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.     

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.     

Two Alleles of Maize ALCOHOL DEHYDROGENASE 1 Have 3' Structural and Poly(a) Addition Polymorphisms

Two standard electrophoretic alleles of the maize alcohol dehydrogenase 1 locus (Adh1-1S and Adh1-1F) have been isolated and characterized. Restriction endonuclease mapping shows that a region of less than 5 kb is conserved in both alleles and is flanked both 5' and 3' by regions highly polymorphic for restriction sites. Nucleotide sequence comparison of these two alleles reveals that polymorphism in the 3' flanking region is due to rearrangements including tandem duplications, a transposable element-like insertion and a deletion. S1 nuclease analysis shows that both the Adh1-1S and the Adh1-1F alleles contain multiple poly(A) addition sites; four sites are observed for the Adh1-1S alleles and seven sites for the Adh1-1F allele. Only two of these poly(A) addition sites appear to be identical in the two alleles. No consensus signal for poly(A) addition is observed near any of these sites.     

Biochemical selection of immature,haploid embryos of Zea mays L.

Summary A method was devised for the biochemical selection of immature, haploid Zea mays embryos using Adh1 ^– and either the Stock 6 or indeterminate gametophyte (ig in W23) high haploid-inducing systems. Haploid (Adh1 ^–) embryos survived exposure to levels of allyl alcohol which killed diploid (Adh1 ⁺/Adh1 ^–) embryos. Of the total surviving embryos which were examined cytologically 15% (using ig) and 22% (using Stock 6) were haploid. In two experiments with Stock 6, 100% of the surviving embryos were haploid. To obtain maximum effectiveness of Stock 6 and ig, Adh1 ^– was transferred to stock 6 and W23 backgrounds. Immature, haploid embryos are being used to develop haploid, morphogenic tissue cultures of Zea mays.     

Three alcohol dehydrogenase genes in wild and cultivated barley: Characterization of the products of variant alleles

Barley (Hordeum vulgare) and its wild progenitor (H. spontaneum) have three loci for alcohol dehydrogenase (EC 1.1.1.1; ADH). The Adh1 locus is constitutively expressed in seed tissues, whereas expression of the loci Adh2 and Adh3 requires anaerobic induction. The Adh3 gene is well expressed in aleurone and embryo tissues kept under N₂ for 2–3 days. Using N₂-treated embryos, a diverse collection of H. spontaneum was screened in starch gels for electrophoretic variants at the Adh3 locus. Four variants were found: two were conventional mobility variants (Adh3 S, Adh3 V); one was a null variant (Adh3 n); and the fourth (Adh3 I) variant lacked active homodimers and showed reduced heterodimer activity. The ³⁵S-labeled monomers induced under N₂ in the lines homozygous for Adh1, Adh2, or Adh3 variants were immunoprecipitated with antiserum raised against maize ADH. Fluorography after separation by SDS-PAGE and by urea-isoelectric focusing indicated that the Adh3 n allele was CRM- and that the Adh3 I gene product was smaller than normal. The Adh1 and Adh3 variants showed independent segregation.     

The effect of insertion of the maize transposable elementMutator is dependent on genetic background

A secondary mutant, derived from an allele of maize alcohol dehydrogenase 1 (Adh1) carrying a Mutator transposable element (Mu1) in its first intron, was reported to exhibit a threefold decrease in ADH enzymatic activity and steady-state RNA levels compared to the original mutant. The original mutant,Adh1-S3034 (abbreviatedS3034), was previously characterized at the molecular level. The derivative, abbreviatedS3034b, has now been cloned; at the DNA sequence level the insertion and surroundingAdh1 sequences are indistinguishable fromS3034. Furthermore, in our lines there is no difference in relative ADH activities between products of the two putative alleles. A comparison of gene expression in heterozygotes obtained by crossing to different tester lines reveals a correlation between the measured decrease in levels of ADH polypeptide produced by the mutant allele and the background in which it is measured; this effect is distinct from any background-related variation in the expression of the progenitor allele. It does not appear to be attributable to alternative patterns of DNA modification. It appears to reflect a background-associated difference in the level of normalAdh1-RNA produced. Thus the previously reported distinction betweenS3034 andS3034b may be due to differences in the extent to which the mutant allele and a given genetic background interact to produce functionalAdh1-RNA.This research was supported by United States Public Health Service Grant GM38616 and United States Department of Agriculture Grant 87-CRCR-1-2500 to J.S. D.O. was supported by an NIH predoctoral training grant to the Department of Genetics.     

Molecular Differences between Alleles Adh1-F and Adh1-Sin Sugar Beet Beta vulgaris L.

