sequences are represented by a stippled rectangle and BamHI linkers are shown as small filled rectangles. Arrows designate the direction of transcription of the U0 and white genes. The boxed P’s indicate the P-element inverted repeats. Restriction enzyme sites: Av, Aval; Bg, Bglll; Bm, BamHl; P, Pstl. The Materials and Methods give a detailed description of the steps diagrammed here. ”MP 107 q R2 '0 U0 an“ 1 Hal ll llflmt Av -. 89 FL'A——I 00 l AVII dlqsst Av FL-j 00 l dNTPs, Klonw l BImill llrtkIrI, T4llgIII n.9,” 00 1 ”11.81 034llmrlzod with 80mm, T4119». 52-ng Pf 6m 00 hr! 1 Pstl digest _E}! Figure M1. hr! with 108 into M13mp18 and sequenced to verify that no base deletions or changes had occurred during the linkering process since the U0 reading frame must be maintained for subsequent steps. The BamHI fragment was isolated from puc119 was also cloned into the BamHI site of the vector pMLB1034 which contains the E. coli lacZ gene (Shapira et al., 1983). Appropriate restriction digests were made to select clones in which the BamHI fragment was oriented such that the U0 coding sequences were in-frame with the lac-Z coding sequences. The UO-lacZ fusion was excised from pMLB1034 by digestion with Pstl (Figure M1) and cloned into the Pstl site of CaSpeR (Pirrotta, 1988). g. P[(w"'A)deI(-138,-126)DmUO-IacZ]. The M13mp18 subclone of the BamHI fragment containing the U0 gene (described above) was subjected to site-directed mutagenesis using the method of Vandeyar et al. (1988). The site-directed mutagenesis method is described in further detail below. In this case, synthetic oligonucleotide #18 (T able A) was used to delete 13 bp of the distal DR element of the D. melanogaster U0 gene (positions -138 to -126, Figure 1). Possible deletion mutants were screened by performing the T-sequencing reaction of single stranded DNA isolated from the M13 plaques and comparing the sequence ladder to the “T reaction” from the M13 BamHl subclone containing the “wild type “ UO gene. Out of 20 plaques screened, two contained the deletion. Plasmid DNA was prepared from an M13 subclone containing the deletion and the BamHI fragment containing the U0 gene was isolated. This fragment was fused in-frame with the feel gene and the fusion gene was excised with Pstl and cloned into CaSpeR. 109 h. P[(w+A)dei(+11,+23)omuo-iaczl. The steps to make this construct were identical to those for the construct P[(w+A)deI(-138,-126)DmUO-lacZ] except that synthetic oligonucleotide #19 (Table A) was used to delete the proximal DR element of the D. melanogaster UO gene (positions +11 to +23, Figure 1). Three out of 20 plaques were deleted for this region. i. P[(w+A)del(-138,126)(+11,+23)DmUO-Iac2]. The steps to make this construct were identical to those used to make P[(w+A)del(-138,-126) with one exception. The parental single stranded template DNA was an M13 subclone containing a deletion of the DR element from positions +11 to +23. By creating a deletion using primer 19, subclones with both of the DR elements deleted were obtained. Four out of 16 plaques screened had deletions for both the proximal and distal DR elements. 13. Site directed mutagenesis to create large 5' U0 deletions. Large deletions within the 5’ region of the D. melanogaster UO gene were made using the site-directed mutagenesis method of Vandeyar et al. (1988). Single stranded M13mp18 DNA containing the U0 BamHI fragment (section above) was used as the parental DNA for mutagenesis. The steps taken to produce deletions within the 5’ flanking DNA of the U0 gene are diagramed in Figure M2. Mutagenesis reactions were as described in United States Biochemical T7-GEN In Vitro Mutagenesis Instruction Manual. Phosphorylated synthetic primers of 34 nucleotides were independently annealed to the template DNA. Each 17-mer “arm” of a primer hybridized to positions approximately 100 bp apart along the template DNA (Figure 12). After hybridization, the primer was extended in the presence of dATP, dGTP, dTI'P, 110 5-methyl-dCTP and T7 DNA polymerase. Products which extended along the M13 template to the 3' end of the primer were ligated using T4 DNA ligase. The final double stranded product containing a “looped out” region to be deleted (Figure M2) was digested with Mspl and Hhal. These restriction enzymes nick only the nonmethylated parental strand which is subsequently removed by digestion with Exolll. The remaining methylated strand which has a deletion for the material between the two primer “arms” was transformed into the non- restrictive host E. coli SDM (United States Biochemical), replicated and then infected into an E. coli host susceptible to M13 infection. E. coil“ MV1193 was used here. Deletion mutants were screened by sequencing the “T-reaction” from single stranded DNA prepared from M13 plaques. Examples of deletions spanning approximately 100 bp are rare and considered to be difficult to obtain due to the increased distance between the sites in which the two “arms” of the primer bind. However, in the experiments reported here, large deletions occurred at high