Mouse t haplotype-specific double insertion of B2 repetitive sequences in the Tcp-1 intron 7.
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Animals↗
Biomedical subjects
A Matsushiro
Publications and source records attributed to A Matsushiro.
Expression and secretion of an Arthrobacter dextranase in the oral bacterium Streptococcus gordonii.
We have constructed a plasmid to express and secrete dextranase in the oral bacterium Streptococcus gordonii. The dextranase gene from Arthrobacter sp. strain CB-8 was linked to a promoter and a DNA sequence encoding the signal peptide of Streptococcus downei glucosyltransferase I (gtfI) followed by the Escherichia coli rrnBt1t2 terminator and inserted in the shuttle vector pVA838. S. gordonii transformed with this plasmid (pMNK-4) expressed and secreted mature Arthrobacter dextranase. The transformant was found to repress the firm adherence of water-insoluble glucan in a coculture experiment with cariogenic bacteria, Streptococcus sobrinus, in the presence of sucrose. Such genetically engineered oral bacteria could provide a therapy to prevent dental caries.
Amino Acid Sequence↗
Cloning of a cDNA encoding the Tcp-1 (t complex polypeptide 1) homologue of Arabidopsis thaliana.
We isolated a plant homologue of Tcp-1 (t complex polypeptide 1) cDNA from Arabidopsis thaliana. It encodes a 545-amino acid (aa) protein, which has extensive aa and nucleotide sequence homology to the corresponding proteins of mouse, yeast, fruit fly and archaebacteria.
Amino Acid Sequence↗
Structure and expression of the gene encoding mouse t-complex polypeptide (Tcp-1).
The nucleotide (nt) sequence of the structural gene (Tcp-1) encoding mouse t-complex polypeptide 1 (TCP-1) has been determined. The nt sequence extending to 10,043 bp shows that the Tcp-1 gene is divided into 12 exons, 11 introns and 5'- and 3'-flanking regions. The Tcp-1 gene has a tight cluster of major transcription start points (tsp). Two GC boxes, one CCAAT box and some other possible regulatory elements are located in the region upstream from the tsp, but no TATA box was found. Extending from the 5'-flanking region to the first intron, a CpG dinucleotide-rich cluster is located. In addition, Tcp-1 gene transcripts in mouse organs, embryos and cultured cells were analyzed by Northern blotting. The Tcp-1 mRNA is enriched not only in testes, but also in early post-implantation embryos and some cultured cell lines, as compared with mouse organs other than the testis. The amount of Tcp-1 mRNA in embryos decreases during development. These results suggest that the expression of the Tcp-1 gene may be regulated spatially and temporally in embryonic and adult mice by transcriptional control or by mRNA stability.
Amino Acid Sequence↗
Nucleotide sequence of a mouse Tcp-1 pseudogene: a nucleotide record for a t complex gene carried by an ancestor of the mouse.
We have isolated clones of a processed pseudogene of mouse t complex polypeptide 1 (Tcp-1) and determined the nucleotide sequence of the pseudogene. The pseudogene was 1363 bp long and had no intron. The Tcp-1 pseudogene had 88.4% or 88.3% nucleotide identity to the mouse Tcp-1 cDNA of wild-type (Tcp-1b) or t haplotype (Tcp-1a), and 87.5% identity to the rat Tcp-1 cDNA. On 12 nucleotide positions where the open reading frames (ORFs) of mouse Tcp-1b and Tcp-1a cDNAs have bp substitutions, the Tcp-1 pseudogene had 6 bp identical to Tcp-1b, 5 bp identical to Tcp-1a and 1 bp not identical to neither. On ten amino acid positions where TCP-1B and TCP-1A polypeptides have substitutions, deduced amino acids of the Tcp-1 pseudogene had four amino acids identical to TCP-1B, five amino acids identical to TCP-1A and one amino acid identical to neither. These results suggest that the ancestral mouse Tcp-1 gene would have had no significant difference between the resemblance to Tcp-1b and that to Tcp-1a before they were diverged and that amino acids of TCP-1B and TCP-1A would have been substituted in similar high rates.
