Abstract:
A method of diagnosing, assessing susceptibility, and/or treating schizophrenia involving the observation of regulator of G-protein signaling 4 (RGS4) levels in a subject. Embodiments of the present invention include increasing RGS4 expression levels in the cortex, either by chemical means or by genetic complementation (e.g. gene therapy).

Description:
[0001] This invention was made with United States Government support in the form of Grant Nos. MH45156, MH01489, MH56242, MH53459, and MH45156 from the National Institute of Mental Health. The United States Government may have certain rights in the invention. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The present invention relates generally to the field of neurological and physiological dysfunctions associated with schizophrenia. The invention further relates to the identification, isolation, and cloning of genes which, when mutated or varied, are associated with schizophrenia. The present invention also relates to methods for diagnosing and detecting carriers of the genes and to diagnosis of schizophrenia. The present invention further relates to the construction of animal models of schizophrenia.  
         BACKGROUND OF THE INVENTION  
         [0003]    Schizophrenia is a serious brain disorder that affects approximately 1% of the human population. The cause of this complex and devastating disease remains elusive, although genetic, nutritional, environmental, and developmental factors have been considered. A combination of clinical, neuroimaging, and postmortem studies have implicated the dorsal prefrontal cortex (PFC) as a prominent site of dysfunction in schizophrenia.  
           [0004]    Schizophrenia is typically characterized as a disorder of thinking and cognition, as contrasted to other disorders of mental faculties, such as mood, social behavior, and those affecting learning, memory, and intelligence. Schizophrenia is characterized by psychotic episodes during which an individual may lose the ability to test reality or may have hallucinations, delusions, incoherent thinking, and even disordered memory. There are varying forms of schizophrenia differing in severity, from a schizotypal disorder to a catatonic state. A review of schizophrenia can be found in  Principles of Neural Science,  3 rd  ed., 1991, Kandel, Schwartz, and Jessel (Eds.), Connecticut: Appleton &amp; Lange, pp.853-868; of which Chapter 55 is incorporated herein by reference.  
           [0005]    Diseases of organ systems, such as those of the heart, lung, and kidney, are usually confirmed by tissue pathology. A demonstrable pathology includes identifying and defining a structural abnormality in the organ, along with an associated alteration in organ function. This type of diagnosis is also utilized in certain neurological diseases. However, there are few psychiatric disorders in which clinical manifestations and symptoms can be correlated with a demonstrable pathology. The majority of mental illnesses are evaluated by observing changes in key behaviors such as thinking, mood, or social behavior. These alterations are difficult to ascertain and nearly impossible to quantify. However, progress is being made in diagnosing mental illness and in determining the neuropathology of mental illnesses.  
           [0006]    The Diagnostic and Statistical Manual of Mental Disorders, Third Edition (DSM-III-R) and the updated DSM-IV, published by the American Psychiatric Association, represent the progress made in providing a basis for objective and rigorous descriptive criteria for categories of psychiatric disorders. While the DSM-III-R is very thorough and detailed, it is also quite lengthy. Thus, the process of reviewing the categories and applying them to data from a patient is also very time-consuming and arduous. In addition, there is no mechanism by which a patient can be diagnosed either as having or being susceptible to schizophrenia prior to the expression of symptoms. Thus, there is a longstanding need for an easy and definitive method for diagnosing schizophrenia. A diagnostic tool that can be applied prior to the expression of symptoms would also have great utility, providing a basis for the development of therapeutic interventions.  
           [0007]    There is strong evidence for a genetic linkage of schizophrenia. Historically, there have been a number of studies on monozygotic twins of schizophrenics that indicated that 30-50% of the twins also had schizophrenia. The fact that this number is not 100% indicates that there are other factors involved in this disease process that may protect some of these individuals from the disease. It is apparent from a number of studies that the patterns of inheritance in most forms of schizophrenia are more complex than the classical dominant or recessive Mendelian inheritance. Recently, locus 1q21-22, a chromosome region containing several hundred genes, has been strongly linked to schizophrenia as shown by Brzustowicz et al.,  Science  288, 678-82, 2000, which is hereby incorporated by reference.  
           [0008]    Until the 1950&#39;s there were no specific, effective treatments for schizophrenia. Antipsychotic drugs were identified in the 1950&#39;s, and these drugs were found to produce a dramatic improvement in the psychotic phase of the illness. Reserpine was the first of these drugs to be used and was followed by typical antipsychotic drugs including phenothiazines, the butyrophenones, and the thioxanthenes. A new group of therapeutic drugs, typified by clozapine, has been developed and were referred to as “a typical” antipsychotics. Haloperidol has been employed extensively in the treatment of schizophrenia and is one of the currently preferred options for treatment. When these drugs are taken over the course of at least several weeks, they mitigate or eliminate delusions, hallucinations, and some types of disordered thinking. Maintenance of a patient on these drugs reduces the rate of relapse. Since there is no way of determining if an individual is susceptible to schizophrenia, it is currently unknown if these antipsychotic compounds are useful in the prophylactic treatment of schizophrenia.  
           [0009]    Signal transduction is the general process by which cells respond to extracellular signals (e.g. neurotransmitters) through a cascade of biochemical reactions. The first step in this process is the binding of a signaling molecule to a cell membrane receptor that typically leads to the inhibition or activation of an intracellular enzyme. This type of process regulates many cell functions including cell proliferation, differentiation, and gene transcription.  
           [0010]    One important mechanism by which signal transduction occurs is through G-proteins. Receptors on the cell surface are coupled intracellularly to a G-protein that becomes activated, when the receptor is occupied by an agonist, by binding to the molecule GTP. Activated G-proteins may influence a large number of cellular processes including voltage-activated calcium channels, adenylate cyclase, and phospholipase C. The G-protein itself is a critical regulator of the pathway by virtue of the fact that GTPase activity in the G-protein eventually hydrolyzes the bound GTP to GDP, restoring the protein to its inactive state. Thus, the G-protein contains a built-in deactivation mechanism for the signaling process.  
           [0011]    Recently, an additional regulatory mechanism has been discovered for G-protein signaling that involves a family of mammalian gene products termed regulators of G-protein signaling, or RGS (Druey et al., 1996, Nature 379: 742-746 which is hereby incorporated by reference). RGS molecules play a crucial modulatory role in the G-protein signaling pathway. RGS proteins bind to the GTP-bound Gα subunits with a variable Gα specificity as a substrate. RGS molecules shorten the GTP binding of the activated Gα subunits by acting as GTPase activating proteins (GAPs), accelerating GTP hydrolysis by up to one hundred fold. By the virtue of this GAP action and by making available the GDP-bound Gα to re-attach to βγ dimers, RGS proteins shorten the duration of the intracellular signaling. RGS proteins are expressed in nearly every cell; however, they show a tissue-specific expression across the body and cell type-specific expression in the brain. For example, RGS4 is strongly expressed in the central nervous system, moderately expressed in the heart, and slightly expressed in skeletal muscle (Nomoto et al., 1997, Biochem. Biophys. Res. Commun. 241(2):281-287 which is herein incorporated by reference).  
           [0012]    Several members of the G-protein signaling pathways, most located downstream of RGS4 modulation, have been implicated in schizophrenia. Gil, Gq and Golf messenger RNA (mRNA) and protein levels all have been reported to be altered in various brain regions of the schizophrenic subjects. Furthermore, changes in expression of adenylate cyclase, phospholipase C, and protein kinases, as well as DARPP (dopamine- and cAMP-regulated phosphoprotein) phosphorylation changes are expected to be influenced by RGS regulation of Gα signaling. In addition, RGS modulation changes are expected to have significant effects on the signal transduction effected by neurotransmitters including dopamine, serotonin, GABA, glutamate, and norepinephrine.  
           [0013]    An additional genetic marker of schizophrenia has been identified by Meloni et al. (U.S. Pat. No. 6,210,879).  
           [0014]    These investigators found that an allele of the microsatellite HUNTH01 in the tyrosine hydroxylase gene correlated with the expression of schizophrenia.  
           [0015]    However, the allele only appears to be present in sporadic schizophrenias.  
           [0016]    There has been a long-standing need for a definitive and easy method for diagnosing schizophrenia as well as for an effective treatment with minimal side effects.  
           [0017]    Further, a need has been recognized in connection with being able to detect schizophrenia prior to the expression of noticeable symptoms.  
           [0018]    A need has been recognized in connection with overcoming the various limitations to the current implementation of a method for diagnosing and assessing the susceptibility to schizophrenia are addressed through the use of the current invention.  
         SUMMARY OF THE INVENTION  
         [0019]    In accordance with at least one embodiment of the present invention, there is provided a system and method for diagnosing and determining the susceptibility to schizophrenia.  
           [0020]    In summary, one aspect of the present invention provides an isolated and substantially purified DNA sequence corresponding to SEQ ID NOS: 3, 4, 5, 6, 7, 8, and contiguous portions thereof.  
           [0021]    Another aspect of the present invention is a polynucleotide sequence that is complementary to a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID  
           [0022]    NO: 7, SEQ ID NO: 8, and contiguous protions thereof.  
           [0023]    A further aspect of the present invention is an expression system comprising a DNA sequence that corresponds to SEQ ID NO:3.  
           [0024]    A yet further aspect of the present invention is a method for diagnosing schizophrenia in a human comprising obtaining a DNA sample comprising a RGS4 gene from a patient and detecting a variation in the RGS4 gene indicating schizophrenia.  
           [0025]    A still further aspect of the present invention is a method for determining the susecptiblity to schizophrenia comprising obtaining from a patient a DNA sample comprising a RGS4 gene and detecting a variation in said RGS4 gene indicating susceptibility to schizophrenia.  
           [0026]    An additional aspect of the present invention is a method for daignosing schizophrenia comprising obtaining from a patient to be tested for schizophrenia a sample of tissue, measuring RGS4 mRNA levels in said sample, and determing if there is a reduced level of RGS4 mRNA in the sample.  
           [0027]    A still additional aspect of the present invention is a method of determing susceptibility to schizophrenia comprising obtaining from a patient to be tested for susceptibility to schizophrenia a sample of tissue, measuring RGS4 mRNA levels in said sample, and determing if there is a reduced level of RGS4 mRNA in the sample.  
           [0028]    A yet further aspect of the present invention is A method of determining susceptibility to schizophrenia comprising obtaining from a patient to be tested for susceptibility to schizophrenia a sample of tissue, measuring RGS4 protein levels in said sample, and determining if there is a reduced level of RGS4 protein in the sample.  
           [0029]    Yet another aspect of the present invention is A method of treating schizophrenia, said method comprising measuring RGS4 protein or mRNA levels in a patient, and altering said RGS4 protein levels to provide the patient with an improved psychiatric function.  
           [0030]    Another aspect of the present invention is a kit for diagnosising schizophrenia in a patient, said kit comprising antibodies to RGS4, and a detector for ascertaining whether said antibodies bind to RGS4 in a sample.  
           [0031]    Another aspect of the present invention is a kit for diagnosising schizophrenia in a patient, said kit comprising a detect of RGS4 transcript levels in a patient, and a standard to ascertain altered levels of RGS4 transcript in the patient.  
           [0032]    A still further aspect of the present invention is the DNA sequence of SEQ ID NO: 3 containing variations as described in the text below.  
           [0033]    A yet further aspect of the present invention is a transgenic mouse whose genome comprises a disruption of the endogenous RGS4 gene, wherein said disruption comprises the insertion of a transgene, and wherein said disruption results in said transgenic mouse not exhibiting normal expression of RGS4 protein.  
           [0034]    A still additional aspect of the present invention is a transgenic mouse wherein a transgene comprises a nucleotide sequence that encodes a selectable marker.  
           [0035]    These and other embodiments and advantages of the present invention will be better understood with reference to the following figures and detailed description. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0036]    The present invention and its presently preferred embodiments will be better understood by reference to the detailed disclosure hereinbelow and to the accompanying drawings, wherein:  
         [0037]    [0037]FIG. 1A displays the design of microarray immobilized probes and in situ probes for RGS4, wherein numbers on the RGS4 nucleic acid fragments denote nucleotide position in relationship to the RGS4 mRNA, as currently in the NCBI database;  
         [0038]    [0038]FIG. 1B is a pseudocolored intensity view of a single RGS4 feature from the 516 control/547 schizophrenic PFC comparison after a dual-fluorescent hybridization; both images represent the same spot under cy3 and cy5 excitation, respectively; the balanced cy3 signal intensity α-control subject) was 6.2-fold brighter than the cy5 signal intensity (s-schizophrenic subject);  
         [0039]    [0039]FIG. 1C displays changes in RGS expression in the PFC of schizophrenic and control subjects reported by cDNA microarray analysis;  
         [0040]    [0040]FIG. 2A shows in situ hybridization results for PFC RGS4 expression levels which are decreased in 9 of 10 schizophrenic subjects;  
         [0041]    [0041]FIG. 2B shows the in situ hybridization data from 10 PFC pairwise comparisons which were quantified using film densitometry;  
         [0042]    [0042]FIG. 3A shows that 632 G-protein signalling-related genes were detected out of 1644 possible detections (274 genes/microarray×six microarrays);  
         [0043]    [0043]FIG. 3B shows that 239 1q21-22 locus-related genes were detected out of 420 possible detections (70 genes/mircoarray×six microarrays); RGS4 contribution to the transcript distribution is denoted by a hatched bar;  
         [0044]    [0044]FIG. 4A shows high power photomicrographs of VC tissue sections from the same matched pair of schizophrenic and matched control subjects represented in FIG. 2A, viewed under darkfield illumination;  
         [0045]    [0045]FIG. 4B shows a graph of 10 supragranular VC SCH pairwise comparisons, in which schizophrenic subjects showed a comparably significant RGS4 transcript reduction to the PFC comparisons;  
         [0046]    [0046]FIG. 4C shows high power photomicrographs of MC tissue sections from the same matched pair of schizophrenic and matched control subjects represented in FOG. 2A, viewed under darkfield illumination;  
         [0047]    [0047]FIG. 4D shows a graph in which schizophrenic subjects across the same 10 subject pairs across the MC had comparably decreased RGS4 expression levels (mean=−34.2%, F 1,15 =10.18; p=0.006) to VC and PFC;  
         [0048]    [0048]FIG. 5 shows a scatter plot of relative RGS4 expression changes across the experimental groups.  
         [0049]    [0049]FIG. 6 displays the genomic organization that is derived from available sequences for clone NT — 022030, as well as the sequence analyses presented here; five exons were identified from the coding sequence for RGS4 (approximately 8.5 kb); the critical RGS domain is encoded by exons 3 to 5; the SNPs that were analyzed are listed in the top panel; * (a small star) indicates SNPs identified by re-sequencing the RGS4 gene and * (a large star) indicates SNPs used for association analysis. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0050]    The present invention focuses on the genetic underpinnings of schizophrenia. In the first phase of the research, cDNA microarrays were used to investigate potential alterations in transcript expression in six pairs of schizophrenic subjects. RGS4 was determined to be the most significantly and consistently changed transcript. In situ hybridization was also used to verify the microarray findings and to examine the regional and disease-related specificity of this change. Out of the several hundred genes on locus 1q21-22, the present studies indicate that RGS4 is a strong candidate for a major susceptibility gene on this locus. Genetic association and linkage studies were conducted using two samples independently in Pittsburgh and by the NIMH Collaborative Genetics Initiative. Using the Transmission Disequilibrium Test (TDT), significant transmission distortion was observed in both samples, albeit with different haplotypes. In support of the TDT results, increased sharing of alleles, identical by descent was observed for polymorphisms in this region among affected siblings of the NIMH cases, though associations were not observed when the cases were compared to a limited number of population-based controls. These analyses are consistent with the possibility that inheritable polymorphisms in the flanking untranslated regions (UTR) of the RGS4 gene confer susceptibility to schizophrenia.  
         [0051]    Expression Studies  
         [0052]    Two groups of human subjects, consisting of six and five pairs of schizophrenic and control subjects, were used in the present studies. Subject pairs were completely matched for sex (18 males and 4 females). The mean (±SD) difference within pairs was 4.6±3.5 years for age and 4.4±2.7 hours for post mortem interval (PMI). The entire group of schizophrenic and control subjects did not differ in mean (±SD) age at time of death (46.5±10.7 and 45.1±11.5 years, respectively), PMI (19.4±7.1 and 17.7±5.0 hours, respectively), brain pH (6.85±0.29 and 6.81±0.15, respectively), or tissue storage time at −80° C. (45.4±12.3 and 37.7±13.1 months, respectively) when the studies initiated. Nine of the schizophrenic subjects were receiving antipsychotic medications at the time of death, five had a history of alcohol abuse or dependence, and one died by suicide. Also studied were 10 subjects with major depressive disorder (MDD), each of whom were matched to one normal control subject. The MDD subject pairs were also completed matched for sex (18 males and 2 females). The mean (S.D.) difference within pairs was 1.2+1.4 years for age and 2.5±2.1 hours for PMI. The depressive and control subjects did not differ in mean (±S.D.) age at time of death (52.7±13.1 and 52.1±13.1 years, respectively), PMI (14.9±5.3 and 15.7±5.5 hours, respectively), brain pH (6.81±0.17 and 6.72±0.30), or tissue storage time at −80° C. (39.0±17.4 and 39.9±13.2 months, respectively). Two of the depressed subjects had a history of alcohol dependence, and six died by suicide. Two of the control subjects had also been matched to subjects with schizophrenia (685c, 604c). Consensus DSM-IIIR diagnoses were made for all subjects using data from clinical records, toxicology studies, and structured interviews with surviving relatives.  
         [0053]    RCS4 Transcript Analysis  
         [0054]    A Human Multiple Tissue Northern Blot (Clontech) and a  32 P-labeled cDNA probe were used to confirm the size of the RGS4 transcript reported previously (Druey, et al., 1996). However, our results reported the presence of single dark bands of ˜3 kB in lanes from multiple brain regions (whole cerebral cortex, frontal pole, occipital pole, temporal lobe), with much fainter or absent bands observed in lanes from other brain regions (cerebellum, medulla, spinal cord, putamen). Because the UniGene entry for the RGS4 cDNA (U27768) contained only the truncated transcript (800 bp), we designed custom PCR primers based on the BAC clone sequence containing the RGS4 gene (NT — 022030) to rapidly obtain the full-lenght RGS4 transcript sequence. For this analysis, mRNA from a control human brain was purified, DNased, and re-purified prior to first strand cDNA synthesis using Superscript II (Gibco) with an oligo dT primer. The resulting cDNA-mRNA mixture was diluted and used in a standard PCR reaction using AmpliTaq Gold (see above). All reaction products yielded single bright bands on 2% agarose/ethidium bromide-stained gels, and were subsequently purified and sequenced. Alignment of these sequences produced &gt;99% identity matches with the BAC clone sequence containing RGS4. The 3′ UTR for RGS4 obtained in this manner also aligned &gt;99% with a cDNA entry (AL137433.1) that contains both a poly A signal and a poly A attachment site, confirming that the human RGS4 transcript is 2949 bp without the poly A tail and includes a cDNA entry not previously associated with the human transcript in the NCBI database (see below; FIG. 6).  
         [0055]    Microarray Experiments  
         [0056]    Fresh-frozen human tissue was obtained from the University of Pittsburgh&#39;s Center for the Neuroscience of Mental Disorders Brain Bank. Area 9 from the right hemisphere was identified and isolated and sectioned into tubes at −24° C. as described previously by Glantz, L. A. and Lewis, D. A. in Arch Gen Psychiatry 54: 943-952, 2000, which is herein incorporated by reference. Total RNA and mRNA were isolated according to manufacturer&#39;s instructions using Promega (Madison, Wis.) kit #Z5110, RNAgents® Total RNA Isolation System and Qiagen (Valencia, Calif.) kit #70022, Oligotex mRNA Kits, respectively. The volume was adjusted using Microcon columns YM-30 #42409 to 50 ng/μl. The quality and purity of the mRNA used in the reverse transcription labeling reactions was evaluated by size distribution on a 1% non-denaturing agarose gel (&gt;50% of mRNA smear over 1 kb; integrity of rRNA bands) and optical density (OD) measurements (260/280&gt;1.80), respectively.  
         [0057]    Sample Labeling, Microarrays, Hybridization, and Data Analysis  
         [0058]    Labeling was performed at Incyte Genomics, Inc. (Fremont, Calif.). Two hundred nanograms of mRNA was reverse transcribed using cy3- or cy5-labeled fluorescent primers; appropriate matched control and schizophrenic sample pairs were combined, and hybridized onto the same UniGEM-V cDNA microarray. Each UniGEM-V array contained over 7,000 unique and sequence-verified cDNA elements mapped to 6,794  UniGene Homo sapiens  annotated clusters found at the following NIH website: “http://www.ncbi.nlm.nih.gov/UniGene/index.html”. Hybridization and washing was performed using proprietary Incyte protocols. If a gene or expressed sequence tag (EST) was differentially expressed, the cDNA feature on the array bound more of the labeled probe from one sample than the other, producing either a greater cy3 or cy5 signal intensity. The microarrays were scanned under cy3-cy5 dual fluorescence, and the resulting images were analyzed for signal intensity. If the cy3 vs. cy5 signal intensity was within three fold, and the microarray detected spiked-in control standard less abundant than 1 copy in 50,000, the raw data were exported to a local SQL server database. On the server, the data were further analyzed using GemTools (Incyte&#39;s proprietary software) and MS-Excel 2000. Note that the operators performing the labeling, hybridization, scanning, and signal analysis were blind to the specific category to which each sample belonged.  
         [0059]    Gene Expression Criteria  
         [0060]    A gene was considered to be expressed if the DNA sample was successfully amplified by PCR, produced signal from at least 40% of the spot surface, and had a signal/background ratio over 5-fold for either the cy3 or cy5 probe. Based on Incyte&#39;s control hybridization studies (“http://www.incyte.com/reagents/gem/products.shtml/GEM-reproducibility.pdf”) and control experiments, array data reliability and reproducibility cutoffs were established as follows:  
         [0061]    1. Genes were comparably expressed between the control and experimental samples if the cy3/cy5 ratio or cy5/cy3 ratio was &lt;1.6.  
         [0062]    2. Gene expression was changed between the two samples at the 95% confidence level (95% CL) if the cy3/cy5 or cy5/cy3 signal was 1.6-1.89.  
         [0063]    3. Gene expression was changed between the two samples at the 99% confidence level (99% CL) if the cy3/cy5 or cy5/cy3 signal was &gt;1.9. In the control experiments, &lt;0.5% of the observations fell into this category.  
         [0064]    Gene Group Analysis  
         [0065]    Of the genes represented on the array, a G-protein group was created for data analysis, and included transcripts on the microarray for G-protein-coupled receptors (GPCR), heterotrimeric G-protein subunits, Ras proteins, regulator of G-protein signaling (RGS) molecules, and G-protein-dependent inward rectifying potassium channels (GIRKs), totaling 274 genes.  
         [0066]    At least two genes, RGS4 (Unigene cluster Hs 227571) and RGS5 (Unigene cluster Hs 24950) were mapped to the cytogenetic band 1q21-22. In order to determine whether there is altered expression of multiple genes mapped to this locus, a 1q21-22 group was created from genes represented on the microarray locus. The 1999 NCBI database human 1q21-22 map is represented by 70 genes on the microarray, although some of them are not expressed in the central nervous system.  
         [0067]    RGS4 Sequences  
         [0068]    The RGS4 microarray immobilized probes sequence matched the entry in the NCBI database (accession number U27768, UniGene cluster Hs.227571). Of the 800 bp full-length mRNA, the double-stranded DNA microarray immobilized probe was complementary to the 3′ region of 571 nucleotides, as shown in FIG. 1A. The anti-sense, in situ hybridization probe was derived from the mRNA region spanning nucleotides 39-739, resulting in a 700 nucleotide long cRNA probe (see below). The RGS4 cDNA sequence, as determined from the complete mRNA coding sequence is listed as follows:  
                               gtacgctcaa agccgaagcc acagctcctc ctgccgcatt tctttcctgc ttgcgaattc   60                   caagctgtta aataagatgt gcaaagggct tgcaggtctg ccggcttctt gcttgaggag   120               tgcaaaagat atgaaacatc ggctaggttt cctgctgcaa aaatctgatt cctgtgaaca   180               caattcttcc cacaacaaga aggacaaagt ggttatttgc cagagagtga gccaagagga   240               agtcaagaaa tgggctgaat cactggaaaa cctgattagt catgaatgtg ggctggcagc   300               tttcaaagct ttcttgaagt ctgaatatag tgaggagaat attgacttct ggatcagctg   360               tgaagagtac aagaaaatca aatcaccatc taaactaagt cccaaggcca aaaagatcta   420               taatgaattc atctcagtcc aggcaaccaa agaggtgaac ctggattctt gcaccaggga   480               agagacaagc cggaacatgc tagagcctac aataacctgc tttgatgagg cccagaagaa   540               gattttcaac ctgatggaga aggattccta ccgccgcttc ctcaagtctc gattctatct   600               tgatttggtc aacccgtcca gctgtggggc agaaaagcag aaaggagcca agagttcagc   660               agactgtgct tccctggtcc ctcagtgtgc ctaattctca cctgaaggca gagggatgaa   720               atgccaagac tctatgctct ggaaaacctg aggccaaata ttgatctgta ttaagctcca   780               gtgctttatc cacattgtag cctaatattc atgctgcctg ccatgtgtga gtcacttcta   840               cgcataaact agatatagct tttggtgttt gagtgttcat cagggtggga ccccattcca   900               gtccaatttt cctaagtttc tttgagggtt ccatgggagc aaatatctaa ataatggcct   960               ggtaggtctg gattttcaaa gattgttggc agtttcctcc tcccaacagt tttacctcgg   1020               gatggttggt tagtgcatgt cacatgacat ccacatgcac atgtattctg ttggccagca   1080               cgttctccag actctagatg tttagatgag gttgagctat gatatgtgct tgtgtgtatg   1140               tctatgtgta tatattatat atacattaga cacacatata cattatttct gtatatagat   1200               gtctgtgtat acatatgtat gtgtgagtgt atgtatacac acacacacac acacacacac   1260               acacttttgc aagagtgatg ggaaagaccc taggtgctca taactagagt atgtgtatgt   1320               acttacatgg gtgttttgat ctctgttctt tcatactaca tttgaacagg gcaaaatgaa   1380               ctaactgcca tgtaggctaa gaaagaaatg ctaacctgtg gaaagttggt tttgtaaaat   1440               tccatggatc ttgctggaga agcatccaag gaacttcatg cttgatttga ccactgacag   1500               cctccacctt gagcactatt ctaaggagca aataccttag ctcccttgag ctggttttct   1560               ctgatggcac ttttgagctc ctaagctgcc agccttccct tcttttcctg ggtgctcagg   1620               gcatgcttat tagcagctgg gttggtatgg agttggcaga caggatgttc aacttaatga   1680               agaaatacag ctaaggcctt gccagcaaca cctgccgtaa gttactggct gagtgagggc   1740               atagaagtta aaggttactg tttttatcct ctatcctttt ttcctttcct gatcaaggtg   1800               ctcttctcat tttttcctga gaaccttagc catcagatga ggctccttag tttattgtgg   1860               ttggttgttt tttctttata atggctctgg gctatatgcc tatatttata aaccagcagc   1920               aggggaaaga ttatatttta taagagggaa caaattttca caatttgaaa agcccacata   1980               agttttctct tttaaggtag aatcttgtta atttcattcc aaacatcggg gctaacagag   2040               actggaggca tttcttttta ggctctgaga ctaaatgaga ggaaaagaaa agaaaaaaaa   2100               aatgattgtc taaccaattg tgagaattac tgtttgaaac ttttcaaggc acattgaaat   2160               acttgaaaac ttctcattta tgttatttat gatgttattt tgtacgtgtt attattatta   2220               tattgtttta taaatggagg tacaggatat cacctgaatt attaatgaat gcccaggaag   2280               taattttctt ctcattcttc taaaactact gcctttcaaa gtgcacacac acgcgtccac   2340               atacactgca ttcgttgctc cagtataaat tacatgcatg agcacctttc tggcttttaa   2400               gccaatataa tgggctgcaa aatgaagaca ccagagtgta tgcatacaaa tctcactgta   2460               ttaaagatgc aggttttcta attgtaccct tcttgtctct ctggcaatct tgcccttaat   2520               atccctggag ttcctcatca gtgtcatttt ctgttataca cagttccaca attttgtctc   2580               tagttgactt caaatgtgta actttattgg tcttgcccta ttataattgt catgactttc   2640               agattgtatc tgaactcaca gactgctgtc ttactaatag gtctggaagg tcacgctgaa   2700               tgagaagtaa attattttat gtaatacatt tttgagtgtg tttttcagtt gtatttccct   2760               gttatttcat cactatttcc aatggtgagc ttgcctgctc atgctccctg gacagaatac   2820               tccttccttt tgcatgcctg tttctatcat gtgcttgata ggcctcaaag ctaatgcttc   2880               cagtgaaaca cacgcatctt aataataagg gtaaataaac gctccatatg aaac   2934          
 
