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J Trapman

Publications and source records attributed to J Trapman.

125 records · Page 7Linked to original sources
PubMed1985

Organization, structure and expression of murine interferon alpha genes.

Using a human interferon-alpha probe we have isolated recombinant phages containing murine interferon-alpha (Mu IFN-alpha) genes from a genomic library. One of these phages contained two complete Mu IFN-alpha genes and part of a third gene. The insert of a second phage held two IFN genes. This indicates that the Mu IFN-alpha genes are clustered in the genome as is the case for the analogous human genes. The nucleotide sequences of these 5 genes were determined. They show that the genes are all different, albeit highly homologous. The deduced amino acid sequences show that four of the five genes contain a putative glycosylation site. Three genes were transiently expressed in COS cells and they gave rise to protein products showing antiviral properties. The expression of the five Mu IFN-alpha genes and the Mu IFN-beta gene was studied in virus-induced mouse L cells. The individual mRNAs were visualized in a nuclease S1 experiment, using a specific probe for each gene. In RNA preparations from induced cells mRNAs for each of the five alpha genes and the beta gene were present. However, substantial differences in the amounts of the individual mRNAs were observed.

Animals↗
PubMed1985

Mouse interferon alpha and beta genes are linked at the centromere proximal region of chromosome 4.

In order to determine the chromosomal localization of the murine interferon-alpha (MuIFN-alpha) and murine interferon-beta (MuIFN-beta) genes the DNAs of a panel of somatic cell hybrids were analysed by Southern blot hybridization. The hybrid cells were derived from E36 Chinese hamster cells and GRSL or GR MaTu mouse cells and retained all hamster chromosomes but segregated mouse chromosomes. The MuIFN-alpha probe used was a 0.7 kb HindIII-EcoRI fragment derived from the MuIFN-alpha 1 gene which hybridized with both mouse and hamster DNA. However, four fragments present in EcoRI digests of mouse DNA were clearly absent from the hybridization profile of EcoRI-digested hamster DNA and could be used for detection of MuIFN-alpha sequences in the hybrid cells. The MuIFN-beta probe, a 0.5 kb BglII-BamHI fragment derived from the MuIFN-beta gene, hybridized with a 2.6 kb EcoRI fragment of mouse DNA and only weakly cross-hybridized with a 4.8 kb EcoRI fragment in hamster DNA. Southern blot analysis of DNA from mouse/hamster hybrids compared with the analysis of chromosome markers showed that both the MuIFN-alpha and the MuIFN-beta genes are located on chromosome 4. Analysis of DNA from hybrids that contained only part of chromosome 4 indicated that the MuIFN-alpha gene family and the MuIFN-beta gene are situated at the centromere-proximal region of the chromosome.

Animals↗
PubMed1985

Constitutive expression of a murine interferon alpha gene in hamster cells and characterization of its protein product.

The coding part of a murine interferon alpha (MuIFN-alpha) gene was cloned into an expression plasmid containing the simian virus 40 early promoter and the rabbit beta-globin polyadenylation signal. This construct was transfected into Chinese hamster ovary cells, together with a plasmid containing the Ecogpt gene as a selection marker. Resulting colonies were assayed for constitutive interferon production and analysed for integration of MuIFN-alpha genes. There was no obvious correlation between the number of genes integrated and the amount of interferon produced. The highest producer, designated CHO-pSV10EF-3, contained four copies of the mouse gene and constitutively secreted up to 100 000 International Units of interferon per ml per day. The MuIFN-alpha subspecies produced by this clone was characterized by analysis of its antiviral activity on heterologous cells, heparin-Sepharose affinity chromatography and chromatofocusing. The results obtained indicate that it is identical or closely related to a minor component present in conventional MuIFN-alpha preparations.

Animals↗
PubMed1984

Isolation and characterization of subspecies of murine interferon alpha.

Interferon produced by mouse L-929 cells by incubation with poly(rI).poly(rC) is known to be composed of a mixture of MuIFN-alpha and MuIFN-beta. The alpha component was separated from the bete species by affinity chromatography over a monoclonal anti-MuIFN-beta agarose column and partially purified by gel filtration. MuIFN-alpha, prepared by this method was separated into at least five subspecies by chromatofocusing. The approximate pI values of these components are greater than or equal to 7.5, 6.5, 6.2, 5.9 and 5.6, respectively. Component 3 (pI 6.2) was the most prominent subspecies present in our MuIFN-alpha preparations, representing 40 to 50% of the total antiviral activity. Component 1 (pI greater than or equal to 7.5) which accounted for about 5% of the antiviral activity on mouse cells, differed in some properties from the other interferon subspecies. It showed a relatively high antiviral activity on heterologous cells and it was eluted from a Sephadex column after the other alpha subspecies. Furthermore, it showed a diminished binding to heparin as compared to the other MuIFN-alpha subspecies, indicating a lower affinity for polynucleotides.

