Mammalian ets-1 and ets-2 genes encode highly conserved proteins.

Cellular ets sequences homologous to v-ets of the avian leukemia virus E26 are highly conserved. In mammals the ets sequences are dispersed on two separate chromosomal loci, called ets-1 and ets-2. To determine the structure of these two genes and identify the open reading frames that code for the putative proteins, we have sequenced human ets-1 cDNAs and ets-2 cDNA clones obtained from both human and mouse. The human ETS1 gene is capable of encoding a protein of 441 amino acids. This protein is greater than 95% identical to the chicken c-ets-1 gene product. Thus, the human ETS1 gene is homologous to the chicken c-ets-1 gene, the protooncogene that the E26 virus transduced. Human and mouse ets-2 cDNA clones are closely related and contain open reading frames capable of encoding proteins of 469 and 468 residues, respectively. Direct comparison of these data with previously published findings indicates that ets is a family of genes whose members share distinct domains.     

Avian carcinoma virus MH2 contains a transformation-specific sequence, mht, and shares the myc sequence with MC29, CMII, and OK10 viruses.

Avian carcinoma virus MH2 has been grouped together with MC29, CMII, and OK10, because all of these viruses share a transformation-specific sequence termed myc. A 5.2-kilobase (kb) DNA provirus of MH2 has been molecularly cloned. The complete genetic structure of MH2 is 5''-delta gag(1.9-kb)-mht(1.2-kb)-myc(1.3-kb)-delta env(?) and noncoding c-region (0.2-kb)-3''. delta gag, delta env, and c are genetic elements shared with nondefective retroviruses, whereas mht is a unique, possibly MH2 transformation-specific, sequence. Hybridizations with normal chicken DNA and cloned chicken c-myc DNA indicate that the mht sequence probably derives from a normal cellular gene that is distinct from the c-myc gene. The genetic structure of MH2 suggests that the delta gag and mht sequences function as a hybrid gene that encodes the p100 putative transforming protein. The myc sequence of MH2 appears to encode a second transforming function. Therefore, it seems that MH2 contains two genes with possible oncogenic function, whereas MC29, CMII, and OK10 each carries a single hybrid delta gag-myc transforming gene. It is remarkable that, despite these fundamental differences in their primary structures and mechanisms of gene expression, MH2 and MC29 have very similar oncogenic properties.     

Avian erythroblastosis virus E26: only one (myb) of two cell-derived coding regions is necessary for oncogenicity.

The oncogene hypothesis postulates that mutated cellular genes, termed proto-onc genes, function as cancer genes because they are related to retroviral onc genes. However, in contrast to retroviral onc genes, mutated proto-onc genes from cancers are not sufficient for carcinogenesis. Therefore, it has been proposed that mutated proto-onc genes depend on other proto-onc genes for carcinogenesis. Since the oncogene of the avian leukemia virus E26 includes coding regions derived from two cellular proto-onc genes, proto-myb and proto-ets, this hybrid gene has been proposed to be a model for two-gene-carcinogenesis. Here we set out to test this proposal. For this purpose myb and ets deletion mutants of cloned E26 provirus were prepared, and the corresponding viruses, produced by transfected primary chicken embryo cells, were tested for leukemogenicity in newborn chickens. It was found that an ets deletion mutant was just as leukemogenic as the wild-type virus and that a myb deletion mutant lacked leukemogenicity completely. To eliminate the possibility that our E26 myb deletion mutant failed to be leukemogenic because it failed to replicate, the virus was titered by a quantitative polymerase chain reaction (PCR) method. By this method, E26 from the plasma of infected chickens was first allowed to reverse-transcribe viral RNA to cDNA in vitro, and then the cDNA concentration was determined from the lowest dilution that gave a positive signal after amplification of E26 cDNA by the PCR method. Virus titers of about 10(5) per ml were found for wild type and for myb and ets deletion mutants of E26. It is concluded that the ets region is not essential for carcinogenesis, and E26 derives transforming function from overexpression of its proto-myb coding region via the retroviral promoter. Thus, E26 is a single-hit carcinogen and, like all other oncogenic retroviruses, is not a model for two-gene-carcinogenesis. Viral ets probably reflects a genetic accident that transduced sequences of proto-ets together with proto-myb in generating E26.     

