Molecular cloning and characterization of the rat cytochrome c oxidase subunit Vb gene.

We have isolated two non-overlapping clones containing the genes for subunit Vb of cytochrome c oxidase (COXVb) from a rat genomic library in Charon 4A using a newly isolated full-length cDNA as a probe. One of the two genomic clones, designated as lambda COXVb741, contained a functional gene (COXVb-1), while the other one, designated as lambda COXVb211, contained an intronless processed pseudogene (COXVb-2). The COXVb-1 gene spans approximately 1.8 kb and consists of four exons interrupted by three introns. The nucleotide sequences of all exons are completely identical to the corresponding sequences of the rat liver and brain COXVb cDNAs, indicating that this gene is actually expressed. The 5'-flanking region of the gene lacks conventional TATA and CAAT boxes, but exhibits strong promoter activity in the chloramphenicol acetyltransferase (CAT) assay. Deletional analysis and gel shift assay of the 5'-flanking region suggested that the binding of nuclear factor Sp-1 could be essential for transcription of the gene. Southern blotting analysis implied the occurrence of multiple COXVb genes in the rat genome. However, the results of the present experiments suggest that only the COXVb-1 gene is expressed in rat tissues.

High level expression of rat gamma-D-crystallin in Escherichia coli.

Gamma-crystallins have been implicated in various kinds of cataracts. In order to facilitate studies elucidating the molecular mechanism of cataractogenesis, large quantities of rat recombinant gamma-D-crystallin were produced in E coli. A full length cDNA clone coding for gamma-D-crystallin was isolated from a rat lens lambda gt11 cDNA library using a synthetic oligonucleotide as a probe. The coding region of this cDNA was inserted into a cloning vector pKK233-2 under the control of the trc promoter. The resulting construct, pKKCR91, was transfected into E coli to produce rat gamma-D-crystallin in an amount of 10-15% of the total bacterial proteins. The crystallin produced was purified to an apparent homogeneity as judged by SDS gel electrophoresis. The sequence of the N-terminal 11 amino acids of the purified crystallin was determined, showing that it is completely identical to that predicted from the cDNA sequence. Measurements of the far-UV CD spectra also revealed that recombinant rat gamma-D-crystallin thus produced retains a native conformational structure.

A point mutation in the 3,5,3'-triiodothyronine-binding domain of thyroid hormone receptor-beta associated with a family with generalized resistance to thyroid hormone.

A tight linkage between generalized resistance to thyroid hormone (GRTH) and the thyroid hormone receptor-beta (TR beta) gene is indicated. We evaluated a family with GRTH for the TR beta gene. We found that a new point mutation, consisting of a cytosine to adenine replacement at nucleotide position 1642, resulted in substitution in codon 448 in the T3-binding domain of TR beta. This base substitution was found in only one allele of affected members, but not in unaffected members of the family. The in vitro translation products of this mutant TR beta gene demonstrated significantly reduced T3-binding affinity. Previously, others have reported a kindred with GRTH, in that the same codon was subjected to proline to histidine replacement due to a mutation consisting of a cytosine to adenine replacement at nucleotide position 1643. There appeared to be a significant phenotypic difference between our kindred and that described by others.

Transforming genes and chromosome aberrations in therapy-related leukemia and myelodysplastic syndrome.

The presence of activated transforming genes was investigated in four patients with therapy-related leukemia and in three with therapy-related myelodysplastic syndrome. DNA of bone marrow cells from six of the patients exhibited transforming activity in the tumorigenicity assay. Five of the six patients who were positive in the tumorigenicity assay contained activated N-ras oncogenes, and three contained activated K-ras oncogenes. Thus, concurrent activation of N-ras and K-ras oncogenes was observed in two patients. In vitro DNA amplification followed by oligonucleotide dot-blot analysis was used to investigate mutations in codons 12, 13, and 61 of the N-ras and K-ras oncogenes. Two patients exhibited an N-ras mutation, substituting aspartic acid (GAT) for glycine (GGT), and three patients exhibited an N-ras codon 13 mutation, substituting valine (GTT) for glycine. Two patients exhibited K-ras codon 12 mutations, substituting aspartic acid (GAT) or cysteine (TGT) for glycine (GGT), respectively, and one case exhibited a K-ras codon 61 mutation, substituting lysine (AAA) for glutamic acid (CAA). Cytogenetic analysis revealed that loss of chromosome 7 was frequent (four patients: 57%). Our data indicate that activation of N-ras and K-ras genes, as well as loss of heterozygosity for specific alleles on chromosome 7, plays a more important role in the leukemogenesis of both therapy-related leukemia and myelodysplastic syndrome.

