Journal of Neuroscience Research 27:153-158 (1990) Patterns of Cerebral Cortex mRNA Expression zyxw J. Bernal, M. Godbout, K.W. Hasel, G.H. Travis, and J.G. Sutcliffe Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla. California A pool of 163 clones, isolated by screening 60,000 members of a zyxwvutsr Macaca fascicularis cerebral cortex cDNA library with a cortex-minus-cerebellum sub- tracted probe prepared by the phenol enhancement method, was analyzed by Northern blot hybridization studies. One hundred fifty-three of these clones cor- responded to 22 RNAs whose abundance was at least 2-fold higher in cerebral cortex poly(A) zyxwvut + RNA sam- ples than in samples of cerebellar poly(A)+ RN4. Seven of these RNAs, represented by 131 clones, were undetectable in cerebellum. Only 10 of the 163 clones proved to be false positives. The abundance of several of these cortex-enriched RNAs was altered in Alzhei- mer's disease brains. Several RNAs that were present in cerebral cortex but undetectable in cerebellum were generally enriched in telencephalon, although none was restricted to the cortex. One of the cortex enriched RNAs, whose nucleotide sequence is presented, en- coded monkey preprocholecystokinin. Overall, this study provides insights into the powers and limitations of subtractive hybridization and into the patterns of gene expression in the central nervous system. Key words: subtractive hybridization, Alzheimer's disease, cholecystokinin INTRODUCTION It is not known to what extcnt differences in mor- phology and function of neurons is the result of differ- ences in each cell's pattern of gene expression. Genes expressed in subsets of neurons may encode proteins whose functions relate directly to the speciali7ed func- tions of those particular cells. An example of this is the limited distribution pattern of the D2 dopamine receptor (Meador-Woodruff et al., 1989) compared to thc pan- neuronal distribution of neurofilament proteins. Previous work from this laboratory has been aimcd toward detect- ing messenger RNAs (mRNAs) whose expression is lim- ited to the brain, detcrmining their structures, then char- acterizing the protein predicted to be encoded by cach To identify brain KNAs whose expression might be restricted to the cerebral cortex, we cmployed the tech- nique of subtractive hybridization. Pilot studies (Travis et a]., 1987) revealed that, in the cynomolgus monkey zy (Macaca ,jh,sciculari,s) brain, the most abundant RNA (rcprcscntcd by clone 1H8a), expressed in the ccrcbral cortex but not detectable in the cerebellum, represented only approximately O.OS-O.lo/C of the cortical poly(A)' RNA mass. This abundance rcprescnts the lower limit of detection of differential colony hybridization. To detect clones of lower abundance, cortex-enriched KNAs. we developed a more sensitive subtractive hybridization pro- cedure utilizing cloned driver and phenol emulsion hy- bridization conditions (PERT). Application of this method led to the isolation of clones of RNAs expressed at levels as low as 0.001% (Travis and Sutcliffe, 1988, detailed method in Travis et al., 1989). From 60,000 members of a monkey cortex cDNA library screened with a cortex-minus-cercbelluni sub- tracted probe prepared by this method, 163 positives were identified, including one clone of the mRNA for preprosomatostatin ('Travis and Sutcliffe, 1988). The goal of thc present study is to elucidate the nature of thc RNAs corresponding to this group of 163 clones so as to appraise further this technology and to gain insights into the patterns of gene expression within the brain. MATERIALS AND METHODS Or thc 163 clones isolated by the cortex-minus- cerebellum PERT subtractive hybridization experiment described in the original publication (Travis and Sutc- Received April 6> 1990: revised May 10, IYYO: acceptcd May I I. I 090. G.H. l'ravia zyxwvu IS now at Department of Psychiatry F5.400, UT South- western Medical Center, 5323 Harry Hines Blvd, Dallas. TX 75235- 9070 'IRNA' Recent work has been directed towards RNAs that are restricted to particular regions of the J. Bernal is now at Instituto Investipaciones Biorriedicas, Facultad de Medicina de la Universidad Autonoma, Arzobispo Morcillo, 4 28U29 brain. Madrid. Spain zyxwvu G 1990 Wiley-Liss, Inc.