Strachan, Fiona Rachel (1986) The autonomy and function of eukaryotic signal sequences : segregation of variants of preprochymosin and prelysozyme in "Xenopus oocytes" and "in vitro". PhD thesis, University of Warwick.

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Abstract

Cloned complementary DNAs encoding the secretory proteins chick prelysozyme and calf preprochymosin were inserted downstream from various viral promoters in modified recombinant “shuttle" vectors. The microinjection of these constructs into the nuclei of Xenopus laevis oocytes resulted in the efficient expression of lysozyme and prochymosin proteins which were segregated into membranes and secreted by the oocyte. The signal sequences of the proteins were correctly processed as judged from molecular weight estimations. Injection of DNA encoding prochymosin without its signal sequence resulted in the synthesis of a prochymosin protein which was localised in the oocyte cytoplasm; whereas when DNA encoding mature chymosin was injected no proteins were detected by immunoprécipitation with prochymosin antisera. The same cDNAs were subsequently cloned into SP6 vectors and synthetic, capped RNA was prepared. Following cytoplasmic injection of SP6 RNA into oocytes the same compartmentation of the proteins was observed but again no chymosin protein was detected following injection of RNA encoding mature chymosin, although translation of this RNA in vitro produced a protein with the expected molecular weight of chymosin which was precipitated by prochymosin antisera.

The expression of preprochymosin messenger RNA, following cytoplasmic injection into oocytes and also using in vitro translation systems, showed the mRNA encoded two preprochymosin proteins specifically precipitated by prochymosin antisera. In the oocyte both forms were processed, segregated and secreted; whilst in vitro the precursors were cleaved on translocation within dog pancreatic microsomes where they became resistant to digestion by exogenous proteases. The translation of preprochymosin mRNA in vitro has previously been reported to produce only one major polypeptide on gel electrophoresis of products precipitated by antiprochymosin sera. The origin and nature of the two electrophoretically distinct species is not certain; but it was noted that the protein product of the cloned preprochymosin cDNA showed the same mobility on SDS-polyacrylamide gels as the faster migrating species encoded by the mRNA.

Two hybrid genes were constructed encoding proteins in which the signal sequence of prelysozyme was replaced with different N-terminal regions of preprochymosin. C₆L contained a fragment from the preprochymosin cDHA which encoded the signal sequence and the first six amino acids of prochymosin; this was fused to codons 8 to 129 of mature lysozyme. The second construct C₆₂L carried a larger portion of preprochymosin, up to codon 62 of prochymosin, with the same C-terminal region of lysozyme. These fusions showed poor and variable expression following nuclear injection of the hybrid genes contained in the shuttle vector. However cytoplasmic injection of the corresponding SP6 RNAs demonstrated that both fusion proteins were synthesized in the oocyte and segregated into membranes, but did not get secreted from the oocyte. The distribution of C₆₂L protein within the oocyte corresponded with that observed for the majority of other secretory proteins including preprochymosin, with most protein fractionating with vesicles. In contrast C₆L displayed the anomalous fractionation previously observed for lysozyme, with approximately equal amounts of the processed protein fractionating with the cytoplasm and the membranes. Relative to the precursor polypeptides produced by in vitro translation of the SP6 RNAs for C₆L and C₆₂L, the respective proteins expressed in oocytes each showed an increased mobility on electrophoresis consistent with the cleavage of the signal peptide. The same processing of the preC₆L and preC₆₂L proteins was observed when in vitro translation was carried out In the presence of pancreatic mlcro6omes. The observed compartmentation and processing of these hybrid proteins Indicates that a eukaryotic signal sequence functions autonomously in Initiating the translocation of secretory proteins, but that other properties of the protein conformation are necessary to achieve subsequent secretion.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QL Zoology Q Science > QP Physiology Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Eukaryotic cells -- Genetics, Xenopus -- Research, Ovum, Proteins -- Secretion, Xenopus laevis
Official Date:	August 1986
Dates:	Date Event August 1986 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Colman, Alan
Sponsors:	Medical Research Council (Great Britain)
Extent:	xii, 179, [49] leaves : illustrations
Language:	eng

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