Structure and dimorphism of c-rel (turkey), the cellular homolog to the oncogene of reticuloendotheliosis virus strain T.

A locus has been identified in turkey DNA that contains nucleotide sequences homologous to the oncogene (v-rel) in the avian retrovirus, reticuloendotheliosis virus strain T. This locus, c-rel, has been molecularly cloned from an apparently heterozygous turkey. c-rel is approximately 23 kilobase pairs in length, with at least seven apparent introns, and contains sequences sufficient to account for all of v-rel. Nucleic acid sequence differences exist between v-rel and homologous regions of c-rel. We examined a population of turkeys to determine whether these sequence differences are the result of polymorphism in the population. Within the turkey population, c-rel is dimorphic in apparent introns and 3' flanking sequences, but polymorphism has not been detected within the regions of the c-rel locus that are homologous to v-rel. Additionally, no nucleic acid sequence differences have been detected between the regions of c-rel in turkeys that are homologous to v-rel and the sequences related to v-rel of a homologous locus in chickens (Chen et al., J. Virol. 245:104-113, 1983). The general organization of introns and flanking sequences is conserved for both c-rel in turkeys and this locus in chickens, indicating that c-rel, like other proto-oncogenes, may have an important development or metabolic function.     

Activation of oncogenicity of the c-rel proto-oncogene.

Reticuloendotheliosis virus strain T (Rev-T) induces a lethal lymphoma in young birds and transforms avian lymphoid cells in vitro. The transforming gene of Rev-T, v-rel, was derived from the turkey proto-oncogene c-rel. Comparison of the nucleotide sequences of v-rel and c-rel indicates that in addition to several internal amino acid changes relative to c-rel, p59v-rel has amino acid sequences at both ends derived from the reticuloendotheliosis virus strain A-related virus env gene (K. C. Wilhelmsen, K. Eggleton, and H. M. Temin, J. Virol. 52:172-182, 1984). In this report, the v-rel sequences important for transformation were defined by constructing recombinant retroviruses in which c-rel sequences replaced the analogous v-rel sequences. These recombinant viruses expressing chimeric proteins were tested for their ability to transform spleen cells in vitro and to induce tumors in young chickens. Activation of the oncogenicity of c-rel in Rev-T required alteration of the amino terminus and the central region of the protein. Deletion of the noncoding sequences 3' to c-rel and of most of the helper virus-related env sequences was necessary for the formation of Rev-T.     

Transforming viruses spontaneously arise from nontransforming reticuloendotheliosis virus strain T-derived viruses as a result of increased accumulation of spliced viral RNA.

The highly oncogenic avian retrovirus reticuloendotheliosis virus strain T (Rev-T) contains a substitution of the oncogene v-rel for much of env and a deletion of gag and pol relative to the helper virus Rev-A. Replacement of gag and pol sequences in Rev-T suppresses transformation by reducing the accumulation of spliced viral mRNA and v-rel protein in infected cells (C. K. Miller and H. M. Temin, J. Virol 58:75-80, 1986). After infection of spleen cells with viruses containing gag and pol sequences, revertant viruses that are strongly transforming were found. Approximately three-fourths of the revertant viruses appeared structurally the same as the parental virus, and approximately one-fourth of the revertant viruses had large deletions (similar in size and location to the deletion in Rev-T). Two revertant viruses that appeared structurally the same as the parental virus were molecularly cloned. The regions sufficient to change the parental virus to a strongly transforming virus were determined by construction of recombinant viruses. In one revertant virus, the region sufficient for transformation contained a 327-base-pair insertion 5' of the 3' splice site used by Rev-T. In the other revertant virus, the region sufficient for transformation contained a 1-base-pair transition and a deletion of one copy of a 9-base-pair direct repeat, both 3' of the 3' splice site used by Rev-T. These differences resulted in the accumulation of increased levels of subgenomic v-rel mRNA and protein, ultimately leading to transformation.     

Transformation of avian lymphoid cells by reticuloendotheliosis virus

Avian reticuloendotheliosis virus (REV-T) is the most virulent of all retroviruses, inducing an invariably fatal leukemia in chickens with a latent period of 7-10 days. Unlike avian cells transformed by other acutely transforming viruses, lymphoid cells transformed by REV-T are immortalized. Furthermore, in vitro derived, REV-T transformed cells which do not produce virus are tumorigenic and induce lethal reticuloendotheliosis when injected into histocompatible birds. Thus REV-T transforms its target cell both in vitro and in vivo. In addition this transformation is independent of any helper virus functions. Like other acute leukemia viruses, REV-T is replication-defective and must co-replicate with a reticuloendotheliosis associated virus (REV-A). During evolution, a substantial portion of its genome has been deleted and replaced with a host-derived genetic sequence, designated v-rel. Presumably, the v-rel oncogene was transduced from a normal turkey DNA locus, c-rel. There are 9 regions of homology between c-rel and v-rel, however, several differences exist between these genes, suggesting that transformation by REV-T results from the production of an altered v-rel protein. The v-rel sequence is distinct from other known oncogenes and encodes a 57-kDa phosphoprotein. In REV-T transformed cells, this pp57v-rel protein is localized in the cytoplasm. The product of the v-rel oncogene is present at a low level, representing only about 0.003% of total methionine-labelled protein. In addition, pp57v-rel is relatively stable, having an estimated half-life of 4-10 h. The v-rel protein when purified close to homogeneity is complexed with a 40-kDa cellular phosphoprotein in transformed lymphoid cells and possesses serine kinase activity. This review discusses the molecular aspects of transformation by REV-T in the context of other oncogene-encoded proteins.     

