The ankyrin repeat-containing adaptor protein Tvl-1 is a novel substrate and regulator of Raf-1.

Tvl-1 is a 269-amino acid ankyrin repeat protein expressed primarily in thymus, lung, and testes that was identified by screening a murine T-cell two-hybrid cDNA library for proteins that associate with the serine-threonine kinase Raf-1. The interaction of Tvl-1 with Raf-1 was confirmed by co-immunoprecipitation of the two proteins from COS-1 cells transiently transfected with Tvl-1 and Raf-1 expression constructs as well as by co-immunoprecipitation of the endogenous proteins from CV-1 and NB2 cells. Tvl-1 interacts with Raf-1 via its carboxyl-terminal ankyrin repeat domain. The same domain also mediates Tvl-1 homodimerization. Tvl-1 was detected by immunofluorescence in both the cytoplasm and the nucleus suggesting that in addition to Raf-1 it may also interact with nuclear proteins. Activated Raf-1 phosphorylates Tvl-1 both in vitro and in vivo. In baculovirus-infected Sf9 insect cells, Tvl-1 potentiates the activation of Raf-1 by Src and Ras while in COS-1 cells it potentiates the activation of Raf-1 by EGF. These data suggest that Tvl-1 is both a target as well as a regulator of Raf-1. The human homologue of Tvl-1 maps to chromosome 19p12, upstream of MEF2B with the two genes in a head to head arrangement.

The Tpl-2 protooncoprotein activates the nuclear factor of activated T cells and induces interleukin 2 expression in T cell lines.

Tpl-2 expression is induced within 30-60 min after ConA stimulation of rat splenocytes, suggesting that it may contribute to the induction of IL-2 during T cell activation. Herein we show that wild-type and carboxyl-terminally truncated (activated) Tpl-2 activate the nuclear factor of activated T cells (NFAT) and induce interleukin 2 (IL-2) expression in EL4 cells. In Jurkat cells the truncated Tpl-2 activates NFAT and induces IL-2, whereas wild-type Tpl-2 activates NFAT only when cotransfected with NFAT expression constructs, suggesting that Tpl-2 may induce NFAT activation signals. Experiments in NIH 3T3 cells revealed that the NFATp isoform, but not the NFATc or NFATx isoform, undergoes nuclear translocation when coexpressed with wild-type Tpl-2 and confirmed this hypothesis. Activation of NFAT by anti-CD3 stimulation but not by phorbol 12-myristate 13-acetate and ionomycin in Jurkat cells was inhibited by the kinase-dead Tpl-2K167M, suggesting that Tpl-2 contributes to the transduction of NFAT activation signals originating in the T cell receptor. The Tpl-2-mediated induction of IL-2 was not observed in T cell lymphoma lines other than EL4 and Jurkat, as well as in normal T cells. NFAT activation by Tpl-2, however, was observed in several cell lines including some of nonhematopoietic origin. The activation of NFAT by Tpl-2 in different cell types defines a molecular mechanism that may contribute to its oncogenic potential.

The Gfi-1B proto-oncoprotein represses p21WAF1 and inhibits myeloid cell differentiation.

Gfi-1 is a cellular proto-oncogene that was identified as a target of provirus integration in T-cell lymphoma lines selected for interleukin-2 (IL-2) independence in culture and in primary retrovirus-induced lymphomas. Gfi-1 encodes a zinc finger protein that functions as a transcriptional repressor. Here we show that Gfi-1B, a Gfi-1 related gene expressed in bone marrow and spleen, also encodes a transcriptional repressor. IL-6-induced G1 arrest and differentiation of the myelomonocytic cell line M1 were linked to the downregulation of Gfi-1B and the parallel induction of the cyclin-dependent kinase inhibitor p21WAF1. Experiments addressing the potential mechanism of the apparent coordinate regulation of these genes revealed that Gfi-1B represses p21WAF1 directly by binding to a high-affinity site at -1518 to -1530 in the p21WAF1 promoter. Forced expression of Gfi-1B, but not of Gfi-1B deletion mutants lacking the repressor domain, blocked the IL-6-mediated induction of p21WAF1 and inhibited G1 arrest and differentiation. We conclude that Gfi-1B is a direct repressor of the p21WAF1 promoter, the first such repressor identified to date, and that sustained expression of Gfi-1B blocks IL-6-induced G1 arrest and differentiation of M1 cells perhaps because it prevents p21WAF1 induction by IL-6.

