Tritrichomonas foetus isolates from cats and cattle show minor genetic differences in unrelated loci ITS-2 and EF-1alpha

The protozoan parasite Tritrichomonas foetus is well known as an important causative agent of infertility and abortion in cattle (bovine trichomonosis). This World Organisation for Animal Health (O.I.E.) notifiable disease is thought to be under control in many countries including Switzerland. In recent studies, however, T. foetus has also been identified as an intestinal parasite that causes chronic large-bowel diarrhoea in cats. Since the feline isolates were considered indistinguishable from bovine isolates, the possibility and risk of parasite transmission from cats to cattle and vice versa has been intensively discussed in current literature. Therefore, we investigated if cat and cattle isolates are genetically distinct from each other or in fact represent identical genotypes. For this purpose, two independent genetic loci were selected that turned out to be well-suited for a PCR sequencing-based genotyping of trichomonad isolates: (i) previously published internal transcribed spacer region 2 (ITS-2) and (ii) a semi-conserved sequence stretch of the elongation factor-1 alpha (EF-1alpha) gene used for the first time in the present study. Respective comparative analyses revealed that both loci were sufficiently variable to allow unambiguous genetic discrimination between different trichomonad species. Comparison of both genetic loci confirmed that T. suis and T. mobilensis are phylogenetically very close to T. foetus. Moreover, these two genetic markers were suited to define host-specific genotypes of T. foetus. Both loci showed single base differences between cat and cattle isolates but showed full sequence identity within strains from either cat or cattle isolates. Furthermore, an additional PCR with a forward primer designed to specifically amplify the bovine sequence of EF-1alpha was able to discriminate bovine isolates of T. foetus from feline isolates and also from other trichomonads. The implications these minor genetic differences may have on the biological properties of the distinct isolates remain to be investigated.

Genetic diversity of EBV-encoded LMP1 in the Swiss HIV Cohort Study and implication for NF-Κb activation

Epstein-Barr virus (EBV) is associated with several types of cancers including Hodgkin's lymphoma (HL) and nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1), a multifunctional oncoprotein, is a powerful activator of the transcription factor NF-κB, a property that is essential for EBV-transformed lymphoblastoid cell survival. Previous studies reported LMP1 sequence variations and induction of higher NF-κB activation levels compared to the prototype B95-8 LMP1 by some variants. Here we used biopsies of EBV-associated cancers and blood of individuals included in the Swiss HIV Cohort Study (SHCS) to analyze LMP1 genetic diversity and impact of sequence variations on LMP1-mediated NF-κB activation potential. We found that a number of variants mediate higher NF-κB activation levels when compared to B95-8 LMP1 and mapped three single polymorphisms responsible for this phenotype: F106Y, I124V and F144I. F106Y was present in all LMP1 isolated in this study and its effect was variant dependent, suggesting that it was modulated by other polymorphisms. The two polymorphisms I124V and F144I were present in distinct phylogenetic groups and were linked with other specific polymorphisms nearby, I152L and D150A/L151I, respectively. The two sets of polymorphisms, I124V/I152L and F144I/D150A/L151I, which were markers of increased NF-κB activation in vitro, were not associated with EBV-associated HL in the SHCS. Taken together these results highlighted the importance of single polymorphisms for the modulation of LMP1 signaling activity and demonstrated that several groups of LMP1 variants, through distinct mutational paths, mediated enhanced NF-κB activation levels compared to B95-8 LMP1.

Mutations of the myeloid transcription factor CEBPA are not associated with the blast crisis of chronic myeloid leukaemia

The transcription factor CEBPA is crucial for normal myeloid differentiation. CEBPA gene mutations have been reported in patients with acute myeloid leukaemia. The inevitable evolution of chronic myeloid leukaemia (CML) in chronic phase (CP) to a fatal blast crisis (BC) is assumed to result from the acquisition of additional genetic changes in the leukaemic clone. Gain of CEBPA mutations might represent a key event causing the differentiation block observed in myeloid CML-BC, but not in CML-CP. Here, no CEBPA mutation in 95 CML-BC patients was found, suggesting a limited role, if any, of CEBPA mutations in this disorder.