         [0069]    For purposes of the present invention, the RGS4 cDNA will be referred to as SEQ ID NO:1.  
         [0070]    The 205 amino acid long sequence of RGS4, as determined and reported by Druey et al. in Nature, 379: 742-746 (1996) which is hereby incorporated by reference in its entirety, is listed as GenBank Accession number P49798 as follows:  
                                   MCKGLAGLPA SCLRSAKDMK HRLGFLLQKS DSCEHNSSHN                       KKDKVVICQR VSQEEVKKWA ESLENLISHE CGLAAFKAFL                       KSEYSEENID FWISCEEYKK IKSPSKLSPK AKKIYNEFIS                       VQATKEVNLD SCTREETSRN MLEPTITCFD EAQKKIFNLM                       EKDSYRRFLK SRFYLDLVNP SSCGAEKQKG AKSSADCASL                       VPQCA          
 
         [0071]    The above amino acid sequence of RGS4 is referred to as SEQ ID NO: 2 for purposes of the present invention.  
         [0072]    Untranslated regions upstream and downstream from the RGS4 coding region are identified in the context of the present invention as being relevant components of the RGS4 gene. The RGS4 coding sequence along with these sequences are found on NT — 022030 as described in greater detail below. This sequence is  
                               agttcaagac cagcctgagc aacatggtga aaccccatct ctactaaaaa tacaaaatta   60                   gacaggcatg gtgatacacg cctgtaatcc cagctacttc ggaggccgag gcaggagaat   120               cacttgaacc tgctgggggt ggaggttgcg gggagcaaga tcatgccatt gcactccagc   180               ccaggcaaca agagcgaaat gtcatctcag aaaaaaaaaa aggcatttta tatatatata   240               tatatatata tacacacaca cacacatata tatatacaca tatatataca catatataca   300               tatatacaca tatatacaca tatatataca catacatatg tacacatata tatacacata   360               tgtatacaca tatatacaca tatatacaca catatataca catatataca cacatatata   420               cacatatata cacatatata cacatataca catatataca catatataca tatatacaca   480               tatatataat atacacacat atatatacac atatatacac acatatatac acatatatac   540               acatatatat acacatatat acacatatat acatatatac acatatatat acatatatac   600               acatatatac atatatacac atatatacat atatacacac atatatacac atacatatac   660               acacacatag atatacatat atatacacat atatatacgt atatatatgt atatatatat   720               gctccagagt tcataagagg tagcagttga ttaccactgg ggatagagga aaagagagtt   780               tgacagcagt gtattgtgag aaggacattt caggttgatg gcaaatagta ggggaaatac   840               ataaatgtgt aataaaacct atctgtaagg tagttaagaa ggtaacacta tatatatata   900               tagtgaaagc agtgtaaacc taaaggatgg gccaaggatt taaatgttat agaagaatgg   960               ctaagatgcc aaagctcagt gtatgtggca gaggcatggt gtagggtgtg tccaggttca   1020               tatattgcat taagtgtgag aacaccctgg agtatgaacc aagaaaatgc aaaagccaga   1080               agtgatggag gaaatgagac acaataatga agatattgag aggagggtgt gggcctagag   1140               tgaagctttt cgtgccagta cttcttttga aggcccagtt ctcttctctc tcgggggctc   1200               cttcatctct catagagtcc acagctttta agggccaaca cttgaggtca gcctggctct   1260               ctcatttgag ctggatagaa cattttagag caccatctat tcttcaagag gaagtttaaa   1320               aataaaagaa ccttgaagag gaaaaaatgt agacattcaa tctaaccttt tcattttact   1380               agccaaagct aaatagaatg caggttacct gtttttcagc caggcaccat catttcctaa   1440               ttgttataaa atttattatt attgttgtta ttattattat ttgccataag aagtttccca   1500               tatcctttta gtataacaaa aacacaattc acaagcatta taaaacccat ggtgtctaac   1560               tattaaaaaa attaagtgga acacacttgt cccagctact ggggaggctg aggagggagg   1620               atcacgtgat cccagggggt caaggttatg gagagctatg attgtgccac tgcactccag   1680               cctgggtgac agggaaagac cctgtctcta aaattttttt taaaaaaact aaactggttt   1740               tattacagag attctggaga cagctacaca taaaagggtg gtatgcctca tattagctac   1800               ccagggaggt ggaatgccaa cttaggtggt gtcaccacta ttaaaaatgc cccaaagcaa   1860               tcaaaactga gaacttcctg ggagcttagc attgtgcaaa agcagcacaa aacacttaaa   1920               caattcacag ttgtgttgga atgggaaggc ctggaaatat aaaccaaaga gtatattgtc   1980               taaattgata gagattacaa ttgcctgaaa gaaaaagttg acttttaact agaatgttca   2040               gagtaggttt acagaagaag ctcttaaact gggctccagt ggatttgtca atgctttgga   2100               agctggtggg gtgggagggt tggagggggc ataaaaagtc atgttggtat gctctgctca   2160               agtctccatt ctgtttcctt ttcctctttt caatgtcatg tcccattatt tcattatggg   2220               cttcccttta tccaggatca atatgccacc tcttggttgt cttttaccta cttctccacc   2280               tcactatgga atcgtccttg ggtagctcct gtgcttggga acctgcacgg gcacttttct   2340               gatgtcttga ttccagcttt actcctaaaa cttaaatgct gaggggccaa caccatggca   2400               gtggtaggga tgggaatggg ggtcttgtaa cacactacat aaactacacg aaataaacta   2460               catgaaactc aacatgtttg caagactcag ttcacatcca tgaggagctc atgcttctcc   2520               ctcctgctcc cctagcacac atgattatct ctatttggaa atgtttggca tttttggtga   2580               agtgaatggt tcaataactt tctccaccat cagaacaaaa gctctttaag gttagggatg   2640               ggatcataca cacttccctt gtccaagtcc ccatcacccc ttatctagac aattgctaca   2700               gtttcctaca cactcttcta acctcttgca gtctattttc ataaaacagc tagagaactt   2760               tgagatgtaa gtcaaaaaat agaacatgtc gctctttccc attgtttttg aaataaagtt   2820               caaccccctt accagggtca acaaggccct gcaatgattt ggtcctgtta aaaattcttt   2880               agccttaact catgctgttc ttccttacac tcactgcatt ctagccattg aggtttctat   2940               gcatcaaact ttttttggtc ccagcactgt gcacatcctt ctgggtagaa tgccccttga   3000               tttgtataat tagcacctcc ttcatcattt aggtcttagt ataactacta ccttcttaga   3060               gaagctctgc ttcttcatcc tataaaaaag taaaattcct taccctgtta ttttttaagt   3120               catccgtgtt tcattctgtt aaagttctta tcacaattta tcattatttt atttacagtc   3180               atgtgccaca taacaatgtt tcagtcaggg atagaacaca aatgtatctg gccccataat   3240               attataagct gagaaatttc tattaactag tgatatcgca gccatcataa gtgtaatgca   3300               ggacattacc ttttctatgt ttagatatgt tagatacaca aatatatttc attgtgttat   3360               aatttcctac agtattcagt acagtaacat gctgtacagg tttgtaacct aggagtaata   3420               ggctatacca tacagcttag gtgtgtagta ggctataacc atctaggttt gtgtaagtac   3480               attctatgat attcccacaa tgatgaaatc acctaactac acatttctca gaatgtttca   3540               ctgttgtgaa gtgacccatg actatatttt cctatatact tgatattttt gtgcatctgc   3600               ccatgagaat gtagtgtaag atcaaaggat gcaagaatgg gttctatcca gtatagtacc   3660               cactacactg gtggatgtca atatgtattt gttagattaa tatctcaaga atgagcacct   3720               ttctcagaca cataaaagat gctcaatata aaagtttgtt gaactgaacg ttattggcaa   3780               atgtaacatg atcggattta aagaggagcg aaacagaggt ctggctcaaa caccatactt   3840               ctagagtgca taagaggtag cagttgatta ccactggcga caggagaaaa aagagcttga   3900               ccgcagggta ctgtgaagac atttcaggtt gatggcacag aacaggggaa atacataaat   3960               gtgtgggaat attcagtggt ctgggatgac tacatagtag aatataatga agaaaagagt   4020               ggaagggaaa gatgaaaagt tggaatgggg atgaattatg aaagtaccag aatgttatgc   4080               taaggaatct agattttaaa atgtgagggc aaattgaagt  c c t gggcacg ttacaaaact   4140               agaggtcata aagtttaccc taatttacca agatttccta gaggatctat aattggaatc   4200               cagatctgcc tctctgtaaa gttcaagcac tttccatgac accatactgt ttctttccac   4260               ctgcacaatg caaatgaact cttatgaaac tgctgtttct atcctgggct aaatgttgca   4320               gaaaaaagat ttaatctttg ggataaggct attttgggtt ttctcct a ct tcttgggaaa   4380               caaggttttc ttcccctggc taattaagtg tggtattgtt cttccaggga aatcagtgat   4440               gcatcacctg ctgctatcaa atgtcagggt tggagttcct gatttattgc atgtgcccac   4500               aaagcttggt gcaaagaatt ggacacattt cccaaaagta agacatactg ggaagtccct   4560               gtttaccttc ctggtataca gcatcctcca gccccatatc tttgcttttt agtcctaaaa   4620                 a tcaataact gaactctcat tgatgtctag gccattgtag taaacaataa agaaggaggg   4680               aggcttctga caactgagag gaaattgtca tctgaagtgg tgcaagcaca gcctggggct   4740               gagccttggc ctacatcctg cccaagtgga ggatcagtg c cccatttaac atctggtaga     4800               actaaagaac gcaac g cctg ccacaatgac ttatttccct gcatttgata ccgtcaatcc   4860               ttgagaaatg ttttcttttg ttctccctga gcaaaggttg gaaaaatttg aaatttacct   4920               agagaccaca catagttcac atcctgctgt gtggctgaat gtctgcccc c  cagtaggaaa   4980               cagttcttct aaagcctatt gtcaacaata ccttccagat gttagcattt tacaatttaa   5040               ggaacttaaa atag c cttca aactttttgc cagtttctct gatatccaat ctattctttt   5100               actctgcctc ccaagctttc tttctagaat gctaacctga tcggcttaag tacttgaact   5160               acctcttctc ctccattaac tacagagtaa attctggtct tcagagtaac aagaaacacc   5220               ctttagttct cagcatattc gtgcaccttc atttatctct ccttctctct caaagctgca   5280               gtaggggtga aaac g tgtga tacattttct cttccatcat aagggtcgca accaaaactc   5340               ctatagtaaa agacaggtta ataagagcaa aacctaacaa atttatttaa tcaaagtttt   5400               acatgacatg ggagtcttca gaaatgaaga cccaaagacc caggggaaac tgtctgtttt   5460               ttttgctgag gttcgatgaa gaatggatag catgtagcca tgtagattag acaaaaggat   5520               atgatctagt ggtaaaggac tcagggggaa acacagcaag gcctgtctat tcagattctt   5580               cttgatctct ctctctctat gtatagcatt ctttcctcct gagtatgggg caggactctt   5640               cttcaatgag ggtcttcaag ggagaaggga gaaagtggcc tttttagatt ttat gg cttg   5700               cttcggggaa gaggagttct agtttctatg acccatcttg gggaagagga attctggttt   5760               ctgtgacttg ctttcatgaa gaaagaggag taagaggcag gagggcagga gatggtcaga   5820               aagagacttg gctgcttctg agggcttccg ctctccttta gttccaagta cttcttagca   5880               taccaaagca ctatactttg gcatatggtt ttctgagctc taacactgca atcatgctaa   5940               actcctctat gaccttcaaa cattccactt gcttttattc tttatggttg tgatggcata   6000               gaggtcaata gcaaagaccc tggagtccca ctgtctgagc tggcataaca ttactaccac   6060               ttaatcaatg tgtaagctca ggtaagtact taagtcctct atgcttcatc tgtaaaatga   6120               gaatcattga agaacattct ctcaggatgg atcatgagga ataagtgaat taactggcat   6180               atagtgctta aaccagtgcc ttgctcagtt agtgacagat aaaatcatct gttattactg   6240               tgcccactat tgtgatgctc ttctcttctt tgtacaacga ctacatctct atttatcatt   6300               ttagggtctc cttgtgaaaa accactccag attcaaaaga ttgagtttaa tctctatcct   6360               ctgtgctttc ctggagtttt gtaaagtaaa tcttcacttg acatcatgga taggttcttg   6420               gaaactacaa cttcaagtga aaggacataa ctaaaccaat ttttttctca tcaacgttat   6480               aatgaaatgg cattgatgaa atgatggcat tcaaggacct gctgtacctt gtttcactta   6540               aagtcactgt ttccaataat ctattgatga cattgaggac ttactatata ataataaata   6600               tatatataat cgacgaaaca ggaatcaaac tgctaactct gctaactggt ctccctgctt   6660               ccacactctg cccactcatc tcagtctttc tttcacaaga gtcagaatga tcagatgaga   6720               cccctcctct gcttctgttt cttccatgga tttccactgc actctgataa agtccagcct   6780               cttgaccaca gcctacaaat ccttgcacga tctatcgttt acttttccat ctccttttat   6840               gctactttca tcttgttctc aattctctag ctatgctggc cccttcttgt tctttcccat   6900               ttttttttaa tttttaaaat ttgtatatat ttatgggtta taagtgaaat ctttttagat   6960               gcataggttg tatagtgata aaatcagggc ttttagggta ttcatcacct gaatgatgta   7020               cattgtaccc cttaagtaat ttctcaccat ccgctgactt cttgccccct gggtattcat   7080               cacctgaatg atgtgcattg taccccttaa gtaatttctc accatccgct gacttcttgc   7140               cccctgggta ttcatcacct gaatgatgtg cattgtaccc cttaagtaat ttctcaccat   7200               ccgctgactt cttgccccct catccttctg aggctccatt gtccatcatt ccacactcta   7260               catctatgtg tacacattat ttagctccta cttataagtg ataacatgca atatttgtct   7320               ttctgtgtct gtcttgtttt acttatgata atggccccca gttctatcta ggct g ctgca   7380               aaaggcatga tttcattctt ttttatggct atgttctttc ccaatttaga taaagaacac   7440               tcgcacttgc tcttacttct atttggaata ctaattccta ggcttcttgc attgctttct   7500               ccttctcacc catcaaatct cattttagat accacctctt caaagagggc tttcctgacc   7560               accttggctg aattagccct tcaccatctg attactctct agcacatcac ctgcccattt   7620               tattcatggt acaggtcaaa atctggaatc acctgatttg tttattttct gactccttct   7680               actgagatga aaactctact agagcggaga ttttatctgc ttgtatcagg tactgcttca   7740               aacagcacct gatacaga g t aggtggtcaa aagatatttc ttaaacaaat gaacaaataa   7800               aaagtagatc ttttgagagt aaagctcttc cacactacca gagtcattca ggaatgacaa   7860               atcatagaat aacagaattt gatgctttgt gcatatcaga gaaagaaggt ggaaggttgt   7920               caaggtatca tgatgtacca gtcctcgcct cctcaaacac aatctgcaag tcccacagtg   7980               aaaaagtaag ttaactcatg tgaagcgttt tacaaacact tttttaaaag tcttaaaact   8040               cctaagaaag caagatttaa tagtcaaaga agtgagtaaa catgaaatgc ctgaacagag   8100               taatgagcta agcacaaagt tagagacatg ttagttaata tgtcttgaaa gcagcagctc   8160               ctgctttcaa ggagcaagaa caaattgggc aagtgaacac tccttgaata aaatgtgtaa   8220               aattaatttt gggttatgtt ctatactgtg tataatagaa tgataaaaat tatttgacta   8280               gcactttgta gtttagaaat atctctattt acacagttta ccttatttga taagactgtt   8340               gagtgatggg atagcatggt ggacaatcca cataactgag tatcgagaca cctgtatctg   8400               gacccagctc tgttagtaag aagctgtaac ctcagcaagt cactttctct ttctgggtct   8460               ctatttcctt tttggtgaaa tgagagtgtt aggctagatt gcctttgaag tcccattttg   8520               tctttaaagt cccatctatt gcagtgattt atatttaact catgacaaat caggcttctc   8580               ttattctaag tgcaa g acat aaaactttta ttgtggaatt tcaggcatca gtaaatcttt   8640               ttgggtactc acttatgttc ctgaaatcaa tctatttgag tgatcactct tttaggtgcc   8700               caggtaaaca aagaaggcca tggtctttct ttgagtgacc ttctttccct tttaattagt   8760               ctgacctctt taatgtcagt tctgactgat tcatttccct ggtccatctt ccttggtctg   8820               agggccttcc tagtttcata ttgcacttca gttccttcca caccaccatc aaggatggct   8880               gtcaacattc atttgttcta tgttataatt caaggaaaag ttgcccagta gctaatccaa   8940               taaatgccct cttatgggcg gctagagact ttttcctata atttaaatgc atcttctgta   9000               gattatggtc cctccaccac tttacatttg tctgctgtct ccttgctctg ctagtcatgg   9060               aacgtgttgg tagtgggggc agtgtgggat gttcaagggc acgtattggg tagggccaca   9120               tatgggcatt gctttgtgcc attctttcta tatttttggt attttgcatc tcactggaac   9180               ccaactattt ttcatctctt ccacctaaac tatttgatgc ctctgtttct tatatataaa   9240               gtatagctca ctgtagccta tgatcaggaa cctatctgct ttctaaatga aagctgtttt   9300               ggtcagatct agcaattaat tcccttcttc cacttatagc tttcctctgt aactctggtg   9360               taggtatttg gtttatggct ataagatgtg aaacacctga atgattctgt ccatgcaggc   9420               atttcagttc atgatattgt atgtaaaaga tactgattgt ctaggtgttc agaaacacct   9480               atagggctta atattcttac aatcagtttg aaggctggtg atacgcaaag caaactacat   9540               atttttctgc ctgctctctc tctttctctc tacatctctc tttctttatc ttttgaaata   9600               tcagtttgga gacttagaat tacataagac ataaacccat ttgatataag aattgctgtg   9660               tatatttgct catctactcc ctcctttggt cctcgagctg ccggtttaga ctttttacag   9720               gacgcaggca tgtgaaggag aaactgtcag tgctaggctg aattctgttg ttaccaagat   9780               ttctagaaaa