Aberrant c-myc RNAs of Burkitt's lymphoma cells have longer half-lives.

BL67 and BL18 are Burkitt's lymphoma cell lines with t(8;14) translocations (the breakpoint is in the first exon and first intron, respectively) in which the mu-heavy chain switch region is fused to the c-myc gene in head to head orientation. In both cell lines only aberrant c-myc RNAs are found. BL67 cells contain two c-myc RNA species of 2.4 and 3.5 kb. The 2.4-kb RNA is initiated at several cryptic promoters in the first intron. The 3.5-kb RNA is transcribed from the immunoglobulin heavy chain anti-sense strand across the breakpoint of the translocation into the first exon of the c-myc gene and is then normally spliced using the physiological splice donor and acceptor sites of the c-myc gene. BL18 contains c-myc RNA of 2.4 kb initiated at cryptic promoters in the first intron and additional RNAs of 0.90 kb and 0.74 kb transcribed from the dual c-myc promoters on the reciprocal fragment of the translocation. The cytoplasmic turnover of these RNAs differs significantly from that of the normal c-myc message. The 3.5-kb RNA of BL67 cells and the 0.90-kb and 0.74-kb RNAs of BL18 cells, which are both hybrid molecules consisting of c-myc and immunoglobulin sequences, have a half-life of several hours in contrast to the normal c-myc message with a half-life of 15 min. The aberrant 2.4-kb c-myc RNAs of BL67 and BL18 cells are also more stable than the normal c-myc message and disappear with a half-life of 50 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Yeast pre-mRNA splicing requires a minimum distance between the 5'' splice site and the internal branch acceptor site.

We have generated several deletions within the intron of a yeast actin gene construct which have lead to different splicing efficiencies as measured by Northern blot (RNA blot) and primer extension analyses. Our data especially demonstrate that a minimum distance from the 5' splice site to the internal branch acceptor site is required for accurate and efficient splicing. In a construct in which splicing was completely abolished, splicing could be restored by expanding the distance from the 5' splice site to the internal branch acceptor site with heterologous sequences. Alternative splicing, i.e., exon skipping and the use of a cryptic 5' splice site, was observed when the mRNA precursor was derived from a tandem repeat of a truncated intron with flanking exon sequences.     

Burkitt lymphoma cell line carrying a variant translocation creates new DNA at the breakpoint and violates the hierarchy of immunoglobulin gene rearrangement.

The Burkitt lymphoma cell line KK124, which contains a reciprocal t(8;22) translocation, was shown to have rearranged in a region 3' to the c-myc proto-oncogene on chromosome 8 and 5' to the lambda constant region on chromosome 22. The breakpoint was cloned and sequenced, revealing that c-myc and a portion of its 3' region abutted a complete lambda variable gene that had undergone V-J recombination. Since this cell line expresses kappa light chain, this lambda rearrangement violates the previously proposed hierarchy of immunoglobulin gene rearrangement. A novel duplication of normal chromosome 8 sequences was also found at the breakpoint. The first exon of c-myc and its flanking sequence from the translocated allele was sequenced and compared with a normal counterpart. Extensive mutation was found within the first exon in contrast to its 3' and 5' flanking regions. S1 nuclease analysis revealed that it was the translocated c-myc being expressed and that there was a promoter shift from P2 to P1. The detailed structural analysis of this cell line provides clues concerning mechanisms of chromosomal translocation and c-myc deregulation in Burkitt lymphomas.     

Clustered somatic mutations in and around first exon of non-rearranged c-myc in Burkitt lymphoma with t(8;22) translocation.

We have examined the restriction map of the c-myc gene in 15 BL cell lines carrying the variant t(8;22) translocation in which c-myc is known to remain on chromosome 8. Using 3 restriction enzymes cutting outside the c-myc domain (EcoRI, BamHI, HindIII), we found no evidence for a c-myc/Ig lambda rearrangement in 14 BL cell lines. In the last one, BL 37, the 3' flanking region was rearranged corresponding to the already identified breakpoint located 400 pb downstream from the c-myc gene (9). Using 4 restriction enzymes cutting inside the c-myc gene (PvuII, PstI, SacI, HincII) we looked for discrete abnormalities within the gene limits, and we found in 9 BL cell lines several abolished and created sites, compatible with multiple independent somatic mutations. They are significantly clustered in the 5' non coding region, with a striking prevalence at the end of exon 1. The role of mutations in the non-coding first exon region for the deregulation of c-myc expression is discussed.     

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.     

The green fluorescent protein gene functions as a reporter of gene expression in Phanerochaete chrysosporium

The enhanced green fluorescent protein (GFP) gene (egfp) was used as a reporter of gene expression driven by the glyceraldehyde-p-dehydrogenase (gpd) gene promoter and the manganese peroxidase isozyme 1 (mnp1) gene promoter in Phanerochaete chrysosporium. Four different constructs were prepared. pUGGM3' and pUGiGM3' contain the P. chrysosporium gpd promoter fused upstream of the egfp coding region, and pUMGM3' and pUMiGM3' contain the P. chrysosporium mnp1 promoter fused upstream of the egfp gene. In all constructs, the egfp gene was followed by the mnp1 gene 3' untranslated region. In pUGGM3' and pUMGM3', the promoters were fused directly with egfp, whereas in pUGiGM3' and pUMiGM3', following the promoters, the first exon (6 bp), the first intron (55 bp), and part of the second exon (9 bp) of the gpd gene were inserted at the 5' end of the egfp gene. All constructs were ligated into a plasmid containing the ura1 gene of Schizophyllum commune as a selectable marker and were used to transform a Ural1 auxotrophic strain of P. chrysosporium to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. The transformants containing a construct with an intron 5' of the egfp gene (pUGiGM3' and pUMiGM3') exhibited maximal fluorescence under the appropriate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that the insertion of a 5' intron resulted in more egfp RNA than was found in transformants carrying an intronless egfp. These results suggest that the presence of a 5' intron affects the expression of the egfp gene in P. chrysosporium. The expression of GFP in the transformants carrying pUMiGM3' paralled the expression of endogenous mnp with respect to nitrogen and Mn levels, suggesting that this construct will be useful in studying cis-acting elements in the mnp1 gene promoter.     

Cryptic intron activation within the large exon of the mouse polymeric immunoglobulin receptor gene: cryptic splice sites correspond to protein domain boundaries.

The fourth exon of the mouse polymeric immuno-globulin receptor (pIgR) is 654 nt long and, despite being surrounded by large introns, is constitutively spliced into the mRNA. Deletion of an 84 nt sequence from this exon strongly activated both cryptic 5' and 3' splice sites surrounding a 78 nt cryptic intron. The 84 nt deletion is just upstream of the cryptic 3' splice site; the cryptic 3' splice site was likely activated because the deletion created a better 3' splice site. However, the cryptic 5' splice site was also required to activate the cryptic splice reaction; point mutations in either of the cryptic splice sites that decreased their match to the consensus splice site sequence inactivated the cryptic splice reaction. The activation and inactivation of these cryptic splice sites as a pair suggests that they are being co-recognized by the splicing machinery. Interestingly, the large fourth exon of the pIgR gene encodes two immunoglobulin-like extracellular protein domains; the cryptic 3' splice site coincides with the junction between these protein domains. The cryptic 5' splice site is located between protein subdomains where an intron is found in another gene of the immunoglobulin superfamily.