Anaerobic tolerant null: a mutant that allows Adh1 nulls to survive anaerobic treatment

Anaerobic tolerant null (ATN) is a recessive factor that allows alcohol dehydrogenase-1 (ADH1) null individuals of Zea mays L. to survive 24 h of anaerobic conditions. ADH1 null lines that do not possess this factor survive only a few hours of anoxia. We studied ADH activity levels in protein extracts from the primary root tissue of ATN. ADH levels were similar in ATN and other ADH1 null lines, suggesting that ADH activity does not account for differences in the ability of ATN to survive anaerobic treatment. The ATN survival trait segregated as a single recessive locus in crosses between ATN and double null (Adh1-S5657, Adh2-33). We also made crosses between ATN and 1s2p, an inbred line with ADH1 activity that carries an electrophoretic mutation of Adh2, to determine whether atn increases the number of survivors over that which would be expected from the segregation of Adh1 alone and to use the Adh2P allele to study the cosegregation of Adh2 and atn. The observed number of survivors in that cross exceeded the expected number of survivors by a margin consistent with a single recessive gene adding to the ADH+ survivors. Extracts from the primary root or scutellum of induced F2 seedlings from the above crosses were assayed for ADH activity by native polyacrylamide gel electrophoresis (PAGE) and simultaneously scored for survival to determine whether Adh2 and atn were segregating independently. We screened the (ATN x 1s2p)F2 progeny for ADH1 activity by staining root tips with an ADH-specific stain to select Adh1 null individuals prior to gel assay. Atn was found to be assorting independently of Adh1 and Adh2 in both crosses.     

Transposon-mediated mutations in the untranslated leader of maize Adh1 that increase and decrease pollen-specific gene expression.

The unstable mutation Adh1-Fm335 contains a Dissociation (Ds1) transposable element at position +53 in the untranslated leader of the maize Alcohol dehydrogenase-1 (Adh1) gene. Excision of Ds1 is known to generate new alleles with small additions and rearrangements of Adh1 DNA. We characterized 16 revertant alleles with respect to ADH1 activity levels in scutellum (nutritive tissue of the seed), anaerobic root, and pollen. Whereas gene expression was not different from the wild type in the sporophytic tissues of the scutellum and anaerobic root, there were strong allelic differences in pollen. One allele underexpressed pollen ADH1 at 48% of the wild-type level, and another overexpressed pollen ADH1 at 163% of the wild-type level. Quantitative RNase protection assays demonstrated that the mutant phenotypes reflected changes in the levels of steady state mRNA in pollen. These data provide a definitive demonstration of an overexpression mutant in plants and further show that marked increases in mRNA levels can follow minor alterations in central untranslated leader sequences. The nucleotide sequence of 12 new revertant alleles and the molecular mechanisms responsible for pollen-specific gene expression are discussed.     

Genetic determination of the developmental program for maize scutellar alcohol dehydrogenase: Involvement of a recessive,trans-acting,temporal-regulatory gene

The developmental program of alcohol dehydrogenase (ADH) activity in the scutellum of maize strain R6-67 is different from that of W64A. The level of scutellar ADH activity in R6-67 remains relatively high during the course of early sporophytic development as compared to the commonly observed pattern. In the typical inbred strain W64A, the activity of ADH declines substantially during that period. The variance values from the crosses between R6-67 and W64A reveal that the trait is under genetic control. Detailed genetic analysis suggests that a single gene is responsible for the altered developmental program of ADH activity in R6-67. This gene meets the criteria for temporal regulatory genes and is different from Adh2, the structural gene which codes the ADH-2 isozyme. We have designated this gene as Adr1 (alcohol dehydrogenase regulator, #1). Adr1 is unlinked to Adh2. There is no de novo synthesis of ADH in the scutellum during germination, and the difference in the activity level reflects the difference in the amount of enzyme protein as demonstrated by density labeling and rocket immunoelectrophoresis. Thus, it appears that Adr1 may regulate the degradation of ADH.     