Nucleotide sequence of avian carcinoma virus MH2: two potential onc genes, one related to avian virus MC29 and the other related to murine sarcoma virus 3611.

The 5.2-kilobase (kb) RNA genome of avian carcinoma virus MH2 has the genetic structure 5'-delta gag (0.2 kb)- mht (1.2 kb)-myc (1.4 kb)-c (0.4 kb)-poly(A) (0.2 kb)-3'. delta gag is a partial retroviral core protein gene, mht and myc are cell-derived MH2-specific sequences, and c is the 3'-terminal retroviral vector sequence. Here we have determined the nucleotide sequence of 3.5 kb from the 3' end of delta gag to the 3' end of molecularly cloned proviral MH2 DNA, in order to elucidate the genetic structure of the virus and to compare it with other mht - and myc-containing oncogenic viruses as well as with the chicken proto-myc gene. The following results were obtained: (i) delta gag- mht forms a hybrid gene with a contiguous reading frame of 2682 nucleotides that terminates with a stop codon near the 3' end of mht . The 3' 969 nucleotides of mht up to the stop codon are 80% sequence related to the onc-specific raf sequence of murine sarcoma virus 3611 (94% homologous at the deduced amino acid level). (ii) The myc sequence is preceded by an RNA splice acceptor site shared with the cellular proto-myc gene, beyond which it is colinear up to a 3'-termination codon and 40 noncoding nucleotides with the myc sequences of avian retrovirus MC29 and chicken proto-myc. Thus, myc forms, together with a 5' retroviral exon, a second MH2-specific gene. (iii) myc is followed by the 3'-terminal c region of about 400 nucleotides, which is colinear with that of Rous sarcoma virus except for a substitution near the 5' end of the long terminal repeat. It is concluded that MH2 contains two genes with oncogenic potential, the delta gag- mht gene, which is closely related to the delta gag-raf transforming gene of MSV 3611, and the myc gene, which is related to the transforming gene of MC29. Furthermore, it may be concluded that the cellular proto-onc genes, which on sequence transduction become viral onc genes, are a small group because among the 19 known onc sequences, 5 are shared by different taxonomic groups of viruses of which the mht /raf homology is the closest determined so far.     

Transformation of salivary gland secretion protein gene Sgs-4 in Drosophila: stage- and tissue-specific regulation, dosage compensation, and position effect.

The Sgs-4 gene of Drosophila melanogaster encodes one of the larval secretion proteins and is active only in salivary glands at the end of larval development. This gene lies in the X chromosome and is controlled by dosage compensation--i.e., the gene is hyperexpressed in males. Therefore, males with one X chromosome produce nearly as much Sgs-4 products as females with two X chromosomes. We used a 4.9-kilobase-pair (kb) DNA fragment containing the Sgs-4d coding region embedded in 2.6 kb of upstream sequences and 1.3 kb of downstream sequences for P-element-mediated transformation of the Sgs-4h underproducer strain Kochi-R. Sgs-4d gene expression was found in all 15 transformed lines analyzed, varying with the site of chromosomal integration. The transposed gene was subject to tissue- and stage-specific regulation. At X-chromosomal sites, the levels of gene expression were similar in both sexes, signifying dosage compensation. At autosomal sites, it was on average 1.5 times higher in males than in females. The results indicate that the transforming DNA fragment contains all sequences necessary for tissue- and stage-specific regulation and for hyperexpression in males.     

Nucleotide sequence of two overlapping myc-related genes in avian carcinoma virus OK10 and their relation to the myc genes of other viruses and the cell.

