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Biomedical subjects

L C Hannah

Publications and source records attributed to L C Hannah.

30 records · Page 2Linked to original sources
PubMed1990

Identification and molecular characterization of shrunken-2 cDNA clones of maize.

Mutation at the shrunken-2 (Sh2) locus of maize, a gene described more than 40 years ago, greatly reduces starch levels in the endosperm through its effect on the starch synthetic enzyme ADP-glucose pyrophosphorylase, an enzyme thought to be regulatory in this biosynthetic pathway. Although our previous work has suggested that Sh2 is a structural gene for this enzyme, we have also reported data compatible with Sh2 acting post-transcriptionally. In this study, we took advantage of a transposable element-induced Sh2 allele, its progenitor, and revertants to identify a clone for this locus. Although the cloning and identification were done independently of any knowledge concerning the product of this gene, examination of the deduced amino acid sequence revealed much similarity to known ADP-glucose pyrophosphorylase subunits of plants and bacteria, including regions involved in substrate binding and activator binding. Little sequence similarity, however, was found at the DNA level. These observations provide direct evidence that Sh2 encodes a subunit for endosperm ADP-glucose pyrophosphorylase. Analysis of several phenotypically wild-type alleles arising from a mutable sh2-Ds allele revealed one unexpected case in which DNA sequences of Sh2 were rearranged in comparison with the progenitor Sh2. In contrast to wild type, the Ds-induced sh2 allele conditions at least two transcripts in the endosperm.

Alleles↗
PubMed1989

Increased gene expression by the first intron of maize shrunken-1 locus in grass species.

The first intron of the shrunken-1 (Sh1) locus of maize was incorporated into constructs containing the chloramphenicol acetyltransferase gene (CAT) coupled with the nopaline synthase 3' polyadenylation signal. Transcription was driven with the 35S promoter of the cauliflower mosaic virus (CaMV) or the Sh1 promoter of maize. Transient gene expression was monitored following electroporation into protoplasts of Panicum maximum (guineagrass), Pennisetum purpureum (napiergrass), or Zea mays (maize). The 1028 base pair intron increased gene expression in cells of each species when transcription was driven with the 35S promoter. Eleven to 91-fold increases were observed. Expression levels observed in maize were two and eight times those observed in napiergrass and guineagrass, respectively. The 35S promoter gave CAT activity 10 to 100 times that observed with the Sh1 promoter. Whereas expression driven by the 35S promoter was reproducible, that observed with the Sh1 promoter proved quite variable. In similar constructs the first intron of the alcohol dehydrogenase-1 (Adh1) gene of maize led to increased gene expression of only 7 to 10% of that observed with the Sh1 first intron. The increased level of gene expression caused by the Sh1 first intron is approximately 10 times higher than that caused by any other plant introns that have been used. Thus, the Sh1 first intron may prove quite useful in increasing expression of foreign genes in monocots and possibly other plants.

Journal Article↗
PubMed1988

The mutation bronze-mutable 4 derivative 6856 in maize is caused by the insertion of a novel 6.7-kilobase pair transposon in the untranslated leader region of the bronze-1 gene.

The Ds-controlled allele, bz-m4 Derivative 6856 [bz-m4 D6856], is reported to have an altered temporal- and tissue-specific pattern of gene expression. We have cloned this allele and have characterized it at the molecular level. The mutation was caused by the insertion of a complex transposon-like structure 36 base pairs downstream from the Bz mRNA cap site. The insert is 6.7-kbp long. Ds elements, each approximately 2 kbp in length, are at both ends of the insert. The sequence between the Ds elements is a partial duplication of flanking sequences from the 3' end of the Bz gene. These data suggest that Ds initially inserted near the 3' end of the gene and mobilized adjacent sequences as it transposed.

Base Sequence↗
PubMed1988

Promoter strength comparisons of maize shrunken 1 and alcohol dehydrogenase 1 and 2 promoters in mono- and dicotyledonous species.

Promoter strengths of two maize alcohol dehydrogenase genes, Adh1 and Adh2, and the maize shrunken-1 gene, Sh1, were evaluated by transient expression in cultured protoplasts of Panicum maximum, Triticum monococcum, and Daucus carota. Promoter elements were ligated in correct and opposite orientations as transcriptional gene fusions to the chloramphenicol acetyl transferase gene containing the nopaline synthase 3' polyadenylation signal. The relative levels of gene expression were compared to the cauliflower mosaic virus 35S promoter. The full length Adh1 promoter (-1100 to +15) functioned in all species, but at a reduced level in D. carota. An Adh1 promoter deletion from -304 to -1100 did not express at detectable levels in any species nor did the Sh1 promoter construction. The Adh2 promoter (-860 to +90) only expressed in D. carota. The full length Adh1 promoter gave the highest level of CAT expression in the monocot cells but at levels which were approximately 30% compared to the CaMV 35S promoter. This was reduced further in D. carota to approximately 4%. These data suggest that at least some of the regulatory factors responsible for promoter function are somewhat species specific and that these differences should be considered in gene expression studies.

Journal Article↗
PubMed1988

Shrunken-1 encoded sucrose synthase is not required for sucrose synthesis in the maize endosperm.

