X-linkage is not a general inhibitor of tissue-specific gene expression in Drosophila melanogaster.

As a consequence of its difference in copy number between males and females, the X chromosome is subject to unique evolutionary forces and gene regulatory mechanisms. Previous studies of Drosophila melanogaster have shown that the expression of X-linked, testis-specific reporter genes is suppressed in the male germline. However, it is not known whether this phenomenon is restricted to testis-expressed genes or if it is a more general property of genes with tissue-specific expression, which are also underrepresented on the X chromosome. To test this, we compared the expression of three tissue-specific reporter genes (ovary, accessory gland and Malpighian tubule) inserted at various autosomal and X-chromosomal locations. In contrast to testis-specific reporter genes, we found no reduction of X-linked expression in any of the other tissues. In accessory gland and Malpighian tubule, we detected higher expression of the X-linked reporter genes, which suggests that they are at least partially dosage compensated. We found no difference in the tissue-specificity of X-linked and autosomal reporter genes. These findings indicate that, in general, the X chromosome is not a detrimental environment for tissue-specific gene expression and that the suppression of X-linked expression is limited to the male germline.

'Escaping' the X chromosome leads to increased gene expression in the male germline of Drosophila melanogaster.

Genomic analyses of Drosophila species suggest that the X chromosome presents an unfavourable environment for the expression of genes in the male germline. A previous study in D. melanogaster used a reporter gene driven by a testis-specific promoter to show that expression was greatly reduced when the gene was inserted onto the X chromosome as compared with the autosomes. However, a limitation of this study was that only the expression regulated by a single, autosomal-derived promoter was investigated. To test for an increase in expression associated with 'escaping' the X chromosome, we analysed reporter gene expression driven by the promoters of three X-linked, testis-expressed genes (CG10920, CG12681 and CG1314) that were inserted randomly throughout the D. melanogaster genome. In all cases, insertions on the autosomes showed significantly higher expression than those on the X chromosome. Thus, even genes whose regulation has adapted to the X-chromosomal environment show increased male germline expression when relocated to an autosome. Our results provide direct experimental evidence for the suppression of X-linked gene expression in the Drosophila male germline that is independent of gene dose.

Patterns of DNA sequence variation suggest the recent action of positive selection in the janus-ocnus region of Drosophila simulans.

Levels of nucleotide polymorphism in three paralogous Drosophila simulans genes, janusA (janA), janusB (janB), and ocnus (ocn), were surveyed by DNA sequencing. The three genes lie in tandem within a 2.5-kb region of chromosome arm 3R. In a sample of eight alleles from a worldwide distribution we found a significant departure from neutrality by several statistical tests. The most striking feature of this sample was that in a 1.7-kb region containing the janA and janB genes, 30 out of 31 segregating sites contained variants present only once in the sample, and 29 of these unique variants were found in the same allele. A restriction survey of an additional 28 lines of D. simulans revealed strong linkage disequilibrium over the janA-janB region and identified six more alleles matching the rare haplotype. Among the rare alleles, the level of DNA sequence variation was typical for D. simulans autosomal genes and showed no departure from neutrality. In addition, the rare haplotype was more similar to the D. melanogaster sequence, indicating that it was the ancestral form. These results suggest that the derived haplotype has risen to high worldwide frequency relatively recently, most likely as a result of natural selection.

Stacking and stability of RNA duplexes containing fluorobenzene and fluorobenzimidazole nucleosides.

Six different fluorobenzene or fluorobenzimidazole ribonucleosides and one abasic site were incorporated in oligoribonucleotides. Individual contributions of base stacking and solvation of the modified nucleosides could be determined. In fluorobenzene.fluorobenzimidazole-modified base pairs a duplex stabilizing force was found that points to a weak F...H hydrogen bond. The lipophilicity of the unprotected nucleosides were investigated by determination of 1-octanol water partition coefficients.

Molecular evolution of the ocnus and janus genes in the Drosophila melanogaster species subgroup.

Genes involved in male fertility are potential targets for sexual selection, and their evolution may play a role in reproductive isolation and speciation. Here we describe a new Drosophila melanogaster gene, ocnus (ocn), that encodes a protein abundant in testes nuclear extracts. RT-PCR indicates that ocn transcription is limited to males and is specific to testes. ocn shares homology with another testis-specific gene, janusB (janB), and is located just distal to janB on chromosome 3. The two genes also share homology with the adjacent janusA (janA) gene, suggesting that multiple duplication events have occurred within this region of the genome. We cloned and sequenced these three genes from species of the D. melanogaster species subgroup. Phylogenetic analysis based on protein-encoding sequences predicts a duplication pattern of janA --> janA janB --> janA janB ocn, with the latter event occurring after the divergence of the D. melanogaster and Drosophila obscura species groups. We found significant heterogeneity in the rates of evolution among the three genes within the D. melanogaster species subgroup as measured by the ratio of nonsynonymous to synonymous substitutions, suggesting that diversification of gene function followed each duplication event and that each gene evolved under different selective constraints. All three genes showed faster rates of evolution than genes encoding proteins with metabolic function. These results are consistent with previous studies that have detected an increased rate of evolution in genes with reproductive function.

