Three major forms of adenylate kinase from adult and fetal rat tissues.

The major enzymatic forms of adenylate kinase have been purified to homogeneity from fetal liver and adult brain of the rat. The two enzymes differ with respect to isoelectric points, Km (ATP), Km (AMP), and Ka (citrate). Antibody to adult liver adenylate kinase does not inhibit either enzyme, while entibody to adult skeletal muscle enzyme inhibits the brain enzyme but not the fetal liver enzyme. It is therefore probable that there are three major forms of adenylate kinases in fetal and adult rat tissues.     

The fastest contracting muscles of nonmammalian vertebrates express only one isoform of the ryanodine receptor.

The skeletal muscles of chickens, frogs, and fish have been reported to express two isoforms (alpha and beta) of the sarcoplasmic reticulum calcium release channel (ryanodine receptor or RYR), while mammals express only one. We have studied patterns of RYR isoform expression in skeletal muscles from a variety of fish, reptiles, and birds with immunological techniques. Immunoblot analysis with a monoclonal antibody that recognizes both nonmammalian RYR isoforms and a polyclonal antibody specific to the alpha isoform show two key results: (a) two reptilian orders share with mammals the pattern of expressing only the alpha (skeletal) RYR isoform in skeletal muscle; and (b) certain functionally specialized muscles of fish and birds express only the alpha RYR isoforms. While both isoforms are expressed in the body musculature of fish and birds, the alpha isoform is expressed alone in extraocular muscles and swimbladder muscles. The appearance of the alpha RYR isoform alone in the extraocular muscles and a fast-contracting sonic muscle in fish (toadfish swimbladder muscle) provides evidence that this isoform is selectively expressed when rapid contraction is required. The functional and phylogenetic implications of expression of the alpha isoform alone are discussed in the context of the mechanism and evolution of excitation-contraction coupling.     

Molecular cloning of three major sequence species from Rainbow trout protamine mRNA.

Double stranded cDNA molecules complementary to purified Rainbow trout protamine mRNA have been cloned in the bacterial plasmid pBR322. In order to circumvent the problems associated with a heterogeneous cDNA probe when identifying recombinants, a comparative hybridisation technique was used which can resolve between closely related cloned sequences. Using this technique, selected recombinants were shown to carry sequences corresponding to separate major fractions of protamine mRNA. Partial nucleotide sequences of the inserts in two clones confirms this conclusion.     

Expression and characterization of pea chloroplastic glyceraldehyde-3-phosphate dehydrogenase composed of only the B-subunit.

A cDNA fragment coding for the pea (Pisum sativum L.) chloroplastic glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) B-subunit and a truncated form corresponding in length to the A-subunit have been cloned into an expression vector, expressed in the absence of the A-subunit in a gap- Escherichia coli strain, purified, and studied. Like the isolated enzyme from higher plant chloroplasts, the recombinant enzymes have dual specificity for NADPH and NADH. The recombinant glyceraldehyde-3-P dehydrogenases have the same optimal pH as the enzyme isolated from pea chloroplasts. Like the native chloroplast enzyme, the recombinant B-subunit has a marked tendency to form large aggregates, whereas the truncated B-subunit exists as the tetramer. The recombinant B-subunit glyceraldehyde 3-P dehydrogenase is more sensitive to dithiothreitol than its truncated form. It seems likely that a different pair of cysteines is responsible for the redox sensitivity of the activity of the enzyme composed of B-subunits than the cysteine residues implicated in the modulation of the activity of the enzyme composed of A-subunits by previous work in this laboratory.     

Complete nucleotide sequence of a cloned cDNA derived from the major adult alpha-globin mRNA of X. laevis.

The complete sequence of a cloned cDNA derived from the major adult alpha-globin mRNA of Xenopus laevis (the South African Clawed Toad) is presented. The sequence contains the complete coding and 3' non-coding regions of the mRNA and part of the 5' non-coding region. The amino acid sequence of the encoded alpha-globin polypeptide has been deduced and is compared to other alpha-globin polypeptides. We find that the sequence is equally diverged from a bullfrog tadpole alpha-globin polypeptide and human alpha-globin polypeptide suggesting that these three sequences may have diverged from a common ancestral sequence several hundred million years ago.     

Differential expression and sequence analysis of the maize glyceraldehyde-3-phosphate dehydrogenase gene family.

Two cDNA clones for maize cytosolic glyceraldehyde-3-phosphate dehydrogenase are described. One is about 97% similar in coding capacity to a previously published clone [Brinkmann et al. (1987). J. Mol. Evol. 26, 320-328], while the other shows only 88% similarity. Evidence points toward the three cDNAs being the products of three genes, to be called Gpc1, Gpc2, and Gpc3. When the least similar clone, corresponding to Gpc3, was used to analyze RNA gel blots, anaerobic treatment for 6 hours induced RNA accumulation in the shoots 15.6-fold, while a 1-hour shift from 28 degrees C to 40 degrees C increased accumulation 5.1-fold. Roots had a higher basal level of expression, leading to a 6.0-fold anaerobic induction, and a 2.4-fold heat stress induction. RNA gel blot analysis using the clone corresponding to Gpc2 showed decreased RNA accumulation within 6 hours of anaerobiosis, while analysis with the previously published clone, corresponding to Gpc1, showed a decrease within 24 hours. Neither Gpc1 nor Gpc2 showed heat stress induction, while some other known anaerobic genes did. Through the use of hybrid selection, in vitro translation, and immune precipitation, the relative expression of the three genes is shown. The role of the observed changes in gene expression is discussed in relation to stress physiology.     