Correct developmental expression of a cloned alcohol dehydrogenase gene transduced into the Drosophila germ line

We have used P-element-mediated transformation to introduce a cloned Drosophila alcohol dehydrogenase (Adh) gene into the germ line of ADH null flies. Six independent transformants expressing ADH were identified by their acquired resistance to ethanol. Each transformant carries a single copy of the cloned Adh gene in a different chromosomal location. Four of the six transformant lines exhibit normal Adh expression by the following criteria: quantitative levels of ADH enzyme activity in larvae and adults; qualitative tissue specificity; the size of stable Adh mRNA; and the characteristic developmental switch in utilization of two different Adh promoters. The remaining two transformants express ADH enzyme activity with the correct tissue specificity, but at a lower level than wild type. These results demonstrate that an 11.8 kb chromosomal fragment containing the Adh gene includes the cis-acting sequences necessary for its correct developmental expression, and that a variety of chromosomal sites permit proper Adh gene function.     

Mutant alleles that are altered in quantitative,organ-specific behavior

Three new mutant alleles of maize alcohol dehydrogenase-1 (Adh 1) were recovered following allyl alcohol selection of pollen. Each is altered in quantitative, organ-specific, regulatory properties. All mutant sites act in cis to the structural gene component. One mutant arose spontaneously, one followed indirectly from irradiation with high Z accelerated particles, and one was induced by an autonomous mutator system. Each mutant is assessed in three organs by utilizing ADH allozyme ratios that were quantified at the level of ADH enzyme activity and either [³H]-Leu incorporation into newly synthesized ADH 1 subunits or direct protein determinations. One mutation simultaneously raises Adh 1 expression in one organ and lowers it in another, another affects expression in one organ only, and another is extremely underexpressed in all organs but is unstable. This unstable allele has generated derivative mutant alleles that have less or zero ADH expression. We do not yet know whether or not coding sequences are involved in these mutants. We conclude that information for organ specificity and quantitative behavior resides near or within Adh 1 coding sequences.     

Hypoxic Induction of Anoxia Tolerance in Roots of Adh1 Null Zea mays L

Seedlings of alcohol dehydrogenase 1 null mutants (Adh1-) of Zea mays L., which fail to synthesize alcohol dehydrogenase 1 (ADH1) isozymes, were hypoxically acclimated by 18 h of exposure to an atmosphere of 4% (v/v) O2 in N2 at 25[deg]C. Their ability to tolerate subsequent anoxia by exposure to anaerobic (O2-free) conditions was compared with that of unacclimated seedlings that were transferred immediately from an atmosphere of 40% (v/v) O2 to anaerobic conditions. Only 10% of the root tips of unacclimated seminal roots survived 6 h of anoxia, whereas 70% of the hypoxically acclimated root tips were viable at 24 h. During anoxia, acclimated root tips had enhanced ADH activity compared with unacclimated root tips, through induction of Adh2. Despite this, enzyme activity was still only about 5% that of acclimated, wild-type root tips and about half that of unacclimated, wild-type root tips. During anoxia, acclimated Adh1- root tips showed a higher rate of anaerobic respiration and ethanol production, greater concentrations of ATP and total adenylates, and a greater adenylate energy charge compared with unacclimated root tips. These results suggest that although enhanced ADH activity may have raised fermentation rates in acclimated Adh1- tissues and thereby contributed to energy metabolism and viability, the high levels of ADH activity inducible in acclimated, wild-type maize root tips appear to be in excess of that required to increase rates of fermentation.     

