PubMed Health⌕ Search

Biomedical subjects

M Fuke

Publications and source records attributed to M Fuke.

At least 19 recordsLinked to original sources
PubMed1989

High-level expression, purification, and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris.

Human tumor necrosis factor (TNF) alpha/cachectin was expressed in the methylotrophic yeast Pichia pastoris at high levels (greater than 30% of the soluble protein) by placing the TNF cDNA under the control of regulatory sequences derived from the alcohol oxidase gene. Batch fermentor cultures at cell densities of 50 and 85 g dry cell weight/L contained approximately 6 X 10(10) and 10(11) units/L TNF bioactivity (6 and 10 g/L TNF), respectively. TNF productivity of 0.108 g L-1 h-1 was obtained in the continuous mode on glycerol- and methanol-mixed feed at 25 g dry cell weight/L cell density. TNF contained in the yeast cell lysate was soluble, displayed full cytotoxic activity, and was recognized by antibodies prepared against TNF derived from Escherichia coli. TNF was purified to greater than 95% purity with greater than 75% recovery by using three sequential chromatographic steps with a coordinated effluent-affluent buffer scheme which allowed one eluate to also serve as the loading buffer for the succeeding column. The amino acid composition, NH2-terminal amino acid sequence, isoelectric point, and minimal molecular weight determined for TNF corroborated those properties predicted from the nucleotide sequence. Sedimentation data indicated that TNF in the native form is a compact trimer held by noncovalent interactions. Circular dichroic spectra of TNF resemble those of proteins with high beta structure. TNF exhibited cachectic activity on mouse 3T3-L1 cells at about the same equivalence as the cytotoxic activity toward mouse L929 cells. In the criteria examined, TNF derived from P. pastoris closely resembles TNF derived from recombinant E. coli and human HL-60 cells.

Animals↗
PubMed1987

Invertase gene (SUC2) of Saccharomyces cerevisiae as a dominant marker for transformation of Pichia pastoris.

A two-step method for the selection of transformants of prototrophic industrial strains of the methylotrophic yeast Pichia pastoris has been developed. This method is based on our observation that P. pastoris cannot use sucrose as the sole carbon source (Suc-) and that introduction of the invertase gene (SUC2) of Saccharomyces cerevisiae renders P. pastoris Suc+. P. pastoris was transformed with a plasmid which contains the SUC2 gene of S. cerevisiae and an autonomously replicating sequence PARS1 from P. pastoris. The transformants were initially allowed to regenerate on medium containing dextrose and the regenerated cells were pooled and plated on sucrose medium to screen for Suc+ transformants. It was shown that the Suc+ transformants of P. pastoris with the autonomously replicating plasmid were highly unstable with respect to the plasmid maintenance, even when grown on sucrose as the sole carbon and energy source. This high instability was attributed to an efficient cross-feeding by Suc- segregants on glucose and fructose generated due to hydrolysis of sucrose by the invertase enzyme secreted by Suc+ cells. Spontaneous integration of the plasmid DNA resulting in a stable Suc+ phenotype was also observed. However, stable Suc+ transformants were obtained more readily by integration of SUC2 into P. pastoris genome following transformation with a linearized plasmid with the ends homologous to P. pastoris HIS4 locus. All such integrants were completely stable for Suc+ phenotype after 20 generations of growth in a nonselective medium.

Animals↗
PubMed1986

N-band proteins of nucleolar organizers: chromosomal mapping, subnucleolar localization and rDNA binding.

