Variation in alcohol dehydrogenase activity in vitro in flies from natural populations ofDrosophila melanogaster

Alcohol dehydrogenase (ADH) activity variation in male flies taken directly from seven natural populations ofDrosophila melanogaster is largely accounted for by segregation of alleles at theAdh structural gene locus. There was little overlap in the ADH activities ofAdh ^F andAdh ^s homozygotes. Body weights varied only slightly betweenAdh genotypes and contributed little to ADH variation. Between and within population variation in ADH activity and ADH protein in flies in the wild is mainly due to the relative frequencies ofAdh ^F andAdh ^s.     

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.     

Meiotic mutations from natural populations ofDrosophila melanogaster

Two meiotic genes from natural populations are described. A female meiotic mutation,mei(1)g13, mapped to 17.4 on the X chromosome, causes nondisjunction of all homologs except for the fourth chromosomes. In addition, it reduces recombination by 10% in the homozygotes and causes 18% increased recombination in the heterozygotes. A male meiotic mutation,mei-1223 ^m144 , is located on the third chromosome. Although this mutation causes nondisjunction of all chromosomes, each chromosome pair exhibits a different nondisjunction frequency. Large variations in the sizes of the premature sperm heads observed in the homozygotes may reflect irregular meiotic pairing and the subsequent abnormal segregation, resulting in aneuploid chromosome complements.     

Nonlatitudinal environmental correlations for alcohol dehydrogenase in Southern African populations ofDrosophila melanogaster

The relation between alcohol dehydrogenase (ADH) allozyme frequencies and several environmental variables, as well as latitude and longitude, was examined in 17 natural populations of Drosophila melanogaster in Namibia, South Africa, and Zimbabwe. Unlike several other studies, we found no latitudinal allele-frequency clines and no significant correlation with temperature. We found that the frequency of the most common allele (AdhS) was positively correlated with an increase in rainfall. We suggest that substrate-induced toxicity may explain the geographic variability in our results.     

Isolation and genetic characterization of alcohol dehydrogenase thermostability variants occurring in natural populations of Drosophila melanogaster

Drosophila melanogaster collected from natural populations were examined fo thermostability variants within electrophoretic mobility classes of two enzymes. In alcohol dehydrogenases, two discrete forms of the "slow" allozyme and three discrete forms of the "fast" allozyme were revealed by postelectrophoretic treatments ranging from 15 sec at 40 C to 40 sec at 43 C. All variants have been mapped to within 0.7 unit of the Adh locus. Results of a geographic survey indicate that two alleles giving rise to fast-moderate and slow-moderate allozymes are common everywhere; other variants have a collective frequency ranging from 0% to 7%. In a test of the possibility that the rare Adh alleles could be generated by intragenic recombination between the two common alleles, electrophoresis and heat treatment of progeny recombinant for flanking markers of Adh revealed no new allozymes. Among 27 stocks containing slow alpha-glycerophosphate dehydrogenase allozymes and 109 fast stocks, heat treatments revealed no additional variation.     

Hybrid dysgenesis in natural populations ofDrosophila melanogaster in Japan

The P-M system of hybrid dysgenesis inDrosophila melanogaster was investigated on the basis of gonadal dysgenesis, using 1,590 strains from 28 natural populations in Japan, and 20 populations from Southeast Asia, the Pacific area and Africa. Strong P strains were found sporadically in several populations in Japan. Few strong M strains were observed. Q strains were present at a high frequency in most populations. Thus, most populations in these areas were regarded as Q populations. The distribution of the P element and the evolution of P, Q and M populations are also discussed.     

