Expression of the bovine leukemia virus X region in virus-infected cells.

Bovine leukemia virus, like its closest relatives the human T-cell leukemia virus types I and II, contains a 1.8-kilobase X region between the env gene and the 3' long terminal repeat. In this communication, we report the detection and characterization of a subgenomic mRNA from which this X region is presumably translated. This mRNA was produced by a complex splicing mechanism which resulted in juxtaposition of the 5' end of the env gene and the two overlapping X-region open reading frames. Translation of this mRNA could yield at least two distinct proteins depending on which initiation codon is used. Detection of the protein encoded by the BLV X-region long open reading frame has been reported (N. Sagata, J. Tsuzuku-Kawamura, M. Nagayoshi-Aida, F. Shimizu, K.-I. Imagawa, and Y. Ikawa, Proc. Natl. Acad. Sci. USA 82:7879-7883, 1985). Using synthetic peptide antisera, we detected a protein encoded by the short open reading frame in virus-infected cells. The protein migrated in sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 19,000. It is a nuclear phosphoprotein.     

Saccharomyces cerevisiae positive regulatory gene PET111 encodes a mitochondrial protein that is translated from an mRNA with a long 5'' leader.

The yeast nuclear gene PET111 is required specifically for translation of the mitochondrion-coded mRNA for cytochrome c oxidase subunit II. We have determined the nucleotide sequence of a 3-kilobase segment of DNA that carries PET111. The sequence contains a single long open reading frame that predicts a basic protein of 718 amino acids. The PET111 gene product is a mitochondrial protein, since a hybrid protein which includes the amino-terminal 154 amino acids of PET111 fused to beta-galactosidase is specifically associated with mitochondria. PET111 is translated from a 2.9-kilobase mRNA which, interestingly, has an extended 5'-leader sequence containing four short open reading frames upstream of the long open reading frame. These open reading frames exhibit an interesting pattern of overlap with each other and with the PET111 reading frame.     

Differential splicing in mouse thymus generates two forms of terminal deoxynucleotidyl transferase.

A new form of TdT mRNA has been identified by screening a mouse thymus cDNA library. It contains an open reading frame of 1527 base pairs corresponding to a protein containing 509 aminoacids, whereas the previously identified mouse TdT mRNA is composed of 1587 base pairs and encodes a protein of 529 aminoacids. Analysis of a mouse genomic clone containing the 3' portion of the TdT gene shows that these twenty additional aminoacids are encoded by an additional exon located between exons X and XI. Both forms of TdT mRNA are present in the thymus and could be generated by alternative splicing. The cDNA reported here corresponds to the major form of TdT mRNA in Balb/c mice and closely resembles human and bovine TdT cDNA. Expression of this cDNA in mammalian cells shows that it encodes a functional protein capable of catalysing N region insertions at the recombination junction of an episomic recombination substrate.     

Identification of a potential protease-coding gene in the genomes of bovine leukemia and human T-cell leukemia viruses

The genomes of bovine leukemia and human T-cell leukemia viruses both contain an unidentified region between the gag and pol genes. These regions harbor an open reading frame that is in a different phase from the reading frames of the gag and pol genes. Based on the deduced amino acid sequences, we show here that they potentially encode a gag precursor-cleaving protease, which is known to be fused to the gag and pol products of avian and murine retroviruses, respectively. This finding raises the interesting question of the expression and evolution of retroviral genes.     

Nucleotide sequence of AKV murine leukemia virus.

AKV is an endogenous, ecotropic murine leukemia virus that serves as one of the parents of the recombinant; oncogenic mink cell focus-forming viruses that arise in preleukemic AKR mice. I report the 8,374-nucleotide-long sequence of AKV, as determined from the infectious molecular clone AKR-623. The 5'-leader sequence of AKV extends to nucleotide 639, after which lies a long open reading frame encoding the gag and pol gene products. The reading frame is interrupted by a single amber codon separating the gag and pol genes. The pol gene overlaps the env gene within the 3' region of the AKV genome. The nucleotide sequence of the 5' region of AKV reveals the following features. (i) The 5'-leader sequence lacks any AUG codon to initiate translation of gPr80gag, suggesting that gPr80gag is not required for the replication of AKV. (ii) A short portion of the leader region diverges in sequence from the closely related Moloney murine leukemia virus and appears to be related to a sequence highly repeated in eucaryotic genomes. (iii) As in Moloney murine leukemia virus, there is a potential RNA secondary structure flanking the amber codon that separates the gag and pol genes. This structure might function as a regulatory protein binding site that controls the relative levels of synthesis of the gag and pol precursors. The nucleotide sequence of the 3' region of AKV is compared with sequences reported previously from both infectious and noninfectious molecular clones of AKV.     

malM, a new gene of the maltose regulon in Escherichia coli K12. I. malM is the last gene of the malK-lamB operon and encodes a periplasmic protein

