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N B Raj

Publications and source records attributed to N B Raj.

At least 37 records · Page 2Linked to original sources
PubMed1988

Human fibroblast but not lymphoid cells have unusually long polyadenylated interferon-beta 1 mRNAs.

The transcription unit of human interferon-beta 1 (IFN-beta 1) mRNA was examined by chain elongation of nascent RNA in isolated nuclei of human fibroblasts and lymphoid cells induced to produce IFN. In fibroblasts, transcription proceeds beyond 2,400 nucleotides downstream from the poly(A) site of mature mRNA and appears to terminate in the region rich in Alu sequences. Northern hybridization showed the presence of a minor polyadenylated RNA species, about 3,200 nucleotides long, that hybridized to the probes derived from 3'-flanking regions of IFN-beta 1 mRNA. S1 nuclease analysis established that this long polyadenylated transcript represents a mixture of three RNA molecules with defined 3' termini. In all three mRNAs, as in mature IFN-beta 1 mRNA, the polyadenylation site was located within a few nucleotides downstream from the AAUAAA hexanucleotide consensus sequence. Surprisingly, in Namalva lymphoblastoid cells no transcription beyond the polyadenylation site of mature IFN-beta 1 mRNA could be detected either in isolated nuclei or total RNA.

Base Sequence↗
PubMed1987

Activation of human immunodeficiency virus by herpesvirus infection: identification of a region within the long terminal repeat that responds to a trans-acting factor encoded by herpes simplex virus 1.

Herpes simplex virus 1 (HSV-1) infection induces transcription of the chloramphenicol acetyltransferase (CAT) gene directed by the long terminal repeat (LTR) of human immunodeficiency virus (HIV) in both transiently and permanently transfected cells containing the HIV-LTR/CAT hybrid gene. To define the mechanism by which HSV-1 stimulates the HIV LTR, we examined the effects of isolated regulatory genes from HSV-1. The results of cotransfection assays with the immediate-early (IE) genes of HSV-1, IE110 (ICP0) and IE175 (ICP4), showed that the IE110 protein, either alone or in combination with the IE175 protein, can activate the HIV LTR. Cotransfection with the IE175 gene alone or with the Vmw65 gene (coding for a virion transcription factor) alone did not lead to HIV-LTR activation. The lack of requirement for the IE175 or Vmw65 gene products in transient-expression assays was confirmed in permanent cell lines containing the HIV-LTR/CAT hybrid gene by using temperature-sensitive mutants defective in the IE175 gene product or in uncoating functions. By deletion analysis, we localized a 73-bp-long region (positions -104 to -32) from the HIV LTR that responded to HSV-1 activation; when this region, which is distinct from the previously identified trans-activating responsive (TAR) region, was ligated to a heterologous, HSV-1-nonresponsive gene (alpha 4-interferon/CAT), it conferred inducibility by both HSV-1 infection and IE110/175 cotransfection. Both simian and human cytomegalovirus also induced the HIV-LTR/CAT hybrid gene. However, we failed to detect specific upstream sequence requirements for induction by cytomegalovirus. Our results indicate that infection with unrelated viruses can alter the expression of HIV in an infected cell.

Acetyltransferases↗
PubMed1987

Methylation as a modulator of expression of human immunodeficiency virus.

When Vero or murine cells were stably transfected with the human immunodeficiency virus (HIV) long terminal repeat (LTR) that directs the chloramphenicol acetyltransferase (CAT) gene (pU3R-III-CAT), expression was suppressed. Treatment with the nucleoside analog 5-azacytidine (5-azaC) restored CAT expression. S1 nuclease analysis and a nuclear run-on assay demonstrated that activation of the latent HIV LTR by 5-azacytidine occurred at the transcriptional level. Southern blot analysis demonstrated that this activation was due to the demethylation of cytosine residues in the LTR enhancer. Thus, the HIV LTR appears to be susceptible to transcriptional inactivation by methylation, a process that is proposed to play a modulatory role in viral latency.

Acetyltransferases↗
PubMed1986

Synthesis of fusion and mature murine alpha interferons in Escherichia coli.

Four murine interferon-alpha (MuIFN-alpha) genes (alpha 1, alpha 4, alpha 5, alpha 6) were previously identified and characterized. The coding regions of these IFN-alpha genes were inserted into bacterial expression vectors behind the lpp promoter under the control of the lac promoter-operator region, resulting in fusion peptides containing additional N-terminal amino acids (aa). Plasmids coding for the expression of mature IFN-alpha 1 and alpha 5 were also constructed using the same vector system, by inserting a 30-bp synthetic oligodeoxynucleotide, which contains a stop codon for the lpp gene, a ribosome-binding sequence and an ATG start codon for the IFN peptides. The amounts of IFN polypeptides synthesized in Escherichia coli were estimated in the maxi-cell system and their biological activities were measured on mouse and other mammalian cells. The yields of mature IFN produced in this vector were 2 to 4 X 10(6) units/liter; the antiviral activity of the majority of the MuIFNs on human and bovine cells was 100- to 1000-fold lower than on mouse cells. IFN-alpha 4, which contains an internal deletion of 5 aa, showed a lower antiviral activity than other MuIFNs on mouse cells.

