Analysis in Neisseria meningitidis and other Neisseria species of genes homologous to the FKBP immunophilin family

The immunophilin family of FK506-binding proteins (FKBPs), involved in eukaryotic protein folding and cell regulation, have recently been found to have prokaryotic homologues. Genes with sequences homologous to those encoding human FKBPs were examined in Neisseria species. An FKBP DNA sequence was present, as shown by the polymerase chain reaction and Southern blotting experiments, in the chromosome of Neisseria meningitidis (14 strains) and in all 11 different commensal Neisseria spp. studied, but was not found in Neisseria gonorrhoeae (11 strains tested) or in Moraxella catarrhalis. The nucleotide and predicted protein sequences of the FKBP-encoding domain from five of the meningococcal strains were highly conserved (e.g. ≥97% homologous). The meningococcal nucleotide sequence was ≥93% homologous and the consensus meningococcal protein sequence was ≥97% homologous to FKBP sequences found in seven different commensal Neisseria spp. The meningococcal nucleotide and predicted protein sequences were ≥59% homologous to the conserved C-terminus of the human FKBP gene family. The FKBP nucleotide sequence was present as a single copy in the chromosome of commensal Neisseria spp. and in most strains of N. meningitidis. The FKBP gene was linked to the silent pilin locus, pilS, in class II-piliated meningococcal strains. In meningococcal strains expressing class I pili, the FKBP gene was linked to one of several pilS loci but not the pilE locus present in these strains. FKBP genes found in commensal Neisseria spp. were not linked to known pilin loci.     

Analysis of the chromocenter DNA composition in polytene chromosomes of <Emphasis Type="Italic">Drosophila orena</Emphasis> ovarian nurse cells

Chromocenter DNA fragments of polytene chromosomes of Drosophila orena ovarian nurse cells were cloned from a region-specific library (Dore1) in a plasmid vector to yield 133 clones. A total of 76 clones were selected and sequenced. The total length of the sequenced fragments was 23940 bp. Analysis with several software packages revealed various repetitive sequences among the fragments of the Dore1 library, including mobile genetic elements (25 fragments homologous to various LTR retrotransposons, five fragments homologous to LINEs, three fragments homologous to Helitrons, one fragment homologous to Polinton, and one fragment homologous to the mini-me non-LTR retrotransposon), four minisatellites, a satellite (SAR_DM), the (TATATG)n simple sequence repeat, and a low-complexity T-rich repeat. Sequences homologous to protein-coding genes were also found in the Dore1 library. Various repetitive DNA sequences and gene homologs were identified as conserved sequences of pericentric heterochromatin of polytene chromosomes of ovarian nurse cells in nine species of the melanogaster species subgroup.     

Chromosomal locations of two DNA segments that flank ribosomal insertion-like sequences in Drosophila: Flanking sequences are mobile elements

We report the chromosomal locations of two repetitive DNA sequences that flank ribosomal insertion-like sequences in Drosophila melanogaster. The chromocentric region of D. melanogaster contains many copies of sequences that are homologous to type 1 ribosomal insertions. These insertion-like elements are interspersed with other DNA segments that we call flanking sequences. Two distinct flanking sequences derived from the same cloned DNA molecule pDmI 101, the HindIII fragments 101E and 101F, were studied. Whole genome Southern blots with DNA from the D. melanogaster stocks Oregon R (P2), gt-1, and gt-X11 showed complex restriction patterns that differed substantially between the three stocks. This and other data show that flanking sequences are members of diverged repetitive sequence families. In situ hybridization to salivary gland chromosomes of gt-1 and gt-X11 showed that both sequences are homologous to the chromocenter and to about 5 to 8 (101E) or 25 to 30 (101F) euchromatic sites in each stock. Most, if not all, of these sites differed in gt-1 and gt-X11. Both 101E and 101F are homologous to the chromocenter and very few euchromatic bands in D. simulans, but 101F is homologous to numerous bands in D. mauritiana. We conclude that the flanking sequences represented by 101E and 101F are mobile elements within the genome of Drosophila. These two sequences differ in several structural features from mobile DNA elements previously described in this organism.We dedicate this paper to Professor W. Beermann at the occasion of his 60th birthday     

Ab initio modeling of small proteins by iterative TASSER simulations

Background  

Predicting 3-dimensional protein structures from amino-acid sequences is an important unsolved problem in computational structural biology. The problem becomes relatively easier if close homologous proteins have been solved, as high-resolution models can be built by aligning target sequences to the solved homologous structures. However, for sequences without similar folds in the Protein Data Bank (PDB) library, the models have to be predicted from scratch. Progress in the ab initio structure modeling is slow. The aim of this study was to extend the TASSER (threading/assembly/refinement) method for the ab initio modeling and examine systemically its ability to fold small single-domain proteins.     

