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S Chen-Kiang

Publications and source records attributed to S Chen-Kiang.

33 records · Page 2Linked to original sources
PubMed1989

Interleukin 6 induces secretion of IgG1 by coordinated transcriptional activation and differential mRNA accumulation.

The molecular mechanism by which interleukin 6 (IL-6) induces terminal differentiation of B cells was investigated in a subpopulation of the clonal human B-lymphoblastoid cell line CESS selected for high density of cell surface IgG1. Induction of CESS cells with IL-6 resulted in a 15-fold preferential accumulation of secreted-specific gamma 1 (gamma 1s) mRNA but not of the alternatively processed membrane-specific gamma 1 (gamma 1m) mRNA. Similarly, microseconds mRNA but not the microns mRNA of the nonproductively rearranged mu heavy-chain allele was also increased. Accompanying the differential accumulation of gamma 1s mRNA was a 4.5-fold increase in lambda light-chain mRNA, leading to secretion of IgG1. Analyses of transcription in isolated nuclei demonstrated that transcriptional activation was the primary mechanism for quantitative increase of immunoglobulin mRNAs (5.5-fold for gamma 1 and mu and at least 2-fold for lambda). Since polymerase loading is diminished by 75% before reaching the downstream gamma 1m polyadenylylation site in CESS cells, irrespective of IL-6 induction, transcriptional pausing/termination appears intrinsic and contributes to the selection of gamma 1s and gamma 1m polyadenylylation sites in activated B cells. Furthermore, differential mRNA stabilization is likely to contribute to the alteration of the gamma 1s/gamma 1m mRNA ratio at IL-6 induction.

B-Lymphocytes↗
PubMed1987

Productive infection of cultured human lymphoid cells by adenovirus.

We investigated infection of cultures from established human B- and T-cell lines by adenoviruses. Infection by adenovirus type 2 or 5 was productive by the criteria of viral DNA replication, RNA synthesis, immunofluorescent staining of viral proteins, and assembly of biologically active virions. Whereas the kinetics of infection were reproducible and characteristic for each cell line, there appeared to be no correlation between the kinetics of infection and the origin from which the cell lines were established. In a myeloma and a T-cell line, the kinetics of infection approached those in HeLa cells. The presence of the Epstein-Barr virus genome in B lymphoid cells was not a prerequisite for adenoviral infection. Furthermore, expression of the E1A gene was repressed in myeloma cells in comparison with HeLa cells.

Adenoviruses, Human↗
PubMed1986

Cell-type-specific synthesis of murine immunoglobulin mu RNA from an adenovirus vector.

The mouse immunoglobulin heavy-chain mu constant region gene was cloned into the early region 1B of an adenovirus type 5 vector to allow reproducible kinetics of expression of the mu gene in the presence of continuous host protein synthesis after infection by the recombinant. The immunoglobulin-adenovirus recombinant is helper independent in infecting human fibroblastic and B- and T-cell lines and expresses mu in a cell-type-specific manner. By Northern blot analysis, correctly polyadenylated and spliced E1B-mu S and E1B-mu m mRNAs are found to be equally abundant at steady state in fibroblasts. In contrast, and appropriately, only E1B-mu S mRNAs accumulate in a lambda light-chain-secreting myeloma cell line. Analysis of nascent transcripts pulse labeled in isolated nuclei demonstrates equimolar polymerase loading throughout the mu region in all cell types infected by mu-Ad. Thus, correct polyadenylation and splicing of E1B-mu S and E1B-mu m in fibroblasts does not require transcription termination in the region separating the mu S and mu m polyadenylation sites. Furthermore, differential expression of mu transcripts in the background of myeloma cells is regulated at the level of RNA processing and does not require the presence of the immunoglobulin heavy-chain enhancer or promoter element.

Adenoviruses, Human↗
PubMed1984

Pausing and premature termination of human RNA polymerase II during transcription of adenovirus in vivo and in vitro.

The major late transcriptional unit of adenovirus type 2 has served as a model for studying transcription in eukaryotes. We report that pausing and premature termination are intrinsic to the transcription of this transcriptional unit by RNA polymerase II. In vivo and in isolated nuclei, transcription pauses at discrete sites proximal to the initiation site and can prematurely terminate at nucleotide 175 and possibly also at nucleotide 120. The prematurely terminated RNAs are not associated with the transcription complexes and accumulate in the cell nucleus in vivo, whereas paused RNAs remain associated with the transcription complexes and elongate into full-length transcripts. Pausing is also reproduced in the transcription complexes in a soluble system. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole enhances pausing but not premature termination, and its action is reversible. The proposed premature termination site at nucleotide 175 in adenovirus type 2 bears sequence homology to the tR1 site in coliphage lambda.

Adenoviruses, Human↗
PubMed1984

Premature termination by human RNA polymerase II occurs temporally in the adenovirus major late transcriptional unit.