frequencies. Mutagenesis with primer #20 (T able A6) yielded three out of eight M13 plaques with a 103 bp deletion, mutagenesis with primer #21 yielded three out of eight M13 plaques with a 235 bp deletion and finally, mutagenesis with primer #23 yielded eleven out of eighteen M13 plaques with a 114 bp deletion. Combinative deletions were made to examine the effects of simultaneous multiple deletion on the regulation of the U0 gene. To construct combinative deletions, M13 template DNA, having a deletion of 5' U0 DNA from a previous mutagenesis, was subjected to a second round of mutagenesis with a different primer than the one used to make the original deletion. The primers were designed such that the right “arm” of one primer hybridized to the same seuqence that the left “arm” of another primer hybridized to, in order to make contiguous deletions which would only have the primer binding sites remaining between the deleted regions (Figure 12). 111 Figure M2. Diagram of the steps to prepare P-element constructs containing combinative oligonucleotide-directed large deletions of the 5' flanking DNA of the D. melanogaster UO gene. The M13mp18 sublone contains the Canton-S Bglll-Aval BamHl-linkered restriction fragment of the U0 gene. UO, lacZ and white coding sequences are represented by an open rectangle, a cross- hatched rectangle and a stippled rectangle, respectively. Arrows indicate the direction of transcription of the U0 and white genes. BamHl linkers are represented by small filled rectangles. The primer used for in Vito mutagenesis is shown as a short curved line. M’s decorating the double stranded M13mp18 symbolize the presence of 5-methyl-dCTP residues in the strand containing the deletion mutation. A deletion in the 5’ flanking DNA of the U0 gene after the mutagenesis steps is indicated by a gap. Restriction enzyme sites: Av, Aval; Bg, Bglll; Bm, BamHl; P, Pstl. A detailed description of the steps diagrammed here is within the text of Materials and Methods. 112 MT? 0611’ NW S-motlwl -£TP l'l Will ‘ HhIl IftitIIl primer T7Apolumsrm digestion ———" ——'" f1 —-9 \ H 00 ...’ O... M J Int P But .w’ :’ 41: Is" stratification ID .x’ a", A” trInIformSDI‘l infect MV1193 8111 "P an |‘-l R —'=Afl UO 1 pl‘lLBI 0341100014206 with Built“, T4 liom 8m P 6m P 8m I‘—-I (___—W— 1 4. uo Md 1 Patidlqut P 6m P 00 III-Z 1 CISpIR lliiIIrilel with Pstl, Tallow P But P _ -[fl-L--l W ll 00 III-Z vhllI Figure M2. 113 14. P-eiement transformation. a. Preparation of DNA for micro-injection into D. melanogaster embryos. Having “clean” DNA, free of all chemicals used in the purification procedure, is essential for obtaining high transformation frequencies. DNA should be purified in a cesium chloride gradient and subsequently dialyzed for at least three days (Rubin and Spradling, 1982) as described for a large scale plasmid DNA preparation (above). After dialysis, the P-element construct DNAs were precipitated in the presence of 0.2 M NaCI with 2.5 volumes of ethanol at -20°C. The DNA was recovered by centrifugation at 4°C for 15 minutes and the pellet was washed once with 0.2 M NaCl in 70% ethanol and once with 70% ethanol. The pellet was vacuum dried and resuspended in 50 ml of injection buffer (5 mM KCII 0.1 mM NaH2P04, pH 6.8). A solution of P-element construct DNA at 300 nglpl of injection buffer and P- element helper plasmid pir25.7 “wings clipped” at 50 nglpl of injection buffer was used for micro-injection into D. melanogaster embryos. Just prior to injection, the mixture of P-element construct and P-element helper was centrifuged for 10 minutes at room temperature and the solution was transferred to a new tube. This last centrifugation was used to remove debris and precipitation which was not detectable by eye, but capable of clogging embryo injection needles. b. Embryo injection needle preparation. Needles used for injections were made from glass capillary tubing with an outside diameter of 1.0 mm and an inside diameter of 0.78 mm (Sutter Instruments 00., #BF100-78-10). Capillary tubes were made into needles using a Flaming Brown micropipette Pluller (Sutter Instruments Co., Model P80lPC) 114 P80lPC) using Program 9. Program 9 was designed by Sutter Instruments given our needle specifications and consisted of the following settings: heat, 32,000 milliamps of current to the filament; pull, 400 milliamps to the pull solenoid; velocity, 230 millivolts of transducer output; time, 5 milliseconds. A boxed heating filament (2 mm x 1.5 mm) and a setting of 50 psi for the nitrogen was used. Needles pulled in this fashion were closed at the ends. In order to break the tip of the needle to make a 1-2 micrometer jagged opening, the needles were gently stroked across the curvilinear surface of a freshly broken microscope slide using a micromanipulator. The microscope edge of the microscope slide was broken using needle nose pliers. c. Collection of appropriately staged embryos. The host strain for all P-element transformations was the white deficient stock Df(1)w,yw67°23(2), which was made homozygous for the second