Genetic properties influencing the evolvability of gene expression.

Identifying the properties of gene networks that influence their evolution is a fundamental research goal. However, modes of evolution cannot be inferred solely from the distribution of natural variation, because selection interacts with demography and mutation rates to shape polymorphism and divergence. We estimated the effects of naturally occurring mutations on gene expression while minimizing the effect of natural selection. We demonstrate that sensitivity of gene expression to mutations increases with both increasing trans-mutational target size and the presence of a TATA box. Genes with greater sensitivity to mutations are also more sensitive to systematic environmental perturbations and stochastic noise. These results provide a mechanistic basis for gene expression evolvability that can serve as a foundation for realistic models of regulatory evolution.

Marginal fitness contributions of nonessential genes in yeast.

Analysis of the complete genome sequence of Saccharomyces cerevisiae confirms and extends earlier evidence that a majority of yeast genes are not essential, at least under laboratory conditions. Many fail to yield a discernible mutant phenotype even when disrupted. Genes not subject to natural selection would accumulate inactivating mutations, so these "cryptic" genes must have functions that are overlooked by the standard methods of yeast genetics. Two explanations seem possible: (i) They have important functions only in environments not yet duplicated in the laboratory and would have conditional phenotypes if tested appropriately. (ii) They make small, but significant, contributions to fitness even under routine growth conditions, but the effects are not large enough to be detected by conventional methods. We have tested the second "marginal benefit" hypothesis by measuring the fitnesses of a random collection of disruption mutants in direct competition with their wild-type progenitor. A substantial majority of mutant strains that lack obvious defects nevertheless are at a significant selective disadvantage just growing on rich medium under normal conditions. This result has important implications for efforts to understand the functions of novel genes revealed by sequencing projects.

Morphogenesis of denticles and hairs in Drosophila embryos: involvement of actin-associated proteins that also affect adult structures.

We sought components that function in morphogenetic events downstream from the segmentation pathway in Drosophila embryos, so we examined mutations that affect development of adult hairs and/or bristles to identify a subset that also affect hairs and denticles on the cuticle of first instar larvae. Mutations at 4 of 23 loci surveyed cause distinct abnormalities in these larval structures, and two other loci have more subtle, variable effects. In particular, forked and singed mutants produce complex, allele-specific phenotypes. These loci encode actin-associated proteins and, consistent with that information, mutations cause abnormalities in actin bundles that support nascent hairs and denticles in stage 14-16 embryos. We suggest that interactions between these and other actin-associated proteins are important in generating the diverse shapes of the cuticular specializations seen in both larvae and adults.

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

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. affinidisjuncta (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.

Evolution of regulatory genes and patterns: relationships to evolutionary rates and to metabolic functions.

In an effort to understand the forces shaping evolution of regulatory genes and patterns, we have compared data on interspecific differences in enzyme expression patterns among the rapidly evolving Hawaiian picture-winged Drosophila to similar data on the more conservative virilis species group. Divergence of regulatory patterns is significantly more common in the former group, but cause and effect are difficult to discern. Random fixation of regulatory variants in small populations and/or during speciation may be somewhat more likely than divergence driven by selection. Within the picture-winged group, we also have compared enzymes that fulfill different metabolic roles. There are highly significant differences between individual enzymes, but no obvious correlations to functional categories.

CONSERVATION OF MOLECULAR PREPATTERNS DURING THE EVOLUTION OF CUTICLE MORPHOLOGY IN DROSOPHILA LARVAE.

We are using patterns of cuticle specialization in Drosophila larvae as models to investigate the molecular, genetic, and developmental bases of morphological evolution. Members of the virilis species group differ markedly from one another in the distribution of hairs on the dorsal surface of first instar larvae. In particular, characteristic bands of hairs cover about 20% of each trunk segment in some species but about 70% in others. These major types do not correlate with recently proposed phylogenetic relationships, suggesting that similar phenotypes have arisen independently in different lineages. The patterns of expression of several genes that control or reflect intrasegmental patterning are indistinguishable in species with very different cuticle morphologies. We conclude that, in this case, morphology probably has evolved via altered response to a conserved molecular prepattern.