Calmodulin-binding transcription activator 1 (CAMTA1) alleles predispose human episodic memory performance

Little is known about the genes and proteins involved in the process of human memory. To identify genetic factors related to human episodic memory performance, we conducted an ultra-high-density genome-wide screen at > 500 000 single nucleotide polymorphisms (SNPs) in a sample of normal young adults stratified for performance on an episodic recall memory test. Analysis of this data identified SNPs within the calmodulin-binding transcription activator 1 (CAMTA1) gene that were significantly associated with memory performance. A follow up study, focused on the CAMTA1 locus in an independent cohort consisting of cognitively normal young adults, singled out SNP rs4908449 with a P-value of 0.0002 as the most significant associated SNP in the region. These validated genetic findings were further supported by the identification of CAMTA1 transcript enrichment in memory-related human brain regions and through a functional magnetic resonance imaging experiment on individuals matched for memory performance that identified CAMTA1 allele-specific upregulation of medial temporal lobe brain activity in those individuals harboring the 'at-risk' allele for poorer memory performance. The CAMTA1 locus encodes a purported transcription factor that interfaces with the calcium-calmodulin system of the cell to alter gene expression patterns. Our validated genomic and functional biological findings described herein suggest a role for CAMTA1 in human episodic memory.

No role of matrixmetalloproteinase-3 genetic promoter polymorphism 1171 as a risk factor for cirrhosis in alcoholic liver disease

BACKGROUND: As only a minority of alcoholics develop cirrhosis, polymorphic genes, whose products are involved in fibrosis development were suggested to confer individual susceptibility. We tested whether a functional promoter polymorphism in the gene encoding matrix metalloproteinase-3 (MMP-3; 1171 5A/6A) was associated liver cirrhosis in alcoholics. METHODS: Independent cohorts from the UK and Germany were studied. (i) UK cohort: 320 alcoholic cirrhotics and 183 heavy drinkers without liver damage and (ii) German cohort: 149 alcoholic cirrhotics, 220 alcoholic cirrhotics who underwent liver transplantation and 151 alcoholics without liver disease. Patients were genotyped for MMP-3 variants by restriction fragment length polymorphism, single strand confirmation polymorphism, and direct sequencing. In addition, MMP-3 transcript levels were correlated with MMP-3 genotype in normal liver tissues. RESULTS: Matrix metalloproteinase-3 genotype and allele distribution in all 1023 alcoholic patients were in Hardy-Weinberg equilibrium. No significant differences in MMP-3 genotype and allele frequencies were observed either between alcoholics with or without cirrhosis. There were no differences in hepatic mRNA transcription levels according to MMP-3 genotype. CONCLUSIONS: Matrix metalloproteinase-3 1171 promoter polymorphism plays no role in the genetic predisposition for liver cirrhosis in alcoholics. Stringently designed candidate gene association studies are required to exclude chance observations.

Genetic testing for glucokinase mutations in clinically selected patients with MODY: a worthwhile investment

The differential diagnosis for children with diabetes includes a group of monogenic diabetic disorders known as maturity-onset diabetes of the young (MODY). So far, six underlying gene defects have been identified. The most common subtypes are caused by mutations in the genes encoding the transcription factor HNF-1a (MODY 3) and the glycolytic enzyme glucokinase (GCK) (MODY 2). MODY 2 is the most benign form of diabetes as the threshold for glucose sensing is elevated resulting in mild, regulated hyperglycemia. MODY 2 may usually be treated with diet alone without risk of microvascular complications. Patients with MODY usually present as children or young adults. Genetic testing for MODY in diabetic subjects is often not performed because of the costs and its unavailability in Switzerland. We describe the impact of the genetic analysis for MODY 2 on diabetes management and treatment costs in a five-year-old girl. The patient and her diabetic mother were both found to have a heterozygous missense mutation (V203A) in the glucokinase gene. The five-year-old girl was started on insulin therapy for her diabetes but because her HbA1c remained between 5.8-6.4% (reference 4.1-5.7%) and her clinical presentation suggested MODY insulin was discontinued. She is now well controlled on a carbohydrate controlled diet regimen only. Omission of insulin treatment made regular blood glucose monitoring unnecessary and removed her risk of hypoglycemia. Costs for the genetic analysis were 500 Euro. At our centre costs for diabetes care of a patient with type 1 diabetes are approximately 2050 Euro/year compared to 410 Euro/year for the care of a patient with MODY 2. In addition, a diagnosis of MODY 2 may reassure patients and their families, as microvascular complications are uncommon. Thus there are both health and financial benefits in diagnosing MODY 2. We recommend genetic testing for MODY 2 in clinically selected patients even though this analysis is currently not available in Switzerland and costs are not necessarily covered by the health insurances.

