erg, a human ets-related gene on chromosome 21: alternative splicing, polyadenylation, and translation.

The avian acute leukemia virus E26 induces a mixed erythroid-myeloid leukemia in chickens and carries two distinct oncogenes, v-myb and v-ets. Recently, a novel gene named erg, closely related to the v-ets oncogene, was identified in human COLO 320 cells and the nucleotide sequence of its approximately 5.0-kilobase transcript, erg 1 was determined. In the present study, the nucleotide sequence of the alternatively spliced transcript, erg 2, was found to differ from erg 1 by a splicing event that causes a coding frameshift near the amino terminus, resulting in an additional 99-amino acid insertion at the amino-terminus. Expression of complementary DNAs for the two transcripts in vitro resulted in synthesis of polypeptides of approximately 41 and 52 kilodaltons, suggesting the use of alternative translation initiation codons in the case of erg proteins. The erg gene was localized by somatic cell genetic analysis to human chromosome 21. It is proposed that alternative sites of splicing and polyadenylation, together with alternative sites of translation initiation, allow the synthesis of two related polypeptides from a single erg gene transcriptional unit.

High-level expression in Escherichia coli of enzymatically active Harvey murine sarcoma virus p21ras protein.

The gene for the Harvey murine sarcoma virus (Ha-MuSV) p21ras protein was fused to the amino-terminal portion of the bacteriophage lambda cII gene on the expression vector pJL6. The fusion was such that transcription was controlled by the well-regulated phage lambda pL promoter, and translation initiated in the cII gene continued in frame into the ras gene sequences that code for p21. When the pL promoter was derepressed, the Escherichia coli cells harboring the fusion plasmid synthesized 23,000-dalton protein, which represented more than 10 percent of the total cellular protein. This protein was chimeric and contained 14 residues, which were specified by the vector; these residues were followed by all of the amino acids that make up Ha-MuSV p21ras except for four residues at the amino-terminal end. The protein appears similar to Ha-MuSV p21ras in that it undergoes immunoprecipitation by monoclonal antibodies directed toward that protein, binds guanosine diphosphate, and is capable of autophosphorylation.

Identification of HTLV-III/LAV sor gene product and detection of antibodies in human sera.

The nucleotide sequence of the genome of HTLV-III, the infectious agent etiologically associated with the acquired immune deficiency syndrome, predicts a small open reading frame, termed sor, located between the pol and env genes. A DNA segment containing 82 percent of the sor region was inserted into a prokaryotic expression vector, pJL6, to determine whether sor encodes a viral protein and to gain some insight into its possible function. The bacterially synthesized sor protein reacted with sera from individuals infected with HTLV-III, indicating that sor is expressed as a protein product or products that are immunogenic in vivo. Antibodies to the purified, bacterially synthesized sor protein were found to react specifically with the same protein and also with a protein of molecular weight 23,000 (23K) in HTLV-III-infected H9 cell extracts. The 23K protein comigrated with a protein immunoprecipitated by the serum of a hemophiliac patient with antibodies to HTLV-III, suggesting that this protein is probably the sor gene product.

A common onc gene sequence transduced by avian carcinoma virus MH2 and by murine sarcoma virus 3611.

A common cellular sequence was independently transduced by avian carcinoma virus MH2 (v-mht) and murine sarcoma virus (MSV) 3611 (v-raf). Comparison of the nucleotide sequences of v-mht and v-raf revealed a region of homology that extends over 969 nucleotides. The homology between the corresponding amino acids was about 95 percent with only 19 of 323 amino acids being different. With this example, 5 of the 19 known different viral onc genes have been observed in viruses of different taxonomic groups. These data indicate that (i) the number of cellular proto-onc genes is limited because, like other viruses of different taxonomic groups, MH2 and MSV 3611 have transduced the same onc gene-specific sequences from different cell species and (ii) that specific deletion and linkage of the same proto-onc sequences to different viral vector elements affect the oncogenic potential of the resulting viruses. The difference in transformation capabilities of MH2 and MSV 3611 serves as an example.

