The effects of radiation on the expression of a newly cloned and characterized rat cyclin B mRNA.
OBJECTIVE
To evaluate the hypothesis that the radiation induced G2 delay in the cell cycle is associated with radiation induced effects on cyclin B expression in a rodent cell system.


METHODS
Two rodent, rat and Chinese hamster, cyclin B cDNAs were cloned and characterized. The two rodent species were 85% and 86% identical, respectively, when compared to the human cyclin B, indicating that they are the rodent homologous of cyclin B. 3.7 cells (rat embryo cells transformed by H-ras and v-myc) were synchronized and then irradiated. Flow cytometry and Northern blots were performed to evaluate the effects of radiation on cyclin expression in relation to phase of the cell cycle.


RESULTS
Examination of the rodent cyclin B sequences revealed only two regions with significant divergence to the human sequence, one in the lysine rich region adjacent to the cyclin destruction box, which is the putative site for ubiquitination, and one at the C terminal end. Although many of the amino acids diverged in the lysine rich region, the positions of the lysines themselves were virtually invariant suggesting their potential importance in ubiquitination. Both rodent species were also noted to have a PEST-like sequence which occurs in the human, but not in non-mammalian cyclins cloned to date and could also potentially contribute to rapid destruction. The rat and Chinese hamster mRNAs contain much longer 3' untranslated regions than the published human sequence with multiple AUUUA and AUUU motifs which are seen in other mRNAs with rapid turnover times. This feature has not been previously found in cyclin mRNAs. In addition we have found that in the 3' region of the rodent cDNAs we find two potential polyadenylation sites suggesting that this gene may have several transcripts. Our studies suggest that multiple mechanisms of control of mammalian cyclin B destruction exist, both at the mRNA and protein level. Evidence is also provided that the levels of rat cyclin B mRNA peaks during G2/M. Irradiation is shown to induce a G2 delay in synchronized 3.7 cells, compared to unirradiated controls, and the delay is temporally related to decreased levels of cyclin B mRNA expression. Since the G2 delay induced by ionizing radiation may contribute to the ability of cells to survive irradiation, cyclin B expression may be a key component in the determination of sensitivity or resistance to radiation therapy.


CONCLUSIONS
The isolation and characterization of two rodent cyclin B's confirm that multiple mechanisms of control of mammalian cyclin B destruction exist. Our studies show that rat cyclin B expression is influenced by radiation and is temporally related to the delay in the G2 phase induced by radiation.
Markiewicz
D A
DA
Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia 19104.
McKenna
W G
WG
Flick
M B
MB
Maity
A
A
Muschel
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D016213:Cyclins / Q000235:genetics*; D016195:G2 Phase / Q000235:genetics; D015870:Gene Expression / Q000528:radiation effects*; D006801:Humans; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats
D016213:Cyclins; D012333:RNA, Messenger