gtattcctca gtcaggttga ttacagatat agcaaatcta tttttcctag   9840               ggtagtttct gtatgctgcc gggcttataa ctgtctgtca tccagctatt t c tctccacc   9900               ttcttgtttg cataacaacc aaggcaactt ccgcaaatca ctgcgtggag acgatgatcc   9960               tg c cagctcc cttttggaaa tcgtgaggat cagatcttgg accatgtata atatgatgct   10020               tctaatccaa aagaggaaag gcattgggag tcagctccta agtaagctcc agaattcctg   10080               ctggtacttt tccttccagg aagcaacttc cttgatattt tttttttaca g g catatgaa   10140               taaaaactat attttgcagc attgtacact ttttttcctt ttctagaaat tctaaacctc   10200               tgacattggt ggagacattg agtacatttt ttcccatatc cctacttttc agaaggattt   10260               tctctgctcg ttcacttaac attgctgatg cgtcagtctt ttcttcctca tctctttcag   10320               gggctggaga ggcagaggga gacagaggag ctggtactgc agagcggtcg tctgattggc   10380               tggacggtcg tagctgggct ataaaagaga cccctacagg cttagcagga agacgctcag   10440               aggattctga caatatcttt accggagaag aggcaaagta cgctcaaagc cgaagccaca   10500               gctcctcctg ccgcatttct ttcctgcttg cgaattccaa gctgttaaat aagatgtgca   10560               aagggcttgc aggtctgccg gcttcttgct tgaggaggta agattgcttt cagccattaa   10620               ccatattaaa cttttggcta gactttctca gttatttaca tgttgtactt actaacctag   10680               ttctgtgcaa ttagaaacag tgtggtcagg agagcacgac tttctaactt tcctccaaga   10740               ctagctagat attgtgactt aagacatgtg ctccccaaat ttcagccctt atgtgttgtt   10800               ttgtgtgacc tcagttttga gaactgttct attctttaag ccaggtctaa gaaagctagt   10860               tttaattaag aagcgagatg aggtttgagg ctatgtacag tgatctgtaa tatctccatc   10920               tgtgattact actgctattt gagcatccct ggagtacata gaagcctggc tctgggcttt   10980               ctgattgtat gctacaactt gtttcaggaa aggtacccca gaatgaggtt tggctccatc   11040               atcagaaagg cacta t gctt tccgtgtggt ggtgcagtaa ctttcactct  c tatgttctt   11100               ataag c aaat gttacaatga gatatgagtt ttaaagccag atcttcctta tctctctgcc   11160               ccatctctag ttcttgaagt gtctcatatg agtttggttg agaaatattg atcattacaa   11220               atcagttaat agttttgtag aagatctcat cttaaagaca ttgttttgtt aatatactcc   11280               cttgattttt ttaaaagacc ttacagacat acagctattc atttgttttt ggtttgttca   11340               aaaaaggtat aaagaaatgc attcagagaa agatcatata ttagccagtt gaaaattaaa   11400               cacaaaatga gtgcatatta cattacttaa tcttgcagtc aaaggtaaaa agtcaaccta   11460               aaggtatact acctgctttc ttatcgcact gcaaatagaa attaccacaa attttatttt   11520               ggaaataatc tcagaaaaca taatttttta tgtactatta aaacatttac tttccaaata   11580               ttctgtcatt caggagtatg gaagtatcga tggcttcttt aaaatgaagc aggagggtct   11640               ggcagagagt atctatgaaa taagttcctc tgaccttcac gcttaatttt ctgaatggag   11700               tggagcaaat tacttcaagc ttcacttaac ttgcatatga aatgaaccgt acaaaaatac   11760               aagagtgtca gga g aaagtt atgctctggt aatatttttg caaaacagat aaaagataat   11820               actagagctc tgtcctcaaa gagttaagca gctaatctaa ggaggtaaac tctatgtcag   11880               caggatgaac tgctcttccc tttcctcctc aataaattgc aaatcatcta gtccaacatc   11940               tttaccacca gtgcctgagg ctccagagga gccattgcct tctcaaggtc acataggtgg   12000               tgggtgagtt aggaccaaat ctagaattcc tgactccagt aacttctgaa gtcattttgt   12060               tttttatttt tatggtttta ttataagaat acttgctaag cacacttacc ccctgcattg   12120               attaataact ctaggatctc ag g t g gatcc agcacataga aatatgaatt cgtttctatt   12180               tggacttcat gatatattta cattatcacc ttggaatcac cctaacattc aggattgtat   12240               cttgttataa tcaaaaagga tgttgcatcc cctgaacagt catcagtcag ggaagcagag   12300               gagggaaagt aatcttgcga ggaagagaaa atactattta agggacagtc agagaacata   12360               atggaattca aactttctgg gaaaacctac atacataaat gtattagtgg ccatcctaaa   12420               tgtctttata tctttgaggc tttattttcc ctactccaaa tagacacatt tagttattca   12480               tttcttttaa aatggtattt ctctttttaa actatttctt gactttttta ataaaaagag   12540               atgcaagcaa gaggatattt aataaaaagt aagagagttg agcttaaggc ttattaaaag   12600               accccctttt tctagttagt caggagctct aatgtgccct ggctacctat taaatggtgg   12660               caataaactg gaagctcagt gatgactcta gcctgcttct cctaatagct gttaagcctc   12720               aaatgccctt tagagtgtgt atgtccttta aagtagctat taagaaggaa agcagcagca   12780               gcagatattg tctagaaaga agccccaaga agctgaggtt tcagcttggg catttgtttt   12840               cgccatccca tgctccattt ccctctgctg gaactgtgca cctcagtgta ttctccctct   12900               atacctcaca gcaggaactg cttgcccccc cccccccccc ccaacataca tggctggaac   12960               tgaatagact tttactttcc cgaggtgctt ctacagttcc ctctgccagc aggggaacag   13020               atggaaatag caatcacctg ccagaaggtg gcgtgcagca aggatgtgca tcttttgccg   13080               ctactgcttt ctgattccta aaaattactc agagatcact catgtgttca gtgattcagg   13140               ttctgttgaa gataccaaag atattcggtt ggtcaaaatg acgggcatat aaaggcttct   13200               caggtttctg aggtaaactg aagggtcaga attccagttg tggatgaagg aaatggtgtt   13260               atgactgcct caaggttttg tagcaagtca tagggaacca agaggaatct tgttttcctc   13320               agaggtcatg ccaactccaa ctcccgttcc ctaaactgtc tctgagccat agactagtaa   13380               tggactcttc aagctctacc attaggtatc ttttaaagaa agctggttat tactatttat   13440               tcattttttt ctcttctgtg cagtgcaaaa gatatgaaac atcggctagg tttcctgctg   13500               caaaaatctg attcctgtga acacaattct tcccacaaca agaaggacaa agtggttatt   13560               tgccagaggt aagagaaaag gccttggtga agatgtactt agtattaact atctgatgat   13620               ggggatgttc tgtgagaagg aacttgtgct cctagttaag ccagatttgg atcaagatag   13680               cctccatttt catggagatc ataactacat ttgaaatttc tatacattta gtgaaaaact   13740               gccctcatca ataacatatt ttgtcataac gatggaaaat aaaatctttg ccttcattca   13800               ggatcttaga tttcttgccc caattttttt accatggcat tccaattatt ctgtttctct   13860               ctattttttc tagagtgagc caagaggaag tcaagaaatg ggctgaatca ctggaaaacc   13920               tgattagtca tgaatgtaag tctgacagca acctgggatg aggtactctg gataagacaa   13980               gttatattat gctggtctaa tagaaactgc agcaaggcct ggcttctttc tgatgttcag   14040               actcaggaga ctctttaggt cttaaattca gtctgtttaa aattttaata tgccctagag   14100               ctttgtgata tacaatgaaa agtttatgca ggaaccatgt ggaaaaccat ctctctcatc   14160               acaaggaaaa acggaagaga gaaaaaaaat gataaatatc aataccttct tgcaaaatca   14220               atctcagttt ctctttccca aattgacctt ggtaattgat agctgcatag gcatttcaga   14280               agcaaaatac ttccttgaaa gaggcttcca acttgagtaa gaatcattag gtagaactgg   14340               gaaccactgg atatcaaaca cagatt a ggg ttacctgact ccaggtgact tgaaaaaagc   14400               aggggaaaaa gggattgctt gaatccatgc tttatccccc aagtacctca gctttatgtg   14460               aaatagcata tccaagaggc caaccagtgt gatgacaact gtggtccttt ctcctgtatc   14520               ataggtgggc tggcagcttt caaagctttc ttgaagtctg aatatagtga ggagaatatt   14580               gacttctgga tcagctgtga agagtacaag aaaatcaaat caccatctaa actaagtccc   14640               aaggccaaaa agatctataa tgaattcatc tcagtccagg caaccaaaga ggtaggtttt   14700               ttatggatac ataaaaattg tacgtattta tggagtatgt gtgatatttt gatacatgca   14760               tacaatgtga taacaatcaa atcagggcaa ttgctatata catatctcaa acatttatta   14820               tttctacgtg ttgagaacat tccaaatctc ctcttctagc tatcttaaaa tatacaataa   14880               actattgata actatatcac cctaatgtgc tatcaaacac tagaacctat tccctctacc   14940               caactttcta tctattcctt ctacccatta gccaacctga ccaaaaaggt aagcttttat   15000               ggcagagaac tctctggatc ttagtgaagg ttcctagaat agtggagctg actatcataa   15060               tcttgacaac cccaaataaa tcagtttttt aaaaaatctc ttttatccat gtggcttacc   15120               ataacctccc tgcatgaatt tttctgatga atctccccaa tttgttagac agaacagaag   15180               atcttgccct gctctctcta aagcagaaag gttcattctg aacctttcat actctctcac   15240               atgtgccaag gaggacccca atgtcacttt tgttttttgc ttctgaaata cagagggtgc   15300               actgccactt acaagtcact acaaagcata caggcttgca tcctcaacag ggatataggt   15360               ctaatgaagc cttggccttt gcccctcagg tgaacctgga ttcttgcacc agggaagaga   15420               caagccggaa catgctagag cctacaataa cctgctttga tgaggcccag aagaagattt   15480               tcaacctgat ggagaaggat tcctaccgcc gcttcctcaa gtctcgattc tatcttgatt   15540               tggtcaaccc gtccagctgt ggggcagaaa agcagaaagg agccaagagt tcagcagact   15600               gtgcttccct ggtccctcag tgtgcctaat tctcacctga aggcagaggg atgaaatgcc   15660               aagactctat gctctggaaa acctgaggcc aaatattgat ctgtattaag ctccagtgct   15720               ttatccacat tgtagcctaa tattcatgct gcctgccatg tgtgagtcac ttctacgcat   15780               aaactagata tagcttttgg tgtttgagtg ttcatcaggg tgggacccca ttccagtcca   15840               attttcctaa gtttctttga gggttccatg ggagcaaata tctaaataat ggcctggtag   15900               gtctggattt tcaaagattg ttggcagttt cctcctccca acagttttac ctcgggatgg   15960               ttggttagtg catgtcacat gacatccaca tgcacatgta ttctgttggc cagcacgttc   16020               tccagactct agatgtttag atgaggttga gctatgatat gtgcttgtgt gtatgtctat   16080               gtgtatatat tatatataca ttagacacac atatacatta tttctgtata tagatgtctg   16140               tgtatacata tgtatgtgtg agtgtatgta tacacacaca cacacacaca cacacacact   16200               tttgcaagag tgatgggaaa gaccctaggt gctcataact agagtatgtg tatgtactta   16260               catgggtgtt ttgatctctg ttctttcata ctacatttga acagggcaaa atgaactaac   16320               tgccatgtag gctaagaaag aaatgctaac ctgtggaaag ttggttttgt aaaattccat   16380               ggatcttgct ggagaagcat ccaaggaact tcatgcttga tttgaccact gacagcctcc   16440               accttgagca ctattctaag gagcaaatac cttagctccc ttgagctggt tttctctgat   16500               ggcacttttg agctcctaag ctgccagcct tcccttcttt tcctgggtgc tcagggcatg   16560               cttattagca gctgggttgg tatggagttg gcagacagga tgttcaactt aatgaagaaa   16620               tacagctaag gccttgccag caacacctgc cgtaagttac tggctgagtg agggcataga   16680               agttaaaggt tactgttttt atcctctatc cttttttcct ttcctgatca aggtgctctt   16740               ctcatttttt cctgagaacc ttagccatca gatgaggctc cttagtttat tgtggttggt   16800               tgttttttct ttataatggc tctgggctat atgcctatat ttataaacca gcagcagggg   16860               aaagattata ttttataaga gggaacaaat tttcacaatt tgaaaagccc acataagttt   16920               tctcttttaa ggtagaatct tgttaatttc attccaaaca tcggggctaa cagagactgg   16980               aggcatttct ttttaggctc tgagactaaa tgagaggaaa agaaaag a aa aaaaaaatga   17040               ttgtctaacc aattgtgaga attactgttt gaaacttttc aaggcacatt gaaatacttg   17100               aaaacttctc atttatgtta tttatgatgt tattttgtac gtgttattat tattatattg   17160               ttttataaat ggaggtacag gatatcacct gaattattaa tgaatgccca ggaagtaatt   17220               ttcttctcat tcttctaaaa ctactgcctt tcaaagtgca cacacacgcg tccacataca   17280               ctgcattcgt tgctccagta taaattacat gcatgagcac ctttctggct tttaagccaa   17340               tataatgggc tgcaaaatga agacaccaga gtgtatgcat acaaatctca ctgtattaaa   17400               gatgcaggtt ttctaattgt acccttcttg tctctctggc aatcttgccc ttaatatccc   17460               tggagttcct catcagtgtc attttctgtt atacacagtt ccacaatttt gtctctagtt   17520               gacttcaaat gtgtaacttt attggtcttg ccctattata attgtcatga ctttcagatt   17580               gtatctgaac tcacagactg ctgtcttact aataggtctg gaaggtcac g ctgaatgaga     17640               agtaaattat tttatgtaat acatttttga gtgtgttttt cagttgtatt tccctgttat   17700               ttcatcacta tttccaatgg tgagcttgcc tgctcatgct ccctggacag aatactcctt   17760               ccttttgcat gcctgtttct atcatgtgct tgataggcct caaagctaat gcttccagtg   17820               aaacacacgc atcttaataa taagggtaaa taaacgctcc atatgaaact atttgcttgg   17880               aaacacatta atgatccaga gacatgctat gagaaacatc agggtgtagg gtgactttag   17940               aaaaatactc atactgagtc tttaatccct cctgtgccag tgaactctgg gaaagaaagt   18000               acaaactgaa tattgtttat tctttagttc atgccactgc tctgcttggc tctactcata   18060               gaaccaaggc aatcttagct tcagagactg caaaacagat taagtgattt gcttgcagat   18120               tctcaatcaa ttttcaaggg atagagttca ccttccagag ccattctttt atttccagtt   18180               acccgcctgt ttgagagatg atagagcagt gggaaattga gagagttgaa aggagatata   18240               gattcttacc caaacttcaa aaatccttcc ctcccttttg ttaattctct ttcctggaaa   18300               agaggtcata aaatgttcac atcctcagta ataggccctg tgctgtgtct attatgtcat   18360               gagactccca tttcctgacc cttctttccc attgtaagag tagtagttac aaggtgttaa   18420               ggatagatga tcttcaacac ttttgagaaa tagatccatt tacggatctg gtaaaaacta   18480               tggaccgaac catcttttaa gaaaaaaatt cagagaggaa tctaaatttt gtgtgctttg   18540               aggggaaact ctcagaatct cccctcaaaa ctatcattct tctcttatac tatagatgtg   18600               tcagactctc actgggactg tatagttgct gctccctgta tttgataata tctatcaaga   18660               actgcagggt aattcaaagt cacgctatta gcagcaagtg tgagcagtgt tggtttcccc   18720               agtctctaca tccctcatcc tttctttctt ctttatggtt gtctattaaa gaaataaaaa   18780               aaaatattgg ctgaccgttt ttctgaagat aatgtatatc aaggaccacc ttttgaaaaa   18840               cactcattat tcgagaacaa agacacaaca tacgagaatc tctgggatac attcaaagca   18900               gtgtgtagag ggaaatttat agcactaaat gcccacaaga gaaagcagga aagatctaaa   18960               attgataccc taacatcaca attaaaagaa ctagaaaagc aagagcaaac acattcaaaa   19020               gctagcagaa gacaagaaat aactaagatc agagcagaac tgaaggaaat agagacacaa   19080               aaaacccttc aaaaaattaa tgaatccagg agctggtttt ttgaaaagat taacaaaatt   19140               gatagactgc tagcaagact aataaagaag aaaagagaga agaatcaaat agacacaata   19200               aaaaatgata aaggggatat caccaccgat cccacagaaa tacaaactac catcagagaa   19260               tactataaac acctctacgc aaataaacta gaaaatctag aagaaatgga taaattcctc   19320               gatacataca ccctcccaag accaaaccag gaagaagttg aatctctgaa tagaccaata   19380               acaggctctg aaattgaggc aataatcaat agcttaccaa ccaaaaaaag tccaggacca   19440               gatggattca cagctgaatt ctaccagacg tacaaagagg agctggtacc attccttctg   19500               aaactattcc aatcaataga aaaagaggga atcctcccta actcatttta tgaggccagc   19560               atcatcctga taccaaagcc tggcagagac acaaccaaaa aagagaattt tagaccaata   19620               tccttgatga acattgatgc aaaaatcctc aataaaatac tggcaaaccg aatccagcag   19680               cacatcaaaa agcttatcca ccatgatcaa gtgggtttca tccctgggat gcaaggctgg   19740               ttcaacatac gcaaatcaat aaatgtaatc cagcatataa acagaaacaa agacaaaaac   19800               cacatgatta tctcaataga tgcagaaaag gcatttgaca aaatttaaca actcttcatg   19860               ctaaaaactc tcaatcaatt aggtattgat gggacgtatc tcaaaataat aagcactatc   19920               tatgacaaac tcacagccaa tatcatactg aatgggcaaa aactggaagc attccctttg   19980               aaaacgggca caagacaggg atgccctctc tcaccactcc tattcaacat agtgttggaa   20040               gctctggcca gggcaattag gcaggagaag gaaataaagg gtattcaatt aggagaagag   20100               gaagtcaaat tgtccctgtt tgcagatgac atgattgtat atctagaaaa ccccatcgtc   20160               tcagcccaaa atctccttaa gctgataagc aacttcagca aagtctcagg atacaaaatc   20220               aatgtacaaa aatcacaagc actcttatac atcaataaca gacaaacaga gagccaaatc   20280               atgagtgaac tcccattcac 20300          
 