We compared nucleotide sequences of exon 4 and part of exon 5 of alleles F and S of the Adh1 locus controlling alcohol dehydrogenase in sugar beet. The Adh1-F and Adh1-S sequences of the examined fragment were shown to differ by two nucleotides. Adenine (A) and cytosine (C) of Adh1-F were substituted by respectively thymine (T) and adenine (A) in Adh1-S. Consequently, glutamine and asparagine from the F subunit of ADH1 are replaced by valine and lysine, respectively. Because of differences in the amino acid content, the F subunit is by two elementary charges more negatively charged electrically than the S subunit, which correlates with differences in their electrophoretic mobility. Comparison of the examined Adh1 fragment of sugar beet with its counterparts in other plants showed that the sites bearing substitutions in the former species are classed as variable.     

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     

Transient gene expression after electroporation of protoplasts derived from embryogenic maize callus

Protoplasts isolated from embryogenic callus cultures derived from immature embryos ofZea mays L. are suitable for analysis of transient gene expression using electroporation-mediated DNA transfer. Expression of introduced genes is comparable to the levels obtained with protoplasts from Black Mexican Sweet suspension cultures. Two different promoters, that directing synthesis of the 35S RNA of cauliflower mosaic virus and the maizeAdh1 promoter were placed in front of the luciferase reporter gene to assess protoplast gene expression and the impact of an intron on expression level.Abbreviations 35S promoter isolated from CaMV - CaMV cauliflower mosaic virus - Adh1 maize gene encoding Alcohol dehydrogenase-1 enzyme - BMS suspension cultures of the Black Mexican Sweet maize variety     

Nucleotide polymorphism in the Adh2 region of the wild rice Oryza rufipogon

DNA variation in the alcohol dehydrogenase (Adh2) region of the wild rice Oryza rufipogon and its related species was analyzed to clarify maintenance mechanisms of the DNA variation in these species. A dimorphic pattern was detected in the Adh2 region of O. rufipogon. The silent nucleotide diversity (π) in the Adh2 region in O. rufipogon was 0.011, which was higher than that of the Adh1 region in O. rufipogon. Especially, a high nucleotide diversity was detected at synonymous sites of the catalytic domain 1. Average nucleotide diversity at silent sites within each of the dimorphic sequence types of the Adh2 region was similar to that in the Adh1 region, indicating that the high level of silent polymorphism in the Adh2 region was caused by the difference between the dimorphic sequence types. On the other hand, the level of replacement polymorphism in the Adh2 region was as low as that in the Adh1 region. The neutrality test of Fu and Li indicated significantly negative deviation from the neutral mutation model for the replacement sites of the Adh2 region. This result suggests purifying selection on the replacement sites of the Adh2 region, as detected for the Adh1 region. Significant linkage disequilibria (16.4% of the tests) were detected between the Adh1 and Adh2 regions. Even when nonrandom association was tested for the strains belonging to one of the divergent sequence types of the Adh2 region, significant interlocus linkage disequilibria were detected. The close physical distance and/or epistasis between the two Adh regions could be invoked to explain these nonrandom associations.     

Phenotype Dependence of the Combining Ability in Mutant Sugar Beet

Repetitive gametic selection for a higher frequency of the Adh1-Ssemilethal mutant allele of the alcohol dehydrogenase (ADH) gene yielded viable homozygotes Adh1-SS. The plants varied in phenotype from weak mutant to nearly normal (restored). Phenotypically different plants were individually tested for combining ability. This parameter was high in plants with the mutant phenotype and tended to decrease, rather than further increase, in plants with a restored normal phenotype. The results are discussed in terms of viability restoration mechanisms in homozygotes for semilethal mutant alleles.     

Dissociation-recombination of intergenic sunflower alcohol dehydrogenase isozymes and relative isozyme activities

Two unlinked genes, Adh ₁ and Adh ₂, control the production of alcohol dehydrogenase (ADH) in seeds of the annual sunflower (Helianthus annuus). Each gene is polymorphic, having F and S alleles. Starch gel electrophoretic zymograms of the four possible double homozygotes have three bands, representing two homodimers and an intermediately migrating intergenic isozyme. Zymograms of double heterozygotes consist of nine bands produced by ten isozymes: six intragenics and four intergenics, two of which are coincident. Results of dissociation-recombination (D-R) experiments are reported which demonstrate the subunit composition of the intergenic isozymes, thus supporting the relationships suggested by genetic studies. Densitometric tracings of the zymogram of a cleared gel and measurements of activities of homodimer isozymes eluted from gels following D-R of an intergenic isozyme showed that the Adh ₂ isozymes were more than twice as active as those of Adh ₁. Measurements of activities of crude extracts from the four possible double homozygous genotypes indicated that the seeds of the genotype Adh ₁ ^F /Adh ₁ ^F , Adh ₂ ^S /Adh ₂ ^S produced more activity than the other three. This genotype is the most common one found in wild and cultivated stocks. Isozymes eluted following electrophoresis of the same extracts had averages of 19%, 70%, and 11% of total activity contributed by the Adh ₁, Adh₂, and intergenic isozymes, respectively. A simple but efficient method of isozyme elution from starch gels is described which resulted in nearly full expected recovery (approximately 46%) of the ADH activity in the applied sample.Supported by Graduate School and BioMed grants and by NSF Grant GB35853.     

