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Biomedical subjects

K Itakura

Publications and source records attributed to K Itakura.

At least 127 records · Page 7Linked to original sources
PubMed1982

Isolation of a cDNA clone for the murine transplantation antigen H-2Kb.

A library of cloned cDNAs constructed from the poly(A)+RNA of the murine thymoma cell line EL4 (b haplotype) was screened with a probe encoding a short region of the H-2Kb transplantation antigen. One of the clones isolated, pH202, contains a region that can code for a transplantation antigen with an amino acid sequence 98% homologous to that previously published for H-2Kb. Based on this high degree of homology, pH202 appears to encode the H-2Kb antigen from amino acid 66 through the carboxy terminus, including 386 nucleotides of 3'-untranslated sequence. The amino acid sequence deduced from pH202 suggests that the H-2Kb antigen is actually 2 amino acids longer than previously reported (a total of 348 residues). Four other differences in amino acid assignments are seen. Analysis of the DNA sequences of pH202 and other H-2 clones previously described in the literature suggests that alternative routes of splicing at the 3' end of the coding region are involved in the production of different transplantation antigen mRNAs.

Animals↗
PubMed1982

Role of positive charge on the amino-terminal region of the signal peptide in protein secretion across the membrane.

The positively charged amino-terminal region of the signal peptide has been proposed to have an important role at an initial step of protein secretion across the membrane (loop model). To test this hypothesis, the charge on the amino-terminal region of the signal peptide of the prolipoprotein of the Escherichia coli outer membrane was altered by using synthetic oligonucleotides from +2 to +1, 0, and -1 by guided site specific mutagenesis of a plasmid DNA carrying an inducible lipoprotein gene. The wild-type sequence of this sectio, Met-Lys-Ala-Thr-Lys (+2), was thus changed to Met-Lys-Asp-Thr-Lys (I-1; +1), Met-Ala-Thr-Lys (I-2; +1), Met-Asp-Thr-Lys (I-3; 0), and Met-Glu-Asp-Thr-Lys (I-4; -1). After induction of lipoprotein production, cells were pulse labeled with [35S]methionine for 10 sec. The lipoprotein of I-1, I-2, and I-3 was assembled in the membrane, although the rates of lipoprotein production progressively decreased as the charge on the signal peptide became more negative. Conversely, in the case of I-4, only a small amount of lipoprotein assembled in the membrane while a large amount of glycerol-unmodified prolipoprotein accumulated in the cytoplasm. This soluble prolipoprotein was gradually and posttranslationally secreted across the membrane to be modified and assembled in the membrane. These results indicate that the positively charged amino-terminal region of the signal peptide plays an important role in efficient protein secretion across the membrane.

Amino Acid Sequence↗
PubMed1982

Identification of the 45-base-long primordial building block of the entire class I major histocompatibility complex antigen gene.

Within the 885-base-long incomplete coding sequence for the mouse class I major histocompatibility complex (MHC) antigen H-2Kb heavy chain, we have identified 32 recurring base oligomers (1 decamer, 4 octamers, 9 heptamers, and 18 hexamers), the decamer being T-C-A-C-C-C-T-G-A-G. The compilation of these recurring base oligomers led to the identification of the 45-base-long primordial building block for this class of genes which is: C-C-T-G-C-G-G-A-C-A-A-T-G-T-C-A-C-C-C-T-G-A-G-C-T-G-C-T-G-G-G-C-T-C-T-G-G-G-C-G - T-T-G-A-C. Inasmuch as these base oligomers that recurred within the mouse H-2Kb coding sequence proper also recurred within the adjacent, transcribed noncoding sequence, we conclude that the entire approximately equal to 4,000-base-long germ-line DNA segment containing eight or nine separate exons of the coding sequence for class I MHC antigen heavy chains can be construed as having evolved from tandem repeats of this one 45-base-long primordial building block.

Amino Acid Sequence↗
PubMed1982

Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function.

We have used oligonucleotide-directed mutagenesis to make a specific change in the beta-lactamase (EC 3.5.2.6) (ampicillin resistance) gene of the plasmid pBR322. Evidence suggests that the active site for this enzyme may include a serine-threonine dyad (residues 70 and 71). By priming in vitro DNA synthesis with a chemically synthesized 16-base oligodeoxyribonucleotide, we have inverted the Ser-Thr dyad to Thr-Ser and thereby generated a mutant with an ampicillin-sensitive phenotype. This "double-mismatch" method is relatively simple and also very general because detection of mutants is at the level of DNA and involves only colony hybridization. Accordingly, the procedure can be applied to any DNA sequence and does not depend on the phenotype of the mutant.

Amino Acid Sequence↗
PubMed1981

Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA.

A nonadecanucleotide has been used both as a site specific mutagen to introduce a T leads to A transversion mutation in the human beta-globin gene cloned in pBR322 as well as a probe to screen transformed colonies for the desired mutant. The specificity of the oligonucleotide as a mutagen and as a hybridization probe provide a general method for producing site specific mutations in DNA cloned in plasmid vectors such as pBR322.

