Mos Genes Research Articles

Gene expression and demographic analyses in women with the poor ovarian response: a computational approach.

Poor response to ovarian stimulation (POR) typicallyis reflected asdecreased follicular response and low estradiol (E2) levels followingovarian stimulation by FSH/HMG. Many genes are involved in oocyte maturation, and demographic features and lifestyle can affect the oocyte maturity and developmental competence. The present study was conducted to investigate the magnitude of gene expression and lifestyle habits in POR women as compared to healthy women, using different statistical and computational methods. Fifty women in the two groups were studied. The study groups included POR women (n = 25) with 1-9 released oocytes, and the control group (normal women, n = 25) with 9-15 released oocytes. Quantitative PCR was used to estimate the expression of FIGLA, ZAR1, WNT4, LHX8, APC, H1FOO, MOS, and DMC1 genes in granulosa cells. The results showed no significant difference in the magnitude of the studied genes' expression and linear discriminant analysis did not differentiate the studied groups based on all the genes together. Redundancy analysis (RDA) and latent factor mixed model (LFMM) results produce no significant association between the genes' expression magnitude and the geographical variables of the patients' local habitat. Linear discriminant analysis (LDA) of the demographic features differentiated the two groups of women. Our results indicate that demographic features may have an effect on sample gene expression levels.

Changes in aberrant DNA methylation after Helicobacter pylori eradication: A long‐term follow‐up study

Changes of DNA methylation in gastric mucosae after eradication of Helicobacter pylori have not been clarified yet. From this background, we investigated time course of DNA methylation following H. pylori eradication in 221 successfully H. pylori eradicated subjects with endoscopic follow-up at least for 6 months, including 114 controls, 53 subjects with gastric dysplasia and 54 patients with early gastric cancer. All dysplasia and gastric cancer patients underwent endoscopic resection at the time of enrollment. The methylation levels in LOX, APC and MOS genes from noncancerous gastric mucosae using quantitative methylation-specific PCR, as well as the histologic findings of gastric mucosae, were compared before and after eradication. Average follow-up duration was 26.0 months (range: 6 to 76 months). H. pylori eradication decreased methylation levels in LOX (p-value for slope < 0.001) but not in APC. In MOS, decrease of its methylation level following H. pylori eradication was significant among controls without intestinal metaplasia (IM) (p-value for slope < 0.05); however, it was not observed among patients with IM or those with dysplasia or gastric cancer. After H. pylori eradication, methylation level in MOS persistently increased in patients with dysplasia or gastric cancer (p < 0.01). In conclusion, H. pylori eradication decreases aberrant DNA methylation with gene-specific manner. Methylation level in MOS is associated with IM and may be used as a surrogate marker for gastric cancer risk, regardless of H. pylori eradication history.

A Rolling Stone Gathers No Moss, but Resistant Plants Must Gather Their MOSes

The detection of pathogenic microbes by plant resistance (R) proteins and the subsequent activation of R protein-mediated immunity constitute an important layer in the plant innate immune system. Most R genes encode proteins with nucleotide-binding (NB) and leucine-rich repeat (LRR) domains. The autoimmune mutant suppressor of npr1, constitutive 1 (snc1), that constitutively activates resistance signaling, is a unique model used in our laboratory to dissect the details of TIR (Toll/Interleukin1 receptor)-NB-LRR, protein-mediated defense responses. Suppressor screens of snc1 yielded 15 modifier of snc1 (mos) complementation groups containing second-site mutations, and resulted in the identification of 13 novel MOS genes via either positional cloning or T-DNA tagging. Characterizations of the mos mutants have revealed important roles for transcriptional regulation, RNA processing, protein modifications, and nucleocytoplasmic trafficking in R protein-mediated immunity. The MOS genes have taught us a great deal about the complex mechanisms surrounding R protein activation. Future in-depth genetic and biochemical analyses will further enhance our knowledge of how R proteins are deliberately activated and how specific, targeted immunity is achieved in plants.

