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Y Hayashizaki

Publications and source records attributed to Y Hayashizaki.

15 recordsLinked to original sources
PubMed1992

A new method for constructing NotI linking and boundary libraries using a restriction trapper.

We have developed a novel method for constructing NotI linking and boundary libraries using a modified "solid-supported ligation primer" (restriction trapper). The restriction trapper could be used to purify the DNA fragments with a specific restriction enzyme cutting site(s) at their ends. The method uses a ligation and recutting reaction with double-stranded DNA ends of a hairpin-shaped oligolinker which is connected covalently to the surface of the latex beads. Selectivity is based on the specificity of the restriction enzyme for its recognition site, resulting in efficient purification. We applied this technique to the construction of high-quality NotI linking and NotI boundary libraries, which contain almost all the NotI sites of the genome and, in addition, are free of illegitimately ligated clones.

Base Sequence↗
PubMed1991

A genomic scanning method for higher organisms using restriction sites as landmarks.

We have developed a powerful genomic scanning method, termed "restriction landmark genomic scanning," that is useful for analysis of the genomic DNA of higher organisms using restriction sites as landmarks. Genomic DNA is radioactively labeled at cleavage sites specific for a rare cleaving restriction enzyme and then size-fractionated in one dimension. The fractionated DNA is further digested with another more frequently occurring enzyme and separated in the second dimension. This procedure gives a two-dimensional pattern with thousands of scattered spots corresponding to sites for the first enzyme, indicating that the genome of mammals can be scanned at approximately 1-megabase intervals. The position and intensity of a spot reflect its locus and the copy number of the corresponding restriction site, respectively, based on the nature of the end-labeling system. Therefore, this method is widely applicable to genome mapping or detection of alterations in a genome.

Animals↗
PubMed1990

Chromosome regional mapping for the human thyroid stimulating hormone beta subunit (TSHB) gene.

The human thyroid stimulating hormone beta subunit (TSHB) gene, located on chromosome 1, was studied to determine its subregional location by in situ hybridization and Southern blot analysis of human x mouse hybrid cells. The results allowed localization of TSHB to the proximal portion of 1p22, which is in the region of localization of the linkage group including amylase (AMY), nerve growth factor beta subunit (NGFB), and NRAS, which are conserved in humans and rodents.

Blotting, Southern↗
PubMed1990

Deoxyribonucleic acid analyses of five families with familial inherited thyroid stimulating hormone deficiency.

Five families with familial inherited TSH deficiency, reported to date, were examined for the TSH beta gene at the nucleotide level. The first family carries a single base substitution in the 29th codon which lies in the so-called CAGYC region; GCA (glycine) is replaced by AGA (arginine). This substitution induces conformational changes of the beta-polypeptide which make it unable to associate with the alpha-subunit. This mutation generates a new cleavage site for a restriction endonuclease MaeI, a new marker that can be used for DNA diagnosis. The second and third families were found to carry the same nucleotide substitution. Also, all three families were associated with an additional single base substitution in intron 2 as a polymorphic change, suggesting that these three families may have originated from the same single founder from Shikoku Island in Japan. The nucleotide sequence from the fourth and fifth families showed no alterations in the TSH beta gene from the about -200 basepair up-stream region to the polyadenylation site.

Amino Acid Sequence↗
PubMed1989

The role of the carboxyl-terminal 6 amino acid extension of human TSH beta subunit.

The nucleotide sequence of human thyroid stimulating hormone (hTSH) gene can encode a protein of 138 amino acids. However, the mature polypeptide is lacking 6 amino acids of the carboxyl-terminus (C-terminus), suggesting posttranslational cleavage of these residues. To analyze a possible function of these 6 amino acids, we expressed two hTSH beta cDNAs with or without the 6 codons for C-terminal extension, together with alpha subunit cDNA in CHO cells, and determined the amino acid sequence of C-terminus of hTSH beta. hTSH beta propeptides without C-terminal extension were glycosylated, associated with alpha subunit and secreted, as normal propeptides were, and its heterodimer with alpha subunit showed normal TSH bioactivity in FRTL-5 bioassay. These data indicate that the 6 amino acid C-terminal extension is not necessary for the hTSH maturation in the process of the biosynthesis and for its bioactivity.

Amino Acid Sequence↗
PubMed1989

Thyroid-stimulating hormone (TSH) deficiency caused by a single base substitution in the CAGYC region of the beta-subunit.

Congenital isolated thyroid-stimulating hormone (TSH) deficiency is an autosomal recessive disease that manifests as hypothyroidism (cretinism), causing severe mental and growth retardations. Patients were found to have a single base substitution in the codon for the 29th amino acid of the TSH beta subunit gene. The alteration is in the center of the so-called CAGYC region, which consists of an amino acid sequence conserved among all of the known glycoprotein hormone beta subunits. No other nucleotide substitutions have been found in the gene thus far sequenced. Microinjection of the mutated beta mRNAs into Xenopus laevis oocytes led to the formation of conformationally altered beta polypeptides that could not associate with alpha subunits. The mutation created a new recognition site for the enzyme MaeI. Southern blot hybridization of genomic DNA digested with MaeI showed that the patients were homozygous and their parents were heterozygous for the mutation. This test was also used to examine other family members for the disease.

Amino Acid Sequence↗
PubMed1988

The structure of the human thyrotropin beta-subunit gene.