First Evidence for Homologous Recombination-mediated Large DNA Inversion on the Bacillus subtilis 168 Chromosome

A Bacillus subtilis 168 strain carrying an inversion of about 1600kb-long chromosomal DNA was isolated. Physical and genetic analyses demonstrated that the inversion was generated as a result of homologous recombination between two homologous sequences integrated at the met and leuB loci. This is the first clear evidence of a large stable chromosomal inversion induced by homologous recombination in B. subtilis.     

DNA probes for species identification of mosquitoes in the Anopheles gambiae complex

Abstract. Identification of species within the Anopheles gambiae Giles species complex is essential for the correct evaluation of malaria vector ecology studies and control programmes. The development of DNA probes to distinguish species of the An.gambiae complex is described. Genomic libraries were prepared for four members of the An.gambiae complex. These were screened using radiolabeled DNA from different species of An. gambiae sensu lato and a number of clones selected on the basis of their species specificity. These clones could be divided into two groups, each containing homologous sequences. Sequences homologous to group 1 inserts are highly reiterated in the genomes of Anopheles arabiensis Patton and Anopheles merus Dönitz, present in low copy number in Anopheles melas Theobald, but were not detected in Anopheles gambiae sensu stricto. Studies on the organization of this sequence in the genome of An.arabiensis show that homologous sequences are male specific and interspersed within the chromatin. Sequences homologous to group 2 inserts are highly repeated in the genomes of An.merus and An.melas, but present in low copy number in An.gambiae s.s. and An.arabiensis. Group 2 homologous sequences are not sex-specific in the species tested and appear to be tandemly repeated. When used as hybridization probes, these sequences provide a sensitive means for the identification of species within the Anopheles gambiae complex.     

Characterization and Comparative Analysis of DNA from the Pericentric Heterochromatin of Chromosome 2 of Anopheles atroparvus V. Tiel (Culicidae, Diptera)

The library containing DNA sequences from the diffuse pericentric heterochromatin from the right arm ofAnopheles atroparvus V. Tiel (Culicidae, Diptera) chromosome 2 (2R) was generated by use of chromosome microdissection technique. Southern-blot hybridization of the library fragments with the labeled genomic DNA of A. atroparvus and analysis of their primary structure showed that this heterochromatin region contained repeated DNA sequences differed by their primary structure and the number of copies. These were mostly AT-rich sequences harboring the features characteristic of the S/MAR regions. Based on the clones homology to the sequences from the A. gambiae and Drosophila melanogaster genomes, it was demonstrated that the pericentric heterochromatin from the right arm of A. atroparvus chromosome 2 contained gypsy-like transposable elements, as well as the sequences homologous to the structural genes. In situ hybridization with the chromosomes of A. atroparvus and of the two representatives of the Anopheles maculipennis species complex, A. messeae and A. beklemishevi, showed that pericentric regions of all these chromosomes contained DNA sequences homologous to the sequences from the region-specific library. Cloned fragments of conserved repetitive DNA revealed upon interspecific Southern-blot hybridization of the clones with the labeled genomic DNA of A. messeae can be utilized in further investigations of evolutionary rearrangements of the pericentric heterochromatin within the Anopheles maculipennis species complex.     

