Kilobase Insert Research Articles

NaCl Regulation of Tonoplast ATPase 70-Kilodalton Subunit mRNA in Tobacco Cells

A cDNA clone encoding the 70-kilodalton subunit of the tobacco (Nicotiana tabacum var Wisconsin 38) tonoplast ATPase has been isolated. The 1.656 kilobase insert contains only open reading frame that represents more than 80% of the carrot cDNA coding region. The deduced amino acid sequence has greater than 95% sequence identity with the homologous carrot sequence. A transcript of approximately 2.7 kilobase was detected on Northern blots of tobacco poly(A)(+) selected or total RNA using labeled probe produced from this clone. The gene was expressed throughout the growth cycle in unadapted and 428 millimolar NaCl adapted cells. Transcription of the 70-kilodalton subunit gene or mRNA stability was induced by short-term NaCl treatment in NaCl adapted cells or by abscisic acid treatment in both adapted and unadapted cells. Southern analysis indicated the presence of up to four genes encoding the 70-kilodalton subunit.

CDNA Clones for Corn Leaf NADH:Nitrate Reductase and Chloroplast NAD(P)+:Glyceraldehyde-3-Phosphate Dehydrogenase

cDNA clones were selected from a corn (Zea mays L.) leaf lambda gt11 expression library using polyclonal antibodies for corn leaf NADH:nitrate reductase. One clone, Zmnrl, had a 2.1 kilobase insert, which hybridized to a 3.2 kilobase mRNA. The deduced amino acid sequence of Zmnrl was nearly identical to peptide sequences of corn leaf NADH:nitrate reductase. Another clone, Zm6, had an insert of 1.4 kilobase, which hybridized to a 1.4 kilobase mRNA, and its sequence coded for chloroplastic NAD(P)(+):glyceraldehyde-3-phosphate dehydrogenase based on comparisons to sequences of this enzyme from tobacco and corn. When nitrate was supplied to N-starved, etiolated corn plants, nitrate reductase, and glyceraldehyde-3-phosphate dehydrogenase mRNA levels in leaves increased in parallel. When green leaves were treated with nitrate, only nitrate reductase mRNA levels were increased. Nitrate is a specific inducer of nitrate reductase in green leaves, but appears to have a more general effect in etiolated leaves. In the dark, nitrate induced nitrate reductase expression in both etiolated and green leaves, indicating light and functional chloroplast were not required for enzyme expression.

Human uroporphyrinogen III synthase: molecular cloning, nucleotide sequence, and expression of a full-length cDNA.

Uroporphyrinogen III synthase [URO-synthase; hydroxymethylbilane hydro-lyase (cyclizing), EC 4.2.1.75], the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 x 10(6) recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The authenticity of this clone was established by colinearity of the predicted amino acid sequence with 81 microsequenced residues from the purified enzyme. In addition, high levels of enzymatic activity and immunoreactive protein were expressed when a blunt-ended 971-base-pair Ava II cDNA fragment containing the entire coding region was inserted into vectors for expression in Escherichia coli. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria.

Cloning of fragments of lambda dapB2 DNA and identification of the dapB gene product.

DNA of the specialized transducing phage lambda dapB2 has been digested with the restriction endonucleases Bam I, HindIII, or both together to generate fragments originating from the bacterial substitution on the phage. Seven such fragments ranging in size from 0.8 to 7.1 kilobases and encompassing the entire bacterial substitution of 18 kilobases of DNA have been covalently ligated to the vector pBR322. The recombinant plasmids so constructed have been tested for their ability to complement the dapB17 allele in Escherichia coli strain AT999. Only pGM4, which contains a 7.1 kilboase fragment generated by Bam I cleavage of lambdadapB2 inserted into pBR322, relieves this strain's requirement for DL-diaminopimelic acid and restores dihyrodipicolinic acid reductase activity to wild type levels. Deletions were obtained in pGM4 by two methods. None of the resultant shortened plasmids were proficient in complementation of the dapB17 allele. The proteins encoded by the parental plasmid and by those of seven deletions derived from it have been identified by coupled transcription and translation of plasmid DNA templates in vitro or by the stimulation of protein synthesis promoted by these plasmids in an ultraviolet irradiated host. The parent encodes four proteins unique to the 7.1 kilobase insert whose apparent molecular weights are 48,000, 36,000, 32,000, and 17,000. Of these, the protein of 32,000 is consistently missing when noncomplementing pasmids harboring deletions are used as templates. This protein is tentatively identified as the product of the dapB gene. The role of the other three proteins whose genes are closey linked to the dapB gene is unknown. There appear to be at least two transcriptional units within this cluster of genes, however, suggesting independent regulation and, possibly, function.