Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Multiple initial culture conditions enhance the establishment of cell lines from primary ovarian cancer specimens

Summary To increase the efficiency of stable cell line establishment from primary ovarian cancer specimens, we simultaneously initiated cultures under, multiple conditions, varying extracellular matrices and the inclusion of supplements (e.g., serum or serum albumin), while minimizing exposure to xenogeneic antigens (e.g., fetal calf serum). Primary cultures were initiated from 30 specimens; cell lines were established from 10 of these for a success rate of 33%. In some instances, multiple cell lines were established from the same specimen. Five lines were characterized extensively with respect to growth properties, antigen expression, and genomic alterations. Although these lines are all low-passage, marked heterogeneity was observed, even between lines derived from the same specimen. The culture approach outlined herein will facilitate generation of reagents useful for many aspects of ovarian cancer biology. Equal contribution.     

Evaluation of the hemoglobin-binding activity of Actinobacillus pleuropneumoniae using fluorescein-labeled pig hemoglobin and flow cytometry

In the present study, the hemoglobin (Hb)-binding activity of Actinobacillus pleuropneumoniae was examined using fluorescein-labeled pig Hb and flow cytometry. Comparison of the Hb-binding activity of A. pleuropneumoniae serotype 1 strain 4074 grown under iron-restricted conditions with cells grown under iron-sufficient conditions indicated that iron-restriction in A. pleuropneumoniae promotes the expression of Hb receptors, and that Hb-binding activity is, at least in part, iron-repressible. Hb-binding activity was also observed in representative strains of A. pleuropneumoniae belonging to serotypes 1 and 2. In addition, A. pleuropneumoniae serotype 1 LPS or capsule isogenic mutants were tested in flow cytometry in order to understand the influence of surface polysaccharides on Hb-binding activity. Experiments with an acapsulated mutant indicated that surface molecules with Hb-binding activity are more exposed at the cell surface in the absence of capsular polysaccharides. However, the Hb-binding activity of LPS mutants analyzed in this study was unchanged compared to the parent strain. The outer membrane proteins profile of A. pleuropneumoniae serotype 1 grown under iron-restricted or iron-sufficient conditions was also evaluated by polyacrylamide gel electrophoresis. Iron-regulated outer membrane proteins were observed under iron-restricted growth conditions which suggests that one or more of these outer membrane proteins may play a role in the Hb-binding activity detected by flow cytometry.     

Species cohesion of an extremophyte (Carex angustisquama,Cyperaceae) in solfatara fields maintained under interspecific natural hybridization

Background and AimsHybridization is the main driver of plant diversification, and gene flow via hybridization has multifaceted effects on plant evolution. Carex angustisquama is an extremophyte that grows on soils heavily acidified by volcanism. Despite its habitat distinct from that of other species, this species is known to form interspecific hybrids, implying interspecific gene flow. It is crucial to verify the extent and direction of interspecific gene flow between C. angustisquama and closely related species to understand the evolutionary process of an extremophyte in solfatara fields.MethodsIn this study, expressed sequence tag–simple sequence repeat markers were utilized to infer the extent and direction of interspecific gene flow between C. angustisquama and closely related species.Key ResultsBayesian clustering and simulation analyses revealed that all individuals of the three hybrid species were classified into the first hybrid generation or first backcross to C. angustisquama; therefore, current interspecific gene flow is limited. Moreover, in the Bayesian inference of historical gene flow based on multispecies samples, the model that assumed no interspecific gene flow was the most strongly supported across all species pairs, including phylogenetically close but ecologically distinctive species pairs.ConclusionsOur results revealed that interspecific gene flow between C. angustisquama and its related species has been limited both currently and historically. Moreover, our results of Bayesian inference of historical gene flow indicated that extrinsic, rather than intrinsic, factors probably act as isolating barriers between Carex species, with hybrid breakdown via microhabitat segregation being the probable potential barrier. Overall, our findings provide insights into the evolutionary process of an extremophyte in solfatara fields and offer an important example of the mechanisms of diversification of the speciose genus Carex.     

The microbial community composition of a nitrifying-denitrifying activated sludge from an industrial sewage treatment plant analyzed by the full-cycle rRNA approach

The composition of the microbial community present in the nitrifying-denitrifying activated sludge of an industrial wastewater treatment plant connected to a rendering facility was investigated by the full-cycle rRNA approach. After DNA extraction using three different methods, 94 almost full-length 16S rRNA gene clones were retrieved and analyzed phylogenetically. 59% of the clones were affiliated with the Proteobacteria and clustered with the beta- (29 clones), alpha- (24), and delta-class (2 clones), respectively. 15 clones grouped within the green nonsulfur (GNS) bacteria and 11 clones belonged to the Planctomycetes. The Verrucomicrobia, Acidobacteria, Nitrospira, Bacteroidetes, Firmicutes and Actinobacteria were each represented by one to five clones. Interestingly, the highest 'species richness' [measured as number of operational taxonomic units (OTUs)] was found within the alpha-class of Proteobacteria, followed by the Planctomycetes, the beta-class of Proteobacteria, and the GNS-bacteria. The microbial community composition of the activated sludge was determined quantitatively by using 36 group-, subgroup-, and OTU-specific rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH), confocal laser scanning microscopy and digital image analysis. 89% of all bacteria detectable by FISH with a bacterial probe set could be assigned to specific divisions. Consistent with the 16S rRNA gene library data, members of the beta-class of Proteobacteria dominated the microbial community and represented almost half of the biovolume of all bacteria detectable by FISH. Within the beta-class, 98% of the cells could be identified by the application of genus- or OTU-specific probes demonstrating a high in situ abundance of bacteria related to Zoogloea and Azoarcus sensu lato. Taken together, this study provides the first encompassing, high-resolution insight into the in situ composition of the microbial community present in a full-scale, industrial wastewater treatment plant.     

