High molecular weight glutenin subunit in durum wheat (T. durum)

Summary The diversity of high molecular weight (HMW) glutenin subunits of 502 varieties of durum wheat (Triticum durum) from 23 countries was studied using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Twenty-nine types of patterns were observed with 18 mobility bands. A total of 18 alleles were identified by comparing the mobilities of their subunits to those previously found in hexaploid wheat (T. aestivum) and in Triticum turgidum var. dicoccum. Five new alleles were detected: two on the Glu A1 and three on the Glu B1 locus. Comparison of the frequency of alleles in the three species T. aestivum, T. dicoccum and T. durum was investigated. Significant differences exist between each of these species on the basis of the frequency distributions of their three and four common alleles at the Glu A1 and Glu B1 locus, respectively. The Glu B1c allele occuring very frequently in hexaploid wheats was not found in the two tetraploid species. More than 83% of the T. durum analysed were found to have the Glu A1c (null) allele.     

Genetic control of alcohol dehydrogenase isozymes in maize

By means of horizontal gel electrophoresis and the zymogram technique, genetic variants and the formation of a hybrid molecule of the enzyme alcohol dehydrogenase (ADH) have been found in Zea mays. Each inbred homozygous stock examined showed two types of ADH isozyme patterns: a fast faint zone and a slower deeply staining zone, both anode-migrating at pH 8.5. The variants found differed in that each of the ADH zones varied in its electrophoretic mobility when compared to its counterpart in the other strain. When appropriate genetic crosses were made, the resulting heterozygotes showed the parental ADH zones, and, in addition, a band of intermediate mobility was formed between the deep-staining ADH bands. However, in the fast-moving zone only the parental isozymes were represented in the heterozygote. The formation of the hybrid molecule and the apparent gene dosage effects support the hypothesis that ADH-2 in maize exists as a dimer, whereas ADH-1 may exist as a monomer.This work was supported by the U.S. Atomic Energy Commission, under contract No. AT(11-1)-1338.     

Characteristics of somatic cell hybrids (mouse X Chinese hamster) with different ratios of parental species chromosome sets. IV. Electrophoretic analysis of several enzymes of the dehydrogenase class]

Electrophoretic mobilities in polyacrylamide gel of five dehydrogenases: NADP-dependent malate dehydrogenase (NADP-MDH), 6-phosphogluconate dehydrogenase (6PGD), alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GDH) were investigated in a series of mouse X Chinese hamster somatic cell hybrids. Seven hybrid lines with different ratio of chromosome sets of hamster and mouse: 1:1, 2:1, 3:1 and 1:2 respectively were studied. NADP-MDH and 6PGD of both parental species and intermediate hybrid bands were present in all hybrids except two lines. These lines had only hamster MDH due to the elimination of mouse chromosomes. A correlation was found between the gene dose and the intensity of the expression of the MDH bands. The mouse type ADH was detected in all hybrids. The hamster ADH was found in one of the hybrid lines that lost all mouse chromosomes during cultivation. It is suggested that hamster ADH activity was suppressed in hybrids by the mouse genome. The species origin of GDH and G6PD could not be established due to similarity of electrophoretic mobilities of respective enzymes in parental cells.     

Mutational study of the alcohol dehydrogenase-1 FC m duplication in maize

The alcohol dehydrogenase-1 FCm (Adh-FCm) duplication in maize was subjected to ethyl methanesulfonate (EMS) mutagenesis. Of the mutants recovered, eight produced ADH polypeptides with altered electrophoretic mobility. Four produced new mobilities of the progenitor F with no change of the Cm molecule; the remainder altered only the Cm enzyme. No cases were found in which the electrophoretic mobilities of the two types of subunits were simultaneously altered, and no complete nulls lacking both F and Cm were recovered. These observations confirm the duplicate nature of the FCm complex.     

Introduction of Alcohol Dehydrogenase in Lettuce Seedlings by Flooding Stress

Alcohol dehydrogenase (ADH), its isozyme profiles and ethanol concentration in lettuce (Lactuca sativa L.) seedlings subjected to flooding stress were determined. Flooding stress caused increases in ADH activity and ethanol concentration. By 48 h, ADH activity and ethanol concentration in the flooded seedlings increased 3.2- and 7.0-fold, respectively, in comparison with those in non-stressed seedlings. Five electrophoretically separable ADH bands were found in extract of the flooded seedlings, whereas only two or three ADH bands were found in extract of non-stressed seedlings. These results indicate that lettuce ADH may have a system of three-gene and six-isozyme, and the increase in ADH activity in the flooded seedlings may be due to increased synthesis of the enzyme.     

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.     

