DNA insertion in the first intron of maize Adh1 affects message levels: cloning of progenitor and mutant Adh1 alleles.

Abstract

A genetically unstable mutant allele of maize alcohol dehydrogenase-1 (Adhi) causes reduced levels of messenger RNA. This mutant was derived from a Robertson's mutator genetic background and previously was shown to be associated with an approximately 1.5-kilobase insertion. This report compares a genomic clone of the allele, Adhi-S3034, with a clone of its progenitor nonmutant allele, Adhi-S Gerlach et al. (4) to obtain a cDNA clone from ADH1 mRNA. By using this ADH1 cDNA clone as a probe for the maize genome, it was found that Adhl may be identified on Southern blots as a single-copy gene (5). Among the several AdMl mutants that seem to alter regulatory rather than coding functions is a series of unstable quantitative alleles originating from a single mutant: Adhl-53034 (6). The mutant was selected as an allyl alcohol-resistant pollen grain shed from a genetically marked line carrying Robertson's mutator activity (7) and the Adhl-S target allele. The mutant allele, S3034, supports about 40% normal ADH1 translation rates, and is about 100 times more unstable genetically (at two revertants per 104 pollen grains) than ethyl methanesulfonate-induced mutants of Adhl-S (6). Mutant derivatives of S3034 were selected that express 13% and 0% ADH1 protein, and these quantitative alleles lowered expression in scutellum (of the seed), anaerobic root, and pollen to about the same extent (6). The availability of a cDNA probe for Adhl sequence allowed the demonstration that the low expression of S3034 and its derivative alleles reflected poly(A)+ RNA levels and that each allele carries a DNA insertion of about 1.5 kilobases (kb) just 5' to the region of genome recognized by the cDNA clone (5).This report focuses on a comparison of restriction maps between two genomic clones: one containing the insertion mutant S3034 and the other containing the nonmutant progenitor allele, S. We locate the site of insertion into the first intron of Adhl by sequence comparison and discuss the possible significance of our result.MATERIALS AND METHODS Biologicals. The spi vector, X1059, and recipient Escherichia coli strains Q358 and Q359 (8) were provided by S. Brenner. E. coli strains BHB2688 and BHB2690 were provided by B. Hohn (9). Maize lines containing Adhl-S and Adhi-S3034 alleles have been described elsewhere (5). Plant HV594-1 was the origin of S3034 DNA.Reagents. Restriction enzymes, kinase, and ligase were from the Bethesda Research Laboratories (Rockville, Maryland). Radioactive materials were from Amersham.Maize DNA was purified once by CsCI/ethidium bromide centrifugation by the technique of Rivin et al. (10) from leaves and immature tassels. The DNA was digested with a 2-fold excess of BamHI and was size-fractionated in sucrose density gradients (11). Fractions containing DNA fragments of 9-20 kb (for Adhl-S) or 10-22 kb (for Adhl-S3034) were pooled and ethanol-precipitated without carrier.Packaging extracts were prepared from E. coli strains BHB2688 and BHB2690 by the technique of Hohn (9). Extracts were used only if t...

Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondrial isozyme of alcohol dehydrogenase in Saccharomyces cerevisiae.

“…mammalian liver (20). maize (5), Bacillius stearotherlnophilus (20). and Drosophila melanogaster (22) have been previously reported.…”

Somatically heritable switches in the DNA modification of Mu transposable elements monitored with a suppressible mutant in maize.

“…Mul elements from Mu-active plants contain only unmodified HinfI sites, whereas a high proportion of those from Mu-inactive plants have modified sites. Unmodified Mul homologous elements fall into two size classes, Mul and Mul.7, which yield fragments of 1.3 and 1.7 kb, respectively, when digested with HinfI and hybridized with internal probes from Mul (Barker et al 1984;Bennetzen et al 1984). In contrast, modified elements are present on larger HinfI fragments, as the HinfI sites in the inverted repeats are not sensitive to digestion.…”

Biochemical and cytological characterization of wheat/Aegilops ventricosa addition and transfer lines carrying chromosome 4MV

“…ventricosa, (33) transfer line H-93-33. The probé used was a 2.3 kbp HindIII fragment subcloned (pB428) from a maize alcohol dehydrogenase gene by Bennetzen et al (1984) and was labeled with [a-32 P] dATP by the method of Feinberg and Vogelstein (1983). The horizontal arrowhead points to a hybridization band specific for Ae.…”

“…Restriction digestión, agarose gel electrophoresis and Southern blotting to nylon membranes (Hybond N, Amersham) were performed according to Maniatis et al (1982) and to the manufacturen instructions. A maize probé for alcohol dehydrogenase was supplied by M. Freeling (Bennetzen et al 1984). The probé was made radioactive by the oligo-labelling method (Feinberg and Vogelstein 1983), hybridized overnight at 65 °C and washed twice at 50 °C.…”