Structural analysis of normal and transforming mil(raf) proteins: effect of 5'-truncation on phosphorylation in vivo or in vitro

The phosphorylation sites of the cellular proto-oncogene product p71/73c-mil(raf) from quail and from human cells were analyzed by two-dimensional peptide mapping and compared to the sites phosphorylated in proteins encoded by three transforming alleles of c-mil(raf). These alleles all were 5'-truncated resulting from either retroviral transduction (v-mil, v-raf) or promoter insertion mutagenesis (LTR-c-raf). The normal cellular proteins each were phosphorylated in vivo on three major sites, two of which were identical in the two protein species. MH2 p100gag-mil, murine sarcoma virus 3611 p75gag-raf, and LTR-c-raf p45-50 delta c-raf were phosphorylated in vivo on several sites. One site was shared between these transforming proteins and was also conserved in both avian and human p71/73c-mil(raf). All normal and transforming mil(raf) proteins were phosphorylated on serine in vivo while p100gag-mil and p75gag-raf occasionally also contained low levels of phosphothreonine. No specific phosphorylation of p71/73c-mil(raf) was detected in vitro under conditions that readily revealed presumed autophosphorylation of p100gag-mil, p75gag-raf, and p45-50 delta c-raf. However, the in vitro phosphorylated sites of these proteins were different to each other and to the sites phosphorylated in vivo. In contrast to the predominant threonine phosphorylation of the two viral proteins, only phosphoserine could be detected in p45-50 delta c-raf phosphorylated in vitro.     

raf oncogenes in carcinogenesis

There are three active raf genes in man and at least two in Xenopus and Drosophila. The mammalian c- and A-raf genes have 16 coding exons, which span 40 and 20 kb, respectively. B-raf is larger and extends over greater than 46 kb. Human c-raf-1 maps to chromosome 3p25 and A-raf-1 to Xp21. c-raf-1 RNA is present in many tissues, while A-raf and B-raf expression is restricted. A- and c-raf encode cytoplasmic ser/thr protein kinases of 68 and 74 kDa, which contain three conserved regions (CR). CR1 and 2 are in the amino terminal half, CR1 comprises the presumed ligand binding site, and CR3 represents the carboxy terminal kinase domain. All three genes can be artificially activated by deletions, provided CR3 is preserved. However, only c-raf-1 occurs naturally in truncated versions, such as v-raf and v-mil in the acutely transforming retroviruses 3611-MSV and MH2. raf transformation can also be affected by point mutation, suggesting that this mechanism may activate c-raf-1 as an oncogene in carcinogenesis.     

The transforming activity of the chicken c-myc gene can be potentiated by mutations

It was previously demonstrated that four different avian v-myc oncogenes harbor several point mutations. At least one of these leads to an amino acid substitution located in the proximity of position 61 in the second exon, whereas additional substitutions are found in exon 3. We have investigated whether these mutations affect the transforming activity of myc. By constructing avian retroviral genomes expressing hybrid gag-myc oncogenes, in which all or parts of the v-myc domains were replaced by corresponding parts of c-myc, we show here that a substitution of threonine 61 of c-myc for a methionine (as in v-mycmc29) significantly enhances the fibroblast transforming capacity of the recombinant oncogene. However, such a hybrid v/c-myc gene is still several fold less active than the v-mycmc29 oncogene. We have also expressed c-myc from subgenomic retroviral mRNAs: in these constructions the AUG of gag in the RNA leader sequence is in the same reading frame as that of c-myc, apparently leading to the production of a myc protein with 11 N-terminal amino acids encoded by gag and non-coding c-myc sequences. These myc proteins also transform chicken embryo fibroblasts, albeit with a lower efficiency than v-myc, again suggesting that mutations can increase the transforming capacity of myc.     

A retroviral sequence of the Chinese hamster ovary cell line.

