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F S Pedersen

Publications and source records attributed to F S Pedersen.

At least 73 records · Page 4Linked to original sources
PubMed1992

Molecular cloning of osteoma-inducing replication-competent murine leukemia viruses from the RFB osteoma virus stock.

We report the molecular cloning of two replication-competent osteoma-inducing murine leukemia viruses from the RFB osteoma virus stock (M. P. Finkel, C. A. Reilly, Jr., B. O. Biskis, and I. L. Greco, p. 353-366, in C. H. G. Price and F. G. M. Ross, ed., Bone--Certain Aspects of Neoplasia, 1973). Like the original RFB osteoma virus stock, viruses derived from the molecular RFB clones induced multiple osteomas in mice of the CBA/Ca strain. The cloned RFB viruses were indistinguishable by restriction enzyme analysis and by nucleotide sequence analysis of their long-terminal-repeat regions and showed close relatedness to the Akv murine leukemia virus.

Animals↗
PubMed1992

Matrix protein of Akv murine leukemia virus: genetic mapping of regions essential for particle formation.

Type C retroviruses assemble at the plasma membrane of the infected cell. Attachment of myristic acid to the N terminus of the Gag precursor polyprotein has been shown to be essential for membrane localization and virus morphogenesis. Here, we report that the matrix (MA) protein contains regions that in conjunction with myristylation are important for Gag protein stability and the assembly of murine leukemia viruses. We identified these domains by generating a series of Akv murine leukemia virus mutants carrying small in-frame deletions within the coding region of the MA protein encompassing 129 amino acids. Studies show that mutants with deletions within the segment encoding the first 102 amino acids were all replication defective, whereas the C-terminal residues 103 to 124 seem not to have any critical function in virus maturation. Cells expressing the replication-defective genomes did not release any detectable Gag proteins. In one mutant, deletion of 3 amino acids in the N terminus resulted in an inefficiently myristylated, stable Gag polyprotein. The remaining defect genomes encoded unstable Gag proteins, although they were modified with myristic acid. The results suggest that the matrix domain plays an important role in stabilizing the Gag polyprotein.

Amino Acid Sequence↗
PubMed1992

Murine helix-loop-helix transcriptional activator proteins binding to the E-box motif of the Akv murine leukemia virus enhancer identified by cDNA cloning.

The enhancer region of Akv murine leukemia virus contains the sequence motif ACAGATGG. This sequence is homologous to the E-box motif originally defined as a regulatory element in the enhancers of immunoglobulin mu and kappa genes. We have used double-stranded oligonucleotide probes, corresponding to the E box of the murine leukemia virus Akv, to screen a randomly primed lambda gt11 cDNA expression library made from mouse NIH 3T3 fibroblast RNA. We have identified seven lambda clones expressing DNA-binding proteins representing two different genes termed ALF1 and ALF2. The results of sequencing ALF2 cDNA suggests that we have recovered the gene for the basic-helix-loop-helix transcription factor A1, the murine analog of the human transcription factor E47. The cDNA sequence of ALF1 codes for a new member of the basic-helix-loop-helix protein family. Two splice variants of ALF1 cDNA have been found, differing by a 72-bp insertion, coding for putative proteins of 682 and 706 amino acids. The two ALF1 mRNAs are expressed at various levels in mouse tissues. In vitro DNA binding assays, using prokaryotically expressed ALF1 proteins, demonstrated specific binding of the ALF1 proteins to the Akv murine leukemia virus E-box motif ACAGATGG. Expression in NIH 3T3 fibroblasts of GAL4-ALF1 chimeric protein stimulated expression from a minimal promoter linked to a GAL4 binding site, indicating the existence of a transcriptional activator domain in ALF1.

3T3 Cells↗
PubMed1991

Tagging the genome of the murine leukemia retrovirus SL3-3 by a bacterial lac operator sequence.