Molecular analysis of genetic differences among inbred mouse strains controlling tissue expression pattern of alcohol dehydrogenase 4

The ADH gene family in vertebrates is composed of at least seven distinct classes based upon sequence comparisons and enzyme properties. The Adh4 gene product may play an important role in differentiation and development because of its capacity to metabolize retinol to retinoic acid. Allelic gene differences exist among inbred mouse strains which control structure and tissue-specific regulation of Adh4. C57BL/6 mice are unique and have no detectable ADH4 enzyme activity in epididymis and low levels in seminal vesicle, ovary and uterus compared to other strains. C57BL/6 mice express Adh4 in stomach at levels similar to other strains. The goal of this research was to investigate this genetic variation at the molecular level. Northern analysis revealed that the content of ADH4 mRNA in tissues correlate with the enzyme expression pattern. Interestingly, C57BL/6 mice express an ADH4 mRNA in stomach which is smaller than expressed in C3H and other mice. An analysis of the 5'- and 3'-ends of the mRNA using RACE analysis determined that the ADH4 mRNA in C57BL/6 mice is truncated in the 3'-untranslated region. Sequence analysis of RACE products showed that the truncation is due to a single nucleotide mutation which produces an early polyadenylation signal. Additional RACE and Northern analysis revealed that at least five different polyadenylation sites are used in the Adh4 gene. Using 3'-end polymorphisms found between C57BL/6 and C3H strains and RT-PCR, it was shown that the lack of expression in epididymis in C57BL/6 mice is cis-acting in F(1) hybrid animals. The DNA sequence of the proximal promoter (-600/+42 nt) was determined in several mouse strains differing in tissue-specific expression patterns and did not reveal any nucleotide substitutions correlating with expression pattern suggesting further upstream or downstream sequences may be involved.     

Natural variants of pearl millet (Pennisetum typhoides) with altered levels of set II alcohol dehydrogenase activity

Two linked genes, Adh1 and Adh2, specify three sets of ADH isozymes in pearl millet. Set I is a homodimer specified by Adh1, Set III is a homodimer specified by Adh2, and Set II is a heterodimer consisting of one ADH1 subunit and one ADH2 subunit. Dry seeds exhibit only Sets I and II. Anaerobic treatment of seeds greatly increases the activity of Sets I and II and causes the Set III isozymes to be expressed. In the investigation reported here, the ADH zymogram phenotypes of 112 inbred pearl millet lines were analyzed. Two kinds of naturally occurring ADH variant strains were observed: in the low-activity variant, Set II activity is low in the dry seed, and no Set III activity is present upon anaerobic treatment. In the high-activity variant, Set II activity is high and Set III isozymes are expressed in the dry seed. The mutation in the high-activity strain appears to affect the product of Adh2 and not the product of Adh1. Dominance tests show that the mutations in both types of variant strains act in cis. These observations and linkage tests indicate that the mutations are closely linked to or at the Adh2 locus.This work was supported by a PHS National Research Service Award Training Grant in Genetics to the Biology Department of the University of Oregon.     

Excessive vitamin A toxicity in mice genetically deficient in either alcohol dehydrogenase Adh1 or Adh3.

Alcohol dehydrogenase (ADH) deficiency results in decreased retinol utilization, but it is unclear what physiological roles the several known ADHs play in retinoid signaling. Here, Adh1, Adh3, and Adh4 null mutant mice have been examined following acute and chronic vitamin A excess. Following an acute dose of retinol (50 mg.kg(-1)), metabolism of retinol to retinoic acid in liver was reduced 10-fold in Adh1 mutants and 3.8-fold in Adh3 mutants, but was not significantly reduced in Adh4 mutants. Acute retinol toxicity, assessed by determination of the LD(50) value, was greatly increased in Adh1 mutants and moderately increased in Adh3 mutants, but only a minor effect was observed in Adh4 mutants. When mice were propagated for one generation on a retinol-supplemented diet containing 10-fold higher vitamin A than normal, Adh3 and Adh4 mutants had essentially the same postnatal survival to adulthood as wild-type (92-95%), but only 36% of Adh1 mutants survived to adulthood with the remainder dying by postnatal day 3. Adh1 mutants surviving to adulthood on the retinol- supplemented diet had elevated serum retinol signifying a clearance defect and elevated aspartate aminotransferase indicative of increased liver damage. These findings indicate that ADH1 functions as the primary enzyme responsible for efficient oxidative clearance of excess retinol, thus providing protection and increased survival during vitamin A toxicity. ADH3 plays a secondary role. Our results also show that retinoic acid is not the toxic moiety during vitamin A excess, as Adh1 mutants have less retinoic acid production while experiencing increased toxicity.     

