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PubMed1994

Human casein kinase II: structures, genes, expression and requirement in cell growth stimulation.

Casein kinase II (CKII) is an ubiquitous Ser/Thr protein phosphotransferase in control of a variety of crucial cellular functions including metabolism, signal transduction, transcription, translation and replication. CKII levels are consistently higher in neoplastic tissues. The human CKII is composed of subunits alpha, alpha', and beta with molecular masses of 43, 38 and 28 kDa, respectively, that form heterotetrameric holoenzymes (alpha 2 beta 2; alpha alpha' beta 2, alpha'2 beta 2) showing autophosphorylation particularly at subunit beta and hence suspected to play a regulatory role. The amino acid sequences of subunits indicate high evolutionary conservation. Employing the complete set of tissue-derived (placenta) and recombinant (expressed in E. coli) subunits and CKII holoenzymes, the catalytic function of alpha and alpha' and the several-fold stimulation by beta is shown to occur comparably in tissue-derived and recombinant CKII and the autophosphorylation of beta is shown by site-directed mutagenesis to be not decisive for the tuning of CKII activity. The human genome contains two genes encoding CKII alpha. First, there is a processed (pseudo)gene which is 99% homologous to the CKII alpha cDNA and which possesses a promoter-like region adjacently upstream with TATA and CAAT boxes so that transcription cannot be excluded. Second, there is an active gene of which we have characterized so far a 18.9 kb long central fragment which contains 8 exons comprising bases 102-824 of the CKII alpha coding region. The gene fragment contains repetitive elements, most prominently 16 Alu repeats. The genome further contains one as yet uncharacterized CKII alpha' gene and one gene encoding CKII beta. The CKII beta gene has been characterized as a 4.2 kb spanning gene composed of seven exons which possesses three transcription start sites and the translation start site in the second exon. The first intron harbors an Alu repeat also. The promoter region of the CKII beta gene contains elements such as multiple GC boxes, a CpG island, and nonstandard-positioned CAAT boxes but lacks a TATA box thus characterizing the gene as a housekeeping gene. The CKII genes are not clustered at a certain chromosome but rather are distributed over the whole human genome. Using the genomic clones as the probes for in situ hybridization, the active CKII alpha gene was mapped to chromosome 20p13, the processed CKII alpha (pseudo)gene to chromosome 11p15, and the CKII beta gene to chromosome 6p21. (The CKII alpha' gene has been localized on chromosome 16 with a cDNA probe.).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Tissue distribution, elimination and metabolism of [3H]-leukotriene C4 in the American bullfrog, Rana catesbeiana.

Tissue distribution, elimination, and metabolism of [3H]-leukotriene C4 were studied at 2.5 hours after injection in the conscious and anesthetized American bullfrog, Rana catesbeiana. Conscious frogs were injected via the dorsal lymph sac or the sciatic vein. Anesthetized frogs were injected via the abdominal vein. The organs containing the greatest percent of injected radioactivity at 2.5 hours after injection were liver, small intestine and kidney. Route of injection and anesthesia appears to alter distribution and elimination of leukotrienes. [3H]-leukotrienes were eliminated into bladder water and bile. In addition, 7.8 +/- 2.2 and 5.2 +/- 2.5 percent of the injected radioactivity was found in the pan water bathing the ventral surface of the venously and dorsally injected conscious frogs, respectively, suggesting transfer of radioactivity across the skin. At 2.5 hours, polar metabolites represented 50% of the radioactivity found in liver, bile, and bladder water. These polar metabolites were determined to be 18-carboxy-19,20-dinor-leukotriene E4, 20-carboxy-leukotriene E4, and 20-hydroxy-leukotriene E4. Of the non-oxidized leukotrienes, bile contained mainly LTD4 while bladder water contained primarily LTE4. N-acetyl LTE4 was not detected in any samples. The tissue distribution, elimination and metabolism of leukotrienes in the bullfrog was similar to mammalian studies and suggests evolutionary conservation of leukotriene processing.

