Genetic sexing in Drosophila melanogaster using the alcohol dehydrogenase locus and a Y-linked translocation

Summary By incorporating ethanol (4% v/v) into the larval rearing medium of a specially constructed Drosophila melanogaster strain it was possible to produce only male adults; the female larvae died.In this strain, the male determining chromosome was linked with a positive Alcohol dehydrogenase (ADH) allele by a translocation. The females were homozygous for the null allele and hence sensitive to ethanol.This genetic sexing method is discussed in relation to its use in the genetic control of insects.     

Isolation and biochemical analysis of ethyl methanesulfonate-induced alcohol dehydrogenase null mutants ofArabidopsis thaliana (L.) Heynh

Several mutants have been isolated at theArabidopsis thaliana (L.) Heynh. alcohol dehydrogenase (ADH) gene locus using allyl alcohol selection on ethyl methanesulfonate (EMS)-mutagenized seeds. Eleven mutants were isolated in theADH1-A electrophoretic allele, and 21 in theADH1-S allele. These null mutants are characterized by the absence of measurable ADH activity and genetic data showed that the mutations were confined to theADH1 gene locus ofArabidopsis. Eleven mutants in theADH1-A background were further characterized at the protein and mRNA level. These experiments revealed striking differences in the ADH protein and mRNA content. Some of the mutants did not synthesize any mRNA or ADH-like protein, whereas some of them had a nearly normal level of ADH protein and mRNA. Others had a very low level of both protein and mRNA. ADH null mutants differed physiologically from the wild type by their higher sensitivity to anaerobic treatment in plants and significantly reduced resistance to acetaldehyde in suspension cultures.This research was supported by the Geconcerteerde Onderzoeksactie, Grant 86/91–103, and the Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw (IWONL), Grant 4972A.     

Approaches for the Isolation of Arabidopsis adh1 Regulatory Mutants Using Allyl Alcohol Selection

Allyl alcohol, a suicide substrate for the alcohol dehydrogenase enzyme (EC.1.1.1.1), has been frequently used as a negative selection method for the isolation of alcohol dehydrogenase mutants in plants, animals and microorganisms. This approach led to the isolation of mutants that mapped to the ADH gene itself. We attempted to use allyl alcohol selection for the isolation of adh1 regulatory mutants in Arabidopsis. First we selected at plantlet level on ADH1–GUS transgenic plants. This enabled us to use GUS staining to discriminate between structural and regulatory mutants. Allyl alcohol selection of 50000 EMS-treated seeds did not yield any potential mutants. Secondly we selected EMS and -ray-treated seeds of a transgenic line transformed with an additional copy of the ADH1 gene including its own promoter. Fifteen allyl alcohol-resistant plants were selected from the mutagenized seed. Genetic analysis of three putative mutants (adr8, adr10, and adr15) indicated that the ADH1-null phenotype was due to monogenic trans-recessive mutations. But treatment with the demethylating agent 5-azacytidine and analysis of methylation levels of the ADH1 gene indicated that these mutant candidates have increased levels of methylation in the promoter and coding region of ADH1. These results suggest that the allyl alcohol resistance of adr8, adr10, and adr15 is due to silencing of ADH1 rather than to a mutation of a regulatory locus.     

A simple method for the isolation of allelic series using male-linked translocations

Summary Using Adh null alleles a genetic sexing technique is being developed in Ceratitis capitata. In order to facilitate the isolation of a whole series of null alleles at this locus a technique utilizing male-linked translocations is described. It provides a simple efficient and general method for the isolation of any allelic series in species where little genetic information is available.     

Isolation ofCaenorhabditis elegans mutants lacking alcohol dehydrogenase activity

Alcohol dehydrogenase (ADH) and the genes encoding this enzyme have been studied intensively in a broad range of organisms. Little, however, has been reported on ADH in the free-living nematodeCaenorhabiditis elegans. Extracts of wild-typeC. elegans contain ADH activity and display a single band of activity on a native polyacrylamide gel. Reaction rate for alcohol oxidation is more rapid with higher molecular weight alcohols as substrate than with ethanol. Primary alcohols are preferred to secondary alcohols.C. elegans is sensitive to allyl alcohol, a compound that has been used to select for ADH-null mutants of several organisms. Allyl alcohol-resistant mutant strains were selected from ethylmethanesulfonate (EMS)-mutagenized nematode populations. ADH activity was measured in extracts from eight of these strains and was found to be low or nondetectable. These results form a basis for molecular and genetic characterization of ADH expression inC. elegans.     

Cloning and sequence analysis of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases.