GENETIC ANALYSIS OF THE FUNCTION OF THE DROSOPHILA DOUBLESEX-RELATED FACTOR dmrt93B

DMRT (Doublesex and Mab-3 related transcription factor) proteins generally associated with sexual differentiation in many organisms share a common DNA binding domain and are often expressed in reproductive tissues. Aside from doublesex, which is a central factor in the regulation of sex determination, Drosophila possesses three different dmrt genes that are of unknown function. Because the association with sexual differentiation and reproduction is not universal and some DMRT proteins have been found to play other developmental roles we chose to further characterize one of these Drosophila genes. We carried out genetic analysis of dmrt93B, which was previously found to be expressed sex-specifically in the developing somatic gonad and to affect testis morphogenesis in RNAi knockdowns. In order to disrupt this gene, the GAL4 yeast transcriptional activator followed by a polyadenylation signal was inserted after the dmrt93B start codon and introduced into the genome by homologous recombination. Analysis of the knock-in mutation as well as a small deletion removing all dmrt93B sequence demonstrate that loss of function causes partial lethality at the late pupal stage. Surprisingly, these mutations have no significant effect on gonad formation or male fertility. Analysis of GAL4-driven GFP reporter expression indicates that the dmrt93B promoter activity is highly specific to neurons in the suboesophageal and proventricular ganglion in larva and adult of both sexes suggesting a possible role in digestive tract function. Using the Capillary Feeder (CAFÉ) assay to measure daily food intake we find that reduction in this gene’s function leads to an increase in food consumption. These results suggest dmrt93 plays an important role in the formation or maintenance of neurons that affect feeding and support the idea that dmrt genes may not be restricted to roles in sexual differentiation.

Genetic networks controlling retinal injury.

PURPOSE: The present study defines genomic loci underlying coordinate changes in gene expression following retinal injury. METHODS: A group of acute phase genes expressed in diverse nervous system tissues was defined by combining microarray results from injury studies from rat retina, brain, and spinal cord. Genomic loci regulating the brain expression of acute phase genes were identified using a panel of BXD recombinant inbred (RI) mouse strains. Candidate upstream regulators within a locus were defined using single nucleotide polymorphism databases and promoter motif databases. RESULTS: The acute phase response of rat retina, brain, and spinal cord was dominated by transcription factors. Three genomic loci control transcript expression of acute phase genes in brains of BXD RI mouse strains. One locus was identified on chromosome 12 and was highly correlated with the expression of classic acute phase genes. Within the locus we identified the inhibitor of DNA binding 2 (Id2) as a candidate upstream regulator. Id2 was upregulated as an acute phase transcript in injury models of rat retina, brain, and spinal cord. CONCLUSIONS: We defined a group of transcriptional changes associated with the retinal acute injury response. Using genetic linkage analysis of natural transcript variation, we identified regulatory loci and candidate regulators that control transcript levels of acute phase genes.

A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development.

The molecular mechanisms controlling bone extracellular matrix (ECM) deposition by differentiated osteoblasts in postnatal life, called hereafter bone formation, are unknown. This contrasts with the growing knowledge about the genetic control of osteoblast differentiation during embryonic development. Cbfa1, a transcriptional activator of osteoblast differentiation during embryonic development, is also expressed in differentiated osteoblasts postnatally. The perinatal lethality occurring in Cbfa1-deficient mice has prevented so far the study of its function after birth. To determine if Cbfa1 plays a role during bone formation we generated transgenic mice overexpressing Cbfa1 DNA-binding domain (DeltaCbfa1) in differentiated osteoblasts only postnatally. DeltaCbfa1 has a higher affinity for DNA than Cbfa1 itself, has no transcriptional activity on its own, and can act in a dominant-negative manner in DNA cotransfection assays. DeltaCbfa1-expressing mice have a normal skeleton at birth but develop an osteopenic phenotype thereafter. Dynamic histomorphometric studies show that this phenotype is caused by a major decrease in the bone formation rate in the face of a normal number of osteoblasts thus indicating that once osteoblasts are differentiated Cbfa1 regulates their function. Molecular analyses reveal that the expression of the genes expressed in osteoblasts and encoding bone ECM proteins is nearly abolished in transgenic mice, and ex vivo assays demonstrated that DeltaCbfa1-expressing osteoblasts were less active than wild-type osteoblasts. We also show that Cbfa1 regulates positively the activity of its own promoter, which has the highest affinity Cbfa1-binding sites characterized. This study demonstrates that beyond its differentiation function Cbfa1 is the first transcriptional activator of bone formation identified to date and illustrates that developmentally important genes control physiological processes postnatally.