Diagnostic potential for human malignancies of bacterially produced HTLV-I envelope protein.

Two regions of the gene for the human T-cell leukemia virus subgroup I (HTLV-I) envelope were expressed in Escherichia coli by use of the vector pJLA16. One corresponds to the carboxyl terminal region of the major envelope protein p46, and the other corresponds to the transmembrane protein p21E. Reactivity of the expressed protein with human serum was tested by the Western blot procedure. Each of 11 sera tested that had been shown to contain antibodies to HTLV-I or HTLV-II by an enzyme-linked immunosorbent assay recognized the bacterially synthesized envelope proteins. There was no reaction detected when 17 control sera were tested. This system will be useful for large-scale seroepidemiological surveys for HTLV-I and related human retroviruses.

Nucleotide sequence of the transforming gene of avian myeloblastosis virus.

Avian myeloblastosis virus is defective in reproductive capacity, requiring a helper virus to provide the viral proteins essential for synthesis of new infectious virus. This virus arose by recombination of the nondefective helper virus and host cellular sequences present within the normal avian genome. These latter sequences are essential for leukemogenic activity. The complete nucleotide sequence of this region is reported. Within the acquired cellular sequences there is an open reading frame of 795 nucleotides starting with the initiation codon ATG (adenine, thymine, guanine) and terminating with the triplet TAG. This open reading frame could code for the putative transforming protein of 265 amino acids with a molecular weight of approximately 30,000.

Sequence-specific binding of human Ets-1 to the T cell receptor alpha gene enhancer.

Expression of the human T cell receptor (TCR) alpha gene is regulated by a T cell-specific transcriptional enhancer that is located 4.5 kilobases (kb) 3' to the C alpha gene segment. The core enhancer contains two nuclear protein binding sites, T alpha 1 and T alpha 2, which are essential for full enhancer activity. T alpha 1 contains a consensus cyclic adenosine monophosphate (cAMP) response element (CRE) and binds a set of ubiquitously expressed CRE binding proteins. In contrast, the transcription factors that interact with the T alpha 2 site have not been defined. In this report, a lambda gt11 expression protocol was used to isolate a complementary DNA (cDNA) that programs the expression of a T alpha 2 binding protein. DNA sequence analysis demonstrated that this clone encodes the human ets-1 proto-oncogene. Lysogen extracts produced with this cDNA clone contained a beta-galactosidase-Ets-1 fusion protein that bound specifically to a synthetic T alpha 2 oligonucleotide. The Ets-1 binding site was localized to a 17-base pair (bp) region from the 3' end of T alpha 2. Mutation of five nucleotides within this sequence abolished both Ets-1 binding and the activity of the TCR alpha enhancer in T cells. These results demonstrate that Ets-1 binds in a sequence-specific fashion to the human TCR alpha enhancer and suggest that this developmentally regulated proto-oncogene functions in regulating TCR alpha gene expression.

Requirement of ets-2 expression for Xenopus oocyte maturation.

A molecular clone of the Xenopus laevis ets-2 gene was isolated from an oocyte complementary DNA library. The amount of messenger RNA (mRNA) in each oocyte or embryo was almost constant during oogenesis and was maintained until the blastula stage of embryonic development, indicating that the observed 3.2-kilobase transcript is a maternal message. The only normal adult tissue in which ets-2 mRNA was detected was the ovary. Injection of antisense oligonucleotides homologous to the ets-2 sequence into oocytes led to degradation of the mRNA and blocked hormone-induced germinal vesicle breakdown. The ets-2 product is thus required for the meiotic maturation of Xenopus oocytes.

Hu-ets-1 and Hu-ets-2 genes are transposed in acute leukemias with (4;11) and (8;21) translocations.

