The amiloride resistance gene, car1, of Schizosaccharomyces pombe

Amiloride, an inhibitor of various sodium transporters, is toxic to Schizosaccharomyces pombe at low concentration in minimal but not in rich media. Amiloride-resistant mutants were isolated and shown to represent a new locus (car1 for changed amiloride resistance) on chromosome I. The carl gene was cloned and sequenced. Sequence analysis revealed an open reading frame of 526 amino acids with a predicted molecular weight of 58 545 Da. It has 52% hydrophobic residues and belongs to the class of 12-transmembrane-domain transport proteins. Gene disruption of carl results in increased amiloride resistance. earl has sequence similarity to proteins from Candida associated with resistance to benomyl, methotrexate and cycloheximide. No single physiologically identifiable component of sodium transport appeared to be lost. We propose that earl serves an uptake function, perhaps as a symport with an unknown substrate and this carrier may transport amiloride into the cell. Further, we suggest that amiloride toxicity at low concentrations is not due to its effect on sodium transport but, rather, depends on intracellular interference with an unknown biosynthetic pathway.     

Pseudomonas mosselii E1铁载体合成相关基因cysI的克隆与功能初步分析

从棉花根际分离的铁载体产生菌E1,其16SrDNA与Pseudomonas mosselii ATCCBAA-99的同源性为100%。采用三亲本杂交方法将携带转座子Tn5-1063的质粒pRL1063a导入E1中进行转座子插入诱变。利用CAS法,从1000个突变株中,筛选到一株铁载体合成缺失突变株E1-185。利用TAIL-PCR方法,扩增位于Tn5-1063两端的侧翼序列。测序结果表明,转座子插入到E1的cysI基因内。该基因与Pseudomonas entomophila L48的cysI同源性为96%,其CysI氨基酸序列相似性为97%。该基因与半胱氨酸的合成密切相关,而在加有半胱氨酸的CAS平板上,突变株恢复了铁载体产生能力,证明cysI在E1铁载体合成过程中具有重要作用。据推测,cysI可能与铁载体合成途径中关键蛋白acyl-S-PCPs的形成有关。     

Characterisation of the gene encoding a protective antigen from Babesia microti identified it as η subunit of chaperonin containing T-complex protein 1

Passive immunisations with a monoclonal antibody termed 1-5H showed a partial but significant inhibition of parasitaemia against Babesia microti challenge infection. By immunoscreening with 1-5H, a clone (termed p58 gene) was obtained from a cDNA expression library of B. microti and the complete nucleotide sequence was determined. A protein homology search showed significant amino acid identities to the η subunit of the chaperonin containing T-complex protein 1 (CCT) of human (59%), mouse (58%) and Plasmodium falciparum (62%). Genomic analyses indicated that the p58 gene is present as a single copy gene and contains a total of approximately 400-bp introns in the genome of B. microti. The mAb 1-5H recognised a 58-kDa protein of B. microti and was found to cross-react with a 60-kDa protein of Babesia rodhaini. These results suggest the possibility that the p58 protein is the CCT η subunit of B. microti and functions as a chaperonin.     

Cloning and characterization of a novel human TEKTIN1 gene

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. A new member of the tektin gene family was cloned from the human fetal brain cDNA library. We hence named it the human TEKTIN1 gene. TEKTIN1 cDNA consists of 1375 bp and has a putative open reading frame encoding 418 amino acids. The predicted protein is 48.3 kDa in size, and its amino acid sequence is 82% identical to that of the mouse, rat, and dog. One conserved peptide RPNVELCRD was observed at position number 323–331 of the amino acid sequence, which is a prominent feature of tektins and is likely to represent a functionally important protein domain. TEKTIN1 gene was mapped to the human chromosome 17 by BLAST search, and at least eight exons were found. Northern blot analysis indicated that TEKTIN1 was predominantly expressed in testis. By in-situ hybridization analysis, TEKTIN1 mRNA was localized to spermatocytes and round spermatids in the seminiferous tubules of the mouse testis, indicating that it may play a role in spermatogenesis.     

The Telomere-Binding Protein Taz1p as a Target for Modification by a SUMO-1 Homologue in Fission Yeast

In fission yeast (Schizosaccharomyces pombe) the homologue of the mammalian SUMO-1 ubiquitin-like modifier is encoded by the pmt3 gene. A two-hybrid screen using the telomere-binding protein Taz1p as bait identified Pmt3p as an interacting factor. In vitro experiments using purified components of the fission yeast Pmt3p modification system demonstrated that Taz1p could be modified directly by Pmt3p. The amino acid sequence of Taz1p contains a close match to the consensus modification site for SUMO-1, and a PEST sequence similar to those found in established SUMO-1 targets. Although previous experiments have identified an increase in telomere length as one consequence of the pmt3– genotype, we could not detect Pmt3p modification of Taz1p in protein extracts made from exponentially growing haploid cells or any effect of Pmt3p on the localization of GFP-Taz1p at discrete foci in the haploid cell nucleus.     