棉花DUR3基因的鉴定及进化分析

植物DUR3同源蛋白属于钠离子/溶质共运蛋白家族的尿素高亲和力运输蛋白，在植物体对外源尿素的主动吸收及内源尿素的再分配过程中具有重要作用。为明确棉花DUR3基因的结构和进化情况，基于生物信息学的方法，从全基因组水平鉴定陆地棉和雷蒙德氏棉的DUR3基因，并对基因结构、跨膜结构域、基序分布、进化关系等进行分析。结果表明：(1)从陆地棉A亚组和D亚组染色体各鉴定出1个DUR3基因，从雷蒙德氏棉基因组鉴定出1个DUR3基因。这3个棉花DUR3同源蛋白同其他植物DUR3同源蛋白一样，具有15个跨膜结构域，具有3个位置一致、高度保守的基序。(2)基因结构分析表明，双子叶植物DUR3基因的外显子个数明显多于单子叶植物，这3个棉花DUR3基因的外显子个数亦是如此。(3)根据物种间种属亲缘关系，对不同物种DUR3氨基酸序列构建的进化树显示，棉花的同双子叶植物的聚在一起。(4)DUR3直系同源基因和旁系同源基因的K_a/K_s比值普遍均大于1,说明这些基因在进化过程中主要受到正向选择的作用。该研究结果为深入研究棉花DUR3同源蛋白提供了理论基础。     

Localisation of reiterated nucleotide sequences in Drosophila and mouse by in situ hybridisation of complementary RNA

The location of highly reiterated nucleotide sequences on the chromosomes has been studied by the technique of in situ hybridisation between the DNA of either Drosophila melanogaster salivary gland chromosomes or mouse chromosomes and tritium labelled complementary RNA (c-RNA) transcribed in vitro from appropriate templates with the aid of DNA dependent RNA polymerase extracted from Micrococcus lysodeikticus. The location of the hybrid material was identified by autoradiography after RNase treatment. — When Drosophila c-RNA, transcribed from whole DNA, was annealed with homologous salivary chromosomes in the presence of formamide the well defined labelling was confined to the chromocentre. With heat instead of formamide denaturation there was evidence of discontinuous labelling in various chromosome regions as well, apparently associated with banding. Xenopus ribosomal RNA showed no evidence of annealing to Drosophila chromosomes with the comparatively short exposure times used here. — When mouse satellite DNA was used as template the resulting c-RNA showed no hybridisation to Drosophila chromosomes but, when annealed with mouse chromosomes, the centromeric regions were intensely labelled. The interphase nuclei showed several distinct regions of high activity which suggested aggregation of centromeric regions of both homologous and non-homologous chromosomes. The results of annealing either c-RNA or labelled satellite DNA to homologous chromosomes were virtually indistinguishable. Incubation of Drosophila c-RNA with mouse chromosomes provided no evidence of localisation of grains. — It is inferred that both in mouse and Drosophila the centromeric regions of all chromosomes are enriched in highly reiterated sequences. This may be a general phenomenon and it might be tentatively suggested that the highly reiterated sequences play some role in promoting the close physical approximation of homologous and non-homologous chromosomes or chromosome regions to facilitate regulation of function.     

十字花科多肽激素类基因RALF的同源基因克隆及进化分析

多肽激素类基因是对植物生长发育起重要作用的一类基因,RALF是其中的重要一员,而十字花科在中国蔬菜作物中是重要的一大类群。为了摸清十字花科多种蔬菜作物的RALF同源基因信息,该文根据前期研究从油菜中扩增到的多肽激素类基因RALFbn的序列设计引物,从提取的十字花科芸薹属、萝卜属、蔊菜属、山芥属的7份重要蔬菜作物的基因组DNA中分别克隆到了RALF的同源序列。结果表明:7种蔬菜作物的RALF同源基因编码区均在300 bp左右,且无内含子,编码的蛋白质由79个氨基酸组成,说明RALF在十字花科4个属内的保守性较强;对RALF同源基因在十字花科4个属中的表达分析表明,该类基因在根、茎、叶、花序轴等营养器官中不表达或弱表达,但主要在生殖器官中表达,其中在总花蕾和开放花中的表达量普遍高于嫩角果中的表达量,表明该类基因在十字花科中的生理活跃期是花发育时期。同时,构建了十字花科4个属中RALF同源基因的系统树,芸薹属的油菜、甘蓝和芥蓝形成一个分支,而茎瘤芥和分蘖芥形成另外一个分支,且与萝卜属、山芥属、蔊菜属的材料聚在一支,该基因的进化途径在一定程度上也反映出这几种作物的遗传背景关系。该研究结果丰富了RALF家族信息,增添了十字花科植物的分子进化数据。     

Comparative FISH analysis of C-positive regions of chromosomes of wood mice (Rodentia,Muridae, <Emphasis Type="Italic">Sylvaemus</Emphasis>)