Recent origin for a thermostable alcohol dehydrogenase allele ofDrosophila melanogaster

Summary The nucleotide sequence of theFast-Chateau Douglas isolate of the thermostable alcohol dehydrogenase allele is compared with the sequences of theSlow andFast alleles ofDrosophila melanogaster. Conceptual translation of theFChD sequence indicates that the thermostable polypeptide has the diagnostic FAST amino acid replacement at residue 192 and an additional replacement of serine for proline at residue 214. This suggests aFast origin for the thermostableAdh allele. However, some of the biochemical properties of the FCHD protein resemble those of the SLOW rather than the FAST polypeptides. The serine for proline replacement confers upon the thermostable polypeptide substrate specificities and some kinetic parameters similar to the SLOW protein. The same replacement substitution within the third coding exon also appears to alter the ADH protein concentration to a level similar to the SLOW polypeptide and the probable effect is at the level of mRNA concentration. The low level of nucleotide sequence variation, other than that leading to the amino acid substitution, suggests a recent origin for the thermostable allele. The time since divergence of theFChD sequence fromFast is estimated to be approximately 260,000–470,000 years.     

Geographical differentiation of allozymic variability in natural Indian populations ofDrosophila melanogaster

Fourteen Indian populations ofD. melanogaster collected along a 22° latitudinal range were analyzed electrophoretically to compare the geographical patterns of allozymic variation at 13 loci. The data show higher genetic differentiation on the basis of moderate to higherF _ST values and significant statistical correlation of allelic frequencies at six polymorphic loci with latitude. The results add support to the hypothesis that the occurrence of parallel or complementary latitudinal clines across different continental populations provides evidence of natural selection maintaining such clinal variation.     

Analysis of Aldox n alleles isolated from natural populations of Drosophila melanogaster

Aldox null alleles which were isolated from natural populations in Great Britain and North Carolina were analyzed for complementation. No complementation was observed between any combinations of null alleles for aldehyde oxidase (AO) specific activity in late third-instar larvae and newly emerged adults. AO immunologically cross-reacting material (AO-CRM) was quantitated in all homozygous stocks at both developmental stages as well as all allelic combinations in newly emerged adults. When the adult organism contains only Aldox ⁿ alleles, the polypeptides are not immunologically recognizable or may be rapidly degraded. Larvae and adults have different abilities to degrade mutationally altered enzymatically inactive AO polypeptide or synthesize them differentially. This is indicated by easily measurable AO-CRM levels in late third-instar larvae of Aldox ⁿ homozygotes, while newly emerged adult Aldox ⁿ homozygotes have very little, if any, AO-CRM. Newly emerged adult heterozygotes of Aldox ⁿ /Aldox ⁺ do have increased AO-CRM, indicating that the Aldox ⁿ alleles can code for a polypeptide which can be rescued if Aldox ⁺ gene product is present. Heterozygotes containing an Aldox ⁺ allele with a deficiency for the Aldox region produce 74.2% of the AO-CRM found in Aldox ⁺ homozygotes. This may indicate the presence of trans-acting factors which serve to activate gene expression in a system in which each gene copy is not maximally expressed.This work was supported by an Alberta Heritage Foundation for Medical Research Establishment Grant and a Natural Sciences and Engineering Research Council of Canada Operating Grant.     

The partial purification and characterization of cytosol alcohol dehydrogenase from Astasia

1. The cytosol alcohol dehydrogenase (alcohol-NAD oxidoreductase, EC 1.1.1.1) of Astasia longa was partially purified and characterized from cells grown in the presence of air+CO(2) (95:5) or of O(2)+CO(2) (95:5). 2. Under both these growth conditions, the cells contained a fraction, ADHII, which was characterized by its electrophoretic properties, by a high degree of resistance to heat inactivation, by a sharp pH optimum at 8.2 and by its kinetic properties. The estimated molecular weight of this fraction was approx. 150000, which is similar to that of yeast alcohol dehydrogenase. 3. Cells grown in air+CO(2) (95:5) contain another fraction, ADHI, which can be further separated into two subfractions by polyacrylamide-gel electrophoresis and by DEAE-cellulose chromatography. This was termed fraction ;ADHI-air'. 4. In addition to fraction ADHII, cells grown in the presence of O(2) have a twofold increase in fraction ADHI-air activity as well as two new fractions that could not be demonstrated in air-grown cells. These new fractions which we have called fraction ;ADHI-O(2)', account for about 10% of the total activity. 5. The ADHI fractions (air) and (O(2)) have similar broad pH-activity curves and similar kinetic properties, both having a lower K(m) for ethanol and NAD than fraction ADHII. However, they differ from each other with respect to their activity with various substrates. The estimated molecular weight of these two ADHI fractions and their chromatographic behaviour on hydroxyapatite and on DEAE-cellulose also distinguish them.     

