Molecular organization of the alcohol dehydrogenase loci of Drosophila grimshawi and Drosophila hawaiiensis

GENE AN INTERNATIONAL *JOURNAL GENES AND G E N O M E 5

ELSEVIER

ON

Gene 181 (1996) 51-55

Molecular organization of the alcohol dehydrogenase loci of Drosophila grimshawi and Drosophila hawaiiensis M a r k D. Brennan a,., Patrick A. Thorpe 1,b, Jie Hu a, W.J. Dickinson b a Department of Biochemistry, School of Medicine, The University of Louisville, Louisville, KY40292, USA b Department of Biology, University of Utah, Salt Lake City, UT84112, USA Received 16 February 1996; revised 19 April 1996; accepted 29 April 1996

Abstract To determine sequences involved in conserved and species-specific aspects of alcohol dehydrogenase (Adh) gene expression in Hawaiian Drosophila, a 3644-base pair (bp) region containing the D. grimshawi gene and the homologous 3335-bp region containing the D. hawaiiensis Adh gene were sequenced. These genes have the two-promoter and exon-intron structure seen for many Drosophila Adh genes. Analysis of putative and known regulatory sequences of the D. grimshawi and D. hawaiiensis genes in comparison to those of D. affinidisjuneta (the only other Hawaiian species for which the promoter organization is known) highlighted elements likely to be involved in conserved aspects of Adh gene expression as well as sequences that may account for ~species-specific differences in tissue-specific expression. Sequence comparisons, in the context of regulatory roles previously assigned to particular gene fragments, indicated that multiple insertions and deletions in the promoter regions are responsible for differences in tissue-specific regulation displayed by these genes.

Keywords: Gene structure; Regulatory variation; Promoter elements; Dna footprints; Transcriptional regulation; Conserved introns

1. Introduction A number of studies documenting evolutionary alterations in developmental patterns of gene expression have focused on the picture-winged Drosophila of the Hawaiian islands (Dickinson, 1980; Thorpe and Dickinson, 1988; Thorpe et al., 1993). Of the various gene-enzyme systems studied in these species, the alcohol dehydrogenase (Adh) genes from D. affinidisjuncta, D. grimshawi, and D. hawaiiensis are the best understood mechanistically. The regulatory differences that these genes display in their native species are conserved in D. melanogaster germ-line transformants carrying the genes (Brennan and Dickinson, 1988; Brennan et al., 1988; Wu et al., 1990). Germ-line transformation analysis using chimeric genes shows that the sequences responsible for these regulatory differences m a p to the putative pro* Corresponding author. Tel. + 1 502 5887732; Fax + 1 502 5886222; e-mail: [email protected] 1 Present address: BiologyDepartment, Grand Valley State University, Allendale, MI 49401-9403, USA. Abbreviations: Adh, Alcohol dehydrogenase (gene); Bp, base pair(s); Myr, million years; Nt, nucleotide(s). 0378-1119/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PHS0378-1119(96)00459-3

moter regions of the three genes (Fang and Brennan, 1992; Fang et al., 1991; Wu and Brennan, 1993). Moreover, m a n y redundant elements, including a downstream promoter duplication, play roles in expression of the D. affinidisjuncta gene in the larval fat body (McKenzie et al., 1994). Thus, it was of interest to determine the sequences of the D. grimshawi and D. hawaiiensis genes and to compare these to that previously reported for D. affinidisjuncta (Rowan and Dickinson, 1988). By this means, sequences involved in conserved aspects of Adh gene expression (particularly expression in the larval and adult fat bodies) and those responsible for species-specific regulatory differences can be identified.