Amino Acid Sequence↗
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↗
Nucleotide sequence of mouse Tcp-1a cDNA.
We have isolated complete cDNA clones encoding the mouse t-complex polypeptides 1A and 1B (TCP-1A and TCP-1B) from t-haplotype and wild-type (wt) mice, respectively. The complete nucleotide (nt) sequence of the Tcp-1a cDNA was determined. The Tcp-1a cDNA has an open reading frame (ORF) encoding a 60-kDa protein of 556 amino acids (aa). A comparison of nt sequences between the Tcp-1a and Tcp-1b cDNAs revealed that the 1786-bp regions upstream from their polyadenylation signals differed by 17 substitutions and that Tcp-1a had different polyadenylation sites from Tcp-1b. In these ORFs, 15 bp were substituted between the two alleles, occurring in 14 codons and resulting in eleven single-aa substitutions. Among these 15 substitutions, twelve were nonsynonymous (aa change) and three were synonymous (no aa change). The aa substitution in TCP-1 has occurred at least 20 times faster between t-haplotype and wt than between mouse and human or mouse and Drosophila.
Amino Acid Sequence↗
Sequence of EndoA gene encoding mouse cytokeratin and its methylation state in the CpG-rich region.
A genomic clone obtained from mouse liver DNA using a mouse cytokeratin EndoA cDNA probe revealed the complete sequence of the EndoA gene. The gene is divided into nine exons and the exon-intron pattern has been conserved compared to that of other type-II cytokeratin-encoding genes. The 5' upstream, 3' downstream and first and third introns contain potential regulatory sequences, including polyoma virus enhancer motifs (PEA1 and PEA3) and AP-1 elements. The 5' regions upstream of the EndoA, EndoB and Ck8 genes contain homologous sequences surrounding the TATA boxes. In addition, a CpG dinucleotide cluster region was located around the first exon. This CpG cluster region was found to be hypomethylated in endodermal PYS-2 cells, retinoic acid-treated F9 cells, and F9 embryonal carcinoma cells, but hypermethylated in BALB/C 3T3 fibroblast cells that do not express EndoA. These findings may provide a clue to understanding the molecular mechanisms of EndoA gene expression.
Animals↗
The promoter of the endo A cytokeratin gene is activated by a 3' downstream enhancer.
Mouse cytokeratin EndoA is an intermediate filament subunit of the type II cytokeratin class which initiates expression in trophectoderm cells of blastocyst during embryogenesis. To identify the regulatory elements of the endo A gene, we constructed a series of CAT expression vectors and transfected them into PYS-2 cells. We found an enhancer element locating 1 kb downstream from the endo A gene which acts on both the endo A and SV40 promoters. This enhancer consists of six direct repeated sequences with homology to the PEA3 motif in polyoma virus alpha enhancer core. In undifferentiated F9 embryonal carcinoma cells, expression of the construct containing the enhancer was not detected. These results indicate that one of the regulatory mechanisms of endo A gene expression is the 3' downstream enhancer.
Animals↗
Polyadenylated and 3' processed mRNAs are transcribed from the mouse histone H2A.X gene.
We have isolated a cDNA clone encoding a mouse histone H2A.X from a cDNA library of teratocarcinoma F9 cells. The predicted amino acid sequence of this clone is 97% identical to human histone H2A.X. The first 119 residues of the mouse H2A.X were very similar (96-97%) to those of the major H2A histones (H2A.1 and H2A.2) of mouse and the long carboxy terminal sequence of H2A.X was homologous with those of several lower eukaryotes. Northern blot analysis revealed that this cDNA hybridized with two mRNAs in different sizes, 0.5 kb and 1.4 kb. The two mRNAs were present in tissue culture cells, and in spleen, thymus and testes of mice, but the ratio of abundance of the two transcripts differed in different cells and tissues. The shorter mRNA contained the highly conserved palindromic sequence typical of the 3' end of replication-dependent histone genes. The amount of this transcript was coupled to DNA synthesis and rapidly decreased in culture cells. It was synthesized just after the beginning of S-phase and degraded just after the end of S-phase. On the other hand, the longer mRNA was polyadenylated at 0.9 kb downstream from the palindromic sequence. This transcript was very stable when compared with the shorter one. These results indicate that these two mRNAs are transcribed from a single gene and maintained differently during the cell cycle, perhaps to maintain a partially replication-dependent level of histone H2A.X.