Insertion of several different DNAs in reticuloendotheliosis virus strain T suppresses transformation by reducing the amount of subgenomic mRNA.

The highly oncogenic retrovirus reticuloendotheliosis virus (Rev) strain T (Rev-T) has, relative to its helper virus Rev strain A, a substitution of the oncogene v-rel for most of the env gene and a large deletion of gag and pol sequences. When the helper virus sequences that are deleted in Rev-T are replaced, the recombinant virus is nontransforming (I. S. Y. Chen and H. M. Temin, Cell 31: 111-120, 1982). We show that suppression of transformation occurs when several different DNA sequences are inserted in Rev-T and that suppression is correlated with a reduction in the amount of v-rel mRNA and v-rel protein in infected cells. The reduced amount of v-rel protein is insufficient for transformation.     

v-rel oncoproteins in the nucleus and in the cytoplasm transform chicken spleen cells.

The transforming protein encoded by the v-rel oncogene of the highly oncogenic avian retrovirus reticuloendotheliosis virus strain T (Rev-T) is a 59,000-dalton protein, p59v-rel. The mechanism by which p59v-rel induces transformation of early lymphoid cells is unknown. As a step towards understanding the mechanism of v-rel-induced transformation, we sought to establish the subcellular site of action of p59v-rel. In this report, we show that p59v-rel contains sequences that are necessary for its efficient localization in the nucleus of infected chicken embryo fibroblasts. These v-rel sequences when added to the normally cytoplasmic protein, beta-galactosidase, directed that protein to the nucleus. A mutation in the v-rel nuclear-localizing sequence did not affect the transforming function, although it did alter the nuclear-localizing function. The addition of a supplemental nuclear-localizing sequence from simian virus 40 large T-antigen to v-rel resulted in the expression of a transforming rel protein which was located exclusively in the nucleus of transformed spleen cells, in contrast to wild-type p59v-rel, which was largely cytoplasmic in transformed spleen cells. Our results support the hypothesis that v-rel encodes a protein which can act either in the nucleus or in the cytoplasm to transform spleen cells.     

The c-sis gene encodes a precursor of the B chain of platelet-derived growth factor.

The relationship between platelet-derived growth factor (PDGF) and the proto-oncogene c-sis has been determined by amino acid sequence analysis of PDGF and nucleotide sequence analysis of c-sis genomic clones. The nucleotide sequences of five regions of the human c-sis gene which are homologous to sequences of the transforming region (v-sis) of simian sarcoma virus (SSV) were determined. By alignment of the c-sis and v-sis nucleotide sequences the predicted amino acid sequence of a polypeptide homologous to the putative transforming protein p28sis of SSV was deduced. Both predicted sequences use the same termination codon and additional coding sequences may lie 5' to the homologous regions. Amino acid sequence analysis of the PDGF B chain shows identity to the amino acid sequence predicted from the c-sis sequences over 109 amino acid residues. Polymorphism may exist at two amino acid residues. These results suggest that c-sis encodes a polypeptide precursor of the B chain. A partial amino acid sequence of the PDGF A chain is also described. This chain is 60% homologous to the B chain and cannot be encoded by that part of c-sis which has been sequenced but could be encoded by sequences which lie 5' to the five regions of v-sis homology in c-sis, or at a separate locus.     

p59v-rel, the transforming protein of reticuloendotheliosis virus, is complexed with at least four other proteins in transformed chicken lymphoid cells

Previous studies have identified the protein product of v-rel, the oncogene carried by reticuloendotheliosis virus (REV), as a 59,000-dalton phosphoprotein located predominantly in the cytosol of transformed chicken lymphoid cells. In immune precipitates of p59v-rel, there is a closely associated protein kinase activity. In chicken lymphoid cells that do not contain REV, p68c-rel is found free in the cytosol not associated with other proteins and not detectably phosphorylated. In this study, we found that immune precipitates of 59v-rel from REV-transformed cells contain at least four other proteins, of approximate molecular weights 124, 115, 68, and 36 kilodaltons (kDa). The 124-, 115-, and 36-kDa proteins are apparently unrelated to p59v-rel in sequence, and their coprecipitation suggests that they are complexed with p59v-rel. The coprecipitating 68-kDa protein was found to be p68c-rel, which, like the other three proteins, precipitates by virtue of its association with p59v-rel. Glycerol gradient analysis suggested the presence of more than one type of complex: one containing p115, p68c-rel, p59v-rel, and p36, and another containing p124, p115, p59v-rel, and possibly p68c-rel. In vitro kinase activity was found in all size classes, coinciding with the distribution of p115 and p59v-rel. The complex(es) was stable under a variety of conditions, including a wide range of ionic strengths, chelators, and detergents, and through multiple cycles of immune precipitation and elution. This suggests a specific and functionally significant interaction among the members that may be of direct relevance to the mechanism of REV-induced transformation.     