Avian carcinoma virus OK10 has the genetic structure gag-delta pol-myc-delta env. It shares the transformation-specific myc sequence with three other avian carcinoma viruses (MC29, MH2, CMII) and also with a normal chicken gene proto-myc and the gag, pol, and env elements with non-transforming retroviruses. Unlike the other myc-containing viruses, which synthesize singular myc proteins, OK10 synthesizes two different myc-related proteins of 200 and 57 kDa. Here we have sequenced the myc region of an infectious OK10 provirus to investigate how OK10 synthesizes two different proteins from the same myc domain and to identify characteristic differences between the normal proto-myc gene and the myc-related viral transforming genes. It was found that the 1.6-kilobase myc domain of OK10 is colinear and coterminal with the myc domains of MC29, MH2, and the terminal two exons of proto-myc. It is preceded by the same splice acceptor as the myc sequence of MH2 and as the second proto-myc exon. From this and the known structure of retroviruses, it follows that the OK10 gene encoding the 57-kDa protein is discontinuous with a small 5' exon that includes six gag codons and a large 3' myc exon (delta gag-myc). This gene and the delta gag-myc gene of MH2 are isogenic. The proto-myc-derived intron preceding the myc domain of OK10 is in the same reading frame as the adjacent delta pol and myc domains and, hence, is part of the gag-delta pol-myc gene encoding the 200-kDa protein. Sequence comparisons with proto-myc and MC29 and MH2 indicate that there are no characteristic mutations that set apart the viral myc domains from proto-myc. It is concluded that transforming function of viral myc-related genes correlates with the lack of a viral equivalent of the first proto-myc exon(s) and conjugation of the viral myc domains with large or small retroviral genetic elements rather than with specific point mutations. Because OK10 and MH2 each contain two genes with potential transforming function (namely, delta gag-myc and gag-delta pol-myc or delta gag-mht, respectively), it remains to be determined whether the delta gag-myc genes have transforming function on their own or need helper genes. The possible helper requirement cannot be very specific because the two potential helper genes are very different.     

Nucleotide sequence analysis of the chicken c-myc gene reveals homologous and unique coding regions by comparison with the transforming gene of avian myelocytomatosis virus MC29, delta gag-myc.

Myelocytomatosis virus MC29 is a defective avian retrovirus with a hybrid transforming gene (delta gag-myc) consisting of a 1,358-base pair (bp) sequence from the retroviral gag gene and a 1,568-bp sequence (v-myc) shared with a cellular locus, termed c-myc. We have subjected to sequence analysis 2,735 bp of the cloned c-myc gene, which includes the v-myc-related region of 1,568 bp, an intervening sequence of 971 bp, and unique flanking sequences of 45 bp and 195 bp at the 5' and 3' ends, respectively. Analysis of the genetic information and alignment of the c-myc sequence with the known sequence of MC29 indicates that: (i) the two myc sequences share the same reading frame, including the translational termination signal; (ii) there are nine nucleotide changes between c-myc and v-myc that correspond to seven amino acid changes; (iii) the 971-bp intervening sequence of c-myc can be defined as an intron by consensus splice signals; (iv) the unique 5' sequence of c-myc could either extend its reading frame beyond the homology with v-myc or could be an intron because its junction with the myc region of the locus is a canonical 3' splice-acceptor site; (v) the v-myc contains 10 nucleotides at its 5' end not shared with the c-myc analyzed here and also not with known gag genes, probably derived from an upstream exon; and (vi) the c-myc locus can generate a mRNA whose termination signals have been identified to be located 83 bp and 119 bp from the point of divergence between the v-myc and c-myc. We conclude that the gene of the c-myc locus of the chicken and the onc gene of MC29 share homologous myc regions and differ in unique 5' coding regions and we speculate, on this basis, that their protein products may have different functions. The hybrid onc gene of MC29 must have been generated from the c-myc gene by deletion of the 5' cellular coding sequence, followed by substitution with the 5' region of the viral gag gene.     

Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication.

In wild-type diploid cells of Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) at the MAT locus can be efficiently repaired by gene conversion using the homologous chromosome sequences. Repair of the broken chromosome was nearly eliminated in rad52delta diploids; 99% lost the broken chromosome. However, in rad51delta diploids, the broken chromosomes were repaired approximately 35% of the time. None of these repair events were simple gene conversions or gene conversions with an associated crossover, instead, they created diploids homozygous for the MAT locus and all markers in the 100-kb region distal to the site of the DSB. In rad51delta diploids, the broken chromosome can apparently be inherited for several generations, as many of these repair events are found as sectored colonies, with one part being repaired and the other part being lost the broken chromosome. Similar events occur in about 2% of wild-type cells. We propose that a broken chromosome end can invade a homologous template in the absence of RAD51 and initiate DNA replication that may extend to the telomere, 100 or more kb away. Such break-induced replication appears to be similar to recombination-initiated replication in bacteria.     

