A prespore gene, Dd31, expressed during culmination of Dictyostelium discoideum

During culmination of Dictyostelium fruiting bodies, prespore and prestalk cells undergo terminal differentiation to form spores and a cellular stalk. A genomic fragment was isolated by random cloning that hybridizes to a 1.4-kb mRNA present during culmination. Cell type separations at culmination showed that the mRNA is present in prespore cells and spores, but not in prestalk or stalk cells. After genomic mapping, an additional 3 kb of DNA surrounding the original 1-kb fragment was cloned. The gene was sequenced and named Dd31 after the size of the predicted protein product in kilodaltons. Accumulation of Dd31 mRNA occurs immediately prior to sporulation. Addition of 20 mM 8-Br-cAMP to cells dissociated from Mexican hat stage culminants induced sporulation and the accumulation of Dd31 mRNA, while 20 mM cAMP did not. Dd31 mRNA does not accumulate in the homeotic mutant stalky in which prespore cells are converted to stalk cells rather than spores. Characterization of Dd31 extends the known temporal dependent sequence of molecular differentiations to sporulation.     

人端粒酶逆转录酶(hTERT)基因第3内含子的克隆和分析

从端粒酶活性呈阳性的永生细胞株人肺腺癌细胞SPC A 1中分离了总RNA ,以此为模板 ,结合RT PCR技术和长模板PCR技术 ,用hTERT基因特异性引物扩增到一长约 2 .2kb的cDNA片段。将该片段纯化后克隆到通用测序载体T easyvector上得到重组质粒。用测序引物SP6和T7对该片段进行部分双向测序。经序列分析和同源比较推测该片段包含了hTERT基因的第 3内含子。该结果提示了RT PCR技术和长模板PCR技术用于真核生物基因内含子克隆的可行性。进一步的分析表明 ,该片段在不同细胞的RT PCR产物中的产量不同 ,提示hTERT基因前体mRNA中的第 3内含子可能在不同细胞中有不同的剪接效率。     

Plasmid-borne cadmium resistance genes in Listeria monocytogenes are similar to cadA and cadC of Staphylococcus aureus and are induced by cadmium.

pLm74 is the smallest known plasmid in Listeria monocytogenes. It confers resistance to the toxic divalent cation cadmium. It contains a 3.1-kb EcoRI fragment which hybridizes with the cadAC genes of plasmid pI258 of Staphylococcus aureus. When introduced into cadmium-sensitive L. monocytogenes or Bacillus subtilis strains, this fragment conferred cadmium resistance. The DNA sequence of the 3.1-kb EcoRI fragment contains two open reading frames, cadA and cadC. The deduced amino acid sequences are similar to those of the cad operon of plasmid pI258 of S. aureus, known to prevent accumulation of Cd2+ in the bacteria by an ATPase efflux mechanism. The cadmium resistance determinant of L. monocytogenes does not confer zinc resistance, in contrast to the cadAC determinant of S. aureus, suggesting that the two resistance mechanisms are slightly different. Slot blot DNA-RNA hybridization analysis showed cadmium-inducible synthesis of L. monocytogenes cadAC RNA.     

Expression of the cytochrome b-URF6-URF5 region of the mouse mitochondrial genome

The nature of RNA coded by the only light-strand (L-strand) open-reading frame unidentified reading frame 6 (URF6) was studied by using a variety of single- and double-strand DNA subclones derived from the 3.6-kilobase (kb) cytochrome b (cyt b)-URF5 coding region of the mouse mitochondrial genome. Northern blot experiments using single-strand-specific M13 clones indicate that both the heavy (H) and L strands of this genomic region are symmetrically transcribed and processed into poly(adenylic acid) [poly(A)] RNAs of comparable size. The 1.2- and 2.4-kb RNAs coded by the H strand, putative mRNAs for cyt b and URF5 reading frames, respectively, are derived from a common precursor of 3.6-kb RNA. The L-strand-coded 1.15-kb RNA, on the other hand, is derived from a short-lived precursor of 3.6-kb RNA by a multiple-step processing involving a 2.4-kb intermediate RNA. The S1 nuclease protection experiments using both the 3'- or 5'-end-labeled DNA probes and also affinity-purified 32P-labeled RNA probes indicate that the 1.15-kb RNA maps between the start of the URF6 reading frame (3' end) and a region 590-600 nucleotides to the 5' end of this reading frame. The 1.15-kb RNA thus contains the entire URF6 coding sequence and an about 590-nucleotide-long 3' untranslated region. The molar abundance of the three mRNAs in the steady-state mitochondrial RNA varies markedly. The 1.15-kb URF6 mRNA is only one-tenth the level of 1.2-kb cyt b mRNA, although it is nearly as abundant as the 2.4-kb URF5 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Tetrahymena rRNA intron self-splices in E. coli: in vivo evidence for the importance of key base-paired regions of RNA for RNA enzyme function

We have developed an in vivo RNA splicing assay for the self-splicing rRNA intron of Tetrahymena thermophila using E. coli as the host. A DNA fragment containing the intron sequence has been cloned into M13mp83 so that expression of the beta-galactosidase alpha-fragment is dependent upon intron excision from the mRNA precursor. Plaque phenotypes correlate well with levels of excised intron RNA. Point mutations were made by oligonucleotide-directed mutagenesis in conserved sequences P, Q, and S. All showed reduced splicing, agreeing with mitochondrial genetic data for S and providing the first direct evidence that P and Q are functionally important. The results support the hypothesis that base-pairing of R with S and P with Q is important for intron structure and function.