Nucleotide sequence of the genomic region encoding alcohol dehydrogenase in Drosophila affinidisjuncta.

The DNA sequence of a 3886-bp genomic region containing the alcohol dehydrogenase (Adh) gene from Drosophila affinidisjuncta, and the RNA sequences of the D. affinidisjuncta Adh transcripts, are presented. These data support the conclusion that two Adh promoters generate distinct, developmentally regulated Adh transcripts. Correlations between these sequences and the transcription map are discussed. Comparisons between these and equivalent data from D. melanogaster are also presented. We note the following observations: (1) Except at the extreme 3' end, the two genes are identically organized. (2) Drosophila Adh protein accumulates amino acid replacements at the rate of approximately 0.5 per million years. (3) Among the non-protein-coding DNA sequences, putative homologies occur in the two promoter regions.

Complex developmental regulation of the Drosophila affinidisjuncta alcohol dehydrogenase gene in Drosophila melanogaster.

During development, the alcohol dehydrogenase genes of Drosophila melanogaster and D. affinidisjuncta are expressed in similar, yet distinct, tissue- and stage-specific patterns. Transcripts from both of these genes arise from two promoters (distal and proximal) that also display tissue and stage specificity. We used P-element-mediated transformation to introduce the D. affinidisjuncta Adh gene into the germ line of D. melanogaster. We show that the D. affinidisjuncta Adh gene is expressed at comparable overall levels in both species and that the tissue- and stage-specific expression for this gene (including promoter utilization) is similar in the donor and the host species. However, in some details, the expression of the D. affinidisjuncta gene in D. melanogaster resembles the host pattern, and one novel tissue-specific expression phenotype is displayed by transformants. In general, our results suggest that there has been strong conservation of cis- and trans-acting regulatory factors since the divergence of the two species but that this conservation has not been perfect.

Developmentally regulated RNA transcripts coding for alcohol dehydrogenase in Drosophila affinidisjuncta.

The organization of the gene coding for alcohol dehydrogenase (Adh) in Drosophila affinidisjuncta has been determined by physically mapping Adh RNA transcripts to cloned genomic DNA. Two distinct transcript types accumulate with developmental specificity. Because only a single genomic Adh locus is detected in D. affinidisjuncta, and since all Adh transcripts appear to be identical except at their termini, the two Adh RNA types are products of the same gene. One type of transcript, abundant in adults, contains a small 5' terminal exon that is completely lacking in the other type of transcript, which accumulates in larvae. This 5' end difference suggests that the D. affinidisjuncta Adh gene, like the homologous gene from the distantly related species D. melanogaster, is expressed from two promoters. According to the transcription map, these D. affinidisjuncta promoters are separated by approximately 560 base pairs of genomic DNA sequence. D. affinidisjuncta Adh transcripts also resemble D. melanogaster Adh transcripts in both their overall organization and their developmental distribution. Multiple 3' ends are responsible for the size heterogeneity of both types of D. affinidisjuncta Adh RNA, and some of these also appear with stage specificity.

Two alternative transcripts coding for alcohol dehydrogenase accumulate with different developmental specificities in different species of picture-winged Drosophila.

Two alternate transcripts of the single copy Alcohol dehydrogenase (Adh) gene accumulate with developmental specificity in all of 12 species of Hawaiian picture-winged Drosophila which have been examined. Relative to the paradigm species D. affinidisjuncta, the Adh transcript normally restricted to larvae is found to accumulate in both larval and adult tissues in D. formella. The other Adh transcript, which normally accumulates only in adults, accumulates in third-instar D. prostopalpis larvae as well. In species hybrids, the D. formella phenotype shows additive inheritance. These observations document the existence of a novel type of genetic variability. Furthermore, such variants suggest specific properties for the biological systems that regulate ADH expression in Drosophila, and they should facilitate further experimental investigations.

