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Cloning of mitichondrial cytochrome b gene from Pseudourostyla cristata by heat shock
Author(s) -
LIU XINYING,
MAO YONGZHEN,
DANIEL LEE KAMLEN,
JIN LIPEI
Publication year - 2005
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.2005.05202003_1_50.x
Subject(s) - biology , gene , paramecium , complementary dna , microbiology and biotechnology , genetics , untranslated region , cloning (programming) , heat shock protein , gene expression , paramecium caudatum , sequence analysis , messenger rna , computer science , programming language
The total RNA was extracted from Pseudourstyla cristata after 2 h of heat shock, and the cDNA was synthesized directly using Smart LD‐PCR. The amplified cDNA products showed a strong band around 1.5 kb. The products were purified, cloned and sequenced. The resulting full cDNA sequence had a total length of 1538 bp including 1068 bp of cytb gene and UTR at both sides and showed all of the expected features for a fully functional CYB. The predicted protein is 49% (30% identical) similar to Paramecium tetraurelia . The sequence predicts a UAA stop codon 207 nucleotides from the end of the transcript suggesting a 3′ UTR, unlike that of Paramecium tetraurelia . Prediction of transmembrane helices in the analysis of proteins indicates the expected eight transmembrane domains. The cytb gene expression profiles after heat shock and before heat shock were generated using quantitative real‐time PCR. After 2 h of heat shock, the cytb gene expression was significantly enhanced. This is the first report that shows complete cytb gene among hypotrichous ciliates. Currently, we are investigating whether the method of cloning cytb rapidly are fit for other hypotrichous ciliates so as to analyze their phylogenetic relationship. This work was supported by a grant from NSFC (No: 30370210) to L. P. JIN.