Cloning and characterization of a cDNA encoding the beta subunit of human casein kinase II

Previous studies [Summercorn et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8834-8838; Klarlung & Czech (1988) J. Biol. Chem. 263, 15872-15875] have indicated that Balb/c 3T3 cells and 3T3-L1 adipocytes incubated with insulin show increased casein kinase II activity within minutes, implicating this serine/threonine kinase as an early step in an insulin signaling pathway. We recently reported the isolation of a cDNA encoding an alpha subunit of human casein kinase II [Meisner et al. (1989) Biochemistry 28, 4072-4076] as an initial step toward examining the regulation of this enzyme. We now describe a HepG2 cell casein kinase II beta subunit cDNA of 2.57 kb containing 96 bases of 5' untranslated sequence, 645 bases of open reading frame, and 1832 bases of 3' untranslated sequence with two polyadenylation consensus signal sequences and two poly(A) stretches. The open reading frame of the human beta subunit cDNA was 77% and 87% identical with the Drosophila sequence at the nucleotide and amino acid levels, respectively, and 99% identical with the bovine amino acid sequence. RNA analysis of HepG2 cell RNA utilizing HepG2 beta subunit cDNA fragments as probes revealed one major band migrating at 1.2 kb and two minor bands migrating at 3.0 and 4.2 kb. Results from DNA analysis of HepG2 genomic DNA, consistent with results utilizing Drosophila genomic DNA, suggest the presence of a single gene for the beta subunit of casein kinase II.     

Cloning and characterization of the gene encoding beta subunit of mitochondrial processing peptidase from the basidiomycete Lentinula edodes

We have isolated the gene encoding the beta subunit of mitochondrial processing peptidase (β-MPP) from the shiitake mushroom Lentinula edodes. It is a nuclear gene with two small introns. Comparison with known β-MPP genes revealed that the L. edodes gene is most closely related with that from Neurospora crassa, with 60.8% identities and 87% similarity in the amino-acid sequences. The deduced L. edodes β-MPP peptide sequence contains the inverse zinc-binding motif (H–X–E–H) that has been found in a large family of zinc-binding metalloproteinases including bacterial proteinases, insulin degrading enzymes and β-MPPs. The two histidines are thought to contribute two of the three residues for zinc binding. The expression of L. edodes β-MPP is higher during the development of the fruiting bodies, suggesting that higher mitochondrial activities may be required to meet the energy demand in the rapid growth of the fruiting bodies.     

Cloning of the gene encoding the catalytic subunit of casein kinase II from the yeast Yarrowia lipolytica

Casein kinase II from the yeast Yarrowia lipolytica is a heterotetramer of the form αα′β₂. We report on the cloning and sequencing of a partial cDNA and of the complete genomic DNA coding for the catalytic α subunit of the casein kinase II from this yeast species. The sequence of the gene coding for this enzyme has been analyzed. No intron was found in the gene, which is present in a single copy. The deduced amino acid sequence of the gene shows high similarity with those of α subunit described in other species, although, uniquely, Y. lipolytica CKIIα lacks cysteines. We find that the α subunit sequence of Y. lipolytica CKII is shown greater homology with the corresponding protein from S. pombe than with that from S. cerevisiae. We have analyzed CKIIα expression and CKIIα activity. We show that expression of this enzyme is regulated. The catalytic subunit is translated from a single mRNA, and the enzyme is present at a very low level in Y. lipolytica, as in other yeasts.     

Cloning, sequencing, and mapping of an alpha-actinin gene from the nematode Caenorhabditis elegans.

The dense-bodies in the body wall muscle of the nematode Caenorhabditis elegans function to anchor the actin thin filaments to the adjacent sarcolemma. One of the major components of the dense-bodies is the actin-binding protein alpha-actinin. To facilitate a genetic analysis of alpha-actinin, we have cloned a cDNA encoding the nematode protein, identified its position on the nematode physical map, and developed a unique PCR based approach to test the position of the cloned gene relative to known genetic deletions. The peptide sequence deduced from the cDNA shows that, apart from a few exceptional regions, the nematode protein shows strong similarity to other known alpha-actinins. Its position on the genetic map shows that none of the known muscle affecting mutations identified in C. elegans are in this alpha-actinin gene. This gene has been given the name atn-1 (alpha-actinin-1).     

