Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but lack a 5'-cap structure

Few genes in the divergent eukaryote Trichomonas vaginalis have introns, despite the unusually large gene repertoire of this human-infective parasite. These introns are characterized by extended conserved regulatory motifs at the 5' and 3' boundaries, a feature shared with another divergent eukaryote, Giardia lamblia, but not with metazoan introns. This unusual characteristic of T. vaginalis introns led us to examine spliceosomal small nuclear RNAs (snRNAs) predicted to mediate splicing reactions via interaction with intron motifs. Here we identify T. vaginalis U1, U2, U4, U5, and U6 snRNAs, present predictions of their secondary structures, and provide evidence for interaction between the U2/U6 snRNA complex and a T. vaginalis intron. Structural models predict that T. vaginalis snRNAs contain conserved sequences and motifs similar to those found in other examined eukaryotes. These data indicate that mechanisms of intron recognition as well as coordination of the two catalytic steps of splicing have been conserved throughout eukaryotic evolution. Unexpectedly, we found that T. vaginalis spliceosomal snRNAs lack the 5' trimethylguanosine cap typical of snRNAs and appear to possess unmodified 5' ends. Despite the lack of a cap structure, U1, U2, U4, and U5 genes are transcribed by RNA polymerase II, whereas the U6 gene is transcribed by RNA polymerase III.     

E5 open reading frame of bovine papillomavirus type 1 encodes a transforming gene.

We have previously shown that the early region of the bovine papillomavirus type 1 genome contains two nonoverlapping segments that can independently induce the morphological transformation of cultured cells. The transforming gene from the 5' end of the early region is encoded by the E6 open reading frame. The second transforming segment was previously localized to a 2.3-kilobase fragment (2.3T) from the 3' end of the early region. To determine which of the four open reading frames (E2, E3, E4, and E5) located within 2.3T encodes a transforming gene, we have now introduced a series of insertion and deletion mutations into a clone (pHLB1) in which 2.3T is activated by the Harvey viral long terminal repeat, and we tested the mutants for their ability to induce focal transformation. Our results indicate that the E5 open reading frame, which could encode a low-molecular-weight hydrophobic peptide, is required for pHLB1-induced transformation of NIH 3T3 cells, but that the E2, E3, and E4 open reading frames are not.     

A 25-bp ancient spliceosomal intron in the TvRab1a gene of Trichomonas vaginalis

Spliceosomal introns play a key role in eukaryotic genome evolution and protein diversity. A large Rab GTPase family has been identified in a unicellular eukaryote Trichomonas vaginalis. However, the characteristics of introns in Rab genes of T. vaginalis have not been investigated previously. In this study, we identified a 25-bp spliceosomal intron in the T. vaginalis Rab1a (TvRab1a) gene, the smallest intron in T. vaginalis to be characterized to date. This intron contains a canonical splice site at both 5' (GT) and 3' (AG) ends, and a putative branch-point sequence (TCTAAC) that matches the Trichomonad consensus sequence of ACTAAC except for the first nucleotide. The position and phase of the TvRab1a intron are evolutionarily conserved in Rab1 homologous genes across at least five eukaryotic supergroups, including Opisthokonta, Amoebozoa, Excavata, Chromalveolata, and Plantae. These results strongly suggest that the TvRab1a intron is likely to be an ancient spliceosomal intron, and it can therefore be used as a phylogenetic marker to evaluate particular eukaryotic groupings. Identification and characterization of the TvRabla intron may provide an insight into the evolution of the large Rab repertoire in T. vaginalis.     

赤麂SRY基因的测序及其与部分偶蹄目动物SRY基因序列的比较

采用人SRY基因的一段保守序列的引物,通过PCR在雄性赤麂中扩增出了赤麂SRY基因的特异片段,通过DNA斑点杂交证实其扩增产物与人SRY基因探针进行菌落杂交筛选出赤麂SRY基因的阳性克隆,并对其进行了,将其序列与基因库中录入的所有偶蹄目动物的SRY基因序列进行同源性比较,用UPGMA法构建了其系统进化树,从分类和进化上对赤麂SRY基因进行分析。     

'VIT1', a novel gene associated with vitiligo.

