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D Skup

Publications and source records attributed to D Skup.

36 records · Page 2Linked to original sources
PubMed1988

An embryonic DNA-binding protein specific for a region of the human IFN beta 1 promoter.

Embryonal carcinoma (EC) cells are unable to make interferon in response to inducing agents. This block disappears after differentiation. We have found that nuclear extracts from undifferentiated P19 EC cells contain a DNA-binding activity which specifically recognizes a region within the human interferon-beta 1 promoter. This activity is absent from differentiated cell types, both of EC and non-EC origin. The binding of the factor in undifferentiated EC cells leads to dramatic changes in the overall protein binding pattern of the interferon promoter as compared with differentiated cells, and may be responsible for repression of the endogenous interferon-beta gene prior to differentiation.

Animals↗
PubMed1988

In vitro synthesis of the active tissue inhibitor of metalloproteinases encoded by a complementary DNA from virus-infected murine fibroblasts.

We have recently described the characterization and expression of a murine gene highly homologous to the human tissue inhibitor of metalloproteinases/erythroid potentiating activity (TIMP/EPA) gene. We have also reported that expression of this gene is regulated in response to virus infection. In the present report we describe the use of a cDNA clone derived from mRNA isolated from Newcastle disease virus-induced murine cells to direct in vitro synthesis of proteins encoded by this murine TIMP/EPA gene. This approach was used to analyze structural and functional parameters of the TIMP/EPA protein. Translation experiments using microsomes revealed a murine protein similar in size to that of human TIMP: Mr of approximately 22,000 for the core protein and 28,000 for the processed protein. Processing in microsomes involved N-glycosylation and cleavage of the signal peptide. Both the processed and unprocessed proteins were able to inhibit degradation of collagen by collagenase but unable to inhibit virus replication. Synthesis of truncated TIMP proteins showed that the collagenase-inhibiting activity was not encoded within a delimited portion of the molecule. This result suggests that conformation is probably essential for TIMP activity.

Animals↗
PubMed1988

Specific induction of cellular gene transcription in herpes simplex virus type 2-transformed cells.

In spite of much work, the mechanism of oncogenic transformation by herpes simplex virus (HSV) is as yet unknown. It has been proposed that HSV type 2 (HSV-2) can transform cells by a 'hit and run' mechanism. In the past we have demonstrated that several polypeptides can be immunoprecipitated from HSV-2-transformed cells, but not from control cells or adenovirus-transformed cells, by rabbit hyperimmune sera to HSV-2. It is possible that the expression of these proteins might be the result of activation of cellular genes during transformation. We have now isolated cDNAs representing transcripts of genes that are expressed at higher levels in HSV-2-transformed hamster embryo fibroblasts than in the parental cells. Cytoplasmic transcripts and genomic sequences homologous to three clones (pAA8, pHD1 and pLC7) were analysed. Northern blot analyses showed that 0.75 kb transcripts which hybridize to the three cDNAs were present in HSV-2-transformed cells and were completely absent or present at low levels in control hamster fibroblasts. These transcripts were not present in mouse cells transformed by other DNA viruses or by a chemical carcinogen. The expression of these transcripts seemed to be confined to certain HSV-2-transformed cell lines. Southern blot analysis suggested that the 0.75 kb transcripts corresponding to these cDNAs may have arisen from a single gene. Nuclear run-off experiments indicated that activation occurred at the level of transcription. The activation of the gene or genes corresponding to these cDNAs may be an integral part of the mechanism of transformation by HSV-2.

Animals↗
PubMed1988

Regulation of interferon gene expression: mechanism of action of the If-1 locus.

