Structural changes in the promoter region mediate transvection at the sn-glycerol-3-phosphate dehydrogenase gene of Drosophila melanogaster.

Trans effects at the sn-glycerol-3-phosphate dehydrogenase locus (Gpdh) of Drosophila melanogaster give rise to an increase in GPDH activity and mRNA from the wild-type allele in heterozygotes with some low-activity alleles. Either the low-activity alleles that induce the effect have a defective P-element inserted between the promoter and a downstream intronic enhancer element or the promoter region is deleted. The trans effect is pairing dependent, characteristic of transvection at some other loci. The defective P-elements that mediate transvection are located between the promoter and at least up to 6 bp downstream of the transcription start site. Transvection at Gpdh appears similar to the "enhancer action in trans" mode at the yellow locus.

A KP element inserted between the two promoters of the alcohol dehydrogenase gene of Drosophila melanogaster differentially affects expression in larvae and adults.

An allele of the Drosophila melanogaster alcohol dehydrogenase (Adh) gene has a 1.15-kb KP element inserted, in the same orientation as Adh transcription, five nucleotides upstream of the distal transcription start site. The target site--GTCCAAGT--in Adh between nucleotides -13 and -6 is duplicated at both ends of the insertion. Adult flies with either one or two copies of the allele have less than 12% of the alcohol dehydrogenase (ADH) activity of the controls. Activity levels are also reduced in larvae, but by a much smaller amount. Quantitative Northern analyses showed that the low activity level in adults was caused by reduced transcript levels from the distal promoter. 5' RACE experiments indicated that adult transcripts were mostly initiated downstream of the distal promoter but some skipped the first adult exon. In late third-instar larvae the transcripts present were from the proximal promoter. After the KP element was deleted by an appropriate breeding program, the distal transcript increased in adults to the control level, but ADH activity increased to only 50% of the control. No nucleotide changes were found in the gene that could explain this difference.

Molecular analysis of a Drosophila melanogaster sn-glycerol-3-phosphate dehydrogenase allozyme variant that has cold labile activity.

A rare naturally occurring allele, GpdhACb62, at the sn-glycerol-3-phosphate dehydrogenase locus in Drosophila melanogaster, encodes an enzyme with an electrophoretic mobility that is more cathodal than that produced by the common slow electrophoretic allele. After electrophoresis and staining of extracts of single adult flies there is a single band of activity corresponding in position to GPDH-1, but, using highly concentrated extracts, a faint band corresponding to GPDH-3 is observed. In GpdhACb62 homozygotes there is about 26% of the normal level of activity in adults, and less than 6% in third instar larvae. The reduction in activity is significantly greater than the decrease in GPDH immunologically cross-reacting material (CRM). Northern analyses, and rapid amplification of the cDNA ends (RACE) of the 3' regions of the transcripts, show that the levels and structures of the poly(A)+RNAs are similar in homozygotes for GpdhACb62 and for a normal activity allele GpdhAC8. Hybridization to oligonucleotide probes specific for the GPDH-1 and GPDH-3 transcripts was of a similar intensity in GpdhACb62 and GpdhAC8 adult flies. In third instar larvae the main transcript is for GPDH-3 and again the hybridization signals were similar in each line. The activity of the enzyme produced by GpdhACb62 was unstable both at 50 degrees C and at 0 degrees C. The activity lost at 0 degrees C was recovered by incubation at 20 degrees C. The complete GpdhACb62 gene, and the partial Gpdh tandem duplication 3' to this gene, were cloned and sequenced. Comparisons with two normal activity GpdhF genes revealed 31 unique changes in the first copy of GpdhACb62. In exon 4, a T to G substitution changes cysteine to glycine and may disrupt a disulphide bond and be responsible for the distinctive properties of GPDH-ACb62.

Molecular relationships between alcohol dehydrogenase null-activity alleles from natural populations of Drosophila melanogaster.