Amino Acid Sequence↗
PubMed1985

Efficient expression in Escherichia coli of two species of human interferon-alpha and their hybrid molecules.

A new type of interferon (IFN)-alpha cDNA (IFN-alpha I') was identified in a cDNA library constructed from Namalva cells infected with Sendai virus. The nucleotide sequence of this cDNA showed homology, with the exception of two nucleotides in the coding region, with the previously identified IFN-alpha I gene (Lawn et al., 1981). An expression plasmid which directs the synthesis of the mature IFN-alpha I' peptide was constructed using vectors carrying the lpp/lac promoter and "runaway" replicon. Furthermore, hybrid genes were constructed by in vitro recombination of IFN-alpha I' and IFN-alpha A at a common restriction endonuclease site located at amino acid positions 121-122. While the specific antiviral and anticellular activities of IFN alpha A and IFN-alpha I' on human cells were comparable, the antiviral activity of IFN-alpha I' on mouse cells was 125-fold higher than that of IFN-alpha A. The specific antiviral activities of the hybrid IFNs on human and bovine cells were similar to that of the amino-terminal parental IFN peptide, while the anticellular activities on human cells of the alpha A/alpha I' hybrid were higher and that of the alpha I'/alpha A hybrid were lower than the parental IFN-alpha A and IFN-alpha I'.

Amino Acid Sequence↗
PubMed1985

Differential regulation of interferon synthesis in lymphoblastoid cells.

We have examined the molecular mechanisms involved in the induction and regulation of expression of alpha and beta 1 human interferons (HuIFN) in Namalva cells. Cloned IFN-alpha and -beta 1 cDNAs, and antisera to purified IFN-alpha and -beta 1 were used as specific probes to determine the expression of HuIFN genes both on the RNA and protein levels. The rates of gene transcription were correlated with the relative levels of HuIFN mRNA present in induced cells and with the amounts of HuIFN peptides synthesized by these cells. The comparative rate of transcription of HuIFN-alpha and -beta 1 genes was measured in nuclei isolated from Namalva cells before and after induction. No transcription of HuIFN-alpha and -beta 1 genes was detected in nuclei isolated from the uninduced cells. The correspondence in the rate of HuIFN-alpha and -beta 1 genes transcription after virus infection with the relative levels of HuIFN mRNA in the induced cells indicates that the stimulation of HuIFN synthesis by viral infection results from the activation of the transcription of HuIFN genes. The relative levels of alpha and beta 1 induced transcripts were the same in spite of the differences in the number of copies of HuIFN-alpha and -beta 1 genes indicating that the beta 1 gene is transcribed more efficiently than the alpha genes. The steady-state levels of HuIFN-alpha and -beta 1 mRNAs in induced Namalva cells are comparable, however, the overall amount of HuIFN-beta 1 synthesized (as determined by radioimmunoassay and biological activity) is approximately 10-fold lower than that of IFN-alpha. No evidence has been found that would indicate that HuIFN-beta 1 mRNA induced in Namalva cells is different from that induced in human fibroblasts. The data indicate, however, that in Namalva cells, the IFN-beta 1 polypeptide has a higher turnover rate and slower rate of release into medium than the HuIFN-alpha polypeptides, indicating that the observed difference in the overall amounts of these two types of interferons present in the medium is due to regulation on posttranslational level.

Animals↗
PubMed1983

Two levels of regulation of beta-interferon gene expression in human cells.

We cloned alpha- and beta-interferon cDNA and used them as specific probes to determine the relative levels of interferon mRNA in human fibroblasts cells induced with poly(rI).poly(rC) or Newcastle disease virus to synthesize interferon. Both inducers activated only the beta-interferon gene; however, the half life of beta-interferon mRNA in cells induced with virus was substantially longer than in poly(rI).poly(rC)-induced cells. The transcription rate of beta-interferon RNA sequences was examined in nuclei isolated from poly(rI).poly(rC)-induced cells; it was found that the induction leads to transcriptional activation of the beta-interferon gene and that the shutoff period when no interferon synthesis or cytoplasmic betamRNA are detected. Thus, the synthesis of beta interferon in poly(rI).poly-(rC)-induced human fibroblasts is controlled both by activation of transcription of the beta-interferon gene and by alteration of the beta-interferon mRNA stability.

