Inversion of a chicken ets-1 proto-oncogene segment in avian leukemia virus E26.

The segment of the avian leukemia virus E26 genome near the termination of the p135gag-myb-ets open reading frame contains an inversion of the chicken ets-1 sequence. The inversion contains at least 41 bp and may be as large as 46 bp. This results in the replacement of 13 amino acids of chicken ets-1, with 16 amino acids derived from reverse complement of the normal ets-1 coding strand or read-through into E26 env sequences. At least 13 of these codons are specified by the inverted ets sequences. This represents the first reported occurrence of inverted oncogene sequences in a natural retrovirus. The inverted ets sequences are immediately followed by sequences homologous to the Rous sarcoma virus Prague B env gene. Since the E26 env sequence is more closely related to subgroup B avian retroviruses than to avian retroviruses from subgroups A, C, D, or E, the progenitor of E26 was a virus belonging to avian retrovirus subgroup B.     

Multiple domains for the chicken cellular sequences homologous to the v-ets oncogene of the E26 retrovirus.

We have investigated the structure of chicken genomic DNA homologous to v-ets, the second cell-derived oncogene of avian retrovirus E26. We isolated a c-ets locus spanning ca. 30.0 kilobase pairs (kbp) in the chicken genome with homologies to 1,202 nucleotides (nt) of v-ets (total length, 1,508 nt) distributed in six clusters along 18.0 kbp of the cloned DNA. The 5'-distal part of v-ets (224 nt) was homologous to chicken cellular sequences contained upstream within a single 16.0-kbp EcoRI fragment as two typical exons but not found transcribed into the major 7.5-kb c-ets (or 4.0-kb c-myb) RNA species. Between these two v-ets-related cellular sequences we found ca 40.0 kbp of v-ets-unrelated DNA. Finally, the most 3' region of homology to v-ets in the cloned DNA was shown to consist of a truncated exon lacking the nucleotides coding for the 16 carboxy-terminal amino acids of the viral protein but colinear to one of the two human c-ets loci, c-ets-2.     

Identification in chickens of an evolutionarily conserved cellular ets-2 gene (c-ets-2) encoding nuclear proteins related to the products of the c-ets proto-oncogene.

In chicken cells, we previously identified a set of proteins (p58-64) structurally related to, but distinct from, the products encoded by the c-ets proto-oncogene. We report here the isolation and nucleotide sequence of a cDNA encoding nuclear products of mol. wt 58, 60, 62 and 64 kd, indistinguishable from those detected in chicken cells. The p60 and p64 species appear to represent phosphorylated versions on serine and threonine residues of p58 and p62. The homology of p58-64 to other ets-related proteins, including the v-ets encoded domain of the transforming protein of avian leukemia virus E26 and p54c-ets, the translation product of the chicken (Ck) c-ets gene, is confined to two regions of 175 and 96 amino acid residues localized respectively at the carboxy-terminal domain and close to the amino-terminal domain of these molecules. This cDNA corresponds to a gene localized in a locus distinct from that of c-ets which is transcribed as a 4.0-kb RNA species in most chicken tissues. We also identified the human (Hu) c-ets-2-encoded products as two proteins of 60 and 62 kd, highly related to chicken p58-64. This, together with the fact that the amino acid sequence of the cDNA encoding p58-64 is 95% identical to the reported partial sequence of a Hu-c-ets-2 cDNA, indicates that p58-64 are the translation products of the Ck-c-ets-2 gene.     

Alternative splicing of RNAs transcribed from the chicken c-mil gene.

Two distinct c-mil-related cDNA clones have been isolated from a chicken embryo cDNA library. Results presented here show that the single chicken c-mil gene is coding for two c-mil mRNA species, different by at least 60 base pairs and generated by an alternative splicing mechanism. These mRNA molecules can be translated into two distinct proteins of 73 and 71 kilodaltons.     

Alternative splicing variants of the human DBL (MCF-2) proto-oncogene

The DBL (MCF-2) proto-oncogene is a prototype guanine nucleotide exchange factor (GEF) that modulates the Rho family of GTPases. In this communication we describe the isolation of three novel splicing variants of Dbl. The prototype Dbl gene (designated var.1 here) contains 25 exons, while splicing variant 2 (var.2) lacks exons 23 and 24. Var.3 contains additional 3 exons from 5(')-UTR in place of exon 1, while var.4, var.2, and var.3 contain a 48bp insertion between exons 10 and 11, resulting in the insertion of 16 amino acids. We found that var.1 was expressed only in brain, whereas var.3 was expressed in heart, kidney, spleen, liver, and testis, and var.4 in brain, heart, kidney, testis, placenta, stomach, and peripheral blood. The Dbl protein was detectable in brain, heart, kidney, intestine, muscle, lung, and testis. An assay for GEF activity revealed that the var.2 exhibits decreased GEF activity towards Cdc42, var.3 exhibits a weak but significant activity toward Rac1 and Cdc42, var.4 exhibits significant activity toward RhoA and Cdc42, while var.1 exhibits no activity toward RhoA, Rac1, or Cdc42. In summary, we describe 4 splicing variants of the human DBL proto-oncogene that show different tissue distributions and GEF specificities.     