Kernels of wild-type maize (Zea mays L.) shrunken-1 (sh1), deficient in the predominant form of endosperm sucrose synthase and shrunken-2 (sh2), deficient in 95% of the endosperm ADP-glucose pyrophosphorylase were grown in culture on sucrose, glucose, or fructose as the carbon source. Analysis of the endosperm extracts by gas-liquid chromatography revealed that sucrose was present in the endosperms of all genotypes, regardless of carbon supply, indicating that all three genotypes are capable of synthesizing sucrose from reducing sugars. The finding that sucrose was present in sh1 kernels grown on reducing sugars is evidence that shrunken-1 encoded sucrose synthase is not necessary for sucrose synthesis. Shrunken-1 kernels developed to maturity and produced viable seeds on all carbon sources, but unlike wild-type and sh2 kernels grown in vitro, sucrose was not the superior carbon source. This latter result provides further evidence that the role of sucrose synthase in maize endosperm is primarily that of sucrose degradation.

Journal Article↗
PubMed1986

The cloning, genetic mapping, and expression of the constitutive sucrose synthase locus of maize.

Two differentially expressed genes encode isoenzymes of sucrose synthase in Zea mays. A clone of the shrunken 1 (Sh1) locus, the structural gene for the major endosperm form of sucrose synthase, was used to isolate a genomic clone of constitutive sucrose synthase (Css), the structural gene for the isoenzyme expressed in embryo and other tissues. The Css clone was positively identified by RNA blot analysis of RNA from wild type and a sh1 deletion stock and by analysis of the in vitro translation product of hybrid-selected mRNA. Southern blot analysis of DNA from monosomic plants derived from an r-x1 stock, coupled with restriction fragment length polymorphism mapping, placed the Css gene 32 map units from Sh1 on chromosome 9. In seedling tissues, Css mRNA is present at higher levels than Sh1 mRNA. Expression of both Sh1 and Css in root tissue is enhanced by anaerobic conditions, although Css is induced to a lesser extent than is Sh1. Thus, Css appears to be expressed constitutively, whereas Sh1 is expressed at high levels only in response to specific developmental and environmental stimuli.

Journal Article↗
PubMed1986

Sugar utilization by developing wild type and shrunken-2 maize kernels.

To characterize the movement of sugars during kernel development in maize, a newly devised in vitro kernel development scheme was utilized. Viable seeds of wild type maize (Zea mays L.) as well as the mutant shrunken-2 (sh2) were found to mature when grown in culture with reducing sugars or sucrose as the carbon source. However, wild type and sh2 kernels had greater germination, starch content, and seed weight when sucrose, rather than reducing sugars, was the carbon source. By the use of labeled sucrose it was shown that sucrose can move into endosperm tissue without intervening degradation and resynthesis. These results show that when grown in vitro the maize seed can utilize reducing sugars for development, but it prefers sucrose.

Journal Article↗
PubMed1976

Characterization of ADP-glucose pyrophosphorylase from shrunken-2 and brittle-2 mutants of maize.

Electrophoretic characterization of adenosine diphosphate glucose pyrophosphorylase from the developing endosperms of nine shrunken-2 and four brittle-2 mutants revealed that (1) all mutants had low but detectable levels of activity, (2) mutation at either locus decreased activity of pyrophosphorylases A and B, and (3) differences in mobility were not found. However, pyrophosphorylase B extracted from several shrunken-2 and brittle-2 mutants differed from normal in extent of urea denaturation, Km (glucose-1-phosphate) or type of glucose-1-phosphaociation with the sh2 locus) appears to differ from normal in Km (glucose-1-phosphate).

Adenosine Diphosphate Glucose↗
PubMed1976

Comparison of chemical and microbiological methods in the estimation of methionine in cowpea (Vigna unguiculata) seeds.

1. Meals were prepared from the seeds of fifteen varieties of cowpea (Vigna unguiculata), one of lima bean (Phaseolus lunatus) and one of yam bean (Sphenostylis stenocarpa), and their methionine content was determined by six different methods. 2. Total methionine content was determined by two chemical methods (ion-exchange chromatography and a colorimetric procedure) and by two microbiological methods. The 'available' methionine content was determined by microbiological assay with Streptococcus zymogenes. 3. All the different methods for total methionine determination gave similar results, with much the same high extent of precision. 4. The value for 'available' methionine content were similar to or marginally higher than the corresponding microbiological assay value for total methionine content. There was no indication that the methionine in any of the test samples was not completely available.

Biological Assay↗
PubMed1975

Characterization of adenosine diphosphate glucose pyrophosphorylases from developing maize seeds.

Electrophoretic examination of 22-day-old, normal maize (Zea mays L.) endosperm extracts revealed two zones of adenosine diphosphate glucose pyrophosphorylase activity. The enzymes are identical in terms of Km for glucose 1-phosphate and the effect of 3-phosphoglyceric acid on apparent Km for glucose 1-phosphate. Both enzymatic activities increase with increasing doses of the functional alleles at the shrunken-2 and brittle-2 loci. Molecular weight differences between the two electrophoretic species were inferred from sucrose gradient centrifugation. It is suggested that the two bands of activity represent different aggregation states of the same enzyme because under different extraction conditions, only one enzyme is found. Molecular weight estimates of 237,000 and 253,000 were obtained for the smaller enzyme. It is suggested that this enzyme is an aggregate of several subunits. Comparison of the embryo and endosperm pyrophosphorylases showed the embryo activity to be more heat stable and probably independent of direct shrunken-2 or brittle-2 control.

Journal Article↗