Cloning of the gene encoding the catalytic subunit of casein kinase II from the yeast Yarrowia lipolytica

Casein kinase II from the yeast Yarrowia lipolytica is a heterotetramer of the form αα′β₂. We report on the cloning and sequencing of a partial cDNA and of the complete genomic DNA coding for the catalytic α subunit of the casein kinase II from this yeast species. The sequence of the gene coding for this enzyme has been analyzed. No intron was found in the gene, which is present in a single copy. The deduced amino acid sequence of the gene shows high similarity with those of α subunit described in other species, although, uniquely, Y. lipolytica CKIIα lacks cysteines. We find that the α subunit sequence of Y. lipolytica CKII is shown greater homology with the corresponding protein from S. pombe than with that from S. cerevisiae. We have analyzed CKIIα expression and CKIIα activity. We show that expression of this enzyme is regulated. The catalytic subunit is translated from a single mRNA, and the enzyme is present at a very low level in Y. lipolytica, as in other yeasts. Received: 20 December1997 / Accepted: 19 June 1997     

Cloning and characterization of antioxidant enzyme methionine sulfoxide-S-reductase from Caenorhabditis elegans

Methionine (Met) residues in proteins are susceptible to oxidation. The resulting methionine sulfoxide can be reduced back to methionine by methionine sulfoxide-S-reductase (MsrA). The MsrA gene, isolated from Caenorhabditis elegans, was cloned and expressed in Escherichia coli. The resultant enzyme was able to revert both free Met and Met in proteins in the presence of either NADPH or dithiothreitol (DTT). However, approximately seven times higher enzyme activity was observed in the presence of DTT than of NADPH. The enzyme had an absolute specificity for the reduction of l-methionine-S-sulfoxide but no specificity for the R isomer. K(m) and k(cat) values for the enzyme were approximately 1.18 mM and 3.64 min(-1), respectively. Other kinetics properties of the enzyme were also evaluated.     

Cloning and characterization of a Caenorhabditis elegans cDNA encoding a new insulin/IGF-like peptide

A Caenorhabditis elegans cDNA encoding a new insulin/IGF-like peptide was cloned and examined. The predicted peptide shows significant sequence similarity with the peptide Ceinsulin-1 reported previously and contains a characteristic insertion consisting of three residues in the putative B domain as with the Ceinsulin-1. The gene expression pattern during development is almost identical to that of Ceinsulin-1. The predicted tertiary structure of the peptide is quite similar to that of Ceinsulin-1, and their predicted receptor-recognition surfaces also closely match. These facts suggest that both peptides could recognize the same receptor.     

Cloning of a yolk protein gene family from Caenorhabditis elegans

A novel family of large, imperfectly repeated DNA sequences has been found in Escherichia coli. Two members of this family, rhsA and rhsB, occur as direct repeats, flanking the pit glyS xyl segment of the chromosome. Unequal sister-chromatid crossing over between rhsA and rhsB accounts for the frequent tandem duplication of the glyS locus that has been observed by various workers. This unequal recombination is recA-dependent. The rhsA locus is operationally defined as the segment between xyl and mtl that is repeated at other chromosomal locations. Using this definition, rhsA extends minimally 5500 base-pairs; 3800 base-pairs of rhsA are sufficiently homologous to rhsB to form an S1 nuclease-resistant heteroduplex with it. The rhsA sequence also exhibits internal repetition. At least one additional rhs sequence occurs in the E. coli chromosome unlinked to either rhsA or rhsB. Southern analysis of restriction digests of genomic DNA from E. coli strains C and B/5 showed that both of these strains have rhs hybridizable patterns similar to strain K-12, but the rhs sequence is absent in Salmonella typhimurium. The function of the rhs sequences has not been discovered. In the course of this work we developed a technique, termed "transductional walking", by which chromosomal DNA adjacent to a previously cloned DNA segment can be cloned through genetic procedures.     

Cloning and characterization of the gene encoding Aspergillus nidulans DNA topoisomerase II.

We have determined the complete nucleotide sequence of a 5544bp genomic DNA fragment from Aspergillus nidulans that encodes DNA topoisomerase II (topo II). It contains a single open reading frame of 4740bp that codes for 1579 amino acid residues with a molecular weight of 178kDa; when expressed in Escherichia coli and Saccharomyces cerevisiae the molecular weight was 180kDa. The gene (TOP2) is divided into three exons. Two introns, 54bp and 60bp in length, are located at nucleotide positions 187 and 3214 respectively. Comparison of the deduced amino acid sequence with other eukaryotic topo II sequences showed a higher degree of identity with other fungal enzymes than the human topo IIalpha. One of monoclonal antibodies raised against human topo II, 6H8, can cross-react with Aspergillus topo II.