Identification 、Characterization and Sequence Analysis of smt3 Related Genes fo Caenorhabditis elegans

• Main Authors: Ming-Chang Yang, 楊明昌
• Other Authors: Chung-Long Cho
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/28751825823213314324

碩士 === 國立中山大學 === 生物學系 === 87 === Caenorhabditis elegans is a 1-mm long, cylinder shape, soil-living nematode. It has been used as a model organism for biological research since 1963. Its life cycle take about 3 ~ 4 days, and consists of six stages include eggs, embryo, first-stage larvae (L1), second-stage larvae (L2), third-stage larvae (L3), fourth-stage larvae (L4), adults and dauer larvae. The size of genomic DNA of C. elegans is approximately 97 Mega base pair nucelotides within its six chromosomes. We used C. elegans as an experimental organism and try to identify hsmt3 family gene homologues. Using RT-PCR strategy, we obtained a hsmt3b homologue of C. elegans and termed cesmt3b. There were 3 different residues between hsmt3b and cesmt3b through alignment of the amino acid sequence. The different amino acids are threonine , glutamic acid 69, and leucine 84 instead of Ala12, Asp69, and Leu84, respectively. The smt3b protein was identical in human, bovine, porcine, mouse and rat. The residued difference between hsmt3b and cesmt3b revealed that this gene highly conserved. The situation is the same as ubiquitin. The protein-coding region of cesmt3b was 285 bp and deduced 95 amino acids. The amino acid identity of cesmt3b with hsmt3a, hsmt3b, hsmt3c were 91%, 96%, 45%, respectively. The transcribed size was 2.0 kb and was expressed in L2, L3, adult, dauer larvae stages. Northern blotting data showed additional 3.4, 8.0 kb hybridization signals were that supposed to be mRNA expression signals of cesmt3b fusion genes. The molecular weight of cesmt3b expressed fusion protein was 14 kDa. The expression of cesmt3b was inhibited by 12 hours taxol treatment at dauer larvae stage. This 3.4 kb signal also was disappeared after DMSO, heat shock, taxol, PAsO, nocodazole, vinblastine treatment. Another fragment was amplified by PCR method based on the sequence data of hsmt3a. The DNA sequence was submitted to BLAST search program and shown identity to a gene that is similar to ETS family oncoprotein DNA binding domain. Both cesmt3b and ETS-like fragment might play important roles in mitosis, microtubule integrity, centromere / kinetochore structure maintenance mechanisms.