cis-acting sequences involved in exon selection in the chicken beta-tropomyosin gene.

The chicken beta-tropomyosin gene contains an internal pair of mutually exclusive exons (6A and 6B) that are selected in a tissue-specific manner. Exon 6A is incorporated in fibroblasts and smooth muscle cells, whereas exon 6B is skeletal muscle specific. In this study we show that two different regions in the intron between the two mutually exclusive exons are important for this specific selection in nonmuscle cells. Sequences in the 3' end of the intron have a negative effect in the recognition of the 3' splice site, while sequences in the 5' end of the intron have a positive effect in the recognition of the 5' splice site. First, sequences in exon 6B as well as in the intron upstream of exon 6B are both able to inhibit splicing when placed in a heterologous gene. The sequences in the polypyrimidine stretch region contribute to splicing inhibition of exons 5 or 6A to 6B through a mechanism independent of their implication in the previously described secondary structure around exon 6B. Second, we have identified a sequence of 30 nucleotides in the intron just downstream of exon 6A that is essential for the recognition of the 5' splice site of exon 6A. This is so even after introduction of a consensus sequence into the 5' splice site of this exon. Deletion of this sequence blocks splicing of exon 6A to 6B after formation of the presplicing complex. Taken together, these results suggest that both the mutually exclusive behavior and the choice between exons 6A and 6B of the chicken beta-tropomyosin gene are trans regulated.     

Evolutionary conservation of kinetochore protein sequences in plants

The evolutionary conservation of structural/functional kinetochore proteins has been studied on isolated nuclei and pro-/metaphase chromosomes of mono- and dicot plants. The cross-reactivities of antibodies against human CENPC, CENPE and CENPF, and against maize CENPCa with the centromeric regions of mitotic chromosomes of Vicia faba and/or Hordeum vulgare are shown. Putative homologs of the kinetochore protein SKP1 (suppressor of kinetochore protein 1p of yeast) were found in both species and of CBF5p (centromere binding factor 5 of yeast) in barley. Antibodies against synthetic peptides derived from partial sequences encoding these proteins were produced and recognized the centromeric regions on mitotic chromosomes as detected by indirect immunofluorescence.     

Variant chromatin structure of the oriP region of Epstein-Barr virus and regulation of EBER1 expression by upstream sequences and oriP

Most of the Epstein-Barr virus genome in latently infected cells is in a standard nucleosomal structure, but the region encompassing oriP and the Epstein-Barr virus-encoded small RNA (EBER) genes shows a distinctive pattern when digested with micrococcal nuclease. This pattern corresponds to a previously mapped nuclear matrix attachment region. Although the EBER genes are adjacent to oriP, there is only a two- to fourfold effect of oriP on EBER expression. However, sequences containing a consensus ATF site upstream of EBER1 are important for EBER1 expression.     

Molecular cloning of the 25 kbp region upstream of exon 0 of the human Ki-ras oncogene and its conservation in transformed mouse NIH 3T3 cells

Human sequences associated with the Ki-ras oncogene of the mammary tumour cell line, H-466B have been cloned from a tertiary NIH3T3 mouse transfectant. These sequences are located 5' upstream of exon 0 of the Ki-ras oncogene, span over 25 kbp of DNA and are conserved in half of the primary transfectants obtained with the Ki-ras gene of different types of tumours. No gross alterations were observed in the sequences upstream of the Ki-ras gene. The partial or total deletion of these sequences in the other half of primary transformants argues that they are not absolutely required for the transforming activity of the Ki-ras oncogene. The even distribution of the human-mouse junction points in primary transformed mouse cells suggests the absence of a specific region of recombination in the 5' flanking region of Ki-ras.     

cis-acting translational effects of the 5'' noncoding region of c-myc mRNA.

