CXCR4 homologues of gibbon ape, African green monkey, squirrel monkey, and cotton-top marmoset.

CXCR4 gene homologues were isolated from an ape (gibbon), an Old World monkey (African green monkey), and two New World monkeys (squirrel monkey and cotton-top marmoset), and their DNA sequences determined. The squirrel monkey and cotton-top marmoset CXCR4 sequences more closely resemble homologues from apes than Old World monkeys, a pattern not seen for the related chemokine receptor CCR5. The African green monkey CXCR4 gene is similar to its homologue in baboon, a pattern that has also been seen among CCR5 homologues. The gibbon CXCR4 contains the first polymorphisms recognized in ape homologues, the human and chimpanzee CXCR4 proteins being identical, and two of these three differences are also observed in one or more Old World monkey homologues. While 18 positions within CXCR4 are now known to be polymorphic in primates, 7 of these polymorphisms have been observed in multiple examples and 11 have been observed only once.

Human and fungal 3' splice sites are used by Trypanosoma brucei for trans splicing.

In Trypanosoma brucei, pre-mRNAs are joined to a 5' 39 nt spliced leader sequence by trans splicing, a process that has not been well characterized. We have asked whether the 3' splice site regions of human and yeast introns are able to substitute in vivo for the 3' spliced leader acceptor regions of trypanosome pre-mRNA sequences. The ability of heterologous sequences to participate in trans splicing in trypanosomes was assayed by chloramphenicol acetyltransferase (CAT) enzyme activity and/or the detection of spliced CAT mRNA. Four out of the six heterologous 3' splice site regions (human beta-globin intervening sequence (IVS)2, human c-myc IVS2, human factor-VIII IVS1, and yeast actin IVS) functioned as 3' spliced leader acceptor regions in T. brucei, while two did not show significant or detectable levels of CAT activity (human beta-globin IVS1 and human c-myc IVS1). In the case of the human beta-globin IVS1 however, lengthening of the polypyrimidine tract as a result of single purine to pyrimidine transversions produced an active acceptor in which the spliced leader addition site coincides with the 3' splice site of the beta-globin exon 2. These studies indicate that some, but not all 3' acceptor regions in humans can function as spliced leader addition sites in trypansomes.

Mitochondrial minicircle DNA supports plasmid replication and maintenance in nuclei of Trypanosoma brucei.

In a search for trypanosome DNA sequences that permit replication and stable maintenance of extrachromosomal elements, a 1-kilobase-pair (kbp) fragment from a mitochondrial kinetoplast DNA (kDNA) minicircle of Trypanosoma brucei was isolated and characterized. The plasmid pTbo-1, carrying the kDNA element, is maintained in T. brucei as a supercoiled concatemer containing approximately seven to nine pTbo-1 monomer units (5.6 kbp each) in a head-to-tail orientation. The concatemer is found in approximately one copy per cell when procyclic trypanosomes are cultured in the presence of 100 micrograms of hygromycin per ml; however, in the absence of continuous hygromycin selection, the plasmid is lost from the population with a t1/2 of approximately 8.7 days (17 cell generations). A second unrelated kDNA minicircle was also able to serve as an autonomously replicating sequence (ARS) element in T. brucei, suggesting that this is a general property of kDNA minicircles. Replication of mitochondrial DNA in the nucleus may be due to either a specific consensus sequence (such as in yeast ARS elements) or nonspecific sequence characteristics (such as the degree of A&T-richness or bent DNA).

Ribosomal protein L25 from Trypanosoma brucei: phylogeny and molecular co-evolution of an rRNA-binding protein and its rRNA binding site.

The gene encoding ribosomal protein L25, a primary rRNA-binding protein, was isolated from the protozoan parasite Trypanosoma brucei. Hybridization studies indicate that multiple copies of the gene are present per T. brucei haploid genome. The C-terminal domain of L25 protein from T. brucei is strikingly similar to L23a protein from rat, L25 proteins from fungal species, and L23 proteins from eubacteria, archaebacteria, and chloroplasts. A phylogenetic analysis of L23/25 proteins and the putative binding sites on their respective LSU-rRNAs (large subunit rRNAs) provides a rare opportunity to study molecular co-evolution between an RNA molecule and the protein that binds to it.

Levels of Epstein-Barr virus DNA in lymphoblastoid cell lines are correlated with frequencies of spontaneous lytic growth but not with levels of expression of EBNA-1, EBNA-2, or latent membrane protein.

The process of Epstein-Barr virus (EBV)-induced transformation of human B lymphocytes results in a cell line that is a mixture of latently and lytically infected cells, with the lytic cells composing roughly 5% to less than 0.0001% of the overall population. A set of nine normal lymphoblastoid cell lines that span a 100- to 200-fold range in average EBV DNA content were studied, and the frequency with which these cells entered a lytic phase of viral growth correlated with their EBV DNA copy number (as a population average). However, neither factor correlated with the levels of expression of transcript for the viral genes EBNA-1, EBNA-2, and latent membrane protein, nor did they correlate with the levels of EBNA-2 protein and latent membrane protein. The rate at which a cell line enters into lytic growth spontaneously is therefore not dependent on the overall steady-state levels of expression of these latent-phase genes.

Relative rates of RNA synthesis across the genome of Epstein-Barr virus are highest near oriP and oriLyt.

The rates of Epstein-Barr virus transcription were measured in isolated nuclei from marmoset and human lymphoblasts transformed in vitro. In B95-8, a marmoset B-lymphoid cell line, the most frequently transcribed viral genes are the EBERs (small nuclear RNAs) and BHLF-1 (encoding a lytic-phase gene product). The EBERs and BHLF-1 genes are separated by nearly 50 kilobase pairs on the Epstein-Barr virus genome and lie adjacent to (less than 300 base pairs from) oriP and oriLyt, respectively. oriP and oriLyt are putative origins of viral DNA replication, and each is associated with a transcriptional enhancer element. Among the human B-lymphoblastoid cell lines tested, only the transcription of EBERs predominates.

Characterization of an antigen whose cell surface expression is induced by infection with Epstein-Barr virus.

Metabolically labeled monoclonal antibodies were used to measure the number of determinants per cell for an Epstein-Barr virus (EBV) cell surface antigen (EBVCS) (C. Kintner and B. Sugden, Nature [London] 294:458-460, 1981) which is expressed on the surface of EBV-transformed cells. The antigenic determinants were present approximately 5 X 10(5) times per in vitro-transformed cell. Immunoprecipitation followed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate indicated that four independent monoclonal antibodies to EBVCS recognized a protein of 47,000 daltons. The identification of EBVCS isolated from EBV-transformed cells grown in tunicamycin demonstrated that the antigen when isolated from cells grown without this drug was glycosylated. Finally, preclearing experiments with monoclonal antibodies to EBVCS or to HLA (class I products of the human major histocompatibility locus) and to beta 2-microglobulin indicated that EBVCS is not a major histocompatibility type 1 antigen.