Analysis of p60v-src mutants carrying lesions involved in temperature sensitivity.

Analysis of the src genes of three temperature-sensitive (ts) mutants of Rous sarcoma virus (tsNY68, tsNY72-4, and PA104) showed that each has two C-terminal mutations in the kinase domain required for temperature sensitivity, as assayed by morphological alteration and anchorage-independent growth. In all three mutants, one of the mutations is a valine-to-methionine change at position 461. To assess the contribution of each mutation to the biochemical properties of the src protein, we analyzed the kinase activity and the interaction with cellular proteins p50 and p90 of recombinant src gene products in which only one mutation was combined with wild-type src sequences. Chimeric src protein containing only the Met-461 mutation was indistinguishable from the wild type by all criteria examined, while the effect of the second C-terminal mutation alone varied with the defectiveness of the parental ts mutant. The second mutation alone, while not sufficient to cause ts transformation, altered p60src complex formation with cellular proteins p50 and p90 and altered the in vitro thermolability of src kinase activity. The results indicate that these biochemical properties of p60src are more sensitive to mutation than others, such as in vivo kinase activity, which require more profound structural alterations.     

Linker insertion-deletion mutagenesis of the v-src gene: isolation of host- and temperature-dependent mutants.

The host cell regulators and substrates of the Rous sarcoma virus transforming protein pp60v-src remain largely unknown. Viral mutants which induce a host-dependent phenotype may result from mutations which affect the interaction of pp60v-src with host cell components. To isolate such mutants and to examine the role of different regions of src in regulating pp60v-src function, we generated 46 linker insertion and 5 deletion mutations within src. The mutant src genes were expressed in chicken embryo fibroblasts and in rat-2 cells by using retrovirus expression vectors. Most linker insertions within the kinase domain (residues 260 to 512) inactivated kinase activity and transforming capacity, while most insertions in the N-terminal domain and at the extreme C terminus were tolerated. A number of mutations generated a host-dependent phenotype. Insertions after residues 225 and 227, within the N-terminal regulatory domain (SH2), produced a fusiform transformation in chicken embryo fibroblasts and abolished transformation in rat-2 cells; a similar phenotype also resulted from two deletions affecting SH2 (residues 149 to 174 and residues 77 to 225). Insertions immediately C terminal to Lys-295, which is involved in ATP binding, also produced a conditional phenotype. Insertions after residues 299 and 300 produced a temperature-sensitive phenotype, while insertions after residues 304 and 306 produced a host cell-dependent phenotype. An insertion which removed the major tyrosine autophosphorylation site (Tyr-416) greatly reduced transformation of rat-2 cells, a property not previously observed with other mutations at this site. We conclude that mutations at certain sites within src result in conditional phenotypes. These sites may represent regions important in interactions with host cell components.     

src Genes of Ten Rous Sarcoma Virus Strains, Including Two Reportedly Transduced from the Cell, Are Completely Allelic; Putative Markers of Transduction Are Not Detected

