Variation in culture,isoenzyme patterns and plastid DNA in the genusDaucus

Summary To identify markers for fusion and transformation studies, cell suspension cultures of four members of theDaucus genus were examined to determine differences in culture conditions, isoenzyme patterns, and plastid DNA. The four were:D. carota subsp.sativus cv. Danvers,D. carota subsp.gummifer, D. capillifolius, andD. pusillus. Under appropriate conditions, all four grew well as liquid cell suspension cultures and regenerated from protoplasts into plants. Enzyme activities of homoserine dehydrogenase (HSDH) and alcohol dehydrogenase from cell culture extracts were analyzed on electrophoretic gels. Although only one form of HSDH was present in eachDaucus line, the rate of migration of HSDH from cv. Danvers was different from that of the other cell lines. Multiple isoenzymic forms of ADH were present in eachDaucus cultivar. Camparison of endonuclease restriction fragment patterns from plastid DNAs digested by BamHI revealed only small differences between plastid DNAs of cv. Danvers and subsp.Gummifer, whereas large differences were observed between cv. Danvers andD. pusillus plastid DNA patterns. No differences were found between cv. Danvers andD. capillifolius plastid DNA patterns when examined using eight different restriction enzymes. The data indicate that specific isoenzyme and organelle DNA restriction fragment patterns will be useful markers for precise identification of genomes of differentDaucus species in somatic hybridization experiments. This research was supported by the U.S. Department of Agriculture under Agreement 59-2246-1-1-737-0.     

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.     

Genetic polymorphism of sorbitol dehydrogenase in the brown hare and the distribution of the variation in Central Europe

A genetic polymorphism of sorbitol dehydrogenase (SDH; EC 1.1.1.14) for two allozymes is demonstrated in the brown hare (Lepus europaeus) by means of horizontal starch gel electrophoresis. Segregation analysis was performed in a sample of four matings with 12 offspring and confirmed the genetic hypothesis: two alleles at an autosomal locus. The calculated gene frequencies in the brown hare breed studied areSdh ^a =0.712 andSdh ^b =0.288. The distribution of the polymorphism in free-ranging brown hare populations from Austria, Czechoslovakia, Hungary, and Poland is described.This research was supported by a grant from the Fonds zur Förderung der Wissenschaftlichen Forschung, Austria (project P6767B).     

Genotypic differences and alterations of protein patterns of tomato explants under copper stress

In vitro response of six tomato genotypes to different copper concentrations was studied. Cu was toxic to tomato explants at a relatively high concentration (100 μM), which reduced callus growth and shoot regeneration. Peto-86 followed by UC-97-3 were more tolerant to copper than the other genotypes. Cu (100 μM) induced the synthesis of eight new proteins (70.86 - 14.78 kD) in Peto-86 and six in Western Improve (46.43 - 14.78 kD) and UC-97-3 (77.69 - 14.78 kD). Cu-stress reduced the expression of some enzymatic bands of alcohol dehydrogenase and esterase, meanwhile, one peroxidase band at the locus Prx-1 was newly expressed under Cu-treatment. This revised version was published online in July 2006 with corrections to the Cover Date.     

Alcohol dehydrogenase polymorphism in Apis mellifera

A polymorphic system of ADH isozymes is described in the honeybee Apis mellifera. Three and six different electrophoretic patterns were found, respectively, in drone and worker pupae analysis. The data indicate that the ADH isozymes are controlled by three alleles, Adh-1 ¹, Adh-1², and Adh-1 ³. The frequency of the Adh-1 alleles is different in two analyzed subspecies, Apis mellifera adansonii (African bees) and Apis mellifera ligustica (Italian bees). In the African bees, the frequencies are 0.256 and 0.697 for Adh-1 ¹ and Adh-1², respectively. In the Italian bees, these values are shown to be 0.902 and 0.098, respectively. The allele Adh-1 ³ was not detected in the Italian bee population. The effect of NAD on the resolution of this system was investigated, and only one region of ADH activity was obtained in drone pupae analysis when NAD was used in the gels. However, two different regions of activity were observed in the same samples, in the absence of the coenzyme. ADH activity was not detected in young larvae, but it increased to a maximum in prepupal and white-eyed pupal phases. It then declined progressively to total absence in the emerging bees.This work was supported by the Brazilian Research Council (CNPq) and S~ao Paulo State Research Foundation (FAPESP). This study was performed by the senior author in partial fulfillment of the requirements for a master's degree and was directed by Dr. M. A. Mestriner, Genetics Department, University of São Paulo at Ribeirão Preto.     

