Charles Hedgcoth

Nucleotide sequence of a γ gliadin gene: comparisons with other γ gliadin sequences show the structure of γ gliadin genes and the general primary structure of γ gliadins

Abstract A low stringency screening of a wheat ( Triticum aestivum L.) genomic library produced three types of γ gliadin clones. The sequence of one clone, λ10–20, encoded a γ gliadin of 34.3 kDa. Comparisons of this protein with the proteins encoded by other γ gliadin DNA sequences revealed a general γ gliadin structure: a 19-residue signal peptide; 12-residue mature amino terminus; 12–16 copies of a proline- and glutamine-rich heptapeptide repeat; a 76-residue region high in glutamine containing most of the cyysteines and charged residues: a 6–16-residue polyglutamine region; and the 41-residue carboxyl terminus. Comparisons of the 5′ and 3′ flanking regions of several γ gliadins reveals the high homology and general structure of γ gliadin genes. A further comparison of the 5′ flanking regions with the 5′ flanking regions of other prolamin genes showed that γ gliadin genes contain three copies of a conserved sequence seen within approx. 600 b.p. upstream of the translation start sites of prolamin genes.

A CHIMERIC GENE (ORF256) IS EXPRESSED AS PROTEIN ONLY IN CYTOPLASMIC MALE-STERILE LINES OF WHEAT

Mitochondria derived from Triticum timopheevi have a chimeric gene, orf256, immediately upstream from coxI. Antibodies to a peptide corresponding to a part of the encoded amino acid sequence of orf256 detect a 7 kDa protein on western blots of mitochondrial proteins from cytoplasmic male-sterile (cms) wheat (T. aestivum nucleus, T. timopheevi mitochondria) but not in mitochondrial proteins from T. aestivum, T. timopheevi, or cms plants restored to fertility by introduction of nuclear genes for fertility restoration. The 7 kDa protein appears to serve as a marker for cms wheat. Its occurrence as an integral protein of the inner membrane may indicate a cms effect through an influence on mitochondrial membrane function.

Determination of nucleoside composition of ribonucleic acid by gas-liquid chromatography

Abstract A procedure for analyzing microgram quantities of RNA for nucleoside composition, including pseudouridine, is described. It involves enzymically digesting RNA to the constituent nucleosides, reducing the reaction mixture to dryness, and forming trimethylsilyl derivatives by treating with N,O -bis(trimethylsilyl)acetamide without first removing salt and protein. Gas-liquid chromatography of an aliquot of nucleoside derivatives containing 10 μg or less of total nucleosides is performed with linear temperature programming. Chromatography time is less than 40 min.

Heptapeptide repeat structure of a wheat γ-gliadin

Abstract A γ gliadin cDNA clone has been sequenced. The clone encodes 251 amino acid residues beginning with a 19 residue signal peptide. The putative processed amino terminus is characteristic of a γ gliadin. Fourteen imperfect repeats of a heptapeptide occur near the amino terminus. Data from hybrid selected translation suggest that the cDNA contains sol3 4 of the mRNA coding region. These results provide the first description of the amino terminus of a γ gliadin precursor.

A chimeric open reading frame associated with cytoplasmic male sterility in alloplasmic wheat with Triticum timopheevi mitochondria is present in several Triticum and Aegilops species, barley, and rye

Abstract. Mitochondrial DNA from Triticum timopheevi has a chimeric gene, orf256, upstream of coxI. This gene is cotranscribed with coxI in cytoplasmic male sterile plants and produces a 7-kDa protein which is not produced in fertile or fertility-restored plants. T. aestivum, the nuclear donor in sterile plants, does not have orf256. Analysis by polymerase chain reaction of DNA from barley, rye, Aegilopsbicornis, Ae. searsii, Ae. sharonensis, Ae. speltoides, Ae. tauschii, T. monococcum, and T. turgidum was done with oligonucleotide primers designed to detect orf256 or coxI sequences. Except for T. turgidum, these plants have various elements of the orf256 sequence over a 1-kb length of DNA immediately upstream of coxI in exactly the same arrangement as is found in the coxI region of T. timopheevi. Only T. timopheevi and Ae. speltoides have orf256 transcripts, and only cytoplasmic male-sterile plants involving these two species as maternal donors produce a protein from orf256. Part of an orf256-like sequence is present in T. turgidum but is at least slightly different in arrangement relative to coxI, as compared with the sequence in T. timopheevi. Neither maize nor sorghum have the orf256 sequence.