Summary Treatment of tomato seeds with ethyl methanesulphonate (EMS) followed by allyl alcohol selection of M₂ seeds has led to the identification of one plant (B15-1) heterozygous for an alcohol dehydrogenase (Adh) null mutation. Genetic analysis and expression studies indicated that the mutation corresponded to the structural gene of the Adh-1 locus on chromosome 4. Homozygous Adh-1 null mutants lacked ADH-1 activity in both pollen and seeds. Using an antiserum directed against ADH from Arabidopsis thaliana, which crossreacts with ADH-1 and ADH-2 proteins from tomato, no ADH-1 protein was detected in seeds of the null mutant. Northern blot analysis showed that Adh-1 mRNA was synthesized at wild-type levels in immature seeds of the null mutant, but dropped to 25% in mature seeds. Expression of the Adh-2 gene on chromosome 6 was unaffected. The potential use of the Adh-1 null mutant in selecting rare transposon insertion mutations in a cross with mutable Adh-1 ⁺ tomato lines is discussed.     

Ethanol-resistant mutants of Nicotiana plumbaginifolia are deficient in the expression of pollen and seed alcohol dehydrogenase activity

Summary Six independent mutant lines ofNicotiana plumbaginifolia resistant to ethanol, designated E3, E8, E101, E112, E144 and E251, were isolated as germinating seedlings on selective medium. In all cases, resistance to ethanol was conferred by a single recessive nuclear mutation at the same locus. Mutant seeds and pollen lacked detectable ADH activity, with the exception of E251 where a residual activity was detected. An antiserum directed againstArabidopsis thaliana ADH detected an ADH-related polypeptide of 44 kDa present in wild-type seeds and, to a lesser extent, in the seeds of the leaky mutant E251. No ADH-related polypeptide could be detected in seeds of the other mutants. However, all of them had a nearly normal level of ADH mRNA except one which did not synthesize any mRNA. These results suggest that these ethanol-resistant mutants are impaired in one of the structural genes coding for alcohol dehydrogenase. The corresponding locus has been designatedAdh1.Abbreviations ADH alcohol dehydrogenase - EMS ethyl methane-sulfonate - MTT dimethyl thiazol tetrazolium - NAD nicotinamide adenine dinucleotide - NBT nitro blue tetrazolium - p-cells protoplast-derived cells - PMS phenazine methosulfate - SDS sodium dodecyl sulfate     

Towards a male-only release system for SIT with the Queensland fruit fly,Bactrocera tryoni,using a genetic sexing strain with a temperature-sensitive lethal mutation

Flies that are homozygous for the recessive autosomal mutation bent wingshave a limited ability to fly and are less tolerant of high temperatures than normal flies in both the egg and puparial stages. The differences between the mutant and normal flies were found sufficient to be the basis of a genetic sexing strain. Genetic sexing strains were created using translocations of the autosome bearing the wild-type allele of bent wings(chromosome 2) to the Y chromosome, and crossing male flies carrying the translocation to mutant bent wingsfemales. In the resulting strain, the females were homozygous for the bent wingsmutation and the males were phenotypically normal for wing characters. Several translocations were recovered after irradiation, but only one translocation involving chromosome 2 was both stable and expressed in a stock that was vigorous enough for long-term viability. Unfortunately, all stocks containing the translocation showed high levels of temperature-dependent lethality, including, inexplicably, both males and females. Translocation stocks showing this effect included bent wings, another second chromosome mutation, white marks, and an otherwise normal stock. This phenomenon is probably rare, as it has not been reported before. It is likely that bent wingscould be suitably used with another translocation.     

Induction of alcohol dehydrogenase null mutants in the mediterranean fruit fly Ceratitis capitata

Alcohol dehydrogenase null mutants have been induced with X rays in Ceratitis capitata, for use in a genetic sexing system. A combination of selective medium and electrophoretic screening was used to identify these mutants. The results indicate that they are probably large deletions since no homozygotes were found for any of the induced mutants.     

Sexual competitiveness of a transgenic sexing strain of the Mediterranean fruit fly, Ceratitis capitata

The sterile insect technique (SIT), when used for the control of the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), generally relies on the release of sterile flies of only the male sex. Male selection is achieved through the use of a genetic sexing strain (GSS) in which females are killed by heat treatment in the generation prior to release. Transgenic sexing strains (TSS) have been developed that perform the same function of female-lethality, this time by withholding tetracycline (or related compounds) from the larval diet. The use of TSS may allow for certain problems associated with conventional GSS, such as strain instability and reduced productivity in mass-rearing, to be avoided. The performance, and principally the sexual competitiveness, of released male flies is important for the success of an SIT control programme. This study describes field cage experiments in which the competitiveness of males from a TSS (OX3376B) was compared with that of a conventional GSS (VIENNA-8) and two wild-type strains (TOLIMAN and ARG). When competing for female mates with wild-type males, OX3376B male performance was acceptable. When OX3376B males competed directly for mates with VIENNA-8 males, VIENNA-8 slightly outperformed the TSS males. Parallel tests, in which wild-type males competed with either OX3376B or VIENNA-8 males, showed that males from both sexing strains were highly competitive with wild-type males. These results suggest that OX3376B in particular, and TSS in general, show sufficiently good mating competitiveness to merit further research into their suitability for eventual use in SIT programmes.     

