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...
A cloned DNA fragment from the maize allele Adhl-S3034 contains all of Mul, an insertion element involved in Robertson's Mutator activity. The element is 1367 base pairs (bp) long and is flanked by nine bp direct repeats of insertion site DNA. It has inverted terminal repeats of 215 and 213 bp showing 95% homology. Within the element are two direct repeats of 104 bp showing 96% homology. Four open reading frames (ORFs) were found, two in each DNA strand. Mul can be divided into two halves, each containing one terminal inverted repeat, an internal direct repeat, and two overlapping ORFs. The GC content of each half is high (70%), while that of a central 60 base portion of the element is low (26%). The central region contains the only sequence resembling the TAATA Goldberg and Hogness eukaryotic promoter signal. Multiple copies of DNA sequences related to Mul found in Mutator maize plants are generally similar in organization to the cloned element. A larger version containing a discrete 300 to 400 base pair insertion was found in some Mutator lines.
The early steps of symbiotic nodule formation by Rhizobium on plants require coordinate expression of several nod gene operons, which is accomplished by the activating protein NodD. Three different NodD proteins are encoded by Sym plasmid genes in Rhizobium meliloti, the alfalfa symbiont. NodD1 and NodD2 activate nod operons when Rhizobium is exposed to host plant inducers. The third, NodD3, is an inducer-independent activator of nod operons. We previously observed that nodD3 carried on a multicopy plasmid required another closely linked gene, syrM, for constitutive nod operon expression. Here, we show that syrM activates expression of the nodD3 gene, and that nodD3 activates expression of syrM. The two genes constitute a self-amplifying positive regulatory circuit in both cultured Rhizobium and cells within the symbiotic nodule. We find little effect of plant inducers on the circuit or on expression of nodD3 carried on pSyma. This regulatory circuit may be important for regulation of nod genes within the developing nodule.
The uptake of theophylline and caffeine was determined in isolated pancreatic islets employing a dual isotope procedure with sucrose as an extracellular marker. Islets rapidly accumulated caffeine and theophylline with apparent dissociation constants of approximately 23 and 6 mM, respectively. Theophylline inhibited the uptake of caffeine and caused displacement of caffein from islets. These results indicated a competition by theophylline and caffeine for a common site (binding and/or transport carrier). In addition, theophylline and caffeine inhibited D-glucose transport in a dose-dependent manner and within the limits of the experimental system, this inhibition appeared to be non-competitive. (Bu)2cAMP under similar experimental conditions exerted no effect on D-glucose transport. These results present evidence for a rapid uptake of theophylline and caffeine in pancreatic islets, which is compatible with their immediate cellular effects. In addition, these results demonstrate a direct effect by theophylline and caffeine on D-glucose transport which appears independent of their ability to alter intracellular cAMP levels.
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