The ribosomal DNA(rDNA)-containing chromatin in eukaryotes forms a unique architecture called the "secondary constriction" or "nucleolus organiser region (NOR)" on mitotic chromosomes. To gain more insight into non-histone chromosomal proteins (NHCP), termed "N-band proteins", that are specifically associated with the NOR in a wide variety of eukaryotes, we attempted to: identify the NHCP responsible for N-band staining; determine their stoichiometry; map them on metaphase chromosomes; determine their subnucleolar localization and examine their possible ability to bind rDNA. Based on several criteria, including chromosomal localization, solubility, association with chromatin, and intra-nuclear localization, two of the nucleolus-rich NHCP, termed component B of mol.wt 55,000 and component C of mol.wt. 41,000, were tentatively identified as N-band proteins. Immunological studies using a polyclonal, monospecific antibody raised against component C show that this protein is in fact associated with the chromosomal telomeres where NORs are located. In nucleoli, N-band proteins appear to be compartmentalized into a structure that probably corresponds to fibrillar components. Both components B and C are among several NHCPs that showed, under in vitro conditions, a strong affinity for rDNA cloned in lambda phage but not for calf thymus genomic DNA or phage vector DNA. The antibody against component C effectively suppressed in vitro transcription by RNA polymerase I in nucleoli and nucleolar chromatin. Component C appears to exist in the nucleus at 3.75-5.13 X 10(3) copies per rDNA unit or 0.09-0.13 copy per nucleotide. These findings support the hypothesis that the NOR is a chromosomal site, architecturally not only unique but also different from other chromatin regions in that constituent DNA, i.e., rDNA, is organized in a specific manner by interacting with specific NHCP, i.e., N-band proteins.

Animals↗
PubMed1985

Cloning and sequencing of a human 18S ribosomal RNA gene.

A clone containing an 18S ribosomal RNA (rRNA) gene has been isolated from a human genomic library constructed in lambda Charon 4A. This gene was sequenced and found to be 1868 bp long. The sequence divergencies in the human 18S rRNA gene and the previously sequenced mouse and rat genes are found in one G + C-rich region of 110 bp located in the 5' domain of the molecule. Except for this variable region, extensive homology exists among these three mammalian genes. Overall, the human 18S rRNA gene is 98.8% homologous with those of rat and mouse.

Animals↗
PubMed1984

Pseudogene IFN-alpha L: removal of the stop codon in the signal sequence permits expression of active human interferon.

Biologically active interferon (10(6)-10(7) units/liter) was produced in Escherichia coli from modified human alpha interferon (IFN-alpha) pseudogene L. IFN-alpha pseudogene L has a stop codon in the signal peptide coding region. The region that contains the stop codon was replaced with the corresponding region of another human IFN-alpha gene, WA, that does not have a stop codon and was previously engineered for expression by fusion to the M13mp11 lac promoter. The interferon L fusion product was induced with IPTG after infecting E. coli JM103 with the M13 bacteriophage that contained the modified human IFN-alpha pseudogene L. Hence, the IFN-alpha L mature interferon coding sequence, which is not identical to any other alpha-interferon gene, has been conserved for active interferon coding information.

Amino Acid Sequence↗
PubMed1984

Human genomic library screened with 17-base oligonucleotide probes yields a novel interferon gene.

A method is presented that has permitted a human genomic library to be screened for low-copy genes using 17-base synthetic oligonucleotides as probes. Parallel screening with two different 17-base probes permitted the unambiguous identification of clones containing interferon-alpha (IFN-alpha) genes. The isolated human IFN-alpha genes were sequenced, and one appears to be IFN-alpha L; the other is one not previously described, which we have designated IFN-alpha WA. The IFN-alpha WA sequence differs from those of IFN-alpha genes A-L at approximately equal to 10% of the positions and is most similar to IFN-alpha C, -alpha F, and -alpha H. IFN-alpha WA has been found to encode amino acids that differ from those conserved at each of five positions in all previously reported IFN-alpha species. The IFN-alpha WA gene codes for an active interferon, which has been expressed in Escherichia coli using an M13-lacZ fusion as an expression vector. About 5 X 10(6) units of IFN-alpha WA were obtained per liter of bacterial culture. The described screening procedure using short probes should permit the isolation of genes for which sequence information is available from animal or plant genomic libraries.

Base Sequence↗
PubMed1983

The complete nucleotide sequence of the rat 18S ribosomal RNA gene and comparison with the respective yeast and frog genes.