Alcohol dehydrogenase null-activity alleles extracted from a number of natural populations of Drosophila melanogaster in Tasmania were shown to be molecularly similar by probing, with an oligonucleotide specific to an inserted region in intron 2 of the gene, genomic DNA amplified by the polymerase chain reaction. This insertion had previously been shown to be the cause of the loss of activity in one of the null alleles whose DNA sequence was known. Three Adh null alleles from mainland populations did not contain the insertion. Two of these null alleles, extracted from the Coffs Harbour population in different years, were cloned, and their DNA sequences showed that they were identical and that both had a 438-bp deletion which removed most of exon 2. The third null allele, identified in a sample of flies from Chateau Tahbilk, was shown by 4-bp restriction-endonuclease mapping to contain a 320-bp insertion in intron 1, although this may not be the cause of the loss of activity. The data show that at least three different Adh null alleles have been found in Australian populations and that at least two have been maintained as heterozygotes over a period of years.

Aberrant splicing of a naturally occurring alcohol dehydrogenase null activity allele in Drosophila melanogaster.

The DNA sequence of a naturally occurring alcohol dehydrogenase null activity allele, AdhnAC14, has eight extra nucleotides (in two groups of four) in the second intron, commencing six bases 3' from the 5' splice site. A stop codon was also found in exon 2. S1 nuclease protection experiments have shown that the insertions in intron 2 disrupt the correct splicing of intron 2. The null allele produces a transcript approximately 100 bases longer than the normal mature adult transcript, and the amount of the null allele transcript is only about 10% of the normal level.

Molecular similarity of Drosophila melanogaster alcohol dehydrogenase thermostable alleles from populations on different continents.

Allele specific oligonucleotide probes have been used to show that DNA, amplified by the polymerase chain reaction, from eleven thermostable Adh alleles extracted from populations on different continents contains the triplet TCC, which distinguishes AdhFChD (Fast Chateau Douglas) from AdhF (Fast). The molecular similarity of Adh thermostable alleles suggests that they had a common origin, and it is argued that the mutation probably occurred in China where high frequencies of AdhFChD are found.

Molecular structure of a naturally occurring alcohol dehydrogenase null activity allele in Drosophila melanogaster.

An alcohol dehydrogenase null activity allele, AdhnAH52, extracted from a natural population of Drosophila melanogaster has been cloned and sequenced. Compared with the wild-type consensus sequence, the nucleotide sequence of AdhnAH52 contains eight extra bases in intron 2, adjacent to the 5' splice site. It seems likely that the extra bases result from two structural changes, with a 10-base pair insertion at the same site as a 2-base pair deletion. The insertion includes an 8-base pair duplication of an adjoining region. This structural change alters transcription to give rise to an mRNA which is longer than normal and at 10% of the wild-type level.

The alcohol dehydrogenase polymorphism of Drosophila melanogaster in relation to environmental ethanol, ethanol tolerance and alcohol dehydrogenase activity.

Ethanol levels in Drosophila breeding sites were higher in a winery storing fortified wines than in nearby grape pressings or in orchard fruits. The relative abundance of D. simulans to D. melanogaster was negatively correlated with ethanol levels. In D. melanogaster there were no significant differences in AdhF frequency between the orchard and winery populations. The ethanol tolerance of wild caught D. melanogaster males paralleled the levels of ethanol in the breeding sites but Adh alleles and ethanol tolerance segregated largely independently of each other. Levels of ADH activity were positively associated with the ethanol tolerance of the different populations and with levels of ethanol in the breeding sites, but it is argued that the ethanol levels are not causative. Flies from inside the winery had higher ADH levels due mainly to greater amounts of ADH-F. The difference in activity persisted for at least one generation in the laboratory. After ten generations of laboratory culture the differences in ethanol tolerance were still present but there were no significant differences in ADH activity.

Transcription analysis of alcohol dehydrogenase null alleles from natural populations of Drosophila melanogaster.

Southern analysis of 19 Adh null activity alleles isolated from Tasmanian populations of Drosophila melanogaster have shown that there are no detectable insertions or deletions in an 11.8-kb region that contains the gene. Northern blot analyses of the null alleles have shown that they all produce a transcript about 100 bases longer than that produced by the normal allele and they accumulate a precursor of 1800 bases. The amount of the major transcript produced by the null alleles is about 10% of that produced by normal alleles. The molecular properties of the null alleles suggest that they share a common origin.