The complete sequence of a full length cDNA for human liver glyceraldehyde-3-phosphate dehydrogenase: evidence for multiple mRNA species.

A recombinant M13 clone (O42) containing a 65 b.p. cDNA fragment from human fetal liver mRNA coding for glyceraldehyde-3-phosphate dehydrogenase has been identified and it has been used to isolate from a full-length human adult liver cDNA library a recombinant clone, pG1, which has been subcloned in M13 phage and completely sequenced with the chain terminator method. Besides the coding region of 1008 b.p., the cDNA sequence includes 60 nucleotides at the 5'-end and 204 nucleotides at the 3'-end up to the polyA tail. Hybridization of pG1 to human liver total RNA shows only one band about the size of pG1 cDNA. A much stronger hybridization signal was observed using RNA derived from human hepatocarcinoma and kidney carcinoma cell lines. Sequence homology between clone 042 and the homologous region of clone pG1 is 86%. On the other hand, homology among the translated sequences and the known human muscle protein sequence ranges between 77 and 90%; these data demonstrate the existence of more than one gene coding for G3PD. Southern blot of human DNA, digested with several restriction enzymes, also indicate that several homologous sequences are present in the human genome.     

The glyceraldehyde-3-phosphate dehydrogenase gene family in the nematode, Caenorhabditis elegans: isolation and characterization of one of the genes

The isolation and genomic sequence of one of possibly four glyceraldehyde-3-phosphate dehydrogenase genes in the nematode, Caenorhabditis elegans is presented. The complete nucleotide sequence of the coding as well as the noncoding flanking regions of this gene has been determined. The deduced amino-acid sequence agrees with the sequence of typical glyceraldehyde-3-phosphate dehydrogenase enzymes and its molecular weight of 36,235 agrees with its size determined previously (Yarbrough, P. and Hecht, R. (1984) J. Biol. Chem. 259, 14711-14720). That this isolated gene encodes a nematode glyceraldehyde-3-phosphate dehydrogenase is additionally confirmed by demonstrating its immunoreactivity to an anti-nematode glyceraldehyde-3-phosphate dehydrogenase antibody after its expression as a fusion protein with dihydrofolate reductase. Codon utilization follows a pattern typical of other expressed nematode genes. The gene is split by two introns that are highly conserved in comparison to other introns observed in C. elegans. The placement of one of these introns is conserved with respect to the chicken glyceraldehyde-3-phosphate dehydrogenase gene. Within the 5' flanking sequence homology to actin and the homology 2 block of the major myosin gene (unc-54) is noted. It is of interest that the 3' flanking region contains a CAAAT box, followed by a TATAAT box, before an open reading frame of a closely linked gene that also contains a small AT-rich intron with the nematode consensus splice junction.     

Expression of only one of two types of mRNA transcribed from the serine dehydratase gene is repressed in hepatoma cells.

The expressions of mRNA for serine dehydratase in H4IIE, HTC, and HepG2 hepatoma cells were investigated. Of the two types of mRNA transcribed from the single copy of the rat serine dehydratase gene, expression of only that encoding 35 kDa serine dehydratase is repressed in these cells. The other type coding the 8.9 kDa truncated translation product is expressed at a similar level to that in hepatocytes in primary culture and is not under hormonal control.     

Growth hormone induces two mRNA species of the serine protease inhibitor gene family in rat liver

In order to study the molecular actions of growth hormone on gene expression, we have cloned and characterized two unique, but related, cDNA sequences from rat liver, lambda Spi-1 and lambda Spi-2. These two cDNA sequences are complementary to rat hepatic mRNA species previously designated as Spots 3 and 20 when assayed by in vitro translation and two-dimensional gel electrophoresis. By Northern blot, the two mRNAs are both 1900 bases in length and growth hormone administered to hypophysectomized rats increases the levels of both of these mRNAs. In contrast, the combined administration of thyroxine, corticosterone, and dihydrotestosterone to hypophysectomized rats did not augment these mRNAs. The simultaneous administration of all four hormones resulted in a level greater than that observed for animals treated with growth hormone alone. Analysis of genomic DNA suggests the presence of two similar, but not identical, genes. DNA sequencing of lambda Spi-1 and lambda Spi-2 revealed that they were 90% homologous at the nucleotide level and 87% homologous at the amino acid sequence level. lambda Spi-2 has 78% homology with mouse contrapsin, 60% with human alpha 1-antichymotrypsin, and 51-55% with alpha 1-antitrypsins, all members of the serine protease inhibitor gene family. The nucleotide and deduced amino acid sequences of lambda Spi-1 and lambda Spi-2 which align with the reactive centers of known members of this family differ substantially from each other and from other members of the family. The difference in the reactive center suggests that the specificity or function of these proteins may differ from other members of serine protease inhibitor gene family.     

Effect of anions on the structure of glyceraldehyde-3-phosphate dehydrogenase from rat skeletal muscle

Glyceraldehyde-3-phosphate dehydrogenase from rat skeletal muscle undergoes reversible partial dissociation into 4.2–4.5 S fragments at +4°C and at protein concentrations as high as 8–9 mg/ml, depending upon the anion species present. The order of effectiveness of various anions in dissociating activity corresponds to their position in the Hofmeister series: bromide > nitrate > chloride > acetate > sulfate > phosphate=citrate. Quite similar is the order of activity of anions in their influence on the thermal stability of the dehydrogenase.