Protein complex and esterase isoenzyme patterns ofAllium sativum L. cultivars and clones-regenerants

Protein complex patterns of cloves and esterase isoenzyme patterns of apical buds of cloves were studied with Czechoslovak virus-free cultivars ofAllium sativum L. and the wild speciesA. longicuspis Regel, Similarly, four clones-regenerants obtained using explant culture techniques from A.sativum L. cv. Bzenecky paličák (two somaclones and two clones derived from plants regenerated from meristem cultures treatedin vitro with colchicine) differing in their ploidy, morphology, and yields were studied. Immunophoreograms of protein complexes of theA. sativum L. cultivars under investigation differed from one another in the number and mobility of protein fractions in both the cathodic and anodic regions and thus these cultivars can be distinguished. On the basis of esterase isoenzyme patterns, the Czechoslovak cultivars of A. sativum L. can be arranged into three groups - bolting winter cultivars with broad leaves, non-bolting winter cultivars with broad leaves, and non-bolting spring cultivars with narrow leaves. All the clones-regenerants showed the same protein complex and esterase isoenzyme patterns as their original cultivar.A. longicuspis Regel markedly differed in its protein complex and esterase isoenzyme patterns from all the other genotypes studied.Received May 17, 1989: accepted January 5,1990     

Polymorphism of human liver alcohol dehydrogenase: Identification of ADH2 2-1 and ADH2 2-2 phenotypes in the Japanese by isoelectric focusing

Liver homogenate-supernatants from most Japanese exhibit an atypical pH optimum for ethanol oxidation at pH 8.8 instead of 10.5, the typical pH-activity optimum. It has been proposed that atypical livers contain alcohol dehydrogenase isozymes with ₂ subunits while typical livers contain isozymes with ₁ subunits, both produced by the ADH ₂ gene. Because it is difficult to differentiate the atypical ADH₂ 2-2 phenotype from the ADH₂ 2-1 phenotype by starch gel electrophoresis, an agarose isoelectric focusing procedure was developed that clearly separated the atypical Japanese livers into two groups, A1 and A2. The isozymes in A1 and A2 livers were purified. Type A1 livers contained a single isozyme with an atypical pH-rate profile; it was designated ₂₂. Three isozymes were isolated from A2 livers, two of which corresponded to ₁₁ and ₂₂. A third, absent from the typical and the atypical A1 livers, had an intermediate mobility; it was designated ₂₁. Type A1 livers are, therefore, the homozygous ADH₂ 2-2 phenotype, and type A2 livers, the heterozygous ADH₂ 2-1 phenotype. The ADH₂ 2-2 phenotype was found in 53% of 194 Japanese livers, and the ADH₂ 2-1 phenotype, in 31%. Accordingly, the frequency of ADH ₂ ² was 0.68.This study was supported by U.S. Public Health Service Grant AA 02342.     

Comparison of esterase isoenzyme patterns in seeds of someAllium species and in cultivars ofAllium cepa L

Esterase isoenzyme patterns were studied in seeds of 6 cultivars ofAllium cepa L. and of14 species ofAllium, namelyAllium aflatunense B. Fedtsch.,A. altaicum (Pall.) Reyse,A. Cristophii Trautv.,A. fistulosum L.,A. jajlae Vved.,A. Karsianum Fom.,A. nutans L.,A. porrum L. cv. Gigant,A. praemixtum Vved.,A. pskemense Vved.,A. ramosum L.,A.rotundum L.,A. schoenoprasum L.,A. stipitatum Regel. The cultivars differ in their isoenzyme patterns, the cultivar Ka?tická stands apart from all the other cultivars, probably due to the high alkalinity of its seed extract. The examined species, arranged according to their mutual similarity of isoenzyme patterns, form several groups corresponding to individual sections of the genus. Our results corroborate the recognizing of the sectionCepa and the subsectionPhyllodolon. The maintaining ofA. jajlae andA. rotundum as well described species fits better with our results, than degrading them to subspecies ofA. scorodoprason. Our results agree in main features with those obtained by the immunological method. Some few differences appear concerning individual species. It is evident that esterase isoenzyme patterns can be used in chemotaxonomy ofAllium on an infrageneric level, and, at least inA. cepa, also on an infraspecific level.     