The src genes of different Rous sarcoma virus (RSV) strains have been reported to be highly conserved by some investigators using RNA-cDNA hybridization, whereas others using oligonucleotide, peptide, and serological analyses have judged src genes to be variable in 30 to 50% of the respective markers. Moreover, distinctive src oligonucleotides and peptides of so-called recovered RSVs (rRSV's) whose src genes were reported to be experimentally transduced from the cell are thought to represent specific markers of host-derived src sequences. By contrast, we have pointed out previously that these markers may represent point mutations of parental equivalents. Here we have compared the src-specific sequences of eight RSV strains and of two rRSV's to each other and to a molecular clone of the src-related chicken locus. Our comparisons are based on RNase T(1)-resistant oligonucleotides of RNA hybridized to src-specific cDNA, which was prepared by hybridizing RSV cDNA with RNA of isogenic src deletion mutants, or to a cloned cellular src-related DNA. All of the approximately 20 src-oligonucleotides of a given RSV strain were recovered by src-specific cDNA's of all other RSV strains or by cellular src-related DNA. The number of oligonucleotides varied slightly with the length of the src deletion used to prepare src-specific cDNA, thus providing a measure for src deletion mutants. Our data indicate that the src genes of all RSV strains tested, including the two reportedly transduced from the cell, are about 98% conserved and completely allelic with only scattered single nucleotide differences in certain variable regions which are subject to point mutations. Hence, based on the src oligonucleotide markers analyzed by us and others, we cannot distinguish between a cellular and viral origin of rRSV's. However, the following are not compatible with a cellular origin of rRSV's. (i) The only putative oligonucleotide marker which is exclusively shared by the two rRSV's studied and which differs from a parental counterpart in a single base was not detectable in cellular src-related DNA. (ii) The number of different allelic src markers observed by us and others in rRSV's was too large to derive from one or two known cellular src-related loci. (iii) The known absence of linkage of the cellular src-related locus with other virion sequences was extended to all non-src oligonucleotides, including some mapping directly adjacent to src. This is difficult to reconcile with the claim that transformation-defective, partial src deletion mutants of RSV which contain both, one, or, as we show here, possibly no src termini nevertheless transduce at the same frequencies, even though homologous, single or double illegitimate recombinations would be involved. Given (i) our evidence that src genes are subject to point mutation under selective conditions similar to those prevailing when rRSV's were generated and (ii) the lack of absolute evidence for the clonal purity of the transformation-defective, partial src deletion mutants of RSV used to generate rRSV's, we submit that the src genes of rRSV's could have been generated by cross-reactivation of nonoverlapping src deletions or mutation of src variants possibly present in transformation-defective, partial src deletion mutants of RSV. To prove experimental transduction, unambiguous markers need to be identified, or it would be necessary to generate rRSV's with molecularly cloned transformation-defective, partial src deletion mutants of RSV. Although our evidence casts doubt on the idea that specific src sequences of rRSV's originated by transduction, the close relationship between viral src and cellular src-related sequences argues that src genes originated at one time in evolution from the cell by events that involved illegitimate recombination and deletion of non-src sequences that interrupt the cellular src locus.     

Syncytial mutations in the herpes simplex virus type 1 gK (UL53) gene occur in two distinct domains.

Syncytial (syn) mutants of herpes simplex virus cause cell fusion. Many syn mutations map to the syn1 locus, which has been identified with the gK (UL53) gene. In this work, the gK genes of eight syn mutants derived from the KOS strain were sequenced to identify residues and, possibly, domains important for the fusion activity of mutant gK. DNA sequencing showed that six mutants (syn30, syn31, syn32, syn102, syn103, and syn105) had single missense mutations in the gK gene. Two of these, syn31 and syn32, had identical mutations that caused the introduction of a potential site for N-linked glycosylation. syn31 gK was analyzed by in vitro translation and found to utilize the novel glycosylation site. Two other mutants, syn8 and syn33, had three mutations each, resulting in three amino acid substitutions in syn8 and two substitutions in syn33. Of the 10 gK syn mutant sequences known, 8 have mutations in the N-terminal domain of gK, suggesting that this domain, which is likely to be an ectodomain, is important for the function of the protein. The other two mutants, syn30 and syn103, have mutations near the C terminus of gK.     

Generation of nondefective Rous sarcoma virus by asymmetric recombination between deletion mutants.

A replication-defective deletion mutant of Prague Rous sarcoma virus (RSV), which lacks functional gag, pol, and env genes, was crossed with a transformation-defective deletion mutant derived from Schmidt-Ruppin RSV. Transformation- and replication-competent viruses were generated in the cross. Characterization of one of these rescued viruses indicated that it was a nondefective recombinant containing the src gene of the replication-defective mutant plus the replicative genes of the transformation-defective virus. These results indicate that, contrary to previous reports, asymmetric recombination between RSV deletion mutants can result in the formation of nondefective RSV.     

