A temperature-sensitive mutant of the myeloproliferative sarcoma virus, altered by a point mutation in the mos oncogene, has been modified as a selectable retroviral vector

Abstract

The myeloproliferative sarcoma virus (MPSV) is a mos-oncogenic retrovirus which induces an acute myeloproliferative disease in adult mice. The isolation and molecular cloning of two mutants of MPSV temperature sensitive (ts) for mos transformation (Kollek et al., J. Virol. 50:717-724, 1984) have been described previously. In this report, we describe the biological activity of these clones, the molecular basis of the ts lesion of one clone, and the construction of a selectable vector based on the MPSV ts genome. Both molecular clones, ts159 and ts124, proved to have retained the ts phenotype, the former being tighter for the induction and maintenance of the transformed phenotype. A single transition (G-*A) at position 1888 in the mos coding region, resulting in the change of Gly to Arg at position 307, was responsible for the ts phenotype of clone ts159. Substitution of sequences carrying this mutation with the corresponding sequences of the * Corresponding author. t Present address: Whitehead

v-mos proteins encoded by myeloproliferative sarcoma virus and its ts159 mutant

“…The possible reason for this earlier sequencing error could be the high GC content in the region surrounding codon 262. The point mutations reported earlier for MPSV and ts159 MPSV v-mos sequences (6,31) were, however, 6. Nucleic acid sequence analysis around codon 7 of the env-mos coding region in MPSV (clone 18-663).…”

“…The sequence was determined with a sense strand primer (5'-ACCAGGGAGGTGCCTTACTC-3'). The sequences read from the regions between the arrows are shown in panels C and D. (C) Contrary to the previous reports (6,31), codon 262 was found to be present in MPSV v-mos. (D) The sequencing of codons 327 to 348 confirmed the sequences reported earlier (6,34).…”

“…In order to provide convincing evidence that the MPSV v-mos protein is similar in size and protein kinase activity to p37env-mos, we utilized an in vitro transcription-translation system. Both the wild-type and the ts159 forms of MPSV v-mos genes (the XbaI-HindIII fragment spanning the fulllength v-mos gene excised from plasmids p18-663 and p20-159, representing the wild-type and the ts159 mutant [6], respectively) were cloned into the transcription vector pSPT18 (Boehringer Mannheim, Indianapolis, Ind.). RNA was synthesized in vitro with T7 RNA polymerase.…”

The Chicken Lysozyme 5′ Matrix Attachment Region Increases Transcription from a Heterologous Promoter in Heterologous Cells and Dampens Position Effects on the Expression of Transfected Genes

“…Kb, Kilobase. plasmid tk-neo containing the neomycin resistance gene as a selectable gene, and 5 x i05 rat-2 cells (34) were transfected as described previously (12). (In pioneering experiments with solely plasmid tk-neo, linearization did not increase the frequency of G418-resistant clones.)…”

“…G418-resistant clones were isolated 14 days later. Tissue culture conditions used have been previously described (12). Cellular DNA was purified and analyzed (23,33).…”