Molecular mechanisms of deletion formation in Escherichia coli plasmids. I. Deletion formation mediated by long direct repeats.

Summary Derivatives of plasmid pBR327 with the tet gene interrupted by 165 pb or 401 by direct repeats were constructed. In cells harboring these plasmids, deletions which restored the wild-type tet gene gave rise to tetracycline-resistant colonies, thereby allowing a simple phenotypic test for deletion formation. The frequencies of deletions in these plasmids were measured in Escherichia coli strains proficient or deficient in general recombination. The structure of plasmid DNA isolated from tetracycline-resistant transformants was analyzed by agarose gel electrophoresis, restriction mapping and sequencing. The data presented here demonstrate that deletion formation is always associated with dimerization of plasmid DNA. Dimeric plasmids were of two types. Those which carried both a deletion and a compensating duplication were the major type in a Rec⁺ background and were rare in recA, recF, recJ and recO backgrounds. Dimers of the second type contained deletions, but no compensating duplications, and their formation was RecA-independent. The data presented demonstrate that deletion formation mediated by long direct repeats is mainly the result of unequal crossing-over between two plasmid molecules.     

Mating systems of Cuphea laminuligera and Cuphea lutea

Summary A series of RFLP and isozyme markers were followed in the progenies of two alien addition lines of Brassica campestris-oleracea. One of the lines, carrying the C genome chromosome 4 as the alien chromosome, was surveyed for six markers. Fifty-four percent of the plants carrying alien chromosomes displayed all the expected makers, whereas the rest had one to five markers missing. The second line for C genome chromosome 5 displayed a similar behavior when surveyed for three markers. All three markers were transmitted together in 46% of the plants carrying alien chromosomes, whereas the rest carried only one or two of the markers. The loss of markers was associated with reduced chromosome size caused by deletions. The observed chromosome deficiencies permitted deletion analysis for a rough physical mapping and ordering of the markers on the two C genome chromosomes. The deletions observed may represent another mechanism for molding the chromosomes of the Brassica genomes during their evolution.     

Plasmids in carboxydotrophic bacteria: physical and restriction analysis

Twenty species and strains of aerobic CO-oxidizing bacteria were screened for the occurrence of plasmids. Six of them harbored plasmids between 45 and 558kb. Megaplasmids of 428 and 558 kb were resolved in Alcaligenes carboxydus. Restriction digest patterns of plasmids from different carboxydotrophic bacteria were dissimilar. However, the patterns obtained with the plasmids from the strains OM5, OM4 and OM2 of Pseudomonas carboxydovorans were very much the same. The nine cured mutants of P. carboxydovorans OM5, as well as the deletion mutant OM5-29, could not grow chemolithotrophically with CO or H₂ plus CO₂, as they were devoid of CO dehydrogenase, hydrogenase and ribulose bisphosphate carboxylase. The deletion mutant OM5-24 retained the ability to grow with CO. It could not grow with H₂ plus CO₂ and was devoid of H₂ase. The data suggest the residence of structural and/or regulatory genes of CODH, H₂ase and RuBPCx on plasmid pHCG3 of P. carboxydovorans.Abbreviations CODH carbon monoxide dehydrogenase - CRM cross reacting material - EMS ethyl methane sulfonate - H₂ase hydrogenase - kb kilobase - NTG N-methyl-N-nitro-N-nitrosoguanidine - RuBPCx ribulose bisphosphate carboxylase - SDS sodium dodecylsulfate     

On the toleration of duplications and deletions by the Vicia faba genome

Summary From eight pairs of crosses between differently reconstructed diploid karyotypes of Vicia faba, the progeny after selfing of plants heterozygous for both parental chromosome reconstructions were inspected for occurrence and transmission of duplications and deletions of defined chromosome segments, comprising together about one third of the metaphase genome length. The duplications and deletions studied involved either one or more chromosome segments of the respective karyotype (0.8%–9.1% of the metaphase length). They arose during meiosis in double heterozygotes by crossing over between partially homologous chromosomes or by mis-segregation from multivalents. While most duplications, provided they were not accompanied by deletions and in dependence on the segment involved, were viable and transmissible, even in homozygous state, deletions had lethal effects on gametes of both sexes.     