The complete nucleotide sequence of the rat 18S ribosomal RNA gene has been determined. A comparison of the rat 18S ribosomal RNA gene sequence with the known sequences of yeast and frog revealed three conserved (stable) regions, two unstable regions, and three large inserts. (A,T) leads to (G,C) changes were more frequent than (G,C) leads to (A,T) changes for three comparisons (yeast leads to frog, frog leads to rat, and yeast leads to rat). GC pairs were inserted preferentially over AT pairs for the same three comparisons. These two factors contribute to the progressively higher GC content of 18S ribosomal RNA of yeast, frog, and rat.

Animals↗
PubMed1983

Nucleotide sequence of the 5'-terminal region of rat 18S ribosomal DNA.

The 5'-terminal 597 base-pairs (bp) of the Sprague-Dawley rat 18S ribosomal RNA gee and 10 bp of the adjoining transcribed spacer have been sequenced. Previously sequenced 10 large oligonucleotides of rat 18S RNA were located in this region. This mammalian sequence has been compared with the known sequences of yeast and frog 18S rDNA's. The analysis indicates that 534 bp of the 597 bp (89%) are conserved between rat and frog sequences but only 75% of the nucleotides are conserved between rat and yeast in this region. Two large and two small sections have been identified where insertions have been introduced during evolution. Of these 58 bp long inserted sections of the rat rDNA sequence, 50 bp (86%) were G-C base-pairs.

Base Sequence↗
PubMed1981

Nucleotide sequence of the 3'-terminal region of rat 18S ribosomal DNA.

The 3'-terminal 230 base-pairs (bp) of the gene for 18S rRNA and 40 bp of the adjoining spacer have been sequenced for the Sprague-Dawley rat. This mammalian sequence has been compared with the known sequences of yeast, fruit fly, silkworm, and frog. This study has shown that the nucleotide-sequence differences between rat and frog are the smallest among and longer species, probably reflecting their evolutionary closeness and longer maturation time compared to the others. There is little similarity in the nucleotide sequences of the transcribed spacer regions of the five species compared.

Animals↗
PubMed1981

Characterization of cloned rat ribosomal DNA fragments.

Two Charon 4A lambda bacteriophage clones were characterized which contain all and part o the 18S ribosomal DNA of the rat. One clone contained two Eco RI fragments which include the whole 18S ribosomal RNA region and part of 28S ribosomal RNA region. The other clone contained an Eco RI fragment which covers part of 18S ribosomal RNA region. There were differences between the two clones in the non-transcribed spacer regions suggesting that there is heterogeneity in the non-transcribed spacer regions of rat ribosomal genes. The restriction maps of the two clones were compared to the restriction map of the cloned mouse ribosomal DNA. Eco RI, Hind III, Pst I, and Bam HI sites in 18S ribosomal RNA regions were in the same places in mouse and rat DNA but the restriction sites in the 5'-spacer regions were different.

Animals↗
PubMed1979

Comparison of nucleotide sequences of large T1 ribonuclease fragments of 18S ribosomal RNA of rat and chicken.

Nucleotide sequences of large T1 ribonuclease fragments of 18S ribosomal RNA of Novikoff rat ascites hepatoma cells and chicken lymphoblastoid cells were determined and compared. Among the 19 large T1 ribonuclease fragments examined of rat 18S ribosomal RNA, 12 fragments were found to be the same in chicken 18S ribosomal RNA. Three fragments of rat 18S ribosomal RNA were not found among large T1 ribonuclease fragments of chicken 18S ribosomal RNA. Four fragments of rat 18S ribosomal RNA were found to be changed in chicken 18S ribosomal RNA. All the changes were point mutations except the change in the largest T1 ribonuclease fragment 1 which is 21 nucleotides long. 2'-0-methylation at the center of the fragment was lost in chicken 18S ribosomal RNA; all the other nucleotides were the same.

Animals↗