chromosome from the wild type D. melanogaster Canton-S strain. This stock is referred to as Df(1)w,yw67°23(2), Df(1)w and chs within the text. The sequence of the U0 gene on the Canton-S second chromosome contains several nucleotides in the 3' untranslated region that are different from those of the U0 gene isolated from a stock containing the temperature sensitive 20-hyroxyecdysone mutation, eod1 (Figure 1). This difference in UO sequence allowed for discrimination between the message from the U0 gene introduced via the P-eiement and the endogenous Canton-S UO message of the host strain (see Northern analyses in Figures 8 and 9). Newly emerged white deficient adults were collected, fed a rich diet of yeast- honey paste and aged four to five days prior to egg collections. Approximately 300 aged adults were placed in small collection chambers (Santamaria et al., 115 1986) and allowed to lay eggs on a lucite tray with a small amount of grape juice-agarose medium supplemented with yeast-honey paste. Collections of eggs were made every half hour by replacing the collection tray with a fresh one. Embryos from the first two half hour collections were discarded due to the lack of synchrony in egg laying. The goal was to obtain stage 2 embryos (Wieschaus and Ni‘isslein-Voihard, 1986) which had not yet cellularized. Each half hour thereafter, embryos were collected and chemically dechorionated in a 1:1 dilution of bleach in water for 15 seconds with swirling of the solution and the eggs. Dechorinated embryos were aligned on double sticky tape affixed to a coverslip with their posterior end hanging over the edge of the tape. After alignment, embryos were desiccated in a petri dish of Drierite for one to two minutes and then covered with Halocarbon oil (Series 700, Halocarbon Products Corporation). d. Embryo injections. Properly prepared embryos covered in Halocarbon oil were viewed for injection through a Wild compound microscope with a 10X objective. Needles were backfilled with the mixture of the P-element construct and the P-eiement helper plasmid (see above) using a Hamilton syringe and placed into a holder attached to a Leitz micromanipulator. The DNA solution was injected into the pole piasm of the posterior portion of the embryo which gives rise to the germ cells. The solution was driven out of the needle and into the embryos by a pulse of nitrogen (20-25 psi) for 20-40 milliseconds delivered by a Picospritzer ll (General Valve Corporation). Only embryos which were at a developmental stage prior to pole cell formation were injected. Appropriately staged embryos have an easily discernable clear area at their posterior end (Wieschaus and Nusslein-Volhard, 1986). Embryos which were too advanced in development for injection or those which “bled” after injection were discarded. The remainder 116 of the embryos, all successfully injected, were kept under Halocarbon oil and incubated in humidified petri dishes at 18°C or room temperature. After approximately 24 to 32 hours, first instar larvae hatched and were removed from the Halocarbon oil and placed in groups of 30 per vial of Drosophila media and allowed to develop at 25°C. The Go adults were backcrossed to the white deficient host strain in single pair matings. The G1 progeny from these crosses were screened for pale yellow to wild type brick red eye color. Transformed G1 adults were backcrossed to the white deficient host as single pair matings. The 62 progeny from a single vial which were heterozygous for a P-element insertion were crossed in single pair matings to each other. G3 progeny which were homozygous for the P-element insertion usually had a darker eye color than that of the heterozygous G3 progeny and thus, homozygous G3 adults from a single vial were mated in pairs to create a homozygous stock. Some stocks possessing P-element integrations that were lethal when homozygous were analyzed for U0 transgene expression as heterozygotes. 15. Histochemical staining for p-galactosidase activity. Whole larvae pupae and adults as well as hand-dissected Malpighian tubules were stained for p-galactosidase activity according to Raghavan et al. (1986). Whole animals were splayed open in 10 mM phosphate buffer, pH 8 and transferred with forceps to the staining solution (0.066 ml 5% X-gal; 0.020 ml 100 mM potassium ferrocyanide; 0.020 ml 100 mM potassium ferricyanide; 0.050 ml 1.0 M sodium phosphate, pH 8, 0.850 ml 35% Ficoll-400). Malpighian tubules were dissected in 10 mM phosphate buffer, pH 8 and transferred in 10- 20 pl of dissecting buffer to the staining solution. The tissue was stained for one to six hours and then transferred to a drop of 35% Ficoll on a microscope slide. 117 A coverslip was carefully overlayed and the preparations were photographed with an Olympus Vanox-S phase contrast microscope using a 10X objective. 