         [0073]    For purposes of the present invention, this DNA sequence will be referred to as SEQ ID NO:3. The location of the SNPs discussed further below is indicated by bold and larger font letters. Several additional sequences of DNA that are upstream from SEQ ID NO:3 are identified as relevant to the present invention. These DNA sequences are also found on NT — 022030 and are  
                                                                                                                                                                                                                                                 ggattaatca tgacaaaagt aatctaaatc tcgttaagac   60                tacttaatga tcaatctttc                            cctctgtttt ccctgactat agggaagtga attgccccaa   120                tccttctcta tcacccccct                            gcagccatgc caatgcctta cctctgttat attcagccat   180                aggggaagct tattctcata                            gaatcagggg ttggcatg t a gtcactagct attcttggtg   240                agactagtga agatgagtga                            aggaaaatat tgcataggtg aaatctcata ggcacaaata   300                ggtgtttgtg agagtaacaa                            taaaagaaag tcattcccat actctagtag atgactcatt   360                ttctcctcat tttttttttt                            tcaaggcgtt ctctacaacg gttaacctag taccaaaaat   420                ccttctcttt tttcttggac                            aaatcctgtt caagttagca tggcatttac tacgtccaag   480                acattgtcca gatgctgtgg                  
 
         [0074]    For purposes of the present invention, this DNA sequence will be referred to as SEQ ID NO:4.  
                                                                                                                                                                                                                     agagaaagaa aggcaggcag caaggagaaa aaacattttt   60                taaaaaaaga aaattaaaat                            ccatgtaatg tctgatatct gttctgctgt atgtgtagat   120                ctttccatat accaactcat                            tagccttatt ttacaggtga ggaaaatgag ac c gagagtc   180                cttcttactt gaccaagttc                            acacagcaag atcacacatg gtagaaccaa tgttagaacc   240                taggtgtata cttgctcatt                            caatatgtac aataattgca aaagtttcca taggtcttat   300                tatatatcag gcactataaa                            tgctatgcat gtgtcaacta atttaaacct aagcaatatt   360                ataaggaagg tactattata                            gaaatctcag ccttacaggt aagggaacag gaataaagag   420                atgtgaggta atggcccaag                  
 
         [0075]    For purposes of the present invention, this DNA sequence will be referred to as SEQ ID NO:5.  
                                                                                                                                                                                     ataatctcct ttcaagtttt tatcctgtca cttgctagtt   60                gtgtgatttg ggacaaatca                            tttaactcct tgtaaaggga gagaag g aag gctgtaaaaa   120                aattaagtaa taaaaagata                            aactccttgt ggtatatttt gttattgttc aaaaatattt   180                attgcccctc ttaggatgtc                            ttaggtcatt cttgcattgc tataaagaaa tacccaagtc   240                tgggtaattt ataaagaata                            gaggttaaat tggctcacag ttctgcaggc tgcacaggaa   300                gcatcccact ggcgtctact                            cacttctggt gaggactcag aaagcttttg cttatgacag   360                caggctaagt gagagcaggt                  
 
         [0076]    For purposes of the present invention, this DNA sequence will be referred to as SEQ ID NO:6.  
         [0077]    Several additional sequences of DNA that are downstream from SEQ ID NO:3 are identified as relevant to the present invention. These DNA sequences are also found on NT — 022030 and are  
                                                                                                                                                                                                                     catggtattt ttactaccca ttgccttcta ggaaagggta   60                taacaaatag gaaatattaa                            tatttttaat gcctttgagg gtgttaaaaa gcacaactct   120                aaggactgtt tgtaaattc c                              aggtcaaatg ttgtttctcc ttctctattt cctaccttgg   180                tgatggcctg atcttatatg                            gagtcactcc aactagaaac cacagaatca tccctagttc   240                ctacttctga ctcactccat                            acactcaaaa gtcacctgac tctgcagaat ttctctagaa   300                aaactctatg aaaacctatt                            cctgcctctc cacctgcata gatgtagctt catccaggct   360                cttatggtgc atggcctcgg                            ttactgcctt atcctttcta ctggcctctc aatctcccat   420                ctgataccca ttaatgtact                  
 
         [0078]    For purposes of the present invention, this DNA sequence will be referred to as SEQ ID NO:7.  
                                                                                                                                                                                         ccaaatactt tttaggcaca ctgggaagtt acattgtttc   60                ttgcaagtga caggttgtcc                            tttaattagt tctttctctc aaaaagagac tgctgactcc   120                aaactgggaa gaaacccact                            caccagcaaa atgctgctga attcactctg atagttttct   180                aatctctcat cagtagatga                            caataatgaa gccagtattg ttaccacaag actcagatat   240                  g tctatcacc caagatgatt                            tctctttaag acgcaataaa agggaacttt tctccccatt   300                tattagcaac taagatgaaa                            tgagagccag agaaataaag tgaggaagga aagagaattt   360                actaccttta caagctgaaa                  
 