Cloning and characterization of the histidine kinase gene<Emphasis Type="Italic"> Dic1</Emphasis> from<Emphasis Type="Italic"> Cochliobolus heterostrophus</Emphasis> that confers dicarboximide resistance and osmotic adaptation

A gene for a putative two-component histidine kinase, which is homologous to os-1 from Neurospora crassa, was cloned and sequenced from the plant-pathogenic fungus Cochliobolus heterostrophus. The predicted protein possessed the conserved histidine kinase domain, the response regulator domain, and six tandem repeats of 92-amino-acids at the N-terminal end that are found in histidine kinases from other filamentous fungi. Introduction of the histidine kinase gene complemented the deficiency of the C. heterostrophus dic1 mutant, suggesting that the Dic1 gene product is a histidine kinase. Dic1 mutants are resistant to dicarboximide and phenylpyrrole fungicides, and they are sensitive to osmotic stress. We previously classified dic1 alleles into three types, based on their phenotypes. To explain the phenotypic differences among the dic1 mutant alleles, we cloned and sequenced the mutant dic1 genes and compared their sequences with that of the wild-type strain. Null mutants for Dic1, and mutants with a deletion or point mutation in the N-terminal repeat region, were highly sensitive to osmotic stress and highly resistant to both fungicides. A single amino acid change within the kinase domain or the regulator domain altered the sensitivity to osmotic stress and conferred moderate resistance to the fungicides. These results suggest that this predicted protein, especially its repeat region, has an important function in osmotic adaptation and fungicide resistance.Communicated by C. A. M. J. J. van den Hondel     

巨桉BRI1基因的克隆和表达分析

为了解BRI1基因在巨桉中的功能,采用PCR技术克隆了EgrBRI1基因,分析了EgrBRI1的生物信息学和亚细胞定位,并对EgrBRI1基因响应激素和胁迫的差异表达进行了分析。结果表明,EgrBRI1基因全长3 893 bp,编码1 197个氨基酸。EgrBRI1蛋白稳定,空间结构复杂,存在3个motifs,主要定位于细胞膜。茉莉酸甲酯和油菜素内酯(BR)处理后,EgrBRI1基因在叶片中的表达上升,而水杨酸处理则没有明显的变化。盐胁迫和冷胁迫下,EgrBRI1基因表达表现为先下降后上升的趋势。因此,EgrBRI1基因能快速对外施激素做出响应,并在巨桉抗逆方面发挥重要作用,这可能是通过对BR信号的响应来实现的。     

A second generation snp-derived Escherichia coli-Streptomyces shuttle expression vector that is generally transferable by conjugation

An Escherichia coli-Streptomyces shuttle vector (pJN100) was constructed, by inserting an origin of transfer (oriT), derived from the E. coli broad host range plasmid RK2, into pANT1202, a high-copy-number vector for gene expression in Streptomyces. The resulting conjugably transferable vector contains the pANT1202-derived SnpR (LysR-like protein) activated snpA promoter that drives strong heterologous expression of proteins. We initially demonstrated that plasmid pJN100 was transferred with high frequency (10(-5-7) exconjugants per recipient) into several Streptomyces strains that were refractory to transformation by other means. Plasmid pJN100 was also shown to be stable in E. coli and Streptomyces. We confirmed functional protein expression by using a pJN100 derivative to complement a mutant of Streptomyces griseus with a disrupted chromosomal copy of the gene nonM, a gene encoding an essential reductase in the nonactin biosynthesis gene cluster. High levels of protein expression were confirmed using Western blotting to assess the production of the serine esterase NonR, an enzyme responsible for nonactin resistance in the nonactin producer S. griseus.     

Identification and fine mapping of <Emphasis Type="Italic">Pi33</Emphasis>, the rice resistance gene corresponding to the Magnaporthe grisea avirulence gene <Emphasis Type="Italic">ACE1</Emphasis>

Rice blast disease is a major constraint for rice breeding. Nevertheless, the genetic basis of resistance remains poorly understood for most rice varieties, and new resistance genes remain to be identified. We identified the resistance gene corresponding to the cloned avirulence gene ACE1 using pairs of isogenic strains of Magnaporthe grisea differing only by their ACE1 allele. This resistance gene was mapped on the short arm of rice chromosome 8 using progenies from the crosses IR64 (resistant) × Azucena (susceptible) and Azucena × Bala (resistant). The isogenic strains also permitted the detection of this resistance gene in several rice varieties, including the differential isogenic line C101LAC. Allelism tests permitted us to distinguish this gene from two other resistance genes [Pi11 and Pi-29(t)] that are present on the short arm of chromosome 8. Segregation analysis in F₂ populations was in agreement with the existence of a single dominant gene, designated as Pi33. Finally, Pi33 was finely mapped between two molecular markers of the rice genetic map that are separated by a distance of 1.6 cM. Detection of Pi33 in different semi-dwarf indica varieties indicated that this gene could originate from either one or a few varieties.Communicated by D.J. Mackill     

