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Biomedical subjects

S Silverstein

Publications and source records attributed to S Silverstein.

86 records · Page 5Linked to original sources
PubMed1978

Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor.

Previous studies from our laboratories have demonstrated the feasibility of transferring the thymidine kinase (tk) gene from restriction endonuclease-generated fragments of herpes simplex virus (HSV) DNA to cultured mammalian cells. In this study, high molecular weight DNA from cells containing only one copy of the HSV gene coding for tk was successfully used to transform L+K-cells to the tk+ phenotype. The acquired phenotype was demonstrated to be donor-derived by analysis of the electrophoretic mobility of the tk activity, and the presence of HSV DNA sequences in the recipient cells was demonstrated. In companion experiments, we used high molecular weight DNA derived from tissues and cultured cells of a variety of species to transfer tk activity. The tk+ mouse cells transformed with human DNA were shown to express human type tk activity as determined by isoelectric focusing.

Animals↗
PubMed1978

The transfer and stable integration of the HSV thymidine kinase gene into mouse cells.

Treatment of mutant mouse cells (Ltk-) deficient in thymidine kinase with Bam I restriction endonuclease-cleaved HSV-1 DNA results in the appearance of numerous surviving colonies which stably express thte tk+ phenotype. Through a series of electrophoretic fractionations in concert with transfection assays, we isolated a 3.4 kb fragment which contains the thymidine kinase gene and which alone is competent in the biochemical transformation of Ltk- cells. In this report, we have examined the distribution of tk sequences in the DNA of several transformed clones following stable gene transfer. A series of complementary experiments involving reassociation kinetics in solution and annealings with tk DNA to restriction-cleaved cellular DNA following electrophoresis and transfer to filters allow us to make the following general conclusions concerning the fate of the tk gene in all clones examined: the tk gene is present in all cells at a frequency of one copy per chromosomal complement; the tk gene is stably integrated in the DNA of all transformants; and integration is not site-specific and occurs at different loci in the DNA of all transformants examined. The existence of a single active tk gene in tk+ transformants now facilitates an analysis of the sequence organization of tk- mutant cells and provides a useful model system for studies on the transfer of cellular genes.

Cell Line↗
PubMed1978

Alterations in the protein synthetic apparatus of Friend erythroleukemia cells infected with vesicular stomatitis virus or herpes simplex virus.

We have compared the effects of infection with herpes simplex virus (HSV) and vesicular stomatitis virus (VSV) on the protein synthetic apparatus of Friend erythroleukemia cells. Previous studies demonstrated that infection with HSV rapidly shuts off the synthesis of globin and other cellular polypeptides (Y. Nischioka and S. Silverstein, 1977, Proc. Natl. Acad. Sci. U.S.A. 74: 2370-2374). In contrast to these findings, globin synthesis persists in Friend erythroleukemia cells infected with VSV. Physical measurements of the size of bulk-infected cell mRNA, using hybridization with polyuridylic acid, demonstrated that there was no detectable change in the size of mRNA's after infection with VSV. A comparison of the kinetics of hybridization of cytoplasmic RNA extracted from cells infected with either HSV or VSV with globin complementary DNA revealed that by 4 h postinfection with HSV only about 15% of the globin mRNA sequences remained, whereas there was no discernible change in the sequence abundance of globin mRNA in VSV-infected cells.

Cell Line↗
PubMed1978

Requirement of protein synthesis for the degradation of host mRNA in Friend erythroleukemia cells infected wtih herpes simplex virus type 1.

We describe experiments which demonstrate that shortly after infection of Friend erythroleukemia cells with herpes simplex virus (HSV), polyribosomes dissociate and cellular mRNA degrades. Analysis of infected cell extracts on sucrose density gradients demonstrates that the majority of the polyribosomes have dissociated to monoribosomes at 2 h postinfection. Physical measurements of infected-cell RNAs support this conclusion and demonstrate that the polyadenylated RNAs decrease in size. The degradation of mRNA is apparently a stochastic process as judged by the failure to detect a shift in the Crt1/2 when polyadenylated RNA extracted from infected cells at different times is hybridized to globin complementary DNA. In experiments designed to determine whether dissociation of polyribosomes is sufficient to cause degradation of globin mRNA, the amount of globin mRNA in uninfected cells did not change when cells were treated with NaF or pactamycin at concentrations sufficient to dissociate all polyribosomes. In cells infected with UV-irradiated virus polyribosomes dissociate but globin mRNA does not degrade, suggesting that it is possible to separate dissociation from degradation.

Animals↗
PubMed1977

Degradation of cellular mRNA during infection by herpes simplex virus.

The fate of preexisting mRNA sequences was examined after infection by herpes simplex virus. Murine erythroid cells transformed by Friend leukemia virus were used as the host. Such cells, when exposed to 2% dimethyl sulfoxide, produce large amounts of globin and globin mRNA. The protein and its mRNA are easily recognized at 4 days by electrophoresis in high percentage acrylamide gels and by hybridization to cDNA, respectively. Herpes simplex virus replicates in these cells. By 2 hr after infection the rate of protein synthesis decreases to 30% of the level in mock-infected cells and only 49+/-8% (SEM) of the globin mRNA sequences present prior to infection could be detected by hybridization to cDNA. At 4 hr after infection, when the rate of protein synthesis in infected cells is at a maximum, only about 15% of the globin mRNA sequences remained. Control experiments support the hypothesis that globin mRNA sequences are degraded after infection by herpes simplex virus.

Base Sequence↗
PubMed1973

Relationship between post-transcriptional adenylation of herpes virus RNA and messenger RNA abundance.