To define genes associated with the pigmentary disorder vitiligo, gene expression was compared in non-lesional melanocytes cultured from three vitiligo patients and from three control melanocyte cultures by differential display. A basic local alignment search tool search did not reveal homology of six differentially expressed cDNA fragments to previously identified expressed sequence tags; thus, one was used to screen a melanocyte cDNA library. The underlying VIT1 gene maps to chromosome 2p16. The 3' portion of the VIT1 message is complementary to the 3' end of hMSH6 mRNA, enabling the formation of RNA-RNA hybrids, which may interfere with G/T mismatch repair function. Moreover, the aligned cDNA sequence revealed an open reading frame identical to a hypothetical protein expressed in brain, with a similarity to Drosophila calmodulin, and containing a zinc-finger motif partially identical to N-recognin. Expression of ORF mRNA was confirmed for multiple skin cell types, suggesting its importance for skin physiology.     

Organization of the biosynthesis genes for the peptide antibiotic gramicidin S.

A recombinant bacteriophage containing the intact Bacillus brevis gene for gramicidin S synthetase 1, grsA, and the 5' end of the gramicidin S synthetase 2 gene, grsB, was identified by screening an EMBL3 library with anti-GrsA antibodies. This clone, EMBL315, has a 14-kilobase (kb) insert that hybridizes to the previously isolated 3.9-kb fragment of the grsB gene, which encodes the 155-kilodalton ornithine-activating domain of gramicidin S synthetase 2. Deletion and subcloning experiments with the 14-kb insert located the grsA structural gene and its putative promoter on a 4.5-kb PvuII fragment which encoded the full-length 120-kilodalton protein in Escherichia coli. In addition, hybridization analysis revealed that the 5' end of the grsB gene is located approximately 3 kb from the grsA structural gene. Furthermore, these studies indicated that grsA and grsB are transcribed in opposite orientations.     

The ATP-binding cassette proteins of the deep-branching protozoan parasite Trichomonas vaginalis

The ATP binding cassette (ABC) proteins are a family of membrane transporters and regulatory proteins responsible for diverse and critical cellular process in all organisms. To date, there has been no attempt to investigate this class of proteins in the infectious parasite Trichomonas vaginalis. We have utilized a combination of bioinformatics, gene sequence analysis, gene expression and confocal microscopy to investigate the ABC proteins of T. vaginalis. We demonstrate that, uniquely among eukaryotes, T. vaginalis possesses no intact full-length ABC transporters and has undergone a dramatic expansion of some ABC protein sub-families. Furthermore, we provide preliminary evidence that T. vaginalis is able to read through in-frame stop codons to express ABC transporter components from gene pairs in a head-to-tail orientation. Finally, with confocal microscopy we demonstrate the expression and endoplasmic reticulum localization of a number of T. vaginalis ABC transporters.     

Yeast DNA topoisomerase II is encoded by a single-copy, essential gene

The gene TOP2 encoding yeast topoisomerase II has been cloned by immunological screening of a yeast genomic library constructed in the phage lambda expression vector, lambda gt11. The ends of the message encoded by the cloned DNA fragment were delimited by the Berk and Sharp procedure (S1 nuclease mapping) for the 5' end and mapping of the polyA tail portion of a cDNA fragment for the 3' end. The predicted size of the message agrees with the length of the message as determined by Northern blot hybridization analysis. The identity of the gene was confirmed by expressing the gene in E. coli from the E. coli promoter lac UV5 to give catalytically active yeast DNA topoisomerase II. Disruption of one copy of the gene in a diploid yeast creates a recessive lethal mutation, indicating that the single DNA topoisomerase II gene of yeast has an essential function.     

Physical mapping and complete nucleotide sequence of the denV gene of bacteriophage T4.

Phage T4 deletion mutants that are folate analog resistant (far) and contain deletions in the region of the T4 genome near denV have been isolated previously. We showed that one of these mutants (T4farP12) expressed normal denV gene activity, whereas another mutant (T4farP13) was defective in the denV gene. The rII-distal (right) physical endpoints of these deletions defined the limits of the interval in which the rII-proximal (left) endpoint of the denV gene should be located. The deletion endpoints were identified by restriction and Southern hybridization analyses of phage derivatives containing deoxycytidine instead of hydroxymethyldeoxycytidine in their DNAs. The results of these analyses localized the rII-proximal (left) end of the denV gene to a region between 62.4 and 64.3 kilobases on the T4 physical map. denV+ phage resulted from marker rescue with two of five denV- alleles tested, using plasmids containing a 1.8-kilobase fragment from this region or a 179-base-pair terminal fragment derived from it. Sequencing of the 179-base-pair fragment from wild-type DNA showed a 130-base-pair open reading frame with its termination codon at the rII-proximal end. Confirmation that this open reading frame is part of the denV coding sequence was obtained by identifying a TAG amber codon in the homologous DNA derived from a denV amber mutant strain. This mutant strain rescued the denV+ allele from plasmids containing the wild-type sequence. An adjacent overlapping restriction fragment was also cloned, permitting determination of the remaining denV gene sequence. Based on these results, the 3' end of the coding region of the denV locus was mapped to kilobase position 64.07 on the T4 physical map, and the 5' end was mapped to position 64.48.     