Human liver alcohol dehydrogenase: ADHIndianapolis results from genetic polymorphism at the ADH 2 gene locus

New molecular forms of human liver alcohol dehydrogenase (ADH), collectively designated ADH_Indianapolis (ADH_Ind), were recently discovered in 29% of liver specimens from Black Americans [Bosron, W. F., Li, T.-K., and Vallee, B. L. (1981). Proc. Natl. Acad. Sci. USA 77:5784]. Three different ADH_Ind phenotypes have now been identified by starch gel electrophoresis, and four ADH_Ind enzyme forms isolated by affinity and ion-exchange chromatography. The most cathodic ADH_Ind form has a single pH optimum at 7.0 for ethanol oxidation and is a homodimer of a newly discovered subunit, as evidenced by dissociation-recombination studies. The remaining three purified ADH_Ind forms have dual pH optima for ethanol oxidation at 7.0 and 10.0 and generate two new bands on starch gel electrophoresis after dissociation-recombination. They appear to be heterodimers of this new subunit with the known subunits, , ₁, and ₁. Based on the occurrence of these four ADH_Ind isozymes and isozymes containing ₁ subunits in the homogenate supernatants of 135 livers, we conclude that ADH_Ind results from polymorphism at the ADH ₂locus, with the variant ADH ₂ ^Ind allele coding for the _Ind subunit. The frequency of ADH ₂ ^Ind was 0.16 in Black Americans, and this allele was not observed in any of the 63 livers from White Americans. The frequency of the ADH ₃ ¹ and ADH ₃ ² alleles also differed in these two populations.This study was supported by U.S. Public Health Service, Grant AA 02342.     

Molecular analysis of the ADH1-C m allele of maize

The Adh1-C ^mallele and each gene in the Adh1-FC ^mduplication have been cloned and restriction-mapped. Of the C ^mallele 6 kb was sequenced. A single amino acid substitution of aspartate for tyrosine at residue 52 accounts for the altered enzymatic properties of the C ^mprotein. Comparison of the nucleotide sequence to that of Adh1-1F and Adh1-1S shows structural and restriction site polymorphisms in the 3 flanking DNA. C ^mlacks the insertion sequence present in 1F and 1S and contains a complex sequence composed of two direct repeats and an inverted repeat. The two genes of the duplication allele have similar restriction maps to C ^mand each other.     

洛阳市汉族群体ADH2和ALDH2的基因多态性研究

为研究洛阳市汉族群体ADH2和ALDH2基因的多态性分布,应用聚合酶链反应-扩增片段长度多态性（PCR-APLP）分析法,对ADH2基因外显子3和ALDH2基因外显子12的特定片段同时进行特异性扩增,用非变性的聚丙烯酰胺垂直凝胶电泳和DNA银染方法判定基因型。ADH2*1和ADH2*2等位基因频率分别为42.86%和57.14%,ADH2*1/*1、*1/*2和*2/*2的基因型频率分别为22.86%、40.00%和37.14%;ALDH2*1和ALDH2*2的等位基因频率分别为85.24%和14.76%,ALDH2*1/*1、*1/*2和*2/*2的基因型频率分别为71.43%、27.62%和0.95%。洛阳市汉族群体ADH2和ALDH2的等位基因频率和基因型频率不同于台湾人和上海人,ALDH2*1/*1基因型频率明显高于上海人和台湾人的。因而,洛阳市居民对酒精的耐受性比上海人和台湾人强。 Studies of Genetic Polymorphisms of ADH2 and ALDH2 among the Han Population in Luoyang China ZHANG Zhu-mei1,LIU Cha-zhen1,BIAN Jian-chao1,TANG Bo-ming2,JIANG Feng1,WANG Qi-jun2,WANG Qi-min2,ZHU Xin2,SHEN Fu-min1 1.Department of Epidemiology,Public Health School of Fudan University,Shanghai 200032,China; 2.Section Office of Epidemiology,Luoyang Hygiene and Anti-epidemic Center,Luoyang 471000,China Abstract:In order to investigate genetic polymorphisms of ADH2 and ALDH2 among the Han population in Luoyang City,portions of exon 3 of ADH2 and exon 12 of ALDH gene were amplified by using polymerase chain reaction.The amplified products were electrophoresed on 10% undenatured vertical polyacrylamide gels and stained with argentine.Frequencies of ADH2*1 and ADH2*2 alleles are 42.86% and 57.14%.Frequencies of three genotypes of ADH2 are 22.86%、40.00% and 37.14%,respectively.Frequencies of ALDH2*1 and ALDH2*2 alleles are 85.24% and 14.76%.Genotype frequencies of ALDH2 loci are 71.43%、27.62% and 0.95%,respectively.Genetic polymorphisms of ADH2 and ALDH2 among the Han population in Luoyang City are different from those among Taiwanese and Shanghainese.Frequency of ALDH2*1/*1 in Luoyang people is higher than those in Shanghai and Taiwan.Therefore,there is a higher resistance to alcohol drinking in the Han population in Luoyang. Key words：polymerase chain reaction-amplified products length polymorphism; alcohol dehydrogenase 2; aldehyde dehydrogenase 2; genetic     