Animals↗
PubMed1984

Heterogeneous mitochondrial DNA D-loop sequences in bovine tissue.

Mitochondrial DNA from bovine tissue contains heterogeneous sequences located within an evolutionary conserved cytosine homopolymer sequence near the 5' end of the D-loop region. This part of the mammalian mitochondrial genome is known to contain the origin of heavy strand DNA synthesis and the major transcriptional promoter for each strand. Nucleotide sequence analysis of cloned DNA and electrophoretic analysis of appropriate small fragments from animal tissue reveal a population of length polymorphs containing from nine to 19 cytosine residues. No individual length species represents more than 40% of the population. These data imply a state of significant intraanimal mtDNA sequence heterogeneity, which most likely occurs intracellularly as well. The localization of variability to a homopolymer run suggests that replication slip-page generated the sequence population. We also report that when recombinant clones containing this region are repeatedly passaged in E. coli, they begin to regenerate length variation similar to that seen in animal mtDNA.

Animals↗
PubMed1990

The roles of Drosophila cyclins A and B in mitotic control.

We have cloned, sequenced, and characterized the expression of a Drosophila cyclin B gene. The independent evolutionary conservation of A- and B-type cyclins implies that they have distinct roles. Indeed, in mutant embryos deficient in cyclin A, cells that accumulate only cyclin B do not enter mitosis. Thus, in vivo, cyclin B is not sufficient for mitosis. Furthermore, we find that the two cyclins are coexpressed in all proliferating cells throughout development. Though lacking a formal demonstration that cyclin B is essential as it is in other organisms, we propose that each of these proteins fulfills a distinct and essential role in the cell cycle.

Amino Acid Sequence↗
PubMed1992

A mammalian homolog of SEC61p and SECYp is associated with ribosomes and nascent polypeptides during translocation.

SEC61p is essential for protein translocation across the endoplasmic reticulum membrane of S. cerevisiae. We have found a mammalian homolog that shows more than 50% sequence identity with the yeast protein. Moreover, several regions of SEC61p have significant similarities with corresponding ones of SecYp of bacteria, indicating a strong evolutionary conservation of the mechanism of protein translocation. Mammalian Sec61p, like the yeast protein, is located in the immediate vicinity of nascent polypeptides during their membrane passage. It is tightly associated with membrane-bound ribosomes, suggesting that the nascent chain passes directly from the ribosome into a protein-conducting channel. These results define Sec61p as a ubiquitous key component of the protein translocation apparatus.

Amino Acid Sequence↗
PubMed1995

Molecular characterization of a human V lambda VIII germline gene.

Human lambda light chains of the recently recognized variable region (VL) subgroup V lambda VIII can be distinguished from proteins of other V lambda gene families on the basis of distinctive chemical and serologic properties and by their preferential association with certain types of autoantibodies, i.e. rheumatoid factors (RFs). We now report that we have cloned from a human placental library a V lambda VIII-encoding germline gene, designated IGLV8A1, using as a molecular probe a partial V lambda VIII fragment generated by polymerase chain reaction (PCR) from genomic DNA. IGLV8A1 contained all the requisite elements of a potentially functional gene, including a V lambda exon with an open reading frame encoding 103 residues. Its expressed products were identified through analyses of cDNA cloned from two different monoclonal lambda VIII B-cell populations. The primary structure of lambda VIII light chains differed from that of lambda I, lambda II, lambda III, lambda IV and lambda VI proteins by the presence of distinctive residues within the first framework region (FR1) and an 11- rather than 7-residue second complementarity-determining region (CDR2). Remarkably, the IGLV8A1 gene was more homologous to the two functional rabbit V lambda germline genes, RV lambda 2 and RV lambda 3 (including the presence of one extra codon within the leader sequence), and to the murine V lambda x gene. Light chains encoded by the human, rabbit and mouse lambda VIII-related genes shared certain unique primary structural features, notably the four additional CDR2 residues. The evolutionary conserved nature of the human V lambda gene and, in particular, the apparently novel tertiary structural effects induced by an elongated CDR2 provide evidence for the biological and functional importance of the V lambda VIII subgroup.