We have previously shown that the 5' noncoding region of mouse c-myc mRNA has a negative effect on translational efficiency in a rabbit reticulocyte lysate (A. Darveau, J. Pelletier, and N. Sonenberg, Proc. Natl. Acad. Sci. USA 82:2315-2319, 1985). We wanted to localize and characterize the inhibitory translational element(s) in the mRNA and to study its effect in other in vitro and in vivo systems. Here we report that the restrictive element is confined to a 240-nucleotide sequence of the 5' noncoding region of mouse c-myc mRNA and that this sequence acts in cis to inhibit the translation of a heterologous mRNA. In addition, we report that the cis-inhibitory effect is also exhibited in microinjected Xenopus oocytes and wheat-germ extracts but not in HeLa cell extracts. Transfection of corresponding plasmid DNA constructs into several established cell lines did not produce the cis-inhibitory effect. A model to explain these results is presented.     

Evolutionary conservation of the human 7 S RNA sequences

The cloning and characterization of the cytoplasmic 7 S RNAs of HeLa cells has provided pure probes to study the organization of the corresponding genomic DNA sequences. Such analysis has shown that the 7 S L and K RNAs are derived from families of middle repetitive DNA (Ullu & Melli, 1982; Ullu et al., 1982). In this work we analyze the evolutionary conservation of these sequences in the RNA and DNA of distantly related species. Hybridization of the 7 S recombinants to the RNA of rodents, birds, amphibians and echinoderms suggests high conservation of these sequences throughout evolution. Southern blot analysis of genomic DNAs from the same species shows the presence of families of repeated sequences homologous to the 7 S recombinants and Alu DNAs in the genomes of the same species. We were unable to hybridize the 7 S probes to the RNAs of Drosophila melanogaster or Dictyostelium discoideum, although sequence(s) homologous to the 7 S L probe were found in the genome of D. discoideum and to both 7 S L and K probes in the genome of D. melanogaster.     

Evolutionary conservation of homeodomain-binding sites and other sequences upstream and within the major transcription unit of the Drosophila segmentation gene engrailed.

The engrailed (en) gene functions throughout Drosophila development and is expressed in a succession of intricate spatial patterns as development proceeds. Normal en function relies on an extremely large cis-acting regulatory region (70 kilobases). We are using evolutionary conservation to help identify en sequences important in regulating patterned expression. Sequence comparison of 2.6 kilobases upstream of the en coding region of D. melanogaster and D. virilis (estimated divergence time, 60 million years) showed that 30% of this DNA occurs in islands of near perfect sequence conservation. One of these conserved islands contains binding sites for homeodomain-containing proteins. It has been shown genetically that homeodomain-containing proteins regulate en expression. Our data suggested that this regulation may be direct. The remaining conserved islands may contain binding sites for other regulatory proteins.     

Truncation of exon 1 from the c-myc gene results in prolonged c-myc mRNa stability.

The human c-myc gene consists of three exons transcribed from two distinct promoters and the function of the first, noncoding exon is unknown. In COLO 320 cells, there co-exist normal and truncated (i.e., lacking exon 1) c-myc genes, both of which are transcribed. Studies on the turnover of c-myc mRNA show that the normal mRNA has an in vivo half-life of approximately 30 min which is approximately similar to the turnover time of the mRNA in lymphoblastoid cells. However, the truncated mRNA was found to be substantially more stable. This observation was also made with a Burkitt's lymphoma cell line which has a translocated, truncated c-myc gene. Therefore truncation of the c-myc gene can cause the mRNA to be more stable than the full size product suggesting that this can be a crucial factor in the activation of the c-myc oncogene, by exon 1 loss, in chromosomal translocation. The results also suggest a role for exon 1 in the c-myc mRNA degradative mechanism.     

Evolutionary conservation of DNA sequences expressed in sea urchin eggs and early embryos

Summary DNA sequence divergence measurements indicate thatStrongylocentrotus franciscanus is more distinct fromS. purpuratus andS. drobachiensis than these two species are from each other, in agreement with paleontological and morphological evidence. The evolutionary divergence of several classes of expressed DNA sequences was compared with that of total single-copy DNA. BetweenS. franciscanus andS. purpuratus the divergence of cDNA made from gastrula cytoplasmic poly(A)⁺ RNA is about half that of total single-copy DNA. Similar results were obtained for cDNA made from unfertilized egg poly(A)⁺ RNA. In contrast, sequences expressed in gastrula nuclear RNA have diverged almost as much as total single-copy DNA.