elk, tissue-specific ets-related genes on chromosomes X and 14 near translocation breakpoints.

The myb-ets-containing acute leukemia virus, E26, transforms myeloblasts and erythroblasts in culture and causes a mixed erythroid and myeloid leukemia in chicks. Genes (ets-1, ets-2, and erg) with variable relatedness to the v-ets oncogene of the E26 virus have been identified, cloned, and characterized in several species. Two new members (elk-1 and elk-2) of the ets oncogene superfamily have now been identified. Nucleotide sequence analysis of the elk-1 cDNA clone revealed that this gene encodes a 428-residue protein whose predicted amino acid sequence showed 82% similarity to the 3' region of v-ets. The elk or related sequences appear to be transcriptionally active in testis and lung. The elk cDNA probe detects two loci in the human genome, elk-1 and elk-2, which map to chromosome regions Xp11.2 and 14q32.3, respectively. These loci are near the translocation breakpoint seen in the t(X;18) (p11.2;q11.2), which is characteristic of synovial sarcoma, and the chromosome 14q32 breakpoints seen in ataxia telangiectasia and other T cell malignancies. This suggests the possibility that rearrangements of elk loci may be involved in pathogenesis of certain tumors.

Fusion of the bcr and the c-abl genes in Ph'-positive acute lymphocytic leukemia with no rearrangement in the breakpoint cluster region.

Two types of Philadelphia (Ph') chromosome positive acute lymphoblastic leukemias (ALL) have been described. One shows rearrangements within the 5.8 kb breakpoint cluster region (bcr), which forms the mid-portion of the bcr gene, on chromosome 22, while the other carries rearrangements involving a more proximal region on chromosome 22. To understand the nature of the breakpoints on chromosome 22 in bcr rearrangement negative, Ph'-positive ALLs, we have cloned and sequenced the cDNA of the c-abl oncogene in such ALL cells. The 5' ends of the cDNA clones correspond to the normal sequences of the bcr gene first exon with two of the clones extending beyond the GCCATGG consensus sequence for the initiation of translation. The bcr sequence stops at nucleotide 1813 of the coding sequence of the bcr gene, while the c-abl sequence starts at the beginning of the second c-abl exon (nucleotide 227). Thus the joining point between bcr and c-abl is at the boundary between two exons, suggesting intronic fusion and the occurrence of a splicing event. Our current observations indicate that the Ph' translocation in bcr negative ALL involves bcr gene sequences, albeit only a proximal portion of those involved in CML. These genomic differences may be important factors in the pathogenesis of the distinct phenotypes of ALL and CML.

Cloning and characterization of the human PIM-1 gene: a putative oncogene related to the protein kinases.

The mouse PIM-1 gene has been implicated in the evolution of retrovirus-associated mouse lymphomas. We have initiated a study of the human PIM-1 gene because of its potential importance as a human oncogene. We have isolated genomic and cDNA clones for this gene and characterized this locus in detail. The predicted PIM-1 protein is 313 amino acids in length. It has homology to a number of the protein kinases but does not have a transmembrane region. The amino acid corresponding to tyrosine-416 of pp60v-src is a tyrosine (position 198), which is consistent with the hypothesis that PIM-1 is a tyrosine kinase rather than a serine-threonine kinase. The PIM-1 gene was found to have six exons and five introns derived from 5 kb of genomic DNA. The site of transcription initiation was localized by S1 nuclease protection studies which indicated that the mature PIM-1 mRNA was approximately 2.7 kb in length. The promotor of this gene had no TATA or CAAT box but did have multiple GC boxes (CCGCCC) that might bind the Sp1 protein. The PIM-1 gene was expressed in myeloid and B lymphoid cell lines, but not in T lymphoid and nonhemopoietic lines. This initial characterization of PIM-1 will allow us to define its role in normal and malignant hematolymphoid differentiation.

Expression of members of immunoglobulin gene family in somatic cell hybrids between human B and T cells.

Somatic cell hybrids were obtained between human T and B cells and tested for the expression of differentiated traits of both cell lineages. The T-cell parent SUP-T1 is CD3-, CD4+, CD1+, CD8+, is weakly positive for HLA class I determinants, and has an inversion of chromosome 14 due to a site-specific recombination event between an immunoglobulin heavy-chain variable gene and the joining segment of the T-cell receptor alpha chain. The B-cell parent, the 6-thioguanine- and ouabain-resistant mutant GM1500, is a lymphoblastoid cell line that secretes IgG2, kappa chains, and expresses B1, B532, and HLA class I and II antigens. All hybrids expressed characteristics of B cells (Ig+, B1+, B532+, EBNA+, HLA antigens), whereas only CD4 among the T-cell markers was expressed. The level of T-cell receptor beta-chain transcript was greatly reduced and no RNA of the chimeric T-cell receptor alpha-chain joining segment-immunoglobulin heavy-chain variable region was detected. Southern blot analysis indicated that absence of T-cell differentiation markers in the hybrids was not due to chromosomal loss. Rather, some B-cell-specific factor present in the hybrids may account for the suppression.

Characterization of the human PIM-1 gene: a putative proto-oncogene coding for a tissue specific member of the protein kinase family.