Amino Acid Sequence↗
PubMed1986

Immunological classification of a pike lymphoma.

Monoclonal and polyclonal antibodies against mammalian intermediate filament (IF) proteins were used to demonstrate IF expression in tissues and lymphoma tumors of northern pikes, Esox lucius L., from the Aland Islands of Finland by indirect immunofluorescence microscopy. Frozen sections of pike tissues demonstrated IFs in a manner confirming their evolutionary conservation and subclass specificity. Tumor cells showed morphological resemblance to head kidney cells and were positive for vimentin while negative for cytokeratin, desmin, neurofilament proteins, and glial fibrillary acidic protein. The results show that the neoplasm is a mesenchymal as opposed to an epithelial, muscle, neural, or glial tumor, and is probably hemic cell derived. A rabbit anti-pike IgM antiserum showed that up to 90% of mononuclear (MN) cells isolated on Ficoll-Isopaque gradients from peripheral blood, spleen and head kidney were surface- and cytoplasmic-immunoglobulin positive by indirect immunofluorescence, while a maximum of 5% of tumor cells were positive. A maximum of 5% of MN cells from hemic tissues exhibited rosettes when incubated with AET-treated sheep red blood cells; however, only 1% of cells in the tumor formed rosettes. Lymphocyte proliferation assays were performed on MN cells from hemic tissues and tumor using phytohemagglutinin P, concanavalin A, tuberculin purified protein derivative and lipopolysaccharide W in medium supplemented with fetal calf serum or autologous pike plasma. Proliferation indices in hemic tissues were similar in the groups. However, all proliferation indices in tissues were significantly higher than corresponding values in the tumor. These assays show that pike MN cells respond when stimulated by T and B cell mitogens, but that this reactivity is lacking in the tumor.

Animals↗
PubMed1995

Mapping of the linear antigenic determinants from the Leishmania infantum histone H2A recognized by sera from dogs with leishmaniasis.

Antibodies reacting against the H2A histone protein were frequently observed in the sera from dogs naturally infected with the protozoan parasite Leishmania infantum. Using synthetic peptides covering the complete sequence of the protein we have identified the amino terminal region, comprising from amino acids 1 to 20, and the carboxyl terminal region, comprising from amino acids 106 to 132, as conforming the antigenic determinants of the protein. Those regions, exposed in the nucleosome surface, are highly divergent in sequence relative to the mammalian H2A histones. The anti-H2A histone antibodies present in the sera of these dogs specifically recognize the L. infantum H2A histone and they do not react with mammalian histones. The present data indicate that, in spite of the evolutionary conservation of the H2A histone protein among eukaryotic organisms, the humoral response against this protein during natural infection is specifically triggered by the parasite protein antigenic determinants.

Amino Acid Sequence↗
PubMed1986

Assignment of a human DNA-repair gene associated with sister-chromatid exchange to chromosome 19.

The Chinese hamster ovary (CHO) cell mutant, EM9, is defective in rejoining strand breaks, hypersensitive to chlorodeoxyuridine (CldUrd), and has a high frequency of sister-chromatid exchange (SCE). Somatic cell hybrids constructed from fusion of EM9 cells with normal human lymphocytes and fibroblasts, and selected in CldUrd, extensively segregate human chromosomes but preferentially retain markers of human chromosome 19. The SCE frequency in the hybrid clones is low as in normal CHO cells, but in CldUrd-sensitive subclones, which lose the human chromosome 19 markers, SCE frequencies return to mutant levels. We therefore assign a human gene designated repair complementing defective repair in Chinese-hamster (RCC) to chromosome 19. Since this is the second (of two) human genes complementing repair-deficiency mutations in CHO cells assigned to the 19, the assignment and organization of DNA-repair genes is discussed in the light of hemizygosity in CHO cells and the evolutionary conservation of mammalian linkage groups.