We have examined the mechanism of action of the If-1 interferon (IFN) regulatory locus. This locus controls the level of circulating IFN produced in inbred mice in response to intravenous injection of Newcastle disease virus. Mice carrying the If-1h (high) allele show circulating IFN levels 10- to 15-fold higher than those carrying the If-1l (low) allele. In this report we show that induced splenocytes from If-1h and If-1l mice produce IFN at levels which are in the same proportions as those found in the circulation. Higher levels of IFN-specific mRNA were observed in splenocyte populations from If-1h animals. This was due to increased transcription of IFN genes. At the same time, the high- and low-producing populations showed no significant difference in the number of IFN mRNA-containing cells. We conclude that the effect of If-1 in the spleen is to control the levels of transcription of the IFN genes in individual induced splenocytes.

Alleles↗
PubMed1988

Presence of transcription regulatory elements within an intron of the virus-inducible murine TIMP gene.

The expression of the gene for the murine tissue inhibitor of metalloproteinases (TIMP) is induced in response to viruses, growth factors, and phorbol esters. In this report we show that the accumulation of TIMP mRNA after Newcastle disease virus induction is caused by transcriptional activation of the gene. Comparison of the sequences of cDNA and genomic clones along with RNase protection and primer extension analyses revealed that the murine TIMP gene possesses multiple cap sites and that the exon 1 consists exclusively of 5'-noncoding sequences. We observed that DNA regions analogous to those found upstream of the virus-inducible interferon genes are present within intron 1 of the TIMP gene. To investigate the possible role of TIMP intron 1 in gene expression, we used a functional assay based on the transfection of plasmids in which the DNA segment to be tested is placed in proximity to a marker gene driven by the heterologous herpes simplex virus thymidine kinase promoter. Our results indicate that TIMP intron 1 contains DNA sequence elements capable of modulating the activity of a heterologous promoter in two different ways: (i) by enhancing constitutive expression and (ii) by conferring virus inducibility. These results suggest that intron 1 may be involved in the transcriptional regulation of TIMP gene expression.

Animals↗
PubMed1987

Isolation and characterisation of a murine cDNA clone highly homologous to the yeast L29 ribosomal protein gene.

We report here the isolation of a murine cDNA clone (cCL3) which is homologous to the mRNA of the yeast ribosomal protein L29. Comparison of the deduced amino-acid composition of cCL3 to those known for rat ribosomal proteins indicates that this cDNA codes for mammalian ribosomal protein L27'. The gene corresponding to the cDNA is present at approximately 15 copies per genome, some of these probably representing processed pseudogenes. The cDNA hybridizes to an mRNA of 600 nucleotides from various mammals at high stringency, and to an avian transcript of the same size at low stringency. It has been suggested that L29 is involved in peptidyl transferase activity. The strong homology of mammalian L27' to yeast L29 suggests a function which has been conserved throughout evolution, and thus L27' may also be involved in peptidyl transferase activity.

Amino Acid Sequence↗
PubMed1987

Characterization and expression of a murine gene homologous to human EPA/TIMP: a virus-induced gene in the mouse.

A genomic clone encompassing the entire coding region of a murine gene homologous to human erythroid potentiating activity/tissue inhibitor of metalloproteinase (EPA/TIMP) was isolated and sequenced. Based on alignment with human EPA/TIMP cDNAs we deduce a structure comprising five exons and four introns extending over 4.3 kb of DNA. In mouse and hamster cell lines transcription from this gene and interferon genes is induced by Newcastle Disease virus (NDV). Examination of the 5'-flanking sequences of the gene reveals a set of repeated elements with structural similarity to those previously described as inducer-responsive elements in the human IFN-beta 1 gene. The 4.3-kb DNA fragment encompassing the homologous murine EPA/TIMP gene was transfected into human T98G cells and transfectants tested for NDV inducibility. In contrast to the endogenous human gene, the integrated murine EPA/TIMP gene was NDV-inducible and TIMP activity was detectable in the cell culture fluid.

Amino Acid Sequence↗
PubMed1986

Expression of a synthetic human interferon-alpha 1 gene with modified nucleotide sequence in mammalian cells.

