Deconstructing PTI-1: PTI-1 is a truncated, but not mutated, form of translation elongatin factor 1A1, eEF1A1.

The prostate tumor-inducing gene 1 (PTI-1) transcript is detected in various human carcinoma cells. PTI-1 is reported to consist of a 5' untranslated region (5' UTR) homologous to mycoplasma 23S rRNA and a coding region corresponding to a truncated and mutated form of the translation elongation factor 1A, eEF1A. We have found that the PTI-1 transcript may encode a truncated, but not mutated, form of the human isoform eEF1A1.

Gene structure of the murine 2'-5'-oligoadenylate synthetase family.

The 2'-5'-oligoadenylate synthetases (OASs) are members of a family of interferon-induced proteins playing an important role in the antiviral effect of interferons as well as being involved in apoptosis and control of cellular growth. Based on sequence data from the murine BAC clone (RP23-39M18), and a number of EST and IMAGE clones and the Celera Mouse database, we identified twelve Oas genes in the mouse genome, all localized to the chromosome 5F region. In contrast to the single OAS1 gene found in humans, we identified eight closely linked Oas1 genes in the murine genome, together with the genes of Oas2 and Oas3.

Gene structure and function of the 2'-5'-oligoadenylate synthetase family.

2'-5'-Oligoadenylate synthetase was among the first interferon-induced antiviral enzymes to be discovered. This family of enzymes plays an important role in the mechanisms of action of interferon antiviral activity, but is also involved in other cellular processes such as apoptosis and growth control. We have reviewed the function and genomic structure of this class of at least nine proteins.

Differential regulation of the human, interferon inducible tryptophanyl-tRNA synthetase by various cytokines in cell lines.

Cytokines regulate the expression of specific sets of proteins which mediate their biological effects. We have comprehensively delineated the regulation of the human tryptophanyl-tRNA synthetase (hWRS) by eight different cytokines (including IFNs) and poly(I).poly(C) in several cell lines.

Spectrophotometric pyrophosphate assay of 2',5'-oligoadenylate synthetase.

A nonradioactive multiwell spectrophotometric assay for the interferon-induced enzyme 2',5'-oligoadenylate synthetase measuring the inorganic pyrophosphate produced during oligoadenylate synthesis has been developed. A coupled enzymatic reaction results in a mole to mole formation of NADPH compared to the inorganic pyrophosphate through the use of the three enzymes UDP-Glc pyrophosphorylase (EC2.7.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 physiology of stringent factor (ATP:GTP 3'-diphosphotransferase) in Escherichia coli.

The enzyme ATP:GTP 3'-diphosphotransferase catalyzes the transfer of the beta, gamma-pyrophosphate of ATP to the 3' position of GTP or GDP. The amounts of enzyme were measured in cell extracts of a relA+ strain of E. coli grown at different growth rates between 0.

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 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.

Analysis of the relA gene product of Escherichia coli.

The relA gene product, ATP: GTP 3'-pyrophosphotransferase (stringent factor) has been isolated in homogeneous form from an Escherichia coli strain polyploid for this gene at a yield of 1 mg/100 g cells and at a specific activity in a ribosome-activated assay at 37 degrees C of 120 mumol guanosine pentaphosphate formed min-1 mg protein-1. The specific activity in a methanol-activated assay at 25 degrees C was found to be 4 mumol guanosine pentaphosphate formed min-1 mg protein-1. These values are about 100 times higher than reported by others.