桃儿七叶绿体比较基因组学分析

为探究桃儿七(Sinopodophyllum hexandrum)不同叶绿体基因组特征,本研究以桃儿七5个叶绿体基因组为研究对象,借助生物信息学工具进行基因组图谱构建、重复序列分析、密码子偏好分析、反向重复序列区(inverted repeat, IR)/单拷贝区(single-copy, SC)边界分析、基因组序列比较分析及系统发育分析。结果表明:桃儿七5个叶绿体基因组全长为157 203–157 940 bp,为典型的叶绿体四分体结构,共注释出133–137个基因,说明桃儿七叶绿体基因组具有多样性。桃儿七不同叶绿体基因组简单重复序列(simple sequence repeat, SSR)位点不同,单核苷酸A/T占主要优势,散在重复序列包括正向重复、回文重复和反向重复3类。密码子偏好分析显示有效密码子(effective number of codon,ENc)值为51.14–51.17,密码子偏好性弱,GC与GC3s所占比例小于50%,密码子偏向使用A和U碱基并且以A和U碱基结尾。桃儿七5个叶绿体基因组IR/SC边界和基因组序列均比较保守。系统发育分析结果表明桃儿七和北美桃儿七亲...     

Two neutral variants segregating at the gametophytic self‐incompatibility locus of European pear (Pyrus communis L.) (Rosaceae,Pyrinae)

Extensive survey of the S‐locus diversity of plant species with RNase‐based gametophytic self‐incompatibility has failed to identify neutral variation segregating within S‐allele specificities. Although this is the expected result according to population genetics theory, it conflicts with recent models of S‐allele evolution, which suggest that new specificities might arise by a continuous process of subtle changes that individually do not alter the specificity of the S‐genes, but whose cumulative effects result in new S‐allele functions. Genomic analysis of S‐RNase sequences associated with the S₁₀₄ (=S₄, =S_b) allele of European pear (Pyrus communis L.) cultivars yielded two distinct variants (named herein S_104‐1 and S_104‐2) that differed at five nucleotide positions within the open reading frame, two of which resulted in changes in the predicted protein sequence. Test‐cross experiments indicated that the S‐alleles associated with the S_104‐1 and S_104‐2RNases exhibit the same pollen and pistil functions, suggesting that they are two neutral variants segregating within the S₁₀₄ haplotype of European pear. These allelic forms might represent transitional states in the process of generating new specificities in the species, in accordance with models that predict S‐function transition through neutral intermediates. This possibility was further evaluated through the pattern of molecular evolution of functionally distinct European pear S‐RNases, which indicated that most recent S‐allele diversification in this species proceeded in the absence of adaptive selective pressure.     

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.     

Genetic analysis of alcohol dehydrogenase isozymes in pearl millet (Pennisetum typhoides)

Pearl millet (Pennisetum typhoides) produces three ADH isozymes, sets I, II, and III, with set III being expressed only in anaerobically treated seeds or seedlings. Variant strains have been identified which produce ADH isozymes with altered electrophoretic mobilities for sets I and II but not for set III activity. Based on genetic analysis of these variants and on dissociation-reassociation experiments, we propose that the three ADH isozymes are dimers of subunits coded by two structural genes, Adh1 and Adh2, with set I being a homodimer specified by Adh1, set III a homodimer specified by Adh2, and set II a heterodimer formed between the products of Adh1 and Adh2.This work was supported by BRSG Grant RR 07080 awarded by the Biomedical Research Grant Program, Division of Research Grants, National Institutes of Health, to D. R. H., and by funds from the Margenroth Endowment to F. B.-B., who is a PHS Research Service Award Trainee in Genetics.     

A Ds-mutation of the Adh1 gene in Zea mays L.

Summary An unstable spontaneous mutation in the maize Adh1 gene, coding for alcohol dehydrogenase, was selected by allyl alcohol poisoning of wild type Adh1 pollen from a maize line carrying Ds at the Bz2 locus and one copy of Ac in an unknown position. The mutant has a null phenotype. No wild type pollen grains were detected in strains devoid of Ac, but in the presence of Ac, wild type pollen grains were detected with a frequency of between 10^-4 and 10^-3. In addition, events have been identified in the aleurone in which reversions of both bz2-m and the unstable adh1 mutation occurred in the same patch of tissue, presumably in response to an alteration of Ac. By these criteria, the Adh1 mutant is caused by Ds. DNA blotting experiments have shown the presence of a 1.3 kb insertion in the Adh1 gene. All or part of this Ds insertion is transcribed, and is detected as an insertion within the ADH1-mRNA. The longer mRNA hybridizes to an authentic Ds probe.This Ds element differs in size from other known Ds insertions.     