Intragenic recombination in maize: pollen analysis methods and the effect of parental adh1 isoalleles

The ability to stain mature pollen grains for the presence of alcohol dehydrogenase (ADH) activity permits the quantitation of ADH( +) gametophytes at frequencies below 10(-6). This resolution allows reversion and genetic fine structure analyses. The rationale of pollen analysis follows Nelson's prototype studies with waxy. As with the waxy gene, revertant frequencies for seven Adh1-deficient ( Adh1(-)) alleles appear to be in excess of microbially derived expectations. Each of the seven Adh1(-) alleles were derived from one of three naturally occurring isoalleles. Based on Schwartz's protein level characterizations of the mutants' products, it was anticipated that the seven Adh1(-) alleles should recombine to yield ADH(+) cistrons in certain pairwise combinations. This expectation was not met. The parental "wild-type" isoalleles from which the mutants were derived appear to be structurally divergent. The discussion interprets these data in view of understanding naturally occurring cistronic variation.     

Distinct retinoid metabolic functions for alcohol dehydrogenase genes Adh1 and Adh4 in protection against vitamin A toxicity or deficiency revealed in double null mutant mice

The ability of class I alcohol dehydrogenase (ADH1) and class IV alcohol dehydrogenase (ADH4) to metabolize retinol to retinoic acid is supported by genetic studies in mice carrying Adh1 or Adh4 gene disruptions. To differentiate the physiological roles of ADH1 and ADH4 in retinoid metabolism we report here the generation of an Adh1/4 double null mutant mouse and its comparison to single null mutants. We demonstrate that loss of both ADH1 and ADH4 does not have additive effects, either for production of retinoic acid needed for development or for retinol turnover to minimize toxicity. During gestational vitamin A deficiency Adh4 and Adh1/4 mutants exhibit completely penetrant postnatal lethality by day 15 and day 24, respectively, while 60% of Adh1 mutants survive to adulthood similar to wild-type. Following administration of a 50-mg/kg dose of retinol to examine retinol turnover, Adh1 and Adh1/4 mutants exhibit similar 10-fold decreases in retinoic acid production, whereas Adh4 mutants have only a slight decrease. LD(50) studies indicate a large increase in acute retinol toxicity for Adh1 mutants, a small increase for Adh4 mutants, and an intermediate increase for Adh1/4 mutants. Chronic retinol supplementation during gestation resulted in 65% postnatal lethality in Adh1 mutants, whereas only approximately 5% for Adh1/4 and Adh4 mutants. These studies indicate that ADH1 provides considerable protection against vitamin A toxicity, whereas ADH4 promotes survival during vitamin A deficiency, thus demonstrating largely non-overlapping functions for these enzymes in retinoid metabolism.     

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.     

Mutations of the Adh1 gene in maize following infection with barley stripe mosaic virus

Summary Mutations at the Adh1 locus in maize were selected from plants infected with barley stripe mosaic virus (BSMV). Pollen from the infected inbred line 1s2p, which is homozygous for Adh1-S (abbreviated S), Adh2-P, c and r was treated with allyl alcohol and applied to silks of a tester stock homozygous for Adh1-F, Adh2-N, C and R. From these pollinations 356 kernels arose on the F₁ ears. Of these eight showed no activity of the S allele in scutellar samples while two exhibited low levels. Five of the putative mutant kernels germinated and two of these contained the contamination markers Adh2-P, c and r. The newly arisen mutations were designated S5446 and S5453. S5453 exhibited an abnormally low level of ADH activity in the F₁ scutellum. In the F₂ generation the mutant reverted at a high frequency with only about 5% of the S5453 alleles expressing low levels. DNA blotting and hybridization analyses showed no alterations in the restriction patterns of S5453 when compared to the progenitor S allele. S5446 which exhibited no ADH activity in the F₁ scutellum is unstable in the pollen; reversion frequencies approaching 10^-2 were observed in samples from some plants. Restriction digestion patterns of DNA from this mutant revealed the presence of a 3.3 kb insertion at Adh. The insert does not appear to contain sequences homologous to the BSMV genome but rigorous analyses remain to be carried out. It is hypothesized that BSMV infection may mobilize endogenous but dormant transposable elements in maize.     