We have constructed a recombinant interferon (IFN) gene whose coding region is a chemically synthesized human (Hu)IFN-alpha 1 gene [Edge et al., Nature 292 (1981) 756-762] encoding the same protein as its natural counterpart while possessing important modifications. These modifications were introduced in order to: change codon usage, destroy integrity of repeated and complementary oligodeoxynucleotides in the coding region, introduce an intron, substitute other sequences for the IFN specific 3'- and 5'-flanking sequences and remove the leader sequence (which encodes for the signal peptide). We report here on the transfection and isolation of murine cells which contain low-copy-number insertions of our recombinant gene. These lines maintain a high steady-state level of biologically active HuIFN-alpha in the cytoplasm, produced from mRNAs of Mr expected for correctly processed transcripts. These results show directly that the leader sequence is essential for secretion of IFN proteins. They also argue against a crucial role of the particular features of the transcribed sequence of the natural HuIFN-alpha 1 gene in regulating its expression.

Animals↗
PubMed1984

Changes in the conformation of the interferon beta gene during differentiation and induction.

DNase I sensitivity was used to investigate the chromatin conformation of the interferon beta gene during differentiation of the mouse teratocarcinoma cell line PC13 . These cells do not produce interferon upon viral induction in their undifferentiated state, but do so on differentiation from stem cells to endoderm. Only in induced differentiated cells were the interferon beta genes digested by DNase I. A similar effect was seen in a line of human cells ( MG63 ) upon induction. We conclude that it is induction of interferon production that brings about the change in the DNase I sensitivity of these genes, rather than differentiation.

Animals↗
PubMed1983

Mapping of murine interferon-alpha genes to chromosome 4.

A cDNA library was constructed from polysomal poly(A)+RNA from Newcastle disease virus (NDV)-induced mouse C243 cells, and screened with a human interferon-alpha (HuIFN-alpha) cDNA probe. A cDNA clone for one of the murine interferon-alpha (MuIFN-alpha) genes was isolated, and sequencing analysis revealed that it was a partial copy which is almost identical to the published sequence for the MuIFN-alpha 2 gene. This partial cDNA clone represents a virus-induced message as seen by Northern blot analysis of RNA from NDV-induced C243 cells, and Southern blot analysis of DNA from BALB/c mouse revealed the presence of a multiple IFN-alpha gene family. The MuIFN-alpha genes were mapped to chromosome 4 by Southern blot analysis of hamster/mouse somatic cell hybrid DNAs.

Animals↗
PubMed1982

Molecular cloning of partial cDNA copies of two distinct mouse IFN-beta mRNAs.

Two cDNA libraries were constructed, using respectively the 12S and the 16S sucrose gradient fractions of polysomal poly (A)+ RNA from mouse C243 cells induced with Newcastle disease virus. Screening of a part of both libraries by mRNA selection hybridization assays revealed the presence of two plasmids hybridizing to an mRNA, whose translation product was characterized as mouse IFN-beta. Blot analysis of RNA indicated that mRNA hybridizing to the DNA from both plasmids could be detected in induced but not in uninduced C243 cells. The two cDNA inserts did not cross hybridize and had distinct restriction maps. Sequencing revealed that both inserts represented the end of the coding region and the entire 3' non coding region of two district mRNAs. Although different, the putative 39 AA and 65 AA carboxy termini of both Mu IFN-beta s display some homology to human IFN-beta 1. Thus there are at least two different murine IFN-beta genes.

Amino Acid Sequence↗
PubMed1982

Expression of a chemically synthesized human alpha 1 interferon gene.

Cells of Escherichia coli containing a chemically synthesized human alpha 1 interferon (IFN-alpha 1) gene, under control of the lac promoter, make a product with biological properties indistinguishable from those of the natural IFN-alpha 1 [antiviral activity, acid stability, species crossreactivity, inactivation by antisera directed against leukocyte or Namalwa cell interferon, and stimulation of (2'-5')oligoadenylate synthetase activity]. Similar levels of IFN synthesis were obtained when the expression unit (lac promoter plus synthetic IFN-alpha 1 gene) was transplanted into the obligate methylotroph Methylophilus methylotrophus.