Replacement of interleukin-2 (IL-2)-generated mitogenic signals by a mink cell focus-forming (MCF) or xenotropic virus-induced IL-9-dependent autocrine loop: implications for MCF virus-induced leukemogenesis.

In earlier studies, we have shown that superinfection of an interleukin-2 (IL-2)-dependent, Moloney murine leukemia virus (MoMuLV)-induced rat T-cell lymphoma line (4437A) with mink cell focus-forming (also called polytropic) murine retroviruses induces rapid progression to IL-2-independent growth. In this report, we present evidence that the vast majority (> 90%) of the IL-2-independent lines established from polytropic or xenotropic virus-infected 4437A cells carry provirus insertions in the 3' untranslated region of the IL-9 receptor gene (Gfi-2 [for growth factor independence-2]/IL-9R). Prior to superinfection, the cells express neither IL-9 nor IL-9R. Following superinfection and provirus insertion in the Gfi-2/IL-9R locus, the cells express high levels of mRNA transcripts with a truncated 3' untranslated region which are predicted to encode the normal IL-9R protein product. The same IL-2-independent cells also express IL-9 which is induced by an insertional mutagenesis-independent mechanism. The establishment of an IL-9-dependent autocrine loop was sufficient to render the cells IL-2 independent, as suggested by the finding that 4437A cells, expressing a stably transfected Gfi-2/IL-9R construct, do not require IL-2 when maintained in IL-9-containing media. Additional experiments designed on the basis of these results showed that IL-9 gene expression is induced rapidly following the infection of 4437A cells by polytropic or xenotropic viruses and occurs in the absence of selection for IL-2-independent growth. Taken together, these data suggest that infection of 4437A cells by mink cell focus-forming or xenotropic viruses induces the expression of IL-9, which in turn rapidly selects the cells expressing the IL-9 receptor through an insertional mutagenesis-dependent mechanism. Given that both the polytropic and xenotropic viruses can induce the IL-9-dependent autocrine loop, the reduced ability of the xenotropic viruses to rapidly induce IL-2 independence in culture and tumors in animals is likely to be the result of their lower growth rates.

Effects of provirus integration in the Tpl-1/Ets-1 locus in Moloney murine leukemia virus-induced rat T-cell lymphomas: levels of expression, polyadenylation, transcriptional initiation, and differential splicing of the Ets-1 mRNA.