Direct determination of usual (Caucasian-type) and atypical (Oriental-type) alleles of the class I human alcohol dehydrogenase-2 locus

Two types of alleles exist in the human alcohol dehydrogenase-2 (ADH ₂) locus. The usualADH ₂ ¹ allele is common in Caucasians, while the atypicalADH ₂ ² allele is predominant in Orientals. TheADH ₂ ² produces the β₂ subunit, which is catalytically far more active than the β₁ subunit produced by theADH ₂ ¹ gene. The racial difference in alcohol-related problems could be related to the genetic differences in ADH and other ethanol-metabolizing enzymes. In order to examine the possibility, a method for determiningADH ₂ genotypes was developed. Two 21-base synthetic oligonucleotides, one complementary to the usualADH ₂ ¹ allele and the other complementary to the atypicalADH ₂ ² allele, were used as specific probes for in-gel hybridization analysis of human genomic DNA from peripheral blood. Under appropriate hybridization conditions, these two probes can hybridize to their specific complementary alleles and, thus, allow the genotyping of theADH ₂ locus. Genotypes of theADH ₂ locus of 49 unrelated Japanese individuals were determined. The frequency of the atypicalADH ₂ ² gene was found to be 0.71 in the Japanese population examined. This research was supported by Grant AA05763 from the National Institutes of Health.     

Alcohol Dehydrogenases: Identification and Names for Gene Families

Plant gene products that have been described as `alcohol dehydrogenases' are surveyed and related to their CPGN nomenclature. Most are Zn-dependent medium chain dehydrogenases, including `classical' alcohol dehydrogenase (Adh1), glutathione-dependent formaldehyde dehydrogenase (Fdh1), cinnamyl alcohol dehydrogenase (Cad2), and benzyl alcohol dehydrogenase (Bad1). Plant gene products belonging to the short-chain dehydrogenase class should not be called alcohol dehydrogenases unless such activity is shown.     

A second polymorphic lens crystallin (LEN-2) in the mouse: Genetic and biochemical analysis of LEN-1 and LEN-2

Two electrophoretic polymorphisms affecting lens crystallins, designated LEN-1 and LEN-2, have been discovered among inbred strains of mice. Analysis by isoelectric focusing demonstrated that both crystallins are monomeric proteins with isoelectric points at or above pH 7. Both proteins eluted in the low molecular weight (LM) fraction upon Sephadex G-200 gel filtration but LEN-2 was shown to be larger than LEN-1 by G75SF gel filtration and denaturing gel electrophoresis. Linkage analysis demonstrated that the genes encoding LEN-1 and LEN-2 assort independently. Amino acid analysis of the allelic products of the two genes revealed that genetic variants of each respective crystallin were very similar in amino acid compositions but that LEN-1 and LEN-2 were dissimilar crystallins.This research was sponsored in part by the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-840R21400 with the Martin Marietta Energy Systems, Inc.     

Alcohol and aldehyde dehydrogenase polymorphisms and risk for suicide: a preliminary observation in the Japanese male population

Epidemiological studies have shown that excessive alcohol consumption is a potent risk factor to develop suicidal behavior. Genetic factors for suicidal behavior have been observed in family, twin, and adoption studies. Because alcohol dehydrogenase (ADH1B) His47Arg and mitochondrial aldehyde dehydrogenase (ALDH2) Glu487Lys single nucleotide polymorphisms (SNPs), which affect alcohol metabolism, have been reported to exert significant impacts on alcohol consumption and on the risk for alcoholism in East Asia populations, we explored associations of the two functional SNPs with suicide using a case–control study of 283 completed suicides and 319 control subjects in the Japanese population. We found that the inactive ALDH2 allele (487Lys) was significantly less frequent in the completed suicides (19.3%) than in the controls (29.3%), especially in males, whereas this was not the case in females. The males bearing alcoholism‐susceptible homozygotes at both loci (inactive ADH1B Arg/Arg and active ALDH2 Glu/Glu genotypes) have a 10 times greater risk for suicide compared with the males bearing alcoholism‐protective homozygotes at both loci. Our data show the genetic impact of the two polymorphisms on suicidal behavior in the Japanese population, especially in males. Because we did not verify the daily alcohol consumption, the association of these SNPs with suicide might be due to alcoholism itself. Further studies using case–control subjects, which verifies the details of current and past alcohol consumption and diagnosis for alcoholism, are required to confirm these findings.     