Amino Acid Sequence↗
PubMed1981

The use of synthetic oligonucleotides as hybridization probes. II. Hybridization of oligonucleotides of mixed sequence to rabbit beta-globin DNA.

Two oligonucleotides 14-bases long were synthesized, one complementary to rabbit beta-globin DNA (R beta G14A) and the other with the same sequence except for a single base change (T for C) (R beta G14B). Hybridization conditions were established such that R beta G14A would hybridize to globin DNA while R beta G14B would not. We also synthesized a mixture of 13-base long oligonucleotides (R beta G13Mix), representing eight of the possible coding sequences for amino acids 15-19 of rabbit beta-globin. One of the eight is complementary to globin DNA. R beta G13Mix was found to hybridize specifically to globin DNA under conditions where oligonucleotides forming single base pair mismatches do not. Furthermore, R beta G13Mix was shown to hybridize specifically to colonies containing a plasmid with a globin DNA insert. These results are discussed with respect to a general procedure for screening recombinant clones for those containing DNA coding for a protein of known amino acid sequence.

Amino Acid Sequence↗
PubMed1981

HLA and rheumatoid arthritis in the Japanese.

Sixty-three Japanese patients with rheumatoid arthritis (RA) were typed for HLA-A, -B, and -DR locus specificities. A significant association was found only with increased frequency of HLA-DR4 in the patients (71.4%) compared with controls (41.8%) (corrected P = 0.007, relative risk = 3.5). No association was found with HLA-A9 or HLA-Bw54, which are in linkage disequilibrium with HLA-DR4 in the Japanese. The association between HLA-DR4 and RA observed in these Japanese patients has also been demonstrated in the white population.

Adult↗
PubMed1981

Identification of an H-2Kb-related molecule by molecular cloning.

Based on the published amino-acid sequence of H-2Kb, we synthesized a mixture of eight 16-base long oligodeoxyribonucleotides representing all possible coding sequences for residues 51-56 (Trp-Met-Glu-Gln-Glu-Gly). The hexadecanucleotide mixture was used as a probe to screen recombinant DNA clones constructed from cytoplasmic PolyA+ RNA isolated from the murine thymoma cell line EL4(b haplotype). Of the 30 000 independent clones screened, one clone was found to hybridize with the probe. DNA sequence analysis showed that the cDNA clone was derived from a portion of an H-2Kb -related mRNA. The clone encodes a protein sequence identical with a region of H-2Kb in 42 consecutive residues (50 through 91). The sequence than diverges from the H-2Kb sequence and, after a single Glu codon, a termination codon is encountered. It is possible that this mRNA codes for a small 92 amino-acid protein with a sequence identical (except for a carboxy-terminal Glu residue) with the amino terminus of H-2Kb. It is further speculated that this mRNA is coded for by the H-2Kb gene and differs from the H-2Kb mRNA in the pattern of posttranscriptional splicing.

Amino Acid Sequence↗
PubMed1981

A set of synthetic oligodeoxyribonucleotide primers for DNA sequencing in the plasmid vector pBR322.

Seven oligonucleotide primers complementary to the plasmid vector pBR322 at positions adjacent to five of the unique restriction endonuclease cleavage sites (EcoRI, HindIII, BamHI, SalI and PstI) have been chemically synthesized. The polarity of the primers is such that any DNA inserted at one or a combination of two of the above restriction sites may be sequenced by the chain termination method using one of the synthetic DNA primers. One of the primers for sequencing inserts at the PstI site of pBR322 is also complementary to the M13 phage vector designated bla6. This set of universal primers is useful for rapid sequence determination of DNA cloned into pBR322 or M13bla6.

Base Sequence↗
PubMed1981

Expression in Escherichia coli of a chemically synthesized gene for a "mini-C" analog of human proinsulin.

A gene has been constructed which codes for an analog of human proinsulin in which the normal 35-amino acid connecting peptide is replaced by a "mini-C" peptide of six amino acids (Arg-Arg-Gly-Ser-Lys-Arg). The gene, composed of oligonucleotide fragments synthesized by the triester method, was cloned and expressed as a beta-galactosidase hybrid protein. The proinsulin analog was separated from beta-galactosidase by cyanogen bromide cleavage and purified. Controlled disulfide exchange in the S-sulfonate of the analog generated a molecule having high-pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) behavior consistent with a proinsulin-like structure.

Amino Acid Sequence↗
PubMed1981

Use of synthetic oligonucleotides as hybridization probes: isolation of cloned cDNA sequences for human beta 2-microglobulin.