A partial cDNA (B52) molecule with the characteristics of retroviral sequences was isolated from the Chinese hamster ovary (CHO) K1 cell line. The B52 cDNA contains 1184 nucleotides. The first 452 nucleotides (nt) are 71% homologous to the env gene of Moloney murine leukemia virus (MMLV) and murine endogenous retroviruses. The 139 amino acids predicted from the 452 nt have 82% homology with the carboxy-terminal amino acids of the env protein of MMLV. The remaining 732 nt have several features of a typical retroviral long terminal repeat (LTR). For example, the first 14 nt are identical to the 5' inverted repeat of the retroviral LTRs. The 41-nt sequence at the 3' end is common to the R region of retroviral LTRs. The 732-nt sequence was shown to have promoter activity. The activity is approximately twofold higher than that of the Rous sarcoma virus LTR, and 1.5-fold lower than that of the early promoter of SV40 virus. Two species of mRNA of 5.2 and 2.7 kb in size were readily detected by B52 cDNA in the CHO K1 cells.     

Disruption of the RanBP17/Hox11L2 region by recombination with the TCRdelta locus in acute lymphoblastic leukemias with t(5;14)(q34;q11).

The t(5;14)(q33-34;q11) translocation constitutes a recurrent rearrangement in acute lymphoblastic leukemia involving the T cell receptor (TCR) delta locus on chromosome 14. Breakpoint sequences of the derivative chromosome 5 were isolated by application of a ligation-mediated PCR technique using TCR delta-specific primers to amplify genomic DNA from the leukemic cells of a patient with t(5;14). Through exon trap analysis, we identified various putative exons of the chromosome 5 target gene of the translocation; compilation of sequence information of trapped exons and available expressed sequence tags (ESTs) from the GenBank database allowed us to assemble 1.2 kb of the cDNA. Full-length cDNAs were isolated from a human testis cDNA library and sequence analysis predicted a putative Ran binding protein, a novel member of the importin-beta superfamily of nuclear transport receptors, called RanBP17. The t(5;14) breakpoint maps to the 3' coding region of the gene. The breakpoint of a second t(5;14) positive patient was mapped about 8 kb downstream of the most 3' RanBP17 exon and 2 kb upstream of the first exon of the orphan homeobox gene, Hox11L2. In both cases TCR delta enhancer sequences are juxtaposed downstream of the truncated or intact RanBP17 gene, respectively on the derivative chromosome.     

The isolation of the human delta chain gene and its expression in normal T cells and T cell leukemias

In this paper we describe a gene that lies 85 kb 5' of the constant region of the human alpha chain and some 30-50 kb 3' of the alpha chain V region (H. Griesser, unpublished observation). This gene undergoes somatic recombination and is transcribed in thymocytes, PHA-stimulated peripheral blood T cells, and some T cell leukemic cell lines. Sequence analysis revealed that the gene has a structure similar to that of immunoglobulin and other T cell antigen receptor genes. Comparison of the sequence to a mouse gene found in a similar location revealed 80% homology at the protein level. Recently, M. Davis and A. Weiss (personal communication) have demonstrated that the protein product of the mouse gene is the delta chain gene. Thus, the gene described in this paper represents the human homolog of the delta chain gene of the T cell antigen receptor.     

Amplification of Both c-myc and c-raf-1 Oncogenes in a Human Osteosarcoma

Fourteen human bone and soft part tumor tissues were screened by Southern blot hybridization using five oncogene probes (c- myc , c-K- ras , c- fos , c- raf -l, and N- myc ). Amplification of c- myc was found in two osteosarcomas and one malignant fibrous histiocytoma. One of these osteosarcomas had amplified c- raf -1 gene. Rearrangement of the amplified gene was not observed. This is the first report of c- raf -1 amplification in human cancer tissues.     

A new variant 15; 16 translocation in mouse plasmacytoma leads to the juxtaposition of c-myc and immunoglobulin lambda.