Electrophoretic patterns of seed albumins in the Old-WorldLupinus species (Fabaceae): Variation in the 2S albumin class

PAGE and SDS-PAGE electrophoretic analysis of 31 accessions of 10 Old-WorldLupinus species, 5 smooth-seeded and 5 rough-seeded, covered total seed albumins and 2S albumins isolated by a solid-phase extraction. PAGE albumin patterns showed a distinct difference between the smooth- and rough-seeded lupins. SDS-PAGE analysis of seed albumins revealed interspecific differences, mainly due to the 2S albumins. The differences were especially marked in the smooth-seeded species. In the rough-seeded lupins the following subgroups were distinguished: (1)L. atlanticus, (2)L. cosentinii andL. digitatus, (3)L. palaestinus andL. pilosus. Evidence was provided that the 2S albumin class contains conglutin , so far classified as a globulin. The results are discussed with reference to taxonomic relationships of the Old-World lupins and characterization of the lupin seed albumin fraction.     

Cyclopropanol in the exploration of bacterial alcohol oxidation

Abstract Cyclopropanol selectively inhibits bacterial alcohol oxidation proceeding via NAD-independent, quinoprotein alcohol dehydrogenases. Thus, for instance, alcohol oxidation by Pseudomonas aeruginosa , grown on ethanol, was inhibited for about 50% by cyclopropanol treatment. Accordingly, cell-free extracts of untreated cells had nearly equal activities of quinoprotein and NAD-dependent alcohol dehydrogenases, whereas only the latter enzyme activity was found in cell-free extracts of cyclopropanol-treated cells. Upon incubation of Hyphomicrobium X with cyclopropanol, oxidation of alcohols was blocked while formaldehyde oxidation was not. Therefore, methanol dehydrogenase in this organism is not specifically involved in formaldehyde oxidation. The examples show that cyclopropanol-derived substrates are potential tools in revealing the physiological role of bacterial alcohol dehydrogenases.     

Variation in the physiological sensitivity of maize inbreds to EMS a possible correlation with alcohol dehydrogenase activity

Three inbred lines of maize, AD-1, AD-7 and AD-19, showed significant differences in their physiological sensitivity to ethyl methanesulfonate (EMS). Among the seedling parameters tested, the width of the leaves was the most sensitive and showed the best correlation with the results obtained at maturity. Plant weight was also a good seedling parameter. The physiological sensitivity to EMS was negatively correlated with alcohol dehydrogenase (ADH) activity in the scutellum of the three lines. A possible explanation for this correlation is discussed; however, it may have been due to chance.     

NAD+ Kinase Activities in Euglena Gracilis and Phaseolus Vulgaris

After an electrophoretic separation of proteins from Euglena gracilis and dry seeds of Phaseolus vulgaris in native conditions in polyacrylamide gels, gels were incubated in mixtures containing NAD+, Mg-ATP2-, glucose 6-phosphate, G6P dehydrogenase, and either phenazine ethosulfate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (PES/MTT) or phenazine methosulfate and nitro blue tetrazolium (PMS/NBT) as coupled redox system for NAD+ kinase activity detection. In the presence of PES/MTT, 4 bands were revealed for E. gracilis, among which two corresponded to NAD+ kinase activity, the other corresponding to a NAD+ reductase activity due to alcohol dehydrogenase (ADH). In the presence of PMS/NBT, only the bands of NAD+ kinase activity were revealed. With Phaseolus vulgaris, 3 bands of ADH were always revealed in both mixtures, and only the use of PMS/NBT allowed the detection of NAD+ kinase as a fourth band. With both materials, NAD+ reductase staining in gels was intensifed in the presence of GTP or ATP and even further with ADP or GDP. The results demonstrate that: 1) the NAD+ kinase and NAD+ reductase are two distinct enzymes; 2) the NAD+ reductase corresponds to ADH.     

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.