Amino Acid Sequence↗
Gene expression of cytokeratin endo A and endo B during embryogenesis and in adult tissues of mouse.
We have examined the pattern of gene expression of mouse cytokeratin endo A and endo B during postimplantational development and in adult organs by Northern blot and in situ hybridization analyses. Both mRNAs localized in the ectoplacental cone, trophoblastic giant cells surrounding the parietal yolk sac, trophoblast cells in placenta, visceral yolk sac, and simple epithelium of the embryo during postimplantational development and in simple or transitional epithelial tissues in adult organs. These results indicate that endo A and endo B are coexpressed and may play some roles in these tissues.
Aging↗
Molecular cloning and nucleotide sequencing of the Arthrobacter dextranase gene and its expression in Escherichia coli and Streptococcus sanguis.
A bacterial strain, which assimilated dextran and water-insoluble glucan produced by Streptococcus mutans, was isolated from soil. The bacterium produced and secreted potent dextranase activity, which was identified as Arthrobacter sp. and named CB-8. The dextranase was purified and some enzymatic properties were characterized. The enzyme efficiently decomposed the water-insoluble glucan as well as dextran. A gene library from the bacteria was constructed with Escherichia coli, using plasmid pUC19, and clones producing dextranase activity were selected. Based on the result of nucleotide sequencing analysis, it was deduced that the dextranase was synthesized in CB-8 cells as a polypeptide precursor consisting of 640 amino acid residues, including 49 N-terminal amino acid residues which could be regarded as a signal peptide. In the E. coli transformant, the dextranase activity was detected mostly in the periplasmic space. The gene for the dextranase was introduced into Streptococcus sanguis, using an E. coli-S. sanguis shuttle vector that contained the promoter sequence of a gene for glucosyltransferase derived from a strain of S. mutans. The active dextranase was also expressed and accumulated in S. sanguis cells.
Amino Acid Sequence↗
Phi 80 transcription antitermination complex: a 15 kDa host-coded protein interacts with the gene N transcription antitermination protein of bacteriophage phi 80.
We purified the transcription antitermination complex of phi 80 (phi 80-pN complex). This complex had a molecular weight of approx. 42,000, and the subunits of this complex could be separated by SDS polyacrylamide gel electrophoresis. The complex was composed of two different polypeptides, one was the phi 80 N gene product (phi 80-pN) with a molecular weight of approx. 12,000, and the other was a host-coded protein with a molecular weight of approx. 15,000. The densitometric analysis after SDS polyacrylamide gel electrophoresis showed that the molar ratio of two proteins was 2 to 1 (phi 80-pN: 15 kDa), and the complex was assumed to be a trimer. Bacteriophage phi 80 could grow in nusA, nusC, and nusE mutants of Escherichia coli, but failed to grow in a nusB mutant. The molecular weight of the nusB gene product is calculated to be 15,689 from the sequencing data. These results suggest that the partner of the N gene product is possibly a host-coded nusB gene product.
Bacterial Proteins↗
Identification of mouse endodermal cytoskeletal protein C (ENDOC) with cytokeratin No. 19.
Using in vitro transcription and translation systems, the synthesized protein products derived from murine cytokeratin no. 19 cDNA were immunoprecipitated by trophectodermal monoclonal antibody (TROMA)-3. Thus, murine trophectodermal cytoskeletal protein EndoC, an antigen for TROMA-3, is identical to murine cytokeratin no. 19.