2. Experimental and discussion 2.1. Major features of locus structure are conserved Fig. 1 shows restriction maps of the sequenced regions from D. hawaiiensis and D. grimshawi in comparison to the homologous genomic segment from D. affinidisjuncta. These genomic segments contain sequences accounting

52

M.D. Brennan et al./Gene 181 (1996) 51-55

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Proximal Transcript Fig. 1. Restriction maps of the regions sequenced from D. hawaiiensis and D. grimshawi and the corresponding region from D. affinidisjuncta. The transcription map of the D. affinidisjuncta gene is shown with thick bars representing exons and thin lines representing introns (Brennan et al., 1984; Rowan et al., 1986; Rowan and Dickinson, 1988). The cloning of the D. grimshawi gene from strain G1 and of the D. hawiiensis gene from strain J14B8 have been reported elsewhere (Rabinow and Dickinson, 1986; Brennan et al., 1988). These maps differ from previously published maps (Fang et al., 1991; Wu and Brennan, 1993) in that the latter show only the single EcoRI site used to generate gene chimeras.

for species-specific differences in the tissue- and stagespecific production of two Adh transcripts (Fang and Brennan, 1992; Wu and Brennan, 1993). The major Adh RNA in adults (distal transcript) arises from an upstream promoter, and the major form in larvae (proximal transcript) arises from a downstream promoter in each species (Rowan and Dickinson, 1986; Rowan et al., 1986). The two transcripts from a given gene differ only in their 5'-nontranslated leader region. Sequences within the distal-specific intron (intron 1) contribute to the 5'-end of the proximal transcript. However, the two transcripts share a portion of the 5'-leader as well as coding and 3'-nontranslated regions. As shown in detail in Fig. 2, the organization of the D. grimshawi and D. hawaiiensis genes is similar to that described for the well-characterized D. affinidisjuncta

gene (Rowan et al., 1986; Brennan and Dickinson, 1988; Green et al., 1989). The inferred intron-exon structure for the D. grimshawi and D. hawaiiensis genes is identical to that for all Drosophila Adh genes described to date (Sullivan et al., 1990). The coding sequences were used to determine the phylogenetic relationships between D. grimshawi, D. hawaiiensis and the other Hawaiian Drosophila for which Adh sequences are available (Rowan and Dickinson, 1988; Thomas and Hunt, 1991; Rowan and Hunt, 1991). In this analysis, D. grimshawi and D. hawaiiensis are grouped as the closest relatives of D. affinidisjuncta regardless of the phylogenetic inference method used. For example, trees based on synonymous substitutions calculated by the method of Li (1993) and using either neighbor-joining/UPGMA ( N E I G H B O R ; Felsenstein, 1993) or Fitch-Margoliash (FITCH; Felsenstein, 1993) methods show D. hawaiiensis branching off immediately before the D. grimshawi/D, affinidisjuncta split. Thus, the D. grimshawi gene is most closely related to the D. affinidisjuncta gene, and both of these are about equally distant from the D. hawaiiensis gene. Given a rate of change for synonymous substitutions of 0.015 substitutions/nucleotide (nt)/million years (Myr) (Rowan and Hunt, 1991), the times of divergence are: 0.6 Myr for D. grimshawi vs. D. affinidisjuncta; 1.7 Myr for D. hawaiiensis vs. D. affinidisjuncta; and 1.9 Myr for D. hawaiiensis vs. D. grimshawi. Both the D. grimshawi and D. hawaiiensis genes carry distal TATA boxes identical to that of D. affinidisjuncta (TATAAAA). These are located between nt 1426 and 1432 in D. grimshawi and between nt 1311 and 1317 in D. hawaiiensis. Similarly, both genes have appropriately positioned proximal TATA boxes (TATAAATA), conforming to the consensus proximal TATA box for Drosophila Adh genes (Sullivan et al., 1990). These are located between nt 2024 and 2031 in D. grimshawi and between nt 1698 and 1705 in D. hawaiiensis. In addition, other upstream sequences with demonstrated or probable regulatory functions are conserved. These include several of the footprinting regions identified by Hu et al. (1995). Notably, both the D. grimshawi and the D. hawiiensis genes contain copies of the sequence TGATAA (beginning at nt 1977 and 1651, respectively). The positions of these, in reference to the