Studies on the sequence of the 3'-terminal region of turnip-yellow-mosaic-virus RNA.

A fragment representing the 3'-terminal 'tRNA-like' region of turnip yellow mosaic (TYM) virus RNA has been purified following incubation of intact TYM virus RNA with Escherichia coli 'RNase P'. This fragment, which is 112+3-nucleotides long has been completely digested with T1 RNase and pancreatic RNase and all the oligonucleotides present in such digests have been sequenced using 32P-end labelling techniques in vitro. The TYM virus RNA fragment is free of modified nucleosides and does not contain a G-U-U-C-R sequence. Using nuclease P1 from Penicillium citrinum, the sequence of 26 nucleotides from the 5' end and 16 nucleotides from the 3' end of this fragment has been deduced. The nucleotide sequence at the 5' end of the TYM virus RNA fragment indicates that this fragment includes the end of the TYM virus coat protein gene.     

The nucleotide sequence at the 5'' end of foot and mouth disease virus RNA.

Foot and mouth disease virus RNA has been treated with RNase H in the presence of oligo (dG) specifically to digest the poly(C) tract which lies near the 5' end of the molecule (10). The short (S) fragment containing the 5' end of the RNA was separated from the remainder of the RNA (L fragment) by gel electrophoresis. RNA ligase mediated labelling of the 3' end of S fragment showed that the RNase H digestion gave rise to molecules that differed only in the number of cytidylic acid residues remaining at their 3' ends and did not leave the unique 3' end necessary for fast sequence analysis. As the 5' end of S fragment prepared form virus RNA is blocked by VPg, S fragment was prepared from virus specific messenger RNA which does not contain this protein. This RNA was labelled at the 5' end using polynucleotide kinase and the sequence of 70 nucleotides at the 5' end determined by partial enzyme digestion sequencing on polyacrylamide gels. Some of this sequence was confirmed from an analysis of the oligonucleotides derived by RNase T1 digestion of S fragment. The sequence obtained indicates that there is a stable hairpin loop at the 5' terminus of the RNA before an initiation codon 33 nucleotides from the 5' end. In addition, the RNase T1 analysis suggests that there are short repeated sequences in S fragment and that an eleven nucleotide inverted complementary repeat of a sequence near the 3' end of the RNA is present at the junction of S fragment and the poly(C) tract.     

人淋巴细胞CD2基因5’侧翼序列的克隆和鉴定

本文报告以ＣＤ２ｃＤＮＡ５’端的片段作探针,从人Ｔ淋巴细胞基因组文库筛选阳性重组克隆,经限制性内切酶降解和Ｓｏｕｔｈｅｒｎ杂交分析,证明其中一个阳性克隆的插入片段中含ＣＤ２基因５’侧翼顺序。经插入片段的亚克隆、限制性内切酶图谱及ＤＮＡ序列分析,鉴定出一含转录起始点及其上游序列的４．０ｋｂ片段。将此片段中含转录起始点和两个ＤＮ（ａｓｅ）Ⅰ高敏感位点的２．５ｋｂ片段定向克隆到以虫萤光素酶为报告基因的表达载体ｐＭＧ３中,并用限制性内切酶对此２．５ｋｂ片段作不同程度缺失,构成一系列突变子。这些重组的表达质粒转染人ＪｕｒｋａｔＴ细胞后,以瞬时表达实验分析各突变子驱动虫萤光素酶基因的表达,结果发现在ＣＤ２基因５’上游具有很弱的启动子活性,初步测定该启动子位于－１．２ｋｂ～－９８ｂｐ域。ＣＤ２基因具有弱启动子、强增强子的特点与Ｔ细胞表面其它抗原分子基因是相似的。