| The GHR cDNA was isolated from Southern catfish (Silurus meridionalis) by reverse transcription polymerase chain reaction followed by rapid amplification of cDNA ends. When using the nuclear acid sequence and the predicted amino acid sequence of scGHR to blast the fugu genome, we found that there were two contigs which shared the most similarity with scGHR.Therefore we realized there maybe were two GHRs encoded by different genes in teleosts.Then,by means of nuclear acid sequence joining,ORF of fGHR1 and fGHR2 were predicted from fugu genomic sequences using cohoGHR isf1, cohoGHR isf2, chGHR and other related GHR amino acid sequence available at NCBI database. In the present study, GHR that was similar to the conventional GHRs was termed as GHR1, and another type was termed as GHR2.Meanwhile, using the same methods, zfGHRl and zfGHR2 were also isolated from zebra fish (Danio rerio) EST and HTG genome sequences. After a comparison of all the GHRs isolated from the present study and others available at NCBI database, we find that GHR2 only exist in teleosts, and there is no such a gene in other vertebrates. A careful comparison about the two GHRs was carried out as follows:ScGHR: The scGHR cDNA consists of 1958bp whose ORF encodes a 602 aa protein,with a polyadnenylation signal and a ply(A)tail. ScGHR has all the characters of conventional GHRs , which include: l)seven conservative cysteines,locating at scC42, scC52, scC85, scC96, scCHO, scC128, scC220 respectively;2)A GHR landmark of FGDFS motif which was believed to fixate the GH:GHR2 tripartite complex;3)A Boxl motif which is the activator of JAK2;4)A very conservative Box2;5) The conservative tyrosine number and position in the cytoplasmic domain of GHR. Semi-quantitative PCR was carried out to study the tissue expression pattern of scGHR, and the results indicate that scGHR is predominantly expressed in the liver and brainan contrast with the fewest expression in heart and muscle.fGHRl and zfGHRl: We failed to find out the 5' ends nuclear acids due to a incomplete genome sequences. The fGHRl consists of 1755bp and encodes a 584 amino acid protein. Its gene includes seven exons(3- 9)and six introns(3 - 8). With respect tozfGHRl, it is also like other GHR1s, which consists of 1578bp and encode a 528 amino acid protein.Its gene only includes eight exons(1-5,8)and seven introns(l-5).Various methods used to find out the transmembrane and Boxl domain of zfGHRl but the result turns out to be fruitless due to unknown reason.fGHR2 and zfGHR2:fGHR2 includes eight exons and seven introns,which consists of 1710 bp and encodes a 569 amino acid protein;zfGHR2 includes eight exons(1-5,8)and seven introns(1-5),which consists of 1758 bp and encodes a 484 amino acid protein.A comparison of fGHR1, fGHR2, zfGHRl and zfGHR2 shows that they all have the typical characters of conventional GHR but distinguished from each other too. The details are as follows: 1) A GHR landmark of FGEFS motif appears in GHR1, fGHR2, zfGHRl, and a FGDFS appears in zfGHR2. Considering that aspartic acid(D)and glutamic acid(E) are very similar to each other, together with the biological function of the landmark,thus that the two landmarks can be regarded as no difference;3)Conservative cysteine in terms of their numbers and positions in the extracellular domain.In GHR1 group, fGHRl and zfGHRl have seven cysteins ,which are the same as that of quadruped.In GHR2 group which includes cohoGHR isf1, cohoGHR isf2, chGHR,fGHR2 and zfGHR2 ,the cysteine number and positon are very similar to each other but are different from GHR1 group. Especially, the fGHR2 has five cysteins that is one more than zfGHR2;3)Conservative Boxl domain. With all the GHRs identified so far, Boxl are comprised of PPXPXP(X represents any amino acid). Other studies show that the last PXP are critical for the signal transduction;4)A WVEFI domain.It is termed as Box2 which are identical in all the GHRs;5)There are also very conservative tyrosine in the cytoplasmic domain of all the GHRs, their phosphorylation is believed...
|
PDF Full Text Request