Fig. 2. Comparison of Adh sequences and locus structure. Species names are abbreviated as follows: gri=D, grimshawi (GenBank accession No. U48714), and haw=D, hawaiiensis (GenBank accession No. U48715). Dots indicate that the same base is present in the two sequences. Dashes represent the absence of any base. The positions of RNA start sites, TATA boxes, distal intron boundaries, the translation start, and the major 3'-end of the mRNA, as determined for the D. affinidisjuncta gene, are designated by asterisks and underlining (Rowan et al., 1986; Green et al., 1989). The entire coding region is also underlined. The positions of footprinting sequences located between the two promoters of the D. affinidisjuncta gene are designated by heavy lines above the D. grimshawi sequence (Hu et al., 1995). Methods: Sequences were determined by sequencing sets of overlapping DNA fragments subcloned into M13mpl8 or mp19 (Messing, 1983; Yanisch-Perron et al., 1985) or into pBluescript (Alting-Mees and Short, 1989). Both strands were sequenced throughout by the dideoxy chain-termination method (Sanger et al., 1977). In some cases, doublestranded plasmid DNAs were sequenced by a modification of this method employing a chemically modified T7 DNA polymerase (Sequenase, US Biochemical Corp.).

M.D. Brennan et al./Gene 181 (1996) 51-55 gzi

53

AAGCTT~AAGAAGATGACGTTGAAATGTTGCATCGTTATCCAAGCCTCCA-~AGTTTACTCCACTTTTATTG~ACACCT~GCT~CCT~TCCCATCAACTTACAA~AATAACTATT~TAATA1 2 0

haw

............................................

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TG-TATAGGTTTCTGTGCATGTATTGTGAGAGAGTGAGGTGTGTCAAGTGTCGAGTGTGTGTGAGCTGCCACGTTTTCAAAATCAAATTGAAACTTAATTTTCACCTGAATGCTCTTTTAT

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.............

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AATTTATAGATTAATAAATTGGACTTTGTTTGTTGTTGTTAAATAGCTGTAATGTAATCTATTGATAATTGTAAAACATTGCTAAAGGCAAACAGCAAACATTTTAAGAACCCTAACTTGT

haw

...................................

gzi

AGTCGGATTAAGAAGCGTGGCGACTT-ATATAATAGATTGACTATAAAGTGTGTTTTATTTCTAGTTTCGTTGACAATCAAATTCAATATGTGTGGCAATAGCGCAGAATAGGTCAGTAAT

..............................

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GCCAGATATCAGG~CATATTGTCGTCAGA~GTCAGAGGTATTTTGTGGTATATTGTATATAAGTCAGGTTCTGCTAA~CATTTCATAAATGAAATGCATTTATT~TTATT~TACTTGAATC

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haw

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CACTTCTCGCACTCTCTGCGCCGTCAGTCAATTTGAATAATAATGCAAATAATGCAACCTGCGAACATAACAACAACAATA

haw

.- .........

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GGTTCGGGGCTTCAAGTGCGTTCCGCTCTCTGCAGCTCAGTTTCATCTCTATCTTATTAATGATCTCCCCTATTTGTTGG-GTTATATTCAAAGTAGTGGCTTACATCTCTTAAGCATTCA

haw

......

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CGATTTCAACATCGACTCGTGTGTGTAAGTATAAAAGGTGTTACTCGCACTGAGAACATTGATTATTGCCATAGCAGTCGACGCACGCAGATCGGACCAGTTACAATATTTTCAAAAAGTA

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TGTCTGCGCCTCGCTCTCACGTCACAATTCATGGTGATTATGTATAATCAGCAACTCGTCTGATTGCGGTTAAGAAGCGACTTATTCCTAACAAGAGTGATAACCCGGACTAGTGTGAGAT

A . . . . . . . . .

263

ACAATTATAAATCGGCTCATATTTTTGTAGTTTCTTGATCAAGCTAATTTAATATAATTAGAATTCTCG

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GGATTTTAAGTATGACCAGATAAAGATAAAGT

...........

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AGTGAAGTTCAGAAAGTCCAAACTAGAACT~GCGCGTCCGGCAAAAAAATTGTAATCTGAATTTTCTAGCAAAAGTTTCCATGGGCACAGGCTAATAGACAATTAAATAAAAGATTAAAAT ......

