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Biomedical subjects

T Y Shih

Publications and source records attributed to T Y Shih.

At least 73 records · Page 4Linked to original sources
PubMed1978

Comparison of the genomic organization of Kirsten and Harvey sarcoma viruses.

Current studies were undertaken to compare the genomes of Kirsten murine sarcoma virus (Ki-MuSV), Harvey murine sarcoma virus (Ha-MuSV), and the replication-defective endogenous rat virus to understand the function of these viral RNAs. Genome organization and sequence homology were studied by fingerprinting large RNase T1-resistant oligonucleotides and by cross-protecting homologous oligonucleotides against RNase A and T1 digestion with complementary DNA prepared from each of the other viral RNA. Ki-MuSV and Ha-MuSV were found to share an extensive series of rat-derived oligonucleotides begining ca. 1 kilobase (kb) from the 3' end and extending to within 1.5 kb of the 5'end of Ki-MuSV RNA. The total map distance covered in ca. 5.5 kb. The eight oligonucleotides covering the 1.5 kb at the 5' end of Ki-MuSV RNA were not found in Ha-MuSV RNA. Five out of these eight oligonucleotides, however, could be designated with certainty to be of rat virus origin. Since Ha-MuSV is 6.5 kb in size and Ki-MuSV is 8 kb in size, the major difference between them is the 1.5 kb from the replication-defective endogenous rat virus sequences at the 5' end of Ki-MuSV not present in Ha-MuSV. Consistent with the difference in the genome structure, these two sarcoma viral RNA'S yielded distinct major translation products in cell-free systems, I.E., A 50,000-dalton polypeptide (P50) from Ki-MuSV and a 22,000-dalton polypeptide (p22) from Ha-MuSV. These polypeptides may provide the necessary protein makers for identifying in vivo virus-coded proteins.

Base Sequence↗
PubMed1978

Heteroduplex analysis of the sequence relations between the RNAs of mink cell focus-inducing and murine leukemia viruses.

The sequence relationships betwen AKR ecotropic virus and an AKR-derived "mink cell focus-inducing" (MCF) isolate (AKR MCF 247), between Moloney murine leukemia virus (M-MLV) and an M-MLV MCF isolate (M-MLV83), and between AKR and M-MLV were studied by electron microscopic heteroduplex analysis. The MCF-specific sequences were found to map from 1.95 kilobases (kb) to 2.75 kb (+/- 0.15 kb) from the 3' end of the RNAs for both MCF isolates. The major sequence nonhomology regions between AKR and M-MLV lie between 0.9 and 3.5 kb from the 3' end. However, the AKR and M-MLV sequences immediately adjacent to the 1.95- and 2.75-kb junctions with MCF-specific sequences are relatively similar in AKR and M-MLV. Our results suggest that the env gene of MLVs maps from 1 kb to 3 kb from the 3' end of the genomic RNA and that the carboxyl end of the glycoprotein of each MCF strain is similar (or identical) to that of its ecotropic parent.

AKR murine leukemia virus↗
PubMed1977

In vitro transcription of Moloney leukemia virus genes in infected cell nuclei and chromatin: elongation of chromatin associated ribonucleic acid by Escherichia coli ribonucleic acid polymerase.

The in vitro transcription of viral specific DNA sequences in nuclei and chromatin isolated from mouse cells chronically infected with Moloney murine leukemia virus (Mo-MuLV) has been studied. The in vitro RNA synthesized by Escherichia coli RNA polymerase has been isolated by sulfhydryl affinity column following reaction in the presence of 5-mercuriuridine triphosphate. By comparison of the Crt curves of the in vitro RNA with that of 70S viral RNA, the content of viral sequences is found to be 1.3% in nuclei product and 0.24% in chromatin product which is lower than the 2.5% found in chromatin associated RNA. This latter value, however, is very close to the in vivo viral RNA content in pulse-labeled [3H]RNA of the infected cells. Unexpectedly, it is observed that over 20% of the chromatin associated RNA prelabeled in vivo with [5-3H]uridine is elongated and tagged with Hg atoms during RNA synthesis catalyzed by the exogenous E. coli RNA polymerase in the presence of Hg-UTP. The elongation reaction is dependent on the presence of all four nucleotide triphosphates and appears to be due to E. coli RNA polymerase per se. It is suggested that most of the viral specific sequences observed in the in vitro RNA products are very likely initiated and derived from the chromatin associated species. The implication of the present findings for in vitro RNA synthesis in nuclei and chromatin as related to regulation of gene expression is discussed.