Mechanisms of deletion formation in Escherichia coli plasmids. II. Deletions mediated by short direct repeats.

Summary A set of plasmids containing 42, 21 and 13 bp direct repeats was used to analyze the effect of repeat length on the frequencies of deletion formation and the structure of the deleted derivatives of different recombination-deficient Escherichia coli strains. Agarose gel electrophoresis of plasmid DNA demonstrated that the formation of deletions in these plasmids was associated with dimerization of plasmid DNA. Restriction analysis of the dimers showed that deletions at short direct repeats arose non-conservatively, that is, the formation of a deletion in one monomeric plasmid unit was not associated with a duplication in the other. Mutations in the recA, recF, recJ and recO genes had no marked effect on either the frequencies of deletion formation or the structure of dimers. In contrast, recB recC mutations greatly increased the frequencies of deletion formation, 6-fold for 42 bp, and 115-fold for 21 by direct repeats. Conversion of DNA replication to the rolling circle mode in a recB recC strain, resulting in the formation of double-stranded ends, is suggested as the stimulatory effector.     

Structural changes in the plastid DNA of rice (Oryza sativa L.) during tissue culture

To investigate the rearrangement of the plastid genome during tissue culture, DNA from rice callus lines, which had been derived individually from single protoplasts isolated from seed or pollen callus (protoclones), was analyzed by Southern hybridization with rice chloroplast DNA (ctDNA) clones as probes. Among 44 long-term cultured protoclones, maintained for 4, 8 or 11 years, 28 contained plastid DNA (ptDNA) from which portions had been deleted. The ptDNA of all protoclones that had been maintained for 11 years had a deletion that covered a large region of the plastid genome. The deletions could be classified into 15 types from their respective sizes and positions. By contrast, no deletions were found in the ptDNA of 38 protoclones that had been maintained for only 1 month. These results indicate that long-term culture causes deletions in the plastid genome. Detailed hybridization experiments revealed that plastid genomes with deletions in several protoclones were organized as head-to-head or tail-to-tail structures. Furthermore, ptDNAs retained during long-term culture all had a common terminus at one end, where extensive rearrangement is known to have occurred during the speciation of rice and tobacco. Morphological analysis revealed the accumulation of starch granules in plastids and amyloplasts in protoclones in which the plastid genome had undergone deletion. Our observations indicated that novel structural changes in the plastid genome and morphological changes in the plastid had occurred in rice cells during long-term tissue culture. Moreover, the morphological changes in plastids were associated with deletions in the plastid genome.     

Rapid sitrapid site-directed domain scanning mutagenesis of enteropathogenic <Emphasis Type="Italic">Escherichia coli espD</Emphasis>

We developed a rapid mutagenesis method based on a modification of the QuikChange® system (Stratagene) to systemically replace endogenous gene sequences with a unique similar size sequence tag. The modifications are as follows: 1: the length of the anchoring homologous sequences of both mutagenesis primers were increased to 16 – 22 bp to achieve melting temperatures greater than 80°C. 2: the final concentrations of both primers were increased to 5–10 ng/µl and the final concentration of template to 1–2 ng/µl. 3: the annealing temperature was adjusted when necessary from 52°C to 58°C. We generated 25 sequential mutants in the cloned espD gene (1.2 kb), which encodes an essential component of the type III secretion translocon required for the pathogenesis of enteropathogenic E. coli (EPEC) infection. Each mutation consisted of the replacement of 15 codons (45 bp) with 8 codons representing a 24 bp sequence containing three unique restriction endonuclease sites (KpnI/MfeI/SpeI) starting from the second codon. The insertion of the restriction endonuclease sites provides a convenient method for further insertions of purification and/or epitope tags into permissive domains. This method is rapid, site-directed and allows for the systematic creation of mutants evenly distributed throughout the entire gene of interest.     