Allelic variation at the level of intragenic recombination

This report examines five different naturally occurring alcohol dehydrogenase-1 alleles via the recombinational behavior of Adh1^- mutants induced within them. Twenty-two biochemically characterized Adh1^- mutants have been assessed for ability to recombine intragenically, using data generated by specifically staining for the presence of ADH in pollen grains. Each of the five naturally occurring Adh1 progenitor isoalleles appears unique. Allelic variation exists in (1) the rate of intragenic recombination sustained by an allele, and (2) the pattern of recombinational success or failure based on the ancestry of each mutant in a heteroallelic pair. In other words, we find quantitative and qualitative Adh1 allelic variation at the level of intragenic recombination. I have experimentally excluded several explanations for recombinational restriction. These results will be related to the structure, function and naturally occurring variability of the gene in higher organisms. Specifically, the "recon" (unit of recombination) has been resurrected as a potentially useful unit of natural selection. The reasonableness of several genres of hypotheses in evolutionary/population genetics, particularly those involving linkage disequilibrium, is called into question.     

Molecular analysis of a somaclonal mutant of maize alcohol dehydrogenase

Summary Plants regenerated from tissue cultures of maize were screened for variants of ADH1 and ADH2. Root extracts of 645 primary regenerant plants were tested, and one stable mutant of Adh1 was detected. The mutant gene (Adh1-Usv) produces a functional enzyme with a slower electrophoretic mobility than that of the progenitor Adh1-S allele, and is stably transmitted to progeny. The mutant was not present among four other plants derived from the same immature embryo, and therefore arose as a consequence of the culture procedure. The gene of Adh1-Usv was cloned and sequenced. A single base change in exon 6 was the only alteration found in the gene sequence. This would translate in the polypeptide sequence to a valine residue substituting for a glutamic acid residue, resulting in the loss of a negative charge and the production of a protein with slower electrophoretic mobility.Abbreviations kb kilobase pairs - ADH alcohol dehydrogenase     

Polymorphism of PCR profiles and expression of alleles at the locus <Emphasis Type="Italic">Adh1</Emphasis> in agamospermous progeny of sugar beet <Emphasis Type="Italic">Beta vulgaris</Emphasis> L.

A modification of the ISSR amplification method based on using a combination of microsatellite and specific unique primer is proposed and tested. This modification simplifies the detected PCR profiles and allows the examination of DNA regions containing definite genes. Combinations of microsatellite primer Mic2 (5′-gacag-acaga-cagac-a-3′) and one of the primers specific to the Adh1 locus, which controls alcohol dehydrogenase (ADH1) in sugar beet, were employed in this work. The microsatellite primer was used in combination with the following specific primers: Adh1f (5′-agagt-gttgg-agagg-gtgtg-ac-3′) containing the binding site at the fourth exon of gene Adh1, or Adh1r (5′-act(ct)a-cagca-ag(ct)cc-(ct)ac(ct)g-ctcc-3′) that binds to the fifth exon of the same gene. In the agamospermous progeny of individual heterozygous diploid plants of sugar beet with the Adh1-F/Adh1-S genotype, polymorphism of PCR profiles obtained in plants of each of three phenotypic classes (FF, FS, and SS) was detected. Among plants of the progeny from an individual plant that represents the heterozygous phenotypic class FS, differences were revealed not only between the PCR profiles but also in the relative activity of allele isozymes of ADH1.