We have molecularly cloned a unique acutely transforming replication-defective mouse type C virus (3611-MSV) and characterized its acquired oncogene. The viral genome closely resembles Moloney (M) murine leukemia virus (MuLV), except for a substitution in M-MuLV in the middle of p30 and the middle of the polymerase gene (pol). Heteroduplex analysis revealed that 2.4 kilobases of M-MuLV DNA were replaced by 1.2 kilobases of cellular DNA. The junctions between viral and cellular sequences were determined by DNA sequence analysis to be 517 nucleotides into the p30 sequence and 1,920 nucleotides into the polymerase sequence. Comparison of the transforming gene from 3611-MSV, designated v-raf, with previously isolated retrovirus oncogenes either by direct hybridization or by comparison of restriction fragments of their cellular homologs shows it to be unique. Transfection of NIH 3T3 cells with cloned 3611-MSV proviral DNA leads to highly efficient transformation and the recovered virus elicits tumors in mice typical of the 3611-MSV virus. Transfected NIH 3T3 cells express two 3611-MSV-specific polyproteins (P75 and P90), both of which contain NH2-terminal gag gene-encoded components linked to the acquired sequence (v-raf) translational product. The cellular homolog, c-raf, is present in one or two copies per haploid genome in mouse and human DNA.Retroviruses act as natural vectors for the transduction of at least some cellular genes, designated proto-oncogenes (1, 2), which bring about malignant transformation of infected cells. The cellular origin of a retroviral transforming gene (v-onc) was first demonstrated for v-src, the oncogene of Rous sarcoma virus (3, 4). Since then, 14 additional v-onc genes present in different acutely transforming retroviruses of avian and mammalian origin have been described, each similarly derived from cellular genes (c-onc) (1, 2). At least six of the v-onc genes code for functionally related tyrosine-specific protein kinases (5, 6), several of which appear to have a common evolutionary origin (7-9). Although the v-onc genes that do not encode protein kinase make up an evolutionarily more diverse group, some, such as v-kis and v-has, also appear to be members of gene families (10).The significance for human disease of c-onc genes had been purely hypothetical prior to the recent demonstration by DNA transfection that active forms of such genes may be associated with human cancers (11,12). Furthermore, human c-onc genes are associated with specific translocations characteristic of certain types of human cancer (13)(14)(15). These findings emphasize the potential importance of onc genes for an understanding of human malignancy and point to a need for a more complete accounting of such genes in human DNA.The isolation of retroviral oncogenes has in the past been sporadic and limited mainly to isolations from spontaneous tumors. More recently we have designed experiments to generate acutely transforming retroviruses by growth of IdUrd-induced endogenous type C viruses in chemicall...

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The ability to archive biological samples for subsequent nucleic acid analysis is essential for tissue specimens and forensic samples. FTA Card is a chemically treated filter paper designed for the collection and room temperature storage of biological samples for subsequent DNA analysis. Its usefulness for the preservation of biological samples for subsequent RNA analysis was tested. Here, we demonstrate that RNA in biological samples stored on FTA Cards is stable and can be used successfully for RT-PCR and northern blot analysis. RNA stability depends on the storage temperature and the type of biological specimen. RNA in mammalian cells stored on FTA Cards is stable for over one year at temperatures at or below -20 degrees C and for two to three months in samples stored at room temperature. For plant leaf, longer storage times (> 5 days) require temperatures at or below -70 degrees C following sample application. FTA Cards may constitute a method not only for convenient collection and storage of biological samples but also for rapid RT-PCR analysis of tissue and cell samples.