The bacterial lactose operator (lacO) was introduced into the PstI site of the long terminal repeat of the SL3-3 murine leukemia virus, generating a virus, SL3-3lacO, that can replicate in NIH3T3 cell cultures. DNA sequences harboring the lacO sequence might be recovered by molecular cloning in Escherichia coli lac+ lacZ+ using bacteriophage lambda or plasmid vectors. The high copy numbers of the lacO sequence titrate out the lac repressor, leading to the induction of the lac operon in the host. We show here that the lacO and the proviral sequences are carried stably together in the genomes of SL3-3lacO-infected cell cultures and in viral particles. This system is designed to facilitate studies on the provirus and the site of viral integration.

3T3 Cells↗
PubMed1991

Analysis of the significance of two single-base-pair differences in the SL3-3 and Akv virus long terminal repeats.

Two single-base-pair differences between the long terminal repeats (LTRs) of the T-lymphomagenic murine retrovirus SL3-3 and nonleukemogenic Akv virus were tested for effects on activity of the LTRs. Evidence was obtained from electrophoretic mobility shift assays for the presence of at least one factor in both T and non-T cells that bound to the region of the viral enhancers that contained the differences. However, no significant differences in activity in expression assays were detected when the two base-pair differences were exchanged between the two LTRs. Therefore, they do not contribute to the higher activity of the SL3-3 LTR in T-lymphoma cell lines.

Animals↗
PubMed1991

SL3-3 enhancer factor 1 transcriptional activators are required for tumor formation by SL3-3 murine leukemia virus.

The transcriptional enhancers of retroviruses that lack an oncogene are important determinants of their oncogenicity. However, no specific cellular transcriptional activator has yet been found to determine the oncogenicity for any of these viruses. The SL3-3 enhancer factor 1 (SEF1) cellular transcriptional activators are expressed preferentially in T lymphocytes. In the SL3-3 murine leukemia virus enhancer, two different sequences can bind SEF1 activators. We show that mutation of the SEF1 binding sites disrupts the disease potential of SL3-3 murine leukemia virus, implying that SEF1 transcriptional activators are required for tumor induction by SL3-3. The SEF1 site mutations did not appear to affect the pathogenicity of SL3-3 by impairment of virus multiplication, but rather by a specific defect in the ability of neoplastic transformation.

Animals↗
PubMed1990

Determination of transient or stable neo expression levels in mammalian cells.

We report an extension of the neomycin phosphotransferase II dot-blot assay to allow more rapid and sensitive quantitative determination of the neo gene product in crude mammalian cell extracts. Our procedure, based upon the dot-blot assay of Platt and Yang [Anal. Biochem. 162 (1987) 502-514], measures both the enzymatic activity and the protein content of a cell extract by scanning with an enzyme-linked immunosorbent assay reader, using the same sample rather than parallel samples for both measurements. We show this assay to be comparable to the chloramphenicol acetyltransferase assay in sensitivity. Therefore, apart from being a useful selectable marker gene, the neo gene is a convenient reporter gene in studies of stable, as well as transient, expression.

Animals↗
PubMed1990

Pathogenicity of BALB/c-derived N-tropic murine leukemia viruses.

N-tropic murine leukemia viruses have been observed in connection with radiation-induced osteosarcomagenesis in BALB/c mice. We have investigated the bone disease-inducing potential of molecularly cloned, BALB/c-derived N-tropic viruses in the random-bred NMRI mouse strain. The germ-line virus and an exogenous virus isolate were found to induce high incidences of osteopetrosis and lymphomas and a lower incidence of osteomas. Two viruses derived from somatically acquired proviruses of independent radiation-induced osteosarcomas induced lower incidence of osteopetrosis and lymphomas. Nucleotide sequence analysis of the long terminal repeat regions and RNase T1 fingerprint analysis revealed only few differences between the isolates. The possible involvement of N-tropic murine leukemia viruses in radiation-induced osteosarcomagenesis in the BALB/c mouse strain is discussed.

Animals↗
PubMed1990

Multiple sequence elements in the U3 region of the leukemogenic murine retrovirus SL3-2 contribute to cell-dependent gene expression.