Enzyme specificity: “pseudopolymorphism” of lactate dehydrogenase in Drosophila melanogaster

An electrophoretic variant in the LDH (l-lactate:NAD oxidoreductase, E.C.1.1.1.27) of Drosophila melanogaster was observed on starch (or polyacrylamide) gels. This variant was found to exhibit an identical isozymic pattern (three isozymes with a decreasing staining density) on starch gel and map position as the Adh locus. On the other hand, anodal polyacrylamide gel electrophoresis in crude extracts has shown LDH to consist of nine bands and ADH of four bands. We have shown that ADH (Alcohol:NAD oxidoreductase, E.C.1.1.1.1) also oxidizes l(+)-lactate or d(–)-lactate with the NAD, while LDH oxidizes ethanol. By using various genetic and biochemical techniques, we have shown that the observed Ldh electrophoretic variant was not a real one and could be attributed to the presence of ADH. We have called this phenomenon pseudopolymorphism, and the problem of enzyme specificity has been examined. The appearance of a band in an assay using lactic acid as a substrate is not sufficient evidence for the presence of LDH. Hence, caution is called for before characterizing an electrophoretic band on a gel as being equivalent to the presence of a genetic locus. Out of the nine electrophoretic zones of activity observed on polyacrylamide gel (or out of the six previously observed) using crude extract, only two (one major and one minor) belong to LDH, as revealed by purified enzyme preparations. Furthermore, purified LDH exhibits activity in two bands on starch gel (out of three observed in crude extracts), which appear in different positions as compared with those of ADH. Finally, one band which responds to the presence of d(–)-lactate but not to l(+)-lactate has been revealed.     

Catalase in Plodia interpunctella I. Genetic control and enzymatic patterns during development

Three electrophoretic patterns of catalase were demonstrated in the adults of the Indian Meal Moth Plodia interpunctella (Hüb) (Lepidoptera: Phycitidae). All the types gave only single bands of catalase activity, which were distinguishable on the gel by mobility. Evidence that the variation between these patterns is controlled by a single sex linked gene with two codominant alleles, was obtained from appropriate genetic crosses. The electrophoretic pattern during ontogenesis of the insect shows that the variation in the enzymic content undergoes progressive alteration, both qualitatively and quantitatively.     

Electrophoretic evidence for the origin ofDryopteris yakusilvicola (Dryopteridaceae)

To investigate the origin of the triploid agamosporous speciesD. yakusilvicola, an electrophoretic analysis was made for five enzymes of theD. sparsa complex.Dryopteris yakusilvicola showed a monomorphic banding pattern for the five enzymes and was heterozygous in all six gene loci coding them. Comparison of enzyme banding patterns suggests that the genome ofD. yakusilvicola was derived through hybridization betweenD. sabaei and either a sexual tetraploid or an agamosporous triploid ofD. sparsa. Cytological evidence (Darnaediet al., 1989) supports the idea that of the two types ofD. sparsa the sexual tetraploid is a parent. The monomorphic pattern implies thatD. yakusilvicola originated from a single hybrid between the parental species, and that it is a neo-endemic of Yakushima Island.     

Identification of human alcohol dehydrogenase isozymes by disc polyacrylamide gel electrophoresis in 7 M urea

Polyacrylamide gel electrophoresis in the presence of 7 M urea provides a simple, reproducible method for the identification of cathodic alcohol dehydrogenase (ADH) isozymes. Treatment of native ADH dimers with 7 M urea and 1 mM dithiothreitol results in a complete dissociation of the 40,000 Mr subunits. Electrophoresis of urea-dissociated ADH isozymes yields a single protein band for homodimers and two bands of equal intensity for heterodimers. The ADH subunits pi, alpha, gamma 2, gamma 1, and beta exhibit electrophoretic mobilities of 0.71, 0.79, 0.88, 0.95, and 1.0, respectively. Thus, the identity of any cathodic ADH isozyme can be determined from the electrophoretic mobilities of its component subunits.     

Studies on isozymes of sorbitol dehydrogenase in some vertebrate species

Summary In swine, the enzyme sorbitol dehydrogenase exhibits a genetically determined polymorphism as identified by multiple electrophoretic bands suggesting a tetrameric structure (Op't Hof, 1969). In order to obtain further information on the genetics of this polymorphism, a number of other vertebrate species were examined. Multiple alleles at the SDH gene locus were found in the Cyprinid fishes Leuciscus and Rutilus, and in the Salmonid fish Salmo trutta. The electrophoretic patterns found in these species are compatible with the model of a tetrameric structure of the enzyme. This model was further verified by dissociation-reassociation studies with the homomeric enzymes of swine and Leuciscus. Since in the heterozygote an intensity gradient of the isozymes was seen, the three phenotypes observed in swine were further characterized by means of activity measurements and application of different substrates. The results point to a differential activity of the two different homomeres.Supported by the Deutsche Forschungsgemeinschaft.     