2',5'-Oligoadenylate Synthetase↗
PubMed1981

In vitro translation in reovirus- and poliovirus-infected cell extracts. Effects of anti-cap binding protein monoclonal antibody.

Monoclonal antibodies directed against rabbit reticulocyte cap binding proteins were used as probes to study cap-dependent translation initiation in mock-infected and virus (poliovirus or reovirus)-infected cells. The antibodies inhibited capped mRNA translation, but did not have any effect on the translation of naturally uncapped mRNAs in uninfected cell-free extracts prepared from either L-cells or HeLa cells. Cell-free extracts prepared from either reovirus-infected L-cells or poliovirus-infected HeLa cells did not support capped mRNA translation. However, translation of naturally uncapped mRNAs in the latter extracts was comparable to that observed in extracts from uninfected cells. Thus, translation of mRNAs in extracts from either reovirus- or poliovirus-infected cells proceeds most probably via a cap-independent mechanism, since it is not inhibited by antibodies directed against cap binding proteins.

Animals↗
PubMed1980

Reovirus-induced modification of cap-dependent translation in infected L cells.

The translational apparatus in cell-free extracts prepared from L cells infected with reovirus undergoes a time-dependent transition from cap dependence to cap independence. Extracts from uninfected L cells translate capped reovirus mRNA at high efficiency and synthesize the expected three size classes of reovirus polypeptides, and the translation is sensitive to m7G(5')ppp. This same extract translates uncapped mRNA at a much lower efficiency. In contrast, extracts from infected L cells translate uncapped reovirus mRNA at high efficiency and synthesize the correct three size classes of polypeptides, and the translation is not sensitive to inhibition by m7G(5')ppp. Infected cell extracts translate capped mRNA at reduced efficiency (a,proximately 25%), the translation is not sensitive to inhibition by m7G(5')ppp, and the correct three size classes of viral polypeptides are not synthesized. These observations may explain how reovirus takes over the host translational apparatus.

Animals↗
PubMed1980

mRNA capping enzymes are masked in reovirus progeny subviral particles.

We examined the enzyme activities associated with progeny subviral particles isolated from L-cells infected with reovirus at 12 h postinfection. Activities normally present in reovirus cores were also found to be present in the progeny subviral particles, with the exception of the capping enzymes. The methylase and guanyl transferase activities, which constitute the capping system, were present in a masked form that could be activated by chymotrypsin digestion. The appearance of these progeny subviral particles in infected cells coincided with the time when mRNA synthesis was maximal, suggesting that viral mRNA synthesized at later times is uncapped.

Adenosine Triphosphatases↗
PubMed1980

Reovirus progeny subviral particles synthesize uncapped mRNA.

Reovirus progeny subviral particles were isolated from L-cells at late times postinfection. It has been shown (D. Skup and S. Millward, J. Virol. 34: 490--496, 1980) that these progeny subviral particles have masked capping enzymes, indicating that mRNA synthesized by these particles should be uncapped. When progeny subviral particles were used for mRNA synthesis in vitro, they failed to incorporate the beta-phosphate of [beta-32P]GTP into the 5' terminal. Direct analysis of reovirus mRNA synthesized by progeny subviral particles in the presence of either [alpha-32P]GTP or [alpha-32P]CTP indicated that the 5' terminal was uncapped, having the structure pGpC... The implications of this finding to the reovirus replicative cycle are discussed.

Chymotrypsin↗
PubMed1977

Highly efficient translation of messenger RNA in cell-free extracts prepared from L-cells.

Micrococcal nuclease was used to eliminate endogenous protein synthesis in extracts prepared from L cells. The nuclease can be inhibited subsequently with 2'-deoxythymidine-3', 5'-diphosphate. Nuclease-treated extracts primed with exogenous reovirus mRNA, synthesized full length polypeptides with linear kinetics for almost two hours leading to stimulation of the order of 10(4) times over endogenous background. On the average, between 40 and 50 molecules of polypeptide were synthesized per molecule of mRNA.

Animals↗