Seasonal Effect on Germinal Vesicle-Stage Bovine Oocytes Is Further Expressed by Alterations in Transcript Levels in the Developing Embryos Associated with Reduced Developmental Competence1

Physiological perturbations of bovine follicle-enclosed oocytes during the lengthy period of follicular development can lead to reduced oocyte developmental competence. It is suggested that heat stress-induced alterations in germinal vesicle (GV)-stage oocytes are further expressed in the transcriptional levels of genes involved in oocyte maturation and early embryonic development. Bovine oocytes were collected during cold (December-April) and hot (May-November) seasons, matured, fertilized, and cultured in vitro. The percentage of fertilized oocytes cleaving to the 2- to 4-cell stage was higher in the cold vs. hot season (89.0% ± 2.63% vs. 75% ± 2.63%, respectively; P < 0.05), as was the percentage of cleaved embryos further developing to blastocysts (26.6% ± 0.9% vs. 10.1% ± 1.8%, respectively; P < 0.05). Total RNA and poly(A) mRNA of oocytes and developing embryos were isolated and subjected to semiquantitative and real-time PCR for MOS, GDF9, and POU5F1 genes. In GV-stage oocytes, their mRNA levels did not differ between the seasons. However, following maturation, mRNA levels were higher in oocytes collected in the cold season (P < 0.05). In 4-cell-stage embryos, GDF9 and POU5F1 showed opposite mRNA patterns between seasons (higher and lower levels, respectively) in the hot season (P < 0.05). In both 8-cell-stage embryos and blastocysts, POU5F1 expression was lower during the hot season (P < 0.05). Exposing the ovarian pool of oocytes to environmental stress appears to impair maternal mRNA storage and/or the mechanism of transcription renewal, in turn affecting embryo gene expression before and after embryonic genome activation. Such impairment might partially explain the carry-over effect of summer heat stress on dairy cow conception rates.

Dissecting plant defence signal transduction: modifiers of snc1 in Arabidopsis †

Plants have evolved sophisticated defence mechanisms against pathogen infection, during which Resistance (R) proteins play a central role in recognizing pathogens and initiating downstream defence cascades. In Arabidopsis, the dominant mutant snc1 was previously identified that constitutively expresses P athogenesis- R elated(PR) genes and displays enhanced resistance against both bacterial and oomycete pathogens. A point mutation in this gene renders the encoded TIR-NB-LRR type R protein constitutively active without interaction with pathogens. Both suppressor screen and activation tagging approaches were used to identify regulators downstream of snc1. From these efforts, 15 complementation groups of m odifier o f s nc1 (mos) mutants were identified. We have cloned 11 of the mutations using map-based approaches, and have fully characterized mos2 through mos8. Analysis of the corresponding MOS genes and their protein products reveals a complicated signalling network downstream of R protein activation that involves nucleo-cytoplasmic trafficking, transcriptional reprogramming, RNA processing and protein modification.

Conserved Functions for Mos in Eumetazoan Oocyte Maturation Revealed by Studies in a Cnidarian

The kinase Mos, which activates intracellularly the MAP kinase pathway, is a key regulator of animal oocyte meiotic maturation. In vertebrate and echinoderm models, Mos RNA translation upon oocyte hormonal stimulation mediates "cytostatic" arrest of the egg after meiosis, as well as diverse earlier events [1-5]. Our phylogenetic survey has revealed that MOS genes are conserved in cnidarians and ctenophores, but not found outside the metazoa or in sponges. We demonstrated MAP kinase-mediated cytostatic activity for Mos orthologs from Pleurobrachia (ctenophore) and Clytia (cnidarian) by RNA injection into Xenopus blastomeres. Analyses of endogenous Mos in Clytia with morpholino antisense oligonucleotides and pharmacological inhibition demonstrated that Mos/MAP kinase function in postmeiotic arrest is conserved. They also revealed additional roles in spindle formation and positioning, strongly reminiscent of observations in starfish, mouse, and Xenopus. Unusually, cnidarians were found to possess multiple Mos paralogs. In Clytia, one of two maternally expressed paralogs accounted for the majority MAP kinase activation during maturation, whereas the other may be subject to differential translational regulation and have additional roles. Our findings indicate that Mos appeared early during animal evolution as an oocyte-expressed kinase and functioned ancestrally in regulating core specializations of female meiosis.