Determination of 5,6-dihydrouridine in ribonucleic acid

Abstract Two simple methods to rapidly and accurately quantify 5,6-dihydrouridine in small amounts of RNA are presented. One involves a colorimetric microassay for dihydrouridine. It is performed directly on RNA in less than one hour. It requires only 25–100 μg of tRNA and is reproducible to ±5%. The other involves enzymic digestion of RNA to nucleosides followed by bidimensional thin-layer chromatography on microcrystalline cellulose. It may be used to quantify dihydrouridine in RNA labeled with a radioactive pyrimidine. It is extremely sensitive, relatively rapid, and allows simultaneous determination of other major and minor pyrimidine nucleosides. The methods have been used to quantify dihydrouridine in tRNA of Escherichia coli, Salmonella typhimurium , yeast, and rat liver, and rRNA of E. coli , with excellent agreement between methods and with literature values.

Seven lysine isoaccepting tRNA's from polyoma virus-transformed cells

Abstract Chromatography of lysyl-tRNA from polyoma virus-transformed mouse fibroblasts on benzoylated diethylaminoethyl cellulose gives four iso-accepting species. One of the isoacceptors is a major species in transformed cells but not in normal cells. In a reverse-phase chromatographic system, five isoacceptors are clearly resolved, one is poorly resolved, and one is observed only with difficulty. A comparison of the results of the two systems reveals a total of seven identifiable isoacceptors of lysyl-tRNA.

Determination of nucleoside composition of ribonucleic acid by thin-layer chromatography

Abstract Separating and quantifying nucleosides of ribonucleic acid on thin layers of microcrystalline cellulose are described. RNA digested enzymically is chromatographed bidimensionally without desalting or removing proteins, which results in complete separation of eight nucleoside residues of acceptor ribonucleic acid including the four major nucleosides, dihydrouridine, inosine, pseudouridine, and ribothymidine. Data indicating quantitative elution of nucleosides from the thin layers and the nucleoside composition of ribonucleic acid are included.

An extra species of lysine transfer ribonucleic acid in polyoma virus-transformed cells in tissue culture☆

Abstract Isoaccepting tRNAs from various mouse cells were fractionated on columns of benzoylated DEAE cellulose. Lysine tRNA from mouse embryo, adult mouse liver and kidney, primary mouse embryo cells in tissue culture, and an established tissue culture line of mouse fibroblasts (3T3) has two peaks of isoaccepting tRNA; lysine tRNA from two established lines of polyoma virus-transformed cells contains an additional peak of lysine tRNA. The extra peak in transformed cells comprises about 25% of the acceptor capacity for lysine. It is stable to denaturation and renaturation and can be chromatographed, stripped of lysine, recharged, and rechromatographed. The extra peak is present in tRNA from transformed cells and absent in tRNA from normal cells regardless of whether the lysyl-tRNA ligase used for aminoacylation is from normal or transformed cells. Isoaccepting tRNAs for arginine, leucine, serine, valine, histidine, and tyrosine reveal similar profiles for the various tRNAs from normal and transformed cells.

Induction of a new species of phenylalanine transfer RNA during lens cell differentiation

Abstract The two major species of phenylalanine transfer RNA (tRNA) of lens cortex have been isolated using BD cellulose and reversed-phase chromatography. Specific activities of 1200 and 1500 pmol of phenylalanine accepted per A260 of tRNA were obtained for phe-tRNA1 and phe-tRNA2 respectively. The fluorescence emission spectra of both phe-tRNAs were identical, with a fluorescence maximum around 420 nm. These measurements, along with chromatographic data, confirm the presence of the Y base in both tRNAs. Nucleotide analysis was carried out on the purified phe-tRNAs. The composition of phe-tRNA2 was almost identical to the phe-tRNA of rabbit liver. Phe-tRNA1, however, had a markedly different major base composition. Phe-tRNA1 therefore, represents a different gene product than phe-tRNA2. This was confirmed by the fact that phe-tRNA1, and phe-tRNA2 chromatograph as separate peaks on a reversed-phase column in the presence of 8·0 m -urea. Therefore the differentiation of a lens epithelial cell into a lens fiber cell is accompanied by a doubling of phe-tRNA as a result of the activation of a new phe-tRNA gene.