MAPPING OF G2 M-PHASE PREVALENCES OF CHAPERONE-ENCODING TRANSCRIPTS BY MEANS OF A SENSITIVE DIFFERENTIAL HYBRIDIZATION APPROACH/

The sensitivity of the differential hybridization approach is signific antly increased by the application of size-selected probes, RNA from e lutriated phase-synchronous Ehrlich ascites tumor (EAT) cells has prev iously been used to prepare cell cycle phase-specific cDNA libraries i n the in-vitro transcription vector pBluescript. PCR amplification of the libraries with vector-fitting primer pairs generates amplified cDN A reflecting the mRNA complexities of cells in G1, S and G2/M phases. Probes with reduced complexities were recovered after side-by-side ele ctrophoresis of equal amounts of PCR-amplified cDNA and elution of pro bes from parallel gel sections. Such size-selected probes release sign ificant differential clones which escape their detection in the conven tional differential hybridization approach. Three clones hybridizing p referentially with the G2/M phase-specific probe were further characte rized. The genes were identified by their nucleotide sequences, They e ncode proteins known to be involved in protein folding: heatshock cogn ate protein, NSC 70; heatshock cognate protein, HSC 73; eta subunit of the chaperonin containing TCP-1 complex, CCT. The G2/M phase-prevalen t expression of these genes were further verified on the mRNA and on t he 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 T CP-1 complex appears to be specifically linked with the S to G2/M phas e transition of the cell cycle. (C) 1997 Academic Press Limited.