Human probes identifying the cellular homologs of the v-ets gene, Hu-ets-1 and Hu-ets-2, and two panels of rodent-human cell hybrids were used to study specific translocations occurring in acute leukemias. The human ets-1 gene was found to translocate from chromosome 11 to 4 in the t(4;11)(q21;23), a translocation characteristic of a subtype of leukemia that represents the expansion of a myeloid/lymphoid precursor cell. Similarly, the human ets-2 gene was found to translocate from chromosome 21 to chromosome 8 in the t(8;21)(q22;q22), a nonrandom translocation commonly found in patients with acute myeloid leukemia with morphology M2 (AML-M2). Both translocations are associated with expression different from the expression in normal lymphoid cells of ets genes, raising the possibility that these genes play a role in the pathogenesis of these leukemias.

c-myc can induce expression of G0/G1 transition genes.

The human c-myc oncogene was linked to the heat shock-inducible Drosophila hsp70 promoter and used to stably transfect mouse BALB/c 3T3 cells. Heat shock of the transfectants at 42 degrees C followed by recovery at 37 degrees C resulted in the appearance of the human c-myc protein which was appropriately localized to the nuclear fraction. Two-dimensional analysis of the proteins of density-arrested cells which had been heat shock treated revealed the induction of eight protein species and the repression of five protein species. All of the induced and repressed proteins were nonabundant. cDNA clones corresponding to genes induced during the G0/G1 transition were used as probes to assay for c-myc inducibility of these genes. Two anonymous sequences previously identified as serum inducible (3CH77 and 3CH92) were induced when c-myc was expressed. In response to serum stimulation, 3CH77 and 3CH92 were expressed before c-myc mRNA levels increased. However, in response to specific induction of c-myc by heat shock of serum arrested cells, 3CH77 and 3CH92 mRNA levels increased after the rise in c-myc mRNA. Therefore, we hypothesize that abnormal expression of c-myc can induce genes involved in the proliferative response.

Phosphorylation of the ETS-2 protein: regulation by the T-cell antigen receptor-CD3 complex.

Phosphorylation of the human ets-2 protein in response to mitogenic signals to T lymphocytes was investigated in Jurkat cells. Activation of the cells by antibodies against the T-cell antigen receptor-CD3 complex or by concanavalin A was followed within 5 min by increased phosphorylation of the protein, as shown by a mobility shift of the protein from 54 to 56 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and increased incorporation of 32P. The Ca2+ ionophores A23187 and ionomycin were able to mimic this effect, suggesting that this phosphorylation is mediated by Ca2+.

A short-lived nuclear phosphoprotein encoded by the human ets-2 proto-oncogene is stabilized by activation of protein kinase C.

The human ets-2 gene is a homolog of the v-ets oncogene of the E26 virus and codes for a 56-kilodalton nuclear protein. The ets-2 protein is phosphorylated and has a rapid turnover, with a half-life of 20 min. When human lymphocytic CEM cells were treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), the level of the ets-2 protein was quickly elevated 5- to 20-fold. This effect of TPA was mimicked by a synthetic diacylglycerol, 1-oleoyl-2-acetyl glycerol, and was blocked by the protein kinase C inhibitor H7, indicating that protein kinase C is involved in the induction. The increase in the ets-2 protein was due to stabilization of the protein, because the protein had a half-life of more than 2 h in the presence of TPA and the ets-2 mRNA level did not increase significantly upon TPA treatment. The protein synthesis inhibitor cycloheximide enhanced the effect of TPA on the ets-2 protein and could itself slow turnover of the protein. Properties of the ets-2 protein, such as nuclear localization, phosphorylation, rapid turnover, and response to protein kinase C, indicate that this protein belongs to a group of oncogene proteins which are generally thought to have regulatory functions in the nucleus (e.g., myc, fos, myb, and p53). Our results suggest that protein kinase C, either directly or indirectly, regulates the level of the ets-2 protein by posttranslational mechanisms.

Hemorrhage, impaired hematopoiesis, and lethality in mouse embryos carrying a targeted disruption of the Fli1 transcription factor.

