5' -and 3'-terminal nucleotide sequences of Tetrahymena pyriformis 17S rRNA.

Ribonuclease T1 oligonucleotides arising from the 5' and 3' termini of the 17S rRNA of Tetrahymena pyriformis were isolated by the diagonal method of Dahlberg (Dahlberg, J. E. (1968), Nature (London) 220, 548), and their nucleotide sequences were determined. The base sequence of the 3'-terminal fragment is (G)AUCAUUAoh, which is identical to that found in other 17S-18S eucaryotic rRNA species. The nucleotide sequence of the 5'-terminal oligonucleotide is pAACCUGp, which is identical in length to that found in other eucaryotes and shows a partial but significant sequence homology to the 5' RNase TI oligonucleotides isolated from other eucaryotic species.     

基于线粒体CO 1基因序列的DNA条形码在鲤科鲌属鱼类物种鉴定中的应用

本研究探讨了线粒体CO1基因作为DNA条形码对鲌属鱼类进行物种鉴定的可行性。研究中获得了鲌属4种鱼类共32个个体长度为816bp的CO1基因序列。利用MEGA软件计算鲌属鱼类种间及种内遗传距离,利用邻接法、最大简约法、最大似然法和Bayesian方法分别构建分子系统树。结果显示,鲌属鱼类的种间遗传距离显著大于种内遗传距离。在系统树中,鲌属鱼类每一物种的个体分别形成各自独立的分支。基于CO1基因的DNA条形码在识别鲌属鱼类物种方面和传统形态学基本一致,而且该基因可以探讨鲌属鱼类种间的系统发育关系。本研究表明以CO1基因作为鲌属鱼类DNA条形码进行物种鉴定具有一定的可行性。     

Tetrahymena australis (Protozoa,Ciliophora): A Well‐Known But “Non‐Existing” Taxon – Consideration of Its Identification,Definition and Systematic Position

A cryptic species of the Tetrahymena pyriformis complex, Tetrahymena australis, has been known for a long time but never properly diagnosed based on taxonomic methods. The species name is thus invalid according to the International Code of Zoological Nomenclature. Recently, a population isolated from a freshwater lake in Wuhan, China was investigated using live observations, silver staining methods and gene sequence data. This organism can be separated from other described species of the T. pyriformis complex by its relatively small body size, the number of somatic kineties and differences in sequences of two genes, namely the small subunit ribosomal RNA (SSU rRNA) and the mitochondrial cytochrome c oxidase subunit I (cox1). We compared the SSU rRNA gene sequences of all available Tetrahymena species to reveal the nucleotide differences within this genus. The sequence of the Wuhan population is identical to two sequences of a previously isolated strain of T. australis (ATCC #30831). Phylogenetic analyses indicate that these three sequences (X56167, M98015, KT334373) cluster with Tetrahymena shanghaiensis (EF070256) in a polytomy. However, sequence divergence of the cox1 gene between the Wuhan population and another strain of T. australis (ATCC #30271) is 1.4%, suggesting that these may represent different subspecies.     

Leishmania major chromosome 3 contains two long convergent polycistronic gene clusters separated by a tRNA gene

Leishmania parasites (order Kinetoplastida, family Trypanosomatidae) cause a spectrum of human diseases ranging from asymptomatic to lethal. The ~33.6 Mb genome is distributed among 36 chromosome pairs that range in size from ~0.3 to 2.8 Mb. The complete nucleotide sequence of Leishmania major Friedlin chromosome 1 revealed 79 protein-coding genes organized into two divergent polycistronic gene clusters with the mRNAs transcribed towards the telomeres. We report here the complete nucleotide sequence of chromosome 3 (384 518 bp) and an analysis revealing 95 putative protein-coding ORFs. The ORFs are primarily organized into two large convergent polycistronic gene clusters (i.e. transcribed from the telomeres). In addition, a single gene at the left end is transcribed divergently towards the telomere, and a tRNA gene separates the two convergent gene clusters. Numerous genes have been identified, including those for metabolic enzymes, kinases, transporters, ribosomal proteins, spliceosome components, helicases, an RNA-binding protein and a DNA primase subunit.     

Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein.

The sequence of a 1,693-base-pair plasmid DNA fragment from Flavobacterium sp. strain ATCC 27551 containing the parathion hydrolase gene (opd) was determined. Within this sequence, there is only one open reading frame large enough to encode the 35,000-dalton membrane-associated hydrolase protein purified from Flavobacterium extracts. Amino-terminal sequence analysis of the purified Flavobacterium hydrolase demonstrated that serine is the amino-terminal residue of the hydrolase protein. The amino-terminal serine corresponds to a TCG codon located 87 base pairs downstream of the presumptive ATG initiation codon in the nucleotide sequence. The amino acid composition of the purified protein agrees well with that predicted from the nucleotide sequence, using serine as the amino-terminal residue. These data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp. Construction in Escherichia coli of a lacZ-opd gene fusion in which the first 33 amino-terminal residues of opd were replaced by the first 5 residues of lacZ resulted in the production of an active hydrolase identical in molecular mass to the hydrolase isolated from Flavobacterium sp. E. coli cells containing the lacZ-opd fusion showed higher levels of hydrolase activity than did cells containing the parent plasmid.     

猪链球菌2型的纤连蛋白/血纤维蛋白原结合蛋白基因的序列分析

目的 为确定猪链球菌2型(SS2)江苏分离株9801是否具有纤连蛋白／血纤维蛋白原结合蛋白基因(fbps)。方法 根据已发表的SS2 fbps序列,设计并合成1对引物,以SS2江苏分离株的基因组为模板,采用聚合酶链反应(PER)方法扩增fbps,克隆于pMD-T18载体。测定阳性质粒插入序列。利用DNAstar软件,比较所测序列与不同来源SS2的fbps和FBPS与链球菌属其他种同源蛋白的同源性。结果 扩增的fbps为1 938 bp,江苏分离株与猪源致病性SS2荷兰分离株的fbps序列有5个碱基不同,与ATCCA3765株有3个碱基不同,同源性均达99．99％以上。推导的氨基酸序列比较,分别有4个和2个氨基酸不同,同源性均为99％以上。纤连蛋白结合蛋白(FBPS)无前导序列,无锚定序列,与链球菌属其他种的同源Fn结合蛋白的同源性为68．8％～76．0％。结论 FBPS是一种无锚的黏附素。     

Nucleotide sequence of the split tRNAleu(UAA) gene from Sorghum bicolor chloroplasts

The nucleotide (nt) sequence of the split tRNAleu(UAA) gene and 328 nt of its flanking regions from sorghum chloroplasts (cp) has been determined. This gene is located in the BamHI-6 fragment in a map position very similar to that of maize. The exon of sorghum tRNAleu gene has an identical nt sequence to its counterpart in maize. Although the 450 nt of intron in sorghum is 8 nt shorter than that of maize, the nt sequence between them shows 97% homology. Like maize and broad bean, the intron from sorghum cp tRNAleu gene could be folded into a secondary structure which is similar to the postulated structure of the intron from the auto-spliceable rRNA precursor of Tetrahymena. Both introns from sorghum and maize contain open reading frames (ORFs) which are conserved at the N terminus. The putative AUG initiation codon for both ORFs is located in the stem region of a 12-bp secondary structure of highly A + T-rich sequences.     

Molecular cloning and sequence analysis of the mouse protein C inhibitor gene

The gene encoding mouse protein C inhibitor (mPCI) was isolated and its nucleotide sequence determined. Alignment of the genomic sequence with that of a cDNA obtained from mouse testis revealed that the mPCI gene (like the human counterpart) is composed of five exons and four introns with highly conserved exon/intron boundaries. It encodes a pre-polypeptide of 405 amino acids, which shows 63% identity with human PCI (hPCI). The putative reactive site is identical to that of hPCI from P5 to P3′, suggesting a similar protease specificity. Also the putative heparin binding sites and `hinge' regions are highly homologous in mouse and hPCI.     