The homology of DNA of C-positive centromeric regions of chromosomes in wood mice of the genus Sylvaemus (S. uralensis, S. fulvipectus, S. sylvaticus, S. flavicollis, and S. ponticus) was estimated for the first time. DNA probes were generated by microdissection from the centromeric regions of individual autosomes of each species, and their fluorescence in situ hybridization (FISH) with metaphase chromosomes of representatives of all studied wood mouse species was carried out. Unlike in the chromosomal forms and races of S. uralensis, changes in the DNA composition of the chromosomal centromeric regions in the wood mouse species of the genus Sylvaemus (including closely related S. flavicollis and S. ponticus) are both quantitative and qualitative. The patterns of FISH signals after in situ hybridization of the microdissection DNA probes with chromosomes of the species involved in the study demonstrate significant differences between C-positive regions of wood mouse chromosomes in the copy number and the level of homology of repetitive sequences as well as in the localization of homologous repetitive sequences. It was shown that C-positive regions of wood mouse chromosomes can contain both homologous and distinct sets of repetitive sequences. Regions enriched with homologous repeats were detected either directly in C-positive regions of individual chromosomes or only on the short arms of acrocentrics, or at the boundary of C-positive and C-negative regions.     

Isolation of resistance gene analogues to powdery mildew resistance sequences in hexaploid wheat

This paper reports the characterization of the powdery mildew resistance homologous genes family of Triticum aestivum. Using degenerate primer pair for wheat resistance genes, we have cloned seven 3′ truncated powdery mildew resistance gene homologous fragments Tpc5a, Tp25a, Tp25b, Tp3a5a, Tp3a5b, Tp4b5a and Tp4b5b. These fragments were sequenced. The deduced amino acid sequences showed that six of them have premature stop codons. All these sequences had a very high level of similarity to known Pm resistance genes such as Pm3a, Pm3b, Pm3d and pm3f in hexaploid wheat. By ignoring the stop codons in the sequences, their deduced protein sequences were of coiled-coil (CC)-nucleotide binding site (NBS)-leucine repeat rich (LRR) structure. These results suggest that there are many powdery mildew resistance gene analogues in both resistant and susceptible wheat. Among them, small insertion/deletion events and point mutations can result in the diversity of wheat Pm resistance homologous genes.     

Homologs of the Genes for Receptor-Like Kinase Proteins Conferring Plant Resistance to Pathogens: Resistance Gene Homologs in Solanum Species

Direct amplification of the genomic DNA from cultivated and wild Solanum species was used to synthesize three groups of NBS-LRR homologs of the genes which encode the pathogen-recognizing receptor-like serine/threonine kinases (RLK): (1) the NBS-kinase regions homologous to the arabidopsis RPS2 gene, the tobacco N gene, and the flax L6 gene (the corresponding GenBank accession nos. U14158, U15605, and U27081); (2) full-size sequences homologous to the Pto gene of Lycopersicon pimpinellifolium (AF220602); and (3) LRR regions homologous to potato genesGpa2/Rx1 (AJ249449 and AJ011801) and the tomato gene Mi1 (AF091048). The nucleotide and deduced amino acid sequences of the cloned fragments of the genes and pseudogenes were compared to the already known genes and their homologs within the family Solanaceae.     

Development and Application of PCR Primers for the Detection of the tfd Genes in Delftia acidovorans P4a Involved in the Degradation of 2,4‐D

Primers specific for the genes tfdD, tfdE and tfdF, derived from conserved amino acid sequence motifs of the corresponding homologous enzymes, and primers specific for the genes tfdA and tfdB as well as tfdC taken from the literature were applied in PCR reactions using the genomic DNA of Delftia acidovorans P4a as the template. PCR products were obtained with all primer pairs that were similar in size to those found with the genomic DNA of strains harbouring plasmid pJP4 as the carrier of tfd genes. The nucleotide sequences and the corresponding amino acid sequences of the PCR products obtained with Strain P4a were compared with the sequence databases. According to BLAST analyses, the partial sequences of tfdA and tfdB exhibited a 94–99% degree of identity with the homologous sequences of the 2,4‐D‐degrading strains Achromobacter xylosoxidans subsp. denitrificans EST4002 (pEST4011), Burkholderia sp. RASC, Variovorax paradoxus TV1 (pTV1) and Burkholderia cepacia 2a (pIJB1), whereas the partial sequences of the tfdC, tfdD, tfdE and tfdF genes revealed a 96–100% degree of identity with the homologous sequences of the chlorobenzene‐utilizing strains Ralstonia eutropha NH9 (pENH91), Pseudomonas chlororaphis RW71 and Pseudomonas sp. P51 (pP51).     