Alternative splicing is associated with decreased expression of the redox proto-oncogene thioredoxin-1 in human cancers

Human thioredoxin-1 (Trx-1) is a small redox protein that is overexpressed in a number of human primary tumors, where it is associated with rapid cell proliferation and inhibited apoptosis. Mutation scanning denaturing high-performance liquid chromatography of Trx-1 mRNA in 58 human tumor cell lines found no evidence for changes in the base sequence of human Trx-1 mRNA. An alternatively spliced form of Trx-1 mRNA lacking exons 2 and 3 was found in 7 of the cell lines but it was not translated. The cell lines having the alternatively spliced Trx-1 mRNA had 73% lower total Trx-1 mRNA than the other cell lines, suggesting that alternative splicing may control the level of Trx-1 mRNA in some cancer cells.     

Structure of a Moloney murine leukemia virus-virus-like 30 recombinant: implications for transduction of the c-Ha-ras proto-oncogene.

Tumor progression locus 2 (Tpl-2) encodes a novel serine-threonine protein kinase which is activated by provirus integration in the late stages of oncogenesis in Moloney leukemia virus (MoMuLV) induced rat T-cell lymphomas. In this report, we present evidence that the provirus integrated in the Tpl-2 locus in 1 of 10 T-cell lymphomas harboring a Tpl-2 rearrangement (2779) is a recombinant between MoMuLV and virus-like 30 (VL30) sequences (Mo-VL30). Recombination between MoMuLV and VL30 may contribute to the transduction of ras, as suggested by the finding that VL30 flanks the ras oncogene in all of the ras transducing viruses isolated from rats to date. The Mo-VL30 recombinant described here represents evidence that recombination between MoMuLV and VL30 can be uncoupled from the transduction of ras, and it may precede the transduction. Sequence comparison between clones of Mo-VL30, Harvey sarcoma virus (Ha-MSV), and genomic c-Ha-ras revealed that all three share a 124-bp region of 87.3% homology. This region was detected at nucleotide positions -1845 to -1720 of c-Ha-ras and 20 bp 5' of the recombination breakpoint between VL30 and ras in Ha-MSV. On the basis of the sequence comparison between VL30, Ha-MSV, and c-Ha-ras, we are proposing a model which explains how VL30 may have facilitated the transduction of c-Ha-ras and perhaps the other ras proto-oncogenes. According to this model, the sequence homology between VL30 and c-Ha-ras targets this gene for transduction by promoting the integration of the provirus in this locus through homologous recombination.     

Conservation of RET proto-oncogene splicing variants and implications for RET isoform function

The RET proto-oncogene encodes a receptor tyrosine kinase required for development of the kidney and neural crest-derived cell types. Alternative splicing of the 3' exons of human RET results in three protein isoforms with distinct C-termini: RET9, RET51, and RET43. These RET isoforms show differential binding to downstream adapter molecules, suggesting they may have distinct signaling functions. We have characterized Ret 3' sequences in mouse and investigated alternative splicing of this region. We found that the organization of Ret 3' sequences is very similar to human RET. The mouse locus also has alternatively spliced C-terminal coding regions, and the sequences corresponding to RET9 and RET51 are highly conserved in both position and sequence with the human locus. Further, we compared the predicted C-terminal amino acids of RET9 and RET51 in seven vertebrate species, and found that they are well conserved. We have identified sequence encoding a putative ret43 isoform in mouse, however the predicted amino acid sequence showed low homology to human RET43. Our data suggest that RET isoforms are evolutionarily highly conserved over a broad range of species, which may indicate that each isoform has a distinct role in normal RET function.     

v-myb and v-ets cooperate for the mitogenic stimulation of primary fibroblasts by avian E26 retrovirus.

By using a series of deletion mutants, we have shown that the stimulation of fibroblast growth by E26 requires the cooperation of the two oncogenes, v-myb and v-ets, fused in the nuclear viral product. Of the two DNA-binding domains, only one must be present to promote anchorage-independent growth, whereas that of v-myb is required to allow growth in low serum medium. Furthermore, the v-ets oncogene comprises multifunctional domains.     

Abnormal localization of proto-oncogene c-ets 1 in acute leukemia with translocation t(1: 11)(q21: q23)

The chromosome localization of a 5.4 kb DNA genomic probe of proto-oncogene c-ets 1 has been analysed in an acute monocytic leukemia with t (1; 11) (q21; q23) translocation. The c-ets probe has been translocated onto the rearranged chromosome 1, suggesting the involvement of the proto-oncogene in leukemias with chromosome rearrangements at band 11 q23.     