We have determined and characterized the entire structure of the human thyrotropin beta-subunit (TSH beta) gene. It is 4.5 kb in size and separated into three exons. Primer extension analysis showed one transcription start point, and S1 mapping analysis showed two blocks of polyadenylation sites in normal pituitaries. The same transcripts were observed in TSH-producing adenomas. The 5'-flanking region contains two 'TATA' boxes, two 'CAAT' boxes, and two sequences similar to the cyclic AMP-responsive elements.

Amino Acid Sequence↗
PubMed1988

The production of active human thyroid-stimulating hormone from alpha and beta mRNAs in Xenopus laevis oocytes.

Active human thyroid-stimulating hormone (hTSH) was produced by Xenopus laevis oocytes following injection of an mRNA mixture of hTSH beta and alpha subunits synthesized by T3 RNA polymerase. Some of the hTSH molecules were secreted into the medium, while others remained in the cells. The active molecules consisted of alpha and beta subunits and were in highly glycosylated form. The Xenopus laevis oocyte-produced hTSH stimulated the rat thyroid cell line FRTL-5 to produce and secrete the cyclic AMP as does authentic hTSH.

Animals↗
PubMed1988

Serum free thyrotropin subunit in congenital isolated thyrotropin deficiency.

In two patients with congenital isolated thyrotropin (TSH) deficiency, serum TSH determined by a sensitive immunoradiometric assay (IRMA) was consistently undetectable. The basal levels of serum free TSH-alpha subunit (TSH-alpha) determined by a specific radioimmunoassay (RIA) were elevated in the hypothyroid state, and decreased to the undectable level during displacement therapy with thyroid hormone. The serum free TSH-alpha significantly increased following intravenous administration of thyrotropin releasing hormone (TRH). Serum free TSH-beta subunit (TSH-beta) was undectable. These findings suggest that TSH deficiency in this disease is not due to absence of thyrotroph in the pituitary gland or deficiency of TSH-alpha, but to abnormalities of the TSH-beta gene.

Adult↗
PubMed1987

Production of human thyroid-stimulating hormone in Chinese hamster ovary cells.

Human thyroid-stimulating hormone (hTSH) has been produced in Chinese hamster ovary (CHO) cells co-transformed with two plasmids: one carrying the alpha subunit cDNA with mouse dihydrofolate reductase gene and the other carrying hTSH beta subunit cDNA. Each cDNA was driven to expression under the control of SV40 early promoter. hTSH and its alpha subunit were secreted into culture media, and their secretion increased with exposure of the cells to increasing concentrations of methotrexate. Gel filtration analysis revealed that the molecular size of the hTSH was the same as that of natural hTSH. Furthermore, the CHO cell-produced hTSH elevated the cyclic AMP level in the rat thyroid cell line FRTL-5 in the same manner as natural hTSH does.

Animals↗
PubMed1986

Expression of human gastrin gene in normal and gastrinoma tissues.

The complete sequence of the human gastrin gene is reported here. This gene consists of three exons. Nine Alu family sequences are found within the gene and in the surrounding region. S1 mapping study showed that the transcription of gastrin gene starts at 60, 57, or 55 bp upstream from the 3' end of the first exon. The mechanism of mRNA synthesis in a gastrinoma tissue was studied to clarify the ectopic production of gastrin. It was found that mRNA synthesis starts from the same three transcriptional start points. Southern blotting profiles for normal gastric antrum and gastrinoma DNA were indistinguishable from each other within the 18-kb region containing the gastrin gene, showing that no genomic rearrangements are associated with the gastrinoma formation. Thus, the overproduction of gastrin in this tumor is likely to be due to an aberrant expression control system of the cell, rather than a change in the control region of the gastrin gene.

Amino Acid Sequence↗
PubMed1986

Effects of corticotropin releasing factor and growth hormone releasing factor on pituitary hormone secretion in patients with congenital thyrotropin deficiency. Abnormal response of growth hormone to corticotropin releasing factor.

Blood concentrations of anterior pituitary hormones, ACTH, GH, TSH, PRL, LH, and FSH were determined in corticotropin releasing factor (CRF) test (synthetic ovine CRF 1.0 microgram per kg body weight) and growth hormone releasing factor (GRF) test (synthetic human pancreatic GRF-44 100 micrograms) in 2 female sibling patients with congenital isolated TSH deficiency, in their mother, in 2 patients with congenital primary hypothyroidism and in 8 normal controls. The patients with isolated TSH deficiency showed normally increased plasma ACTH and serum GH after CRF and GRF, respectively, and also showed an abnormal GH response to CRF. The serum GH showed a rapid increase to maximum levels (12.9 ng/ml) within 30 to 60 min followed by decrease. The possibility of secretion of abnormal GH could be excluded by the fact that on serum dilution, GH value gave a linear plot passing through zero. In addition, serum PRL, LH and FSH levels after CRF administration in case 1 and PRL after GRF in case 2 were also slightly increased but these responses were marginal. The mother of the patients, patients with congenital primary hypothyroidism, and normal healthy controls showed normal responses of pituitary hormones throughout the experiment. Data from the present study and a previous report show that abnormal GH response to the hypothalamic hormones (CRF, TRH and LHRH) may be observed in patients with congenital isolated TSH deficiency.

Adult↗