16. Synthetic Oligonucleotides. Synthetic oligonucleotides were synthesized either by Dr. Chris Somerville on an Applied Biosystems DNA Synthesizer or by the Macromolecular Sequencing Facility at Michigan State University. Some oligonucleotides were purified using an OPC column (Applied Biosystems) according to the manufacturers protocol or by HPLC. Some oligonucleotides used for sequencing and site directed mutagenesis were not further purified after synthesis. The sequence of each primer and method of purification is listed in Table A6. 17. Western Analysis. The Western blotting procedure was similar to that of Kral et al. (1986) and is summarized here. Malpighian tubules were hand-dissected in 0.25 M sucrose, 1.7 mM EDTA, pH 6.9 and transferred in 7.5 ul of dissecting solution to a 1.5 ml microtube and immediately frozen at -70°C. To each frozen sample, 7.5 ul of a two-fold concentrated Laemmli gel loading buffer was added and the samples were boiled for 3 minutes, cooled and loaded onto a 0.1% SDSI 10% polyacrylamide gel (Laemmli, 1970). Following electrophoresis, the proteins were electroblotted onto an Immobilon-P membrane (Millipore). The portion of the membrane containing the size separated protein molecular weight , standards (phosphoryiase b, Mr=94,000; albumin, M,=67,000; ovalbumin, M,=43,000; carbonic anhydrase, Mr=30,000) was stained with Coomasie Blue. The remainder of the membrane was incubated for one hour in blocking solution and then overnight with the primary antibody. The source of primary 118 antibody used to detect D. melanogaster, D. pseudoobscura and D. virilis UO was a polyclonal antiserum raised against purified D. melanogaster UO protein (Friedman and Barker, 1982). Visualization of the U0 proteins occurred by incubating the blot with the secondary antibody, horseradish peroxidase coupled to goat anti-rabbit lgG (BioRad 172-1013), and then 4-chloro-1-napthol (Sigma C-8890) as a substrate for horseradish peroxidase. APPENDIX B Figures and Tables 119 Figure A1. Restriction map and sequencing strategy for the U0 genomic region and twelve independently arising urate oxidase cDNAs. (a) Restriction map of 38 kb of genomic DNA including the Canton-S UO gene. (b) Restriction map of the U0 gene and flanking DNA with the directions and regions sequenced designated by arrows. The transcription initiation site of the U0 gene is at +1 designated by I. The heavy black line represents the coding region of the U0 gene with the open rectangle indicating the 69 base pair UO intron. (c) Composite restriction map and sequencing strategy of twelve UO cDNAs. A UO cDNA (cU02), spans the region between two Pstl sites, ®, which were introduced during cDNA library construction. The numeral above the arrows designates the number of independently arising cDNAs sequenced for that region. The asterisk indicates the location of a synthetic primer used to sequence the extreme 5' end of the U0 cDNAs. AUG and TGA shown on the restriction map indicate the position of the U0 translation start and stop sites, respectively. (d) An autoradiograph of a genomic Southern blot of D. melanogaster Canton-S high molecular weight DNA restricted with Sal I (lane 1), Aval (lane 2), EcoRl (lane 3) and Hindlll (lane 4) was probed with a 5.5 kb Hindlll restriction fragment containing the U0 gene. Restriction sites: A, Alul; Ac, Accl; Av, Aval; B, Bglll; C, Clal; D, Dral; H, Hindlll; Hp,Hpall; N, Nlalil; Pv, Pvull; R, EooRl; S, Sall; Sp, Spel; T, Taql; X, Xholl. The Southern analysis was performed by T. Friedman. Zkb lair—i RR R8 3 IR "IR. 3115 SR H H R RR I! l l ' 1 :1H '1 '1‘; ‘ 11 l l 14] h-mm x 9,, M MM x\x“ mum—o I ‘ 20009 /’ \\\ (bit—4 ,/ 7 a 6,’0 ”Inlay I cc ‘Av\3 I4 I L #1 1 1 V: l 1‘ l #‘l / rm ii. titli‘i w w .. x \ id) 1.23.: n / H——D ——-’——P\ -—'9 l' e—— ,t——+-) ¢—— : ¢\9, l I \ Ina-l / \ Inc-l 1005;. ' ““7" \ "-1 CJ [All it Av A I AIA -1 gill" T ITII TIT I 3 I 1’ l <——— : 1' —D ._u ___. g . _, "7 fl: " E ...-.. 1"- M—1 c “7% Figure A1. 121 Figure A2. Southern analysis revealing restriction enzyme recognition site differences in the 3’ transcribed but untranslated region of the U0 gene from different strains of D. melanogaster. Lanes 1, 5 and 9 contain DNA isolated from sod1 and lanes 2, 6 and 10 contain DNA isolated from Df(1)w,yw°7°23(2). Lanes 3, 7 and 11 contain DNA isolated from Canton-S and lanes 4, 8 and 12 contain DNA isolated for the stock Df(1)w,yw°7°23(2) with the Canton-S second chromosome (see Materials and Methods). The DNA samples in lanes 1-4 were digested with Alul. The DNA samples in lanes 5-8 were digested with Sphl and the samples in lanes 9-12 were simultaneously digested with Sphl and Pvull. The restricted DNA was size separated on an agarose gel, blotted onto nylon and probed with cU02 (Figure A1). Based on sequence data, Alul and Sphl should cut the U0 gene of the acid1 strain, but not the Canton-S strain, in the 3’ transcribed but untranslated region. The approximate 420 bp hybridizing fragment in the DNA samples after digestion with Alul (lanes 1-4) represents an internal fragment within the coding region of U0. The restriction fragment of 610 bp in lane 1 containing ecd1 DNA which hybridized to the U0 probe is the result of cleavage of the 960 from the 1090 bp Alul fragment (lanes 2-4) in the 3’ untranslated region of the U0 gene. Digestion of eod1 DNA within the 3' untranslated region of the U0 gene by Sphl (lane 5) truncated the 6600 bp UO restriction fragment in the Canton-S DNA (lane 7). Similarly, simultaneous digestion of the sod1 DNA by Sphl and Pvull (lane 9) truncated the 1,400 bp UO restriction fragment in the Canton-S DNA (lane 11). 