Site-directed mutagenesis of the src gene of Rous sarcoma virus: construction and characterization of a deletion mutant temperature sensitive for transformation

Transformation of cells by Rous sarcoma virus results from the expression of the viral src gene product, pp60src. Site-directed mutagenesis techniques have been used to construct defined deletion mutations within the src gene of Prague A strain of Rous sarcoma virus. The deletion of DNA sequences at the Bg/II restriction site in the src gene yielded both transformation-defective mutants (tdCH4, 64, and 146) and a mutant temperature sensitive for morphological transformation (tsCH119). The genome of tsCH119 contains an in-phase deletion of approximately 160 base pairs, which mapped to the immediate 3' side of the Bg/II restriction site. Upon infection of chicken cells, tsCH119 encoded a structurally altered src protein, pp53src, containing a deletion of amino acid residues 202 to 255. Immune complexes containing pp53src isolated from tsCH119-infected cells grown at 41 degrees C exhibited only 50% less tyrosine-specific kinase activity than immune complexes isolated from cells grown at 35 degrees C. pp53src immunoprecipitated from tsCH119-infected cells grown at either 35 or 41 degrees C contained phosphoserine and phosphotyrosine. We suggest that tsCH119 represents a class of mutants containing mutations mapping within a functionally important domain of the src protein, distinct from the domain specifying the protein kinase activity.     

Genetic control of galactokinase synthesis in Saccharomyces cerevisiae: evidence for constitutive expression of the positive regulatory gene gal4.

Temperature-sensitive (ts) mutants for the gal80 and gal4 genes of Saccharomyces cerevisiae were isolated and characterized. These mutants were classified into two categories; one showed thermolability (TL) and the other showed temperature-sensitive synthesis (TSS) of the respective products. Both the TL and TSS gal80 mutants are constitutive for galactokinase activity at 35 degrees C and, because they are derived from a dominant super-repressible GAL80s mutant, are uninducible at 25 degrees C. Both the TL and TSS gal4 mutants are galactose negative at 35 degrees C and galactose positive at 25 degrees C. None of the ts gal4 mutations affected the thermolability of galactokinase activity in cell extracts. Induction of galactokinase activity was studied with these mutants. The results indicate that the gal80 gene codes for a repressor and the gal4 gene codes for a positive factor indispensable for the expression of the structural genes or their products. However, striking evidence that the expression of the gal4 gene is constitutive and not under the control of gal80 was provided by a kinetic study with the TL gal4 mutant. The TL gal4 mutant pregrown in glycerol nutrient medium at 35 degrees C showed a prolonged lag period (35 min) in the induction of galactokinase activity at 25 degrees C, whereas the same mutant pregrown at 25 degrees C showed the same lag period as those observed in the wild-type strain and a revertant clone derived from the TL gal4 mutant (15 min).     

Temperature-sensitive mutations in various genes of Escherichia coli K12 can be suppressed by the ssrA gene for 10Sa RNA (tmRNA)

An Escherichia coli strain with a deletion in the ssrA gene that encodes 10Sa RNA (tmRNA) was used to screen for temperature-sensitive (ts) mutants whose ts phenotypes were suppressible by introduction of the wild-type ssrA gene. Mutants in four different genes were isolated. Ts mutants of this type were also obtained in a screen for mutations in thyA, the structural gene for thymidylate synthase. The ThyA activity in crude extracts prepared from the ts mutants was temperature-sensitive. The presence of the ssrA gene caused an increase in the total amount of the temperature-sensitive enzyme expressed, rather than suppressing the ts activity of the enzyme itself. SsrA-DD, a mutant form of 10Sa RNA, suppressed the ts phenotype of a thyA mutant, suggesting that degradation of a tagged peptide was not required for suppression of the ts phenotype. Considering the fact that ssrA-suppressible mutants could be isolated as temperature-sensitive mutants with mutations in different genes, it seems evident that trans-translation can occur on mRNA that is not lacking its stop codon.     