Animals↗
PubMed1980

Kinetoplast DNA and RNA of Trypanosoma brucei.

Kinetoplast DNA (kDNA) and kinetoplast RNA (kRNA) were isolated from bloodstream and procyclic culture forms of two clonal strains of Trypanosoma brucei. No differences were observed in kDNA (maxicircle) restriction profiles between bloodstream or procyclic culture forms of the same strain. Some differences were observed in kDNA maxicircle restriction sites between the two strains. Buoyant density analysis of Pst I digested kDNA showed the release of a minor low density band representing unit length linearized maxicircle DNA. Pst I or Bam H1-linearized maxicircle DNA was isolated by the Hoechst 33258 dye--CsCl method and a restriction enzyme map of the maxicircle was constructed. Closed monomeric minicircles released from kDNA networks by sonication sedimented with a molecular size of around 1100 base pairs. A substantial minor length heterogeneity was evident in acrylamide gel electrophoresis of once cut minicircles. Several minicircle sequence classes and two Hind III maxicircle fragments representing approx. 50% of the maxicircle were cloned in the bacterial plasmid, pBR322, in Escherichia coli. A purified kinetoplast-mitochondrion fraction was isolated from procyclic culture forms by the Renografin flotation method. The major kRNA components were two small RNAs which comigrated with Leishmania tarentolae 9 and 12 S kRNAs in denaturing gels. These RNAs hybridized to the maxicircle component of the kDNA, specifically to the smaller cloned Hind III maxicircle fragment. This cloned fragment had substantial sequence homology with the cloned maxicircle fragment from L. tarentolae which contains the 9 and 12 S RNA genes, implying an evolutionary conservation of the 9 and 12 S gene sequences. Identical kRnAs were observed in cultured bloodstream forms of T. brucei.

Animals↗
PubMed1984

Autonomous replication sequences in the maxicircle kinetoplast DNA of Leishmania tarentolae.

Four fragments from the maxicircle DNA of Leishmania tarentolae cloned into the selectable Saccharomyces cerevisiae shuttle vector, YIp5, exhibited autonomous replicating sequence (ars) activity. Two of the fragments (pSK120, pSK152) produced large yeast transformant colonies and two (pSK30, pSK150) produced small colonies. All yeast transformants contained extrachromosomal self replicating YIp5 hybrid plasmids as shown by mitotic instability in non selective medium and by the transformation of Escherichia coli with yeast minilysates and recovery of the plasmid from the transformed bacteria. The copy numbers of pSK30, pSK150 and pSK152 in the transformed yeast were approximately the same as that of the YRp12 control, which contains the yeast arsl element; the copy number of pSK120, however, was at least 10 fold lower. A 1.87 kb subfragment of the pSK120 fragment also showed strong ars activity. The entire DNA sequences of the pSK120, pSK152 and pSK150 fragments are known, and several yeast 11 mer consensus ars sequences are present within each fragment. In addition there is a sequence (Lt ars 189) within the pSK152 subclone that has 78% similarity with a 189 nt sequence of an ars element from the Crithidia fasciculata maxicircle (Cf ars 189), implying an evolutionary conservation of this putative origin of replication in at least two different kinetoplastid species. The relative positions of the Lt ars 189 sequence in the L. tarentolae maxicircle map and the Cf ars 189 sequence in the C. fasciculata map with respect to the 9 and 12 S ribosomal genes are similar, implying an overall conservation of gene order in this portion of the transcribed regions of these two species and perhaps in all kinetoplastid species.

Animals↗
PubMed1993

Proteolytic processing of neuropeptide Y and peptide YY by dipeptidyl peptidase IV.