The Tpl-1 locus was defined as a genomic DNA region which is targeted by provirus insertion during progression of Moloney murine leukemia virus-induced rat T-cell lymphomas. Using a panel of 156 (Mus musculus x Mus spretus) x Mus musculus interspecific backcross mice, we mapped Tpl-1 to mouse chromosome 9 at a distance of 1.2 +/- 0.9 centimorgans from the Ets-1 proto-oncogene (S.E. Bear, A. Bellacosa, P.A. Lazo, N.A. Jenkins, N.G. Copeland, C. Hanson, G. Levan, and P.N. Tsichlis, Proc. Natl. Acad. Sci. USA 86:7495-7499, 1989). In this report, we present evidence that all the known Tpl-1 provirus insertions occurred immediately 5' of the first exon of Ets-1 (exon A) and that the earlier detected distance between Tpl-1 and Ets-1 was due to the high frequency of meiotic recombination in the region between the site of provirus integration and exon III. Northern (RNA) blot analysis of polyadenylated RNA from normal adult rat tissues and Moloney murine leukemia virus-induced T-cell lymphomas and hybridization to a Tpl-1/Ets-1 probe derived from the 5' end of the gene revealed two lymphoid cell-specific RNA transcripts, of 5.5 and 2.2 kb. Sequence analysis of a near-full-length (4,991-bp) cDNA clone of the 5.5-kb RNA revealed a 441-amino-acid open reading frame encoding a protein identical to the human and mouse Ets-1 proteins with the exception of five and nine species-specific conservative amino acid differences, respectively. The steady-state level of the Tpl-1/Ets-1 RNA and of the Ets-1 protein was modestly elevated in tumors carrying a provirus in the Tpl-1 locus. The relative ratio of the two Ets-1 transcripts, which were shown to arise by differential polyadenylation, was not affected by provirus insertion. Moreover, the major site of transcriptional initiation, which was localized by primer extension 250 bp upstream of the 5' end of the Ets-1 cDNA clone, was shown to be identical in normal cells and tumors carrying a provirus in the Tpl-1 locus. Finally, the differential splicing of Ets-1 exon VII was shown by RNase protection to occur at a rate of 15 to 26% and to remain unaffected by provirus insertion. The subtlety of these effects, in contrast to the strong growth selection of cells with a provirus in the Tpl-1/Ets-1 locus, suggests that provirus insertion may affect the fine regulation of the gene, perhaps during cell cycle progression.

Genomic organization and expression of Tpl-2 in normal cells and Moloney murine leukemia virus-induced rat T-cell lymphomas: activation by provirus insertion.

Tpl-2 is a gene encoding a protein kinase which is primarily expressed in normal spleen, thymus, and lung tissue and is activated by provirus insertion in Moloney murine leukemia virus-induced T-cell lymphomas during the late stages of oncogenesis. Tpl-2 is composed of eight exons and spans a 35-kb genomic DNA region. The provirus integrates reproducibly in the last intron and in the same transcriptional orientation as the Tpl-2 gene. This genetic change leads to the expression of enhanced steady-state levels of a truncated Tpl-2 RNA transcript which is predicted to encode a protein with an altered C-terminal domain. Tpl-2 is transcribed from two alternating promoters, P1 and P2. The RNA transcripts originating in the two promoters harbor different 5' untranslated regions derived from the alternate noncoding exons IA and IB. Utilization of the P2 promoter, which gives rise to exon IB containing Tpl-2 RNA transcripts, was detected primarily in tumor cells. The Tpl-2 protein was expressed in COS-1 cells as an N-terminal fusion with a 12-amino-acid hemagglutinin tag. Immunoprecipitation of transfected COS-1 cell lysates with antihemagglutinin or anti-Tpl-2 antibodies, followed by incubation with [gamma-32P]ATP, confirmed that Tpl-2 possesses protein kinase activity.

Identification of the gene encoding the mitochondrial elongation factor G in mammals.

Protein synthesis in cytosolic and rough endoplasmic reticulum associated ribosomes is directed by factors, many of which have been well characterized. Although these factors have been the subject of intense study, most of the corresponding factors regulating protein synthesis in the mitochondrial ribosomes remain unknown. In this report we present the cloning and initial characterization of the gene encoding the rat mitochondrial elongation factor-G (rEF-Gmt). The rat gene encoding EF-Gmt (rMef-g) maps to rat chromosome 2 and it is expressed in all tissues with highest levels in liver, thymus and brain. Its DNA sequence predicts a 752 amino acid protein exhibiting 72% homology to the yeast Saccharomyces cerevisiae mitochondrial elongation factor-G (YMEF-G), 62% and 61% homology to the Thermus thermophilus and E. coli elongation factor-G (EF-G) respectively and 52% homology to the rat elongation factor-2 (EF-2). The deduced amino acid sequence of EF-G contains characteristic motifs shared by all GTP binding proteins. Therefore, similarly to other elongation factors, the enzymatic function of EF-Gmt is predicted to depend on GTP binding and hydrolysis. EF-Gmt differs from its cytoplasmic homolog, EF-2, in that it contains an aspartic acid residue at amino acid position 621 which corresponds to the EF-2 histidine residue at position 715. Since this histidine residue, following posttranslational modification into diphthamide, appears to be the sole cellular target of diphtheria toxin and Pseudomonas aeruginosa endotoxin A, we conclude that EF-Gmt will not be inactivated by these toxins. The severe effects of these toxins on protein elongation in tissues expressing EF-Gmt suggest that EF-Gmt and EF-2 exhibit nonoverlapping functions. The cloning and characterization of the mammalian mitochondrial elongation factor G will permit us to address its role in the regulation of normal mitochondrial function and in disease states attributed to mitochondrial dysfunction.