Animals↗
Isolation of mutants showing temperature-sensitive cell growth from embryonal carcinoma cells: control of stem cell differentiation by incubation temperatures.
Embryonal carcinoma(EC) cells, the undifferentiated stem cells of teratocarcinomas, have many properties in common with pluripotent embryonic cells, and thus provide an excellent system for studying the early events involved in embryonic development and stem cell differentiation. We have isolated three novel mutants with temperature-sensitive(ts) cell growth that were able to differentiate at a non-permissive temperature for cell growth. These mutations affect the progression of the cell cycle, leading to the transient accumulation of cells in a specific phase, the S phase, of the cell cycle, which is likely to be the primary cause of stem cell differentiation of EC cells at non-permissive temperature. Isolation of these mutants strongly supports the notion that there is a close association between the inhibition of DNA synthesis and EC cell differentiation.
Animals↗
Molecular cloning and characterization of cDNA encoding mouse cytokeratin no. 19.
We have isolated cDNA clones encoding the mouse cytokeratin No. 19 (Ck 19) from an intestinal cDNA library using synthetic oligodeoxyribonucleotides as probes. We obtained four independent clones, which correspond to about 1.4-kb of ck19 cDNA. Nucleotide sequence analysis revealed that these cDNAs encode a protein of 44,541 Da composed of 403 amino acids (aa). The deduced aa sequence defines an alpha-helical central domain, and suggests that the protein lacks a C-terminal non-alpha-helical tail segment, characteristic of the human and bovine 40-kDa keratins (Ck19). The overall aa identity between mouse Ck19 and human and bovine Ck19 is very high, 82.7% and 82.4%, respectively. The coil-forming central domain of mouse Ck19 has 45-65% similarity to other type-I Ck polypeptides, while it displays only 20-30% similarity to type-II Ck polypeptides. Northern blot analysis showed that mouse ck19 mRNA is strongly expressed in adult intestine, stomach and uterus. Interestingly, it is expressed in a placental cell line and a retinoic acid-treated mouse teratocarcinoma cell line (F9), but not in a parietal yolk sac endoderm-like cell line (PYS-2). This pattern of expression is very similar to that for the mouse gene encoding extra-embryonic endodermal cytoskeletal protein C (EndoC), suggesting they may be the same.
Amino Acid Sequence↗
Expression of H-2K and H-2D genes by PYS-2 teratocarcinoma cells.
The parietal yolk-sac carcinoma cell line PYS-2, which has been described to synthesize a trace amount of H-2 antigens, was shown to express low levels of Kb- and/or Db-specific antigens by flow cytometric analysis. Further studies of H-2 cDNA clones from PYS-2 cDNA library revealed 8 out of 10 clones were identified as classical H-2Kb and 2 were H-2Db, which confirmed the result from the immunofluorescent analysis. However, we have not isolated Qa, Tla or other embryonic class I cDNAs from our PYS-2 library.
Animals↗
Nucleotide sequence and structure of the mouse cytokeratin endoB gene.
We report the isolation and nucleotide sequence (7878 bp) of a genomic clone encoding murine cytokeratin EndoB, a type-I intermediate filament protein that is expressed coordinately with EndoA in trophectoderm cells during blastocyst formation. This clone contains the complete endoB gene including the 5' upstream and 3' downstream flanking regions. Sequencing analysis reveals the endoB gene consists of seven exons and six introns. Although the positions of the six introns coincide exactly with those of other type-I cytokeratin genes, the endoB gene has lost the seventh intron, which resides in the sequences coding for tail domains of other type-I cytokeratins. The 5' upstream regions of endoA and endoB genes contain homologous sequences around the respective TATA-like boxes, suggesting their involvement in coordinate regulation of their transcription and/or post-transcription. Surprisingly, this clone contains at least four murine B1 repetitive sequences. One typical B1 repetitive sequence was recognized in the 5' upstream region, and the other three in the 3' flanking region.
Animals↗