TY - JOUR
T1 - Mapping of G2/M-phase prevalences of chaperon-encoding transcripts by means of a sensitive differential hybridization approach
AU - Dittmar, Gunnar
AU - Schmidt, Gebhard
AU - Kopun, Marijana
AU - Werner, Dieter
N1 - Funding Information:
This work was supported by the Deutsche Forschungsgemeinschaft (We 589/2-2).
PY - 1997/6
Y1 - 1997/6
N2 - The sensitivity of the differential hybridization approach is significantly increased by the application of size-selected probes. RNA from elutriated phase-synchronous Ehrlich ascites tumor (EAT) cells has previously been used to prepare cell cycle phase-specific cDNA libraries in the in-vitro transcription vector pBluescript. PCR amplification of the libraries with vector-fitting primer pairs generates amplified cDNA reflecting the mRNA complexities of cells in G1, S and G2/M phases. Probes with reduced complexities were recovered after side-by-side electrophoresis of equal amounts of PCR-amplified cDNA and elution of probes from parallel gel sections. Such size-selected probes release significant differential clones which escape their detection in the conventional differential hybridization approach. Three clones hybridizing preferentially with the G2/M phase-specific probe were further characterized. The genes were identified by their nucleotide sequences. They encode proteins known to be involved in protein folding: heatshock cognate protein, HSC 70; heatshock cognate protein, HSC 73; η subunit of the chaperonin containing TCP-1 complex, CCT. The G2/M phase-prevalent expression of these genes were further verified on the mRNA and on the protein level by Northern and Western blot analysis which confirms the significance of the differential hybridization approach and which indicates that the expression of this group of proteins increases with cell cycle progression. The expression of the chaperonin-containing TCP-1 complex appears to be specifically linked with the S to G2/M phase transition of the cell cycle.
AB - The sensitivity of the differential hybridization approach is significantly increased by the application of size-selected probes. RNA from elutriated phase-synchronous Ehrlich ascites tumor (EAT) cells has previously been used to prepare cell cycle phase-specific cDNA libraries in the in-vitro transcription vector pBluescript. PCR amplification of the libraries with vector-fitting primer pairs generates amplified cDNA reflecting the mRNA complexities of cells in G1, S and G2/M phases. Probes with reduced complexities were recovered after side-by-side electrophoresis of equal amounts of PCR-amplified cDNA and elution of probes from parallel gel sections. Such size-selected probes release significant differential clones which escape their detection in the conventional differential hybridization approach. Three clones hybridizing preferentially with the G2/M phase-specific probe were further characterized. The genes were identified by their nucleotide sequences. They encode proteins known to be involved in protein folding: heatshock cognate protein, HSC 70; heatshock cognate protein, HSC 73; η subunit of the chaperonin containing TCP-1 complex, CCT. The G2/M phase-prevalent expression of these genes were further verified on the mRNA and on the protein level by Northern and Western blot analysis which confirms the significance of the differential hybridization approach and which indicates that the expression of this group of proteins increases with cell cycle progression. The expression of the chaperonin-containing TCP-1 complex appears to be specifically linked with the S to G2/M phase transition of the cell cycle.
KW - Cell cycle prevalence
KW - Centrifugal elutriation
KW - Chaperon
KW - Chaperonin
KW - Differential hybridization
KW - Ehrlich ascites tumor cells
KW - Heat shock cognate proteins
KW - Polymerase chain reaction
UR - http://www.scopus.com/inward/record.url?scp=0031171456&partnerID=8YFLogxK
U2 - 10.1006/cbir.1997.0158
DO - 10.1006/cbir.1997.0158
M3 - Article
C2 - 9268492
AN - SCOPUS:0031171456
SN - 1065-6995
VL - 21
SP - 383
EP - 391
JO - Cell Biology International
JF - Cell Biology International
IS - 6
ER -