122 123456789101112 \ 2.02. Figul'e A2. 123 200bp forces 1 :2 a *B‘Y’E llfiplél’éc _fifiisgi ii A A R R R AVRA R R RA Av ‘— #fl ‘ ‘ # \ \ 3 ‘ ‘—_" F Figure A3. Restriction map and sequencing strategy for a 2.2 kb D. pseudoobscura genomic region containing the U0 gene. The heavy black line represents the transcribed region of the U0 gene with the translation start site (ATG) and translation stop codon (TAA) indicated on the restriction map. The 62 base pair D. pseudoobscura UO intron is represented by an open rectangular area. POiCH3 is a 1.8 kb Hindlll subclone containing the U0 gene. M13 subclones used for sequencing this region are indicated below the restriction map with arrows indicating the direction of sequencing. Restriction sites: A, Alul; Av, Aval; Ac, Accl; C, Clai; E, EcoRl; AE,EooRl in AEMBL3 arm; H,Hindlll; M, anl; R, Rsal; Sp, Sphl; V, Avail; X, Xholl. 124 200 bp Sp 8 PvRHHp Pv Hp D l l 1 L11 1 t | (—-— <———<——— ———> Figure A4. Restriction map and sequencing strategy for a 1.8 kb genomic region containing the D. virilis U01 gene. The heavy black line represents the transcribed region of the U0 gene. M13 subclones used for sequencing this region are indicated below the restriction map with arrows indicating the direction of sequencing. Restriction enzyme sites: B, Bglll; 0, Oral; H, Hindlll; Hp, Hpall; Pv, Pvull; R, EooRl; Sp, Spel. 838388388388988388888883 '826 '806 -739 '738 “672 '605 '604 ‘594 “538 '537 '539 '671 '670 '662 '404 '603 '395 ‘33? '336 '328 '270 ~269: ~261 125 tummcpwccwnaoccaaccr 1u1wnntxxn1wAcTAAAAcccTATAcTAcccTTAcToAACAcwachAAcwccTAAAeTAAAGcom ’11ccGAmwcTmmnanmwnmnnnncnwuumnuvwoamcaeroqAnpccamcweaacncammcwowcnc Lccoeemcacmmrmmecc-r'1''1'ooc-r-roc'rc'1'iteccTAAonwarmmmammrcrmoeem ’AmmaaasIna1ccaaAAAcwcAAcTmAAAcamewomaawTwmTAhoaccamwmncmaeoammmmana _cu1oaaaamaaAAocTcaoAmaAAAaaIeawcAAa1111cn11caaaoocamaacnawcammnrrrr AAAaGGTCTCCTAAAAfl1TmATAAACAAAGTGTCACA!CIAAH1TDAGACGmhhkrmknkrrrrrrrr ccAeawccAAaowccAAAacIcacwsAAa11a11IcTTIeAaoaanmunknauuuuncnmmdccnmw coaAaIaAAaIcAmcmarrrrrrrrrmamaAaamamTmaacwoocauemwccmmamncaamnfififig a; E7 EB 7caaaaa1EIzfliiiiiEflarnnaauuEEaEEazzEEbaaaxnznuunnucamaceoecncnrr 1ccTaAeAmaaweTnAeomamaoaeccc1aawAcccmacmaAacncwwmaaamcmumnenenurrna _Zj3Ej§3a3uumuuKuumamAAcAAawaAAAaTAcrrnMsrnaaruznxxxuuxnmamnanmncc rTTCAABGTTTrANnANTAAEhGTGACTCGwO0GTGAAmAAGAGAGAAASTIAGATTTEAAAAAAGAA cAAama1cAAcTAAaAAmawmAccaAaAAAeAcAeccoaIhaAanamomennpwnaenanaocnama -LCTAAGGCCAGCACCAAGCTACCTTCIChAAflICTCTflGAEGTGCTTCTTCSTEIHCIGTGCT’CACT es lmaAAAaTcAaAeAaeTaaEi5ifi5GGiifiiihTwaccAArrrTcamwwmccccoeaaaewenmecma 55 £8 ammatnaamaemn1cmcAoAc111onwmnwmmmnuacummmmewAcaccrmA l memmmmmnmn-n rAflGGTTAAAACOGCNTTRIIHVIMHflKHCIHflCGThAflflSTGCAAAAASRAGGAETTGGTTAGAJG A—A' AAAAAAAATCCCTA E3 1 126 E5 luufli-Mmmflflmnmmwmwmnmmmmmwwwwmmnmunna GTGAGAGTGATGATfiTNCGA D ACTTGGCACCAGGCCGCAATTATCAGCGCTICTflGTGGTRATTTGAGTTNGACCTTTAAT p '203 '202 ‘136 UV ' .196 LWWTWTMMMWW Dm Op On km In T um . em Imam a nmfifig . a .A G GGC .A D GAE T A. GGC TC AER G GGA C V GTG 'ECQCC .A 8 TCC G A GG H CAC A.G P CCC G E2 I E3 pm“. .mc am” TGGGTAAGCAAGTTflaCHTTIATGATTGTGTGAGTTGAAAGTAAGATTCTGGIGIAETICGTIAGCA 'ft'f (' E2 fimmwwmnnmmwmwmnmmmmw N a ”com o a Y ww r CA L! as 'm ACTACACAAAGNTTHIRFPFGPBA - - m w m w m m w w. mwm mwm mwm C C T G T T +262 2257 0253 Du Dp DV 127 am.” MCTGT N G V O G N C AMMONCAANGAACTGC'“ TCAG GT G C G ARC Tc zma G K C GMAAGTGC TCI‘GT TC V GTC G '1‘ +463 ACC AAC N MT A N +446 +662 am T In W D! #619 CAWAGTWTMTAMMAMMWW AT CCC *602 GACMTWTAflmmm-H- "-735 ’593 TAWAWWWmm-u-uu-uu-m Du DD DV bmc PMGG G amen CC .mcc SMGG .m EMGG a a a DWCG rm vm . ".me .m a 2.. .m oma mwm 128 +886 +862 +851 +908 +966 +953 m +1039 DD +1015 DV +1006 m +1090 Dp +1066 01! +1055 m W W “It DD 0‘! WWTATMCACAAA TAWATWMAW .* TAGATWAWTGTAGTAAATCTAAATQATC’MAATCA I N 8 H L AATMCACC’IG G C T AT AA C T C N AACA GTC m +1141 Dp +1117 DV +1106 7011me mm m AN AN mmmmmmmm “WTWTWWTMTATTWW r CAATAAAT'I‘TATAT'I'IT b WWW h m +1336 FMWMAWWWW but +1202 mem Dp +1245 m DD +1178 DV +1167 m +1269 DV +1236 Dp +1312 129 III +1403 [TATATMMTMAWWMMNHAW W + 137 9 WMWAMWMWAGGAMW m +1470 [TTAGCAAATAACCATTWCCACAAACAWIGACCANACAA I» +1446 MWAWAGCAAAATATC Figure A5. Nucleotide comparisons of the D. melanogaster (Dm), D. pseudoobscura (Dp) and D. virilis (Dv) U0 genes. The complete nucleotide and deduced amino acid sequences of the D. melanogaster UO gene are shown. For the D. pseudoobscura and D. virilis UO genes, only the nucleotides within the coding region that differ from the D. melanogaster UO gene are shown. Dashes (-) represent nucleotides not present based on the alignment of the deduced UO amino acid sequence of these three species (Figure 5). The transcription start site of the U0 gene of each species is designated +1. No attempt was made to align the intron or flanking DNA sequence. Evolutionarlly conserved elements (E1 to E8) of the U0 5’ flanking DNA (Figure 7) are boxed and shaded. The translation stop codon is indicated by. The D. melanogaster U0 sequence has three polyadenylation sites indicated by AN and four potential polyadenylation signal sequences which are overscored. The D. pseudoobscura and D. virilis U0 genes each have a single polyadenylation consensus signal (overscored). g < < {E E I 8 .