The Ets family of transcription factors have been suggested to function as key regulators of hematopoeisis. Here we describe aberrant hematopoeisis and hemorrhaging in mouse embryos homozygous for a targeted disruption in the Ets family member, Fli1. Mutant embryos are found to hemorrhage from the dorsal aorta to the lumen of the neural tube and ventricles of the brain (hematorrhachis) on embryonic day 11.0 (E11.0) and are dead by E12.5. Histological examinations and in situ hybridization reveal disorganization of columnar epithelium and the presence of hematomas within the neuroepithelium and disruption of the basement membrane lying between this and mesenchymal tissues, both of which express Fli1 at the time of hemorrhaging. Livers from mutant embryos contain few pronormoblasts and basophilic normoblasts and have drastically reduced numbers of colony forming cells. These defects occur with complete penetrance of phenotype regardless of the genetic background (inbred B6, hybrid 129/B6, or outbred CD1) or the targeted embryonic stem cell line used for the generation of knockout lines. Taken together, these results provide in vivo evidence for the role of Fli1 in the regulation of hematopoiesis and hemostasis.

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.

Differential expression of S19 ribosomal protein, laminin-binding protein, and human lymphocyte antigen class I messenger RNAs associated with colon carcinoma progression and differentiation.

Three complementary DNA encoding S19 ribosomal protein (S19), laminin-binding protein (LBP), and HLA class I (HLA-I) genes were isolated from a colon tumor-enriched subtraction library. To evaluate this mRNA expression, surgically removed colon tumors as well as matched normal tissue and human colon carcinoma cell lines showing various differentiation states, anchorage dependence, and proliferation states were examined by Northern blot analysis. The mRNA level of S19 mRNA (0.6 kilobase) was higher in primary colon carcinoma tissue than in matched normal colon tissue in 5 of 6 cases. In 2 of 4 cases, the expression of LBP mRNA (1.2 kilobases) was higher in carcinoma than in normal tissue. In 12 human colon cell lines, the level of LBP mRNA was higher in poorly differentiated cells. On the other hand, HLA-I mRNA (1.7 kilobases) was higher in well-differentiated cells. Although the S19 mRNA was expressed in both well- and poorly differentiated cells, a concomitant increase with tumor progression was observed in two pairs of cell lines derived from the same patients (SW480 and SW620; COLO201 and COLO205). Anchorage dependence of butyrate-treated HT29 colon carcinoma cells was correlated with lower levels of S19 and LBP mRNAs and higher levels of HLA-I mRNA expression compared with untreated cells. While the expression of S19 and LBP mRNAs was not changed due to cell growth states, HLA-I mRNA levels were found to be low in proliferating HT29 cells but highly induced in contact-inhibited cells. In summary, therefore, high expression of S19 and LBP combined with low expression of HLA-I were well correlated with colon carcinoma cells of higher malignant potential.

Presence of DNA polymerase in lymphosarcoma in northern pike (Esox lucius).

Northern poke lymphosarcoma DNA polymerase was partially purified from particulate fractions banding at 1.15 to 1.16 g/ml from homogenates prepared from frozen necropsies of tumor-bearing pike. The enzyme behaves as a typical reverse transcriptase, in that it prefers ribotemplates to deoxytemplates. The isoelectric point (pl 5.5) is similar to that of avian myeloblastosis virus polymerase. The pike enzyme elutes from a phosphocellulose column at 0.22 M potassium phosphate, the same as avian myeloblastosis virus DNA polymerase. The enzyme activity is inhibited by pyran, a specific inhibitor of viral DNA polymerases. The most striking difference between the pike lymphoma polymerase and the other viral DNA polymerases tested is the low maximum temperature of 20 degrees, compared to 30 degrees for Rauscher leukemia virus polymerase and 38 degrees for avian myeloblastosis virus and Rous sarcoma virus.

Production of oncogene-specific proteins and human T-cell leukemia (lymphotropic) retrovirus (HTLV-I) envelope protein in bacteria and its potential for use in human cancers and seroepidemiological surveys.

