The Influence of Whole-Arm Trisomy on Gene Expression in Drosophila

The biochemical consequences of extensive aneuploidy in Drosophila have been examined by measuring the levels of specific proteins in larvae trisomic for entire chromosome arms. By far the most common effect is a reduction in gene product levels (per gene template) by one-third from the diploid quantity, consistent with the model that concentration-dependent repressors of these loci reside on the duplicated chromosome arms. Most loci appear sensitive to such repression in one or more of the trisomies examined, suggesting that such regulatory loci might be quite common. Repression of gene-product levels in trisomies may significantly contribute to their inviability. Few loci are activated in trisomies implying that most factors necessary for gene expression are in excess. While autosomal trisomies can repress the expression of both X-linked and autosomal loci, X-chromosomal trisomies have little effect on most autosomal genes. A family of genes coding for larval serum proteins do not respond similarly in trisomies, suggesting that regulation operates on a process which is not common to their coordinate regulation. Finally, Adh genes transposed to new chromosomal positions maintain their ability to be repressed in 3L trisomies suggesting that this response to regulation involves a closely linked cis-acting regulatory element.     

Candidate genes and thermal phenotypes: identifying ecologically important genetic variation for thermotolerance in the Australian Drosophila melanogaster cline

Clinal variation in traits often reflects climatic adaptation; in Drosophila melanogaster clinal variation provides an opportunity to link variation in chromosomal inversions, microsatellite loci and various candidate genes to adaptive variation in traits. We undertook association studies with crosses from a single population of D. melanogaster from eastern Australia to investigate the association between genetic markers and traits showing clinal variation. By genotyping parents and phenotyping offspring, we minimized genotyping costs but had the power to detect association between markers and quantitative traits. Consistent with prior studies, we found strong associations between the clinal chromosomal inversion In(3R)Payne and markers within it, as well as among these markers. We also found an association between In(3L)Payne and one marker located within this inversion. Of the five predicted associations between markers and traits, four were detected (increased heat, decreased cold resistance and body size with the heat shock gene hsr-omega S, increased cold resistance with the inversion In(3L)Payne), while one was not detected (heat resistance and the heat shock gene hsp68). In a set of eight exploratory tests, we detected one positive association (between hsp23a and heat resistance) but no associations of heat resistance with alleles at the hsp26, hsp83, Desat 2, alpha-Gpdh, hsp70 loci, while cold resistance was not associated with Frost and Dca loci. These results confirm interactions between hsr-omega and thermal resistance, as well as between In(3L)Payne and cold resistance, but do not provide evidence for associations between thermal responses and alleles at other clinically varying marker genes.     

Heat shock and developmental regulation of the Drosophila melanogaster hsp83 gene.

In contrast to the hsp70 gene, whose expression is normally at a very low level and increases by more than 2 orders of magnitude during heat shock, the hsp83 gene in Drosophila melanogaster is expressed at high levels during normal development and increases only severalfold in response to heat shock. Developmental expression of the hsp83 gene consists of a high level of tissue-general, basal expression and a very high level of expression in ovaries. We identified regions upstream of the hsp83 gene that were required for its developmental and heat shock-induced expression by assaying beta-galactosidase activity and mRNA levels in transgenic animals containing a series of 5' deletion and insertion mutations of an hsp83-lacZ fusion gene. Deletion of sequences upstream of the overlapping array of a previously defined heat shock consensus (HSC) sequence eliminated both forms of developmental expression of the hsp83 gene. As a result, the hsp83 gene with this deletion mutation was regulated like that of the hsp70 gene. Moreover, an in vivo polymer competition assay revealed that the overlapping HSC sequences of the hsp83 gene and the nonoverlapping HSC sequences of the hsp70 gene had similar affinities for the factor required for heat induction of the two heat shock genes. We discuss the functional similarity of hsp70 and hsp83 heat shock regulation in terms of a revised view of the heat shock-regulatory sequence.     