Isolation and characterization of the two distinct genes for human glutamate dehydrogenase.

Two genomic clones containing a part of the glutamate dehydrogenase gene were isolated from a human genomic library. The restriction map of both clones were distinctly different from one another, although the nucleotide sequences of the three exons that they contained were virtually the same in each clone. Southern blotting analysis of the genomic DNAs from several unrelated human individuals revealed that in every case the probe hybridized with at least two DNA fragments of different sizes, each characteristic to one of the two clones. These results strongly suggest that the two clones presently obtained do not result from polymorphism but are generated from two different gene loci for glutamate dehydrogenase on the human chromosome.

Structural organization of the rat cytochrome c oxidase subunit IV gene.

We have isolated two overlapping clones covering the entire length of the gene of nuclear-encoded sub-unit IV of cytochrome c oxidase (COXIV) from a rat genomic library in Charon 4A and determined the structural organization of the gene. The gene spans approximately 7.0 kilobases and contains five exons interrupted by four introns. Of these exons, exon 2 codes for a whole length of the presequence of the rat COXIV precursor protein, while exons 3 to 5 encode three distinct structural domains of the mature protein. The 5'-flanking region of the gene lacks conventional TATA and CAAT boxes, but has a high G + C content and contains two putative binding sites for transcription factor SP1 and a sequence resembling the AP-4 responsive element. These results indicate that the promoter region of the rat COXIV gene possesses characteristic features common in housekeeping genes. The chloramphenicol acetyltransferase assay performed by constructing an improved phagemid, pBlueCAT3, revealed that a 773-base pair DNA fragment immediately preceding the cap site has a strong promoter activity. An octanucleotide sequence, -TTCTTGGT-, which is very close to the yeast HAP2/HAP3 responsive element, is located in the 5'-upstream region of the present gene.

Molecular cloning and nucleotide sequence of the cDNA for human liver glutamate dehydrogenase precursor.

Two cDNA clones (lambda GDHh1 and lambda GDHn61) for glutamate dehydrogenase (GDH) were isolated from a human liver cDNA library in lambda gt11. The clone, lambda GDHh1, was isolated from the library using a synthetic 45mer oligodeoxy-ribonucleotide, the sequence of which was derived from the known amino acid sequence near the NH2-terminus of human liver GDH. Subsequently, lambda GDHn61 was isolated from the same library using lambda GDHh1 as a probe. The inserts of both clones contained an overlapping cDNA sequence for human liver GDH, consisting of a 5'-untranslated region of 70 bp, an open reading frame of 1677 bp, a 3'-untranslated region of 1262 bp and a 15 base poly(A) tract. The predicted amino acid sequence revealed that the human liver GDH precursor consisted of a total of 558 amino acid residues including the NH2-terminal presequence of 53 amino acids. The sequence deduced for the mature enzyme showed 94% homology to the previously reported amino acid sequence of human liver GDH.

Replacement by site-directed mutagenesis indicates a role for histidine 170 in the glutamine amide transfer function of anthranilate synthase.