Ligand-signaled upregulation of Enterococcus faecalis ace transcription, a mechanism for modulating host-E. faecalis interaction.

Enterococcus faecalis, the third most frequent cause of bacterial endocarditis, appears to be equipped with diverse surface-associated proteins showing structural-fold similarity to the immunoglobulin-fold family of staphylococcal adhesins. Among the putative E. faecalis surface proteins, the previously characterized adhesin Ace, which shows specific binding to collagen and laminin, was detectable in surface protein preparations only after growth at 46 degrees C, mirroring the finding that adherence was observed in 46 degrees C, but not 37 degrees C, grown E. faecalis cultures. To elucidate the influence of different growth and host parameters on ace expression, we investigated ace expression using E. faecalis OG1RF grown in routine laboratory media (brain heart infusion) and found that ace mRNA levels were low in all growth phases. However, quantitative reverse transcription-PCR showed 18-fold-higher ace mRNA amounts in cells grown in the presence of collagen type IV compared to the controls. Similarly, a marked increase was observed when cells were either grown in the presence of collagen type I or serum but not in the presence of fibrinogen or bovine serum albumin. The production of Ace after growth in the presence of collagen type IV was demonstrated by immunofluorescence microscopy, mirroring the increased ace mRNA levels. Furthermore, increased Ace expression correlated with increased collagen and laminin adhesion. Collagen-induced Ace expression was also seen in three of three other E. faecalis strains of diverse origins tested, and thus it appears to be a common phenomenon. The observation of host matrix signal-induced adherence of E. faecalis may have important implications on our understanding of this opportunistic pathogen.

CREB trans-activation of disruptor of telomeric silencing-1 mediates forskolin inhibition of CTGF transcription in mesangial cells.

Connective tissue growth factor (CTGF) participates in diverse fibrotic processes including glomerulosclerosis. The adenylyl cyclase agonist forskolin inhibits CTGF expression in mesangial cells by unclear mechanisms. We recently reported that the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) suppresses CTGF gene expression in collecting duct cells (J Clin Invest 117: 773-783, 2007) and HEK 293 cells (J Biol Chem In press). In the present study, we characterized the involvement of Dot1 in mediating the inhibitory effect of forskolin on CTGF transcription in mouse mesangial cells. Overexpression of Dot1 or treatment with forskolin dramatically suppressed basal CTGF mRNA levels and CTGF promoter-luciferase activity, while hypermethylating H3K79 in chromatin associated with the CTGF promoter. siRNA knockdown of Dot1 abrogated the inhibitory effect of forskolin on CTGF mRNA expression. Analysis of the Dot1 promoter sequence identified a CREB response element (CRE) at -384/-380. Overexpression of CREB enhanced forskolin-stimulated Dot1 promoter activity. A constitutively active CREB mutant (CREB-VP16) strongly induced Dot1 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. Mutation of the -384/-380 CRE resulted in 70% lower levels of Dot1 promoter activity. ChIP assays confirmed CREB binding to the Dot1 promoter in chromatin. We conclude that forskolin stimulates CREB-mediated trans-activation of the Dot1 gene, which leads to hypermethylation of histone H3K79 at the CTGF promoter, and inhibition of CTGF transcription. These data are the first to describe regulation of the Dot1 gene, and disclose a complex network of genetic and epigenetic controls on CTGF transcription.