Molecular cloning and nucleotide sequence of the gene for the ribosomal protein S11 from the archaebacterium Halobacterium marismortui

The gene encoding ribosomal protein S11 (Escherichia coli S15 homologue) from Halobacterium marismortui was cloned employing two synthetic oligonucleotide mixtures, 23 and 32 bases in length, as hybridization probes. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (1300 base pairs) were then determined by the dideoxy chain termination method. Comparison of the nucleotide sequence of the H. marismortui S11 gene with that of the E. coli S15 gene (rpsO) showed that the 3'-end of the S11 gene can be aligned with the entire E. coli S15 gene, sharing 44% identical nucleotides. It has been found that the S11 gene has a higher G + C content (G + C = 65%) than that of the E. coli S15 gene (G + C = 53%). This increase in G + C content specifically shows up as a preference for G + C in the 3rd position of the codon. Upstream of the S11 gene, an archaebacterial promoter sequence (GGACTTTCA) and a putative ribosomal binding site (GCGGT) have been found, 88 and 15 (or 24) base pairs from the initiation codon of the gene. In addition, an open reading frame could be identified immediately after the stop codon for the S11 gene. Northern blotting analysis using the S11 coding region as probe has shown that the S11 gene is located on a 2.4-kilobase mRNA, suggesting that it is cotranscribed with other downstream gene(s).     

Multiple copies of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium which possesses inducible bile acid 7-dehydroxylation activity. Several new polypeptides are produced in this strain following induction with cholic acid. Genes coding for two copies of a bile acid-inducible 27,000-dalton polypeptide (baiA1 and baiA2) have been previously cloned and sequenced. We now report on a gene coding for a third copy of this 27,000-dalton polypeptide (baiA3). The baiA3 gene has been cloned in lambda DASH on an 11.2-kilobase DNA fragment from a partial Sau3A digest of the Eubacterium DNA. DNA sequence analysis of the baiA3 gene revealed 100% homology with the baiA1 gene within the coding region of the 27,000-dalton polypeptides. The baiA2 gene shares 81% sequence identity with the other two genes at the nucleotide level. The flanking nucleotide sequences associated with the baiA1 and baiA3 genes are identical for 930 bases in the 5' direction from the initiation codon and for at least 325 bases in the 3' direction from the stop codon, including the putative promoter regions for the genes. An additional open reading frame (occupying from 621 to 648 bases, depending on the correct start codon) was found in the identical 5' regions associated with the baiA1 and baiA3 clones. The 5' sequence 930 bases upstream from the baiA1 and baiA3 genes was totally divergent. The baiA2 gene, which is part of a large bile acid-inducible operon, showed no homology with the other two genes either in the 5' or 3' direction from the polypeptide coding region, except for a 15-base-pair presumed ribosome-binding site in the 5' region. These studies strongly suggest that a gene duplication (baiA1 and baiA3) has occurred and is stably maintained in this bacterium.     

cDNA cloning of porcine transforming growth factor-beta 1 mRNAs. Evidence for alternate splicing and polyadenylation

Most eukaryotic cells encode principally a 2.5-kilobase (kb) transforming growth factor (TGF)-beta 1 mRNA. However, we have found two major TGF-beta 1 RNA species, 3.5 and 2.5 kb long, in porcine tissues. The 3.5-kb species has a longer 3'-untranslated sequence generated by the selection of an alternate polyadenylation site. There is a 117-nucleotide sequence within this unique 3' region, which is similar to the PRE-1 repetitive sequence of unknown function, reported earlier in the porcine genome. We have also cloned and characterized an alternately spliced mRNA species specific for the TGF-beta 1 gene, in which exons IV and V of the corresponding human TGF-beta 1 gene are deleted. The nucleotide sequence of this cDNA clone predicts a putative precursor protein of 256 amino acids; the N-terminal 211 amino acids of this putative protein are identical to the TGF-beta 1 precursor protein (exons I, II, and III of the human TGF-beta 1 gene), but the C-terminal 45 amino acids are distinct, due to a frameshift in the translation of exons VI and VII. In addition we provide data for the existence of other mRNA species generated in a tissue-specific manner either by alternate splicing or by heterogeneous 5' leader sequences.     

