Tyrosine-specific protein kinases of normal tissues - Molecular cloning, sequencing and expression of A src-Related Tyrosine Kinase Gene

Abstract

Tyrosine - specific protein kinases. have been implicated to occupy prominent positions in the regulation of various functions of the cell. Several lines of evidence show that they are involved in crucial processes like regulation of cell division, malignant transformation, signal transduction, etc. A lot of effort has been directed towards understanding the functioning of the viral tyrosine kinases and those associated with the growth factor receptors. However, the mechanism by which viral tyrosine kinases bring about cell transformation is not known. Similarly the details of the steps involved in the regulation of cell growth, by growth factor receptor kinases are poorly understood. Still less is known about the function of non-receptor type cellular tyrosine kinases. As an initial step to understand the functions of the tyrosine kinases in the normal tissues it is necessary to identify genes {and proteins) for the various tyrosine kinases from a normal cell. eDNA libraries from normal rat spleen were constructed in plasmid pGEM-3Z and Agtll vectors. These libraries were screened with a src-related tyrosine kinase eDNA, to identify related tyrosine kinases. Several positive clones of different sizes were obtained from both the libraries. The largest clone (L115) was 1.94 kb in size. Subcloning of various restriction fragments was done with a view to obtain the complete nucleotide sequence of this clone. The nucleotide sequence obtained using some of the smaller clones and the subclones indicated that all the clones were of the same gene. The complete nucleotide sequence of the largest clone revealed the existence of the conserved features present in the catalytic domain of the src-related tyrosine kinases. It also showed high homology to the murine hck and hence it can be said to be the rat homologue of hck. The L115 eDNA has a continuous open reading frame which could encode a protein of 503 amino acids.