The temperature-sensitive (ts) phenotype of a cold-passaged (cp) live attenuated respiratory syncytial virus vaccine candidate, designated cpts530, results from a single amino acid substitution in the L protein.

cpts530, a candidate live-virus vaccine, is an attenuated strain of human respiratory syncytial virus (RSV). It was derived by subjecting a cold-passaged (cp) strain of RSV to a single round of chemical mutagenesis. cpts530 is a temperature-sensitive (ts) mutant that is attenuated in mice and chimpanzees, and its ts phenotype exhibits a high level of stability during replication in both species. In the present study, the complete nucleotide sequence of cpts530 RSV was determined. The five mutations known to be present in the parent cpRSV were retained in its cpts530 derivative, and one additional nucleotide change was identified at nucleotide (nt) 10060, which resulted in a phenylalanine-to-leucine change at amino acid 521 in the large polymerase (L) protein. To determine if this single amino acid substitution was indeed responsible for the ts phenotype of cpts530, it was introduced alone or in combination with the cp mutations into the full-length cDNA clone of the wild-type A2 RSV. Analysis of infectious viruses recovered from mutant cDNAs indicated that this single mutation specified complete restriction of plaque formation of recombinant cp530 in HEp-2 cell monolayer cultures at 40 degrees C, and the level of temperature sensitivity was not influenced by the presence of the five cpRSV mutations. These findings identify the phenylalanine-to-leucine change at amino acid 521 in the L protein as the mutation that specifies the ts phenotype of cpts530. Furthermore, these findings illustrate the feasibility of using the cDNA-based recovery system to analyze and construct defined attenuated vaccine viruses.     

Role of p60src kinase activity in the induction of neuroretinal cell proliferation by rous sarcoma virus.

Expression of the src gene of Rous sarcoma virus (RSV) in chicken embryo neuroretinal (NR) cells results in morphological transformation and sustained proliferation of a normally resting cell population. We have previously reported the isolation of mutants of RSV which retain full growth-promoting activity while displaying reduced transforming properties. Two such mutants, PA101 and PA104, were used to investigate whether the p60src-associated kinase activity is required for the mitogenic function of src. A comparison of the patterns of phosphorylation of wild-type and mutant p60src revealed that the phosphorylation of tyrosine residues of p60src of PA104 was markedly reduced, whereas the relative amount of phosphotyrosine in p60src of PA101 was comparable to that of the wild-type protein. In vitro kinase activity of p60src immunoprecipitated from NR cells infected with PA101 or PA104 as measured by phosphorylation of the heavy chains of specific immunoglobulin G molecules was 1/10 that of the wild-type molecule. Moreover, when NR cells infected with mutants temperature sensitive for mitogenic capacity were maintained at a temperature either permissive or restrictive for cell growth, quantitation of kinase activity indicated that proliferation of NR cells could not be linked to the absolute level of in vitro kinase activity of p60src. Transformation of NR cells by wild-type RSV resulted in a 10-fold increase in total cellular phosphotyrosine and in the phosphorylation of tyrosine residues of a 34K protein, a possible in vivo substrate for p60src. In contrast, phosphorylation of tyrosine residues of cellular targets was markedly reduced in NR cells infected with PA101 or PA104. These results indicate that the mitogenic capacity of RSV in NR cells does not require elevated levels of p60src kinase activity.     

Stimulation of growth rate of chondrocytes by Rous sarcoma virus is not coordinated with other expressions of the src gene phenotype

Infection and transformation of chondrocytes by Rous sarcoma viruses (RSVs) (Schmidt-Ruppin, Prague) stimulated the rate of cell growth. In contrast, several transformation-defective (td) mutants (tdPRA, tdNY105, tdNY106, tdNY107, and tdNY108) retaining various sizes of the src gene did not stimulate cell growth, indicating that the stimulation of growth of chondrocytes is due to the function of the src gene. With the use of various T (transformation)-class temperature-sensitive (ts) mutants of RSV, growth stimulation of chondrocytes by the src gene was examined. It was found that there are two types of T-class ts mutants with regard to the stimulatory effect on the growth of chondrocytes. One type (tsNY68) stimulates cell growth at both permissive (36 degrees C) and nonpermissive (41.5 degrees C) temperature, as does the wild type of RSV. Another type (ts GI201 [clone 9]) stimulates cell growth only at the permissive temperature. Chondrocytes infected with either of these two types of T-class ts mutants showed ts properties in other transformation markers, such as uptake of 2-deoxy-D-glucose, change of cell morphology, and focus formation. These data indicate that the effect of the src gene on cell growth does not occur coordinately with other transformation markers.     