FHF-2 in the turkey (Meleagris gallopavo)

A cDNA clone homologous to the fibroblast growth factor homologous factor (FHF-2) was isolated and sequenced from the turkey (Meleagris gallopavo). The DNA sequence of the turkey was almost identical to that of the chicken (99% similarity) differing at only 8 of 770 nucleotides in the coding region resulting in a single amino acid difference between these poultry species. The 3'UTR of the turkey FHF-2 gene was 445 nucleotides in length and included an imperfect CT microsatellite (ms) repeat. The sequence of the 3'UTR was amplified from genomic DNA of the chicken and found to be highly conserved differing at only three nucleotides when compared to the turkey. Length of the CT repeat was indifferent in a sample of 52 turkeys (monomorphic) however, the number of CT repeats was greater in the turkey than in the chicken. No inter-individual polymorphism was detected in multiple sequences of the 3'UTR of the FHF-2 gene in the turkey. Based on comparison of the turkey and chicken sequences, the mutation rate for coding and associated non-coding (3'UTR) regions of FHF-2 are approximately equal.     

DNA sequence of the Herpes simplex virus type 2 glycoprotein D gene

We describe a 1635-bp Herpes simplex virus type 2 (HSV-2) DNA sequence containing the entire coding region of glycoprotein D (gD-2). The amino acid sequence of gD-2, deduced from the nucleotide sequence, was compared to that of the analogous Herpes simplex virus type 1 (HSV-1) glycoprotein (gD-1). The two glycoproteins are 85% homologous and contain highly conserved regions of as much as 49 amino acids in length. Comparison of DNA sequences upstream from gD-1 and gD-2 coding regions identified possible conserved regulatory sequences.     

The structure and expression of maize genes encoding the major heat shock protein, hsp70

We have isolated and sequenced two maize genomic clones that are homologous to the Drosophila hsp70 gene. One of the maize hsp70 clones contains the entire hsp70 coding region and 81 nucleotides of the 5' nontranslated sequence. The predicted amino acid sequence for this maize protein is 68% homologous to the hsp70 of Drosophila. The second maize hsp70 clone contains only part of the coding sequence and 1.1 kb of the 5' flanking sequence. This 5' flanking sequence contains two sequences homologous to the consensus heat-shock-element sequence. Both maize genes are thermally inducible and each contains an intron in the same position as that of the heat-shock-cognate gene, hsc1, of Drosophila. The presence of an intron in the maize genes is a distinguishing feature in that no other thermally inducible hsp70 genes described to date contain an intron. We have constructed a hybrid hsp70 gene containing the entire hsp70 coding sequence with an intron, and 1.1 kb of the 5' flanking sequence. We demonstrate that this hybrid gene is thermally inducible in a transgenic petunia plant and that the gene is expressed from its own promoter.     

DNA sequence of the region in the genome of herpes simplex virus type 1 containing the exonuclease gene and neighbouring genes.

We report the sequence of a 7800 base pair region of herpes simplex virus type 1 DNA, representing approximately 0.16 to 0.20 map units in the genome. This contains sequences transcribed into a leftward oriented set of five 3' coterminal mRNAs, together with two rightward transcribed flanking genes. One of the leftward genes encodes the virus's alkaline exonuclease, but the other gene products are uncharacterized. The amino acid sequence of one encoded protein suggested that it is a membrane embedded species. The DNA sequence is densely utilised, with two predicted out-of-frame overlaps of coding sequences, and probably six occurrences of promoter elements within coding sequences. Homologues of five of the genes were found for the distantly related Epstein-Barr virus, with a similar overall relative arrangement.     

The Rhodobacter capsulatus chlorin reductase-encoding locus, bchA, consists of three genes, bchX, bchY, and bchZ.

The bchA locus of Rhodobacter capsulatus codes for the chlorin reductase enzyme in the bacteriochlorophyll synthesis pathway. Previous work has suggested that this locus might encompass a single gene. We have sequenced the bchA locus and found it to contain three coding segments, which we designate bchX, bchY, and bchZ. Each coding segment contains its own translational initiation sequence and follows codon utilization patterns consistent with those of previously published R. capsulatus genes. When various regions of the bchA locus and flanking sequences were subcloned into an expression vector and expressed in Escherichia coli, the three coding segments were all expressed as separate peptides. Finally, conservation of amino acid sequences between bchX and a subunit of the protochlorophyllide reductase (bchL, 34% identity) and the nitrogenase Fe protein (nifH, 30 to 37% identity) suggests structural and mechanistic commonalities among all three proteins.