PROTEIN PHOSPHORYLATION IN AMPHIBIAN OOCYTES AND FERTILIZED EGGS AND (P)SV2CAT DNA EXPRESSION IN THE XENOPUS LAEVIS OOCYTE

EVELYNN EMIKO KAWATA, Purdue University

Abstract

('32)P(,i)-labeled phosphoproteins of amphibian oocytes, induced to mature in vitro, were examined by 2-dimensional gel electrophoresis. From this analysis, we have detected increases in phosphorylation of 17 proteins during oocyte maturation. Examination of the nuclear and cytoplasmic components of the maturing oocyte indicates that the majority of these phosphoprotein changes occur in the cytoplasm. St. IV oocytes, under certain conditions, undergo "partial" maturation, yet exhibit the full spectrum of changes that occur during St. VI oocyte maturation. Of the changes that take place during oocyte maturation, two of these events (MWs = 44kd, pIs = 6.0) may be important for the G2/M transition, in general, since these phosphorylation events take place in the first cell division of a fertilized egg. Furthermore, assuming that the act of fertilization is similar to parthenogenetic activation, we have effectively monitored changes occurring immediately after fertilization by examining those changes that occur upon activation. The second part of this thesis examines the expression of pSV2CAT DNA in the Xenopus oocyte. pSV2CAT is a plasmid containing a chimeric construction composed of the chloramphenicol acetyltransferase gene under the control of the SV40 early gene promoter. Expression of this gene occurs ultimately at the protein level since enzymatically active chloramphenicol acetyltransferase is detected in extracts of DNA-injected oocytes. Analysis of the transcripts produced in pSV2CAT DNA-injected oocytes suggests that the functional transcript is larger than anticipated, based on the known transcriptional and post-transcriptional signals encoded in the gene. 5' end mapping results obtained by others and northern analysis data presented here indicate that 3' end formation of SV2CAT transcripts is anomalous. In addition, variability in the expression of DNA in oocytes from different frogs was investigated by examining protein synthetic rates in, and the steady state SV2CAT RNA transcripts produced by, oocytes from individual frogs. As anticipated, the individual parameters, alone, probably do not account for the vast differences in expression detected among oocytes. This suggests that the variability in DNA expression is due to a combination of the factors involved in eukaryotic gene expression.

Degree

Ph.D.

Subject Area

Biology

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