The structure and expression of the distal part of the malK-lamB operon in Escherichia coli was studied. DNA sequencing was performed as far as a HinfI restriction site located 1313 base-pairs downstream from gene lamB. The open reading frame, formerly called molA, which begins 245 base-pairs downstream from gene lamB, is longer than was initially thought, and was renamed malM. It could encode a protein of 306 amino acid residues. The complete malM open reading frame was cloned under control of the tac 12 promoter. In maxicells, the resulting plasmid permitted tac12-promoted synthesis of two polypeptides, encoded by gene malM, with apparent molecular weights of 37 X 10(3) and 34.5 X 10(3). We provide strong evidence that the 34.5 X 10(3) Mr protein is derived from the 37 X 10(3) Mr protein by processing at the amino-terminal end, and that this processed form is located in the periplasmic space. We show that the chromosomal malM gene is expressed as part of the malK-lamB operon, and that its product is periplasmic. Finally, we demonstrate with nuclease S1 mapping experiments that the mRNA terminates at a typical rho-independent terminator located about 45 base-pairs beyond the end of gene malM, which is thus the last gene of the malK-lamB operon.     

The 3'' region of bovine leukemia virus genome encodes a trans-activator protein.

The genome of bovine leukemia virus (BLV) contains several overlapping, long open reading frames 3' to the envelope gene. Experiments presented here show that the cDNA encompassing the X region open reading frames encodes a trans-activator function capable of increasing the level of gene expression directed by the BLV long terminal repeat sequences. This study provides further evidence of the structural and functional similarities of the bovine leukemia virus and the human T lymphotropic viruses, HTLV-I and HTLV-II.     

Transposition of an intron in yeast mitochondria requires a protein encoded by that intron

The optional 1143 bp intron in the yeast mitochondrial 21S rRNA gene (omega +) is nearly quantitatively inserted in genetic crosses into 21S rRNA alleles that lack it (omega -). The intron contains an open reading frame that can encode a protein of 235 amino acids, but no function has been ascribed to this sequence. We previously found an in vivo double-strand break in omega - DNA at or close to the intron insertion site only in zygotes of omega + X omega - crosses that appears with the same kinetics as intron insertion. We now show that mutations in the intron open reading frame that would alter the translation product simultaneously inhibit nonreciprocal omega recombination and the in vivo double-strand break in omega - DNA. These results provide evidence that the open reading frame encodes a protein required for intron transposition and support the role of the double-strand break in the process.     

Molecular cloning and sequence of the gene for p9Ka. A cultured myoepithelial cell protein with strong homology to S-100, a calcium-binding protein

The gene for p9Ka, a protein of molecular weight 9000 that is expressed in cultured rat mammary myoepithelial cells, has been isolated from a normal rat genomic library in bacteriophage lambda, by its ability to hybridize to a cloned complementary DNA corresponding to p9Ka mRNA. The cloned rat genomic DNA fragment hybridized to translatable p9Ka mRNA. A nucleotide sequence of 2340 base-pairs of genomic DNA surrounding the p9Ka cDNA sequence has been obtained; the gene contains one intervening sequence of 675 nucleotides. The 3' end of the p9Ka mRNA has been identified on the gene sequence to be 13 nucleotides downstream from a poly(A) addition signal AATAAA. The gene contains an open reading frame of 101 amino acid residues, which is the only open reading frame in the entire gene long enough to encode a protein of molecular weight at least 9000. This hypothetical protein sequence shows greater than 40% homology to rat or bovine S-100 protein and over 30% homology to bovine intestinal calcium-binding protein. The results suggest that p9Ka may be related to a family of low molecular weight calcium-binding proteins.     

The secY-rpmJ region of the spc ribosomal protein operon in Escherichia coli: structural alterations affecting secY expression

Summary A unique feature of the spc ribosomal protein operon is that its region distal to the promoter contains a gene (secY) for an integral membrane protein, followed by an open reading frame termed X which has recently been proposed to encode a new ribosomal protein (protein B). We now show that the open reading frame X indeed directs the synthesis of a protein with electrophoretic mobilities similar to the B protein, and this supports the proposal that X may be more appropriately called rpmJ. Insertion of a plasmid sequence into the secY-rpmJ boundary of the chromosome caused a reduced expression of secY probably by destabilizing the secY part of the message. The results of complementation experiments suggested that a normal level of expression of rpmJ is not required for growth or protein secretion.     

Comparison of the entire genomes of bovine leukemia virus and human T-cell leukemia virus and characterization of their unidentified open reading frames.

We have compared the sequence of the entire genomes of bovine leukemia virus (BLV) and human T-cell leukemia virus type I (HTLV-I). Both the gag and pol genes show overall strong homologies indicating the close evolutionary relationship of the two retroviruses. However, a surface glycoprotein portion of the env gene shows no appreciable homology, which probably reflects a difference in their host ranges. The 3' end portion of the BLV genome (designated as pXBL) contains an unidentified long open reading frame that has a typical protein-coding property. The potential product of this open reading frame may be a glycoprotein of approximately 40 000 daltons. We note that its amino acid sequence shows low but appreciable homology, especially in its N-terminal quarter, to that of the HTLV-I counterpart (pX product), and we thus suggest that BLV pXBL and HTLV-I pX have diverged from a common ancestral gene. It is tentatively concluded that both the putative pXBL and pX products are respectively produced from a spliced mRNA.