Animals↗
PubMed1977

Steroid induction of mouse mammary tumor virus: effect upon synthesis and degradation of viral RNA.

Steroid hormones have been demonstrated to induce in tissue culture the production of mouse mammary tumor viral (MMTV) RNA, proteins, and particles 10-fold compared with constitutive levels. However, previous data of increased viral RNA levels did not distinguish between an increased rate of viral-specific RNA synthesis and a slower rate of viral RNA degradation. According to the recently developed assay of Coffin et al. (1974) for measuring rates of viral RNA synthesis, short-term labeling experiments of a mouse mammary tumor cell line indicate that the glucocorticoid hormone dexamethasone stimulates a 3-fold increase in the synthesis of MMTV-specific RNA within 10 min after the addition of hormone and that stimulation of RNA synthesis reaches 5- to 10-fold within 30 to 60 min, while the synthesis of Moloney leukemia virus-specific RNA in the same cell is unaffected by steroids. The decay rates of pulse-labeled and accumulated MMTV RNA in the presence or absence of dexamethasone show this RNA to have a half-life of greater than 8 h. In addition, hormone-stimulated MMTV RNA appears to have an increased rate of decay compared to basal MMTV RNA, thus ruling out an increased stability of MMTV RNA in the presence of steroid hormones as the basis for increased RNA levels. Thus, the magnitude, rapidity, and specificity of hormone action on MMTV RNA synthesis indicate a primary effect upon transcription.

Dactinomycin↗
PubMed1976

Interactions between arginine-rich histones and deoxyribonucleic acids. I. Thermal denaturation.

Physical properties of histone-DNA complexes very often depend upon the method of complex formation. In an attempt to make the studies of histone-DNA interactions more relevant to biological systems, results from thermal denaturation of native chromatin were used as references for determining how closely a given histone-DNA complex approaches its native state in chromatin. In the case of arginine-rich histones H3 (III or f3) and H4 (IV or f2a1), four methods were used for making complexes with calf thymus DNA: (A) NaCl gradient dialysis with urea; (B) NaCl gradient dialysis without urea; (C) direct mixing in 2.5 x 10(-4) EDTA, pH 8.0; and (D) direct mixing in 0.01 M sodium phosphate, pH 7.0. It was observed that a complex made by direct mixing in phosphate (method D) is closer to the native than is one made by direct mixing in EDTA (method C) than the one made by gradient dialysis with urea (method A) or without urea (method B). Regardless of the method used for complex formation, no substantial differences were observed between complexes with histone H3 dimer with disulfide bond(s) and a reduced H3 without disulfide bond, implying that perhaps a dimer with or without disulfide bond is a natural fundamental subunit in our experimental conditions. When the method of direct mixing in EDTA is used, the melting properties of the complexes vary only slightly with any one of the following H3 histones: from calf thymus, H3 without disulfide bond, H3 dimer, and H3 oligomer with disulfide bonds, also, from duck erythrocyte, H3 monomer and dimer. The complexes formed between DNA and a mixture of H3 and H4 by method D have melting properties similar to those of native chromatin. Since an equimolar mixture of histone H3 and H4 in 0.01 M phosphate, pH 7.0, was shown to form a tetramer (D'Anna, J.A., and Isenberg, I. (1974), Biochem. Biophys. Res. Commun. 61, 343), our results suggest that, a tetramer of H3 and H4, likely to be (H3)2(H4)2, formed from one H3 dimer and one H4 dimer, can bind DNA in a manner similar to that in native chromatin.

Animals↗
PubMed1976

Interactions between arginine-rich histones and deoxyribonucleic acids. II. Circular dichroism.