Proposal of Lysobacter pythonis sp. nov. isolated from royal pythons (Python regius)

The bacterial strains 4284/11T and 812/17 isolated from the respiratory tract of two royal pythons in 2011 and 2017, respectively were subjected to taxonomic characterization. The 16S rRNA gene sequences of the two strains were identical and showed highest sequence similarities to Lysobacter tolerans UM1^T (97.2%) and Luteimonas aestuarii DSM 19680^T (96.7 %). The two strains were identical in the sequences of the 16S-23S rRNA internal transcribed spacer (ITS) and partial groEL gene sequences and almost identical in genomic fingerprints. In the ITS sequence Ly. tolerans DSM 28473^T and in the groEL nucleotide sequence Luteimonas mephitis DSM 12574^T showed the highest similarity. In silico DDH analyses using genome sequence based ANIb and gANI similarity coefficients demonstrated that strain 4284/11^T represents a novel species and revealed Ly. tolerans UM1^T as the next relative (ANIb = 76.2 %, gANI = 78.0 %). Based on the topology of a core gene phylogeny strain 4284/11^T could be assigned to the genus Lysobacter. Chemotaxonomic characteristics including polyamine pattern, quinone system, polar lipid profile and fatty acid profile were in accordance with the characteristics of the genera Lysobacter and Luteimonas. Strains 4284/11^T and 812/17 could be differentiated from the type strains of the most closely related species by several physiological tests. In conclusion we are here proposing the novel species Lysobacter pythonis sp. nov. The type strain is 4284/11^T (= CCM 8829^T = CCUG 72164^T = LMG 30630^T) and strain 812/17 (CCM 8830) is a second strain of this species.     

Cloning of the mitochondrial genome of Rana catesbeiana and the nucleotide sequences of the ND2 and five tRNA genes

The entire mitochondrial genome of Rana catesbeiana was cloned into a plasmid vector pBR322 at the unique BamHI site and the nucleotide sequences of the ND2 gene and of its flanking genes were determined. The ND2 gene was encoded by 1,033 base pairs and, as deduced from the nucleotide sequence, the ND2 product consisted of 344 amino acids with a molecular weight of 37,561. This gene was flanked on the 5' side by the tRNA genes for isoleucine, glutamine, and methionine and on the 3' side by those for tryptophan and alanine. These genes were the same in their organization as those found in the mammalian and Xenopus laevis mitochondrial genomes. A comparison of the putative amino acid sequences of the ND2 proteins of different animal species revealed that six regions in the sequence were well conserved during evolution, suggesting that some of these conserved sequences are crucial for biological activity of the ND2 protein. The nucleotide sequence homologies between the five tRNA genes of R. catesbeiana and their counterparts of mammals and X. laevis were in the range of 55 to 85%, depending on the tRNA and animal species.     

Unique sequences in region VI of the flagellin gene of Salmonella typhi

The H1 (now renamed fliC; lino et al., 1988) alleles specifying antigenically different Salmonella flagellins are identical at their ends but differ greatly towards the middle, where there are two hypervariable segments (regions IV and VI). The flagellar antigen, d, of Salmonella typhi, is found also as phase-1 antigen in many other Salmonella species. We cloned the H1-d gene of a strain of S. typhi and determined the nucleotide sequence of its two hypervariable regions. Comparison with gene H1-d of Salmonella muenchen showed substantial differences in region VI: four scattered amino acid differences and ten adjacent amino acids in the inferred S. typhi sequence, all of which differ from the corresponding nine amino acids in the S. muenchen sequence. The results of polymerase chain reaction amplification indicated the presence of the S. typhi version in all of 18 additional S. typhi strains and the presence of the S. muenchen version in all four non-S. typhi species with flagellar antigen d. The difference in amino acid sequence in segment VI may be responsible for the minor serological differences between antigens d of S. typhi and antigen d of S. muenchen.