Tumor progression locus 2 (Tpl-2) encodes a protein kinase involved in the progression of rodent T-cell lymphomas and in T-cell activation.

The Tpl-2 locus, cloned by provirus tagging from one of three sublines of the Moloney leukemia virus-induced rat thymoma 2769, defines a gene encoding a protein kinase associated with progression in 22.5% of the tumors. Tpl-2 is expressed primarily in spleen, thymus, liver, and lung. Provirus integration occurs in the last intron of the gene, leading to the expression of a truncated mRNA that terminates in the proviral long terminal repeat and encodes a protein with an altered C-terminal domain. Strong evidence that this genetic change confers growth advantage to affected cell clones was provided by the finding that, during cultivation of all three sublines derived from tumor 2769, cells were selected that harbored independent provirus insertions in the Tpl-2 locus. Exposure of normal rat spleen cells to Con A induces the expression of enhanced levels of Tpl-2 within the first 60 min from the time of exposure suggesting that, in normal splenocytes, Tpl-2 may be involved in the transition from a quiescent to the G1 phase of the cell cycle.

Structure of a Moloney murine leukemia virus-virus-like 30 recombinant: implications for transduction of the c-Ha-ras proto-oncogene.

Tumor progression locus 2 (Tpl-2) encodes a novel serine-threonine protein kinase which is activated by provirus integration in the late stages of oncogenesis in Moloney leukemia virus (MoMuLV) induced rat T-cell lymphomas. In this report, we present evidence that the provirus integrated in the Tpl-2 locus in 1 of 10 T-cell lymphomas harboring a Tpl-2 rearrangement (2779) is a recombinant between MoMuLV and virus-like 30 (VL30) sequences (Mo-VL30). Recombination between MoMuLV and VL30 may contribute to the transduction of ras, as suggested by the finding that VL30 flanks the ras oncogene in all of the ras transducing viruses isolated from rats to date. The Mo-VL30 recombinant described here represents evidence that recombination between MoMuLV and VL30 can be uncoupled from the transduction of ras, and it may precede the transduction. Sequence comparison between clones of Mo-VL30, Harvey sarcoma virus (Ha-MSV), and genomic c-Ha-ras revealed that all three share a 124-bp region of 87.3% homology. This region was detected at nucleotide positions -1845 to -1720 of c-Ha-ras and 20 bp 5' of the recombination breakpoint between VL30 and ras in Ha-MSV. On the basis of the sequence comparison between VL30, Ha-MSV, and c-Ha-ras, we are proposing a model which explains how VL30 may have facilitated the transduction of c-Ha-ras and perhaps the other ras proto-oncogenes. According to this model, the sequence homology between VL30 and c-Ha-ras targets this gene for transduction by promoting the integration of the provirus in this locus through homologous recombination.

Progression of interleukin-2 (IL-2)-dependent rat T cell lymphoma lines to IL-2-independent growth following activation of a gene (Gfi-1) encoding a novel zinc finger protein.