         [0079]    For purposes of the present invention, this DNA sequence will be referred to as SEQ ID NO:8. In all upstream and downstream sequences (i.e. SEQ ID NOS: 4, 5, 6, 7, and 8), the location of SNPs are indicated by bold and larger font letters.  
         [0080]    In situ Hybridization  
         [0081]    Double-stranded cDNA containing the RGS4 sequence was first amplified from normal human brain cDNA using custom designed primers (Forward primer sequence:  
         [0082]    CCGAAGCCACAGCTCCTC (SEQ ID NO: 3); Reverse primer sequence: CATCCCTCTCCCTTCAGGTG (SEQ ID NO: 4), and “touchdown” PCR with AmpliTaq Gold (PE Biosystems): (94° C. for 10 minutes (min), followed by 10 PCR cycles with a high annealing temperature 94° C. for 30 seconds (sec), 62° C. for 30 sec, and 72° C. for 60 sec), 10 cycles with a medium annealing temperature (94° C. for 30 sec, 60° C. for 30 sec, 72° C. for 60 sec), and 20 cycles at a low annealing temperature (94° C. for 30 sec, 58° C. for 30 sec, 72° C. for 60 sec). The product of this touchdown PCR reaction produced a single bright band on a 2% agarose gel and was purified and ligated into a T/A plasmid cloning vector (AdvanTAge, Clontech) and transformed into competent  Escherichia coli  cells and plated overnight at 37° C. Colony PCR was performed on selected colonies containing the insert, and the products of these reactions were restriction digested and sequenced to verify orientation and insert identity.  
         [0083]    [ 35 S]-labeled riboprobes were synthesized using the T7 Riboprobe In Vitro Transcription System (Promega kit # P1460) and purified using RNeasy kit (Qiagen #74104). A scintillation counter was used to verify the specific radioactivity and yield of the probe. During hybridization, approximately 3 nanograms (ng) of probe was used per slide in a total volume of 90 μl. All other methods used were those described previously in Campbell et al., in Exp. Neurol. 160: 268-278, 1999, which is hereby incorporated by reference.  
         [0084]    Tissue blocks containing the regions of interest (PFC area 9, motor cortex [MC] and visual cortex [VC]) were identified using surface landmarks and sulci (the superior frontal gyrus, the central sulcus and precentral gyrus, and the calcarine sulcus, respectively). After histological verification of the regions, 20 μm sections containing these regions were cut with a cryostat at −20° C., mounted onto gelatin-coated glass slides, and stored at −80° C. until use. The slides were coded so that the investigator performing the analysis was blind to the diagnosis of the subjects.  
         [0085]    Following hybridization and washing, slides were air dried and exposed to BioMax MR film (Kodak) for 8-22 hours and then dipped in emulsion (NTB-2, Kodak), and exposed for 3-5 days at 4° C. High resolution scans of each film image were used for quantification of signal with Image (Scion Corporation, Fredrick, Maryland), version 4.0b), and darkfield images were captured from the developed slides. Throughout all steps and procedures, subject pairs were processed in parallel. Hybridization of sections with sense RGS4 riboprobe, used as a specificity control, did not result in detectable signal.  
         [0086]    Quantification was performed by subtracting the background white matter OD from the average signal OD measured in five non-overlapping rectangular regions on each section (3 sections per tissue block). In PFC and MC, these rectangular regions spanned cortical layers II-VI. Due to the lack of RGS4 signal in layer IV throughout the neocortex, and the great expansion of this layer in VC, the supragranular and infranular signal intensities were analyzed separately in VC. However, there were no significant differences in the levels of signal contained in the supra- and infragranular layers, so they were combined as a measure of overall VC signal intensity.  
         [0087]    Each in situ hybridization was repeated three times in separate hybridization reactions. The resulting ODs were background-corrected and averaged. Visual cortex (V1) OD quantification, due to a bi-laminar transcript distribution, was performed separately for the supragranular and infragranular layers.  
         [0088]    In order to search for novel candidate genes whose expression is consistently altered in schizophrenia, high-density cDNA microarrays (UniGEM-V, Incyte Genomics) were used to examine the expression patterns of over 7,800 genes and ESTs in post mortem samples of prefrontal cortex area 9 from six matched pairs of schizophrenic and control subjects.  
         [0089]    Comparison and Statistical Analyses  
         [0090]    As illustrated in FIG. 1B, a gene was determined to be expressed if the arrayed immobilized probe or target (the design of which is shown in FIG. 1A) was successfully amplified by PCR, produced a signal from at least 40% of the spot surface and had a signal/background ratio over 5-fold for either the cy3 or cy5 probe. Both images represent the same spot under cy3 and cy5 excitation, respectively. In this experiment, the balanced cy3 signal intensity (control or c-subject) was 6.2-fold brighter than the cy5 signal intensity (schizophrenic or s-subject).  
         [0091]    Genes were comparably expressed between the control and experimental samples if the cy3/cy5 ratio or cy5/cy3 ratio was &lt;1.6. Over 80% of observations fell into this class. Gene expression was changed between the two samples at the 95% confidence level (95% CL) if the cy3/cy5 or cy5/cy3 signal was 1.6-1.89. Gene expression was changed between the two samples at the 99% confidence level (99% CL) if the cy3/cy5 or cy5/cy3 signal was 1.9.  
         [0092]    In the microarray analyses, data from experimental subjects were compared to data from matched control subjects in a pairwise design to control for the effects of age, race, sex and PMI on gene expression. To evaluate potential changes in gene group expression on the microarrays, two types of statistical measures were employed: 1) χ-square analysis was performed on the distribution of genes in a group versus the distribution of all genes called present on each individual microarray. The distribution of gene expression ratios was divided into five different bins based on confidence levels for individual gene comparisons: &lt;−1.9, −1.89 to −1.6, −1.59 to 1.59, 1.6 to 1.89 and &gt;1.9. 2) A paired t-test (degrees of freedom=5) was used to compare mean expression ratios for a given gene group to the mean expression ratios for all expressed genes across all six subject pairs. A gene group was considered to be changed only if it reported differential expression by both the χ-square and t-test compared to the mean and distribution of all expressed genes. Microarray changes were also analyzed by descriptive statistics and correlation.  
         [0093]    To mimic the microarray comparisons, the in situ hybridization data were analyzed using ANCOVA with diagnosis as the main effect, subject pair as a blocking factor, and brain pH and tissue storage time as covariates. Furthermore, to verify that the pairing of subjects adequately controlled for sex, age, and PMI, we also conducted an ANCOVA with diagnosis as a main effect, and sex, age, PMI brain pH, and tissue storage time as covariates. Since both models produced similar results, the values from the ANCOVA with subject pair as a blocking factor are reported. Changes between groups were also analyzed by descriptive statistics, Pearson correlation, and Factor analysis.  
         [0094]    Pittsburgh Cases and Parents for Genotyping Analysis  
         [0095]    Inpatients and outpatients were recruited at Western Psychiatric Institute and Clinic, a University of Pittsburgh-affiliated tertiary care center and 35 other treatment facilities within a 500 mile radius of Pittsburgh. The Diagnostic Interview for Genetic Studies (DIGS) was the primary source for clinical information for probands (Nurnberger, et al.  Archives of General Psych.  51, 849-59; discussion 863-4, 1994). Additional information was obtained from available medical records and appropriate relatives, who also provided written informed consent. Consensus diagnoses were established by board certified psychiatrists. There were 93 Caucasian and 70 African-American cases. Genomic DNA, but not clinical information was available from all parents of the Caucasian cases. Cord blood samples were obtained from live births at Pittsburgh and served as unscreened, population-based controls. There were 169 individuals. They included 76 Caucasians and 93 African-Americans.  
         [0096]    National Institute of Mental Health Collaborative Genetics Initiative (NIMH CGI) Sample  
         [0097]    From 1991-98, pedigrees having probands with schizophrenia or schizoaffective disorder, depressed (DSM IV criteria) were ascertained at Columbia University, Harvard University, and Washington University. The DIGS was the primary interview schedule. The families were ascertained if they included two or more affected first degree relatives (Cloninger et al.  Am. J. Med. Gen.  81, 275-81, 1998, which is hereby incorporated by reference). We selected case-parent trios and available affected siblings from this cohort. Thus, 39 cases, their parents and 30 affected sibling-pairs were obtained. They comprised 25 Caucasian families, 10 who reported African-American ethnicity and 4 from other ethnic groups. Transmission disequilibrium test (TDT) analysis utilized only one case/family.  
         [0098]    Written, informed consent was obtained from all participants. Ethnicity was based on self-report (maternal report for neonatal samples).  
         [0099]    DNA Sequencing and Polymorphism Detection  
         [0100]    The genomic sequence for RGS4 was obtained from NT — 022030 (390242 bp), a currently unfinished clone from Human Genome Project, Chromosome 1 database. The annotated data revealed three identified genes, namely, RGS4, MSTPO 32  and RGS5. The genomic organization of RGS4 and RGS5 includes 5 exons which is typical for the RGS family gene.  
         [0101]    A panel of 10 African-American cases and 6 Caucasian controls was initially used to screen for polymorphisms in the exonic, intronic, and flanking genomic sequences of the RGS4 gene. The re-sequenced region included 6.8 kb upstream and 2.9 kb downstream of the coding sequence. The genomic sequence was used to design primers and amplicons ˜500 bp were generated, with overlapping sequences. The amplified fragments were sequenced using an ABI 3700 DNA sequencer. The sequencing panel that was used (n=16) has over 80% power to detect SNPs with minor allele frequency over 5% (Kruglyak et al.  Nature Gen.  27, 234-236, 2001, which is hereby incorporated by reference). We also sequenced cDNA sequences from the post-mortem samples reported on earlier (Mirnics et al.  Mol. Psychiatry  6, 293-301, 2001). The sequences were aligned using Sequencher (version 4.5) and polymorphisms were numbered consecutively. Additional SNPs localized to NT — 022030 were obtained from the NCBI SNP database (“http://www.ncbi.nlm.nih.gov/SNP”). We also obtained genotype data from a prior study of the NIMH sample (“http://zork.wustl.edu/nimh”).  
         [0102]    Polymorphism Analysis  
         [0103]    PCR based assays included primers (5 pmol) with 200 μM dNTP, 1.5 mM MgCl2, 0.5 U of AmpliTaq Polymerase (PE Biosystems), 1× buffer and 60 ng of genomic DNA in 10 or 20 μl reactions. The PCR conditions were 95° C. for 10 min followed by 35 cycles (94° C. for 45 sec, 60° C. 45 sec and 72° C. for 1 min). The final extension at 72° C. for 7 min. The amplified products were digested with restriction endonucleases, electrophoresed on agarose gels, and visualized using ethidium stain. SNPs 4 and 18 were identified as single strand conformational polymorphisms (SSCP) (Orita et al.  DNAS  86, 2766-70, 1989). All genotypes were read independently by two investigators.  
         [0104]    Polymorphisms were detected only in the intronic and flanking sequences of RGS4 (FIG. 6). Among 34 identified SNPs, one was selected from each of six sets which appeared to be in complete linkage disequilibirum in the re-sequenced panel. SNPs were further evaluated for informativeness (minor allele frequency&gt;0.1) and availability of reliable genotyping assays. Among the Caucasian cases from Pittsburgh, deviations from Hardy Weinberg equilibrium (HWE) were noted for SNP 7 (p&lt;0.03) and SNP 13 (p&lt;0.01). Though all maternal genotypes conformed to HWE, deviations were noted at SNPs for the fathers of Pittsburgh cases at SNPs 4 and 18 (p&lt;0.05). For the analysis of IBD sharing among affected sibling-pairs from the NIMH samples, we also used genotypes for markers D1S1595, D1S484, D1S1677, D1S431 and D1S1589 (Faraone et al.  Am. J. of Med. Gen.  81, 290-5, 1998).  
         [0105]    Statistical Analysis  
         [0106]    PEDCHECK software was used to check for Mendelian inconsistencies (O&#39;Connell et al.  Am. J. of Hum. Gen.  63, 259-266, 1998, which is hereby incorporated by reference). χ 2  tests were employed for comparisons between cases and unrelated controls. We also used SNPEM software based on the EM algorithm to estimate and compare haplotype frequencies (Fallin, 2001, which is hereby incorporated by reference). We utilized GENEHUNTER software for TDT analysis of individual SNPs and haplotypes, as well as analysis of identity by descent among affected sibling-pairs (Kruglyak et al.  Am. J. of Hum. Gen.  58, 1347-63, 1996; Spielman et al.  Am. J. of Hum. Gen.  54, 559-60, 1994, both of which are hereby incorporated by reference). We also used TRANSMIT for global tests of association involving multiple haplotypes (Clayton et al.  Am. J. of Med. Gen.  65, 1161-1169, 1999a; Clayton et al.  Am. J. of Hum. Gen.  65, 1170-1177, 1999b, both of which are hereby incorporated by reference).  
         [0107]    Microarray Results  
         [0108]    Single gene transcripts were analyzed across all cDNA microarray comparisons. Across the six microarray comparisons over 90,000 data points were collected, and from these 44,000 were expression-positive observations, resulting in an average of 3,735 expressed genes/microarray. Of the expressed transcripts, 4.8% were judged to be differentially expressed (99% CL) between the schizophrenic and control subjects. The observed differences for any subject pair, in general, were comparably distributed in both directions: 2.6% of the genes were expressed at higher levels in schizophrenic subjects than in the matched controls, whereas 2.2% were expressed at lower levels in the schizophrenic subject.  
         [0109]    Of all the expressed genes, RGS4 transcript reported the most significant decrease across all schizophrenic subjects. In fact, it was the only gene decreased at the 99% CL in all microarray comparisons. The microarray-bound, 571 base pair long, double-stranded cDNA immobilized probe corresponded to the 3′ end of RGS4 and had a less than 50% sequence homology to any other known transcript, including RGS family members. This high binding specificity, coupled with strong cy3 and cy5 hybridization signal intensities, as shown in FIG. 1B, showed that RGS4 was robustly expressed in the human prefrontal cortex. Across the six microarray comparisons, RGS4 mRNA levels were decreased 50-84% in the PFC of schizophrenic subjects, as illustrated in FIG. 1C, while the expression of the ten other RGS family members represented on the microarray were unchanged in the schizophrenic subjects. In the scatter plot shown in FIG. 1C, the X-axis reports subject pairs, the Y-axis reports percent change between schizophrenic and control subjects. Individual symbols represent a gene expression difference between a schizophrenic and control subject in a single pairwise comparison. The black dashed line denotes equal cy3 and cy5 signal intensity (similar expression) between schizophrenic and control subjects (0% change), green dashed line denotes the 95% confidence interval (37.5% change), red dashed line represents 99% confidence interval (47.5% change). Missing symbols in some pairwise comparisons indicate that the corresponding genes&#39; microarray hybridization did not meet expression criteria. Across all the RGS members represented on the microarray, only RGS4 showed a consistent expression change over the 99% CL in schizophrenic subjects.  
         [0110]    To confirm the microarray findings for the RGS4 expression changes, in situ hybridization was performed on the PFC from the same five subject pairs used for the microarray experiments (for pair 794c/665s, no sections were available from the same block of tissue used in the microarray experiment). As a further test of the robustness of the microarray data, five additional subject pairs were added to the in situ hybridization analysis. Radiolabeled cRNA probes designed against RGS4 mRNA were used to localize and quantify relative transcript levels. In the control subjects, RGS4 labeling was heavy in the prefrontal cortex, as shown in FIG. 2A, mimicking previously described labeling in the rat. In the gray matter of prefrontal cortex, the RGS4 riboprobe heavily labeled various size and shape cell profiles, including both projection neurons and interneurons. This labeling was the most prominent in layers III and V, with sparse labeling in the intervening granular layer IV, and appeared to be present over both large pyramidal neurons and smaller cells that could represent interneurons. High power photomicrographs of PFC tissue sections from a schizophrenic (622s) and matched control (685c) subjects were viewed under darkfield illumination. Micrographs for each subject were taken under identical conditions. Roman numbers denote cortical layers. Pial surface is to the left. Strong labeling across all cortical layers except lamina IV was observed, and diminished labeling in the matched schizophrenic subject across all the layers was noted (scale bar=400 μm). White matter labeling was absent.  
         [0111]    Based on optical density analysis, 9/10 subject pairs exhibited a 10.2% to 74.3% decrease in PFC RGS4 expression, as shown in FIG. 2B. The in situ hybridization data from 10 PFC pairwise comparisons were quantified using film densitometry. The X-axis represents subject classes, the Y-axis reports average film OD from 3 repeated hybridizations, measured across all layers. Lines connecting symbols indicate a matched subject pair. Note that in 10 PFC pairwise comparisons, 9 schizophrenic subjects showed RGS4 transcript reduction (mean=−34.5%; F 1,15 =6.95; p=0.019). Specificity of RGS4 expression changes  
         [0112]    To investigate whether RGS4 transcript decrease is a specific alteration in schizophrenia, the same microarray data was analyzed for consistent gene expression changes across other RGS-family members (FIG. 1C). Nine of the eleven RGS family members represented with immobilized probes on the microarrays reported expression in four or more microarray comparisons. RGS13, primarily lung-specific family member, was not expressed in any of the comparisons, while p115-RhoGEF reported expression in only one comparison. RGS4 was the only family member (and the only gene on the microarray) to report a consistent change in expression over the 99% CL in every schizophrenic subject. RGS5 mRNA (a gene also localized to cytogenetic position 1q21-22) was decreased at the 99%CL in one subject pair, at the 95% CL in another subject pair, and unchanged in the remaining 2 pairs that showed detectable RGS5 expression by microarrays. Expression of the other RGS family members did not display any consistent differences across the schizophrenic subjects. The mRNA from pair 567c/537s was analyzed a second time on the newest Incyte microarray, UniGEM-V2, which includes five additional RGS family members (RGSZ, RGS1, RGS7, RGS11, and RGS14). This analysis confirmed that, in the comparisons, RGS4 was the only significantly changed RGS family member.  
         [0113]    Heterotrimeric G-proteins, the main substrates for RGS family members, were assessed for expression patterns. Several reports suggest Gα changes associated with schizophrenia. Thus, it was desirable to assess whether the decrease in RGS4 expression correlated with changes in Gα expression levels. Of the eight Gα RGS substrates represented on the microarrays, only G o  expression was changed beyond the 95% CL in three or more pairwise comparisons. These three subjects with increased Go levels (317s, 547s, and 622s) showed the most robust decrease in RGS4 expression both in the PFC microarray and in situ hybridization assays.  
         [0114]    Expression of 274 genes known to be involved in the G-protein signaling cascades (GPCR, heterotrimeric G-proteins, RGS, GIRKS, G-protein receptor kinases, and mitogen-activated protein kinases) were analyzed in a gene group comparison. An average of 105 genes belonging to this group were expressed in each comparison. The results of microarray analyses showing G-protein and 1q21-22 locus-related expression differences in the PFC of six pairs of schizophrenic and control subjects are shown in FIGS. 3A and 3B. For both gene groups, all expressed genes were classified into signal intensity difference intervals (0.1 bins) according to their cy5/cy3 signal ratio. Transcripts in a “1” bin had identical cy5 vs. cy3 signal intensities. Positive values (to the right) on the X-axis denote higher cy5 signal in schizophrenic subjects (S&gt;C), negative values (to the left) correspond to higher cy3 signal intensity in the control subjects (C&gt;S). The Y-axis reports percentage of expressed genes across the six subject pairs per bin for each gene group. In both panels, the white bars (All genes) denote distribution of all expressed genes across the six PFC pairwise comparisons (n=22,408). Additionally, in both panels, RGS4 contribution to the transcript distribution is denoted by a hatched bar. Note that in both FIG. 3A and FIG. 3B, the cy3/cy5 signal distribution of G-protein and 1q21-22 gene groups was comparable to the distribution of all expressed genes across the six microarray comparisons.  
         [0115]    At the 99% confidence level, 5.6% of G-proteins showed a different distribution between schizophrenic and control subjects, as shown in FIG. 3A: 2.8% of G-proteins were decreased, while 2.8% were increased in the PFC of schizophrenic subjects. Of the 2.8% decrease in schizophrenic subjects, RGS4 observations alone accounted for nearly half of the decrease. When RGS4 was removed from the G-protein group, a gene group analysis by χ 2  test and t-test closely matched the distribution of all expressed genes, suggesting that the majority of different expression levels can be attributed to normal human variability. Except RGS4, no other member of the G-protein gene group was consistently changed across the subject pairs over the 95% or 99% confidence levels.  
         [0116]    The RGS4 gene has been mapped to locus 1q21-22, a novel schizophrenia locus recently implicated by pedigree studies with a linkage of disease score (LOD) of 6.5 as described by Brzustowicz et al. supra. To address if any other genes at this locus displayed altered expression in the PFC of schizophrenic subjects, 70 additional transcripts originating from this cytogenetic region were analyzed. At the 99% CL, 0.4% of 1q2l-22 genes were increased, and 5.9% were decreased in the schizophrenic subjects. Of the transcripts decreased in schizophrenic subjects, RGS4 observations alone accounted for nearly half of the decreases, as shown in FIG. 3B. Furthermore, of all the genes on the 1q21-22 locus, only RGS4 showed a consistent expression change across all the pairwise comparisons over the 95% or 99% confidence levels. Of the remaining genes on this locus, only the ALL1—FUSED gene (AF1q GenBank Accesion #U16954) reported consistent expression change over the 95% CL in the schizophrenic subjects in three or more pairwise comparisons. Furthermore, as a gene group, the expression of the remaining genes on locus 1q21-22 showed the same overall pattern as genes located on non-schizophrenia loci or the overall average gene expression which is shown in FIG. 3B.  
         [0117]    Regional RGS4 Gene Expression Changes  
         [0118]    To test whether RGS4 transcript decrease is specific to the prefrontal cortex or includes a more widespread cortical deficiency, RGS4 expression was assessed by in situ hybridization in the visual cortex (VC) and motor cortex (MC) from the same 10 pairs of control and schizophrenic subjects (for pair 558c/317s MC material was not available, and this pair was substituted with pair 794c/665s). The figure layout for FIGS.  4 A-D is similar to that of FIGS.  2 A-B. In VC, RGS4 in situ hybridization showed heavy labeling under darkfield illumination of diverse cell population in the gray matter, with a very prominent bi-laminar labeling pattern in the supragranular and infragranular layers, as shown in FIG. 4A. Roman numbers denote cortical layers, scale bar=400 μm. There was very sparse labeling in the well-developed layer IV, with very few cellular elements exhibiting detectable levels of RGS4 mRNA. These high power photomicrographs show that RGS4 levels are significantly decreased in the VC region of the schizophrenic subjects. The OD measurements on these two layers were performed separately.  
         [0119]    Across the same ten pairwise comparisons that were examined in the PFC hybridizations, combined RGS4 expression in supragranular and infragranular layers of VC was decreased by 32.8% (F 1,15 =8.24; p=0.012) as shown in FIG. 4B.  
         [0120]    In MC, RGS4 expression was concentrated over the cell-rich layers I-III and V-VI of both control and schizophrenic subjects, as shown in FIG. 4C. High power photomicrographs of MC tissue sections from the same matched pair of schizophrenic and control subject are represented in FIG. 2A and FIG. 4A, viewed under darkfield illumination. Roman numbers denote cortical layers, scale bar=400 μm. Because of the attenuated layer IV in motor cortex, the RGS4 labeling is almost uniform across all layers.  
         [0121]    Similar to the RGS4 transcript decrease observed in supragranular VC, schizophrenic subjects across the same 10 subject pairs were analyzed in MC. The mean RGS4 expression in MC shown in FIG. 4D, measured across all the layers, was decreased by 34.2% across the 10 schizophrenic subjects (F 1,15 =10.18; p=0.006)  
         [0122]    In the PFC, VC, and MC of subjects with schizophrenia, RGS4 expression was consistently decreased compared to the PFC of subjects with the diagnosis of MDD, as shown in the schematic of FIG. 5. In contrast, factor analysis of the pairwise differences in RGS4 gene expression across 3 different cortical areas for all 9 common schizophrenic and control subject pairs revealed that over 84% of the total variance in expression was accounted for by diagnosis (variance proportion=0.848, eigenvalue=2.544, p=0.001. The X-axis represents experimental groups, the Y-axis reports percent RGS4 expression change in PFC, VC, MC, in schizophrenic subjects (SCH) and PFC of subjects with MDD viewed by in situ hybridization. Each symbol represents percent of change between a single pairwise comparison; same symbols represent the same subject pairs. Arrows represent mean expression difference for each group. The same schizophrenic subjects showed a comparable and highly correlated decrease in RGS4 expression across all three cortical regions (PFC-VC: r=0.88, p=0.0003; PFC-MC: r=0.69, p=0.0384; VC-MC: r=0.76, p=0.0144). In contrast, subjects with MDD reported variable RGS4 expression changes when compared to their matched controls.  
         [0123]    The combined data indicate that RGS4 transcript changes are a result of the pathophysiological changes related to schizophrenia and not due to confounds. Furthermore, the RGS4 expression decrease appears to be specific and unique to schizophrenia, and not a hallmark of the major depressive disorder.  
         [0124]    RGS4 labeling in the white matter was comparable to background labeling across all brain regions, suggesting that RGS4 is primarily expressed in neuronal cells. The labeling was abundant in the majority of interneurons and projection neurons. However, in some pyramidal cells and interneurons RGS4 labeling could not be detected. RGS4 labeling was heavy in all cortical layers, except layer IV, where RGS4 expression was both sparse and light. This overall pattern of labeling was comparable across all three cortical regions (PFC, VC, MC). As the granular layer IV is the widest in the primary visual cortex, in this region RGS4 labeling was prominent in supragranular and infragranular layers, separated by a wide zone of mostly unlabeled granular cells. The overall distribution pattern of the RGS4 message does not mimic the known expression patterns of neurotransmitter systems, suggesting that RGS4 regulates many functionally distinct neuronal populations.  
         [0125]    Together, the microarray and in situ hybridization methods suggest decreased RGS4 expression is a consistent characteristic of schizophrenic subjects. Several causes of the reduced RGS4 expression may be offered, including adaptive and genetic changes in schizophrenic patients. It was hypothesized that reduction in RGS4 expression was generated by alterations in the RGS4 gene. In addition, it was contemplated that variations in the DNA upstream and downstream from the coding region of the RGS4 gene may also impact the expression of the RGS4 transcript. These possibilities were investigated by searching for SNPs in the RGS4 gene.  
         [0126]    The specificity of the reduced expression of RGS4 message for schizophrenic patients was confirmed in a series of control experiments. The same reduced level of RGS4 message was not observed in patients suffering from major depressive disorder. In addition, prolonged treatment of non-human primates with the anti-psychotic haloperidol did not result in decreased levels of RGS message in the cerebral cortex. This result indicates that chronic exposure to anti-psychotic drugs are unlikely to be responsible for the depressed levels of RGS4 message observed in schizophrenic patients.  
         [0127]    Genotyping Results  
         [0128]    34 single nucleotide polymorphisms (SNPs) were identified after re-sequencing all exons, introns and flanking 5′ and 3′ UTRs of the RGS4 coding region (FIG. 6). Thirteen SNPs were chosen for analysis using the TDT. SNPs are explicitly defined in Table 1. When the SNPs were tested individually, significantly increased transmission at SNP4 was observed in the Pittsburgh sample. ‘Moving window’ haplotype analyses using two to four contiguous SNPs, revealed significant association for several haplotypes; all but one included SNPs 1, 4, 7, or 18 (Table 2). A global test of association for haplotypes encompassing these SNPs was significant (TRANSMIT software, χ 2 =16.6, 8 df, p=0.035). There were 39 cases with schizoaffective disorder in the sample; these trends remained significant when the sample was restricted to individuals with schizophrenia (χ 2 =13.0, 6 df, p=0.043).  
         [0129]    TDT analysis was conducted next in the ethnically diverse NIMH sample using the same set of SNPs. Significant transmission distortion was observed individually at SNPs 1, 4 and 18 (Table 2). Exclusion of African-American families from the sample also  
                                                         TABLE 1                               Nu-                       cleotide                   identity                   in   Observed           Location of the SNP   SEQ ID   Nucleotide       SNIP #   within the SEQ   NO:3   variation                                27,859   199    {SEQ ID NO:4}   T   C                   34,653   153   {SEQ ID NO:5}   C   T               90,387   87   {SEQ ID NO:6}   G   A               SNP1   4121   {SEQ ID NO:3}   C   T               SNP2   4123   {SEQ ID NO:3}   T   A               SNP3   4368   {SEQ ID NO:3}   A   C               SNP4   4621   {SEQ ID NO:3}   A   C               SNP5   4790   {SEQ ID NO:3}   C   T               SNP6   4816   {SEQ ID NO:3}   G   T               SNP7   4970   {SEQ ID NO:3}   C   T               SNP8   5055   {SEQ ID NO:3}   C   G               SNP9   5295   {SEQ ID NO:3}   G   A               SNP10   5695   {SEQ ID NO:3}   G   A               SNP11   7375   {SEQ ID NO:3}   G   T               SNP12   7759   {SEQ ID NO:3}   G   A               SNP13   8596   {SEQ ID NO:3}   G   A               SNP14   9603-9609   {SEQ ID NO:3}   AGTTTGG   7 bases                       Absent               SNP15   9892   {SEQ ID NO:3}   C   A               SNP16   9963   {SEQ ID NO:3}   C   A               SNP17   10132   {SEQ ID NO:3}   G   A               SNP18   11056   {SEQ ID NO:3}   T   C               SNP19   11091   {SEQ ID NO:3}   C   T               SNP20   11106   {SEQ ID NO:3}   C   A               SNP21   11774   {SEQ ID NO:3}   G   T               SNP22   12143   {SEQ ID NO:3}   G   A               SNP23   12145   {SEQ ID NO:3}   G   T               SNP24   14367   {SEQ ID NO:3}   A   G               SNP25   17028   {SEQ ID NO:3}   A   Base absent               SNP26   17630   {SEQ ID NO:3}   G   T               118740   120   {SEQ ID NO:7}   C   G               130121   221   {SEQ ID NO:8}   G   C                          
 