The oncogenes coding for the Harvey murine sarcoma virus p21ras protein as well as those coding for myc, myb, and mht products were fused to the amino-terminal portion of the bacteriophage lambda cII gene on the expression vector pJL6. In addition two regions of the gene for the human T-cell leukemia virus subgroup I (HTLV-I) envelope were expressed in our bacterial system. Each of 11 human sera tested that had been shown to contain antibodies to HTLV-I or -II by an enzyme-linked immunosorbent assay recognized the bacterially synthesized envelope proteins. No reaction was detected when 17 control sera were tested. This system will be useful for large-scale seroepidemiological surveys for HTLV-I and related human retroviruses. The other oncogene products expressed in our bacterial vector system also demonstrated specific immunoreactivities. In addition to this feature the bacterial ras protein was seen to bind guanosine diphosphate and was capable of autophosphorylation. Taken together these data suggest that the proteins produced with high efficiency by the bacterial expression system can be immunologically recognized as antigens and can in part perform some of their associated biochemical functions.

Oncogenes in hematopoietic and hepatic fish neoplasms.

Neoplastic transformation of cells has often been associated with changes in cellular oncogenes. While much information has been collected in mammalian systems, relatively little is known about the molecular basis of tumor progression in lower vertebrates. For our studies, tumors were collected from feral northern pike (Esox lucius) from Ostego Lake, MI, where the local population exhibited a 15% incidence of large external lymphomas. In laboratory studies, tumors were induced under controlled conditions by known mammalian carcinogens in the Japanese medaka (Oryzias latipes), a small aquarium fish widely used in carcinogenicity studies. DNA isolated from these tumors was assayed for transforming sequences by transfection into NIH3T3 cells. DNAs from the northern pike lymphomas and the chemically induced tumors in the medaka were able to transform NIH3T3 cells and induce tumors in athymic mice. The results of our studies to date are summarized here, together with the current status of oncogene activation in other fish systems.

CaSm: an Sm-like protein that contributes to the transformed state in cancer cells.

A novel gene encoding a protein containing Sm motif-like domains was found to have elevated expression in pancreatic cancer and in several cancer-derived cell lines. CaSm (for Cancer-associated Sm-like) mRNA is up-regulated in 87.5% (seven of eight) of pancreatic tumor/normal pairs. Similarly, cell lines from cancers originating in liver, ovary, lung, and kidney show increased CaSm expression compared to their normal tissue cognates. CaSm encodes a 133-amino acid open reading frame that contains the two Sm motifs found in the common snRNP proteins, with the greatest homology to the Sm G protein (60% similarity). Two hypothetical proteins from Caenorhabditis elegans and Saccharomyces cerevisiae share even greater similarity (72.8 and 67.7%, respectively), suggesting a broad family of proteins containing Sm motifs. Antisense CaSm RNA is able to alter the transformed phenotype of pancreatic cancer cells by reducing their ability to form large colonies in soft agar when compared to untransfected cells. Therefore, CaSm expression appears to be necessary for maintenance of the transformed state.

The c-ets-1 proto-oncogene has oncogenic activity and is positively autoregulated.

The proto-oncogene ets-1 is a member of the ets family of genes that share homology with the viral oncogene, v-ets, of the avian leukemia virus E26. By using expression vectors, we demonstrate that the ets-1 gene transforms NIH3T3 cells and the ets-1 transfected cells form colonies in soft agar and induce tumors in nude mice. We have also determined that the ets-1 protein contains homology with the helix-loop-helix motif of the HLH family proteins, but lacks the basic domain upstream of helix I. Transfection of the NIH3T3 cells with ets-1 vectors results in the activation of the endogenous ets-1 gene. Using hybridization probes that can distinguish between transcripts from endogenous and exogenous templates, we show that the endogenous ets-1 gene is activated by the expression of the transfected exogenous ets-1. In contrast, the expression of transfected ets-2 has no effect on the endogenous ets-1 gene expression. The results indicate that the ets-1 proto-oncogene is positively autoregulated by its product.