During progression of Moloney murine leukemia virus (Mo-MuLV)-induced rat T cell lymphomas, growth selection results in the expansion of cell clones carrying increasing numbers of integrated proviruses. These new provirus insertions reproducibly contribute to enhanced growth, allowing the emergence of cell clones from the initially heterogeneous population of tumor cells. The Mo-MuLV-induced rat T cell lymphoma lines 2780d and 5675d, which are dependent on interleukin-2 (IL-2) for growth in culture (IL-2d), were placed in IL-2-free medium to select for IL-2-independent (IL-2i) mutants. Southern blot analysis of genomic DNA from these mutants, which was hybridized to a Mo-MuLV long terminal repeat probe, revealed that all mutants carried new provirus insertions (from one to four new proviruses per cell line). A locus of integration identified through cloning of the single new provirus detected in one of the IL-2i mutants, 2780i.5, was found to be the target of provirus insertion in 1 additional IL-2i cell line of 24 tested. A full-length cDNA of a gene (growth factor independence-1 [Gfi-1]) activated by promoter insertion in the 2780i.5 cells was cloned and shown to encode a novel zinc finger protein. Gfi-1 is expressed at low levels in IL-2d cell lines cultured in IL-2-containing medium and at high levels in most IL-2i cell lines, including the two harboring a provirus at this locus. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis. In mitogen-stimulated splenocytes, Gfi-1 expression begins to rise at 12 h after stimulation and reaches very high levels after 50 h, suggesting that it may be functionally involved in events occurring after the interaction of IL-2 with its receptor, perhaps during the transition from the G1 to the S phase of the cell cycle. In agreement with this, Gfi-1 does not induce the expression of IL-2. Expression of Gfi-1 in 2780d cells following transfer of a Gfi-1/LXSN retrovirus construct contributes to the emergence of the IL-2i phenotype.

Activation of the prolactin receptor gene by promoter insertion in a Moloney murine leukemia virus-induced rat thymoma.

The prolactin receptor (Prlr) and growth hormone receptor (Ghr) genes and the Moloney murine leukemia virus integration-2 (Mlvi-2) locus were mapped to mouse chromosome 15 and human chromosome 5 bands p12-p14. To examine the potential relationship between Mlvi-2 and the genes encoding the growth hormone receptor and the prolactin receptor, we determined the chromosomal location of all three loci in the rat, using a panel of rat-mouse somatic cell hybrids, and in the mouse, using a panel of (C57BL/6J x Mus spretus)F1 x C57BL/6J interspecific backcross mice. These analyses revealed that Ghr, Prlr, and Mlvi-2 map to chromosome 2 in the rat and to chromosome 15 in the mouse, in close proximity with each other. Pulsed-field gel electrophoresis of rat genomic DNA showed no overlaps between the gene encoding the prolactin receptor and the remaining loci. Moreover, expression of the prolactin receptor was not affected by provirus insertion in Mlvi-2. During these studies, however, we detected one T-cell lymphoma line (2779) in which the prolactin receptor gene was activated by provirus integration. Sequence analysis of polymerase chain reaction-derived cDNA clones showed that the prolactin receptor RNA message initiates at the 5' long terminal repeat and utilizes the splice donor site 5' of the gag gene to splice the viral sequences onto exon 1 of the prolactin receptor. This message is predicted to encode the intact prolactin receptor protein product. Exposure of the T-cell lymphoma line 2779 to prolactin promoted cellular proliferation.

Infection by mink cell focus-forming viruses confers interleukin 2 (IL-2) independence to an IL-2-dependent rat T-cell lymphoma line.