Genetic control of seed proteins in wheat

Summary Electrophoretic profiles of crude protein extracts from seed of F₁ hybrids and reciprocal crosses among diploid, tetraploid and hexaploid wheats were compared with those of their respective parental species. The electrophoretic patterns within each of three pairs of reciprocal crosses, T.boeoticum X T.urartu, T.monococcun X T. urartu and T.dicoccum X T. araraticum, were different from one another but were identical with those of their respective maternal parents. Protein bands characteristic of the paternal parents were missing in F₁ hybrid seed suggesting that the major seed proteins in wheat were presumably regulated by genotype of the maternal parent rather than by the seed genotype. However, in another three pairs of reciprocal crosses, T.boeoticum X T. durum, T.dicoccum X T.aestivum and T. zhukovskyi x T. aestivum, protein bands attributable to the paternal parents were present in the F₁ hybrid seeds indicating that the seed proteins were not always exclusively regulated by the maternal genotype. The expression of paternal genomes is presumably determined by dosage and genetic affinity of the maternal and paternal genomes in the hybrid endosperm. The maternal regulation of seed protein content is probably accomplished through the maternal control over seed size. The seed protein quality may, however, depend upon the extent of expression of the paternal genome.     

Purification and characterization of mouse alcohol dehydrogenase from two inbred strains that differ in total liver enzyme activity

Alcohol dehydrogenase activity in mouse liver homogenate-supernatants is 1.7 times greater in the C57BL/10 strain than in the BALB/c strain, regardless of whether activity is expressed in units per gram liver, total liver, or milligram DNA. The K _m values for ethanol and NAD⁺, approximately 0.4 and 0.03mm, respectively, of enzyme purified from both strains are similar. Moreover, the K _i for NADH, 1 µm, the pH optimum for ethanol oxidation, 10.5, and the V _max for ethanol oxidation, 160 min^–1, for ADH from the C57BL/10 and BALB/c strains are similar. Therefore, the difference in ADH activity in the two strains cannot be due to differences in the catalytic properties of the enzyme. The electrophoretic and isoelectric focusing patterns and two-dimensional tryptic peptide maps of the purified enzyme from both strains are identical. Thus the amino acid sequences of enzyme from C57BL/10 and BALB/c mice must also be identical or very similar. The difference in ADH activity in the two strains is most likely the result of genetic differences in the content of ADH protein in liver.Supported by NIAAA Grant AA 04307.     

B-Chromosomes and E-1 isozyme activity in mosaic bulbs of Scilla autumnalis (Liliaceae)

The electrophoretic patterns of esterase (E-1), alcohol dehydrogenase (ADH), and glutamate oxaloacetate transaminase (GOT) isozymes were studied in two Spanish populations of the lily Scilla autumnalis with B-chromosome carrying individuals. The E-1 isozyme activity appears only in those individuals with B-chromosomes. None of the bulbs free of B's show it. Five bulbs, mosaic for B-content, were identified. Electrophoretic analysis shows that these bulbs are characterised by mosaicism for E-1 isozyme activity. An analysis of individual roots by both electrophoretic and cytological methods shows that tissue mosaicism for B-content correlates with tissue mosaicism for E-1 isozyme activity. The electrophoretic analysis of different roots from bulbs heterozygous for the Est-1 locus indicates that the structural gene for E-1 is not located on the B-chromosome itself. Rather there is a derepressor effect of Bs on E-1 isozyme activity. Since ADH and GOT patterns are unaffected by the presence of B-chromosomes it is clear that they do not exhibit a generalised derepressor effect.     

Allelic inhibition at the autosomally inherited gene locus for liver alcohol dehydrogenase in chicken-quail hybrids

At the gene locus for liver alcohol dehydrogenase (ADH) of the Japanese quail, three alleles which give electrophoretic variants, A, B, and C, exist. This enzyme is autosomally inherited. Allelic polymorphism was not observed in the chicken, but the wild-type ADH of the chicken can readily be distinguished from A, B, and C of the quail by starch gel electrophoresis. In the development of both species, ADH activity reached a near adult level at about the nineteenth day (a few days after hatching in the quail and a few days before hatching in the chicken). Chicken-quail hybrids at the day of hatching (nineteenth day) revealed the presence of maternally derived quail ADH only, and their ADH activities were about half that of both parental species. Those hybrids which received either A or C allele from the mother quail showed three bands of ADH at the third day after hatching. The chicken and quail alleles began to function in synchronous harmony. One 3-day-old and two adult hybrids which received B allele from the quail, however, still revealed complete absence of the paternally derived chicken ADH.This work was supported in part by a grant (CA-05138) from the National Cancer Institute, U.S. Public Health Service, and in part by a research fund established in honor of General James H. Doolittle. Contribution No. 20-67, Department of Biology, City of Hope Medical Center.Dr. Eduardo Castro-Sierra is a fellow of the Institute for Advanced Learning of the City of Hope Medical Center.     