Genetic load and viability variation in korean natural populations ofDrosophila melanogaster

Summary Natural populations of Drosophila melanogaster from Anyang and Susac (suburbs of Seoul) have been analyzed with respect to viability variation on the second chromosome. Homozygotes as well as random heterozygotes for wild chromosomes were studied. The frequency of lethal factors was about 16 per cent, that of drastics 26 per cent. The average viability of homozygotes was 0.650 including lethal lines and 0.858 for quasinormals; that for random heterozygotes was 1.125. Allelism tests have been performed for the lethals. The allelism rate turned out to be as high as 0.036 and 0.0214, respectively. Using a formula by Nei, the effective population size can be estimated from these data. Korean D. melanogaster populations proved as small as 2000 to 3000 individuals. No correlation between homozygous and heterozygous viabilities could be found. According to these observations, along with the fact that partly big clusters of identic lethals could be found in the allelism tests, it is concluded that in Korean populations quite a large part of the hard genetic load is balanced. The connection between population size, population structure and associative or genuine overdominance is discussed.     

The fate ofSepia in small populations ofDrosophila melanogaster

Asepia gene found inD. melanogaster collected in North Carolina, and wildtype flies from North Carolina, Bogotá, Barcelona, and California were used to strt 120 cultures that were maintained by mass transfers of adults every third week for more than a year. The frequency ofsepia was determined in these cultures at the termination of the experiment. Thesepia gene was present in considerable frequency (16%–65%) in all backgrounds except one; in cultures involving wildtype chromosomes from North Carolina, it was virtually eliminated. Each of the wildtype backgrounds exhibited a characteristic final frequency ofsepia, suggesting that they had reached at least quasi-stable equilibria. Although it is likely that the retention ofsepia depended upon the superiority of flies heterozygous for this mutant, the technique does not reveal whethersepia itself was involved in the apparent heterosis.     

Molecular consequences of two formaldehyde-induced mutations in the alcohol dehydrogenase gene ofDrosophila melanogaster

Adhfn23 and Adhfn24 are two formaldehyde-induced, homozygous-viable, alcohol dehydrogenase-null mutants that bear lesions in the gene that codes for the alcohol dehydrogenase (ADH; EC 1.1.1.1) of Drosophila melanogaster. Adhfn23 contains a 34-base pair deletion in the C-terminal coding region of the alcohol dehydrogenase structural gene. By immunological and molecular analysis, we show that the deletion shifts the translation reading frame and results in a prematurely truncated polypeptide product (10 amino acids shorter than wild type) that cross-reacts with antibody raised against ADH. The steady-state level of alcohol dehydrogenase mRNA present in this mutant is close (97%) to that in the wild type, but the steady-state level of alcohol dehydrogenase-like protein is 50% lower. Moreover, the rate of alcohol dehydrogenase synthesis in Adhfn23 flies is reduced to 60% of that found in the wild type. Hence both the rate of synthesis and the rate of degradation of alcohol dehydrogenase are affected. In contrast, Adhfn24 which contains an 11-base pair deletion in the N-terminal coding region of the ADH gene, synthesizes no immunodetectable protein, and the amount of alcohol dehydrogenase mRNA is less than half that of wild-type flies. As with Adhfn23, the deletion in Adhfn24 results in a change in the reading frame. Unlike Adhfn23, however, nucleic acid sequence data indicate that polypeptide chain elongation can proceed for a considerable distance (over 130 amino acids) beyond the deletion. Based upon antigenic binding-site predictions, the resultant aberrant protein (projected 195 amino acids in length) would share few antigenic sites with the alcohol dehydrogenase from the wild type, which may account for the lack of immunoprecipitable material in this mutant. The contrasting effects these two deletions have on the Drosophila ADH mRNA levels and ADH protein levels are discussed.     