- 2 E 3 Y- 1- 0') < bp 12345678 23,130— '. T — - 9,419—3 "‘ " ~- 6,557— 4,371— Figure A6. Example of a Southern blot used to determine the copy number of a P-element insertion. Genomic DNA isolated from three different lines (11F30A, 1M21A and 3M11A) transformed with P[(w+A)DmPstI] and genomic DNA isolated from the D. melanogaster white deficient host strain (chs) was digested with BamHi (lanes 1. 3. 5 and 7) and Xhol (lanes 2, 4, 6 and 8). BamHI and Xhol were used since they restrict the DNA once within the P-element construct. Therefore, a restriction fragment containing the U0 transgene would be derived from a second restriction site within the D. melanogaster genome and would be unique to each P-element integration site. The restricted DNA was size separated on an agarose gel, blotted onto nylon and probed with cU02 (Figure A1). The probe hybridized to an approximate 10,700 bp BamHl DNA fragment and an approximate 11,700 bp Xhol DNA fragment of the chs host (lanes 7 and 8). The DNA isolated from 11 F30A showed two bands in each lane, in addition to the band corresponding to the endogenous UO gene (the larger band in lane 2 is a doublet), which hybridized to the U0 probe (lanes 1 and 2). Two unique bands in this transformed line indicate that there have been two P-element integration events. DNA from strains 1M21A and 3M11 A both show one hybridizing band in each lane, in addition to the band corresponding to the endogenous UO gene (lanes 3-6), and therefore, have a single insertion of the U0 P- element construct. 131 Figure A7. UO enzyme activity during development of D. melanogaster, D. pseudoobscura and D. vin'lis. UO enzyme activity is present exclusively within the Malpighian tubules of Drosophila and is quantified by the amount of 14C allantoin synthesized from 140 uric acid per minute of reaction time per set of Malpighian tubules from a single animal (vertical axis). The first, second and third instar larval stages and white pupal stage are designated 1, 2, 3 and P, respectively. .1” he sex of the larvae (A) assayed for U0 activity was not determined. Adult stages are designated in hours after eclosion with O as males and O as females. The U0 enzyme profile for D. virilis represents activity in strains 1051.0 and 1051.48. and the U0 enzyme activity profile for D. pseudoobscura represents activity in strain AH133. The U0 enzyme assays were performed by T. Friedman. The U0 enzyme activity profile for D. melanogaster Ore-R was taken, in part, from Kral et al. (1982). This figure is taken from Wallrath and Friedman (1991). 132 l Drosophila melanogaster F P mor6msmanch0 >.—._>_.__.U< mw5._>_.PU< wm.:>Fo< 3336 use: ADULT STAGE (hr) PU PAL STAGE LARVAL STAGES 133 Table A1. Drosophila stocks. ___LIQactivlnL—__ Stack Ehenctxne athnstaLlanta adult W Oregon-R wild, We high low CarsonoS wild type high low ecd‘ ts ecdysone high (19°C) low (19°C) deficient ry2 xdri deficient, high high rosy eye color bf(1)w.yw°7°23(2) white eyes, high low yellow body Wm AH133 wild type absent high an: ' 1051.48 wild type high absent 1051.0' wild type high abscent Balm Lindsey and Grell (1967) Lindsey and Greli (1967) ' Garen et al. (1977) Glassman (1965) Pinata 61 al. (1983) 861166116! 61 al. (1987) Species Stock Center Species Stock Center '0. virilis 1051.0 contains a tandem duplication of the U0 gene; all other stocks have a single dopy of the U0 gene per haploid genome. ts - temperature sensitive mutation xdh - xanthine dehydrogenase y - mutant allele of the yellow locus w -mutant allele ofthe whlieiocus Species Stock Center - National Drosophila Species Resource Center, Bowling Green. Ohio 134 Table A2. U0 sequence changes among D. melanogaster D. pseudoobscrua and D. virilis. W D. W 0. nuts Exon 1. base pairs 590 607 585 leader sequence. base pairs. 34 33 42 nucleotide substitutions 14 protein coding region. base pairs 556 574 544 nucleotide substitutions 106 (19.1%) 162 (29.8%) codon position 1 23 (21.7%) 37 (2.8%) codon position 2 18 (17.0%) 32 (19.8%) codon poslion 3 65 (61.3%) 83 (57.4%) transversions 56 86 transitions 50 76 numberolaminoacids 185 191 181 chino acid identities 151 122 synonymous (silent) changes 48 . 53 replacements 33 59 conserved replacements” 29 39 amino acid deletions 7 10 amino acid additions 1 0 lntron. base pass 62 69 55 Exon 2. base pairs d 608. 612 a 629° d protein coding region. base pairs 482 482 482 nucleotide substitutions 79 (16.3%) 113 (23.4%) codon position 1 18 (22.8%) 25 (22.1%) codon position 2 5 (6.3%) 8 (7.1%) codon position 3 . 