Temperature-sensitive transforming mutants of the v-rel oncogene.

By making site-directed mutations in the avian retroviral oncogene v-rel, we created two temperature-sensitive (ts) transforming mutants; these changes were analogous to mutations previously shown to confer a ts function onto the Dorsal protein of Drosophila melanogaster. Chicken spleen cells infected with the ts v-rel mutants formed colonies in agar at 36.5 degrees C but not at 41.5 degrees C. In addition, spleen cells derived from the ts v-rel-transformed colonies could be propagated in liquid culture at 36.5 degrees C but rapidly senesced at 41.5 degrees C. Both mutant v-Rel proteins were also ts for DNA binding in vitro. These mutants may be valuable for identifying genes directly regulated by v-rel.     

Site-directed mutagenesis of the SH2- and SH3-coding domains of c-src produces varied phenotypes, including oncogenic activation of p60c-src.

The products of the viral and cellular src genes, p60v-src and p60c-src, appear to be composed of multiple functional domains. Highly conserved regions called src homology 2 and 3 (SH2 and SH3), comprising amino acid residues 88 to 250, are believed to modulate the protein-tyrosine kinase activity present in the carboxy-terminal halves of the src proteins. To explore the functions of these regions more fully, we have made 34 site-directed mutations in a transformation-competent c-src gene encoding phenylalanine in place of tyrosine 527 (Y527F c-src). Twenty of the new mutations change only one or two amino acids, and the remainder delete small or large portions of the SH2-SH3 region. These mutant alleles have been incorporated into a replication-competent Rous sarcoma virus vector to examine the biochemical and biological properties of the mutant proteins after infection of chicken embryo fibroblasts. Four classes of mutant proteins were observed: class 1, mutants with only slight differences from the parental gene products; class 2, mutant proteins with diminished transforming and specific kinase activities; class 3, mutant proteins with normal or enhanced specific kinase activity but impaired biological activity, often as a consequence of instability; and class 4, mutant proteins with augmented biological and catalytic activities. In general, there was a strong correlation between total kinase activity (or amounts of intracellular phosphotyrosine-containing proteins) and transforming activity. Deletion mutations and some point mutations affecting residues 109 to 156 inhibited kinase and transforming functions, whereas deletions affecting residues 187 to 226 generally had positive effects on one or both of those functions, confirming that SH2-SH3 has complex regulatory properties. Five mutations that augmented the transforming and kinase activities of Y527F c-src [F172P, R175L, delta(198-205), delta(206-226), and delta(176-226)] conferred transformation competence on an otherwise normal c-src gene, indicating that mutations in SH2 (like previously described lesions in SH3, the kinase domain, and a carboxy-terminal inhibitory domain) can activate c-src.     

Intracellular localization and processing of pp60v-src proteins expressed by two distinct temperature-sensitive mutants of Rous sarcoma virus.

The transforming protein of Rous sarcoma virus, pp60v-src, is known to be a tyrosine protein kinase, but the mechanism of cell transformation remains unclear. In further investigating pp60v-src structure and function, we have analyzed two temperature-sensitive (ts) Rous sarcoma virus src gene mutants, tsLA29 and tsLA32. The mutations in tsLA29 and tsLA32 map in the carboxy-terminal region and the amino-terminal half of pp60v-src, respectively, and encode mutant proteins with either temperature-labile (tsLA29) or -stable (tsLA32) kinase activities. Here we examined the intracellular processing and localization of these pp60v-src mutants and extended our characterization of transformation parameters expressed by cells infected by the Rous sarcoma virus variants. No obvious defects in functional integrity of the tsLA32 pp60v-src could yet be demonstrated, whereas the tsLA29 pp60v-src was perturbed not only in kinase activity, but also in aspects of protein processing and localization. Analysis of transformation parameters expressed by infected cells demonstrated the complete temperature lability of both mutants.     