We have synthesized two sets of 15-base-long oligodeoxyribonucleotides corresponding to all possible coding sequences for a small portion of human beta 2-microglobulin. Labeled oligonucleotides were used as hybridization probes to screen bacterial clones containing cDNA sequences primed with oligo(dT) and inserted into the plasmid vector pBR322. One beta 2-microglobulin cDNA clone was detected in the 535 bacterial plasmid clones that were screened. The clone has been characterized by blotting and nucleotide sequence analysis. The cloned beta 2-microglobulin sequence contains 217 base pairs of the 3' untranslated region of the mRNA and 328 base pairs (97%) of the coding region.

Amino Acid Sequence↗
PubMed1981

Mutual interaction between adjacent dG . dC actinomycin binding sites and dA . dT netropsin binding sites on the self-complementary d(C-G-C-G-A-A-T-T-C-G-C-G) duplex in solution.

The Watson-Crick imino protons, the backbone phosphodiester resonances, and the antibiotic exchangeable protons have been used as markers to monitor the separate and simultaneous binding of actinomycin and netropsin to the d(C-G-C-G-A-A-T-T-C-G-C-G) self-complementary duplex in aqueous solution. We demonstrate that intercalation of actinomycin at dG(3'-5')dC sites at either end of the duplex results in a conformational perturbation at the dA . dT tetranucleotide core of the dodecanucleotide duplex. Parallel studies of the groove binding of netropsin at dA . dT sites in the interior of the duplex reveal a conformational perturbation which extends to adjacent dG . dC base pairs in the dodecanucleotide duplex. The NMR markers demonstrate that the d(C-G-C-G-A-A-T-T-C-G-C-G) duplex can accommodate actinomycin and netropsin simultaneously at adjacent dG . dC and dA . dT tetranucleotide blocks along its length with some mutual interaction between neighboring antibiotic binding sites.

Binding Sites↗
PubMed1981

Structure of a B-DNA dodecamer: conformation and dynamics.

The crystal structure of the synthetic DNA dodecamer d(CpGpCpGpApApTpTpCpGpCpG) has been refined to a residual error of R = 17.8% at 1.9-A resolution (two-sigma data). The molecule forms slightly more than one complete turn of right-handed double-stranded B helix. The two ends of the helix overlap and interlock minor grooves with neighboring molecules up and down a 2(1) screw axis, producing a 19 degrees bend in helix axis over the 11-base-pair steps of the dodecamer. In the center of the molecule, where perturbation is least, the helix has a mean rotation of 36.9 degrees per step, or 9.8 base pairs per turn. The mean propeller twist (total dihedral angle between base planes) between A . T base pairs in the center of the molecule is 17.3 degrees, and that between C . G pairs on the two ends averages 11.5 degrees. Individual deoxyribose ring conformations as measured by the C5'-C4'-C3'-O3' torsion angle delta, exhibit an approximately Gaussian distribution centered around the C1'-exo position with delta avg = 123 degrees and a range of 79 degrees to 157 degrees. Purine sugars cluster at high delta values, and pyrimidine sugars cluster at lower delta. A tendency toward 2-fold symmetry in sugar conformation about the center of the molecule is detectable in spite of the destruction of ideal 2-fold symmetry by the molecular bending. More strikingly, sugar conformations of paired based appear to follow a "principle of anticorrelation," with delta values lying approximately the same distance to either side of the center value, delta = 123 degrees. This same anticorrelation is also observed in other DNA and DNA . RNA structures.

Deoxyribose↗
PubMed1981

Location of LyM-1 locus on Mus musculus chromosome 1.

Following segregation of the alleles at the Sas-1, LyM-1 and M1s in the (AKA/J X C3H/HeJ/F1 X C3H/HeJ backcross progeny, we found the locus responsible for the lymphocyte alloantigen LyM-1 to be closely linked to Sas-1 and M1s. The map distances are estimated to be 12.5 +/- 3.9 for Sas-1 to LyM-1, 6.9 +/- 3.0 for LyM-1 to M1s and 19.4 +/- 4.7 for Sas-1 to M1s. These data clearly indicate that the gene order is Sas-1-LyM-1-M1s on Mus musculus chromosome 1.

Animals↗
PubMed1981

Genetic and serologic re-evaluation of LyM-1 antigen specified by a gene closely linked to Mls: establishment of LyM-1 antigen of the mouse.

An alloantiserum prepared against LyM-1 antigen of the mouse has been analysed in detail. This antiserum, C3H/HeJ anti-CBA/J, has turned out to define two antigens which are specified by independently segregating loci. One of these is Lyb-2.1 or similar antigen specified by a gene located on chromosome 4. The other is identified as LyM-1.2 based on the result that a gene determining alloantigenicity in the Lyb-2- strain is closely linked to Mls with recombination frequency of 0.066 +/- 0.028. Since both antigens are expressed preferentially on B lymphocytes and their strain distribution is significantly overlapped, there has been much confusion about LyM-1 antigen. This is overcome by re-evaluating anti-LyM-1.2 serum under the condition in which Lyb-2.1 is not involved in analysis. The strain distribution of LyM-1 antigen, its preferential expression on B lymphocytes and close genetic linkage of LyM-1 to Mls have been established.

Animals↗