The mouse PIM-1 gene is involved in the pathogenesis of virally-induced mouse lymphomas. We have cloned and analyzed the human homologue of the mouse PIM-1 gene to investigate its role in human lymphoma and leukemia. Overlapping cDNA clones from a K562 (human erythroleukemia cell line) library were isolated and sequenced. The deduced amino acid sequence showed significant homology to a number of the protein kinases but did not have a transmembrane region. Genomic clones from the 380 cell line (human B cell leukemia) were analyzed. The PIM-1 transcript was found to derive from 5 Kb of genomic DNA. Six exons and five introns were identified. The promoter region has no TATA or CAAT boxes, but did have multiple potential Sp1 binding sites (CCGCCC). Studies of expression of this gene using Northern blots of human cell lines showed it to be transcribed primarily in B lymphoid and myeloid cell lines. The characterization of the human PIM-1 gene will allow the definition of its role in hemopoietic malignancies and in hematolymphoid differentiation.

Actively transcribed genes in the raf oncogene group, located on the X chromosome in mouse and human.

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-1 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.

Deregulation of c-myc by translocation of the alpha-locus of the T-cell receptor in T-cell leukemias.

Two human T-cell leukemias carrying a t(8;14)(q24;q11) chromosome translocation were studied for rearrangements and expression of the c-myc oncogene. For one leukemia, rearrangement was detected in a region immediately distal (3') to the c-myc locus; no rearrangements of c-myc were observed in the second case (DeF). However, studies with hybrids between human and mouse leukemic T cells indicated that in the leukemic cells of DeF, the breakpoint in chromosome 14 occurred between genes for the variable (V alpha) and the constant (C alpha) regions for the alpha chain of the T-cell receptor. The C alpha locus had translocated to a region more than 38 kilobases 3' to the involved c-myc oncogene. Since human c-myc transcripts were expressed only in hybrids carrying the 8q+ chromosome but not in hybrids containing the normal chromosome 8, it is concluded that the translocation of the C alpha locus 3' to the c-myc oncogene can result in its transcriptional deregulation.

Localization of the human pim oncogene (PIM) to a region of chromosome 6 involved in translocations in acute leukemias.

The human homolog, hpim, of the murine pim-1 gene, which is activated in murine T-cell lymphomas by insertion of retrovirus proviral genomes in the pim-1 region, has been molecularly cloned; the cloned probe has been used to map the hpim locus to human chromosome region 6p21 by somatic cell hybrid analysis and chromosomal in situ hybridization. The hpim gene is expressed as a 3.2-kilobase mRNA in various human cell lines of hematopoietic lineage, most dramatically in the K562 erythroleukemia cell line, which contains a cytogenetically demonstrable rearrangement in the 6p21 region. A characteristic chromosome anomaly, a reciprocal translocation t(6;9)(p21;q33), has been described in myeloid leukemias and could involve the hpim gene.

Heterogeneity of chromosome 22 breakpoint in Philadelphia-positive (Ph+) acute lymphocytic leukemia.

In chronic myelogenous leukemias (CML) with the t(9;22)(q34;q11) chromosome translocation the breakpoints on chromosome 22 occur within a 5.8-kilobase segment of DNA referred to as "breakpoint cluster region" (bcr). The same cytogenetically indistinguishable translocation occurs in approximately 10% of patients with acute lymphocytic leukemias (ALL). In this study we have investigated the chromosome breakpoints in several cases of ALL carrying the t(9;22) translocation. In three of five cases of ALL we found that the bcr region was not involved in the chromosome rearrangement and that the 22q11 chromosome breakpoints were proximal (5') to the bcr region at band 22q11. In addition, we observed normal size bcr and c-abl transcripts in an ALL cell line carrying the t(9;22) translocation. We conclude, therefore, that if c-abl is inappropriately expressed in ALL cells without bcr rearrangements, the genetic mechanism of activation must be different from that reported for CML.

The translocated c-myc oncogene of Raji Burkitt lymphoma cells is not expressed in human lymphoblastoid cells.

We hybridized Raji Burkitt lymphoma cells, which carry a t(8;14) chromosome translocation, with human lymphoblastoid cells to study the expression of the translocated cellular myc oncogene (c-myc) in the hybrid cells. In Raji cells the c-myc oncogene is translocated to a switch region of the gamma heavy chain locus (S gamma). Because of sequence alterations in the 5' exon of the translocated c-myc oncogene in this cell line, it is possible to distinguish the transcripts of the translocated c-myc gene and of the normal c-myc gene. S1 nuclease protection experiments with a c-myc first exon probe indicate that Raji cells express predominantly the translocated c-myc gene, while the level of expression of the normal c-myc gene is less than 2% of that of the translocated c-myc gene. Somatic cell hybrids between Raji and human lymphoblastoid cells retain the lymphoblastoid phenotype and express only the normal c-myc oncogene. This result indicates that the activation of a c-myc oncogene translocated to a S region depends on the stage of B-cell differentiation of the cells harboring the translocated c-myc gene and not on alterations in the structure of the translocated c-myc oncogene.