Successful inactivation of endogenous Oct-3/4 and c- mos genes in mouse preimplantation embryos and oocytes using short interfering RNAs

Understanding oocyte maturation and early development in mammals is very important, especially because these cells serve as a source of materials useful in medical applications, such as ES cells. However, the limited availability of oocytes and embryos hampers the molecular dissection of the very early stage of mammalian development. Recently, the RNA interference technology has been acknowledged to be very effective and useful in diverse groups of cells, including mammalian cells. In this study, we examined whether short interfering RNAs (siRNAs) are applicable to mouse oocytes and preimplantation embryos, by targeting two genes, namely, Oct-3/4 and c- mos. siRNA injections successfully extinguished the production of these target genes. Moreover, the siRNA-injected oocytes and embryos showed phenotypes very similar to those exhibited by Oct-3/4- or Mos-knockout mice in previous studies. Accordingly, we concluded that siRNA is a useful tool in molecular studies on the early development of mouse.

Early production of rhizopine in nodules induced by Sinorhizobium meliloti strain L5-30.

The rhizopine L-3-O-methyl-scyllo-inosamine (3-O-MSI) is metabolized by approximately 10% of the strains of Rhizobium leguminosarum by. viciae and Sinorhizobium meliloti. Rhizopine strains enjoy a substantial competitive advantage in nodulation, which is manifest before 14 days post-inoculation, implying that rhizopine is produced before this time. We were able to detect this compound in the roots of alfalfa (Medicago sativum L. cv. Hunter River) four days after germination (six days post-infection) with S. meliloti strain L5-30 by gas chromatography-mass spectrometry (GC-MS). At four days, nodules were not visible, and the concentration of rhizopine was extremely low, estimated at 67 pg/gfw (picograms/gram fresh weight). The amount increased gradually but remained low until 16 days, when there was a 50-fold increase from day four, by which time nodules were well established. This pattern of synthesis is consistent with previous studies indicating that rhizopine synthesis is regulated by nifA/ntrA regulatory genes, which are maximally expressed in bacteroids at the onset of nitrogen fixation. However, the low level of rhizopine synthesis must be responsible for the early effects on competition for nodulation. Production of rhizopine at this time most likely results from micro-aerobic induction of mos genes in free-living bacteria, either in the infection threads or in the rhizosphere.

HumanE2F5 gene is oncogenic in primary rodent cells and is amplified in human breast tumors

E2F transcription factors (E2F1 to 6) are central players in the control of animal cell proliferation as regulators of genes involved in cell cycle progression and in transformation. In this report, we have investigated the potential involvement of the E2F5 gene in tumorigenesis. We show that E2F5 can promote the formation of morphologically transformed foci in primary baby rat kidney cells (BRK) when it is overexpressed in the presence of its heterodimeric partner DP1 and activated RAS. This suggests that E2F5 behaves like a MYC-type cooperating oncogene in functional assays, prompting us to monitor potential amplifications of the E2F5 gene in primary human tumors. We mapped the human E2F5 gene to 8q21.1-21.3 equidistant from the MOS (8q12) and MYC (8q24) oncogenes. Since the long arm of chromosome 8 is frequently the site of increased gene copy number (ICN) in breast cancer, we screened 442 breast tumor DNAs for gains of E2F5, MOS, and MYC genes. The three genes showed ICN, albeit at variable incidence and levels of amplification, with the ICN of E2F5 occurring concomitantly with those of MOS and/or MYC in almost half of the cases. Moreover, a marked increase of the 2. 5-kb E2F5 transcript was also detected in some tumors and tumor cell lines. In conclusion, the evidence that sustained unregulated expression of E2F5 can cooperate with other oncogenes to promote cell transformation in functional assays, together with the detection of chromosomal amplifications and overexpressions of the E2F5 gene in breast tumors, provides the first indications that E2F5 deregulation may have a role in human tumor development.