Genetic and physical analysis of the bacteriophage P22 tail protein

A plasmid based genetic system was developed for the tail protein of the Salmonella typhimurium bacteriophage P22 and used to isolate and characterize tail protein mutants. The tail protein is a trimeric structural protein of the phage and an endorhamnosidase whose activity is essential for infection. The gene for the tail protein has previously been cloned into a plasmid expression vector and sequenced. A plate complementation assay for tail protein produced from the cloned gene was developed and used to isolate 27 tail protein mutants following mutagenesis of the cloned gene. These mutations were mapped into 12 deletion intervals using deletions which were made on plasmids in vitro and crossed onto P22. The base substitutions were determined by DNA sequencing. The majority of mutants had missense or nonsense mutations in the protein coding portion of the gene; however four of the mutants were in the putative transcription terminator. The oligomeric state of tail protein from the 15 missense mutants was investigated using SDS and nondenaturing polyacrylamide gel electrophoresis of cell lysates. Wild-type tail protein retains its trimeric structure in SDS gels at room temperature. Two of the mutant proteins also migrated as trimers in SDS gels, yet one of these had a considerably faster mobility than wild-type trimer. Its migration was the same as wild-type in a nondenaturing gel, so it is thought to be a trimer which is partially denatured by SDS. Four of the mutants produced proteins which migrate at the position of a monomer in an SDS gel but cannot be seen on a nondenaturing gel. These proteins are thought to be either monomers or soluble aggregates which cannot enter the nondenaturing gel. The remainder of mutants produce protein which is degraded. The mutant tail protein which had normal trimeric mobility on SDS and nondenaturing gels was purified. This protein has essentially wild-type ability to attach to phage capsids, but its endorhamnosidase activity is only 4% of wild-type. ^

Genetic Engineering of Induced Pluripotent Stem Cells and Reprogramming to Motor Neurons to Elucidate Selective Motor Neuron Death in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease, fatal within 1 to 5 years after onset of symptoms. About 3 out of 100’000 persons are diagnosed with ALS and there is still no cure available [1, 2]. 95% of all cases occur sporadically and the aetiology remains largely unknown [XXXX]. However, up to now 16 genes were identified to play a role in the development of familial ALS. One of these genes is FUS that encodes for the protein fused in sarcoma/translocated in liposarcoma (FUS/TLS). Mutations in this gene are responsible for some cases of sporadic as well as of inherited ALS [3]. FUS belongs to the family of heterogeneous nuclear ribonucleoproteins and is predicted to be involved in several cellular functions like transcription regulation [4], RNA splicing [5, 6], mRNA transport in neurons [7] and microRNA processing [8]. Aberrant accumulation of mutated FUS has been found in the cytoplasm of motor neurons from ALS patients [9]. The mislocalization of FUS is based on a mutation in the nuclear localization signal of FUS [10]. However, it is still unclear if the cytoplasmic localization of FUS leads to a toxic gain of cytoplasmic function and/or a loss of nuclear function that might be crucial in the course of ALS. The goal of this project is to characterize the impact of ALS-associated FUS mutations on in vitro differentiated motor neurons. To this end, we edit the genome of induced pluripotent stem cells (iPSC) using transcription activator-like effector nucleases (TALENs) [11,12] to create three isogenic cell lines, each carrying an ALS-associated FUS mutation (G156E, R244C and P525L). These iPSC’s will then be differentiated to motor neurons according to a recently establishe protocol (Ref Wichterle) and serve to study alterations in the transcriptome, proteome and metabolome upon the expression of ALS-associated FUS. With this approach, we hope to unravel the molecular mechanism leading to FUS-associated ALS and to provide new insight into the emerging connection between misregulation of RNA metabolism and neurodegeneration, a connection that is currently implied in a variety of additional neurological diseases, including spinocerebellar ataxia 2 (SCA-2), spinal muscular atrophy (SMA), fragile X syndrome, and myotonic dystrophy.