Animals↗
PubMed1983

Large-scale, one-step purification of murine interferon-beta using a monoclonal antibody.

A monoclonal antibody against murine interferon-beta (MuIFN-beta) was prepared using standard methods. Antibodies were immobilized by coupling to Sepharose and used for large-scale purification of poly(I) . poly(C)-induced mouse L cell IFN. Antibodies isolated from the serum of one nude mouse which was transplanted with the anti-MuIFN-beta antibodies producing hybridoma were able to bind at least 7 X 10(7)U MuIFN-beta. In one single antibody affinity chromatography step MuIFN-alpha was separated from MuIFN-beta and a 1000-fold purification of MuIFN-beta was obtained. The purified material had a specific activity of 5 X 10(8)U/mg protein. The recovery from the antibody column was 100%. SDS-PAGE analysis of the purified material revealed the presence of one single protein band with a molecular weight of 33 kD, representing MuIFN-beta.

Animals↗
PubMed1982

Evidence for a protective role of interferon in resistance to murine cytomegalovirus and its control by non-H-2-linked genes.

Murine cytomegalovirus (MCMV) induces rapid production of a partially pH 2-stable type 1 interferon, the serum level of which is controlled by non-H-2-linked host genes. The production of high, intermediate, and low levels of interferon was found in C3H/He, C57BL/10, and BALB/c mice, respectively, and the use of H-2 congenic mice on the BALB/c or C57BL/10 background showed that H-2-associated genes were not involved. Administration of large (up to 200,000 U) daily doses of partially purified type 1 (alpha plus beta) interferon failed to protect low-producer BALB/c or BALB.K strains from lethal infection. Treatment of the higher (C3H/He) or intermediate (C57BL/10) producer strains with anti-type 1 interferon antibody significantly reduced their resistance to the virus; however, such treatment had no effect on the low-producer BALB/c strain. The decreased resistance of anti-interferon-treated C3H/He mice was accompanied by a transient reduction in serum interferon titers, decreased activation of natural killer cells, a markedly enhanced viremia, and increased viral titers in the liver. These data strongly support a protective role of interferon in defense against MCMV in certain strains of mice. Furthermore, these data suggest that previous observations of a correlation of non-H-2-linked, genetically determined resistance to MCMV with activation of natural killer cells may have its basis in the genetic control of interferon induction by MCMV.

Animals↗
PubMed1981

The effect of high doses of poly(I).poly(C) induced mouse L cell interferon on Rauscher leukemia virus induced erythroleukemia in BALB/c mice.

The effect of high doses of poly(I).poly(C) induced mouse L-cell interferon on the development of Rauscher murine leukemia virus (R-MuLV)-induced erythroleukemia in BALB/c mice was determined. Female mice, 4 to 5 weeks old, were infected with R-MuLV and treated with interferon every 24 h starting 6 h after virus inoculation. Under these conditions injection of 3-5 X 10(4) units of interferon caused a partial inhibition of the leukemia process. Daily application of 3 X 10(5) units completely or almost completely inhibited the erythroleukemia. After 14 days of treatment with these high doses of interferon, spleen weights of interferon-treated infected mice were comparable to those of uninfected animals which received only interferon. Also, no Rauscher cells in spleens and livers of R-MuLV-infected interferon-treated infected animals could be demonstrated and the spleen structure was well preserved in these mice. In interferon-treated infected animals no virus could be detected in the serum as judged from the absence of reverse transcriptase activity in the serum. Moreover, no virus-infected cells could be demonstrated in spleen or liver as deduced from negative immunofluorescence data using anti-p30 and anti-gp70 sera. No virions budding from spleen cell membranes were seen by electron microscopic studies. However, when interferon treatment was stopped the leukemic process was reactivated and all the mice died. In control experiments interferon caused an inhibition of red blood cell formation and a 50 to 100% enlargement of the spleen. Pharmacokinetic data showed that, after intraperitoneal inoculation, maximum amounts of interferon were present in the peripheral blood after 1-2 h. After 12-24 h almost all interferon activity had disappeared from the blood.

Animals↗
PubMed1976

Maturation of ribosomes in yeast. I Kinetic analysis by labelling of high molecular weight rRNA species.