Function-dependent cooperation between oncogene activation and nonrandom chromosome change during tumorigenic conversion of Syrian hamster cells

We determined the tumorigenicity of SHOK cells, a Syrian hamster cell line, morphologically transformed by the introduction of either c-Ha- ras, c-Ki- ras, N- ras, v- mos, v- fgr, or v- src genes. When 1 × 10 6 cells were injected subcutaneously into nude mice, SHOK cells containing one of those oncogenes gave rise to tumors within 3 weeks, while the parental SHOK cells, or SHOK cells containing the neo gene, did not show tumorigenicity even 200 days after injection. From karyotype analysis, the addition of chromosome 3 was consistently found in tumor cells derived from SHOK cells containing the c-Ha- ras, c-Ki- ras, or v- mos genes, and the acquisition of chromosome 9 was a common change observed in tumor cells derived from SHOK cells morphologically transformed by the v- fgr or v- src gene. No consistent chromosomal change was observed in tumor cells containing the N- ras gene. The results obtained from Southern blot analysis showed that amplification of introduced oncogenes was detected in the case of c-Ha- ras, c-Ki- ras, N- ras, v- fgr, and v- src genes. These results indicate that nonrandom chromosome changes, along with the amplification of introduced oncogenes, are required for the tumorigenic conversion. Our findings propose the possibility that secondary chromosomal changes associated with the tumorigenic conversion of morphologically transformed cells occur dependent on the function of the primary activated oncogene introduced.

Characterization of genes for synthesis and catabolism of a new rhizopine induced in nodules by Rhizobium meliloti Rm220-3: extension of the rhizopine concept

Rhizopines are selective growth substrates synthesized in nodules only by strains of rhizobia capable of their catabolism. We report the isolation and study of genes for the synthesis and catabolism of a new rhizopine, scyllo-inosamine (sIa), from alfalfa nodules induced by Rhizobium meliloti Rm220-3. This compound is similar in structure to the previously described rhizopine 3-O-methyl-scyllo-inosamine from R. meliloti L5-30 (P.J. Murphy, N. Heycke, Z. Banfalvi, M.E. Tate, F.J. de Bruijn, A. Kondorosi, J. Tempé, and J. Schell, Proc. Natl. Acad. Sci. USA 84:493-497, 1987). The synthesis (mos) and catabolism (moc) genes for the Rm220-3 rhizopine are closely linked and located on the nod-nif Sym plasmid. The mos genes are directly controlled by the NifA/NtrA regulatory system. A comparison of the sequence of the 5' regions of the two mos loci shows very extensive conservation of sequence as well as strong homology to the nifH coding region. Restriction mapping and hybridization to DNA from the four open reading frames (ORFs) of the L5-30 mos locus indicate the absence of mosA and presence of the other three ORFs (ORF1 and mosB and -C) in Rm220-3. We suggest that the L5-30 mosA gene product is involved in the conversion of scyllo-inosamine to 3-O-methyl-scyllo-inosamine. Restriction fragment length polymorphism analysis of the moc regions of both strains shows that they are very similar. Regulation studies indicate that the moc region is not controlled by the common regulatory gene nifA, ntrA, and ntrC. We discuss the striking similarities in gene structure, location, and regulation between these two rhizopine loci in relation to the rhizopine concept.

Correlation between Physiological and Transforming Activities of the c–mos Proto–oncogene Product and Identification of an Essential Mos Domain for These Activities

Using Xenopus eggs and NIH3T3 cells as assay systems, we have compared the physiological (i.e. maturation–inducing and cleavage–arresting) and in vitro transforming activities of the c–mos genes from various species as well as their mutant genes. These analyses show that the three biological activities all depend upon the intrinsic protein kinase activity of Mos and correlate well with each other. Furthermore, our results demonstrate that a well conserved N–terminal 14–amino acid sequence of Mos, termed the Mos–hox, is essential for all three activities. These results indicate that the in vitro transforming activity of Mos can he ascribed to the same kinase activity of Mos that exerts the physiological activities.

Mos gene transforming efficiencies correlate with oocyte maturation and cytostatic factor activities.