结核分枝杆菌毒素-抗毒素系统 maz EF6缺失突变株的构建及其表型的初步探讨

为构建结核分枝杆菌毒素‐抗毒素系统 m azEF6缺失突变株,并对其表型进行初步探讨,首先用聚合酶链反应（PCR）分别从H37Rv标准株和PUC‐19K质粒扩增出 mazEF6基因的同源臂及卡那霉素抗性基因kan ;然后应用融合PCR技术将 mazEF6基因的同源臂与 kan基因进行杂交拼接,获得目的融合片段,将该融合片段克隆于pMD‐19T（simple）载体形成自杀质粒pMD‐19T‐ΔmazEF6‐kan ,并将自杀质粒转化至大肠埃希菌DH5α中;最后利用电穿孔技术将自杀质粒电转至H37Rv标准株中,在卡那霉素抗性改良罗氏培养基上筛选H37Rv ΔmazEF6缺失突变株单个菌落,提取阳性菌株全基因组DNA为模板,PCR扩增克隆片段并测序。将所获得的H37Rv ΔmazEF6缺失突变株进行遗传稳定性检测后,对其表型进行初步研究。结果显示,该缺失株在15代内未发生回复性突变;与野生株相比,缺失株生长速度缓慢且细菌形态短小。本研究证实,融合PCR技术便于快速获得结核分枝杆菌缺失突变株;结核分枝杆菌在缺失毒素‐抗毒素系统 m azEF6基因后生存能力下降,这为进一步研究毒素‐抗毒素系统的作用奠定了基础。     

Excision of Trpv6 gene leads to severe defects in epididymal Ca2+ absorption and male fertility much like single D541A pore mutation

Replacement of aspartate residue 541 by alanine (D541A) in the pore of TRPV6 channels in mice disrupts Ca(2+) absorption by the epididymal epithelium, resulting in abnormally high Ca(2+) concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilization capacity, raising the possibility of residual activity of channels formed by TRPV6(D541A) proteins (Weissgerber, P., Kriebs, U., Tsvilovskyy, V., Olausson, J., Kretz, O., Stoerger, C., Vennekens, R., Wissenbach, U., Middendorff, R., Flockerzi, V., and Freichel, M. (2011) Sci. Signal. 4, ra27). It is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared with the deletion of the corresponding protein. Therefore, we generated TRPV6-deficient mice (Trpv6(-/-)) by deleting exons encoding transmembrane domains with the pore-forming region and the complete cytosolic C terminus harboring binding sites for TRPV6-associated proteins that regulate its activity and plasma membrane anchoring. Using this strategy, we aimed to determine whether the TRPV6(D541A) pore mutant still contributes to residual channel activity and/or channel-independent functions in vivo. Trpv6(-/-) males reveal severe defects in fertility and motility and viability of sperm and a significant increase in epididymal luminal Ca(2+) concentration that is mirrored by a lack of Ca(2+) uptake by the epididymal epithelium. Therewith, Trpv6 excision affects epididymal Ca(2+) handling and male fertility to the same extent as the introduction of the D541A pore mutation, arguing against residual functions of the TRPV6(D541A) pore mutant in epididymal epithelial cells.     

高致病性猪繁殖与呼吸综合征病毒TJ株致弱过程中遗传变异和致病性分析

为了研制高致病性猪繁殖与呼吸综合征(HP-PRRS)弱毒疫苗,将高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)TJ株进行了致弱驯化,在Marc-145细胞上对其进行了连续传代,每5～10代进行噬斑克隆纯化病毒。对致弱过程中不同代次病毒进行遗传变异及致病性分析。结果表明,TJ株在致弱过程中各基因均存在不同程度的变异,至第140代,共有58个氨基酸发生突变,同时在非结构蛋白nsp2区域,在不连续的30个氨基酸缺失(481位和533～561位)之后又出现连续120个氨基酸的缺失,与VR-2332相比,该缺失位点位于推定氨基酸序列的628～747位。动物接种试验结果表明,TJ株经Marc-145细胞传至第20代时,病毒对猪的致病性明显减弱,推测TJ株在这一传代过程中非结构蛋白nsp2-nsp5、nsp7和结构蛋白GP5所发生的遗传变异对病毒毒力致弱起到一定作用。