Alcohol dehydrogenase isozymes in grain sorghum (Sorghum bicolor): Evidence for a gene duplication

Two sets of alcohol dehydrogenase (ADH) bands are regularly observed in grain sorghum (Sorghum bicolor): set I is a permanent triplet; set II is variable, as either two or three bands. A faint set III is detected only when extracts from seeds subjected to anerobiosis are run in neutralpH gels. Dissociation-reassociation experiments reveal that the central band of the set I triplet is a heterodimer of the other two. Full-sib progeny analysis from selfed plants shows that the set II bands are doublets, with heterozygotes having only three apparent bands instead of four because of the similar mobilities of the fast-migrating isozyme specified by the slow allele and the slow isozyme specified by the fast allele. We propose a three-locus model as the best explanation of these patterns. Set I consists of the products of two loci and their intergenic heterodimer. Set III is specified by a third locus. Set II isozymes are the intergenic heterodimers of the two set I loci and the set III locus. This explanation is similar to that of Schwartz and Freeling for maize but suggests that the evolution ofSorghum includes a gene duplication of the homologue of theAdh-1 locus inZea. Supported by USDA Grant 59-2063-01522 to NCE and KWF.     

Variation in amount of enzyme protein in natural populations

Among strains of Drosophila melanogaster each derived from a single fertilized female taken from natural populations, there is variation in both alcohol dehydrogenase (ADH) activity and the amount of ADH protein. The correlation between ADH activity and number of molecules over all strains examined is 0.87 or 0.96 in late third instar larvae depending on whether the substrate is 2-propanol or ethanol. With respect to the two common electrophoretic allozymic forms, F and S, segregating in these populations, the FF strains on the whole have higher ADH activities and numbers of ADH molecules than the SS strains. Over all strains examined, enzyme extracts from FF strains have a mean catalytic efficiency per enzyme molecule higher than that of enzyme extracts from SS strains when ethanol is the substrate, and much higher when 2-propanol is the substrate. One FF strain had an ADH activity/ADH protein ratio characteristic of SS strains.     

Vanadium-dependent bromoperoxidases from <Emphasis Type="Italic">Gracilaria</Emphasis> algae

Red algae from the Gulf of Thailand were examined for haloperoxidatic activity. Six species, Gracilaria changii, G. edulis, G. firma, G. fisheri, G. salicornia, and G. tenuistipitata, showed bromoperoxidatic activity. Duplicate polyacrylamide electrophoretic gels showed enzyme activity patterns developed by phenol red staining for bromoperoxidatic activity and by 3,3′-diaminobenzidine staining for peroxidatic activity. All algae gave isoenzymic bromoperoxidatic activity bands and peroxidatic activity bands, but there were peroxidatic and bromoperoxidatic activity bands that did not correspond. The bromoperoxidatic activity of the crude enzyme extracts as well as previously dialyzed enzyme solutions was enhanced significantly by incubation with vanadium pentoxide. The three purified bromoperoxidases from G. fisheri contained vanadium, and their relative activities corresponded to the ratio of vanadium to enzyme. In addition, they were not inhibited by H₂O₂. These data confirm that the enzymes are vanadium bromoperoxidases.     

Duplication of the autosomally inherited 6-phosphogluconate dehydrogenase gene locus in tetraploid species of cyprinid fish

Among members of the fish family Cyprinidae,a diploid—tetraploid relationship exists. The present study on electrophoretic patterns of 6-phosphogluconate dehydrogenase indicates that such diploid members as Barbus tetrazonamaintain allelic polymorphism at a single gene locus for this enzyme. Tetraploid members such as the carp and goldfish are endowed with two separate gene loci for 6-PGD. Tetraploid evolution apparently fixed two former alleles of the same locus as two separate gene loci. Furthermore, it appears that after becoming tetraploid, the carp and goldfish developed a separate regulatory mechanism for each locus; thus preferential activation of one or the other 6-PGD locus occurs in different tissues of tetraploid species. This investigation was supported in part by a grant (CA-05138) from the National Cancer Institute, U.S. Public Health Service, and in part by a research fund established in honor of General James H. Doolittle. Contribution No. 4-68, Department of Biology, City of Hope Medical Center.Dr. Bender is a recipient of International Postdoctoral Fellowship 3 F05-TW-01198-0152 from the U.S. Public Health Service.