Neuropeptide Y, peptide YY and pancreatic polypeptide share an evolutionary conserved proline-rich N-terminal sequence, a structure generally known to be inert to the attack of common proteinases, but a potential target for specialized proline-specific aminopeptidases. Purified human dipeptidyl peptidase IV (also termed CD 26) liberated N-terminal Tyr-Pro from both, neuropeptide Y and peptide YY, with very high specific activities and Km values in the micromolar range, but almost no Ala-Pro from pancreatic polypeptide. Other proline-specific aminopeptidases exhibited low (aminopeptidase P, liberation of N-terminal Tyr) or totally no activity (dipeptidyl peptidase II), as was also observed with less-specific aminopeptidases (aminopeptidase M, leucine aminopeptidase). When human serum was incubated with neuropeptide Y or peptide YY at micro- and nanomolar concentrations, Tyr-Pro was detected as a metabolite of both peptides. Formation of Tyr-Pro in serum was blocked in the presence of Lys-pyrrolidine and diprotin A (Ile-Pro-Ile), specific, competitive inhibitors of dipeptidyl peptidase IV. Incubation of neuropeptide Y or peptide YY with immunocytochemically defined, cultivated endothelial cells from human umbilical cord also yielded Tyr-Pro. Dipeptidyl peptidase IV could be immunostained on most endothelial cells by a specific antibody. We suggest that dipeptidyl peptidase IV might be involved in the degradation of neuropeptide Y and peptide YY to N-terminal truncated neuropeptide Y(3-36) and peptide YY(3-36). Since specific binding to Y1, but not to Y2 subtype of neuropeptide Y/peptide YY receptors requires intact N- as well as C-termini of neuropeptide Y and peptide YY, removal of their amino-terminal dipeptides by dipeptidyl peptidase IV inactivates them for binding to one receptor subtype.

Amino Acid Sequence↗
PubMed1987

Monkey (CV-1) mitochondrial DNA contains a unique triplication of 108 bp in the origin region.

A fragment of monkey kidney cells (CV-1) mitochondrial DNA (mtDNA) containing the origin of replication has been cloned and sequenced. The nucleotide sequence, 640 bp, extends from the coding sequence of phenylalanyl tRNA to the flanking sequence upstream of the origin region. A unique triplication of 108 bp, including the evolutionary conserved sequence CSB-3, was found. Comparison between the origin regions of monkey, human and mouse mtDNA is presented.

Animals↗
PubMed1991

Isolation and characterization of the canine thyroglobulin gene promoter region.

The 5' flanking sequences from the canine thyroglobulin gene were isolated by homology screening with the evolutionary conserved sequence from the bovine thyroglobulin promoter and sequenced. Transient expression in primary cultured dog thyrocytes demonstrated that the canine clone contains a functional promoter inducible by cAMP. DNAse I footprinting assays showed that the thyroid-specific transcription factor TTF-1, purified from bovine thyroid, also recognizes the canine thyroglobulin promoter. Similar footprints were obtained with crude nuclear extracts from primary cultured dog thyrocytes.

Animals↗
PubMed1991

Cloning of cDNA encoding rat TCP-1.

We have isolated and sequenced a cDNA encoding a rat homolog of the mouse t-complex polypeptide 1 (TCP-1). Its deduced gene product is a polypeptide of 556 amino acids, with a predicted Mr of 60,341. The similarity between mouse Tcp-1 and the rat homolog is about 94.0% at the nucleotide level and 97.1% at the amino acid level showing the evolutionary conservation of this protein. The similarity of the amino acid sequence of the rat TCP-1 is not significantly biased to any of those from wild (TCP-1B) or from t-haplotype mice (TCP-1A). From a comparison of deduced amino acid sequences of eukaryotic TCP-1 proteins, we found highly conserved domains. Southern blot analysis revealed that there are at least two similar sequences to Tcp-1 in the rat, one is a structural gene and the other seems to be a processed pseudogene.