Fujinami sarcoma virus: An avian RNA tumor virus with a unique transforming gene

The oncogenic properties and RNA of the Fujinami avian sarcoma virus (FSV) and the protein it encodes were investigated and compared to those of other avian tumor viruses with sarcomagenic properties such as Rous sarcoma virus and the acute leukemia viruses MC29 and erythroblastosis virus. Cloned stocks of FSV caused sarcomas in all chickens inoculated and were found to contain a 4.5-kilobase (kb) and an 8.5-kb RNA species. The 4.5-kb RNA was identified as the genome of defective FSV because it was absent from nondefective FSV-associated helper virus and because the titer of focus-forming units increased with the ratio of 4.5-kb to 8.5-kb RNA in virus preparations. This is, then, the smallest known tumor virus RNA with a transforming function. Comparisons with other viral RNAs, based on oligonucleotide mapping and molecular hybridization, indicated that 4.5-kb FSV RNA contains a 5' gag gene-related sequence of 1 kb, an internal specific sequence of about 3 kb that is unrelated to Rous sarcoma virus, MC29, and erythroblastosis virus, and a 3'-terminal sequence of about 0.5 kb related to the conserved C region of avian tumor viruses. The lack of some or all nucleotide sequences of the essential virion genes, gag, pol, and env, and the isolation of FSV-transformed nonproducer cell clones indicated that FSV is replication defective. A 140,000-dalton, gag-related non-structural protein was found in FSV-transformed producer and nonproducer cells and was translated in vitro from full-length FSV RNA. This protein is expected to have a transforming function both because its intracellular concentration showed a positive correlation with the percentage of transformed cells in a culture and because FSV is unlikely to code for major additional proteins since the genetic complexities of FSV RNA and the FSV protein are almost the same. It is concluded that the transforming onc gene of FSV is distinct from that of Rous sarcoma virus and other avian tumor viruses with sarcomagenic properties. Hence, multiple mechanisms exist for sarcomagenic transformation of avian cells.     

Human c-myc onc gene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells.

Human sequences related to the transforming gene (v-myc) of avian myelocytomatosis virus (MC29) are represented by at least one gene and several related sequences that may represent pseudogenes. By using a DNA probe that is specific for the complete gene (c-myc), different somatic cell hybrids possessing varying numbers of human chromosomes were analyzed by the Southern blotting technique. The results indicate that the human c-myc gene is located on chromosome 8. The analysis of hybrids between rodent cells and human Burkitt lymphoma cells, which carry a reciprocal translocation between chromosomes 8 and 14, allowed the mapping of the human c-myc gene on region (q24 leads to qter) of chromosome 8. This chromosomal region is translocated to either human chromosome 2, 14, or 22 in Burkitt lymphoma cells.     

Human thrombopoietin: gene structure, cDNA sequence, expression, and chromosomal localization.

Thrombopoietin (TPO), a lineage-specific cytokine affecting the proliferation and maturation of megakaryocytes from committed progenitor cells, is believed to be the major physiological regulator of circulating platelet levels. Recently we have isolated a cDNA encoding a ligand for the murine c-mpl protooncogene and shown it to be TPO. By employing a murine cDNA probe, we have isolated a gene encoding human TPO from a human genomic library. The TPO locus spans over 6 kb and has a structure similar to that of the erythropoietin gene (EPO). Southern blot analysis of human genomic DNA reveals a hybridization pattern consistent with a single gene locus. The locus was mapped by in situ hybridization of metaphase chromosome preparations to chromosome 3q26-27, a site where a number of chromosomal abnormalities associated with thrombocythemia in cases of acute myeloid leukemia have been mapped. A human TPO cDNA was isolated by PCR from kidney mRNA. The cDNA encodes a protein with 80% identity to previously described murine TPO and is capable of initiating a proliferative signal to murine interleukin 3-dependent BaF3 cells expressing the murine or human TPO receptor.     

Human c-fos oncogene mapped within chromosomal region 14q21----q31.