Murine and human cDNAs, related to but distinct from c-raf-1, have been isolated and designated mA-raf and hA-raf, respectively. The mA-raf and hA-raf cDNAs detect the same murine and human fragments in Southern blots of restriction enzyme-cleaved murine and human cellular DNA. The murine restriction enzyme fragments homologous to mA-raf cDNA cosegregate with mouse chromosome X in a panel of Chinese hamster-mouse hybrid cells, thus localizing the mA-raf locus to mouse chromosome X. Two independently segregating loci, detected by the hA-raf cDNA (or mA-raf cDNA), hA-raf-l and hA-raf-2, are located on human chromosomes X and 7, respectively. The mA-raf locus and the hA-raf-1 locus are actively transcribed in several mouse and human cell lines.The normal precursors to activated oncogenes, the protooncogenes, presumably serve important growth-regulatory functions, since at least 4 of the more than 25 known oncogenes derive from components ofthe signal transduction pathway of growth factors: erbB, which derives from a portion of the receptor gene for epidermal growth factor (1); sis, which is homologous to part of the gene for plateletderived growth factor (2-4); the cellular homolog of the McDonough feline sarcoma oncogene, c-fms, which is related to the receptor for the colony-stimulating factor CSF-1 (5); and myc, which can mediate signal transduction for a variety of growth factors (6-8). Most oncogenes have been isolated as part of the genomes of transducing retroviruses (9-12), by molecular cloning of retrovirus integration sites in virusinduced tumor cells (13), or by DNA transfection using tumor-cell DNA (14). Additional oncogene-related genes (N-myc and r-fos) were identified by virtue of their nucleic acid sequence relatedness to known oncogenes (15-17), and putative oncogenes have been identified by virtue of their presence at the junction between chromosomes involved in specific translocations in leukemia and lymphoma (18)(19)(20).The v-raf oncogene was originally isolated as part of the genome of 3611 murine sarcoma virus, an acutely transforming murine retrovirus (21). Subsequently, the avian homolog of v-raf, v-mil (or mht) was identified in the avian carcinoma virus MH2 (22, 23). v-raf and v-mil derive from the 3'-terminal two-thirds of the same cellular gene, c-raf-1. c-raf-l encodes a 74-kDa protein that is predominantly cytoplasmic (24). The deduced amino acid sequence of c-raf-l shows a distant relationship to the src family of oncogenes (25); however, in contrast to many src-family gene products, which have tyrosine-specific protein kinase activity, the proteins encoded by transforming versions of c-raf-1 have protein kinase activity with specificity for serine or threonine residues (26). A role of c-raf-1 in human tumors is suggested by its chromosomal location at 3p25 (27), a region that is commonly altered in small-cell lung carcinoma (28). Moreover, activation of c-raf-J occurs in primary stomach cancer (29).Recently, on screening a mouse cDNA library with a v-raf oncogene probe, we h...

SUMMARYA new human papillomavirus type (HPV-56) was identified by low stringency Southern blot analysis with an HPV-31 DNA probe, in a cervical intraepithelial neoplasm (CIN). The DNA of this virus was molecularly cloned and shown to be a new HPV type based on the absence of cross-reactivity to HPV types 1 to 55 under highstringency hybridization conditions. At low stringency, HPV-56 was most related to HPV types 30 and 45. The deduced organization of the open reading frames of HPV-56, from hybridization and partial nucleotide sequence analyses, reveals a typical HPV genome. HPV-56 was detected in two of 464 normal cervical tissues, in five of 227 cervical condylomas and CIN, and in two of 84 invasive cancers of the cervix.More than 15 distinct human papillomavirus (HPV) types are known to infect the mucous epithelial lining of the anogenital tract (Beaudenon et al., 1986(Beaudenon et al., , 1987Boshart et al., 1984; de Villiers et al., 1981 ; Dfirst et al., 1983;Gissmann et al., 1982; Lrrincz et al., 1986 Lrrincz et al., , 1987 Lrrincz et al., a, 1989Naghashfar et al., 1987;Nuovo et al., 1988;Shimoda et al., 1988). Some of these HPVs, such as types 16, 18 and 31 (Boshart et al., 1984; Dfirst et al., 1983; Lrrincz et al., 1986) are commonly detected in normal, premalignant and malignant cervical tissues. These three types individually (or, rarely, in combination) are detected in approximately 70 ~o of cervical cancers (Fuchs et al., 1988; L6rincz et al., 1987 b;Reid et al., 1987). The remaining 20 ~o of HPV-positive cancers (as revealed by Southern blot analysis) are infected with a diverse group of HPVs such as types 33, 35, 39, 45, 51 and 52, which are each found in approximately 1 to 5 ~o of such lesions. HPV-56 is a new HPV type which was detected in two of 84 (2.4~) cervical cancers and appears to belong to this group of low-prevalence cancer--associated HPVs.Previously we reported the cloning of six new HPV types from anogenital specimens (Lrrincz et al., 1986(Lrrincz et al., , 1987a(Lrrincz et al., , 1989Naghashfar et al., 1987;Shimoda et al., 1988). Extensive hybridization studies revealed additional putatively new HPVs. The low-stringency Southern blot method used to detect new HPVs has been described in detail (Lrrincz et al., 1986). HPV-56 DNA was originally identified by low-stringency hybridization with an HPV-31 probe (Fig. 1), in a specimen (GU56) of cervical intraepithelial neoplasia (CIN) obtained from a woman in the Washington, D.C. metropolitan area. Cellular DNA from this specimen was digested with BamHI or EcoRI and analysed by Southern blotting. The digest yielded a 7-9 kb HPV DNA fragment with BamHI, and 5 kb and 2.9 kb fragments with EcoRI. In addition, specimen GU56 had several other weaker bands in both BamHI and EcoRI digests; these were later demonstrated to be due to the presence of another HPV type. The HPV-related DNA was molecularly cloned into the BamHI sites of 2L47, as described previously (Lrrincz et al., 1986). Hybridization of filter plaque-lifts from 2 × 105 plaques ...