Cloning and nucleotide sequence of the Salmonella typhimurium pepM gene

Summary The pepM gene coding for a methionine-specific aminopeptidase was cloned from Salmonella typhimurium and its nucleotide sequence determined. The gene encoded a 264 amino acid protein that was homologous to a similar protein from Escherichia coli. The sequence of an overproducer mutant allele, pepM100, contained a single base change in the likely –35 region of the pepM promoter that increased its homology to the consensus promoter sequence. A region downstream from the pepM coding sequence contained extensive inverted repeats and was homologous to sequences found elsewhere in both Salmonella and other bacterial species.     

Functional and structural homology among regulatory cistrons of pili-adhesin determinants in Escherichia coli

Summary Expression of the digalactoside-binding Pap pili involves two trans-acting regulatory genes, papB and papI. Using pap-lac operon fusions and DNA hybridization probes derived from pap DNA we tested whether or not other pili-adhesin determinants from different Escherichia coli strains encode homologs to the pap regulatory genes. Digalactoside-specific clones of serotypes F7₂ and F11 complemented papB and papI mutants of the Pap (serotype F13) clone and DNA hybridization analysis showed that the clones are homologous in the DNA sequences encoding the two regulatory genes. Similar results were obtained with an S-pili determinant which mediates binding to sialic acid-containing receptors and the findings suggest that the regulatory regions may be more conserved than other genes in different pili-adhesin gene clusters. Determinants for type 1-pili (mannose-specific binding) and for pili associated with enterotoxigenic E. coli (K88, K99, CFAI, CFAII) did not appear to contain DNA sequences homologous to papB or papI. E. coli strain J96, which was the origin of the pap DNA, was found to carry two additional copies of papB-papI homologous sequences in the chromosome. In strains expressing more than one kind of pili the trans-active gene products thereby may allow for regulatory interaction between separate pili-adhesin gene systems.     

PCR-based gene targeting of the inducible nitric oxide synthase (NOS2) locus in murine ES cells,a new and more cost-effective approach

Gene targeting by double homologous recombination in murine embryonic stem (ES) cells is a powerful tool used to study the cellular consequences of specific genetic mutations. A typical targeting construct consists of a neomycin phosphotransferase (neo) gene flanked by genomic DNA fragments that are homologous to sequences in the target chromosomal locus. Homologous DNA fragments are typically cloned from a murine genomic DNA library. Here we describe an alternative approach whereby the inducible nitric oxide synthase (NOS2) gene locus is partially mapped and homologous DNA sequences obtained using a long-range PCR method. A 7 kb NOS2 amplicon is used to construct a targeting vector where theneo gene is flanked by PCR-derived homologous DNA sequences. The vector also includes a thymidine kinase (tk) negative-selectable marker gene. Following transfection into ES cells, the PCR-based targeting vector undergoes efficient homologous recombination into the NOS2 locus. Thus, PCR-based gene targeting can be a valuable alternative to the conventional cloning approach. It expedites the acquisition of homologous genomic DNA sequences and simplifies the construction of targeting plasmids by making use of defined cloning sites. This approach should result in substantial time and cost savings for appropriate homologous recombination projects.     

Characterization of six new loci within the swine major histocompatibility complex class III region

A search for new potential coding sequences was conducted within two overlapping cosmid genomic DNA clusters of about 170 and 45 kb from the swine major histocompatibility complex class III region. The sequences were detected with various probes, including pools of swine cDNA, homologous and heterologous genomic sequences, and synthetic oligonucleotides. The 170 kb cluster was centered on the tumor necrosis factor genes (TNF), and the 45 kb cluster contained the heat-shock protein 70 genes (HSP70). The TNF cluster revealed the presence of five new genes: lymphotoxin , BAT1, BAT2, BAT3, and a sequence related to DNA-binding factors. No sequence homologous to B144 was found in the TNF cluster, although other unidentified coding sequences may be present in this cluster. The HSP70 cluster contained a gene identified as BAT6, that is, tRNA-valyl synthetase. These results provide new evidence that the genomic maps of these various genes in the TNF and HSP70 sub-regions are similar in swine and human.