Gene Expression in Adult Metafemales of Drosophila Melanogaster

The expression of selected X-linked and autosomal genes was examined in metafemales (3X:2A) compared to diploid sisters. Three enzyme activities (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, beta-hydroxyacid dehydrogenase) encoded by X-linked genes are not significantly different in the two classes of flies. In contrast, three autosomally encoded enzyme activities (alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, isocitrate dehydrogenase) are reduced in metafemales. Protein and DNA comparisons between metafemales and diploid sisters show a lowered level of total protein whereas the total DNA measurements are similar. Thus, the total cell number in metafemales is basically unchanged but gene expression is reduced. Phenotypic analysis of three autosomal loci, glass (gl), purple (pr) and pink-peach (pp), show that all three have lowered expression in metafemales while the X-linked loci, white-apricot (wa) and Bar (B), are dosage compensated. Quantitative dot blot analysis of messenger RNA levels of the second chromosomal locus, alcohol dehydrogenase (Adh), and the X chromosomal locus, rudimentary (r), show that Adh has reduced expression and r is partially compensated per total RNA in metafemales. It is proposed that the increased dosage of the X chromosome inversely affects both the X and autosomal gene expression but the simultaneous increased dosage of the structural genes on the X results in dosage compensation. The reduced levels of expression of autosomal genes could contribute to the great inviability of metafemales.     

Two closely linked transcription units within the 63B heat shock puff locus of D. melanogaster display strikingly different regulation

We report the isolation and characterization of a cloned DNA of D. melanogaster, Dm4L, that is derived from the major heat shock puff site at 63B. This segment contains two closely linked genes that are each present once per Drosophila haploid genome. One of these, the hsp 83 gene, encodes an abundant heat shock mRNA that, unlike other major heat shock mRNAs, is also abundant in uninduced (23 degrees) kco cells. Although only a slight increase in the level of total hsp 83 RNA can be detected after heat shock in Kco cells, the level of hsp 83 poly(A)+ mRNA increases more than 6-fold and the level of pulse-labeled hsp 83 RNA in total cellular RNA increases 11-fold relative to uninduced cells. In contrast, the levels of total, poly(A)+, and pulse-labeled RNA homologous to the second gene, 63B-T2, are approximately the same in both induced and uninduced cells. Hence, even though these genes are separated by only one thousand base pairs, and, from in situ hybridization to polytene chromosomes, both lie within the heat shock puff, they display strikingly different regulatory properties, These results demonstrate that close linkage of a gene to a heat shock puff is not sufficient to render its expression heat inducible.     

DNA sequence variation and latitudinal associations in hsp23, hsp26 and hsp27 from natural populations of Drosophila melanogaster

Heat shock genes are considered to be likely candidate genes for environmental stress resistance. Nucleotide variation in the coding sequence of the small heat shock genes (hsps) hsp26 and hsp27 from Drosophila melanogaster was studied in flies originating from the Netherlands and eastern Australia. The hsp26 gene was polymorphic for an insertion/deletion of three extra amino acids and two nonsynonymous changes in all populations. The hsp27 gene exhibited two nonsynonymous changes and three synonymous mutations. The hsp26 polymorphism showed a latitudinal cline along the east coast of Australia. This pattern was not confounded by the fact that the shsps are located in the inversion In(3 L)P which also shows a latitudinal cline in eastern Australia. A similar latitudinal cline was found for the previously described variation in hsp23, while frequencies of hsp27 alleles did not change with latitude. These findings suggest that variation at two of the shsps or closely linked loci are under selection in natural populations of D. melanogaster.     

Roles of the trkB and trkC gene products of Escherichia coli in K+ transport

Mutations at the trkB and trkC loci of Escherichia coli produce an abnormal efflux of K+. The mutations are partially dominant in diploids and revert frequently by what appears to be intragenic suppression to the null state. The mutations can be reverted by insertion of Tn10 into the mutated gene, and spontaneous revertants are fully recessive to the mutant allele in diploids. K+ efflux produced by NEM* and by DNP* persists in strains with presumed null mutations at either locus, indicating neither gene product is the primary target for the effect of these inhibitors on K+ efflux. The results are consistent with the view that trkB and trkC encode independent systems for K+ efflux. Mutations at these loci alter regulation of the process so that K+ efflux occurs inappropriately. A second mutation to the null state abolishes this abnormal K+ efflux. These genes may encode K+/H+ antiporters, an activity postulated to mediate K+ efflux and demonstrated to exist in E. coli and other bacteria.     