Deletion of a conserved regulatory element in the Drosophila Adh gene leads to increased alcohol dehydrogenase activity but also delays development.

In vivo levels of enzymatic activity may be increased through either structural or regulatory changes. Here we use Drosophila melanogaster alcohol dehydrogenase (ADH) in an experimental test for selective differences between these two mechanisms. The well-known ADH-Slow (S)/Fast (F) amino acid replacement leads to a twofold increase in activity by increasing the catalytic efficiency of the enzyme. Disruption of a highly conserved, negative regulatory element in the Adh 3' UTR also leads to a twofold increase in activity, although this is achieved by increasing in vivo Adh mRNA and protein concentrations. These two changes appear to be under different types of selection, with positive selection favoring the amino acid replacement and purifying selection maintaining the 3' UTR sequence. Using transgenic experiments we show that deletion of the conserved 3' UTR element increases adult and larval Adh expression in both the ADH-F and ADH-S genetic backgrounds. However, the 3' UTR deletion also leads to a significant increase in developmental time in both backgrounds. ADH allozyme type has no detectable effect on development. These results demonstrate a negative fitness effect associated with Adh overexpression. This provides a mechanism whereby natural selection can discriminate between alternative pathways of increasing enzymatic activity.

Comparative sequence analysis and patterns of covariation in RNA secondary structures.

A novel method of RNA secondary structure prediction based on a comparison of nucleotide sequences is described. This method correctly predicts nearly all evolutionarily conserved secondary structures of five different RNAs: tRNA, 5S rRNA, bacterial ribonuclease P (RNase P) RNA, eukaryotic small subunit rRNA, and the 3' untranslated region (UTR) of the Drosophila bicoid (bcd) mRNA. Furthermore, covariations occurring in the helices of these conserved RNA structures are analyzed. Two physical parameters are found to be important determinants of the evolution of compensatory mutations: the length of a helix and the distance between base-pairing nucleotides. For the helices of bcd 3' UTR mRNA and RNase P RNA, a positive correlation between the rate of compensatory evolution and helix length is found. The analysis of Drosophila bcd 3' UTR mRNA further revealed that the rate of compensatory evolution decreases with the physical distance between base-pairing residues. This result is in qualitative agreement with Kimura's model of compensatory fitness interactions, which assumes that mutations occurring in RNA helices are individually deleterious but become neutral in appropriate combinations.

A highly conserved sequence in the 3'-untranslated region of the drosophila Adh gene plays a functional role in Adh expression.

Phylogenetic analysis identified a highly conserved eight-base sequence (AAGGCTGA) within the 3'-untranslated region (UTR) of the Drosophila alcohol dehydrogenase gene, Adh. To examine the functional significance of this conserved motif, we performed in vitro deletion mutagenesis on the D. melanogaster Adh gene followed by P-element-mediated germline transformation. Deletion of all or part of the eight-base sequence leads to a twofold increase in in vivo ADH enzymatic activity. The increase in activity is temporally and spatially general and is the result of an underlying increase in Adh transcript. These results indicate that the conserved 3'-UTR motif plays a functional role in the negative regulation of Adh gene expression. The evolutionary significance of our results may be understood in the context of the amino acid change that produces the ADH-F allele and also leads to a twofold increase in ADH activity. While there is compelling evidence that the amino acid replacement has been a target of positive selection, the conservation of the 3'-UTR sequence suggests that it is under strong purifying selection. The selective difference between these two sequence changes, which have similar effects on ADH activity, may be explained by different metabolic costs associated with the increase in activity.

RNA secondary structure and compensatory evolution.

The classic concept of epistatic fitness interactions between genes has been extended to study interactions within gene regions, especially between nucleotides that are important in maintaining pre-mRNA/mRNA secondary structures. It is shown that the majority of linkage disequilibria found within the Drosophila Adh gene are likely to be caused by epistatic selection operating on RNA secondary structures. A recently proposed method of RNA secondary structure prediction based on DNA sequence comparisons is reviewed and applied to several types of RNAs, including tRNA, rRNA, and mRNA. The patterns of covariation in these RNAs are analyzed based on Kimura's compensatory evolution model. The results suggest that this model describes the substitution process in the pairing regions (helices) of RNA secondary structures well when the helices are evolutionarily conserved and thermodynamically stable, but fails in some other cases. Epistatic selection maintaining pre-mRNA/mRNA secondary structures is compared to weak selective forces that determine features such as base composition and synonymous codon usage. The relationships among these forces and their relative strengths are addressed. Finally, our mutagenesis experiments using the Drosophila Adh locus are reviewed. These experiments analyze long-range compensatory interactions between the 5' and 3' ends of Adh mRNA, the different constraints on secondary structures in introns and exons, and the possible role of secondary structures in RNA splicing.