56 (70.9%) 80 (70.8%) transversions 34 57 transitions 45 56 numberolaminoacids 161 161 161 chino acid identities 152 134 synonymous (silent) differences 63 ' 61 replacements 9 27 conserved replacements" 8 26 amino acid deletions O 0 amino acid additions 0 0 Exon 1 + Exon 2. protein coding region only base pars 1038 1056 1026 unino acids 346 352 342 amino acid identities 303 (87.6%) 256 (74.8%) deduce M, oi urate oxidase 39.266 39.989 38.680 nucleotide dil‘lerences 185 (17.8%) 275 (26.8%) a The surcharge; em site 01 the D. melanogaster urate oxidase gene was determined experme . bConserved amlnoacldchangesweredelined bythe FASTP program (Upman and Pearson 1985). c more are three polyadenylation shes tor the D. melanogaster U0 gene. dThepolyadenylatlon she(s) has notbeen determined lortheD. pseudoobsorn andD. WrisUOgene and.theretore.thelengthottheU0exon20tthesespeoleslsnotknown 135 Table A3. Codon Usage of the D. melanogster, D. pseudoobscura and D. virilis U0 genes. U00 1“ 8/6/10 UCU s 1/1/0 UAU Y 5/7/9 UGU c 0/0/2 UUC F 10/11/5 UCC s 6/7/5 UAc Y 10/8/5 UGC c 4/5/3 UUA L 0/0/1 UCA s 1/3/5 UAA * 1/0/1 UGA * o/o/o UUG L . 2/4/4 UCG s 10/5/6 UAG * O/l/O UGG w 3/3/3 CUU L 1/1/0 CCU p 2/0/1 CAU H 5/6/6 CGU R 3/1/3 CUC L 1/4/5 CCC P 8/9/6 CAC H 10/12/10 CGc R 5/2/7 CUA L 2/0/1 CCA p 2/5/6 CAA 0 4/5/10 CGA R 0/1/0 CUG L 13/13/16CCG P 6/4/2 CAG Q 17/14/9 CGG R 0/2/0 AUU I 3/6/2 ACU tr 1/6/3 AAU N 7/5/8 AGU s 2/3/5 Ath i 15/10/10Acc 'r 10/13/7 AAc N 19/14/12 Add 5 7/4/7 AUA I 0/3/4 'ACA 'I' 3/1/7 AAA K o/s/e AGA R 1/5/1 AUG M 5/4/5 ACG 'r 7/7/5 AAG K 22/18/15 AGG R 4/3/0 GUU v 1/5/7 GCU A 4/5/2 GAU 13 10/15/14 GGU G 3/2/2 GUC v 13/10/8 Gcc A 10/9/4 GM 13 9/6/5 GGC G 10/10/11 GUA v 1/5/4 GCA A 1/1/3 GAA E 0/5/4 GGA G 2/3/2 006 v 15/15/13GCG A 0/5/7 GAG E 16/12/14 GGG G 2/2/2 One-letter symbol is. used for amino acids. Codon usage for the D. pseudoobscura. D. melanogaster and D. virilis U0 genes are designated left to right. respectively. " designates translation termination codons. 136 Table A4. Silent site substitutions of the U0 genes for threonine, proline, alanine. glycine and valine. Number oi Expected Observed . Amino Bias matching number oi number of Acid Codon (%) residuesa differencesb differences Dp Dv Dp Dv Dp Dv THR ACU 12.7 Add 90.9 19 17 6.21 5.55 7 11 ACA 1.9 ACG 4.5 PRO 000 10.2 CCC 67.7 17 14 8.56 7.05 7 8 CCA 15.2 006 6.9 ALA GCU 22.0 600 69.4 12 12 5.60 7.34 6 9. GOA 4.2 606 4.4 GLY GGU 34.9 600 0 13 12 8.40 7.75 4 4 GGA 22.3 660 42.9 VAL GUU 15.6 AGUC 36.5 29 24 18.40 15.22 13 11 GUA ' 2.5 GUG 45.4 TOTALS 90 79 47.17 42.91 37 43 (78%) (100%) Codon usages tor Drosophila are lrom Shields at al. 1968 (high bias group). ‘Dlllerence expected tor complete random codon usage. bThe expected number or dillerances cortrectad tor codon bias was calculated according to Henikolt and Eghtedarzadeh (1987); expected number or differences - No. matching residues 11 (1-zi(0.01 x bias 9102]}. Values obtained lrom a comparison at D. melanogaster U0 and D. pseudoobscura U0 are listed under 0p and values obtained lrom a comparison at D. melanogaster U0 and D. virilis U0 are listed under Dv. 137 Table A5. lnterspecllic sequence comparisons ol Drosophila genes. Guns alcohol dehydrogenase “100001108 Um WWO sstsrase lushl trazu Glucose dehydrogenase mmmhm SW D. melanogaster. D. simulans. D. mauritiana. D. arena. D. melanogaster. D. aflinid’slmcrs D. moisvensls. D. mulleri D. melanogaster. D. molavsnsls. D. wields/mere D. melanogaster. D. orsns D. melanogaster. D. Modem D. melanogaster. D. Mean 0. virilis. D. grimshawi D. melanogaster. D. subobscura D. virilis. D. grimshawi D. melanogaster. D. We. D. melanogaster. D. sllvsstris. D. hereroneura. D. plsnlllbla. D. subobscure . D. virls, -. D. grimshawi D. melanogaster. D. sunbeam. 0. while. D. grimshawi D. melanogaster. D. subobscln D. virilis. D. aimshawl. Bombyx marl. Anrheraea pamyl. Anthsraes polyphemus D. melanogaster. D. Wis. Ceraritis aspirate melanogaster. 0. Ms melanogaster. D. We melanogaster. - D. vials. melanogaster. D. Minis melanogsrer. D. Walls D. D. D. D. D. D. melanogaster. D. possum D. melanogaster. D. hydsl D. melanogaster. D. pseudoobsctn D. melanogaster. D. ”mucus D. villa D. melanogsrer . D. slmdsns. D mwoobscurs. D. Ws (D. melanogaster. D. pseudoobscus. Garbe a al. (1989) . Iiydel antennae Boomer and Ashbumer (1984) Rowan and Dickenson (1988) Atkinson at al. (1988) Ayer and 8enys)ati (1990)' Moses at al. (1990) Ssegsranszulman(1990) Martinez-Cruzado at al. (1988) Fensrfisn at al. (1989) Mltslsls at al. (1989) Martinez-Crusade (1990) Swtnmer at al. (1980) Shes a al. (1990)' Konsolsld It 81.. (1990) Bray and Hirsh (1 Bray at al. (1988)‘ Johnson et al. (1889)' Kassis at al. (1986) Kassis at al. (1989)' Brady at al. (1990) Meier at al. (1990) Henltotl and Eunedtzadeh (1987) Krasney at al. (1990) Blsdcman and Meselson (1986) 138 Table A5 (cont'd). hunchback D. melanogaster. D. Will; I Treier at al. (1989) period locus g melanogaster. D. pseudoobscura. Colot at al. (1988) . virilis D. melanogaster. D. yakuos Thackeray and Kyriscou (1990) rhosomsl protein rp49 D. melanogaster. D. suoobscws Aguads (1988) due praein age: D. melanogaster. D. widens. Martin at al. (1988) D. erecta. D. yakubs 621% D. melanogaster. D. hydel Michieh at al. (1887) D. melanogaster. D. hydel Michlels at al. (1989)' ultrablhorax D. melanogaster. D. pseudoobsctn. Mid. and Akltl (1 ”7) D. (means. Muses domestics Wis dehydrm D. melanogaster. D. pseiidoooscln Riley (1989) 'Cmmmmtomm-awmrmmmbymdmdmhm iunctiohal tests andlor in vitro protein binding smdles. Hymnal 1A 2“ 3A 4A 5A 9. 