The mos proto-oncogenes from different vertebrate species transform mouse NIH 3T3 cells with markedly different efficiencies. v-mos, mouse (c-mosmu), and chicken (c-mosch) mos transform NIH 3T3 cells 10- to 100-fold more efficiently than do human (c-moshu) and Xenopus (c-mosxc) mos. The mos genes with the highest transforming activity efficiently induce maturation in Xenopus oocytes and mimic cytostatic factor (CSF) by causing mitotic cleavage arrest in embryos. Chimeric v-mos/c-moshu proteins that had high transforming efficiencies in NIH 3T3 cells were also effective in the induction of oocyte maturation and CSF cleavage arrest. We measured the in vitro autophosphorylation activities of the different mos proteins and found that the levels of kinase activity of v-mos, c-mosmu, and c-mosch were much higher than that of c-mosxc. These data indicate that mos gene transforming efficiency and the ability to induce oocyte maturation or mimic CSF activity are correlated with in vitro autophosphorylation activity and suggest that the mos protein plays a similar role in transformed cells and normal oocytes.

Mos Gene Transforming Efficiencies Correlate with Oocyte Maturation and Cytostatic Factor Activities

The mos proto-oncogenes from different vertebrate species transform mouse NIH 3T3 cells with markedly different efficiencies. v-mos, mouse (c-mosmu), and chicken (c-mosch) mos transform NIH 3T3 cells 10- to 100-fold more efficiently than do human (c-moshu) and Xenopus (c-mosxc) mos. The mos genes with the highest transforming activity efficiently induce maturation in Xenopus oocytes and mimic cytostatic factor (CSF) by causing mitotic cleavage arrest in embryos. Chimeric v-mos/c-moshu proteins that had high transforming efficiencies in NIH 3T3 cells were also effective in the induction of oocyte maturation and CSF cleavage arrest. We measured the in vitro autophosphorylation activities of the different mos proteins and found that the levels of kinase activity of v-mos, c-mosmu, and c-mosch were much higher than that of c-mosxc. These data indicate that mos gene transforming efficiency and the ability to induce oocyte maturation or mimic CSF activity are correlated with in vitro autophosphorylation activity and suggest that the mos protein plays a similar role in transformed cells and normal oocytes.

Gene replication in the presence of aphidicolin

DNA replication in the nucleus of eukaryotic cells is restricted to the S phase of the cell cycle, and different genes are duplicated at specific times, according to a well-defined temporal order. We have investigated whether activation of initiation sites, in proximity to genes that are replicated in different portions of the S phase, could be detected when synchronized 10T1/2 cells were maintained in aphidicolin (APC), an inhibitor of DNA polymerases alpha and delta. Cells released from confluence arrest into medium containing 2 micrograms/mL APC progressed into the S phase, and nascent DNA accumulated during incubations of 24 and 32 h. Exposure to APC for 40 or 48 h resulted in growth of the radiolabeled DNA into larger molecules. Replicating DNA was isolated in CsCl gradients and probed with 32P-labeled gene probes for early-replicating genes (e.g., Ha-ras, mos, and myc) and a late-replicating gene (VH Ig). DNA replicated during the 24-h incubation in APC was enriched in Ha-ras gene sequences. The VH Ig gene did not replicate in cells incubated for as long as 56 h with APC. The myc and the mos genes were detected after 32 and 40 h in APC, respectively. The myc gene is replicated in 10T1/2 cells after Ha-ras but before mos. Therefore, the order of activation of these genes was conserved in the presence of APC. The delay in replication of myc and mos correlated well with the slowing of DNA replication by APC.

Activation of thermosensitive RNA splicing and production of a heat-labile P85gag-mos kinase by the introduction of a specific deletion in murine sarcoma virus-124 DNA.

