[Screening and isolation of the cerebellar specific expressed mRNA in mouse: subtractive hybridization by phenol emulsion reassociation technique].
There are extremely complicated and numerous gene expressions in mammalian brains. Each region of the brain contains a set of mRNA expressed only in that region and these mRNA would be related to presumed region of specific function. Genes preferentially expressed in cerebellum (CB) are therefore the reasonable candioate genes for site of genetic lesions in CB neurodegenerative disorders. In this study, the subtractive hybridization was introduced into screening and isolating CB specific mRNA. Testing CB and front cortex (CTX) cDNA libraries were built, which covered most low abundantly expressed mRNA. By applying over 100 fold excessive CTX and liver single strand cDNA to the CB double strand cDNA, subtractive hybridization was carried out by phenol-emulsion-reassociation-technique (PERT), in which the common expressed housekeep genes would be eluted by restrict site ligation, and CB specific cDNA flanking with EcoRI site in both ds cDNA ends could be cloned into lamda gill phage. PERT subtraction reduced CB cDNA libraries from initial 5.15 x 10(6) to remaining 2.5 x 10(3) recombinants, which were most likely CB specific expressed cDNA. Random Twenty recombinants (over 2 kb inserts) were amplified randomly by polymerase chain reaction (PCR) and were hybridized with CB cDNA and liver cDNA probes in dot blot. 14 inserts positively hybridized with CB cDNA probe and only 3 inserts showed signals with liver cDNA probe. Colony blot presented similar results. 10 cDNA clones which affinited to CB cDNA probe were selected for the Northern blot. In five tissures, 2 cDNA clones binded only in CB mRNA channel, 6 were proved preferential expression in CB and low abundant expression in 4 other tissues, 1 clone was a housekeep gene and 1 could not be detected in all tissues. The partially sequencing of clone PC7 and PD8 were introduced into Computer Gene Bank Data Base. No homologous complements were found between these two clones and over ten thousands genes that have been sequenced. The two clones were newly reported. According to our experiment, we believe that: 1) CB specifically expressed mRNAs are much less than expected comparing with other brain regions; 2) most of CB mRNA expressions are in a preferential way other than unique expression.
Wei
J J
JJ
Dept. Med. Genet., Shandong Med. University, Jinan.
Hofstetter
R J
RJ
Hodes
M E
ME
D000818:Animals; D001483:Base Sequence; D002531:Cerebellum / Q000737:chemistry*; D004247:DNA / Q000302:isolation & purification; D004655:Emulsions; D005260:Female; D015870:Gene Expression; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000302:isolation & purification*
D004655:Emulsions; D012333:RNA, Messenger; D004247:DNA