Multiple leader RNAs and messenger RNAs are transcribed from the la crosse virus small genome segment

Nucleotide sequencing has demonstrated that the Small genome segment of Bunyaviruses contains the genetic information for two viral proteins (N and NS_s) in overlapping reading frames (Akashi and Bishop, 1983; Cabradilla et al., in press). Using 3′ end-labeled genome probes, La Crosse virus (LAC) infected cells were shown to contain three leader RNAs, which start at position 1 and terminate at approximate positions 74, 95, and 115 from the 3′ end of the genome. Primer extension and S1 mapping studies have shown that LAC-infected cells contain seven mRNAs, five of which initiate at positions 1, 74, 101, 117, and 123 and two more which initiate at approximate positions 147 and 159 from the 3′ end of the Small genome segment. The existence of multiple leader RNAs and mRNAs from the Small genome segment may allow access to both overlapping reading frames by ribosomes that still initiate only at 5′ proximal AUGs, without the need to splice the mRNA.     

The genes for small nucleolar RNAs in Trypanosoma brucei are organized in clusters and are transcribed as a polycistronic RNA

Because the organization of snoRNA genes in vertebrates, plants and yeast is diverse, we investigated the organization of snoRNA genes in a distantly related organism, Trypanosoma brucei. We have characterized the second example of a snoRNA gene cluster that is tandemly repeated in the T.brucei genome. The genes encoding the box C/D snoRNAs TBR12, TBR6, TBR4 and TBR2 make up the cluster. In a genomic organization unique to trypanosomes, there are at least four clusters of these four snoRNA genes tandemly repeated in the T.brucei genome. We show for the first time that the genes encoding snoRNAs in both this cluster and the SLA cluster are transcribed in an unusual way as a polycistronic RNA.     

Yeast H3 and H4 histone messenger RNAs are transcribed from two non-allelic gene sets

The genes coding for the H3 and H4 histones of Saccharomyces cerevisiae have been isolated by recombinant DNA cloning. The genes were detected in a bacteriophage lambda library of the yeast genome by hybridization with plasmids containing the cloned Psammechinus miliaris sea urchin histone genes (pCH7) and the cloned Drosophila histone genes (cDM500). Two non-allelic sets of the H3 and H4 genes have been isolated. Each set consists of one H3 gene and one H4 gene arranged as a divergently transcribed pair separated by an intergene spacer DNA. The histone genes were located on the cloned yeast fragments by S1 nuclease mapping, as was a gene (SMT1) of unknown function that does not code for a histone but is closely linked to one of the histone sets. Sequence homology between the two non-allelic sets is confined to the coding regions of the respective genes while the flanking DNA and intergene spacer DNA are extensively divergent. Cellular RNA homologous to the histone genes, including transcribed non-coding sequences unique to each of the four genes, was detected by S1 mapping, thus demonstrating that all four genes are transcribed in vegetative cells.     

Fibroblasts, glial, and neuronal cells are involved in extravascular prothrombin activation.

A membrane-associated prothrombin activator (MAPA) was found on various cultured cells derived from non-hematopoietic cells [Sekiya, F. et al. (1994) J. Biol. Chem. 269, 32441-32445]. In this study, we investigated the enzymatic properties of this enzyme using protease inhibitors. While the metalloproteinase inhibitor, o-phenanthroline, had no effect, some Kunitz type serine protease inhibitors attenuated MAPA activity. Recombinant tissue factor pathway inhibitor (rTFPI) also markedly reduced the activity (IC(50), 1. 3+/-0.6 x 10(-10) M). MAPA activity is, therefore, most likely to be due to factor Xa. We evaluated the effect of exogenous factor Xa on MAPA activity. Factor Xa-dependent prothrombin activation was observed on fibroblast cells (apparent K(d), 1.47+/-0.72 nM). Activation was also observed on glial and neuronal cells, which expressed MAPA activity. These results imply that membrane-bound factor Xa results in MAPA activity on these cells. Therefore, we considered the involvement of factor Va, a component of prothrombinase, in this activity. We examined whether or not the prothrombinase complex is assembled on these cells. Prothrombin was activated in a manner dependent on both exogenous factor Xa and factor Va (apparent K(d) of 0.51-1.81 nM for factor Va). These results indicate that the prothrombinase complex forms specifically on various extravascular cells. Although the prothrombinase complex can be assembled on monocytes and lymphocytes, it is not known why these cells can activate prothrombin specifically. These cells which have the capacity for prothrombin activator activity could also activate factor X; i.e. cells with factor X activation activity were able to convert prothrombin. These observations suggest that thrombin was generated via two procoagulant activities; factor X activation and subsequent prothrombinase complex formation on the surface of these cells. This mechanism may explain the various pathological states involving or resulting from extravascular thrombin and fibrin formation.     

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.     

Structure and expression of the rat insulin-like growth factor II (rIGF-II) gene. rIGF-II RNAs are transcribed from two promoters

Insulin-like growth factor II (IGF-II) is a mitogenic polypeptide present in rat plasma at high levels during fetal and early postnatal life and is believed to play an important, although as yet undefined, role in fetal development. Both in humans and rats, expression of the IGF-II gene results in the appearance of several mRNA species. In the present study, cDNA and synthetic oligonucleotide probes were used to isolate and characterize the rat IGF-II gene from genomic libraries. The rat IGF-II gene extends over 12 kilobase pairs and contains two 5'-noncoding exons and three protein-coding exons. The two 5' exons represent alternative 5' regions of different mRNA molecules and are expressed from two distinct promoters. The two promoters are transcribed with different efficiencies but exhibit similar tissue-specific expression and regulation with developmental age.