Mapping of G2/M-phase prevalences of chaperon-encoding transcripts by means of a sensitive differential hybridization approach.
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; eta 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.
Dittmar
G
G
Abteilung Biochemie der Zelle, Deutsches Krebsforschungszentrum, Im Nevenheimer Feld 280-0225, Heidelberg, D-69120, Germany.
Schmidt
G
G
Kopun
M
M
Werner
D
D
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor; D002498:Centrifugation; D018833:Chaperonins / Q000235:genetics*; D018076:DNA, Complementary; D005434:Flow Cytometry; D016195:G2 Phase / Q000235:genetics*; D005820:Genetic Testing; D006360:Heat-Shock Proteins / Q000235:genetics; D051379:Mice; D008938:Mitosis / Q000235:genetics*; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000166:cytology / Q000502:physiology
D018076:DNA, Complementary; D006360:Heat-Shock Proteins; D015347:RNA Probes; D012333:RNA, Messenger; D018833:Chaperonins