CST (BART BARF0) family viral RNAs are expressed in several types of Epstein-Barr virus (EBV) infection, including EBV-associated cancers.Many different spliced forms of these RNAs have been described; here we have clarified the structures of some of the more abundant splicing patterns. We report the first cDNAs representing a full-length CST mRNA from a clone library and further characterize the transcription start. The relative abundance of splicing patterns and genomic analysis of the open reading frames (ORFs) suggest that, in addition to the much studied BARF0 ORF, there may be important products made from some of the upstream ORFs in the CST RNAs. Potential biological functions are identified for two of these. The product of the RPMS1 ORF is shown to be a nuclear protein that can bind to the CBF1 component of Notch signal transduction. RPMS1 can inhibit the transcription activation induced through CBF1 by NotchIC or EBNA-2. The protein product of another CST ORF, A73, is shown to be a cytoplasmic protein which can interact with the cell RACK1 protein. Since RACK1 modulates signaling from protein kinase C and Src tyrosine kinases, the results suggest a possible role for CST products in growth control, perhaps consistent with the abundant transcription of CST RNAs in cancers such as nasopharyngeal carcinoma.In several types of infection, in addition to the well-established EBNA, LMP and EBER genes, Epstein-Barr virus (EBV) has been found to express various spliced RNAs transcribed rightward from the region spanning 150,000 to 161,000 on the B95-8 EBV genetic map. These have been referred to as complementary strand transcripts (CSTs), BamHI A rightward transcripts (BARTs), or the BARF0 RNAs. RNAs of this type were originally identified in cDNA made from the C15 xenograft culture of nasopharyngeal carcinoma (NPC) tissue (17). Similar RNAs were subsequently found in various EBV-positive NPC tumor biopsies and xenografts, Burkitt's lymphoma, lymphoid cell lines (LCLs) (3,6,11,15,19,31,43), and biopsies of oral hairy leukoplakia (24). Expression of CST RNAs has also been demonstrated in peripheral blood of normal human carriers of EBV (5), sera from NPC patients have been found to immunoprecipitate a protein product of the BARF0 open reading frame (ORF) made in vitro (12), and cytotoxic T lymphocytes that respond to a peptide derived from BARF0 have been identified in EBV-infected people (22).A very complicated picture of alternatively spliced CST RNAs has built up (36, 38), but some of the proposed structures have been deduced using very sensitive reverse transcription (RT)-PCR methods or have been only single isolates from cDNA libraries, and thus they may yet represent very minor species within the family of RNAs that can be expressed. Predominant sizes of the RNAs expressed have been deduced from Northern blots, but these have only partly been related to the spliced RNA structures. In addition, the coding content of the CST RNAs remains uncertain. Most attention has focused on the BARF0 ORF, particularly a sp...

Ancient duplications and rearrangements of protein-coding segments have resulted in complex gene family relationships. As a result, gene products may acquire new specificities, altered recognition properties, modified functions, and even loss of functionality. The natural cytotoxicity receptor (NCR) family are natural killer (NK)-activating receptors whose members are NKp46 (NCR1), NKp44 (NCR2), and NKp30 (NCR3). The NCR proteins are putative immunoglobulin superfamily members whose ligands are unknown. The NKp46 gene is present and expressed in human and mouse, NKp44 is only present and expressed in human, and NKp30 is present and expressed in human but is a nonexpressed pseudogene in mouse. By searching databases we have detected alternatively spliced forms of the three NCR members. In addition, we have shown by reverse transcription-polymerase chain reaction (RT-PCR) analysis that the human NKp30 gene presents differential expression patterns in tissues. However, no expressed sequence tags (ESTs) are detected for mouse NKp30, and the genomic sequence contains two premature stop codons, which would encode a severely truncated nonfunctional protein. We have sequenced genomic DNA from 13 mouse inbred and wild strains and discovered that NKp30 is a pseudogene in every mouse strain sequenced except Mus caroli where two single nucleotide polymorphisms (SNPs) abolished the premature stop codons. We observed that the laboratory-inbred strains are, for the exonic sequences, genetically identical, except Mus m. musculus C3H. The Mus musculus strains only have a few SNPs, but the rest of the Mus strains have accumulated gradually several SNPs, mainly in the functional immunoglobulin and intracellular domains. RT-PCR analysis performed on RNA from M. caroli tissue samples identified two transcripts, one of which would encode a putative soluble NKp30 protein, also detected in rat but not in human. We have observed that the intracellular domains of NKp30 (and NKp46) are not conserved among the different species, with the most striking difference when comparing human against mouse and rat. The NKp44 gene is only found in human and shows three different splice forms varying in their "stalk" and intracellular domains. Searching for NKp44 orthologs, we found similarity to ESTs from a novel rodent TREM family member, which we termed TREM6, and not to any possible NKp44 ortholog.