Determination of the U3 sequence of the leukemogenic murine retrovirus SL3-2 revealed close relationships to SL3-3, Akv, and Gross passage A viruses. The SL3-2 and Akv regions showed wide differences in their relative transcriptional activity in four cell lines as determined by U3-driven transient expression assays. The U3 regions of SL3-2 and SL3-3 gave rise to similar but not identical levels of expression. Deletion mapping of the SL3-2 U3 region points to several determinants of expression of different relative importance in the cell lines tested.

Animals↗
PubMed1990

Enhancer functions in U3 of Akv virus: a role for cooperativity of a tandem repeat unit and its flanking DNA sequences.

cis-Acting transcriptional control elements in the U3 region of the murine retrovirus Akv were analyzed in mouse NIH 3T3 fibroblast cells by using a transient expression vector system based upon a complete long terminal repeat with linked flanking sequences. Deletion analysis pointed to the essential role of sequences within the 99-base-pair direct repeats, and a fragment encompassing the two repeats was found to possess orientation-independent enhancer activity when positioned either upstream or downstream of the transcriptional unit. Removal of one copy of the 99-base-pair repeat led to a reduction in activity of about 2.5-fold when located in an intact U3 environment but to reductions of up to 2 orders of magnitude when placed in other sequence contexts. Our studies of enhancer functions in the presence of one versus two copies of the tandem repeat point to duplicate functions of repeat sequences and sequences flanking the repeat region and emphasize the complex overall organization of this U3 region.

Amino Acid Sequence↗
PubMed1990

Involvement of nuclear factor I-binding sites in control of Akv virus gene expression.

The U3 region of Akv murine leukemia virus carries a 99-base-pair repeat that is associated with transcriptional enhancement in murine NIH 3T3 cells. Deletion analysis points to a critical function of a region within the repeat unit related to the recognition sequences for nuclear factor I proteins but distinct from the sites previously analyzed in related viruses. Nuclear proteins binding to the critical site were detected in NIH 3T3 cells and in mouse livers. A protein fraction binding to this site was purified from mouse livers by ion-exchange and DNA affinity chromatography and shown to have nuclear factor I properties. Mutations that caused a partial or complete reduction of the in vitro binding were introduced into an Akv long terminal repeat with one 99-base-pair repeat copy driving a reporter gene, and the expression activities of the mutants in NIH 3T3 cells were found to correspond to their in vitro binding activities. This correlation strongly supports the role of nuclear factor I proteins in Akv expression. Residual expression activity was, however, detected in mutants devoid of in vitro binding. This residual activity may relate to the presence of additional sequences with homology to nuclear factor I binding sites both within and outside the repeat region. The ability of these sites to bind crude and purified protein fractions with nuclear factor I activity was analyzed, and the role of the sites within and outside the repeat region for control of gene expression of Akv and related viruses is discussed.

Animals↗
PubMed1989

Graduated resistance to G418 leads to differential selection of cultured mammalian cells expressing the neo gene.

The effects of Geneticin (G418) selection on the growth and survival of cultured mammalian cells expressing the neomycin-resistance gene (neo) were studied by the analysis of cell clones from two retroviral neo vector-infected populations. We found a correlation between the neo expression level and growth rates in medium containing varying G418 concentrations. This relationship permits the use of differential selection schemes for the isolation of rare cells with increased expression. Comparison, by clone sampling, of vector-positive populations before and after selection with a G418 concentration in the range usually used for selection, showed different expression level and vector copy number distributions for the population infected with the vector of lower LTR activity, but not for the other. Such biasing effects of G418 selection may be important when selected cells are used for quantitative studies of gene expression.

Animals↗
PubMed1989

Different relative expression from two murine leukemia virus long terminal repeats in unintegrated transfected DNA and in integrated retroviral vector proviruses.

