Molekularbiologische Untersuchungen über ein neues Cytoskelett-verwandtes Protein

Abstract

This work describes the initial molecular characterization of a novel protein, p170, from the african claw frog Xenopus laevis. The cDNA encoding p170 was identified in a random manner when an expression library from X.laevis ovary was screened with an affinity purified antibody originally designed to recognize the replication factor cdc6. The original p170 cDNA fragment was completed by RACE ('rapid amplification of cDNA ends'), and the full length cDNA (4441bp) was found to encode a protein of 1335 aa related to certain cytoskeleton associated proteins. p170 has an extraordinary amino acid composition with a high content of leucine, glutamic acid and glutamine, but low content of proline and glycine. Secondary structure analysis of the p170 protein reveals that the protein has a high potential to form coiled coils. To allow a biochemical characterization of the p170 protein, two fragments were subcloned and expressed in E.coli to generate p170-specific antibodies by the immunization of rabbits. With these antibodies, immunofluorescence microscopy and biochemical cell fractionation experiments show that the p170 protein is preferentially localized in the cytoplasm of cultured Xenopus cells. The p170 protein is highly expressed in eggs and the p170 amount decreases during early development. Computer searches have shown that p170 is conserved in vertebrates, and homologues in human, mouse, cow, pig and zebrafish could be identified by expressed sequence tags (ESTs). On the basis of published sequence information the genomic organization of the complete human and mouse p170 genes could be determined The human p170 gene is located on Chromosome 1 and consists of 25 exons over a length of at least 107 kb. Further experiments are in progress to test the function of the p170 protein in cytoskeleton assemby.