Anthranilate synthase is a glutamine amidotransferase that catalyzes the first reaction in tryptophan biosynthesis. Conserved amino acid residues likely to be essential for glutamine-dependent activity were identified by alignment of the glutamine amide transfer domains in four different enzymes: anthranilate synthase component II (AS II), p-aminobenzoate synthase component II, GMP synthetase, and carbamoyl-P synthetase. Conserved amino acids were mainly localized in three clusters. A single conserved histidine, AS II His-170, was replaced by tyrosine using site-directed mutagenesis. Glutamine-dependent enzyme activity was undetectable in the Tyr-170 mutant, whereas the NH3-dependent activity was unchanged. Affinity labeling of AS II active site Cys-84 by 6-diazo-5-oxonorleucine was used to distinguish whether His-170 has a role in formation or in breakdown of the covalent glutaminyl-Cys-84 intermediate. The data favor the interpretation that His-170 functions as a general base to promote glutaminylation of Cys-84. Reversion analysis was consistent with a proposed role of His-170 in catalysis as opposed to a structural function. These experiments demonstrate the application of combining sequence analyses to identify conserved, possibly functional amino acids, site-directed mutagenesis to replace candidate amino acids, and protein chemistry for analysis of mutationally altered proteins, a regimen that can provide new insights into enzyme function.

Nucleotide sequence of yeast GDH1 encoding nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase.

The yeast GDH1 gene encodes NADP-dependent glutamate dehydrogenase. This gene was isolated by complementation of an Escherichia coli glutamate auxotroph. NADP-dependent glutamate dehydrogenase was overproduced 6-10-fold in Saccharomyces cerevisiae bearing GDH1 on a multicopy plasmid. The nucleotide sequence of the 1362-base pair coding region and 5' and 3' flanking sequences were determined. Transcription start sites were located by S1 nuclease mapping. Regulation of GDH1 was not maintained when the gene was present on a multicopy plasmid. Protein secondary structure predictions identified a region with potential to form the dinucleotide-binding domain. The amino acid sequences of the yeast and Neurospora crassa enzymes are 63% conserved. Unlike the N. crassa gene, yeast GDH1 has no introns.

Synthesis of cytochrome c oxidase in the liver of Rana catesbeiana tadpole treated with griseofulvin.

The effect of griseofulvin treatment on the synthesis of cytochrome c oxidase was studied with the liver of the tadpole, Rana catesbeiana. (1) In the liver of tadpole treated with griseofulvin, a ferrochelatase inhibitor, the synthesis of heme a, but not cytochrome c oxidase protein, is inhibited. (2) The apocytochrome c oxidase which is formed in the liver of tadpole treated with griseofulvin is converted to the active holoenzyme by exogenously added heme a.

Cytochrome c oxidase from the liver of bullfrog, Rana catesbeiana and change in its turnover rate during metamorphosis.

1. Cytochrome c oxidase was purified from the liver mitochondria of bullfrog (Rana catesbeiana). The heme a content of the purified enzyme was 13.5 nmol per mg protein. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the enzyme protein was composed of nine polypeptide subunits having molecular weights of 42000, 27000, 25000, 20000, 15000, 13000, 8600, 5400 and 3600. The purified enzyme from the adult frog was immunological identified with that from the tadpole. 2. The ratio of synthesis and degradation of cytochrome c oxidase were 5.2- and 2.0-times higher at metamorphic climax than at premetamorphic stage, respectively. The amount of the enzyme in the liver was highest at metamorphic climax.

Heterogeneity in the liver mitochondria of the tadpole, Rana catesbeiana.

1. The heterogeneity of liver mitochondria of the tadpoles, Rana catesbeiana, undergoing metamorphosis was investigated by a combination of pulse-chase labeling of mitochondria with [methyl-3H]thymidine and centrifugation of mitochondria on a density gradient of metrizamide. 2. The liver mitochondria of tadpole at premetamorphic stage are separated into two populations with mean densities of 1.128 (M2) and 1.112 (M3). 3. At metamorphic stage a population with mean densities of 1.174 (M1) appears additionally. 4. The activity of mitochondria to take up [methyl-3H]thymidine in vivo is 2-3 times higher at metamorphic stage than at premetamorphic stage. 5. The M1 population has a prominently high activity to take up L-[4.5-3H]leucine in vitro and also a high specific activity of cytochrome c oxidase. 6. These findings suggest that the mitochondrial populations found are of alternate stages in the mitochondrial maturation.