Genetic dissection of retinoid dehydrogenases

Biochemical studies indicate that alcohol dehydrogenase (ADH) metabolizes retinol to retinal, and that aldehyde dehydrogenase (ALDH) metabolizes retinal to retinoic acid, a molecule essential for growth and development. Summarized herein are several genetic studies supporting in vivo functions for ADH and ALDH in retinoic acid synthesis. Gene targeting was used to create knockout mice for either Adh1 or Adh4. Both knockout mice were viable and fertile without obvious defects. However, when wild-type and Adh4 knockout mice were subjected to vitamin A deficiency during gestation, the survival rate at birth was 3.3-fold lower for Adh4 knockout mice. When adult mice were examined for production of retinoic acid following retinol administration, Adh1 knockout mice exhibited 10-fold lower retinoic acid levels in liver compared with wild-type, whereas Adh4 knockout mice differed from wild-type by less than 2-fold. Thus, Adh1 plays a major role in the metabolism of a large dose of retinol to retinoic acid in adults, whereas Adh4 plays a role in maintaining sufficient retinol metabolism for development during retinol deficiency. ALDHs were examined by overexpression studies in frog embryos. Injection of mRNAs for either mouse Raldh1 or Raldh2 stimulated retinoic acid synthesis in frog embryos at the blastula stage when retinoic acid is normally undetectable. Overexpression of human ALDH2, human ALDH3, and mouse Aldh-pb did not stimulate retinoic acid production. In addition, Raldh2 knockout mice exhibit embryonic lethality with defects in retinoid-dependent tissues. Overall, these studies provide genetic evidence that Adh1, Adh4, Raldh1, and Raldh2 encode retinoid dehydrogenases involved in retinoic acid synthesis in vivo.     

Molecular analysis of two functional homologues of the S 3 allele of the Papaver rhoeas self-incompatibility gene isolated from different populations

The S ₃ allele of the S gene has been cloned from Papaver rhoeas cv. Shirley. The sequence predicts a hydrophilic protein of 14.0 kDa, showing 55.8% identity with the previously cloned S ₁ allele, preceded by an 18 amino acid signal sequence. Expression of the S ₃ coding region in Escherichia coli produced a form of the protein, denoted S₃e, which specifically inhibited S₃ pollen in an in vitro bioassay. The recombinant protein was ca. 0.8 kDa larger than the native stigmatic form, indicating post-translational modifications in planta, as was previously suggested for the S₁ protein. In contrast to other S proteins identified to date, S₃ protein does not appear to be glycosylated. Of particular significance is the finding that despite exhibiting a high degree of sequence polymorphism, secondary structure predictions indicate that the S₁ and S₃ proteins may adopt a virtually identical conformation. Sequence analysis also indicates that the P. rhoeas S alleles share some limited homology with the SLG and SRK genes from Brassica oleracea. Previously, cross-classification of different populations of P. rhoeas had revealed a number of functionally identical alleles. Probing of western blots of stigma proteins from plants derived from a wild Spanish population which contained an allele functionally identical to the Shirley S ₃ allele with antiserum raised to S₃e, revealed a protein (S ₃ s) which was indistinguishable in pI and M _r from that in the Shirley population. A cDNA encoding S ₃ s was isolated, nucleotide sequencing revealing a coding region with 99.4% homology with the Shirley-derived clone at the DNA level, and 100% homology at the amino acid level.     

Isolation of a 6.2 kb genomic fragment carrying the Adh1 gene of tomato and its expression in transgenic tobacco

An 11 kb Eco RI genomic fragment containing the alcohol dehydrogenase (Adh1) gene was cloned. Cross-hybridization with three Adh2 cDNA clones suggested that the entire coding region of the Adh1 gene was contained on a 6.2 kb Xba I/Hind III subfragment. Using RFLP linkage analysis, the genomic clone was mapped on chromosome 4 between the markers TG 182 and TG 65 in a position corresponding to the Adh1 locus. To further confirm the Adh1 origin of the genomic clone, tobacco plants were transformed with the 6.2 kb Xba I/Hinb III genomic subfragment. Isozyme analysis demonstrated that in transgenic tobacco plants functional tomato specific ADH-1 homodimers were synthesized as well as heterodimers composed of tobacco and tomato subunits.