[In vitro cleavage activity of the ribozymes constructed specifically for the second exons of pro alpha I and III collagen genes].
OBJECTIVE
To study the in vitro cleavage activity and the reaction conditions of the hammerhead ribozymes for the second exons of pro alpha 1I and III collagen genes, and observe the effect of the two ribozymes on collagen synthesis by the fibroblasts in scar tissue.


METHODS
The fragments of the second exons of pro alpha 1I and III collagen genes were cloned into the plasmids pT-I and pT-III and labeled with (32)P during transcription to obtain the target RNA. The transcription products of the plasmids pT-gI and pT-gIII containing specific ribozymes were incubated with the label target RNAs, respectively, under various conditions, and the results observed by electrophoresis autoradiography. The constructed ribozymes were transduced into cultured fibroblasts via liposomes for investigation of their effects on mRNA synthesis of type I and III collagen protein by image analysis.


RESULTS
The two ribozymes (rgI and rg III) could efficiently cleave their target RNAs at both 37 degrees Celsius and 42 degrees Celsius, in the presence of Mg(2+) within a relatively wide concentration range form 10 to 20 mmol/L. When the temperature was lowered from 65 degrees Celsius to 37 degrees Celsius and maintained at 37 degrees Celsius, the cleavage activity of the ribozymes reached the maximum. The expression of mRNA of I and III collagen decreased accompanied by reduced collagen synthesis.


CONCLUSIONS
Hammerhead ribozymes for the fragments of the second exons of pro alpha 1I and III collagen genes can cleave its target RNAs in vitro and effectively inhibit the collagen synthesis by the scar-derived fibroblasts.
Zhu
Jia-yuan
JY
Department of Burns, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China. zhujiayuan@163.net
Zhu
Bin
B
Xu
Bing
B
Zhang
Tao
T
Chen
Dong
D
Tang
Bing
B
Li
Xin-qiang
XQ
D002921:Cicatrix / Q000378:metabolism / Q000628:therapy; D003094:Collagen / Q000096:biosynthesis; D024042:Collagen Type I / Q000235:genetics*; D005091:Exons; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D016337:RNA, Catalytic / Q000494:pharmacology*
D024042:Collagen Type I; D016337:RNA, Catalytic; D003094:Collagen