Murine sarcoma virus ts110 (MuSVts110) is a conditionally transformation-defective MuSV mutant lacking 1,487 bases found in its wild-type parent, MuSV-349 (MuSV-124). Expression of the MuSVts110 v-mos gene product, P85gag-mos, requires splicing of the viral transcript to align the gag and mos genes in frame. However, this splice event is restricted to growth temperatures of 33 degrees C or lower. No splicing of the viral RNA, no production of P85gag-mos, and, hence, no cell transformation is observed at growth temperatures above 33 degrees C. To determine whether thermosensitive splicing is an intrinsic property of To determine whether thermosensitive splicing is an intrinsic property of MuSVts110 RNA specified by the 1,487-base deletion or a result of a cellular defect, we examined an "equivalent" or MuSVts110 DNA (designated ts32 DNA) constructed by combining wild-type MuSV-124 DNA fragments with a synthetic oligonucleotide to yield an otherwise wild-type viral DNA containing the same 1,487-base deletion as authentic MuSVts110. As observed in control cells (6m2 cells) infected with the authentic MuSVts110 virus, NIH 3T3 cells transfected with ts32 DNA appeared morphologically transformed when grown at 33 degrees C, but were converted to a more normal, flattened shape within a few hours of a shift to 39 degrees C. In concert with these morphological changes, both the processing of the ts32 RNA transcripts and the production of ts32 p85gag-mos kinase were found to be optimal at growth temperatures from 28 to 33 degrees C, but dramatically reduced at 37 to 41 degrees C. Like authentic P85gag-mos, the ts32 P85gag-mos kinase activity was rapidly inactivated by brief exposure to 39 degrees C. These results suggested that the MuSVts110 equivalent is functionally indistinguishable from authentic MuSVts110 and that the novel temperature-sensitive splicing of MuSVts110 transcripts is specified by an intrinsic property of the viral RNA.

Possible role of c- fos, c-N- ras and c- mos proto-oncogenes in muscular development

Time course analyses of various proto-oncogene transcripts compared with cytoskeleton-specific and muscle-specific messenger RNAs (mRNAs) were carried out during growth and differentiation of a clonal line of rat myoblasts that retain the capacity to form non-contractile fibres in vitro. Throughout their growth phase, these cells express consistent levels of c- fos, c- myc, c-Ki- ras and c-N- ras RNA and no c- mos RNA. When the cultures approach confluency the level of c- fos RNA rises sharply 3–4-fold, peaks, and rapidly declines when muscle-specific transcripts start accumulating, to become negligible in myotube-forming cells. These changes occur whatever the concentration in seric factors. By contrast, the level of c-N- ras RNA rises up to 3-fold and both c- myc and c-Ki- ras RNAs are slowly eliminated during the myogenic process, whereas no c- mos RNA is detectable. However, skeletal muscles from prenatal fetuses and adult animals were reproducibly found to contain both low and high levels of c- mos RNA respectively. These data and the demonstration that inactivation of the c- fos gene correlates with the loss of myogenic capability in six lines of neoplastic myoblasts, including four lines transformed by the v- fos oncogene, suggest a physiological function for this proto-oncogene during early stages of myogenesis and for the c-N- ras and c- mos genes in later stages of muscular development.

Analysis of the transforming potential of the human homolog of mos

The human homology, c- mos hu, of the mouse cellular mos proto-oncogene (c- mos mu) transforms NIH 3T3 cells at low efficiency. Furthermore, the c- mos hu-induced foci are less distinct, and transformed cells contain a high level of human mos protein. The transforming activity of hybrid mos genes derived from human and mouse sequences reveals three domains within the coding region, as well as a negative regulatory sequence upstream from the c- mos hu ORF that reduces its transforming efficiency. The mos C-terminal region, however, which contains the src-kinase homology domain, appears to have the greatest influence on transforming efficiency. The low transforming efficiency of c- mos hu may provide a selective advantage to the host, but it also may indicate a reduced or modified function of mos in humans.

Molecular basis underlying phenotypic revertants of Moloney murine sarcoma virus MuSVts110.

