| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 85 === In response to the environmental change, cells can transduce
signals by using kinases to transfer g-phosphate of ATP or GTP
to the hydroxyl groups(Ser, Thr or Tyr) of their protein
substrates. Hundreds of kinases sequence have been determined.
Comparison between amino acid sequence of these kinase revealed
that the kinases share a common structure, the kinase domain.
The kinase domain consists of 250-300 amino acid residues and
impart catalytic activity. The kinase domain has 3 specific
roles: 1) to bind and orientate ATP and GTP with divalent cation
(usually Mg2+ or Mn2+). 2) to bind and orientate the substrate.
3) to transfer g-phosphate of ATP or GTP to the hydroxyl groups(
Ser, Thr or Tyr) of their protein substrate. The kinase domain
are further divided into 12 subdomains. Subdomains VIB,VIII and
IX are particularly well conserved among the individual members
of different protein kinase families.Many oncogenes and proto-
oncogenes are known to be tyrosine kinases, and tyrosine kinase
play important roles in cell growth of normal and tumor cells.
Therefore we wish to study the tyrosine kinase in bladder
cancer. RT-PCR amplified of RNA from BFTC 905 human bladder
cancer cell line were performed with two degenerate primers, R1
and F3, which were designed to the correspond region of
subdomain VII and IX. The 180 bp PCR products were ligated into
pCRTMII vector with TA cloning kit and transformed into E. coli.
DNA sequence was performed with insert-positive clones. The DNA
sequence of all these clones revealed the expression pattern of
tyrosine kinase and possible novel kinase genes. Among the 460
clones screened by PCR, 354 clones were insert-positive .
Sequencing of 100 clones revealed that:1) The highest expression
kinases are trkE and arg. The sequence of trkE gene appealed in
24 clones of the 100 clones (26%), and arg gene appealed in 16
clones (16%). 2) The DNA sequence of two clones, 240 and 246
can't be matched in BLAST research. Clone 240 is significantly
homologous to a serine/threonine kinase Sok-1 in BLAST search.In
1996, Thomas Force identified a human Ste20 like serine/
threonine kinase Sok-1(Ste20/oxidant stress response kinase-1).
Sok-1 was not activated by growth factors, alkylating agents,
cytokines or environmental stress including heat shock and
osmolar stress, but activated by oxidant stress like H2O2 or
menadione.As Sok-1 plays an important role in tranducing signals
in response to environmental stress, our clone Sok-1 like gene
may also play an important role in a similar pathway. We then
clone the full-length cDNA of the novel Sok-1 like kinase with
the use of RACE method (rapid amplification of cDNA ends). The
DNA sequence of the novel clone is 88 % homology with Sok-1 in
kinase domain.
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