Cell Line↗
PubMed1982

Induction of human beta-interferon synthesis with poly(rI . rC) in mouse cells transfected with cloned cDNA plasmids.

Human genomic DNA and plasmids carrying portions of the cDNA gene for human beta-interferon have been introduced into mouse Ltk- cells by cotransfection with a herpes simplex virus thymidine kinase (TK) gene. One plasmid contains 840 base pairs of human DNA complementary to pre-beta-interferon mRNA inserted into pBR322, whereas the other plasmids have hybrid genes containing only the 560-base pair coding region inserted under the transcriptional control of the TK promoter. Constitutive interferon production could not be detected in any of the mouse TK+ cell lines tested. Nevertheless, synthesis of interferon could be induced by poly(rI . rC) treatment in at least 16 of these cell lines, including clones transfected with genomic DNA, the beta-interferon cDNA, and the TK-beta-interferon cDNA hybrid gene. The interferon produced was specific for human cells and could be neutralized by antiserum against human beta-interferon. In contrast to human fibroblast cells, in which the synthesis of induced beta-interferon is transient, the poly(rI . rC)-induced TK+ lines continued to produce beta-interferon for prolonged periods of time and did not respond to superinduction conditions. Therefore, in transfected mouse cells, the coding DNA sequence from the human beta-interferon gene, without any of the adjacent 3' or 5' flanking human DNA sequences, was sufficient both to direct synthesis of biologically active product and to respond to the specific induction system that operates in human cells. However, the mechanism that switches off the synthesis of induced interferon in human cells appears not to operate in mouse cells transfected with beta-interferon cDNA.

Cell Line↗
PubMed1982

Radioimmunoassay of human beta interferon.

A solid phase radioimmunoassay (RIA) for human fibroblast-derived beta (beta) interferon is described using dinitrophenylated (DNP) IgG from rabbit anti-interferon serum and quantitation of the antigen-bound antibodies by subsequently measuring the attachment of 125I-labeled anti-DNP. The sensitivity limit of the assay is 50 u of interferon corresponding to 0.5 ng of interferon protein. Preparations of alpha leukocyte-derived interferon show only marginal cross-reactivity (less than 0.01%) while the mouse interferons (mixture of alpha and beta)are negative in this assay. The simplicity, reproducibility and the possibility of using 125I-labeled anti-DNP as a universal reagent for different anti-interferon sera may widen the development of RIA tests for interferons both in research and clinical areas.

Animals↗
PubMed1982

Human beta interferon gene localization and expression in somatic cell hybrids.

The human fibroblast interferon gene beta 1 was mapped to human chromosome 9. Sequence homology with a beta 1 cDNA clone was detected in both genomic DNA and induced mRNA of human/mouse or human/hamster somatic cell hybrids containing human chromosome 9, but not in lines lacking this chromosome or those retaining a complex translocation involving chromosomes 9 and 11. Interferon mRNA that did not share sequence homology with the beta 1 cDNA clone was detected in lines containing human chromosomes 2 and 5 but lacking chromosome 9, suggesting the presence of other unlinked interferon sequences in the human genome.

Animals↗
PubMed1981

Analysis of interferon mRNA in human fibroblast cells induced to produce interferon.

The levels of interferon mRNA as a function of interferon induction by poly(rI) . poly(rC) in human fibroblast cells were determined by RNA hybridization using a cloned beta interferon cDNA and by translation in Xenopus oocytes. Whereas previous studies analyzed mixtures of interferons, the availability of the cloned beta interferon cDNA and the antiserum to purified beta interferon enabled us to focus on the expression of only one class (beta) of interferon genes. The induction of interferon synthesis depends primarily on the accumulation of interferon beta mRNA in the cells, and the interferon beta mRNA rapidly disappears several hours after its appearance in the cytoplasm. No detectable interferon beta mRNA sequences are present in uninduced cells. The degradation of interferon beta mRNA in the induced cells requires ongoing protein synthesis; accumulation of interferon beta mRNA was observed in the continuous presence of cycloheximide. The interferon beta mRNA detected at the early stages of induction is 1100 nucleotides long and its size progressively decreases with time. By both the hybridization and the translational assay in Xenopus oocytes, only one size of interferon beta mRNA and one species of beta interferon could be identified.

Cells, Cultured↗
PubMed1981

Interferon heterogeneity resulting from differences in glycosylation.

35S methionine-labeled interferon synthesized in mouse C-243 induced by NDV in the presence and absence of glycosylation was analyzed after immunoprecipitation by gel electrophoresis. Interferons produced in the induced cells show large size heterogeneity in the region of 24,000-35,000-daltons. Interferon synthesized in the tunicamycin treated induced cells show two major bands of 15,000 and 18,000 daltons, which synthesis was also coded for by the interferon mRNA in cell-free system. The results indicate that the size heterogeneity of mouse interferons is due to the variability in the carbohydrate moiety of interferon polypeptides (alpha, beta).

