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Inverse PCR (inverse + pcr)
Selected AbstractsHeterotrophic symbionts of phototrophic consortia: members of a novel diverse cluster of Betaproteobacteria characterized by a tandem rrn operon structureENVIRONMENTAL MICROBIOLOGY, Issue 11 2007Kristina R. Pfannes Summary Phototrophic consortia represent the most highly developed type of interspecific association of bacteria and consist of green sulfur bacterial epibionts attached around a central colourless rod-shaped bacterium. Based on 16S rRNA gene sequencing, the central bacterium of the consortium ,Chlorochromatium aggregatum' was recently shown to represent a novel and phylogenetically isolated lineage of the Comamonadaceae within the ,-subgroup of the Proteobacteria. To date, 19 types of phototrophic consortia are distinguished based on the different 16S rRNA gene sequences of their epibionts, but the diversity and phylogenetic relationships of the heterotrophic partner bacteria are still unknown. We developed an approach based on the specific rrn (ribosomal RNA) operon structure of the central bacterium of ,C. aggregatum' to recover 16S rRNA gene sequences of other central bacteria and their close relatives from natural consortia populations. Genomic DNA of the central bacterium of ,C. aggregatum' was first enriched several hundred-fold by employing a selective method for growth of consortia in a monolayer biofilm followed by a purification of the genome of the central bacterium by cesium chloride-bisbenzimidazole equilibrium density gradient centrifugation. A combination of inverse PCR, cloning and sequencing revealed that two rrn operons of the central bacterium are arranged in a tandem fashion and are separated by an unusually short intergenic region of 195 base pairs. This rare gene order was exploited to screen various natural microbial communities by PCR. We discovered a diverse and previously unknown subgroup of Betaproteobacteria in the chemoclines of freshwater lakes. This group was absent in other freshwater and soil samples. All the 16S rRNA gene sequences recovered are related to that of the central bacterium of ,C. aggregatum'. Fluorescence in situ hybridization indicated that two of these sequences originated from central bacteria of different phototrophic consortia, which, however, were only distantly related to the central bacterium of ,C. aggregatum'. Based on a detailed phylogenetic analysis, these central bacterial symbionts of phototrophic consortia have a polyphyletic origin. [source] The human complement C9 gene: structural analysis of the 5, gene region and genetic polymorphism studiesINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 5 2001K. Witzel-Schlömp Summary C9 is the last of the human complement components creating the membrane attack complex. The single chain serum protein is encoded by a gene located on chromosome 5p13 that is composed of 11 exons. With the aid of inverse PCR, the hitherto unknown regions flanking exon 1 and the 3, part of exon 11 (3,UTR) have been sequenced. A computer-based analysis of the 300-bp region located just upstream of the AUG start codon showed homologies to known DNA modules which affect the transcriptional regulation of certain genes. The most striking of these is a sequence that may substitute the missing TATA box in initiating C9 transcription. In the 3,UTR, three successive polyadenylation signals were found. Although the C9 protein is invariant, four different single nucleotide polymorphisms (SNPs) have been observed at the DNA level by exon-specific PCR and direct sequencing. None of them changes the amino acid composition of the mature protein. Due to a C , T transition in exon 1 at cDNA position 17, the fifth amino acid of the leader peptide may be either an arginine or a tryptophane. Using either PCR/RFLP analysis (exons 1 and 11) or allele-specific PCR (intron 1 and exon 4), each polymorphism can be characterized without sequencing. All of the exon 1, intron 1 and exon 11 variants could be detected in small population samples of European, Thai or South American Indian origin. In contrast, the exon 4 C variant was observed only once in a European. The first three SNPs can be combined to designate eight different ,C9 alleles'. Of these, six have actually be found. These data provide strong evidence that several mutation and recombination events occurred in the course of C9 gene evolution. [source] Study of mRNA Expression by Real Time PCR of Cpkk1, Cpkk2 and Cpkk3, three MEKs of Cryphonectria parasitica, in Virus-free and Virus-infected Isogenic IsolatesJOURNAL OF PHYTOPATHOLOGY, Issue 6 2010Laura Rostagno Abstract Cpkk1 and Cpkk2 are two previously characterized Mitogen-activated protein kinase kinases (MEK) from Cryphonectria parasitica. For the characterization of the third MEK, primers designed to a conserved region of the known fungal MEK sequences were used in a PCR reaction to amplify genomic DNA from C. parasitica. The sequence of the resulting amplicon was compared to known sequences in the database using a Blast search. Results of the sequence comparison indicated that the initial fragment obtained encoded for a new MEK from C. parasitica, that had highest homology to Pbs2 from Saccharomyces cerevisiae. By inverse PCR we obtained a genomic fragment spanning the entire coding sequence of this MEK, which was named Cpkk3. The cDNA of Cpkk3 was obtained by compiling the sequences of RT-PCR products resulting from the amplification of purified mRNA. TaqMan® Probes were designed to analyse the expression of Cpkk1, Cpkk2 and Cpkk3 mRNA through RT-Real Time PCR. This protocol allowed the expression of Cpkk3 to be successfully compared to the expression of Cpkk1 and Cpkk2, two previously cloned C. parasitica MEKs. No variation in expression was associated with the presence of a virus after 2 days of growth in standard conditions whereas an increase in the expression level of all the three MEKs was shown after 4 days of growth. [source] Rapid amplification and cloning of Tn5 flanking fragments by inverse PCRLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2000G. Huang A simple approach is described to efficiently amplify DNA sequences flanking transposon Tn5 insertions. The method involves: (i) digestion with a restriction enzyme that cuts within Tn5; (ii) self-ligation under conditions favouring the production of monomeric circles; (iii) four parallel PCR reactions using primers designed to amplify left or right flanking sequences, and to distinguish target amplicons from non-specific products. This reveals the number of Tn5 insertions and the size of flanking genomic restriction fragments, without Southern blot analysis. The amplified product contains restriction sites that facilitate cohesive-end cloning. This rapid method is demonstrated using Tn5 and Tn5-Mob tagged DNA sequences involved in albicidin biosynthesis in Xanthomonas albilineans. It is generally applicable for efficient recovery of DNA sequences flanking transposon Tn5 derivatives in insertional mutagenesis studies. [source] |