Taxonomy of the glycerol fermenting clostridia and description of Clostridium diolis sp. nov

Six Clostridium strains which ferment glycerol to 1,3-propanediol were tested for their taxonomic and phylogenetic relatedness. All but one were known as C butyricum. By physiological tests, 16S rDNA sequences and fatty acid composition two groups were distinguished. The first comprised the strains VPI 3266, DSM 2478 and DSM 523 (C. "kainantoi") and was consistent with the type strain of C. butyricum in almost all characters. The second group comprising the strains DSM 5430, DSM 5431 and E5 was related to C. beijerinckii. The 16S rDNAs of these strains were almost identical with that of the type strain of C. beijerinckii, DSM 791. The DNA-DNA hybridization value of DSM 5431 and ES with C. beijerinckii DSM 791 was markedly but not decisively lower (67 and 72%, respectively). However, there were significant physiological differences to C. beijerinckii which suggested to describe the strains as a separate species, Clostridium diolis with strain SH1 (= DSM 5431) as the type strain. The new species is distinguished from C. beijerinckii, which requires complex nutrients, by its ability to grow in glucose mineral medium with biotin as the only growth factor and by differences in substrate utilization. "C. kainantoi" Takeda and Matsui was recognized as a later synonym of C. butyricum.     

小麦,玉米接种耐氨固氮菌试验初步分析

１９８９—１９９２年,在广东省部分县市进行了小麦、王米施用耐氨固氮菌大田对比试验,小麦接种耐氨固氮菌后,接菌区小麦的穗氏、有效穗、每穗粒数均比不接菌区明显增加,株高、成穗率、千粒重比对照略有增加,平均每亩比对照增产１６．７ｋｇ干麦,平均增产率为８．１％。玉米接种耐氨固氮菌后,接菌区玉米的茎粗、收获时的青叶片数、单苞重均比对照区明显增加,秃顶度比对照区明显减小,晒干率：出米率略有增加,平均每亩比对照增产５２．８ｋｇ玉米,平均增产率为１１．８９％。     

黄土高原根瘤菌数值分类及DNA-DNA杂交

黄土高原位于我国内陆,气候比较干旱,生长的植被较少,水土流失严重,而有些豆科植物如锦鸡儿(Caragana sinica)、苦豆子(Sophora alopecuroides)、甘草(Glycyrrhiza uralensis)、苦马豆(Swainsoniasalsula)、洋槐(Robinia pseudoacacia)等却能很好生长.这些豆科植物的生长,在防风固沙、保持水土、绿化环境、作为饲用牧草等方面起着很重要的作用.但人们对于黄土高原野生豆科植物根瘤菌的研究尚很少.为此,作者在地处黄土高原的陕西、宁夏及甘肃的部分地区进行了广泛的根瘤菌资源调查.在此基础上,对分离的部分菌株进行了数值分类和DNA同源性分析.     

Evolution of the tetraploid Anemone multifida (2n = 32) and hexaploid A. baldensis (2n = 48) (Ranunculaceae) was accompanied by rDNA loci loss and intergenomic translocation: evidence for their common genome origin

Background and Aims

In the genus Anemone two small groups of taxa occur with the highest ploidy levels 2n = 6x = 48, belonging to the closely related clades: the montane/alpine Baldensis clade and the more temperate Multifida clade. To understand the formation of polyploids within these groups, the evolution of allohexaploid A. baldensis (AABBDD, 2n = 6x = 48) from Europe and allotetraploid Anemone multifida (BBDD, 2n = 4x = 32) from America was analysed.

Methods

Internal transcribed spacer and non-transcribed spacer sequences were used as molecular markers for phylogenetic analyses. Cytogenetic studies, including genomic in situ hybridization with genomic DNA of potential parental species as probe, fluorescence in situ hybridization with 5S and 18S rDNA as probes and 18S rDNA restriction analyses, were used to identify the parental origin of chromosomes and to study genomic changes following polyploidization.

Key Results

This study shows that A. multifida (BBDD, 2n= 4x = 32) and A. baldensis (AABBDD, 2n = 6x = 48) are allopolyploids originating from the crosses of diploid members of the Multifida (donor of the A and B subgenomes) and Baldensis groups (donor of the D subgenome). The A and B subgenomes are closely related to the genomes of A. sylvestris, A. virginiana and A. cylindrica, indicating that these species or their progeny might be the ancestral donors of the B subgenome of A. multifida and A and B subgenomes of A. baldensis. Both polyploids have undergone genomic changes such as interchromosomal translocation affecting B and D subgenomes and changes at rDNA sites. Anemone multifida has lost the 35S rDNA loci characteristic of the maternal donor (B subgenome) and maintained only the rDNA loci of the paternal donor (D subgenome).

Conclusions

It is proposed that A. multifida and A. baldensis probably had a common ancestor and their evolution was facilitated by vegetation changes during the Quaternary, resulting in their present disjunctive distribution.