Distinct electrophoretic polymorphism pattern at alcohol dehydrogenase (Adh) locus of Drosophila melanogaster natural populations from Turkey

Small number of Drosophila melanogaster populations from two distinct geographical regions, Central Anatolia and Black Sea, of Turkey were studied. Populations sampled were electrophoresed for a single locus, alcohol dehydrogenase (Adh) to assess population differentiation. Both the magnitude of genetic differentiation levels and the population structure based on hierarchical F-statistics allow populations to be grouped on two genetically divergent area, Central Anatolian and Black Sea. One ecological correlate, average yearly maximum rainfal. Ryear, seems to track this Adh genetic variation pattern. The study also shows that a typical pattern of geographical Adh polymorphism can emerge with a handfull of populations sampled across a relatively small distance.     

Purification and partial characterization of alcohol dehydrogenase from wheat.

Further support for hypotheses proposed earlier for the genetic control and subunit composition of the alcohol dehydrogenase of Triticum has been obtained through the purification and partial characterization of the enzyme. The alcohol dehydrogenase of the wheat T. monococcum was purified 103-fold to a specific activity of 55,900 units/mg. Purification was achieved using streptomycin sulfate precipitation, gel filtration chromatography, DEAE-cellulose anion-exchange chromatography, and preparative isoelectric focusing. The native enzyme has a molecular weight of 116,000 and a dimeric subunit structure. The apparent Michaelis constants are 1.2 × 10^?2m for ethanol and 1 × 10^?4m for NAD. The substrate specificity of wheat alcohol dehydrogenase differs significantly from the substrate specificities of the enzymes of horse and yeast.     

Recessive genetic variants affecting mating activity of males in natural populations ofDrosophila melanogaster

Mating activity of 115 wild males was compared with 88 homozygotes and 42 heterozygotes for their second chromosomes. Wild males, 48–96 hours old, inseminated on the average, 4.4±0.1 females per 24 hours. The hetero- and homozygotes for their second chromosomes (other chromosomes being randomly combined with those from the laboratory strain), inseminated on the average 2.8±0.2 and 2.0±0.2 females/24 h. respectively. There is no correlation between homozygotes and heterozygotes for the second chromosome and their wild ancestors which carried these chromosomes. Wild second chromosomes which in homozygous condition produced total sterility of their carriers, and some others which made for an unusually high activity in homozygous males, had on an average similar effects in wild carriers.This ariicle is warmly dedicated to Professor Theodosius Dobzhansky.     

Distribution of polymorphic gene duplication at theGpdh locus in natural populations ofDrosophila melanogaster

Summary The glycerol-3-phosphate dehydrogenase (GPDH, E. C. 1.1.1.8) gene ofDrosophila melanogaster contains a tandem duplication of a 4.5-kb-long DNA fragment. Survey of theGpdh gene region by the Southern blot analysis revealed the following features of this gene duplication: (1) The duplication was not observed in chromosome lines that carryIn(2L)t, a cosmopolitan chromosomal inversion in this species. The duplication and the inversion are in linkage disequilibrium. (2) The duplication is polymorphic in the Japan and US natural populations examined. Its frequency is 0.26 on an average inIn(2L)t-free chromosomes. (3) Triplication is absent or has not become frequent in the populations surveyed. Possible evolutionary factors of this duplication polymorphism are discussed.     

Purification and partial characterization of alcohol dehydrogenase,fructose-1,6-bisphosphate aldolase and the cytoplasmic form of malate dehydrogenase from Drosophila melanogaster

A purification scheme for the cytoplasmic form of malate dehydrogenase (s-MDH) of Drosophila melanogaster is presented which is superior to any previously reported method. In addition, this scheme can also be used to obtain alcohol dehydrogenase (ADH) and FDP aldolase. Gel filtration experiments reveal an oligomeric molecular weight of 69,000 for s-MDH, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate indicates subunit molcular weights of 32,100 for s-MDH, 24,600 for ADH and 34,000 for FDP aldolase. The amino acid composition of Drosophila melanogaster s-MDH and FDP aldolase are reported.