Mouse plasmacytomas (MPCs) induced by pristane oil, or by a combination of pristane oil and Abelson virus, carry one of two chromosomal translocations. The typical 12; 15 translocation leads to the juxtaposition of c-myc and immunoglobulin heavy-chain sequences, whereas the 6; 15 translocation links the kappa light-chain locus with the pvt-1 (plasmacytoma variant translocation) locus, located at least 75kb 3' of c-myc [Cory, S., Graham, M., Webb, E., Corcoran, L. & Adams, J. (1985). EMBO J., 4, 675-681]. Unlike the human Burkitt's lymphoma-associated translocation, the lambda/myc juxtaposed variant translocation has not been found previously in MPCs. Using unconventional MPC induction systems in which the tumor precursor cell was induced to proliferate in a secondary host, we have recently identified a 15; 16 translocation in six of the derived MPCs [Wiener, F., Silva, S., Sugiyama, H., Babonits, M. & Klein, G. (1990). Genes Chromosomes Cancer, 2, 36-43]. Chromosome 16 harbors the lambda light-chain gene. To explore whether the 15; 16 translocation represents the lambda/myc juxtaposition, we have mapped the breakpoints on chromosomes 15 and 16 by pulsed-field gel electrophoresis (PFGE). The pvt-1 region was mapped to approximately 220 kb 3' of c-myc. The breakpoint on chromosome 15 in ABPC-Ch-163-10, one of the six 15; 16 translocation-carrying MPCs, was situated approximately 80 kb 3' of c-myc and 140 kb 5' of pvt-1b, the major breakpoint cluster region of the previously analysed 6; 15 variant MPCs. The breakpoint on chromosome 16 was found to cut between the V1 and C3 regions of the lambda locus. Co-migration experiments showed that the C3 and the myc gene were juxtaposed head to tail on the 15; 16 translocation chromosome. On the reciprocal product V1 was juxtaposed to pvt-1.     

Isolation of antibodies specific for avian viral and cellular myc proteins

The myc gene has been implicated in the genesis of various neoplasms in birds, mice, and humans and was originally identified as the cellular homologue of the transforming gene (v-myc) of the avian myelocytomatosis virus MC29. For specific antisera to be obtained for the myc gene product, a bacterial expression vector was constructed in which the coding sequences for approximately 20 kd of MC29 p110gag-myc (amino acid residues 502 to 678) were placed between the coding sequences for the amino terminal 13 kd of Rous sarcoma virus pp60src and the coding sequences for 112 kd of beta-galactosidase. Expression of this tripartite gene was driven by a hybrid trp-lac promoter under lac repressor control. Induction of expression resulted in the production of a 145-kd hybrid protein containing src, myc, and beta-galactosidase sequences. The hybrid protein was purified and injected into rabbits to produce antisera. The resultant antisera immunoprecipitated p110gag-myc and p58myc -p60myc from MC29- and MH2-infected nonproducer quail fibroblasts, respectively. In addition, the antisera also immunoprecipitated a 58-kd protein from the bursal lymphoma cell line BK25, which was identified as chicken c (cellular)-myc gene product.     

The TP1 isolate of feline sarcoma virus encodes a fgr-related oncogene lacking gamma actin sequences

We have isolated a new feline sarcoma virus, TP1-FeSV. The virus encodes a myristilated 83 kD gag-onc fusion protein displaying tyrosine kinase activity. We have established nonproducer cell lines lacking the TP1-FeSV associated helper virus (FeLV) and TP1-FeSV transfected NIH cell lines. Southern Blot analysis of genomic DNA and Northern Blot analysis of RNA isolated from these cell lines revealed that the oncogene of the TP1-FeSV isolate is related to the fgr oncogene of the GR-FeSV, but shows no hybridization to the gamma actin homologous sequences of the GR-FeSV. We have isolated TP1-FeSV specific clones from a genomic library. Restriction enzyme and sequence analysis showed that the TP1-FeSV genome consists of the first 1651 nucleotides of the gag gene, followed directly by fgr sequences. The TP1-FeSV fgr sequence starts 43 nucleotides after the beginning of the GR-FeSV fgr sequence. In contrast to the GR-FeSV fgr which has lost 13 amino acids of the c-fgr carboxy terminus, the TP1-FeSV fgr contains the complete carboxy terminus of the cellular fgr gene. The TP1-FeSV fgr sequence is followed by a unique 328 nucleotide long sequence of unknown origin. The 3' recombination site occurs within the pol gene, 460 nucleotides from the start of the env leader sequence. Comparison of the subcellular localization of the transforming proteins of TP1-FeSV and GR-FeSV show no striking difference; both molecules are in part associated with subcellular membrane/cytoskeletal fractions and form complexes with the cellular pp90 and pp50.     