         [0130]    revealed significant results for these SNPs (p=0.023, 0.011 and 0.033 respectively). However, the transmitted alleles differed from the Pittsburgh sample. Moving window haplotype analyses revealed preferential transmission for more extensive chromosomal segments than the Pittsburgh sample. Like the Pittsburgh sample, all but one of haplotypes with significant increased transmission included SNPs 1, 4, 7 or 18. A global test for association was also significant for haplotypes encompassing these SNPs (TRANSMIT analysis; χ 2 =18.8, p =0.016, 8 df).  
         [0131]    If the significant TDT results were due to linkage, it was reasoned that the affected sibships in the NIMH sample should yield evidence for increased haplotype sharing. For 30 available affected sib-pairs, the proportion of 0, 1, or 2 haplotypes identical by descent (IBD) were elevated over expectations of 0.25, 0.50, 0.25; namely 0.11, 0.44, 0.45 respectively (for SNPs 1, 4, 7 and 18 analyzed in conjunction with 5 flanking short tandem repeat polymorphisms genotyped previously). Increased IBD sharing was also observed when these sets of SNPs or STRPs were analyzed separately.  
         [0132]    Association at the population level was assessed by comparing Caucasian cases from each sample separately with two independent groups of Caucasian community-based controls. Since SNPs 1, 4, 7 and 18 appeared to be critical for transmission distortion in both samples, genotypes and allele frequencies for these SNPs were analyzed. Haplotypes frequencies were estimated using an expectation-maximization algorithm (EM), paying particular attention to haplotypes VI and XI, the haplotypes with excess transmission in the NIMH and Pittsburgh samples, respectively (Table 3). SNP 14 was informative only among African-Americans and so was analyzed separately using 70 African-American cases and 93 control individuals from Pittsburgh. Significant case-control differences were not noted for any of the comparisons. The failure to detect association may reflect superior power for the TDT in the context of population sub-structure.  
               TABLE 2                       Haplotype based comparisons among cases and unrelated controls. The       Caucasian cases from Pittsburgh (n = 93) and NIMH (n = 25) were       compared separately with unscreened Caucasian controls from Pittsburgh       (n = 76). Bonferoni corrections have been applied for the Pittsburgh case-       control comparisons, but not for comparisons involving the NIMH cases.       An omnibus test based on likelihood ratios was used to estimate overall       differences in haplotype frequencies (Fallin et al., Gen. Res. 11, 143-51,       2001) and was significant for both comparisons (χ 2  = 88.7, p &lt; 0.0001       and χ 2  = 30.1, p &lt; 0.0003 respectively for Pittsburgh and NIMH cases).       Similar significant differences based on 3 SNP haplotypes were       present, but are not shown. For each SNP, ‘o’ represents allele 1       and ‘•’ represents allele 2. OR—Odds ratio; NS—Not significant.                                                                        
 