Restriction endonucleases variation in the region of the alcohol dehydrogenase gene: a comparison of null and normal alleles from natural populations of Drosophila melanogaster.

The restriction endonuclease variation in the 12 kb region surrounding twelve Adh null alleles extracted from three Tasmanian populations has been compared with normal alleles from the same populations. Each of the null alleles had the same haplotype as revealed by digestions with eight hexanucleotide restriction enzymes. This haplotype also occurred in 4 of the 46 chromosomes bearing normal alleles which were tested; these four chromosomes with the null allele haplotype carried the AdhS allele. The data suggest that the Adh null alleles from geographically separate populations share a common ancestry and are derived from the same mutation in an AdhS allele.

Variation in the biochemical properties of the Drosophila alcohol dehydrogenase allozymes.

Thirteen Drosophila Adh variants have been characterized with respect to gene expression, substrate preference, thermostability, and specific activity. The results suggest that the variants may be grouped into two biochemical classes, typified by the properties of the two most common enzyme forms, ADH-F and ADH-S. Membership of these classes cannot be predicted from electrophoretic mobility, nor is any simple classification possible with regard to the characteristics of level of gene expression (in terms of ADH activity or ADH protein) or thermostability of the gene product.

An electrophoretically cryptic alcohol dehydrogenase variant in Drosophila melanogaster. III. Biochemical properties and comparison with common enzyme forms.

The biochemical properties of the heat-stable alcohol dehydrogenase variant ADH-FCh.D. have been investigated and compared with those of the two common enzyme forms ADH-F and ADH-S. The results show that ADH-F and ADH-S differ with respect to substrate specificity, their response to high concentrations of secondary alcohols and their apparent Michaelis constants for three alcohols in two different buffer systems. In all these tests the enzyme ADH-FCh.D. resembles ADH-S much more closely than ADH-F. It is concluded that if natural selection is to distinguish between the alleles AdhS and AdhFCh.D. then it most probably does so on the basis of the superior thermostability of ADH-FCh.D. The biochemical properties of all three enzymes are discussed in relation to the role of alcohol dehydrogenase in the exploitation of alcohol by D. melanogaster.

Genetic variation at the alcohol dehydrogenase locus in Drosophila melanogaster in relation to environmental variation: Ethanol levels in breeding sites and allozyme frequencies.

Ethanol levels in Drosophila breeding sites in seepages of unfortified wine inside wineries have been found to be similar to those in many decaying fruits and vegetables. Fortified wine seepages inside wineries have ethanol levels on average three times as high as other breeding sites. However there was no evidence that this variation in ethanol levels was associated with differences in Adh F frequencies in D. melanogaster at sites either within wineries or outside wineries. D. simulans was at lower frequencies at sites inside wineries compared to sites outside although this difference may not be related to ethanol levels. It is concluded that adaptation to natural levels of environmental ethanol by D. melanogaster does not necessarily modify Adh frequencies.

An electrophoretically cryptic alcohol dehydrogenase variant in Drosophila melanogaster. I. Activity ratios, thermostability, genetic localization and comparison with two other thermostable variants.

The alcohol dehydrogenase (ADH) variant ADH-FCh.D. has a secondary alcohol/primary alcohol activity ratio characteristic of ADH-S although it has an electrophoretic mobility inseparable from ADH-F. ADH-FCh.D. is distinguished from these two common ADH variants by being much more thermostable. Genetic analysis suggests tht ADH-FCh.D. is specified by an allele at the Adh locus. Biochemical comparisons show that ADH-FCh.D. has the same electrophoretic mobility, activity ratio and thermostability as the two other heat-resistant variants which have been reported, ADH-F71K in Europe and ADH-Fr in North America. The geographically widespread distribution of a thermostable ADH variant within the ADH-F electrophoretic class indicates that it should be considered in attempts to explain the Adh polymorphism in natural populations.