The development of T-cell lymphomas in rodents infected with type C retroviruses has been linked to the generation of a class of envelope (env) recombinant viruses called mink cell focus-forming viruses (MCF viruses) in the preleukemic thymus. To determine whether infection by MCF viruses altered the growth phenotype of retrovirus-induced T-cell lymphomas, a Moloney murine leukemia virus-induced interleukin-2 (IL-2)-dependent rat T-cell lymphoma line (4437A) was infected with MCF-247, modified MCF-V33 (mMCF-V33), or NZB-xenotropic (NZB-X) virus. The effects of virus infection on the IL-2 dependence of these cells was examined by cultivating them in the absence of IL-2. After IL-2 withdrawal, the uninfected and NZB-X-infected cells went through a crisis period characterized by massive death. All the independently maintained cultures of MCF- and mMCF-V33-infected cells, on the other hand, became IL-2 independent without a crisis. All the polytropic virus-infected IL-2-independent cultures contained a population of cells that was polyclonal with regard to polytropic provirus integration. Over this polyclonal background each culture produced multiple clones of cells that were selected rapidly after IL-2 withdrawal. Furthermore, the resulting MCF- or mMCF-V33-infected IL-2-independent cells retained the expression of IL-2 receptor. These data show that MCF and mMCF-V33 viruses may alter the growth phenotype of a T-cell lymphoma line and suggest that their effect on cell growth may be due to the direct interaction of the MCF envelope glycoprotein with cellular components, perhaps the IL-2 receptor.

The rat leukocyte antigen MRC OX-44 is a member of a new family of cell surface proteins which appear to be involved in growth regulation.

Moloney murine leukemia virus (MoMuLV)-induced rat T-cell lymphomas express discrete 1.8-, 2.2-, and 4-kb mRNA transcripts hybridizing under conditions of reduced stringency to a probe derived from a region upstream of the first exon of the Tpl-1/Ets-1 gene. Screening a cDNA library from one rat T-cell lymphoma with this genomic probe yielded 15 cDNA clones which were derived from 10 different genes. One of these genes, defined by the cDNA clone pRcT7a, was expressed as a 1.8-kb mRNA transcript in spleen and thymus but not in other normal rat tissues. Expression of the gene defined by the pRcT7a cDNA clone in a series of MoMuLV-induced rat T-cell lymphomas showed a perfect correlation with the expression of the rat leukocyte antigen MRC OX-44. Because of this observation, the pRcT7a clone was sequenced and it was shown to identify a gene coding for a 219-amino-acid protein. The homology between pRcT7a and the Tpl-1 probe used for its detection mapped within the 3' untranslated region of the pRcT7a cDNA clone. The pRcT7a protein, which exhibits four putative transmembrane regions and three putative glycosylation sites, contains a region which is nearly identical in sequence to a peptide derived from the rat leukocyte antigen MRC OX-44. This finding suggested that the pRcT7a cDNA clone defines the gene coding for OX-44. To confirm this finding, a pRcT7a construct in the retrovirus vector pZipNeo was introduced into the OX-44- T-cell lymphoma line 2788. Immunostaining with the MRC OX-44 monoclonal antibody followed by flow cytometry revealed that following gene transfer, the 2788 cells became OX-44+. Sequence comparisons revealed that pRcT7a/MRC OX-44 is a member of a family of genes which includes the melanoma-specific antigen ME491; the human leukocyte antigen CD37; the protein TAPA-1, which is expressed on the surface of human T cells and appears to be involved in growth regulation; the human gastrointestinal tumor antigen CO-029; and the Schistosoma mansoni-associated antigen Sm23.

Activation of multiple genes by provirus integration in the Mlvi-4 locus in T-cell lymphomas induced by Moloney murine leukemia virus.