Malignant transformation and EGFR activation of immortalized mouse liver epithelial cells caused by HBV enhancer-X from a human hepatocellular carcinoma

We have previously observed that all human hepatocellular carcinomas (HCCs) from HBV carriers examined had the integrated X region. In this study, HBV DNA was isolated from an integration site in one HCC that had a single, very small integrated viral DNA including the X region, but it had no expression of X gene as poly(A)RNA. It was found that HBV DNA was present between alphoid repetitive sequences, and it included Enhancer and X regions, encompassing the adr sequence from 910 to 1811. Nucleotides for 8 amino acids at the 3' end, a stop codon of X gene and a poly(A) signal downstream of X gene were lost by integration, and nucleotides for 7 amino acids and a stop codon were substituted by a connected alphoid sequence. When this cloned HBV DNA was transfected with an expression vector to an immortalized mouse liver epithelial cell line, MLE-10, malignant transformation occurred. Transformants having expressed poly(A)RNA of the X gene showed anchorage-independent growth in soft agar and tumor formation in the subcutis of nude mice. The mRNA level of EGFR was found to be remarkably enhanced in X-transformed cells, in contrast with the absence of this mRNA in parental and ras-transformed MLE-10. Our data provide evidence that the Enhancer-X region alone is the key contributor to the malignant change of pre-malignant liver cells in HBV carriers through activation of some specific genes, such as EGFR.     

Transformation of murine myelomonocytic cells by myc: point mutations in v-myc contribute synergistically to transforming potential

The v-myc oncogenes of chicken retroviruses (including MC29) bear point mutations relative to chicken c-myc. These mutations result in several amino acid differences in the encoded proteins. We have used recombinant murine retroviruses containing various myc alleles to analyse the myelomonocytic transforming potential of the myc oncogene. The myc alleles used were MC29 v-myc, chicken c-myc, chimeric genes combining 5' sections of v- or c-myc with 3' sections of c- or v-myc, and mouse c-myc. The same retroviral vector (based on the genome of Moloney leukemia virus) was used for each allele and the genes were translated from genomic message. By infecting the primary mouse tissues, bone marrow, peritoneal-derived macrophages and mixed embryonic tissue with the recombinant viruses, variation was found in the transforming efficacy of these alleles: v-myc was most effective, followed by the two chimeric genes, whereas c-myc (chicken or mouse) was least effective in eliciting myelomonocytic transformation. Viral gag sequences were not necessary for this transformation. In each case, the transformed monocytes were growth factor-dependent and non-immortal. However, v-myc transformed monocytes (though not monocytes transformed by other myc alleles) were able to progress to an immortal, growth factor-independent phenotype. Our results indicate that v-myc is far more effective than c-myc in eliciting myelomonocytic transformation; that this is due to combinatorial effects of 5' and 3' mutations in the v-myc gene; and that secondary events in addition to these mutations are required for transformation of myelomonocytic cells to an immortal, tumorigenic phenotype.     

Complete nucleotide sequence of mouse mammary tumor virus from jyg chinese wild mice: Absence of bacterial insertion sequences in the cloned viral gag gene

Mammary tumors of a newly isolated strain of Chinese wild mouse (JYG mouse) harbor exogenous mouse mammary tumor virus (MMTV). The complete nucleotide sequence of exogenous JYG-MMTV was determined on the proviral 5' long terminal repeat (LTR)(partial)-gag-pol-env-3' LTR (partial) fragment cloned into a plasmid vector and the cDNA sequence from JYG-MMTV producing cells. Similarly to the other MMTV species the LTR of JYG-MMTV contains an open reading frame (ORF). The amino acid sequence of the JYG-MMTV ORF resembles that of SW-MMTV (92% identity) and endogenous Mtv-7 (93% identity) especially at the C-terminal region. Thus, a functional similarity in T-cell receptor V beta recognition as a superantigen is implicated among these MMTV species. Analysis of the viral gag nucleotide sequence revealed that this gene is not disrupted by the bacterial insertion sequence IS1 or IS2, which have been reported to be present in the majority of the plasmids containing the gag region. Comparison of amino acid sequences of JYG-MMTV with those of BR6-MMTV showed that over 96% of the amino acids of gag, pol, protease and env products are identical. These results suggest the intact nature of the nucleotide sequence of the near full-length MMTV genome cloned in the plasmid.