In situ reverse transcription-PCR for monitoring gene expression in individual Methanosarcina mazei S-6 cells.

TY - JOUR

T1 - In situ reverse transcription-PCR for monitoring gene expression in individual Methanosarcina mazei S-6 cells.

AU - Lange, M

AU - Tolker-Nielsen, Tim

AU - Molin, S

AU - Ahring, B K

N1 - Keywords: Cell Wall; DNA Primers; Digoxigenin; Escherichia coli Proteins; HSP70 Heat-Shock Proteins; Indicators and Reagents; Methanosarcina; Molecular Chaperones; Muramidase; RNA, Archaeal; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic

PY - 2000

Y1 - 2000

N2 - An in situ reverse transcription-PCR protocol for detecting specific mRNA in Methanosarcina mazei S-6 is described. This method allowed us to detect heat shock-induced increases in the intracellular levels of the transcript of the universal stress gene dnaK. The cell walls of paraformaldehyde-fixed cells were permeabilized by a thermal cycling procedure or by lysozyme treatment, and the cellular DNA was removed with DNase. The cells were subjected to a seminested reverse transcription-PCR protocol in which a digoxigenin-labeled primer was used. Detection of the reporter molecule was based on the 2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate-Fast Red detection system and binding of anti-digoxigenin-alkaline phosphatase conjugate. Fluorescence in permeabilized cells increased after a heat shock compared to fluorescence in non-heat-shocked cells, and the increase corresponded to an increase in the level of the dnaK transcript.

AB - An in situ reverse transcription-PCR protocol for detecting specific mRNA in Methanosarcina mazei S-6 is described. This method allowed us to detect heat shock-induced increases in the intracellular levels of the transcript of the universal stress gene dnaK. The cell walls of paraformaldehyde-fixed cells were permeabilized by a thermal cycling procedure or by lysozyme treatment, and the cellular DNA was removed with DNase. The cells were subjected to a seminested reverse transcription-PCR protocol in which a digoxigenin-labeled primer was used. Detection of the reporter molecule was based on the 2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate-Fast Red detection system and binding of anti-digoxigenin-alkaline phosphatase conjugate. Fluorescence in permeabilized cells increased after a heat shock compared to fluorescence in non-heat-shocked cells, and the increase corresponded to an increase in the level of the dnaK transcript.

M3 - Journal article

C2 - 10788341

SN - 0099-2240

VL - 66

SP - 1796

EP - 1800

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

IS - 5

ER -