Results of transient-expression studies have suggested a correlation between tissue-specific pathogenicity of murine leukemia viruses and the relative transcriptional activities of their long terminal repeats in various cell types. To test whether transient-expression ratios are representative of those of integrated proviruses, we developed a system for generation of retroviral transmission vectors differing only in U3. Vectors with the long terminal repeats of leukemogenic SL3-3 and nonleukemogenic Akv viruses were used for infection of a lymphoid cell line. We then compared expression in infected cells with transient expression after DNA transfection. In contrast to a high SL3-3/Akv reporter gene expression ratio in the transient assays, the ratio in stably infected populations was low. Sets of random cell clones from the two infected populations showed wide variation, with a mean value ratio identical to the population ratio but a considerably higher ratio between lowest values. We suggest that the lower expression levels, like transient expression, reflect inherent enhancer strength and that the higher levels represent chromosomal influence. The different pathogenicity, despite the moderate difference in average expression, may then relate to a different capacity for insertional oncogene activation owing to the different inherent enhancer strengths revealed by the transient-expression assays and the least active proviruses.

Animals↗
PubMed1988

An MuLV transmission vector system designed to permit recovery in E. coli of proviral and cellular flanking sequences.

We have introduced a bacterial suppressor gene (supF) into the long terminal repeat of a molecular clone of the murine leukemia virus (MuLV) SL3-3. A panel of replication competent virus was derived that replicates to high titers in NIH3T3 cells in culture. The tRNA gene is stably carried in the provirus. The supF and viral sequences are present in equimolar amounts in the RNA genome of the expressed recombinant virus. The proviral sequences containing supF can be recovered by cloning into a lambda vector carrying amber mutations. The DNA sequences in the recovered lambda recombinants show a high degree of stability. The presented system should facilitate the study of the interaction between proviral and cellular sequences flanking the integration site.

Bacteriophage lambda↗
PubMed1988

A nucleotide substitution in the gag N terminus of the endogenous ecotropic DBA/2 virus prevents Pr65gag myristylation and virus replication.

The endogenous ecotropic provirus Emv-3 present in DBA/2 mice is poorly expressed in the animal, as well as in cell cultures. Transfection of proviral DNA into NIH 3T3 cells localized the expression defect to the 5' region of the viral genome, spanning the untranslated region and the N-terminal part of the gag gene. Comparison of the nucleotide sequence of the Emv-3 provirus with the sequence of the highly infectious Akv murine leukemia virus revealed three nucleotide differences within the gag coding region. One of these differences was found in codon 3 of the gag polyprotein, where a Gln codon is seen in Akv and a Pro codon is differences was found in codon 3 of the gag polyprotein, where a Gln codon is seen in Akv and a Pro codon is seen in Emv-3. By site-directed mutagenesis, we showed that the defect of Emv-3 expression indeed is localized to codon 3 of the gag gene. The gag polyprotein of mammalian type C retrovirus contains myristic acid covalently linked to the N-terminal glycine. This myristylation is not seen in the Emv-3-coded gag polyprotein. We showed that the in vitro-mutagenized Emv-3 genome containing a Gln codon at position 3 of the gag gene yields a myristylated gag polyprotein. Thus, it seems most likely that the defect of expression of the Emv-3 provirus is due to the presence of a proline is position 3 of the gag polyprotein, preventing the myristylation.

Animals↗
PubMed1986

Oncogenic retrovirus from spontaneous murine osteomas. II. Molecular cloning and genomic characterization.

An N-ecotropic murine leukemia virus (OA MuLV), originally isolated from spontaneous osteomas of strain 101 mice, was molecularly cloned. The virus induces osteomas, osteopetrosis, and malignant lymphomas in NMRI mice. The cloned virus was analyzed by heteroduplex analysis, restriction enzyme mapping, and oligonucleotide mapping. The data show a very close relationship to the endogenous Akv prototype virus with some differences in the gag and the env region. The nucleotide sequence of the U3 region of OA MuLV LTR revealed a structure within the presumable enhancer region very similar to the U3 sequences of the FBJ murine sarcoma virus and its associated helper virus. The significance of these specific structures for the oncogenicity of the virus and the development of the typical disease pattern is discussed.

Animals↗