A cDNA clone encoding the mRNA for the highly polymorphic human enzyme phosphoglucomutase 1 (PGM1; EC 5.4.2.2) has been isolated and characterized. This was achieved indirectly by first isolating a rabbit cDNA from an expression library using anti-rabbit PGM antibodies. A comparison of the nucleotide sequences shows that the homologies between human and rabbit PGM1 mRNAs are 92% and 97% for the coding nucleotide sequence and the amino acid sequence, respectively. The derived rabbit amino acid sequence is in complete agreement with the published protein sequence for rabbit muscle PGM. A physical localization of the human PGMI gene to chromosome lp3l has been determined by in situ hybridization. Analysis of DNA from a wide variety of vertebrates indicates a high level of PGMI sequence conservation during evolution.Phosphoglucomutase (PGM; EC 5.4.2.2) catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate and thus has a pivotal role in glycolysis and gluconeogenesis. The human enzyme is encoded by three autosomal unlinked structural loci, designated PGMJ, PGM2, and PGM3 (1, 2), and there is evidence for a fourth locus expressed in human milk (3). On the basis of protein studies, it is assumed that the loci arose by gene duplication and evolved separately to provide the present day isozymes, showing distinctive properties, such as molecular size (4), thermostability (5), substrate specificity (6), tissue distribution (7), and sulfhydryl group reactivity (8). Multiple isozymes, attributable to multiple PGM loci have been identified in a very wide range of species and it is evident that the initial PGM gene duplication(s) occurred in the remote evolutionary past.Isozyme polymorphism of human PGMI was identified more than 25 years ago (9) and the locus has since been shown to exhibit a very high incidence of both common and rare allelic variation in all populations (10). The PGMI locus is an important anchor point for linkage analysis and for positioning markers on human chromosome lp and its homologues in other species. The heterozygosity demonstrable by isozyme analysis of human erythrocytes [0.81 in the Centre d'Etude Polymorphisme Humain (CEPH) family panel for example] is comparable to several of the variable number tandem repeat loci and exceeds most of the restriction fragment length polymorphisms recognized by other probes on chromosome 1. The PGM1 isozyme polymorphism is a particularly useful marker of individuality in forensic science (11, 12) since the discriminating power of the PGMI locus (1 -probability of a match) is about 0.75 for the common isozyme phenotypes revealed by isoelectric focusing (12). Furthermore, the isozymes are found in a variety of body fluids and enzyme activity is retained for prolonged periods in stains recovered from scenes of crimes.

In view of its central role in glycolysis and gluconeogenesis and its polymorphic genetic variability, the phosphoglucomutase 1 (PGM1) gene in man has been the target of protein structural studies and genetic analysis for more than 25 years. We have now isolated genomic clones containing the complete PGM1 gene and have shown that it spans over 65 kb and contains 11 exons. We have also shown that the sites of the two mutations which form the molecular basis for the common PGM1 protein polymorphism lie in exons 4 and 8 and are 18 kb apart. Within this region there is a site of intragenic recombination. We have discovered two alternatively spliced first exons, one of which, exon 1A, is transcribed in a wide variety of cell types; the other, exon 1B, is transcribed in fast muscle. Exon 1A is transcribed from a promoter which has the structural hallmarks of a housekeeping promoter but lies more than 35 kb upstream of exon 2. Exon 1B lies 6 kb upstream of exon 2 within the large first intron of the ubiquitously expressed PGM1 transcript. The fast-muscle form of PGM1 is characterized by 18 extra amino acid residues at its N-terminal end. Sequence comparisons show that exons 1A and 1B are structurally related and have arisen by duplication.