Sex allocation and clutch size in the gregarious larval endoparasitoid wasp, Cotesia glomerata

The ability of the gregarious larval endoparasitoid Cotesia glomerata L. (Hymenoptera: Braconidae) to adjust progeny sex ratio and clutch size was investigated. The sex ratios (proportion of males) of field clusters were diverse, but many (70%) were female-biased. Nearly 10% yielded males only, suggesting a low percentage of unmated females in the field. In over half of the clusters containing females, the sex ratio was below 0.3. Superparasitism was common in the field, and females were believed to increase progeny sex ratio when attacking previously-parasitized hosts. However, in a single oviposition bout, sex allocation was not precisely controlled both in the field and laboratory. In the laboratory, the number of eggs laid in a day tended to decrease with increasing female age. For females which were offered two hosts per day and for those offered three hosts per day, this value became nearly the same several days after the start of oviposition. The total number of hosts which a female could parasitize during her lifetime was often less than 40. Some of the old females which attacked more than 40 hosts produced male-biased clutches; this was due to sperm depletion, because sperm remained viable throughout a female's lifetime. The amount of sperm used in a single oviposition bout seemed fixed and was not dependent on the number of eggs laid. Females with much oviposition experience did not produce new eggs to compensate for deposited eggs, and the efficiency of egg use (deposited eggs/total eggs) was more than 80%.     

KlADH3, a gene encoding a mitochondrial alcohol dehydrogenase, affects respiratory metabolism and cytochrome content in Kluyveromyces lactis

A Kluyveromyces lactis strain, harbouring KlADH3 as the unique alcohol dehydrogenase (ADH) gene, was used in a genetic screen on allyl alcohol to isolate mutants deregulated in the expression of this gene. Here we report the characterization of some mutants that lacked or had highly reduced amounts of KlAdh3p activity; in addition, these mutants showed alterations in glucose metabolism, reduced respiration and reduced cytochrome content. Our results confirm that the KlAdh3p activity contributes to the reoxidation of cytosolic NAD(P)H feeding the respiratory chain through KlNdi1p, the mitochondrial internal transdehydrogenase. The low levels of KlAdh3p in two of the mutants were associated with mutations in KlSDH1, one of the genes of complex II, suggesting signalling between the respiratory chain and expression of the KlADH3 gene.     

Functional mutants of yeast alcohol dehydrogenase affecting kinetics,cellular redox balance,and electrophoretic mobility

Repeated selection of petite (respiratorily incompetent) Saccharomyces cerevisiae on medium containing allyl alcohol, both on plates and in the turbidostat, results in mutants with a remarkably similar response. Most of the mutations affect the constitutive alcohol dehydrogenase, resulting in enzymes with a cathodal shift in electrophoretic mobility, and none shows a significant anodal shift. The genetics, kinetics, and physiological effect of three of the mutants have been investigated in detail, and while all confer resistance to allyl alcohol through a shift in the NAD/NADH ratio, they do so in slightly different ways. The potential of this system for exploring the range of short-term adaptations open to this organism is discussed.This work was supported by Grant GM 19967 from the National Institutes of Health, U.S. Public Health Service.     

Homology of Saccharomyces cerevisiae ADH4 to an iron-activated alcohol dehydrogenase from Zymomonas mobilis

Summary Insertion of the transposable element Ty at the ADH4 locus results in increased levels of a new alcohol dehydrogenase (ADH) activity in Saccharomyces cerevisiae. The DNA sequence of this locus has been determined. It contains a long open reading frame which is not homologous to the other ADH isozymes that have been characterized in S. cerevisiae nor does it show obvious homology to Drosophila ADH. The hypothetical ADH does, however, show strong homology to the sequence of an iron-activated ADH from the bacterium Zymomonas mobilis. Thus ADH4 appears to encode an ADH structural gene which, along with the Zymomonas enzyme, may define a new family of alcohol dehydrogenases.Now The Plant Cell Research Institute, Inc., 6560 Trinity Court, Dublin, CA 94568, USA     