Long-range base pairing in Drosophila and human mRNA sequences.

The potential for long-range base pairing between the 5' and 3' ends of mRNA molecules was examined for 134 Drosophila and 204 human sequences collected from the GenBank database. Each sequence was divided into two parts, a 5' sequence taken from the start of the protein-encoding region and a 3' sequence taken from the end of the transcript. The strongest RNA pairing stem between each pair of 5' and 3' sequences was identified and scored using an alignment program modified to incorporate RNA base pairing. The observed pairing scores were then compared with a random distribution of scores generated by aligning each 5' sequence to random permutations of its corresponding 3' sequence. For both the Drosophila and the human mRNAs, the observed pairing scores were significantly biased toward the upper tail of the random distributions, with 61% of the Drosophila sequences and 64% of the human sequences falling within the upper half of the random distributions. This suggests that a pattern of long-range base pairing may be a common feature of eukaryotic mRNAs. We have also analyzed a subset of Drosophila and human mRNAs which show the greatest potential for long-range pairing. The human pairings appear to be stronger and localized to more specific regions near the ends of the mRNA sequence than those of Drosophila.

Site-directed mutations reveal long-range compensatory interactions in the Adh gene of Drosophila melanogaster.

Long-range interactions between the 5' and 3' ends of mRNA molecules have been suggested to play a role in the initiation of translation and the regulation of gene expression. To identify such interactions and to study their molecular evolution, we used phylogenetic analysis to generate a model of mRNA higher-order structure in the Adh transcript of Drosophila melanogaster. This model predicts long-range, tertiary contacts between a region of the protein-encoding sequence just downstream of the start codon and a conserved sequence in the 3' untranslated region (UTR). To further examine the proposed structure, site-directed mutations were generated in vitro in a cloned D. melanogaster Adh gene, and the mutant constructs were introduced into the Drosophila germ line through P-element mediated transformation. Transformants were spectrophotometrically assayed for alcohol dehydrogenase activity. Our results indicate that transformants containing a silent mutation near the start of the protein-encoding sequence show an approximately 15% reduction in alcohol dehydrogenase activity relative to wild-type transformants. This activity can be restored to wild-type levels by a second, compensatory mutation in the 3' UTR. These observations are consistent with a higher-order structure model that includes long-range interactions between the 5' and 3' ends of the Adh mRNA. However, our results do not fit the classical compensatory substitution model because the second mutation by itself (in the 3' UTR) did not show a measurable reduction in gene expression.

Molecular evolution of the metallothionein gene Mtn in the melanogaster species group: results from Drosophila ananassae.

Three distinctly different alleles of the metallothionein gene Mtn have been identified in natural Drosophila melanogaster populations: Mtn.3, Mtn1, and Dp(Mtn1), where the latter designates a tandem duplication of Mtn1. In Drosophila simulans, only Mtn.3-type alleles have been found. It has been suggested that Mtn.3 is the ancestral allele and demonstrated that a presumed two-step transition from Mtn.3 to Mtn1 to Dp(Mtn1) is accompanied by an approximate 5-fold increase in RNA levels. We analyzed the evolutionary genetics of the Mtn locus of Drosophila ananassae, a distant relative of D. melanogaster and D. simulans within the melanogaster species group. The Mtn gene of D. ananassae is most similar to Mtn.3: (i) it is identical with Mtn.3 at the amino acid level, but differs from Mtn1 in its terminal codon; (ii) its 3' UTR contains a characteristic extra DNA segment of about 50 bp which is present in Mtn.3, but lacking in Mtn1; (iii) duplications of Mtn were not found in a worldwide sample of 110 wild D. ananassae chromosomes. However, the intron of the Mtn gene in D. ananassae is only 69 bp long, whereas the length of the Mtn.3 and Mtn1 introns is 265 bp; and it lacks a polypyrimidine stretch upstream of the 3' splice site in contrast to the much greater pyrimidine-richness found in the Mtn.3 and Mtn1 introns. A short intron (67 bp) was also identified in a D. pseudoobscura Mtn allele, suggesting that the short intron is the ancestral form and that the transition from the short to the long intron occurred within the melanogaster species group.(ABSTRACT TRUNCATED AT 250 WORDS)