         [0133]    [0133]                                                                                                   TABLE 3                           Pair-wise linkage disequlibrium between SNPs at RGS4. Population based control       individuals (n = 76) were used to estimate linkage disequilibrium. The figures above the       diagonal represent D′ and estimates for statistical significance (p values) are below the diagonal.            SNP   27859   90387   snp1   snp4   snp7   snp18   snp23   118740   130121                    27859       0.096   0.064   0.076   0.287   0.009   0.000   0.000   0.000       90387   0.132       0.000   0.000   0.000   0.000   0.000   0.001   0.627       snp1   −0.123   −0.501       0.000   0.000   0.000   0.000   0.450   0.477       snp4   0.101   −0.501   −1.000       0.000   0.000   0.000   0.128   0.515       snp7   −0.075   0.783   0.970   −0.961       0.000   0.000   0.012   0.068       snp18   0.177   0.377   −0.677   0.989   −0.961       0.000   0.000   0.041       snp23   0.527   −0.302   −1.000   1.000   −0.847   0.674       0.499   0.002       118740   0.385   0.163   0.048   −0.083   0.172   −0.233   0.042       0.000       130121   −0.505   0.049   −0.059   0.046   −0.163   0.174   −0.154   −0.956                    
         [0134]    [0134]               TABLE 4                       SNPs and Haplotypes at RGS4 with increased transmission distortion. TDT analysis of case-parent trios       included 93 families from Pittsburgh and 39 families from the NIMH cohort. Only statistically significant       increased transmissions are shown. The shaded haplotypes correspond to haplotypes VII and X,       respectively from Table 2. T/NT-Transmitted/not transmitted; o-Allele 1, -Allele 2 at each SNP;       /-Allele not specified at this locus; *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.005                                                                                                            
         [0135]    The demonstration of the association between these SNPs and schizophrenia offers a large number of applications in the diagnostic and therapeutic fields. Thus, embodiments of the present invention offer the possibility of diagnosing schizophrenia by means of a biological test and no longer exclusively by means of clinical evaluations. Embodiments of the present invention can also be applied to diagnosing pathologies of the schizophrenia spectrum, such as, in particular, schizotypy, schizoid individuals, etc. Embodiments of the present invention make it possible to refine the criteria for diagnosing these pathologies, which is currently entirely established clinically. Furthermore, embodiments of the invention also makes it possible to demonstrate susceptibility to schizophrenia by means of identifying a genetic vulnerability in the families of patients who posses the identified SNPs in the RGS4 coding region and flanking regions. Once individuals have been identified as being susceptible to schizophrenia, the utility of prophylactic treatment may be investigated.  
         [0136]    The DNA sample to be tested can be obtained from cells that have been withdrawn from the patient. These cells are preferably blood cells (e.g. mononucleated cells), that are easily obtained by the simple withdrawal of blood from the patient. Other cell types, such as fibroblasts, epithelial cells, keratinocytes, etc., may also be employed. The DNA may then extracted from the cells and used to detect the presence of SNPs in the RGS4 coding region and flanking regions.  
         [0137]    Most preferably, the DNA extract is initially subjected to one or more amplification reactions in order to obtain a substantial quantity of material corresponding to the region carrying the RGS4 coding region and flanking regions. The amplification can be achieved by any technique known to the skilled person, and in particular by means of the so-called PCR technique as described above. To this end, embodiments of the present invention also relate to specific primers which make it possible to amplify DNA fragments that are of small size and which carry the RGS4 gene, flanking regions thereof, or portions thereof generated from SEQ ID NOS. 3, 4, 5, 6, 7, or 8. Portion of a polynucleotide sequence is specifically intended to refer to any section of SEQ ID NOS. 3, 4, 5, 6, 7, or 8 that can be used in the practice of this invention, such as use as a primer to identify the presence of SEQ ID NOS. 3, 4, 5, 6, 7, or 8 or variations thereof in a patient or a section of SEQ ID NOS. 3, 4, 5, 6, 7, or 8 that can be used to amplify the entire sequence. The phrase contiguous portion is meant to refer to a series of bases that are adjacent to one another within a polynucleotide sequence. In the context of the present invention, the word gene is intended to mean the protein coding region, the proximal 5′ and 3′ untranslated regions, as well as any distal and proximal regulatory domains. The phrase gene-coding region is meant to refer to the stretch of DNA that begins at the transcription initiation site and includes all exionic and intrionic sequences that encode a protein.  
         [0138]    Embodiments of the present invention may also involve isolating DNA sequences and ligating the isolated sequence into a replicative cloning vector which comprises the isolated DNA of the RGS4 gene, based upon or derived from the cDNA of SEQ ID NOS. 3, 4, 5, 6, 7, or 8 and a replicon operative in a host cell. Additional embodiments include an expression system which comprises isolating DNA of the RGS4 gene, based upon complimentarity to SEQ ID NOS. 3, 4, 5, 6, 7, or 8 and operably linking this DNA to suitable control sequences. Recombinant host cells can be transformed with any of these replicative cloning vectors and may be used to overproduce the RGS4 protein.  
         [0139]    Embodiments of the present invention also include kits that will facilitate the diagnosis of schizophrenia through the amplification of segments of the 1q21-22 locus. Several methods providing for this amplification are described including: at least a pair of single-stranded DNA primers wherein use of said primers in a polymerase chain reaction results in amplification of a portion of the RGS4 gene fragment, wherein the sequence of said primers is derived from the regions of the cDNA defined by or complementary to SEQ ID NOS: 1, 3, 4, 5, 6, 7, or 8. Similarly, embodiments of the invention also provide for a pair of single-stranded DNA primers wherein use of said primers in a polymerase chain reaction results in amplification of an RGS4 gene fragment, wherein the sequence of said primers is based on the exon regions of chromosomal DNA derived from SEQ ID NOS:1 or 3.  
         [0140]    Various nucleic acid probes and primers specific for RGS4 (derived from or complementary to SEQ ID NOS. 3, 4, 5, 6, 7, or 8) may also be useful in diagnostic and therapeutic techniques and are included within the present invention. Among these are a nucleic acid probe complementary to portions or the entirety of human RGS4 gene as well as a nucleic acid probe complementary to human altered RGS4 gene sequences wherein said nucleic acid probe hybridizes to a variant of the RGS4 gene under hybridization conditions which prevent hybridizing of said nucleic acid probe to a wild-type RGS4 gene. Probes that are complementary to portions or the entirety of the RGS4 coding region and flanking regions that contain SNPs may also be used in these diagnostic tests. Any primer which makes it possible to amplify a fragment of the RGS4 coding region or flanking regions also forms part of the present invention. The primers that are used within the context of the invention can be synthesized by any technique known to the skilled person. The primers can also be labeled by any technique known to the skilled person.  
         [0141]    The invention may also be practiced through detection of SNPs in the RGS4 coding region or flanking regions by a variety of techniques. The techniques which may preferably be employed are DNA sequencing and gel separation.  
         [0142]    Any sequencing method known to the skilled person may be employed. In particular, it is advantageous to use an automated DNA sequencer. The sequencing is preferably carried out on double-stranded templates by means of the chain-termination method using fluorescent primers. An appropriate kit for this purpose is the Taq Dye Primer sequencing kit from Applied Biosystem (Applied Biosystem, Foster City, Calif.). Sequencing the SNPs in the RGS4 coding region and the flanking regions makes it possible to identify directly the SNPs that are present in the patient.  
         [0143]    An additional preferred technique for demonstrating the SNPs in the RGS4 coding region and flanking regions is that of separation on a gel. This technique is based on the migration, under denaturing conditions, of the denatured DNA fragments in a polyacrylamide gel. The bands of DNA can be visualized by any technique known to the skilled person, with the technique being based, such as by using labeled probes that are complementary to the entirety or portions of the RGS4 coding region and flanking regions. Alternatively, the bands may be visualized by using ethidium bromide or else by means of hybridization with a radiolabeled probe.  
         [0144]    In addition, measuring the expression of RGS4 message in peripheral tissue allows the diagnosis and determination of the susceptibility to schizophrenia in humans. As a matter of convenience, the reagents employed in the present invention can be provided in a kit packaged in combination with predetermined amounts of reagents for use in determining and/or quantifying the level of RGS4 expression. For example, a kit can comprise in packaged combination with other reagents any or all of the following components: appropriate detectors, buffers, deoxynucleotide triphosphates, ions provided by MgCl 2  or MnCl 2 , and polymerase(s). The diagnostic kits of the invention may further comprise a positive control and/or a negative control as well as instructions for quantitating RGS4 expression.  
         [0145]    Additionally, an embodiment of the present invention relates to ascertaining levels of the RGS4 protein. The level of RGS4 protein can be detected by analyzing binding of a sample from a subject with an antibody capable of binding to RGS4. An embodiment of this detection method utilizes an immunoassay. The sample from a subject may preferably be a biopsy of skeletal muscle, though any tissue accessible to biopsy may be used.  
         [0146]    In addition to providing generally useful diagnostic kits and methods, embodiments of the present invention may provide a method for augmenting traditional treatments by supplying the RGS4 protein to a subject and/or augmenting the subject&#39;s medication, such as antipsychotic drugs, and providing an improved therapeutic outcome.  
         [0147]    Further embodiments of the present invention may relate to the construction of an animal model of schizophrenia. Transgenic mice technology involves the introduction of new or altered genetic material into the mouse germ line by microinjection, retroviral infection or embryonic stem cell transfer. This results in lineages that carry the new integrated genetic material. Insertional mutagenesis occurs when integration of the microinjected genetic material into the host genome alters an endogenous gene resulting in a mutation. Methods of transferring genes into the germline, the expression of natural and hybrid genes and phenotypic changes that have occurred in transgenic mice are described by Palmiter and Brinster in Ann. Rev. Genet. 20 (1986) 465-499. Methods of foreign gene insertion, applications to foreign gene expression, and the use of transgenic mice to study immunological processes, neoplastic disease and other proliferative disorders are described by Gordon in Intl. Rev. Cytol. 115, 1989, 171-299 both of which are hereby incorporated by reference. A further example of genetic ‘knock-in’ technology may be found in Nebert, et al., Ann. N.Y. Acad. Sci. 919, 2000, 148-170 which is hereby incorporated by reference. The insertion of SEQ ID NO:3 containing some or all of the described SNPs into a mouse germ line may be expected to result in adult mice that may be used as an experimental model of schizophrenia. The insertion of SEQ ID NO:3 containing one or more of the variations listed in Table 1 with standard on:off regulatory domains will allow for the creation of mice deficient in RGS4 expression at specified times, and may be used as an experimental model of schizophrenia.  
         [0148]    Having now fully described embodiments of the present invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the invention.  
     
       
       
         1 
         
           
             8  
           
           
             1  
             2934  
             DNA  
             Homo sapiens  
           
            1 

gtacgctcaa agccgaagcc acagctcctc ctgccgcatt tctttcctgc ttgcgaattc     60 

caagctgtta aataagatgt gcaaagggct tgcaggtctg ccggcttctt gcttgaggag    120 

tgcaaaagat atgaaacatc ggctaggttt cctgctgcaa aaatctgatt cctgtgaaca    180 

caattcttcc cacaacaaga aggacaaagt ggttatttgc cagagagtga gccaagagga    240 

agtcaagaaa tgggctgaat cactggaaaa cctgattagt catgaatgtg ggctggcagc    300 

tttcaaagct ttcttgaagt ctgaatatag tgaggagaat attgacttct ggatcagctg    360 

tgaagagtac aagaaaatca aatcaccatc taaactaagt cccaaggcca aaaagatcta    420 

taatgaattc atctcagtcc aggcaaccaa agaggtgaac ctggattctt gcaccaggga    480 

agagacaagc cggaacatgc tagagcctac aataacctgc tttgatgagg cccagaagaa    540 

gattttcaac ctgatggaga aggattccta ccgccgcttc ctcaagtctc gattctatct    600 

tgatttggtc aacccgtcca gctgtggggc agaaaagcag aaaggagcca agagttcagc    660 

agactgtgct tccctggtcc ctcagtgtgc ctaattctca cctgaaggca gagggatgaa    720 

atgccaagac tctatgctct ggaaaacctg aggccaaata ttgatctgta ttaagctcca    780 

gtgctttatc cacattgtag cctaatattc atgctgcctg ccatgtgtga gtcacttcta    840 

cgcataaact agatatagct tttggtgttt gagtgttcat cagggtggga ccccattcca    900 

gtccaatttt cctaagtttc tttgagggtt ccatgggagc aaatatctaa ataatggcct    960 

ggtaggtctg gattttcaaa gattgttggc agtttcctcc tcccaacagt tttacctcgg   1020 

gatggttggt tagtgcatgt cacatgacat ccacatgcac atgtattctg ttggccagca   1080 

cgttctccag actctagatg tttagatgag gttgagctat gatatgtgct tgtgtgtatg   1140 

tctatgtgta tatattatat atacattaga cacacatata cattatttct gtatatagat   1200 

gtctgtgtat acatatgtat gtgtgagtgt atgtatacac acacacacac acacacacac   1260 

acacttttgc aagagtgatg ggaaagaccc taggtgctca taactagagt atgtgtatgt   1320 

acttacatgg gtgttttgat ctctgttctt tcatactaca tttgaacagg gcaaaatgaa   1380 

ctaactgcca tgtaggctaa gaaagaaatg ctaacctgtg gaaagttggt tttgtaaaat   1440 

tccatggatc ttgctggaga agcatccaag gaacttcatg cttgatttga ccactgacag   1500 

cctccacctt gagcactatt ctaaggagca aataccttag ctcccttgag ctggttttct   1560 

ctgatggcac ttttgagctc ctaagctgcc agccttccct tcttttcctg ggtgctcagg   1620 

gcatgcttat tagcagctgg gttggtatgg agttggcaga caggatgttc aacttaatga   1680 

agaaatacag ctaaggcctt gccagcaaca cctgccgtaa gttactggct gagtgagggc   1740 

atagaagtta aaggttactg tttttatcct ctatcctttt ttcctttcct gatcaaggtg   1800 

ctcttctcat tttttcctga gaaccttagc catcagatga ggctccttag tttattgtgg   1860 

ttggttgttt tttctttata atggctctgg gctatatgcc tatatttata aaccagcagc   1920 

aggggaaaga ttatatttta taagagggaa caaattttca caatttgaaa agcccacata   1980 

agttttctct tttaaggtag aatcttgtta atttcattcc aaacatcggg gctaacagag   2040 

actggaggca tttcttttta ggctctgaga ctaaatgaga ggaaaagaaa agaaaaaaaa   2100 

aatgattgtc taaccaattg tgagaattac tgtttgaaac ttttcaaggc acattgaaat   2160 

acttgaaaac ttctcattta tgttatttat gatgttattt tgtacgtgtt attattatta   2220 

tattgtttta taaatggagg tacaggatat cacctgaatt attaatgaat gcccaggaag   2280 

taattttctt ctcattcttc taaaactact gcctttcaaa gtgcacacac acgcgtccac   2340 

atacactgca ttcgttgctc cagtataaat tacatgcatg agcacctttc tggcttttaa   2400 

gccaatataa tgggctgcaa aatgaagaca ccagagtgta tgcatacaaa tctcactgta   2460 

ttaaagatgc aggttttcta attgtaccct tcttgtctct ctggcaatct tgcccttaat   2520 

atccctggag ttcctcatca gtgtcatttt ctgttataca cagttccaca attttgtctc   2580 

tagttgactt caaatgtgta actttattgg tcttgcccta ttataattgt catgactttc   2640 

agattgtatc tgaactcaca gactgctgtc ttactaatag gtctggaagg tcacgctgaa   2700 

tgagaagtaa attattttat gtaatacatt tttgagtgtg tttttcagtt gtatttccct   2760 

gttatttcat cactatttcc aatggtgagc ttgcctgctc atgctccctg gacagaatac   2820 

tccttccttt tgcatgcctg tttctatcat gtgcttgata ggcctcaaag ctaatgcttc   2880 

cagtgaaaca cacgcatctt aataataagg gtaaataaac gctccatatg aaac         2934 

 
           
             2  
             205  
             PRT  
             Homo sapiens  
           
            2 

Met Cys Lys Gly Leu Ala Gly Leu Pro Ala Ser Cys Leu Arg Ser Ala 
1               5                   10                  15 

Lys Asp Met Lys His Arg Leu Gly Phe Leu Leu Gln Lys Ser Asp Ser 
            20                  25                  30 

Cys Glu His Asn Ser Ser His Asn Lys Lys Asp Lys Val Val Ile Cys 
        35                  40                  45 

Gln Arg Val Ser Gln Glu Glu Val Lys Lys Trp Ala Glu Ser Leu Glu 
    50                  55                  60 

Asn Leu Ile Ser His Glu Cys Gly Leu Ala Ala Phe Lys Ala Phe Leu 
65                  70                  75                  80 

Lys Ser Glu Tyr Ser Glu Glu Asn Ile Asp Phe Trp Ile Ser Cys Glu 
                85                  90                  95 

Glu Tyr Lys Lys Ile Lys Ser Pro Ser Lys Leu Ser Pro Lys Ala Lys 
            100                 105                 110 

Lys Ile Tyr Asn Glu Phe Ile Ser Val Gln Ala Thr Lys Glu Val Asn 
        115                 120                 125 

Leu Asp Ser Cys Thr Arg Glu Glu Thr Ser Arg Asn Met Leu Glu Pro 
    130                 135                 140 

Thr Ile Thr Cys Phe Asp Glu Ala Gln Lys Lys Ile Phe Asn Leu Met 
145                 150                 155                 160 

Glu Lys Asp Ser Tyr Arg Arg Phe Leu Lys Ser Arg Phe Tyr Leu Asp 
                165                 170                 175 

Leu Val Asn Pro Ser Ser Cys Gly Ala Glu Lys Gln Lys Gly Ala Lys 
            180                 185                 190 

Ser Ser Ala Asp Cys Ala Ser Leu Val Pro Gln Cys Ala 
        195                 200                 205 

 
           
             3  
             20300  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               A genomic sequence containing RGS4 nucleic 
      acid sequence and sequences upstream and downstream 
      to the RGS4 nucleic acid sequence  
             