Moloney murine leukemia virus-induced rat T-cell lymphomas harbor proviruses integrated near c-myc and near Mlvi-1/Mis-1/Pvt-1, another locus of common integration which maps 270 kilobases 3' of c-myc. In this report, we present the characterization of a new locus of common integration in Moloney murine leukemia virus-induced T-cell lymphomas (Mlvi-4) which maps 30 kilobases 3' of c-myc, between c-myc and Mlvi-1. The Mlvi-4 locus, whose chromosomal map location is conserved in rats, mice, and humans, is also the target of chromosomal rearrangements in a variety of animal and human tumors. Evidence presented elsewhere shows that provirus integration in Mlvi-4 enhances the expression of c-myc and Mlvi-1 by cis-acting mechanisms operating over long distances of genomic DNA. In this manuscript, we show that provirus integration in the Mlvi-4 locus activates, by promoter insertion, one additional gene which maps immediately 3' to the cluster of the Mlvi-4 proviruses and which is transcribed in the same orientation as c-myc, giving rise to 3- and 10-kilobase mRNA transcripts. The Mlvi-4 gene is also expressed in normal thymus and spleen at very low levels, giving rise to 3- and 5.5-kilobase messages. Although Mlvi-4 is expressed in normal thymus, it is not expressed in Moloney murine leukemia virus-induced T-cell lymphomas corresponding to several stages of T-cell differentiation, but lacking a provirus in this locus. This suggests that Mlvi-4 may be expressed only in a subpopulation of T cells. We conclude that provirus insertion in Mlvi-4 activates c-myc and two additional genes, Mlvi-1 and Mlvi-4, whose expression is restricted to, and may be developmentally regulated in, T cells. Since Mlvi-4 is the target of genetic changes in a great variety of human and animal neoplasms, these results are critical for our understanding of oncogenesis.

Provirus insertion in Tpl-1, an Ets-1-related oncogene, is associated with tumor progression in Moloney murine leukemia virus-induced rat thymic lymphomas.

T-cell lymphomas induced in rats by Moloney murine leukemia virus acquire increasing numbers of proviruses in their genome during tumor progression in vivo and passage of tumor cells in vitro. To determine whether the proviruses progressively acquired during tumor progression play a causal role in this process, we cloned one of them from a cell line derived from the primary tumor 2772. A probe from the cellular DNA flanking the provirus was used to analyze 79 DNA samples from primary tumor tissues of 28 tumor-bearing rats and 80 DNA samples from 30 independent tumor cell lines. This analysis revealed a rearrangement in this region in the primary tumor derived from the thymus of one animal but not in a clone of the same tumor segregating in the spleen. Of the cell line DNA samples, three carried a provirus in this region. Two of these integration events had occurred independently in two clonally related sublines derived from tumor 2772, and they were followed by rapid selection in culture. On the basis of these findings this locus was named Tpl-1 (tumor progression locus 1). The Tpl-1 locus was mapped to rat chromosome 8 and to mouse chromosome 9 at a genetic distance of 1.2 +/- 0.9 centimorgans from the Ets-1 protooncogene. Although the genetic distance between Tpl-1 and Ets-1 indicates that they are different genes, analysis of Tpl-1 cDNA clones revealed that the two are closely related.

Activation of the Mlvi-1/mis1/pvt-1 locus in Moloney murine leukemia virus-induced T-cell lymphomas.

The Mlvi-1/mis-1/pvt-1 locus, located approximately 270 kilobase pairs 3' of the c-myc protooncogene, was originally discovered as a common region of provirus integration in Moloney murine leukemia virus-induced rat T-cell lymphomas. The same locus was shown subsequently to be coamplified with c-myc and to be involved in chromosomal translocations in a variety of human and animal neoplasms. Provirus integration in Mlvi-1 in Moloney murine leukemia virus-induced rat T-cell lymphomas activates the c-myc protooncogene. The studies reported here were aimed to determine whether, in addition to the activation of c-myc, provirus integration affected the expression of other neighboring genes. Provirus integration was shown to occur in three clusters separated by regions of uninterrupted DNA. The proviruses in all three clusters had integrated in a single-transcriptional orientation, and they appeared intact. Systematic hybridization of Mlvi-1 clones to rat, mouse, and human genomic DNA revealed three patches of evolutionarily conserved sequences. Two of them were mapped in regions targeted by the provirus, and the third was mapped immediately 5' to the provirus clusters. A probe derived from the conserved sequences 5' of the integrated proviruses detected a tumor-specific RNA transcript in tumors carrying a provirus in Mlvi-1 or in the neighboring Mlvi-4 and c-myc loci. The highest level of RNA transcript expression, however, was seen in a CD4+ CD8+ tumor cell line that was not carrying a provirus in this region. We conclude that provirus insertion in this region activates both c-myc and another gene that is located in the immediate vicinity of the integrated Mlvi-1 proviruses and may be developmentally regulated in T cells.