Analysis of the hybridization kinetics of labeled DNA of herpes simplex virus with unlabeled excess RNA from infected cells showed that viral RNA sequences form two classes differing in molar concentration. The abundant class constituted 93.5-99.3% of total virus-specific RNA and was complementary to 14-16% of the early DNA (2 hr after infection) and to 19-22% of the late DNA (8 hr after infection) in the reproductive cycle of the virus. The early RNA sequences were found to be a subset of the late sequences. The scarce sequences constituted 0.7-6.5% of total virus-specific RNA and were complementary to 28-30% of DNA both early and late in the reproductive cycle. In this study, abundant and scarce sequences were quantitatively separated on the basis of the finding that abundant species are adenylated, i.e., contain post-transcriptionally added poly(A), whereas the scarce RNA is not. Thus, nuclear and polyribosomal adenylated RNA were complementary to 24 and 22%, respectively, of viral DNA and, in abundance competition tests, were found to compete with each other and with abundant RNA from infected cells after 8 hr. The nonadenylated polyribosomal RNA was complementary to 27% of total viral DNA of which 6% was also complementary to adenylated polyribosomal RNA. Hybridization kinetics indicated that each of the fractionated adenylated RNA formed two classes complementary to 6 and 21% of viral DNA.

Adenine Nucleotides↗
PubMed1973

RNA synthesis in cells infected with herpes simplex virus. VII. Control of transcription and of transcript abundancies of unique and common sequences of herpes simplex virus 1 and 2.

Analysis of the kinetics of hybridization in liquid of labeled herpes simplex virus (HSV) 1 and 2 DNAs with excess unlabeled RNA extracted at 2 (early) and 8 (late) h postinfection revealed the following. (i) The RNA transcripts present in the HSV-1-infected cells at 2 and 8 h postinfection are complementary to 44 and 48% of HSV-1 DNA. The RNA transcripts present in the HSV-2-infected cells at 2 and 8 h postinfection are complementary to 21 and 50% of HSV-2 DNA. (ii) The transcripts present in 2-h HSV-1- or HSV-2-infected cells treated with cycloheximide are complementary to 44 and 45% of the respective DNAs. (iii) The RNA transcripts present in the HSV-1-infected cells at 2 h postinfection and in HSV-2-infected cells at 8 h postinfection form 2 classes, abundant and scarce, differing in molar concentrations. The RNA transcripts present in the HSV-2-infected cells at 2 h postinfection form only one abundance class. (iv) The transcripts present in the HSV-1-infected cells at 8 h postinfection are complementary to 24% of HSV-2 DNA and therefore 50% of the transcribed HSV-1 sequences are shared by the two viruses. Of the RNA sequences complementary to HSV-2 DNA, 13% arise from HSV-1 templates specifying abundant RNA and 11% arise from HSV-1 templates specifying scarce RNA. Thus, the DNA sequences shared in common by HSV-1 and HSV-2 DNAs constitute 71% of the HSV-1 templates specifying abundant RNA and 39% of sequences specifying scarce RNA.

Base Sequence↗
PubMed1970

Properties of RNA transcriptase in reovirus subviral particles.

Subviral particles containing reovirus RNA transcriptase have been isolated from extracts of virus-infected mouse fibroblast cells. The purified particles which lacked the outer protein capsomeres of the mature virion had a buoyant density of 1.43-1.44 g/ml in CsCl and contained all of the double-stranded RNA genome of the intact virus. The particles were free of nuclease activity. RNA synthesis required all four ribonucleoside triphosphates and was dependent on magnesium or manganese; optimal activity required potassium or ammonium ions. In the presence of a ribonucleoside triphosphate regenerating system, reaction rates were linear for 20 hr. RNA yields of 40-fold in excess of input template could be obtained. Completed RNA chains were released from the subviral particles. In the course of RNA synthesis, the double-stranded RNA template was fully conserved. The RNA products formed in vitro displayed profiles in sucrose gradients similar to those found for in vitro reovirus mRNA. The RNA products were single-stranded and did not self-anneal. Over 90 percent of the transcriptase products could be annealed with template double-stranded RNA. The annealed products migrated in acrylamide gels as double-stranded RNA, indicating efficient in vitro transcription.

Centrifugation, Density Gradient↗
PubMedDate not supplied

Portability issues for a structured clinical vocabulary: mapping from Yale to the Columbia medical entities dictionary.

OBJECTIVE: To examine the issues involved in mapping an existing structured controlled vocabulary, the Medical Entities Dictionary (MED) developed at Columbia University, to an institutional vocabulary, the laboratory and pharmacy vocabularies of the Yale New Haven Medical Center. DESIGN: 200 Yale pharmacy terms and 200 Yale laboratory terms were randomly selected from database files containing all of the Yale laboratory and pharmacy terms. These 400 terms were then mapped to the MED in three phases: mapping terms, mapping relationships between terms, and mapping attributes that modify terms. RESULTS: 73% of the Yale pharmacy terms mapped to MED terms. 49% of the Yale laboratory terms mapped to MED terms. After certain obsolete and otherwise inappropriate laboratory terms were eliminated, the latter rate improved to 59%. 23% of the unmatched Yale laboratory terms failed to match because of differences in granularity with MED terms. The Yale and MED pharmacy terms share 12 of 30 distinct attributes. The Yale and MED laboratory terms share 14 of 23 distinct attributes. CONCLUSION: The mapping of an institutional vocabulary to a structured controlled vocabulary requires that the mapping be performed at the level of terms, relationships, and attributes. The mapping process revealed the importance of standardization of local vocabulary subsets, standardization of attribute representation, and term granularity.

Clinical Laboratory Information Systems↗