Amino Acid Sequence↗
PubMed1994

Organization of the bovine gene encoding the endothelial nitric oxide synthase.

The bovine endothelial nitric oxide synthase gene plus 2.9 kilobases of 5'-flanking sequence has been isolated and characterized. The gene spans 20 kilobases and contains 26 exons and 25 introns. Two transcription start sites have been determined by primer extension analysis which are located 170 and 240 base pairs upstream, respectively, from the methionine translational initiation codon. Evidence supporting the upstream boundary region for transcriptional initiation was also obtained by reverse transcription-polymerase chain reaction. The 5'-flanking region lacks a typical TATA box but contains numerous putative transcription factor binding sites. These include consensus sequences for an AP-1 site, an NF-1 site, a tumor necrosis factor responsive element, two sterol regulatory elements, 3 acute-phase response element, two sterol regulatory elements, 3 acute-phase response elements, 6 GATA motifs, 16 CACCC boxes, 5 Sp1 sites, 15 estrogen half-palindromic motifs, and 9 fluid shear stress-responsive elements. The isolated gene promoter directs basal transcription of a luciferase reporter gene when transiently transfected into bovine aortic endothelial cells. High sequence homology of the promoter region to the human endothelial nitric oxide synthase gene promoter (75% nucleotide identity in 1.6 kilobases of 5'-flanking sequence) suggests evolutionary conservation of transcriptional regulation. Isolation and characterization of the bovine endothelial nitric oxide synthase gene should facilitate further investigation of mechanisms by which gene expression is regulated.

Amino Acid Oxidoreductases↗
PubMed1995

Correlation between codon usage, regional genomic nucleotide composition, and amino acid composition in the cytochrome P-450 gene superfamily.

The codon usage bias of 110 mammalian cytochrome P-450 genes has been determined and analyzed in relation to a variety of genetic, biochemical, and physiological parameters. In those P-450 genes exhibiting biased usage the preferred codons generally do not differ among the four species examined (rat, rabbit, man, and mouse) or from the predominantly used codons identified for all sequenced genes in a recent data base analysis (Wada et al. (1992) Nucleic Acids Res. 20 (Suppl.), 2111-2118). Codon usage bias does not correlate with evolutionary relationships, evolutionary age, or with the extent of evolutionary conservation of orthologous proteins; there is no obvious correlation with the level of expression of a given P-450, with its inducibility, nor with its physiologic role; and neither the preferred codons nor the degree of bias differ for P-450s expressed in different tissues. Codon usage bias does correlate with the C+G content at the codon third position, and thus preferred codons usually end in C or G; for those P-450s for which gene sequences are available this bias also correlates with the C + G content of the intronic and flanking regions of these genes. Moreover, a lesser increase in the C + G content at the codon first and second positions is also evident in genes located in regions of high C + G content; this leads to predictable differences in the amino acid compositions of P-450 enzymes that correlate with genomic nucleotide composition and the degree of bias in codon usage.

Amino Acids↗
PubMed1980

Immunochemical characterization of neurotensin-like peptides in chicken.

Using radioimmunoassay and 3 region specific antisera toward bovine neurotensin (NT), the NT-like peptides in chicken have been shown to differ from NT but to strongly resemble its COOH-terminal region. Three substances were identified, one of which resembled NT biologically and appeared to share 7 or 8 of its COOH-terminal residues. The two other peptides were smaller than NT but seemed to possess 4-6 residue homologies with it. Tissue distribution studies indicated that the chicken pancreas and thymus had unusually high levels of this material (greater than 200 fold than in rat) and that the 3 substances were distributed differently in tissues. Chromatographic studies showed that the peptides obtained form brain, intestine, thymus, and pancreas were similar. These results, demonstrating evolutionary conservation of the COOH-terminal region of NT, are in keeping with the known importance of this region for biological activity. These finding also suggest the exstence of an NT-family of peptides serving multiple biological roles.

Amino Acid Sequence↗