Circular dichroism (CD) was used to investigate the conformations of arginine-rich histones, H3 (III or f3) and H4 (IV or f2a1), and DNA in the complexes prepared by four different methods: (A) NaCl gradient dialysis with urea; (B) NaCl gradient dialysis without urea; (C) direct mixing in 2.5 x 10(-4) M EDTA, pH 8.0; and (D) direct mixing in 0.01 M sodium phosphate, pH 7.0. Using the CD spectrum of native chromatin as a criterion to judge the closeness of a complex to its native state, it was observed that a complex made by direct mixing at low ionic strength (methods C and D) is better than the ones made by NaCl gradient dialysis with or without urea (methods A and B). It is explained as a result of lack of ordered secondary structures in histones due to the presence of urea in method A or due to nonspecific aggregation in NaCl without urea (method B). Compared with all the earlier reports in literature on the CD of histone-DNA complexes, the CD spectra of arginine-rich histone-DNA complexes prepared by methods C and D are closest to that of native chromatin both in shape and in amplitude. These results imply (a) that arginine-rich histones play an important role in maintaining the conformation of chromatin and (b) that the binding of these two histones to DNA prepared by methods C and D are close to that in native chromatin. Noticeable variation in conformation of free and bound histone and histone-bound DNA has also been observed in histone H3 with one or two cysteine residues, and in reduced or oxidized state even when the complexes were prepared and examined in the same condition. CD spectra of arginine-rich histones in 0.01 M phosphates, pH 7.0, indicate the presence of alpha-helix which could be responsible for a favorable binding of the less basic regions of these histones to DNA under this condition as demonstrated by thermal denaturation (Yu, S. .S, Li H. J., and Shih, T. Y. (1976), Bio-chemistry, the preceding paper in this issue). To preserve or generate alpha-helical structures in histones seems to be a critical step in reconstituting good histone-DNA complexes.

Animals↗
PubMed1976

Isolation of viral specific RNA from SV40 infected cells by viral DNA chemically linked to a cellulose matrix.

SV40 DNA fragments chemically attached to neutral cellulose powder with a water-soluble carbodiimide have been used to isolate late lytic viral specific RNA from virus infected cells. Exhaustive hybridization to SV40 DNA reveals that virtually all of the isolated RNA molecules contain SV40 specific sequences. Comparison with SV40 cRNA prepared with purified Escherichia coli RNA polymerase and a SV40 DNA I template suggests that the purity of the isolated SV40 specific RNA is very close to 100%. The background level for the nonspecific binding of RNA to a purified cellulose matrix is very low. Retention of nonspecific RNA by SV40 DNA-cellulose is only 1.5% of the viral specific RNA isolated under saturating conditions for the column. Sedimentation in neutral sucrose suggests that the major 16S viral specific RNA has been isolated largely intact.

Cell Line↗
PubMed1973

In vitro transcription of the viral-specific sequences present in the chromatin of cells transformed by simian virus 40.

Separated strands of simian virus 40 (SV40) DNA fragments were used in hybridization experiments to study the RNA transcribed by Escherichia coli RNA polymerase from the chromatin of cells transformed by SV40. The template activity of chromatin of the transformed cell line 11A8 (mouse-embryo cells) examined is about 17% that of purified DNA, suggesting that most of the chromatin DNA is repressed by chromosomal proteins. The SV40-specific RNA present in the RNA transcribed in vitro from 11A8 chromatin hybridizes specifically with the minus strand of SV40 DNA. Little or no reaction occurs with the plus strand of viral DNA. The SV40-specific RNA transcribed in vitro from chromatin of transformed cells shares sequences with the RNA produced during the early phase of SV40 lytic infection, and is similar to that present in the 11A8 cell line in vivo. Although the influence of chromosomal proteins on this pattern of transcription was not definitely determined, preliminary evidence indicates that an asymmetric pattern of transcription may also occur when 11A8 DNA is transcribed by E. coli RNA polymerase.

Animals↗
PubMed1973

A general method of gene isolation.

A general method of gene isolation has been developed that involves the chemical linkage of RNA to cellulose by a water-soluble carbodiimide, and the continuous circulation of DNA containing specific sequences complementary to the RNA. The temperature of the cellulose matrix is maintained at 37 degrees (50% formamide, 0.3 M NaCl-0.03 M Na(3) citrate) to allow efficient DNA-RNA interaction in the stationary phase, while unreacted and any reassociated DNA is denatured at 90 degrees and then recirculated into the hybridization chamber. Between 40 and 45% of fragmented (32)P-labeled simian virus (SV)40 DNA was removed from the circulating solution when cellulosebound SV40-specific RNA, assymmetrically transcribed in vitro with Escherichia coli RNA polymerase, was used. In the presence of 10(4)-fold excess of sheared E. coli DNA, nearly half of the [(32)P]SV40 DNA was recovered from the mixture as a DNA-RNA hybrid with negligible contamination by bacterial DNA. The isolation procedure is almost quantitative for the complementary DNA. The efficiency and selectivity of this method permit the isolation of a defined DNA sequence from a large and complex genome.

Carbodiimides↗