The human cellular homolog (c-fos) of the transforming gene of Finkel-Biskis-Jinkins (FBJ) murine osteosarcoma virus was mapped to a single human chromosome. DNA from a series of 31 mouse-human somatic cell hybrid lines was probed with v- and c-fos molecular clones by Southern blotting. Human c-fos segregated with the distal region of the long arm of human chromosome 14. In situ hybridization of 125I-labeled human c-fos probe to normal human metaphase chromosomes independently confirmed these results and localized the c-fos oncogene to region 14q21----q31.     

Acute leukemia viruses E26 and avian myeloblastosis virus have related transformation-specific RNA sequences but different genetic structures, gene products, and oncogenic properties

Replication-defective acute leukemia viruses E26 and myeloblastosis virus (AMV) cause distinct leukemias although they belong to the same subgroup of oncogenic avian tumor viruses based on shared transformation-specific (onc) RNA sequences. E26 causes predominantly erythroblastosis in chicken and in quail, whereas AMV induces a myeloid leukemia. However, upon cultivation in vitro for >1 month, a majority of surviving hemopoietic cells of E26-infected animals bear myeloid markers similar to those of AMV-transformed cells. We have analyzed the genetic structure and gene products of E26 virus for a comparison with those of AMV. An E26/helper virus complex was found to contain two RNA species: a 5.7-kilobase (kb) RNA that hybridizes with cloned AMV-specific proviral DNA and hence is probably the E26 genome; and an 8.5-kb RNA that is unrelated to AMV and represents helper virus RNA. Thus, E26 RNA is smaller than 7.5-kb AMV RNA. Hybridization of size-selected poly(A)-terminating E26 RNA fragments with AMV-specific DNA indicated that the shared specific sequences are located in the 5′ half of the E26 genome as opposed to a 3′ location in AMV RNA. In nonproducer cells transformed in vitro by E26, a gag-related nonstructural 135,000-dalton protein (p135) was found. No gag(Pr76) or gag-pol (Pr180) precursors of essential virion proteins, which are present in AMV nonproducer cells, were observed. p135 was also found in cultured E26 virus producing cells of several leukemic chickens, and its intracellular concentration relative to that of the essential virion proteins encoded by the helper virus correlates with the ratio of E26 to helper RNA in virions released by these cells. p135 is phosphorylated but not glycosylated; antigenically it is not related to the pol or env gene products. It appears to be coded for by a partial gag gene and by E26-specific RNA sequences, presumably including those shared with AMV. Hence, AMV and E26 appear to use different strategies for the expression of related onc sequences: AMV is thought to encode a transforming protein via a subgenomic mRNA, whereas E26 codes for a gag-related polyprotein via genomic RNA. It is speculated that differences in the oncogenic properties of E26 and AMV are due to differences in their genetic structures and gene products.     

Identification of a yeast artificial chromosome clone encoding an accessory factor for the human interferon gamma receptor: evidence for multiple accessory factors.

Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon gamma (Hu-IFN-gamma), as measured by the induction of human HLA class I antigen. Human chromosome 6 encodes the receptor for Hu-IFN-gamma, and human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-gamma receptor. A small region of human chromosome 21 that is responsible for encoding such factors was localized with hamster-human somatic cell hybrids carrying an irradiation-reduced fragment of human chromosome 21. The cell line with the minimum chromosome 21-specific DNA is Chinese hamster ovary 3x1S. To localize the genes further, 10 different yeast artificial chromosome clones from six different loci in the vicinity of the 3x1S region were fused to a human-hamster hybrid cell line (designated 16-9) that contains human chromosome 6q (supplying the Hu-IFN-gamma receptor) and the human HLA-B7 gene. These transformed 16-9 cells were assayed for induction of class I HLA antigens upon treatment with Hu-IFN-gamma. Here we report that a 540-kb yeast artificial chromosome encodes the necessary species-specific factor(s) and can substitute for human chromosome 21 to reconstitute the Hu-IFN-gamma-receptor-mediated induction of class I HLA antigens. However, the factor encoded on the yeast artificial chromosome does not confer antiviral protection against encephalomyocarditis virus, demonstrating that an additional factor encoded on human chromosome 21 is required for the antiviral activity.