Niels Ole Kjeldgaard

The nucleotide sequence of the Akv murine leukemia virus genome.

The nucleotide sequence of an infectious molecular clone of the Akv murine leukemia virus has been determined by the dideoxy chain termination method after subcloning in bacteriophage M13 vectors. The sequence predicts an RNA genome of 8371 nucleotides containing three large open reading frames corresponding to the gag, pol, and env genes. Signal sequences for transcription, splicing, and translation have been identified. The positions of 95 major RNase T1 resistant oligonucleotides of the Akv RNA genome have been located.

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.

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.

Multiple sequence elements in the U3 region of the leukemogenicmMurine 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.

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.

Protein synthesis and formation of guanosinetetraphosphate

Guanosinetetraphosphate (ppGpp) accumulates in the nucleotide pool of tel + strains ofE. coli during a number of conditions where the normal course of protein synthesis is affected [ 1, 2]. The accumulated ppGpp decays after treatment of the cell with several antibiotics which are strong inhibitors of protein synthesis [ 1, 2]. In a previous paper we suggested that an initiation process of protein synthesis was necessary for ppGpp synthesis [2]. Rifampicin induces a decay of the accumulated ppGpp [2-4] and it has been suggested that the effect of rifampicin is a consequence of the breakdown of the mRNA in the cells [2, 4]. Here we report that rifampicin is without effect on the capacity of the cells to form ppGpp if mRNA is present in the cells. This condition was obtained by infection ofE. coli with either R17, T7 wild type or T7 gene 1 mutant bacteriophages.

An MuLV transmission vector system designed to permit recovery inE.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. ThesupF and viral sequences are present in equimolar amounts in the RNA genome of the expressed recombinant virus. The proviral sequences containingsupF 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.

Mammalian expression-and-transmission vector derived from Akv murine leukemia virus

A mammalian transmission-expression vector has been constructed based on the plasmid pBR322 and using the transcriptional signals from the Akv murine leukemia virus (AkvMuLV) to control the expression of the neo gene. The transmission vector pL psi PLneo, when transfected into the psi 2 cell line, confers G-418 resistance on recipient cell clones which produce viral particles encapsidating the transcripts of the vector. Cultures of such clones produce viral particles of titers up to 10(5) cfu/ml. The pL psi PLneo vector has two unique restriction sites which can be used for the insertion of new DNA material.

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.

Transcriptional Control by Retroviral LTR Regions

Murine leukemia viruses represent a polymorphic group of retroviruses. Individual isolates differ in species and tissue tropism and in their specificity and potency of pathogenic properties. Some viruses induce lymphomas with high incidence and a latency period of a few months when injected into newborn mice of various inbred strains, whereas others show a weaker and less specific pathogenicity. The pathogenic effects of these weaker viruses include various lymphomas and leukemias as well as neoplastic and non-neoplastic abnormalities in bone tissues. Recombination mapping between thymomagenic and weakly leukemogenic viruses has localized a major determinant of the oncogenic potency to the long terminal repeat (LTR) region of the viruses, (DesGroseillers et al., 1983; DesGroseiller and Jolicoeur, 1984; Lenz et al., 1984). Recombination mapping between viruses that yield thymic and erythroid leukemias has also localized the determinants for tissue specificity to the LTR region, (Chatis et al., 1983, 1984; Ishimoto et al., 1985; Vogt et al., 1985). The LTR regions contain sequences necessary for the initiation and termination of retroviral transcription including promotor elements, termination signals and enhancers. Mapping studies of the more potent MuLVs point to the transcriptional control region containing an enhancer structure as a disease determinant, (Chatis et al., 1984; Ishimoto et al., 1985; Bosze et al., 1986, (MoMuLV, Friend MuLv); Des Groseillers and Jolicoeur, 1984, (GrossA); Lenz et al., 1984; Celander and Haseltine, 1984, (SL3 - 3)).