盐穗木HcDmc1基因的克隆和表达分析

根据盐穗木盐胁迫下响应的转录组测序结果,克隆获得盐穗木DNA损伤修复基因的cDNA序列,其开放阅读框1 035bp,编码344个氨基酸,命名为HcDmc1。保守结构域分析显示,该基因编码的蛋白具有RecA蛋白家族典型的保守结构域;统进化树分析显示HcDmc1为独立的分支;亚细胞定位于细胞质,为无信号肽、不跨膜的稳定亲水性蛋白。实时荧光定量PCR分析表明,盐穗木在100mmol/L NaCl胁迫7d后,同化枝中HcDmc1基因表达迅速上调并达到最大值,约为对照组的6.58倍;在700mmol/L NaCl胁迫14d后根中HcDmc1基因表达最高,约为对照的1.79倍。研究表明,HcDmc1基因表达受盐胁迫诱导。     

Nucleotide sequence and regulated expression of the Salmonella fljA gene encoding a repressor of the phase 1 flagellin gene

Summary The nucleotide sequence of Salmonella abortus-equi fljA, which together with the phase 2 flagellin gene constitutes the fljBA operon and encodes the repressor for the phase 1 flagellin gene fliC, was determined. The repressor was predicted to be a basic protein consisting of 179 amino acid residues (M_r = 20419 Da) encoded by ORFII. This was confirmed by the fact that host fliC is repressed by plasmid-encoded ORFII, which indeed expresses a 20 kDa product as determined by urea SDS-polyacrylamide gel electrophoresis. An amino acid sequence capable of forming a helix-turn-helix type of structure was predicted in the C-terminal region of FljA. A rho-independent intercistronic terminator was detected between fljB and ftjA. Chloramphenicol acetyltransferase (CAT) assays of fusions indicated that the terminator is capable of reducing expression of fljA to the level of a few percent, relative to fljB in broth cultures and to 1 % in M9 glycerol cultures.     

三浅裂野牵牛ItSGT1基因克隆及分析

SGT1(suppressor of the G2 allele of skpl)是多种植物抗病基因介导的抗病信号途径中的重要元件.该研究利用RT-PCR和RACE方法克隆出甘薯近缘野生种三浅裂野牵牛的SGT1基因,命名为ItSGT1.该基因含有一个长度为1 087 bp的开放阅读框,编码361个氨基酸,分子量约为40.1 kD,等电点为5.05.Blast及多序列比对分析表明,该基因与其他植物中的SGT1具有较高的相似性,且具有SGT1蛋白典型的功能域结构,即TPR区、VR1区、CS区、VR2区和SGS区.Southern杂交结果显示,SGT1基因在三浅裂野牵牛基因组中是多拷贝基因.组织特异性表达分析表明,ItSGT1基因在三浅裂野牵牛的根、茎和叶中均有表达.     

Molecular cloning and expression of a novel CYP26 gene (cyp26d1) during zebrafish early development

Proper restriction of retinoid signaling by Cyp26s is essential for development of vertebrate embryos while inappropriate retinoid signaling can cause teratogenesis. Here, we report cloning and expression analysis of a novel cyp26 gene (cyp26d1) isolated from zebrafish. The predicted protein encoded by cyp26d1 consists of 554 amino acids. It exhibits 54% amino acid identity with human Cyp26C1, 50% with zebrafish Cyp26B1 and 38% with zebrafish Cyp26A1. Whole-mount in situ hybridization shows that cyp26d1 is first expressed in sphere stage, then disappears at 50% epiboly and resumes its expression at 75% epiboly. During segmentation period, cyp26d1 message is found at presumptive hindbrain. Double in situ hybridization with krox20 and cyp26d1 reveals that cyp26d1 is expressed in presumptive rhombomere 2-4 (r2-r4) at 2-somite stage. At 3-somite stage, cyp26d1 gene is expressed in r6 and pharyngeal arch (pa) one in addition to its expression at r2 and r4. At 6-somite stage, cyp26d1 message is present in continuous bands at r2-r6 and in pa1. This expression pattern is maintained from 10-somite stage through 21-somite stage except that the expression level is greatly reduced at r2 and r4. At 21-somite stage, cyp26d1 is also found in a group of cells in telencephalon and diencephalons. At 25-31h post-fertilization (hpf), the zebrafish cyp26d1 expression domain is extended to eyes, otic vesicles and midbrain in addition to its expression in hindbrain, telencephalon, diencephalons, and pharyngeal arches. At 35-48hpf, the expression of cyp26d1 is mainly restricted to otic vesicles, pharyngeal arches and pectoral fins and the expression level is greatly reduced.