Ceratitis capitata—a suitable case for genetic sexing

In the framework of the development of a genetic sexing mechanism male-linked translocations were induced and studied in the Mediterranean fruit fly, Ceratitis capitata.Two-day old males were irradiated with 5 krad of X-rays and mated to virgin females. The induced translocations were selected by identifying linkage between a visible marker (eye colour or pupal colour) and sex. A total of 18 translocations were isolated in three irradiation series. All the lines except one showed complete linkage of the sex with the marker. In the one exception a small percentage of recombinant males was consistently found but they proved to be sterile. Very few recombinant females were found. The origin of these males is at present unclear. Of the six lines which were tested all showed significantly reduced larval survival and 4 had reduced pupal survival. The egg fertility of the lines ranged from 88.39% down to 25.39%.The third irradiation series was used to check if families showing intermediate linkage were present; this could infer that crossing-over was occurring in the male. However, only two families out of 148 tested showed such linkage.It was concluded that crossing-over in the male medfly will not be a serious obstacle to the development of a genetic sexing method using linkage of resistant conditional lethal alleles to the male-determining chromosome.     

Diploid male production correlates with genetic diversity in the parasitoid wasp Venturia canescens: a genetic approach with new microsatellite markers

Sex determination is ruled by haplodiploidy in Hymenoptera, with haploid males arising from unfertilized eggs and diploid females from fertilized eggs. However, diploid males with null fitness are produced under complementary sex determination (CSD), when individuals are homozygous for this locus. Diploid males are expected to be more frequent in genetically eroded populations (such as islands and captive populations), as genetic diversity at the csd locus should be low. However, only a few studies have focused on the relation between population size, genetic diversity, and the proportion of diploid males in the field. Here, we developed new microsatellite markers in order to assess and compare genetic diversity and diploid male proportion (DMP) in populations from three distinct habitat types – mainland, island, or captive –, in the parasitoid wasp Venturia canescens. Eroded genetic diversity and higher DMP were found in island and captive populations, and habitat type had large effect on genetic diversity. Therefore, DMP reflects the decreasing genetic diversity in small and isolated populations. Thus, Hymenopteran populations can be at high extinction risk due to habitat destruction or fragmentation.     

Functional Alcohol Dehydrogenase Mutants of Saccharomyces cerevisiae Conferring Temperature-Conditional Allyl Alcohol Resistance

Selection for allyl alcohol resistance in respiratory incompetent yeast is a highly specific method for isolating functional mutations at ADH1, the gene coding for the cytoplasmic alcohol dehydrogenase, ADHI. Because of the nature of this selection scheme, the ADHI activity of such mutants is retained, but the kinetic characteristics of the enzymes are altered. The high specificity for targeting functional mutations at this locus suggested that selection for enzyme variants with more subtle phenotypic effects might be possible. Here, we describe functional ADHI mutants that are temperature-conditional in their allyl alcohol resistance. Haploid cells of one of these mutants grow well on plates at 10 mM allyl alcohol at 19 degrees, but not at 37 degrees, the restrictive temperature. A second mutant grows well at 10 mM at 37 degrees, but its growth is restricted at 19 degrees. What distinguishes these mutants from other temperature-sensitive mutants is that the temperature-conditional growth phenotypes described here must be due to interactions between allyl alcohol levels and ADHI functional properties and cannot be due to lability of the enzyme at the restrictive temperature. This system shows promise for the investigation of functional enzyme variants that differ by only one or two amino acid residues but have significant temperature- and substrate-conditional effects on growth phenotypes in both the haploids and the diploids.     

A new sex-linked mutant short wing in Aedes aegypti.

A spontaneous recessive sex-linked mutant short wing has been discovered in the mosquito Aedes aegypti. It is situated less than one cross-over unit from the sex determining locus. In homozygous females, flight is impaired and the survival and fecundity is markedly subnormal. Two possible uses of this gene for genetic control operations are envisaged: (a) to provide automatic sexing of males for release and (b) enhancement of the population control potential of other available genetics systems.     

Roles of alcohol dehydrogenases of Zymomonas mobilis (ZADH): characterization of a ZADH-2-negative mutant

Zymomonas mobilis is a potential candidate for fuel ethanol production because of its high ethanol productivity. A key enzyme in ethanol production is alcohol dehydrogenase (ADH). Z. mobilis possesses two isozymes, ZADH-1 and ZADH-2. To clarify their physiological roles, mutants with modified ADH were isolated by selection based on resistance to allyl alcohol. From the physiological characteristics of a ZADH-2-negative mutant, it is suggested that ZADH-1 functions as the major ADH, while ZADH-2 could become functional in the latter stages of fermentation.