Induction of multiple independent T-cell lymphomas in rats inoculated with MOloney murine leukemia virus.

Tumor cell DNA derived from different lymphoid organs of 30 rats serially inoculated at birth with Moloney murine leukemia virus (MoMuLV) was examined by Southern blot analysis and hybridization to the following DNA probes: MoMuLV long terminal repeat (LTR), Moloney leukemia virus integration regions 1, 2, 3, and 4 (Mlvi-1, Mlvi-2, Mlvi-3, and Mlvi-4), T-cell receptor beta locus, and immunoglobulin heavy chain locus. This analysis revealed that the tumors segregating in different lymphoid organs in 10% of the animals were clonally unrelated. These findings are consistent with the hypothesis that the MoMuLV-induced rat thymic lymphomas are polyclonal in origin. At least two factors may be responsible for this phenomenon: (i) increase in the number of the available target cells in virus-infected animals, and (ii) genetic instability associated with provirus integration in the developing premalignant clones.

Mutational analysis of the lambda int gene: DNA sequence of dominant mutations.

We have combined techniques of genetic and physical mapping with rapid DNA sequence analysis to identify the nucleotide change in lambda int mutations. These mutations define two dominant phenotypic classes: (i) recombination that is partially independent of accessory factors, and (ii) inhibition of wild-type Int by missense or nonsense proteins, i.e., negative complementation.

An accessory role for Escherichia coli integration host factor: characterization of a lambda mutant dependent upon integration host factor for DNA packaging.

Bacteriophage lambda grows lytically on Escherichia coli defective for integration host factor, a protein involved in lambda site-specific recombination and the regulation of gene expression. We report the characterization of a mutant, lambda cos154, that, unlike wild-type lambda, is defective for growth in integration host factor-defective E. coli. The cis-dominant mutation in lambda cos154 is a single base pair change in a region of hyphenated dyad symmetry close to the lambda left cohesive end; this mutation prevents DNA packaging. We propose the following two alternative roles for this site in lambda DNA packaging: (i) to bind an E. coli accessory protein required in the absence of integration host factor or (ii) to bind the phage-encoded terminase protein that is essential for DNA packaging.

Analysis of two potential shuttle vectors containing herpes simplex virus defective DNA.

Two potential shuttle vectors which contained the identical herpes simplex virus type 1 (HSV-1) defective particle DNA (dDNA), but prokaryotic DNA of different origins, were examined for their stability when propagated in eukaryotic cells, and for their efficiency as shuttle vectors. Each chimeric molecule contained a 9.5 kilobase-pair (kb) EcoRI fragment (HSV12-7) representing a single unit of a class I HSV-1 dDNA. This dDNA was cloned into the bacteriophage lambda (lambda) vector lambda gtWES X lambda B' to create a 45.3 kb chimeric molecule (lambda gtWES::12-7), and into the plasmid vector pBR325, resulting in a 15.5 kb recombinant DNA molecule (pBR325::12-7). Each of these DNA molecules was transfected independently into African green monkey kidney cells which were then infected with wild-type HSV-1 helper virus. Both chimeric molecules were replicated and packaged into HSV-1 virions. However, regions of the lambda gtWES::12-7 chimeric DNA were rapidly deleted and rearranged, whereas the plasmid/HSV-1 DNA molecules were less rearranged. No intact lambda gtWES::12-7 DNA was recovered from HSV-1 virions as detected by infectivity of in vitro packaged DNA. However, pBR325::12-7 DNA isolated from HSV-1 virions was able to transform E. coli to ampicillin resistance. These results suggest additional considerations when designing single units of HSV-1 dDNA for use as vectors to accommodate large fragments of DNA.