Alternative splicing and imprinting control of the Meg3/Gtl2-Dlk1 locus in mouse embryos

The distal part of the mouse Chr 12 contains a cluster of reciprocally imprinted genes. Recently we found a grandparental origin-dependent, transmission-ratio distortion (TRD) in this region. The TRD resulted from postimplantation loss of embryos that inherited the distal Chr 12 alleles from the maternal grandfather. These data suggested that imprinting of one or more genes in this region was not uniformly well established or maintained in all the embryos. To elucidate the mechanism underlying such a variation, we examined the expression of two genes from the distal Chr 12 imprinted region, the maternally expressed gene 3/gene-trap locus 2 ( Meg3/ Gtl2), and the delta-like homolog 1 ( Dlk1) gene. We demonstrated that the Meg3/ Gtl2 gene had two major mRNA forms. One form, Meg3-proximal ( Meg3p), contained exons 1-3. The second form, Meg3-distal ( Meg3d) did not contain exons 1-3 and was present in oocytes and in 1- and 2-cell embryos. We observed cross-dependent and splice form-specific relaxation of imprinting of the Dlk1 and Meg3d, but not Meg3p. Expression patterns of Dlk1 and Meg3/ Gtl2 in embryos from crosses between different mouse strains suggest that 1). imprinting of the Dlk1 and Meg3/ Gtl2 genes is not strictly coordi- nated; 2). parental origin-dependent expression of these genes is under control of a strain-specific, cis-acting modifier located in a 1.5-Mb region that includes the Meg3/ Gtl2-Dlk1 locus. Biallelic expression of Dlk1 and Meg3d did not affect embryo viability and, therefore, cannot be responsible for the lethal phenotypes in UPD12 embryos or for the transmission-ratio distortion.     

Common mechanism of retrovirus activation and transduction of c-mil and c-Rmil in chicken neuroretina cells infected with Rous-associated virus type 1.

We previously described the isolation of the IC10 retrovirus which transduced the v-Rmil oncogene, a new member of the mil/raf gene family. This virus was generated during serial passaging of Rous-associated virus type 1 (RAV-1) in chicken embryo neuroretina (NR) cells and was selected for its ability to induce proliferation of these nondividing cells. IC10 was isolated after six passages of culture supernatants but was not detected in proliferating NR cells during early virus passages. In this study, we molecularly cloned and sequenced another v-Rmil-containing provirus, designated IC11, from NR cells infected at the third virus passage of the same experiment. Both IC11 and IC10 transduced only the serine/threonine kinase domain of c-Rmil. Comparison of v-Rmil and c-Rmil sequences indicated that amino-terminal truncation is sufficient to activate the mitogenic properties of c-Rmil. IC11 and IC10 have identical 3' ends but differ by their 5' RAV-1-Rmil junctions. The 3' ends of both viruses were generated by recombination between Rmil and env genes, involving partial sequence identity. The 5' RAV-1-Rmil junction of IC11 was formed by a splicing process between the RAV-1 leader and a 37-bp c-Rmil exon located upstream of the kinase domain. NR cells infected with this virus synthesize a unique Rmil protein. IC10 contains most of the gag gene recombined with v-Rmil and encodes a gag-Rmil hybrid protein. Serial passaging of IC11 in NR cells led to the formation of a gag-Rmil-containing retrovirus. These results indicate that IC11 represents an early step in transduction and that this virus further recombined with RAV-1 to generate IC10. They confirm our previously proposed model for the multistep generation of v-mil-transducing retroviruses. Therefore, activation and transduction of c-mil and c-Rmil, in NR cells infected with RAV-1, result from a common mechanism.     

Alternative RNA splicing of the murine alpha A-crystallin gene: protein-coding information within an intron

The eye lens contains a structural protein (alpha-crystallin), composed of two homologous primary gene products, alpha A2 and alpha B2. In certain rodents, there is another minor alpha-crystallin polypeptide, alpha Ains, which is identical to alpha A2 except for a 22 amino acid insert between residues 63 and 64 of the alpha A2 chain. Here we show that the mouse contains a single alpha A-crystallin gene, which has a 1376 bp intron separating codons 63 and 64 of the alpha A2-crystallin mRNA. A sequence encoding a 23 amino acid insert peptide was found 266 bp into the intron. The nucleotide borders of this sequence deviate from the AGGT consensus sequence. The DNA sequence encoding the insert peptide hybridizes to a cytoplasmic 14S RNA, demonstrating that it is transcribed in the lens. We propose that the murine alpha A2-crystallin gene generates both the alpha A2 and the alpha Ains mRNAs by alternative splicing.     

The ets sequence is required for induction of erythroblastosis in chickens by avian retrovirus E26.

E26 is a replication-defective avian retrovirus that causes an erythroblastic leukemia in vivo and transforms hematopoietic precursor cells of both the erythroid and the myeloid lineages in vitro. The E26 genome contains two sets of cell-derived sequences, ets and myb. myb sequences are also present in avian myeloblastosis virus, which transforms myeloblasts exclusively. To determine whether the ets sequence is responsible for the erythroid specificity of E26, we analyzed the transforming activities of several viruses carrying mutations in the ets sequence constructed in vitro. The mutant viruses retained the ability to transform myeloid cells in vitro, indicating that the myb oncogene is sufficient for this viral function. However, the ets-deficient viruses did not cause an overt leukemia in chickens. The results indicate that the ets sequence is required for the induction of erythroblastosis by E26.