We examined the molecular basis for phenotypic reversion in cells infected with a transformation mutant of murine sarcoma virus, MuSVts110. In MuSVts110-infected NRK cells (6m2 cells), the manifestation of the transformed phenotype at 33 degrees C and the normal phenotype at 39 degrees C is governed by thermosensitive splicing of the MuSVts110 primary transcript, a 4.0-kilobase (kb) RNA which contains the gag and mos genes joined out of frame. At 33 degrees C, selectively, the 4.0-kb RNA is processed to a spliced 3.5-kb RNA in which the gag and mos genes are rejoined in a continuous open reading frame, thus allowing synthesis of the P85gag-mos-transforming protein. In contrast, the MuSVts110 revertant cell lines (designated 54-5A4 and 204-3) appear transformed at all growth temperatures from 33 to 39 degrees C and express a P100gag-mos-transforming protein from an apparently unprocessed 4.0-kb viral RNA. In the current study we established both by S1 nuclease analysis and primer extension sequencing that the revertant 54-5A4 and 204-3 4.0-kb viral RNAs suffered a 5-base deletion at the intron-exon border of the 3' splice site. The effect of this deletion is twofold. First, because of the damage to the 3' splice site, the revertant viral 4.0-kb RNAs cannot be processed to the spliced 3.5-kb RNA and, consequently, cannot be translated to P85gag-mos. Second, the 5-base deletion excises an in-frame stop codon positioned at the intron-exon border in the parental RNA and restores the original mos gene reading frame. The net effect is to produce a continuous open reading frame from the gag, alternate mos, and authentic mos gene reading frames which are fused together in the revertant 4.0-kb RNA. This continuous open reading frame can be translated into the P100gag-mos-transforming protein at any growth temperature.

Murine sarcoma virus ts110 RNA transcripts: origin from a single proviral DNA and sequence of the gag-mos junctions in both the precursor and spliced viral RNAs.

Our previous studies have argued persuasively that in murine sarcoma virus ts110 (MuSVts110) the gag and mos genes are fused out of frame due to a approximately 1.5-kilobase (kb) deletion of wild-type murine sarcoma virus 349 (MuSV-349) viral information. As a consequence of this deletion, infected cells grown at 39 degrees C appear morphologically normal, producing a 4-kb viral RNA and a truncated gag gene product, P58gag. At 33 degrees C, however, MuSVts110-infected cells appear transformed, producing two viral RNAs, about 4 and 3.5 kb in length, and two viral proteins, P58gag and P85gag-mos. Recent S1 nuclease analyses (Nash et al., J. Virol. 50:478-488, 1984) suggested strongly that at 33 degrees C about 430 bases surrounding the out-of-frame gag-mos junction and bounded by consensus splice donor and acceptor sites are excised from the 4-kb RNA to form the 3.5-kb RNA. As a result of this apparent splicing event, the gag and mos genes seemed to be fused in frame and allowed the translation of P85gag-mos. In the present study, DNA primers hybridizing to the MuSVts110 4- and 3.5-kb RNAs just downstream of the gag-mos junction points were used to sequence these junctions by the primer extension method. We observed that, relative to wild-type MuSV-349 5.2-kb RNA, the MuSVts110 4-kb RNA had suffered a 1,488-base deletion as a result of the fusion of wild-type gag gene nucleotide 2404 to wild-type mos gene nucleotide 3892. This gag-mos junction is out of frame, containing both TAG and TGA termination codons in the reading frame 42 and 50 bases downstream of the gag-mos junction, respectively. Thus, the MuSVts110 4-kb RNA can only be translated into a truncated gag precursor containing an additional C-terminal 14 amino acid residues derived from an alternate mos gene reading frame. Similar analyses of the MuSVts110 3.5-kb RNA showed a further loss of both gag and mos sequences over those deleted in the original 1,488-base deletion. In the MuSVts110 3.5-kb RNA, we found that gag nucleotide 2017 was fused to mos nucleotide 3936 (nucleotide 2449 in the MuSVts110 4-kb genome). This 431-base excised fragment is bounded exactly by in-frame consensus splice donor and acceptor sequences. As a consequence of this splice event, the TAG codon is excised and the restoration of the original mos gene reading frame allows the TGA codon to be bypassed.(ABSTRACT TRUNCATED AT 400 WORDS)