Animals↗
PubMed1980

The messenger RNA sequences in human fibroblast cells induced with poly rI.rC to produce interferon.

Induction of human fibroblast cells with poly rI.rC induces interferon mRNA which can be translated into interferon precursor in wheat germ cell free system or in Xenopus oocytes into biologically active interferon. The extent of gene expression in the poly rI.rC induced cells was compared to that of the uninduced cells by hybridization of the mRNA to complementary DNA. Homologous template driven hybridization of cDNA revealed the presence of two clearly defined transitions in the total poly A RNA from the induced cells; abundant class and a scarce class comprising approximately 37,000 diverse species of RNA. Heterologus hybridization of the cDNA with total uninduced mRNA showed that the majority of the mRNA sequences are the same in both the induced and uninduced cells. The results of the hybridization using cDNA prepared to the fraction enriched for interferon mRNA, however, showed that about 4% of the sequences present in the interferon enriched fraction are not present in the uninduced cells. These differences may result from the poly rI.rC induced alterations in gene expression.

Fibroblasts↗
PubMed1980

Synthesis of new proteins associated with the induction of interferon in human fibroblast cells.

The relative amounts of translatable cellular mRNAs and newly synthesized cellular proteins were examined in poly(I) x poly(C)-induced human fibroblast cells early during induction. At this time interferon and interferon mRNA synthesis are maximal and cells have not acquired their antiviral thesis are maximal and cells have not acquired their antiviral state. Translation of the mRNA from poly(I) x poly(C)-induced cells in a wheat germ cell-free system led to the synthesis of a [35S]methionine-labeled 22,000-dalton protein that is precipitated by antiserum to highly purified human fibroblast interferon. The synthesis of this protein was detected only with the mRNA preparations that, when translated in Xenopus oocytes, coded for the synthesis of biologically active human interferon. Two-dimensional gel analysis of the [35S]methionine-labeled polypeptides translated from the total mRNA of the induced and uninduced cells revealed the presence of 23 new proteins that were translated from mRNAs of the induced cells but not from the mRNAs of the controls. These polypeptides ranged from 15,000 to 70,000 daltons. Thirteen of these proteins were detected in induced cells labeled with [35S]methionine. It is concluded that, in human fibroblasts, poly(I) x poly(C) induces, in addition to interferon, the synthesis of a variety of "interferon-associated" proteins.

Cell-Free System↗
PubMed1977

Relationship between interferon production and interferon messenger RNA synthesis in human fibroblasts.

Poly(A) containing mRNA prepared from poly(rI)-poly(rC)-induced human fibroblasts stimulated [14C]leucine incorporation into protein in wheat germ cell-free extracts. For the translation of interferon mRNA into a biologically active product, the presence of spermine was essential. The protein synthesized in vitro fulfilled the criteria for human interferon--namely, its antiviral activity was species specific, and its activity was completely neutralized by antiserum to human fibroblast interferon. The amount of interferon synthesized in human fibroblasts induced by poly(rI)-poly(rC) (normal induction) and poly(rI)-poly(rC) in the presence of cycloheximide (superinduction) was compared to the amount of translatable interferon mRNA both in the wheat germ cell-free system and the Xenopus oöcyte system. Although the production of interferon after the termination of transcription by actinomycin D was markedly increased in superinduced cells, the measurable amount of interferon mRNA as assayed in the oöcyte system was only slightly higher in superinduced cells than in cells induced with poly(rI)-poly(rC) alone. When compared in the wheat germ cell-free system, however, the translational product of mRNA preparation from cells induced with poly(rI)-poly(rC) alone was inactive while that from superinduced cells was active.

Animals↗
PubMed1976

Effects of alpha-amanitin, cycloheximide, and thioacetamide on low molecular weight nuclear RNA.

Studies were made on the effects of alpha-amanitin, cycloheximide, and thioacetamide on synthesis and content of low molecular weight nuclear RNA. Cycloheximide, an inhibitor of protein synthesis and the synthesis of 45S pre-rRNA and 5S RNA, also inhibited synthesis of nuclear U1 and U3 RNAs. alpha-Amanitin, an inhibited the synthesis of U1 and U2 low molecular weight nuclear RNA. Thioacetamide, which induces nucleolar hypertrophy and increased nucleolar RNA polymerase activity, markedly increased synthesis of 5.8S RNA and U3 RNA. These results show that syntheses of individual low molecular weight nuclear (LMWN) RNAs are controlled by different regulatory mechanisms. In particular, there appears to be a specific relationship between U3 RNA and functional states of the nucleolus.

Acetamides↗