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AAG-CAAAATATACACACGTACATAAACATTGGAACTA-CAAATACATAAAT-AATAAAAAAAAAAAAAAATGTCTGTATGTGCATGTACATCCAAGAATATCTATTCCAGGTTTTATTAT

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TTGTTTGGTCGATATGACCTTCAATATGTTTAATAATGCTGGACAAAATTAGAATACCCCAATAGAAGAGAATATAATAACACAAATATGTATGTATGCATCTAAATTAAAGCAAGTAGAA

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TGAGAAGAGCCGGCAATATTTGACTGCAATAACCAAAACAACAAATCAAAACAAAAAAAAGTCAAGACCACGAACAGTGCTGATGATCGC-CAACATTACTGATAAACAGCTGCGAGGAAA

haw

........................

gzi

TACTCAAACTTAGCAGACTGTTCGCCTATAAATAGAAGGTATCAACCGGCCAAACTCATATAGTGTTTTGAATTCTCTGTCTGAACAGGTGAACAGAGTTGAGGGTATCACTGAAAAAATG

haw

..............................................

................

C.GAG

......

T..T.AGG

.............

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1790

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BEGIN

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GTTATCGCTAACAGTAACATCATCTTTGTCGCTGGTCTGGGTGGCATTGGCCTGGACACCAGTCGCGAGATTGTCAAGAGTGGCCCCAAGGTAGGTTACAGCCTGTCAATCTCAGGAAAAA

2239

haw

.........................................................................................................................

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GTATGGAAGAATAAAATAAAATTATT

....

ATTATTATTATTTTTTGTTTTTGTTTTTTTCGTTTAGAACCTGGTGGTGCTTGACCGCGTTGACAACCCCGCTGCCATTGCCGAGTTGAAA

2356

haw

..........................

TTTT

.........

2021

gel

GCCCTTAATCCCAAGGTGACCATTACATTCTATCCGTATGATGTAACCGTGCCGTTGGCTGAAACCAAAAAGCTATTGAAGACAATCTTCGACAAGCTGAAGACAGTTGATCTGCTCATCA A ......

.AT..C..C

.................

T .......

T...A

................................

2477

haw

..............

gzx

ATGGCGCTGGTATCCTGGACGATAATCAGATTGAGCGCACAATTGCGGTGAATTTTACAGGTACAGTGAACACCACAACTGCGATCTTGGATTTCTGGGACAAGCGTAAGGGCGGCCCAGG

haw

..............................................

gr1

TGGTGTTGTTGCCAACATTTGCTCAGTAACCGGATTCAACTCCATCTATCAGGTGCCCGTTTACTCTGCCTCAAAAGCGGCTGCTCTAAGCTTCACCACTTCCATTGCGGTAAGTAAATAT

haw

. . . . . . . . . . . . . . . . . .

g=x

ACATATATATATATATATATACTTATCCTTCATGAGGGGA•TACCTATT•AATCT•AACAGAAATTGGCG•ATATTACTGGCGTTAC•GCATATTCCAT•AACCCGGG•ATTACCAAGACT

haw

.T

gz~

GTTCTGGTGCATAAATTCAACTCCTGGCTCGGTGTTGAG~A~GCGTTGC~GAG~TA~TGCTTGAG~ATCC~A~ACAGACAACATTGCAGTGCGCACAGAA~TTTGTGAAGGC~ATTGAGG 2961

haw

. . . . . . .

gzi

CCAA~CAGAATGGTGCCATCTGGAAACTGGATCTTG~CCGTCTGGATGCAATCGAATGGACCAAG~ACTGGGACTCAGGCATCTAAACTGTGCATGAAATTCGTACAAGGTGTGAAA~TGC3 0 8 2

C . . . . . . . . . . .

. . . . . . . . . . . . . . . .

A ....

TCT

G .............

- ..........

---T

....

C .....................................................................................

T ...............

TTG

2142 2598

.......................................................