Structure and Expression of a Heat-Shock Protein 83 Gene of Pharbitis nil

Four cDNA clones representing mRNAs whose levels were affected by a photoperiod that induces flowering in Pharbitis nil were isolated by a differential hybridization screening procedure. The level of mRNAs represented by three clones (12L, 15L, and 17L) increased following a photoperiod that induces flowering and that represented by the fourth clone (clone 27) increased under conditions in which flowering was inhibited. DNA sequence analysis showed that one cDNA, clone 17L, is homologous to members of the 83- to 90-kD heat-shock protein (hsp) gene family. The corresponding gene, hsp83A, was isolated and its DNA sequence was determined. hsp83A encodes a protein that exhibits 70% amino acid identity with Drosophila melanogaster HSP83. The P. nil hsp83A gene contains two introns within the coding region. hsp83A mRNA was not detectable in cotyledons of plants grown in continuous light, but its level increased transiently following a 14-h dark period and reached a maximum 2 h after the lights were turned on. A dramatic increase in the level of hsp83A mRNA was also found 2 h after an end-of-day dark treatment. Genomic Southern blot analysis demonstrated that the P. nil hsp83-90 gene family consists of at least six members, one of which appears to be constitutively expressed in the light.     

A consensus sequence polymer inhibits in vivo expression of heat shock genes.

Promoter function for hsp70 gene expression in Drosophila melanogaster was studied with an in vivo competition assay. A polymer of 40 tandem copies of the pair of regulatory elements of the hsp70 gene was constructed and cloned into a plasmid vector. Various marked genes were cotransfected with the polymer plasmid into Schneider line 2 cells, and their expression was determined by enzyme activity measurements. The polymer dramatically inhibited expression of cotransfected hsp70, hsp26, and hsp83 genes, but not cotransfected copia and histone genes. Our results indicate that in vivo, a trans-acting, positive regulatory factor, which can be titrated by heat shock consensus sequences, is required for activation of heat shock genes and is specific for these genes; the coordinate induction of different heat shock genes appears to be mediated by similar, but not identical, interactions of the trans-acting induction factor and the cis-acting heat shock consensus sequences; and the uninduced or basal level expression of the transfected hsp70 gene is also due to interaction of the consensus sequence with a positively acting factor.     

Mapping and Gene Conversion Studies with the Structural Gene for Iso-1-Cytochrome C in Yeast

We have investigated the order of the four genes cyc1, rad7, SUP4, and cdc8 which form a tightly linked cluster on the right arm of chromosome X in the yeast Saccharomyces cerevisiae. Crossing over and coconversion data from tetrad analysis established the gene order to be centromere-cyc1-rad7-SUP4. Also cdc8 appeared to be distal to SUP4 on the basis of crossovers that were associated with conversion of SUP4. The frequencies of recombination and the occurrence of coconversions suggest that these four genes are contiguous or at least nearly so. Gene-conversion frequencies for several cyc1 alleles were studied, including cyc1-1, a deletion of the whole gene that extends into the rad7 locus. The cyc1-1 deletion was found to be capable of conversion, though at a frequency some fivefold less than the other alleles studied, and both 3:1 and 1:3 events were detected. In general 1:3 and 3:1 conversion events were equally frequent at all loci studied, and approximately 50% of conversions were accompanied by reciprocal recombination for flanking markers. The orientation of the cyc1 gene could not be clearly deduced from the behavior of the distal marker SUP4 in wild-type recombinants that arose from diploids heteroallelic for cyc1 mutations.     

The Heat-Shock Gene hsp83 of Drosophila auraria: Genomic Organization, Nucleotide Sequence, and Long Antiparallel Coupled ORFs (LAC ORFs)

The genomic organization of the hsp83 gene of Drosophila auraria, a far-eastern endemic species belonging to the montium subgroup of the melanogaster species group, is presented here. Based on in situ hybridization on polytene chromosomes, cDNA and genomic clone mapping, nucleotide sequencing, and genomic Southern analysis, hsp83 is shown to be present as a single-copy gene at locus 64B on the 3L chromosome arm in D. auraria. This gene is organized into two exons separated by a 929-bp intron. The first exon represents the mRNA leader sequence and is not translated, while the coding region, having a length of 2,151 bp, is solely included in the second exon. Nucleotide sequence comparisons of D. auraria hsp83 with homologous sequences from other organisms show high conservation of the coding region (88–92% identity) in the genus Drosophila, in addition to the conserved genomic organization of two-exons–one-intron, of comparable size and arrangement. A phylogenetic tree based on the protein sequences of homologous genes from representative organisms is in accord with the accredited phylogenetic position of D. auraria. In the hsp83 gene region, a second case of long antiparallel coupled open reading frames (LAC ORFs) for this species was found. The antiparallel to the hsp83 gene ORF is 1,554 bases long, while the two ORFs overlap has a size of 1,548 bp. The anti-hsp83 ORF does not show significant homology to any known gene sequences. In addition, no similar LAC ORF structures were found in homologous gene regions of other organisms. Received: 18 April 1997 / Accepted: 1 August 1997