Genetic Engineering of Induced Pluripotent Stem Cells and Reprogramming to Motor Neurons to Elucidate Selective Motor Neuron Death in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease, fatal within 1 to 5 years after onset of symptoms. About 3 out of 100’000 persons are diagnosed with ALS and there is still no cure available [1, 2]. 95% of all cases occur sporadically and the aetiology remains largely unknown [3]. However, up to now 16 genes were identified to play a role in the development of familial ALS. One of these genes is FUS that encodes for the protein fused in sarcoma (FUS). Mutations in this gene are responsible for some cases of sporadic as well as of inherited ALS [4]. FUS belongs to the family of heterogeneous nuclear ribonucleoproteins and is predicted to be involved in several cellular functions like transcription regulation, RNA splicing, mRNA transport in neurons and microRNA processing [5] Aberrant accumulation of mutated FUS has been found in the cytoplasm of motor neurons from ALS patients [6]. The mislocalization of FUS is based on a mutation in the nuclear localization signal of FUS [7]. However, it is still unclear if the cytoplasmic localization of FUS leads to a toxic gain of cytoplasmic function and/or a loss of nuclear function that might be crucial in the course of ALS. The goal of this project is to characterize the impact of ALS-associated FUS mutations on in vitro differentiated motor neurons. To this end, we edit the genome of induced pluripotent stem cells (iPSC) using transcription activator-like effector nucleases (TALENs) [8,9] to create three isogenic cell lines, each carrying an ALS-associated FUS mutation (G156E, R244C and P525L). These iPSC’s will then be differentiated to motor neurons according to a recently established protocol [10] and serve to study alterations in the transcriptome, proteome and metabolome upon the expression of ALS-associated FUS. With this approach, we hope to unravel the molecular mechanism leading to FUS-associated ALS and to provide new insight into the emerging connection between misregulation of RNA metabolism and neurodegeneration, a connection that is currently implied in a variety of additional neurological diseases, including spinocerebellar ataxia 2 (SCA-2), spinal muscular atrophy (SMA), fragile X syndrome, and myotonic dystrophy. [1] Cleveland, D.W. et al. (2001) Nat Rev Neurosci 2(11): 806-819 [2] Sathasivam, S. (2010) Singapore Med J 51(5): 367-372 [3] Schymick, J.C. et al. (2007) Hum Mol Genet Vol 16: 233-242 [4] Pratt, A.J. et al. (2012). Degener Neurol Neuromuscul Dis 2012(2): 1-14 [5] Lagier-Tourenne, C. Hum Mol Genet, 2010. 19(R1): p. R46-64 [6] Mochizuki, Y. et al. (2012) J Neurol Sci 323(1-2): 85-92 [7] Dormann, D. et al. (2010) EMBO J 29(16): 2841-2857 [8] Hockemeyer, D. et al. (2011) Nat Biotech 29(8): 731-734 [9] Joung, J.K. and J.D. Sander (2013) Nat Rev Mol Cell Biol 14(1): 49-55 [10]Amoroso, M.W. et al. (2013) J Neurosci 33(2): 574-586.

STRUCTURAL ANALYSIS OF THE TRANSCRIPTION PRODUCTS OF A TEMPERATURE-SENSITIVE PHENOTYPIC MUTANT OF MOLONEY SARCOMA VIRUS--MUSV TS110

Cells infected with a temperature sensitive phenotypic mutant of Moloney sarcoma virus (MuSVts110) exhibit a transformed phenotype at 33('(DEGREES)) and synthesize two virus specific proteins, p85('gag-mos), a gag-mos fusion protein and p58('gag), a truncated gag precursor protein (the gag gene codes for viral structural proteins and mos is the MuSV transforming gene). At 39('(DEGREES)) only p58('gag) is synthesized and the morphology of the cells is similar to uninfected NRK parental cells. Two MuSVts110 specific RNAs are made in MuSVts110-infected cells, one of 4.0 kb in length, the other of 3.5 kb. Previous work indicated that each of these RNAs arose by a single central deletion of parental MuSV genetic material, and that p58('gag) was made by the 4.0 kb RNA and p85('gag-mos) from the 3.5 kb RNA. The objective of my dissertation research was to map precisely the deletion boundaries of both of the MuSVts110 RNAs, and to determine the proper reading frame across both deletion borders. This work succeeded in arriving at the following conclusions: (a) Using S-1 nuclease analysis and primer extension sequencing, it was found that the 4.0 kb MuSVts110 RNA arose by a 1488 base deletion of 5.2 kb parental MuSV genomic RNA. This deletion resulted in an out of frame fusion of the gag and mos genes that resulted in the formation of a "stop" codon which causes termination of translation just beyond the c-terminus of the gag region. Thus, this RNA can only be translated into the truncated gag protein p58('gag). (b) S-1 analysis of RNA from cells cultivated at different temperatures demonstrated that the 4.0 kb RNA was synthesized at all temperatures but that synthesis of the 3.5 kb RNA was temperature sensitive. These observations supported the data derived from blot hybridization experiments the interpretation of which argued for the existence of a single provirus in MuSVts110 infected cells, and hence only a single primary transcript (the 4.0 kb RNA). (c) Analyses similar to those described in (a) above showed that the 3.5 kb RNA was derived from the 4.0 kb MuSVts110 RNA by a further deletion of 431 bases, fusing the gag and mos genes into a continuous reading frame capable of directing synthesis of the p85('gag-mos) protein. These sequence data and the presence of only one MuSVts110-specific provirus, indicate that a splice mechanism is employed to generate the 3.5 kb RNA since the gag and mos genes are observed to be fused in frame in this RNA. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^