T

2719

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ATTTAAAAT-CCATT~CCAAAATTTGATAAGAAGTATATATCCATT~TTAA-CA~CGTTAAGTACGAAAAAACAAATTCTTTTCTTCAGTTT-ATAATTAATGAAATTAACA~AAC.D~`AAA 3200

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AGTGGCGCGTAGGGCTAAACAATTCATAAATCTGAATTTTCTGTCAAACTTTCCCGAGCAAAGGCTGATAAACAAATAAACATTTAAGCATACAAATTTGCAAAATAA~ACAAATCTTTTC

haw

..........

- .................

AA.TT

.

haw

C ....

G ...............................

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...................................

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T ....

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ATCTAATAGTGGCAACTAATGTCTGTATGTGTATTTGCATCCAAGAATATCTATTCCAGGTTTTATTATTAGATTGGTATATAAATTAGAATACCCCAATAGGAGAGTATATAAATAATAT ..T

............................................................................

AAATATGTATGTATGCATCTAAATTAAGCAAGTAGAATGAGA-GAGCCGGTAATATTTGACTG-AGTAGCCAAACCAACACGTTAAAAGCAA

haw

.................................

gri

GATGATCACCAACGTTAGCGATAAACAGCAGCGAGTAAATACGCAATACCTTC

haw

-....G.GA

....

A...CT.-

........

T .....

A .......

C ........

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................ G ....

-.T...-.-

....

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A

. . . . . . . . . .

T ............

A .......... 3'RNA

GCAGACTGTTCGCCTAT

A ......

AAAATAGA-GGT .......

2864

A..T

.......

2984

3442

.... ..........

2743

END*

- ......................................

gri

C ........

.

T ............................

MAJOR

haw

.

3321

A ...................

gri

.

AC

......

........ A...ACAACAGG

A..GAAA

C.C

3104

AAAAACAAATACCACGAGCAGTGCT

3557

.........................

3225

CCAAACTCATATA---TTTTGAATTC

3664

...........

3335

A.GTG

..........

54

M.D. Brennan et al./Gene 181 (1996) 51 55

proximal RNA start site, are identical to those seen for the corresponding sequence in the D. affinidisjuncta gene. The T/AGATAA sequence binds to the transcriptional stimulatory protein ABF (Abel et al., 1993), and is needed for full expression of the D. affinidisjuncta gene in the larval fat body (Hu et al., 1995). Moreover, there is conservation of some of the noncoding sequences within the transcribed region. With the exception of a single nt deletion in D. hawiiensis, the distal-specific mRNA leaders of both species are identical to those of D. affinidisjuncta. It is also noteworthy that the 5'-most 57 nt of intron 2 (beginning at nt 2209 in D. grimshawi) are identical in both genes. Again, comparison to the D. affinidisjuncta sequence shows perfect conservation between the three Hawaiian species. This conservation is consistent with a role for the intron in gene expression. Two other observations support this possibility: the work of Stephan and Kirby (1993) indicates that this sequence contains a conserved stemloop structure. Also, the work of McKenzie, 1994 shows that deletion of this intron results in a pronounced increase in the ratio of RNA to protein in larvae. The presence of a promoter duplication located 3' to the transcribed region appears to be a feature that is unique to the Hawaiian picture-winged species. This duplication is present in both D. grimshawi (beginning at nt 3198) and D. hawaiiensis (beginning at nt 2862). In D. affinidisjuncta, the only picture-winged species for which sequence analysis has extended beyond the 3'-duplication, there is no evidence for another structural gene lying farther downstream (Rowan and Dickinson, 1988). This, coupled with the lack of canonical TATAbox sequences in the 3'-duplications, argues against the presence of a downstream gene in these species. It is likely that the promoter duplication plays a role in expression of the Adh genes in the picture-winged flies, and this may account for its retention in the absence of a downstream structural gene. Using transient transformation of larvae, McKenzie et al. (1994) presented evidence that the 3'-duplication in D. affinidisjuncta may act as an enhancer in the larval fat body - a possibility supported by the negative consequences of deleting this sequence as assayed also by germ-line transformation (McKenzie, 1994).