We have recently demonstrated pausing and premature termination of transcription by eucaryotic RNA polymerase II at specific sites in the major late transcriptional unit of adenovirus type 2 in vivo and in vitro. In further developing this as a system for studying eucaryotic termination control, we found that prematurely terminated transcripts of 175 and 120 nucleotides also occur in adenovirus type 5-infected cells. In both cases, premature termination occurs temporally, being found only during late times of infection, not at early times before DNA replication or immediately after the onset of DNA replication when late gene expression has begun (intermediate times). To examine the phenomenon of premature termination further, a temperature-sensitive mutant virus, adenovirus type 5 ts107, was used to uncouple DNA replication and transcription. DNA replication is defective in this mutant at restrictive temperatures. We found that premature termination is inducible at intermediate times by shifting from a permissive temperature to a restrictive temperature, allowing continuous transcription in the absence of continuous DNA replication. No premature termination occurs when the temperature is shifted up at early times before DNA replication. Our data suggest that premature termination of transcription is dependent on both prior synthesis of new templates and cumulative late gene transcription but does not require continuous DNA replication.

Adenoviridae↗
PubMed1982

Transcription and accurate polyadenylation in vitro of RNA from the major late adenovirus 2 transcription unit.

Nucleoprotein complexes with in vitro transcription activity were isolated from HeLa cells late in lytic infection with Adenovirus type 2 (Ad2). Both polymerase II and polymerase III were active in these extracts, and greater than 85% of the labeled RNA was Ad2-specific. Electrophoretic analyses and Southern blot analyses demonstrated that RNA complementary to the entire 30 kb late transcription unit including RNA near the presumed termination site was synthesized. The addition of DRB (5,6,dichloro-1-beta-D-ribofuranosyl-benzimidazole) in vivo prior to the isolation of the complexes resulted in accumulation of polymerase II at the promoter proximal sites, but nascent chains started in vivo in DRB were successfully elongated in vitro. Approximately 10% of the RNA labeled in vitro contained poly(A), and the length of poly(A) was very similar to that of nuclear RNA isolated from Ad2-infected cells. The in vitro sites of poly(A) addition were specific--labeled poly(A)-terminated RNA molecules ended at a point on the genome coincident with previously mapped poly(A) sites of mRNAs produced in vivo. In addition, the polyadenylation enzyme (or enzymes) cosediment with the nucleoprotein complexes during sucrose gradient centrifugation since gradient purified complexes synthesize poly(A) containing RNA in vitro in the absence of any added nuclear extract.

Adenoviruses, Human↗
PubMed1980

The addition of 5' cap structures occurs early in hnRNA synthesis and prematurely terminated molecules are capped.

After cells were labeled by brief exposure to 3H-methyl-L-methionine, the majority of labeled 5' terminal cap I (m7GpppN1mpN2p) oligonucleotide structures were in nuclear RNA (hnRNA) molecules approximately 750 nucleotides or less in length. After longer label times, the proportion of cap I structures in nuclear molecules longer than mRNA rose to approximately 60% of the total, but approximately 40% of the cap I structures were still in molecules shorter than approximately 750 nucleotides. The cap I structures in both long and short hnRNA chains contained all four 2' methylated nucleotides in the N1 position in about the same proportion as in mRNA. None of the large hnRNA molecules could be demonstrated to contain 5' pppX p termini; the only such terminus in high molecular weight RNA was pppAp which was decreased markedly by low doses of actinomycin and is presumably the terminus of pre-rRNA. These results raise the possibilities that hnRNA chains can initiate with any of the four nucleotides, that capping occurs very close to or at the start of hnRNA chain synthesis and that approximately 40% of the hnRNA chains may be prematurely terminated.

Animals↗
PubMed1978

Expression of collagen biosynthetic activities in lymphocytic cells.

Immunoglobulin-producing Merwin plasma cells, MPC-11, have been found to contain proplyl hydroxylase (prolyl-glycyl-peptide,2-oxoglutarate:oxygen oxidoreductase; EC 1.14.11.2) activity and its crossreacting protein, as well as hydroxyproline and a collagenous protein that could not be classified as type I, II, or III collagen. Friend leukemic cells, on the other hand, contained only prolyl hydroxylase. Thymus-derived (T) lymphocytes and bone-marrow-derived (B) lymphocytes freshly isolated from BALB/c mice expressed low but significant prolyl hydroxylase activity. Upon stimulation with phytohemagglutinin, the enzyme activity in T cells increased 22- to 29-fold. Crossreacting protein was also increased and appeared more stable than the prolyl hydroxylase. The effect of lipopolysaccharide stimulation on B cells uas similar but not as pronounced. Thus, even when not accompanied by other collagen biosynthetic activities, prolyl hydroxylase is present in all cells of hematologic origin.

Animals↗
PubMed1977

Homology between a prolyl hydroxylase subunit and a tissue protein that crossreacts immunologically with the enzyme.

A protein, enzymatically inactive but immunologically related to prolyl hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase; EC 1.14.11.2) (cross-reacting protein), has been purified to near homogeneity from skin of newborn rats. The purified protein has a molecular weight of 60,000 on gel filtration and sodium dodecyl sulfate gel electrophoresis, corresponding to that of the smaller of the two dissimilar subunits of the enzyme. The two subunits of prolyl hydroxylase differ markedly from one another in their amino acid compositions, but crossreating protein and the smaller subunit are very similar in composition. On antibody-affinity chromatography both subunits reacted with the antibody developed against the intact enzyme. Neither crossreacting protein nor the 60,000 molecular weight subunit was adsorbed onto concanavalin A, which adsorbed the intact enzyme as well as the larger subunit. It would appear that crossreacting protein is identical to one of the subunits of prolyl hydroxylase or metabolically related to it.

Amino Acids↗