Complex cis-acting regulators and locus structure of Drosophila tissue-specific ADH variants.

Diverse patterns of tissue-specific expression of alcohol dehydrogenase (ADH) among species of the grimshawi subgroup of Hawaiian picture-winged Drosophila suggests control by complex or multiple, independently acting regulatory elements. These elements act by controlling Adh mRNA accumulation in individual tissue types. Restriction mapping of the Adh loci from these species reveals several insertion/deletion differences, one of which lies just outside the 5' end of the structural sequences and correlates with differences in larval patterns of ADH expression. No tissue-specific rearrangement of Adh sequences was observed.

Introduction of a functional P element into the germ-line of Drosophila hawaiiensis.

When a plasmid carrying a P-transposable element (derived from Drosophila melanogaster) is injected into young embryos of D. hawaiiensis, the P-element sequence from the plasmid transposes into the germ-line chromosomes. The introduction of this P element into D. hawaiiensis provides an opportunity to study the behavior of the transposable element in a novel context. Germ-line transposition and numerical increase of the P elements are readily detected in D. hawaiiensis. Thus these aspects of P-element function do not require chromosomal or cytoplasmic properties that are unique to D. melanogaster. Since D. hawaiiensis is among those Drosophila that are most distantly related to D. melanogaster, these results suggest that P-element-mediated transformation may function in many species.

Regulatory gene evolution: adaptive differences in expression of alcohol dehydrogenase in Drosophila melanogaster and Drosophila simulans.

In Drosophila melanogaster X D. simulans hybrids, the alcohol dehydrogenase (ADH) electromorphs characteristic of the two parents display tissue- and stage-specific differences in relative level of expression. This implies distinct cis-acting regulatory elements associated with the respective Adh alleles. These cis-acting elements account in part, but not completely, for markedly different overall patterns of ADH expression in the two species. The regulatory patterns seem to be adaptively significant since they correlate with species-specific patterns of ethanol tolerance. The activity differences are accounted for by different levels of enzyme protein, but the underlying mechanisms have not been fully analysed and may be complex. Independent evolution of various aspects of the ADH developmental programme may relate to use of different promoters for transcription of the Adh locus in different developmental contexts. This system illustrates the potential importance of regulatory genes in evolution and provides a model for investigating the molecular basis of evolved regulatory differences.

Isolation and initial characterization of the alcohol dehydrogenase gene from Drosophila affinidisjuncta.

Recombinant bacteriophages containing the alcohol dehydrogenase (ADH) gene from Drosophila affinidisjuncta have been isolated by virtue of their cross-hybridization to the previously cloned ADH gene from D. melanogaster. Within the 17 kilobases of cloned DNA represented in the phage genomes, the sequences hybridizing to the D. melanogaster ADH gene lie roughly in the center. The only region of detectable hybridization to cDNA made from templates of D. affinidisjuncta larval poly(A)-containing RNA maps to the same portion of the cloned DNA. Verification that the phages carry the ADH structural gene was obtained by hybrid-selecting ADH mRNA, translating it in vitro, and immunoprecipitating the resulting ADH polypeptide. Analysis of genomic DNA suggests that the ADH gene and most flanking sequences are present only once in the haploid genome. However, 3' to the ADH gene, two separable repetitive elements are found. Both repetitive elements are probably small and poorly conserved in the genome, and neither interferes with localization of the ADH gene, by in situ hybridization, to a position near the base of the third chromosome. Analysis of ADH transcripts demonstrates that there are at least four RNAs produced by the ADH gene. Two size classes of RNA are seen at each stage of development. In addition, ADH transcripts from larvae and adults differ from one another in a reproducible manner.