Reversion of Frameshift Mutations Stimulated by Lesions in Early Function Genes of Bacteriophage T4

Temperature-sensitive (ts) mutants representative of a number of genes of phage T4 were crossed with rII mutants to allow isolation of ts, rII double-mutant recombinants. The rII mutations used were characterized as frameshift mutations primarily on the basis of their revertability by proflavine. For each ts, rII double mutant, the effect of the ts mutation on spontaneous reversion of the rII mutation was determined over a range of incubation temperatures. A strong enhancement in reversion of two different rII mutants was detected when they were combined with tsL56, a mutation in gene 43 [deoxyribonucleic acid (DNA) polymerase]. Three other mutants defective in gene 43 enhanced reversion about fourfold. Two mutations in gene 32, which specifies a protein necessary for DNA replication, enhanced reversion about 5-fold and 18-fold, respectively. Two additional mutations in gene 43 and two in gene 32 had no effect. Fivefold and threefold enhancements in reversion were also found with mutations in genes 44 (DNA synthesis) and 47 (deoxyribonuclease), respectively. No significant effect was found with mutations in seven additional genes. The results of other workers suggest that frameshift mutations arise from errors in strand alignment during repair synthesis occurring at chromosome tips. Our results show that such errors can be enhanced by mutations in the DNA polymerase, the gene 32 protein, and the enzymes specified by genes 44 and 47. This implies that these proteins are employed in the repair process occurring at chromosome tips and that mutational errors in these proteins can lead to loss of ability to recognize and reject strand misalignments.     

Two independent mutations are required for temperature-sensitive cell transformation by a Rous sarcoma virus temperature-sensitive mutant.

We molecularly cloned the src coding region of tsNY68, a mutant of Rous sarcoma virus temperature sensitive (ts) for transformation, and constructed a series of ts wild-type recombinant src genes. DNA containing the hybrid genes was transfected into chicken cells together with viral vector DNA and helper viral DNA, and infectious transforming viruses were recovered. Characterization of these recombinant viruses indicated that at least two mutations are present in the 3' half of the mutant src gene, both of which are required for ts. Nucleotide sequence analysis revealed three differences in the deduced amino acid sequence compared with the parental virus. Two of these changes, a deletion of amino acids 352 to 354 and an amino acid substitution at position 461, are responsible for the ts phenotype.     

Isolation and primary identification of methylotrophic yeast Hansenula polymorpha mutants for peroxisome biogenesis

After exposure of cells of the methylotrophic yeast Hansenula polymorpha HF246 leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45 degrees C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45 degrees C but could grow at optimal temperature (37 degrees C), were isolated. Complementation analysis of mutants using 12 deletion mutants for genes of peroxisome biogenesis (PEX) (available in this yeast species by the beginning of our work) allowed to assign 51 mutants (including 16 ts) to the separate group of mutants unable to complement deletion mutants with defects in eight PEX genes. These mutants were classified into three groups: group 1 contained 10 pex10 mutants (4 ts mutants among them); group 2 included 19 mutants that failed to complement other pex testers: 1 pex1; 2 pex4 (1 ts); 6 pex5 (5 ts); 3 pex8; 6 (3ts)- pex19; group 3 contained 22 "multiple" mutants. In mutants of group 3, hybrids with several testers do not grow on methanol. All mutants (51) carried recessive mutations, except for mutant 108, in which the mutation was dominant only at 30 degrees C, which suggests that it is ts-dominant. Recombination analysis of mutants belonging to group 2 revealed that only five mutants (two pex5 and three pex8) carried mutations for the corresponding PEX genes. The remaining 14 mutants yielded methanol-utilizing segregants in an arbitrarily chosen sample of hybrids with the pex tester, which indicates mutation location in other genes. In 19 mutants, random analysis of ascospores from hybrids obtained upon crossing mutants of group 3 with a strain lacking peroxisomal disorders (ade11) revealed a single mutation causing the appearance of a multiple phenotype. A more detailed study of two mutants from this group allowed the localization of this mutation in the only PEX gene (PEX or PEX2). The revealed disorder of complementation interactions between nonallelic genes is under debate.     

mRNA from mutant Y1 adrenal cells directs the synthesis of altered regulatory subunits of type 1 cAMP-dependent protein kinase

