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Isolation of a Methylocystis strain containing a novel pmoA-like gene (2002)

Dunfield, Peter F. ; Yimga, Merlin Tchawa ; Dedysh, Svetlana N. ; [et al.]

Oxford, UK : Blackwell Publishing Ltd

FEMS microbiology ecology 41 (2002), S. 0

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ISSN: 1574-6941

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology

Notes: A type II methanotrophic bacterium (Methylocystis strain SC2) was isolated from a polluted aquifer and identified based on morphology and on 16S rRNA gene phylogeny. Primers targeting the particulate methane monooxygenase subunit A gene (pmoA) were used to obtain a PCR product from DNA extract of strain SC2. Denaturing gradient gel electrophoresis of this PCR product demonstrated that strain SC2 contained two very different pmoA-like genes. One gene (pmoA1) had very high sequence homology to pmoA genes of other type II methanotrophic bacteria (identical amino acid sequence to pmoA of some other Methylocystis strains). The second gene (pmoA2) possessed only 73% identity with the first gene at the nucleotide level and 68.5% identity (83% similarity) at the amino acid level. The presence of both pmoA-like genes was verified by developing specific oligonucleotide probes for each and using these in Southern hybridisation of genomic DNA. Purity of the culture was exhaustively verified with a variety of methods to ensure that both genes were present in a single genospecies. These included microscopic examination, plating on various media, denaturing gradient gel electrophoresis of PCR products of the 16S rRNA gene (universal to bacteria) and of the methanol dehydrogenase α-subunit gene mxaF (universal to methylotrophic bacteria), and whole-cell hybridisation with fluorescently labelled 16S rRNA-targeted oligonucleotide probes specific for the genera Methylosinus and Methylocystis, or specific for strain SC2. Reverse transcription PCR of extracted RNA suggested that the novel pmoA2 gene was not expressed during growth under standard conditions used for the cultivation of these bacteria. The presence of multiple, diverse pmoA-like genes in a single genospecies of methanotrophic bacteria implies that pmoA must be cautiously applied as a phylogenetic marker in cultivation-independent molecular ecology studies.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1574-6941.2002.tb00962.x

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Invasion marker PAI‐1 remains a strong prognostic factor after long‐term follow‐up both for primary breast cancer and following first relapse (1999)

Harbeck, Nadia ; Thomssen, Christoph ; Berger, Ursula ; [et al.]

Springer

Breast cancer research and treatment 54 (1999), S. 147-157

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ISSN: 1573-7217

Keywords: breast cancer ; cathepsin D ; PAI‐1 ; prognosis ; S‐phase fraction ; uPA

Source: Springer Online Journal Archives 1860-2000

Topics: Medicine

Notes: Abstract In 1991, our group was the first to report the prognostic strength of plasminogen activator inhibitor type 1 (PAI‐1) in primary breast cancer. The prognostic impact of invasion markers PAI‐1 and urokinase‐type plasminogen activator (uPA) on disease‐free survival (DFS) and overall survival (OS) in breast cancer has since been independently confirmed. We now report on the prognostic impact of PAI‐1 and uPA after long‐term median follow‐up of 77 months for our cohort (n=316). Levels of uPA, PAI‐1, and cathepsin D were determined in tumor tissue extracts by immunoenzymatic methods. S‐phase fraction (SPF) was measured flowcytometrically in paraffin sections. Using log‐rank statistics, optimized cutoffs were found for PAI‐1 (14 ng/mg), uPA (3 ng/mg), cathepsin D (41 pmol/mg), and SPF (6%). In all patients, various factors (PAI‐1, uPA, nodal status, SPF, cathepsin D, grading, tumor size, hormone receptor status) showed significant univariate impact on DFS. In Cox analysis, only nodal status (p 〈 0.001, RR: 3.1) and PAI‐1 (p 〈 0.001, RR: 2.7) remained significant. In node‐negative patients (n = 147), PAI‐1, uPA, and SPF had significant univariate impact on DFS, whereas in Cox analysis, only PAI‐1 was significant. PAI‐1 was also significant for DFS within subgroups defined by established factors. In CART analysis, uPA enhanced the prognostic value of PAI‐1 and nodal status for determination of a very‐low‐risk subgroup. For OS, only lymph node status and PAI‐1 were significant in multivariate analysis. PAI‐1 levels in the primary tumor were also a significant prognostic marker for survival after first relapse in both univariate and multivariate analysis.