To study the maturation of ribosomes in Saccharomyces carlsbergensis, protoplasts were pulse labeled with [5-3H]uridine at 15 degrees C. Investigation of the cellular location of pulse-labelled ribosomal RNA precursor and mature ribosomal RNA shows that both the 37-S precursor RNA, common to both 17-S and 26-S rRNA, as well as the 29-S RNA, the direct precursor of 26-S rRNA, are located in the nucleus. Most of the 18-S RNA, the direct precursor of 17-S rRNA, is found in the cytoplasmic fraction. Apart from 37-S and 29-S RNA the nucleus also contains an appreciable amount of 26-S rRNA as well as a small quantity of 18-S RNA. These data indicate that processing of 29-S to 26-S RNA occurs in the nucleus, whereas the conversion of 18-S RNA to 17-S rRNA takes place in the cytoplasm. The kinetics of appearance of pulse-labelled 26-S and 17-S rRNA in the various cytoplasmic ribosomal particles indicate, that newly formed 40-S ribosomal particles are almost immediately incorporated into 80-S ribosomes and polysomes. On the other hand, there appears to exist a fairly large cytoplasmic pool of newly synthesized ribosomal particles containing 26-S rRNA and sedimenting at about 60 S. The kinetics of appearance of newly formed 26-S and 17-S rRNA in mature ribosomes show that the maturation of the large ribosomal subunit takes about twice as much time as that of the small subunit.

Cell Fractionation↗
PubMed1976

Maturation of ribosomes in yeast. II. Position of the low molecular weight rRNA species in the maturation process.

Yeast protoplasts were pulse labelled with [5-3H] uridine and the labelling kinetics of the low molecular weight rRNA species were determined in order to gain more insight into the position of those small rRNAs in the process of ribosome maturation. 7-S RNA, the immediate precursor of 5.8-S rRNA, is found to be present only in the nucleus, indicating that the conversion of 7-S to 5.8-S RNA is a nuclear event. 5.8-S rRNA is observed in the cytoplasm almost immediately after its formation. This as well as the presence of only a small amount of 5.8-S RNA in the nucleus, shows that the ribosomal precursor particles of the large ribosomal subunit are very rapidly transported into the cytoplasm once 5.8-S rRNA is formed. Most of the newly synthesized 5-S RNA is found in the nucleus. This nuclear 5-S rRNA is mainly present in the ribosomal precursor particles. However, a small pool of free 5-S rRNA is probably also present.

Cell Fractionation↗
PubMed1975

Detailed analysis of the ribosomal RNA synthesis in yeast.

In order to study the biosynthesis of ribosomal RNA in Saccharomyces carlsbergensis the labelling kinetics of the various precursor and mature rRNA species were determined using pulse-labelling of protoplasts with [5-3H] uridine at 15 degrees C. Label appears almost immediately in 37 S RNA, the precursor common to both 26 S and 17 S rRNA. Labelled 29 S and 18 S RNA, the immediate precursors of 26 S and 17 S rRNA respectively, were found to appear about 4 min and about 8 min after addition of the isotope respectively. These data indicate that the topography of the 37 S precursor RNA is: 5'-17 S -26 S-3'. The pool size of 29 S RNA is about twice as large as that of either 37 S or 18 S RNA, indicating that under the conditions used processing of 18 S to 17 S rRNA proceeds more rapidly than processing of 29 S to 26 S rRNA. The labelling kinetics of 5.8 S rRNA are in agreement with the existence of a 7 S precursor rRNA, the identity of which was previously established (Trapman, J., de Jonge, P. and Planta, R.J. (1975) FEBS Lett. 57, 26--30) and which, in turn, probably is derived from 29 S precursor rRNA. The labelling kinetics of 5 S rRNA suggest that 5 S RNA sequences, rather than also being part of the common 37 S precursor, are located on a separate primary transcription product. Whether this transcript still contains excess sequences remains to be determined. However, because of the rapid appearance of labelled 5 S RNA, such a precursor would have to be very short lived.

Electrophoresis, Polyacrylamide Gel↗
PubMedDate not supplied

Mechanisms of androgen receptor activation and function.

Androgens play a crucial role in several stages of male development and in the maintenance of the male phenotype. Androgens act in their target cells via an interaction with the androgen receptor, resulting in direct regulation of gene expression. The androgen receptor is a phosphoprotein and modulation of the phosphorylation status of the receptor influences ligand-binding and consequently transcription activation of androgen responsive genes. Androgen binding induces a conformational change in the ligand-binding domain, accompanied by additional receptor phosphorylation. Subsequently the liganded androgen receptor interacts with specific androgen response elements in the regulatory regions of androgen target genes, resulting in stimulation of gene expression. Anti-androgens induce a different conformational change of the ligand-binding domain, which does not or only partially result in stimulation of transactivation. Interestingly, different anti-androgens can induce different inactive conformations of the androgen receptor ligand-binding domain. Recent evidence strongly supports a ligand dependent functional interaction between the ligand-binding domain and the NH2-terminal transactivating domain of the androgen receptor. Two regions in the NH2-terminal domain are involved in this interaction, whereas in the ligand-binding domain the AF-2 AD core region is involved.

Androgens↗