The molecular basis for altered cyclic AMP-dependent protein kinase activity was examined in three different mutant clones (Kin-1, Kin-7, and Kin-8) derived from the Y1 mouse adrenocortical cell line. Parental Y1 cells and the Kin mutants were labeled with L-[35S] methionine and the regulatory subunit of the type 1 cAMP-dependent protein kinase isozyme (RI) was immunoprecipitated from each clone with a specific guinea pig antiserum. When analyzed by electrophoresis on isoelectric focusing gels, the immunoprecipitates from mutant clones exhibited parental forms of RI plus an additional acidic variant form which likely accounted for altered cAMP-dependent protein kinase activity. Poly(A+) RNA was isolated from Y1 and Kin mutant cells and was translated in a cell-free, reticulocyte lysate system in the presence of L-[35S]methionine. The RI synthesized from poly(A+) RNA was immunoprecipitated from the translation mixture and analyzed on isoelectric focusing gels. The poly(A+) RNA from the Kin mutant clones directed the synthesis of parental and acidic variant forms of RI. These results suggest that the altered electrophoretic forms of RI arise from mutations in one of two RI genes rather than from post-translational modifications of the protein. The coexistence of parental and variant forms of RI in the Kin mutants indicate that the mutations are codominant.     

Transformation of chicken embryo fibroblast cells by avian retroviruses containing the human Fyn gene and its mutated genes.

The transforming activity of the human fyn protein, p59fyn, which is a kinase of the src family, was investigated by testing the effect of recombinant avian retrovirus (Fyn virus) expressing p59fyn on chickens or cultured chicken embryo fibroblast (CEF) cells. The Fyn virus did not induce transformed foci. After several passages of the virus stock on CEF cells, however, a few foci were detected in the presence of dimethyl sulfoxide. Chickens inoculated with Fyn virus at the stage of 12-day-old embryos developed fibrosarcomas 3 to 6 weeks after hatching. The viruses obtained from these foci and from one of the tumor tissues showed high transforming activity in the presence of dimethyl sulfoxide, suggesting that these viruses carry spontaneous mutations of the fyn gene. Four fyn genes from CEF DNAs infected with transforming viruses were molecularly cloned, and their products were confirmed to possess transforming activity. DNA sequence analysis of the fyn genes showed that two of the four mutants have Thr instead of Ile at position 338 in the kinase domain. The other two mutants carry deletions of 78 and 108 base pairs, respectively, which result in complete loss of region C of SH2. The overall level of proteins containing phosphotyrosine was significantly higher in transformed cells than in normal CEF cells. Our data indicate that when expressed at high levels in a retrovirus, normal p59fyn cannot cause cellular transformation, but that mutant p59fyn with either a single amino acid substitution in the kinase domain or a deletion including region C produces a transforming protein, perhaps due to enhanced tyrosine kinase activity. This is the first observation that deletion of region C can unmask the potential transforming activity of a src family kinase.     

Recombination between a temperature-sensitive mutant and a deletion mutant of Rous sarcoma virus.

Cells doubly infected with two mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), ts68, which is temperature sensitive for cell transformation (srcts), and a deletion mutant, N8, which is deficient in the envelope glycoprotein (env-), produced a recombinant which carried the defects of both parents. The frequency of formation of such a recombinant was exceptionally high and made up 45 to 55% of the progeny carrying the srcts marker. By contrast, the reciprocal recombinant, which is wild type in transformation (srcts) and contains the subgroup A envelope glycoprotein (envA), was almost undetectable. This remarkable difference in the frequency of the formation of the two possible recombinants suggests that a unique mechanism may be involved in the genetic interaction of the two virus genomes, one of which has a large deletion. When an RNA-dependent DNA polymerase-negative variant of the N8 (N8alpha) was crinants also became deficient in the polymerase. Cells infected by the srctsenv- recombinant were morphologically normal at the nonpermissive temperature (41 degrees C) and susceptible to all subgroups of RSV. The rate by which the wild-type RSV transformed the recombinant-preinfected cells was indistinguishable from that of transformation of uninfected chicken cells by the same wild-type virus. This indicates that no detectable interference exists at postpenetration stages between the preinfected and superinfecting virus genomes and confirms that the expression of the transformed state is dominant over the suppressed state.