13° 14' 15’ 16 17° 18 19 20 21 22 23 24 25 139 Table A6. Synthetic Oligonucieotide Primers. TCCTTGCCGTATCCG TAGCACCGTAGTGGA TCAACTTCGATACGA TACATATATAACTTCGCCGCATGCAATTA GTI'GAGTTTCTGGAATAATC TGATACGGC‘ITACCATGCTCATCCATGCCG CGGAGGGCGAGGAGACG GGGCTGTCTI'TC'ITG TCGTAGATAGCAGGTGAGAT AGAGGCGGTI'I’GCGTAT GGTATAGTG CCCTATI'A AAACATTGTGACTG CATGATACTGATGATGCC TGGGAAATCGTATATCAGAGTATI'AAAGGTCTA CAGATGGATTCGACTGAGTTGGCATAGCAACTGC TTCACGCACGAGTCACTCAGATGGATTCGACTGA TACAGATCACATCTCTG CGTAGGAGATAAGTCGCTTCACGCACGAGTCACT TTCACACTCATCTAACC AGAGTATTAAAGGTCTACGTAGGAGATAAGTCGC ' Designated P2 in Chapter One " purified by HPLC ° purified by OPC column The D. melanogaster U0. 0. pseudoobscura U0 and and Dv. respectively. Application sequence Dm U0 sequence Dm U0 sequence Dm U0 hybridize to ecd1 uo mRNA sequence Dm U0 primer extension experiment sequence Dp U0 sequence Dp U0 sequence Dv U0 sequence Dv U0 sequence 0p U0 deletion of DR element at +11 deletion of DR element at -138 deletion at Dm U0 «808 to -703 deletion at 0m U0 -697 to 452 sequence Dm U0 deletion of 0m U0 ~434 to -302 . sequence Dm U0 deletion at Dm U0 -302 to -156 D. virilis U01 genes are abbreviated Dm. Dp 140 Table A7. P-aiement translormed lines and expression patterns 01 the U0 transgenes U0 expression Itamtunnamine Dunstan 3L 2 A. Mi 1 F138 4- . 4 4 5F1SC 4 - 4 4 1F30A P((w*A)DmUOPstl) + - + + 1 M110 4 . 4 4- 3M 11 4 . 4 4 1 M21 A 4 - 4 ND 3M9 P[(hspw’)DmUOSpel-Stui) - . . - ‘ M‘s C O O 1 F22A 4 . 4 4 3F47A 4 - 4 4 4r-'ssA P[(w’A)DpU0Rl) + . + i 4M1 5A 4 . 4 4 3M2“ 4 - 4 4 3F6A 4 - 4 4 1 FQA 4 . 4- 4 '2F15A P((w*A)DvU01Pstl] #1 . + i 1F19A + . 4 + 3M1A ° . 4 - 4 4 2FSB ' + 4 i . 1 F10!) P((w’A)DvU02Pstl) + + 4- . 4F13D 4 4 4 . 3F60 + . 4 4F14 P[(w*A)DmU0vlacZ) + - + + 1 F1 7 4 _ . 4 4 2F5 Pl(w+A)dal(-138.-126)DmU0vlacZ] + - 4 + 2F26 4 . 4 4 9M 23 - . p 4 111101 P[(w*A)dei(+11.+2s)0muo-ieczi - - p + ‘ M ‘1 8 e e 9 § 1 M161 . - p 4 4F10 . . p 4 21:28 - - . p 4 233: P|(w*°)dei(-136.-126)(+11.+23)0muo-ieczl - - p + O O * 2M23 ‘ . . E 4 U0 mRNA or U0-lacZ expression detected (+) or not detected H by Northern analyses or histochemical staining tor p-galactosidase activity. p-perturbed expression 01 the UOoIacZ transgene with only some adult llias within the stock showing p-galactosidase activity. ND-not determined.3L-third instar larvae. P-pupse. A-aduhs and Nit-Malpighian tubules. 141 Table A8. List of publications from this work. Wallrath, L.L.. J.B. Burnett and TB. Friedman (1990). Molecular characterization of the Drosophila melanogaster urate oxidase gene: An ecdysone- repressible gene expressed only in the Malpighian tubules. Mol. Cell. Biol. 10: 5114-5127. . Wallrath. LL. and TB. Friedman (1991). Species differences in the temporal pattern of DroSOphila UO gene expression are attributed to trans-acting regulatory differences. Proc. Natl. Acad. Sci. USA (in press). Friedman. T.B.. J.B. Burnett. S.L. Lootens and LL. Wallrath (1991). The urate oxidase gene of Drosophila pseudoobscura and Drosophila melanogaster. Evolutionary changes of sequence and regulation. (submitted for publication). Lootens, S.. J.B. Burnett, L.L. Wallrath and TB. Friedman (1991). Genetic and molecular analyses of strains of Dros0phila virilis having either a single copy of a tandem duplication of the urate oxidase gene. (in preparation). Wallrath. LL. and TB. Friedman (1991). Combinative oligonucleotide-directed large internal deletions as a method for surveying the regulatory region of a gene. (in preparation). Wallrath. L.L.. J.B. Burnett and TB. Friedman (1991). Sequence of the Drosophila virilis U0 gene and amino acid sequence comparison to U0 of D. melanogaster and D. pseudoobscura. (in preparation). Additional publications by LL. Wallrath: Friedman, T.B.. K.N. Owens, J.B. Burnett, A.O. Saura and LL. Wallrath (1991). The faint band/interband region 2802 to 2804-5(-) of the Drosophila melanogaster salivary gland polytene chromosome is rich in transcripts. Mol. Gen. Genet. (in press). Anathan. J., L.L. Wallrath, D.H. Johnson, T.B. Friedman and R. Voellmy (1991). Mapping target sites of the embryonic regulator fushi-tarazu on Drosophila chromosomes. (in preparation). LIST OF REFERENCES LIST OF REFERENCES Aguadé, M. (1988). Nucleotide sequence comparison of the rp49 gene region between Drosophila subobscura and D. melanogaster. Mol. Biol. Evol. 5: 433-441. Akerblom, I. 5., E.P. Slater. M. Beato, J.D. Baxter and P. 1.. Mellon (1988). Negative regulation by glucocorticoids through interference with a cAMP responsive enhancer. 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