2.2. Sequence differences account for species-spec!fic regulatory differences Finally, it is of interest to consider the sequences of those gene fragments that determine the tissue-specific regulatory differences between the various Adh genes from the Hawaiian species. Functional assay of chimeric genes in D. melanogaster transformants has shown the importance of upstream regions in this regard (Fang et al., 1991; Fang and Brennan, 1992; Wu and Brennan, 1993).

Sequences between the distal and proximal promoters account for several tissue- and stage-specific expression differences displayed by the proximal promoter. Given earlier findings regarding sequences that play a role in expression in the larval and adult midguts (Fang and Brennan, 1992), the 41-bp deletion in D. hawaiiensis (beginning at nt position 1617) likely reduces expression of the proximal promoter in these tissues. Moreover, the D. hawaiiensis gene lacks the sequence G A T C G C (found at nt position 1962 in D. grimshawi). This sequence is needed for full expression of the D. affinidisjuncta gene in the larval fat body (Hu et al., 1995). Consistently, the proximal promoter of the D. hawaiiensis gene is threeto four-fold less active in larval fat body than is the D. affinidisjuncta gene (Fang and Brennan, 1992). However, the presence of the G A T C G C sequence in the D. affinidisjuncta gene, by itself, cannot account for the strong expression of the proximal promoter in the adult midgut, because the D. grimshawi gene, which contains this sequence, is not expressed at such a high level in this tissue (Wu and Brennan, 1993). Strong expression in the larval Malpighian tubules is conferred to the D. hawaiiensis proximal promoter by replacing sequences from nt 1358 to 1602 with the corresponding region from D. affinidisjuncta (Fang and Brennan, 1992). The 162-bp deletion in D. hawaiiensis (beginning at position 1589) may be responsible for the failure of the D. hawaiiensis gene to be expressed in the larval Malpighian tubules. Neither the D. grimshawi nor the D. affinidisjuncta gene carries this deletion, and both are expressed strongly in this tissue (Dickinson, 1980; Wu et al., 1990). This deletion removes several footprinting sequences that might reflect interactions with relevant trans-acting proteins (Hu et al., 1995). The sequences of the distal promoters shed some light on the nt substitutions responsible for species-specific differences in expression of the distal promoters in the adult Malpighian tubules (Fang and Brennan, 1992). Substitution of D. hawaiiensis sequences between nt 1190 and 1358 with the corresponding D. affinidisjuncta sequences confers expression in the adult Malpighian tubules to the D. hawaiiensis gene, while the reciprocal construction, like the D. hawaiiensis gene, is inactive in this tissue (Fang and Brennan, 1992). Comparison between these two genes reveals no large insertions or deletions in this region. Therefore, one or more of the nt substitutions in this segment (immediately upstream and including the distal RNA start site) are responsible for the regulatory differences. There are seven nt differences between D. affinidisjuncta and D. hawaiiensis within 20 nt upstream of the distal RNA start site, four of which are held in common between D. hawaiiensis and D. grimshawi. Given that the D. grimshawi gene is expressed in the adult Malpighian tubules, either these four substitutions are not important for expression in this tissue, or sequences elsewhere on the D. grimshawi

M.D. Brennan et al./Gene 181 (1996) 51 55

gene compensate to allow expression. We favor the latter possibility because preliminary results indicate that far upstream sequences are needed for expression of the D. grimshawi, but not the D. affinidisjuncta gene, in the adult Malpighian tubules (M. Brennan, unpublished data). Further functional analyses, in the context of the present sequence information, will be needed to clarify the roles of distal promoter sequences in this tissuespecific regulatory difference.

Acknowledgement We thank Dr. J. Felsenstein for providing the PHYLIP software package and Dr. W.-H. Li for providing the FORTRAN program used to estimate synonymous and nonsynonymous substitutions. This work was supported by National Institutes of Health grants HD 10723 to W.J.D. and GM 34961 to M.D.B.

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