Isolation of clones of rat striatum-specific mRNAs by directional tag PCR subtraction.
We report an improved subtractive cDNA cloning procedure, named "directional tag PCR subtraction," for isolating clones of mRNAs enriched in a target tissue compared to a second tissue, the driver. In this method, the target and driver are prepared from directional cDNA libraries constructed in different vectors, and the target cDNA contains tag sequences at both its 5' and 3' ends for PCR amplification. This method avoids several limitations of previous subtraction procedures, and was demonstrated to be technically easy and efficient. Using the directional tag PCR subtraction and improved screening procedures, cDNA clones corresponding to mRNAs expressed in the striatum but not in the cerebellum of the rat brain were efficiently isolated, including mRNAs encoding calmodulin-dependent phosphodiesterase, a transcriptional regulatory protein, and several previously uncharacterized species. Our data suggest that approximately 1% of the striatal polyA+ RNA mass potentially encoding more than 300 distinct proteins corresponds to RNA species reduced in concentration or absent from the cerebellum, of which about one-third are expressed prominently only in the striatum. This unexpected finding suggests that the striatum has a unique biochemical character within the brain, and that characterization of these mRNAs will be important for understanding the biochemical basis of striatal function.
Usui
H
H
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
Falk
J D
JD
Dopazo
A
A
de Lecea
L
L
Erlander
M G
MG
Sutcliffe
J G
JG
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular / Q000379:methods*; D003342:Corpus Striatum / Q000737:chemistry*; D004247:DNA / Q000032:analysis; D015342:DNA Probes; D015870:Gene Expression; D015723:Gene Library; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000302:isolation & purification*; D051381:Rats; D017207:Rats, Sprague-Dawley
D015342:DNA Probes; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D004247:DNA