           
            3 

agttcaagac cagcctgagc aacatggtga aaccccatct ctactaaaaa tacaaaatta     60 

gacaggcatg gtgatacacg cctgtaatcc cagctacttc ggaggccgag gcaggagaat    120 

cacttgaacc tgctgggggt ggaggttgcg gggagcaaga tcatgccatt gcactccagc    180 

ccaggcaaca agagcgaaat gtcatctcag aaaaaaaaaa aggcatttta tatatatata    240 

tatatatata tacacacaca cacacatata tatatacaca tatatataca catatataca    300 

tatatacaca tatatacaca tatatataca catacatatg tacacatata tatacacata    360 

tgtatacaca tatatacaca tatatacaca catatataca catatataca cacatatata    420 

cacatatata cacatatata cacatataca catatataca catatataca tatatacaca    480 

tatatataat atacacacat atatatacac atatatacac acatatatac acatatatac    540 

acatatatat acacatatat acacatatat acatatatac acatatatat acatatatac    600 

acatatatac atatatacac atatatacat atatacacac atatatacac atacatatac    660 

acacacatag atatacatat atatacacat atatatacgt atatatatgt atatatatat    720 

gctccagagt tcataagagg tagcagttga ttaccactgg ggatagagga aaagagagtt    780 

tgacagcagt gtattgtgag aaggacattt caggttgatg gcaaatagta ggggaaatac    840 

ataaatgtgt aataaaacct atctgtaagg tagttaagaa ggtaacacta tatatatata    900 

tagtgaaagc agtgtaaacc taaaggatgg gccaaggatt taaatgttat agaagaatgg    960 

ctaagatgcc aaagctcagt gtatgtggca gaggcatggt gtagggtgtg tccaggttca   1020 

tatattgcat taagtgtgag aacaccctgg agtatgaacc aagaaaatgc aaaagccaga   1080 

agtgatggag gaaatgagac acaataatga agatattgag aggagggtgt gggcctagag   1140 

tgaagctttt cgtgccagta cttcttttga aggcccagtt ctcttctctc tcgggggctc   1200 

cttcatctct catagagtcc acagctttta agggccaaca cttgaggtca gcctggctct   1260 

ctcatttgag ctggatagaa cattttagag caccatctat tcttcaagag gaagtttaaa   1320 

aataaaagaa ccttgaagag gaaaaaatgt agacattcaa tctaaccttt tcattttact   1380 

agccaaagct aaatagaatg caggttacct gtttttcagc caggcaccat catttcctaa   1440 

ttgttataaa atttattatt attgttgtta ttattattat ttgccataag aagtttccca   1500 

tatcctttta gtataacaaa aacacaattc acaagcatta taaaacccat ggtgtctaac   1560 

tattaaaaaa attaagtgga acacacttgt cccagctact ggggaggctg aggagggagg   1620 

atcacgtgat cccagggggt caaggttatg gagagctatg attgtgccac tgcactccag   1680 

cctgggtgac agggaaagac cctgtctcta aaattttttt taaaaaaact aaactggttt   1740 

tattacagag attctggaga cagctacaca taaaagggtg gtatgcctca tattagctac   1800 

ccagggaggt ggaatgccaa cttaggtggt gtcaccacta ttaaaaatgc cccaaagcaa   1860 

tcaaaactga gaacttcctg ggagcttagc attgtgcaaa agcagcacaa aacacttaaa   1920 

caattcacag ttgtgttgga atgggaaggc ctggaaatat aaaccaaaga gtatattgtc   1980 

taaattgata gagattacaa ttgcctgaaa gaaaaagttg acttttaact agaatgttca   2040 

gagtaggttt acagaagaag ctcttaaact gggctccagt ggatttgtca atgctttgga   2100 

agctggtggg gtgggagggt tggagggggc ataaaaagtc atgttggtat gctctgctca   2160 

agtctccatt ctgtttcctt ttcctctttt caatgtcatg tcccattatt tcattatggg   2220 

cttcccttta tccaggatca atatgccacc tcttggttgt cttttaccta cttctccacc   2280 

tcactatgga atcgtccttg ggtagctcct gtgcttggga acctgcacgg gcacttttct   2340 

gatgtcttga ttccagcttt actcctaaaa cttaaatgct gaggggccaa caccatggca   2400 

gtggtaggga tgggaatggg ggtcttgtaa cacactacat aaactacacg aaataaacta   2460 

catgaaactc aacatgtttg caagactcag ttcacatcca tgaggagctc atgcttctcc   2520 

ctcctgctcc cctagcacac atgattatct ctatttggaa atgtttggca tttttggtga   2580 

agtgaatggt tcaataactt tctccaccat cagaacaaaa gctctttaag gttagggatg   2640 

ggatcataca cacttccctt gtccaagtcc ccatcacccc ttatctagac aattgctaca   2700 

gtttcctaca cactcttcta acctcttgca gtctattttc ataaaacagc tagagaactt   2760 

tgagatgtaa gtcaaaaaat agaacatgtc gctctttccc attgtttttg aaataaagtt   2820 

caaccccctt accagggtca acaaggccct gcaatgattt ggtcctgtta aaaattcttt   2880 

agccttaact catgctgttc ttccttacac tcactgcatt ctagccattg aggtttctat   2940 

gcatcaaact ttttttggtc ccagcactgt gcacatcctt ctgggtagaa tgccccttga   3000 

tttgtataat tagcacctcc ttcatcattt aggtcttagt ataactacta ccttcttaga   3060 

gaagctctgc ttcttcatcc tataaaaaag taaaattcct taccctgtta ttttttaagt   3120 

catccgtgtt tcattctgtt aaagttctta tcacaattta tcattatttt atttacagtc   3180 

atgtgccaca taacaatgtt tcagtcaggg atagaacaca aatgtatctg gccccataat   3240 

attataagct gagaaatttc tattaactag tgatatcgca gccatcataa gtgtaatgca   3300 

ggacattacc ttttctatgt ttagatatgt tagatacaca aatatatttc attgtgttat   3360 

aatttcctac agtattcagt acagtaacat gctgtacagg tttgtaacct aggagtaata   3420 

ggctatacca tacagcttag gtgtgtagta ggctataacc atctaggttt gtgtaagtac   3480 

attctatgat attcccacaa tgatgaaatc acctaactac acatttctca gaatgtttca   3540 

ctgttgtgaa gtgacccatg actatatttt cctatatact tgatattttt gtgcatctgc   3600 

ccatgagaat gtagtgtaag atcaaaggat gcaagaatgg gttctatcca gtatagtacc   3660 

cactacactg gtggatgtca atatgtattt gttagattaa tatctcaaga atgagcacct   3720 

ttctcagaca cataaaagat gctcaatata aaagtttgtt gaactgaacg ttattggcaa   3780 

atgtaacatg atcggattta aagaggagcg aaacagaggt ctggctcaaa caccatactt   3840 

ctagagtgca taagaggtag cagttgatta ccactggcga caggagaaaa aagagcttga   3900 

ccgcagggta ctgtgaagac atttcaggtt gatggcacag aacaggggaa atacataaat   3960 

gtgtgggaat attcagtggt ctgggatgac tacatagtag aatataatga agaaaagagt   4020 

ggaagggaaa gatgaaaagt tggaatgggg atgaattatg aaagtaccag aatgttatgc   4080 

taaggaatct agattttaaa atgtgagggc aaattgaagt cctgggcacg ttacaaaact   4140 

agaggtcata aagtttaccc taatttacca agatttccta gaggatctat aattggaatc   4200 

cagatctgcc tctctgtaaa gttcaagcac tttccatgac accatactgt ttctttccac   4260 

ctgcacaatg caaatgaact cttatgaaac tgctgtttct atcctgggct aaatgttgca   4320 

gaaaaaagat ttaatctttg ggataaggct attttgggtt ttctcctact tcttgggaaa   4380 

caaggttttc ttcccctggc taattaagtg tggtattgtt cttccaggga aatcagtgat   4440 

gcatcacctg ctgctatcaa atgtcagggt tggagttcct gatttattgc atgtgcccac   4500 

aaagcttggt gcaaagaatt ggacacattt cccaaaagta agacatactg ggaagtccct   4560 

gtttaccttc ctggtataca gcatcctcca gccccatatc tttgcttttt agtcctaaaa   4620 

atcaataact gaactctcat tgatgtctag gccattgtag taaacaataa agaaggaggg   4680 

aggcttctga caactgagag gaaattgtca tctgaagtgg tgcaagcaca gcctggggct   4740 

gagccttggc ctacatcctg cccaagtgga ggatcagtgc cccatttaac atctggtaga   4800 

actaaagaac gcaacgcctg ccacaatgac ttatttccct gcatttgata ccgtcaatcc   4860 

ttgagaaatg ttttcttttg ttctccctga gcaaaggttg gaaaaatttg aaatttacct   4920 

agagaccaca catagttcac atcctgctgt gtggctgaat gtctgccccc cagtaggaaa   4980 

cagttcttct aaagcctatt gtcaacaata ccttccagat gttagcattt tacaatttaa   5040 

ggaacttaaa atagccttca aactttttgc cagtttctct gatatccaat ctattctttt   5100 

actctgcctc ccaagctttc tttctagaat gctaacctga tcggcttaag tacttgaact   5160 

acctcttctc ctccattaac tacagagtaa attctggtct tcagagtaac aagaaacacc   5220 

ctttagttct cagcatattc gtgcaccttc atttatctct ccttctctct caaagctgca   5280 

gtaggggtga aaacgtgtga tacattttct cttccatcat aagggtcgca accaaaactc   5340 

ctatagtaaa agacaggtta ataagagcaa aacctaacaa atttatttaa tcaaagtttt   5400 

acatgacatg ggagtcttca gaaatgaaga cccaaagacc caggggaaac tgtctgtttt   5460 

ttttgctgag gttcgatgaa gaatggatag catgtagcca tgtagattag acaaaaggat   5520 

atgatctagt ggtaaaggac tcagggggaa acacagcaag gcctgtctat tcagattctt   5580 

cttgatctct ctctctctat gtatagcatt ctttcctcct gagtatgggg caggactctt   5640 

cttcaatgag ggtcttcaag ggagaaggga gaaagtggcc tttttagatt ttatggcttg   5700 

cttcggggaa gaggagttct agtttctatg acccatcttg gggaagagga attctggttt   5760 

ctgtgacttg ctttcatgaa gaaagaggag taagaggcag gagggcagga gatggtcaga   5820 

aagagacttg gctgcttctg agggcttccg ctctccttta gttccaagta cttcttagca   5880 

taccaaagca ctatactttg gcatatggtt ttctgagctc taacactgca atcatgctaa   5940 

actcctctat gaccttcaaa cattccactt gcttttattc tttatggttg tgatggcata   6000 

gaggtcaata gcaaagaccc tggagtccca ctgtctgagc tggcataaca ttactaccac   6060 

ttaatcaatg tgtaagctca ggtaagtact taagtcctct atgcttcatc tgtaaaatga   6120 

gaatcattga agaacattct ctcaggatgg atcatgagga ataagtgaat taactggcat   6180 

atagtgctta aaccagtgcc ttgctcagtt agtgacagat aaaatcatct gttattactg   6240 

tgcccactat tgtgatgctc ttctcttctt tgtacaacga ctacatctct atttatcatt   6300 

ttagggtctc cttgtgaaaa accactccag attcaaaaga ttgagtttaa tctctatcct   6360 

ctgtgctttc ctggagtttt gtaaagtaaa tcttcacttg acatcatgga taggttcttg   6420 

gaaactacaa cttcaagtga aaggacataa ctaaaccaat ttttttctca tcaacgttat   6480 

aatgaaatgg cattgatgaa atgatggcat tcaaggacct gctgtacctt gtttcactta   6540 

aagtcactgt ttccaataat ctattgatga cattgaggac ttactatata ataataaata   6600 

tatatataat cgacgaaaca ggaatcaaac tgctaactct gctaactggt ctccctgctt   6660 

ccacactctg cccactcatc tcagtctttc tttcacaaga gtcagaatga tcagatgaga   6720 

cccctcctct gcttctgttt cttccatgga tttccactgc actctgataa agtccagcct   6780 

cttgaccaca gcctacaaat ccttgcacga tctatcgttt acttttccat ctccttttat   6840 

gctactttca tcttgttctc aattctctag ctatgctggc cccttcttgt tctttcccat   6900 

ttttttttaa tttttaaaat ttgtatatat ttatgggtta taagtgaaat ctttttagat   6960 

gcataggttg tatagtgata aaatcagggc ttttagggta ttcatcacct gaatgatgta   7020 

cattgtaccc cttaagtaat ttctcaccat ccgctgactt cttgccccct gggtattcat   7080 

cacctgaatg atgtgcattg taccccttaa gtaatttctc accatccgct gacttcttgc   7140 

cccctgggta ttcatcacct gaatgatgtg cattgtaccc cttaagtaat ttctcaccat   7200 

ccgctgactt cttgccccct catccttctg aggctccatt gtccatcatt ccacactcta   7260 

catctatgtg tacacattat ttagctccta cttataagtg ataacatgca atatttgtct   7320 

ttctgtgtct gtcttgtttt acttatgata atggccccca gttctatcta ggctgctgca   7380 

aaaggcatga tttcattctt ttttatggct atgttctttc ccaatttaga taaagaacac   7440 

tcgcacttgc tcttacttct atttggaata ctaattccta ggcttcttgc attgctttct   7500 

ccttctcacc catcaaatct cattttagat accacctctt caaagagggc tttcctgacc   7560 

accttggctg aattagccct tcaccatctg attactctct agcacatcac ctgcccattt   7620 

tattcatggt acaggtcaaa atctggaatc acctgatttg tttattttct gactccttct   7680 

actgagatga aaactctact agagcggaga ttttatctgc ttgtatcagg tactgcttca   7740 

aacagcacct gatacagagt aggtggtcaa aagatatttc ttaaacaaat gaacaaataa   7800 

aaagtagatc ttttgagagt aaagctcttc cacactacca gagtcattca ggaatgacaa   7860 

atcatagaat aacagaattt gatgctttgt gcatatcaga gaaagaaggt ggaaggttgt   7920 

caaggtatca tgatgtacca gtcctcgcct cctcaaacac aatctgcaag tcccacagtg   7980 

aaaaagtaag ttaactcatg tgaagcgttt tacaaacact tttttaaaag tcttaaaact   8040 

cctaagaaag caagatttaa tagtcaaaga agtgagtaaa catgaaatgc ctgaacagag   8100 

taatgagcta agcacaaagt tagagacatg ttagttaata tgtcttgaaa gcagcagctc   8160 

ctgctttcaa ggagcaagaa caaattgggc aagtgaacac tccttgaata aaatgtgtaa   8220 

aattaatttt gggttatgtt ctatactgtg tataatagaa tgataaaaat tatttgacta   8280 

gcactttgta gtttagaaat atctctattt acacagttta ccttatttga taagactgtt   8340 

gagtgatggg atagcatggt ggacaatcca cataactgag tatcgagaca cctgtatctg   8400 

gacccagctc tgttagtaag aagctgtaac ctcagcaagt cactttctct ttctgggtct   8460 

ctatttcctt tttggtgaaa tgagagtgtt aggctagatt gcctttgaag tcccattttg   8520 

tctttaaagt cccatctatt gcagtgattt atatttaact catgacaaat caggcttctc   8580 

ttattctaag tgcaagacat aaaactttta ttgtggaatt tcaggcatca gtaaatcttt   8640 

ttgggtactc acttatgttc ctgaaatcaa tctatttgag tgatcactct tttaggtgcc   8700 

caggtaaaca aagaaggcca tggtctttct ttgagtgacc ttctttccct tttaattagt   8760 

ctgacctctt taatgtcagt tctgactgat tcatttccct ggtccatctt ccttggtctg   8820 

agggccttcc tagtttcata ttgcacttca gttccttcca caccaccatc aaggatggct   8880 

gtcaacattc atttgttcta tgttataatt caaggaaaag ttgcccagta gctaatccaa   8940 

taaatgccct cttatgggcg gctagagact ttttcctata atttaaatgc atcttctgta   9000 

gattatggtc cctccaccac tttacatttg tctgctgtct ccttgctctg ctagtcatgg   9060 

aacgtgttgg tagtgggggc agtgtgggat gttcaagggc acgtattggg tagggccaca   9120 

tatgggcatt gctttgtgcc attctttcta tatttttggt attttgcatc tcactggaac   9180 

ccaactattt ttcatctctt ccacctaaac tatttgatgc ctctgtttct tatatataaa   9240 

gtatagctca ctgtagccta tgatcaggaa cctatctgct ttctaaatga aagctgtttt   9300 

ggtcagatct agcaattaat tcccttcttc cacttatagc tttcctctgt aactctggtg   9360 

taggtatttg gtttatggct ataagatgtg aaacacctga atgattctgt ccatgcaggc   9420 

atttcagttc atgatattgt atgtaaaaga tactgattgt ctaggtgttc agaaacacct   9480 

atagggctta atattcttac aatcagtttg aaggctggtg atacgcaaag caaactacat   9540 

atttttctgc ctgctctctc tctttctctc tacatctctc tttctttatc ttttgaaata   9600 

tcagtttgga gacttagaat tacataagac ataaacccat ttgatataag aattgctgtg   9660 

tatatttgct catctactcc ctcctttggt cctcgagctg ccggtttaga ctttttacag   9720 

gacgcaggca tgtgaaggag aaactgtcag tgctaggctg aattctgttg ttaccaagat   9780 

ttctagaaaa gtattcctca gtcaggttga ttacagatat agcaaatcta tttttcctag   9840 

ggtagtttct gtatgctgcc gggcttataa ctgtctgtca tccagctatt tctctccacc   9900 

ttcttgtttg cataacaacc aaggcaactt ccgcaaatca ctgcgtggag acgatgatcc   9960 

tgccagctcc cttttggaaa tcgtgaggat cagatcttgg accatgtata atatgatgct  10020 

tctaatccaa aagaggaaag gcattgggag tcagctccta agtaagctcc agaattcctg  10080 

ctggtacttt tccttccagg aagcaacttc cttgatattt tttttttaca ggcatatgaa  10140 

taaaaactat attttgcagc attgtacact ttttttcctt ttctagaaat tctaaacctc  10200 

tgacattggt ggagacattg agtacatttt ttcccatatc cctacttttc agaaggattt  10260 

tctctgctcg ttcacttaac attgctgatg cgtcagtctt ttcttcctca tctctttcag  10320 

gggctggaga ggcagaggga gacagaggag ctggtactgc agagcggtcg tctgattggc  10380 

tggacggtcg tagctgggct ataaaagaga cccctacagg cttagcagga agacgctcag  10440 

aggattctga caatatcttt accggagaag aggcaaagta cgctcaaagc cgaagccaca  10500 

gctcctcctg ccgcatttct ttcctgcttg cgaattccaa gctgttaaat aagatgtgca  10560 

aagggcttgc aggtctgccg gcttcttgct tgaggaggta agattgcttt cagccattaa  10620 

ccatattaaa cttttggcta gactttctca gttatttaca tgttgtactt actaacctag  10680 

ttctgtgcaa ttagaaacag tgtggtcagg agagcacgac tttctaactt tcctccaaga  10740 

ctagctagat attgtgactt aagacatgtg ctccccaaat ttcagccctt atgtgttgtt  10800 

ttgtgtgacc tcagttttga gaactgttct attctttaag ccaggtctaa gaaagctagt  10860 

tttaattaag aagcgagatg aggtttgagg ctatgtacag tgatctgtaa tatctccatc  10920 

tgtgattact actgctattt gagcatccct ggagtacata gaagcctggc tctgggcttt  10980 

ctgattgtat gctacaactt gtttcaggaa aggtacccca gaatgaggtt tggctccatc  11040 

atcagaaagg cactatgctt tccgtgtggt ggtgcagtaa ctttcactct ctatgttctt  11100 

ataagcaaat gttacaatga gatatgagtt ttaaagccag atcttcctta tctctctgcc  11160 

ccatctctag ttcttgaagt gtctcatatg agtttggttg agaaatattg atcattacaa  11220 

atcagttaat agttttgtag aagatctcat cttaaagaca ttgttttgtt aatatactcc  11280 

cttgattttt ttaaaagacc ttacagacat acagctattc atttgttttt ggtttgttca  11340 

aaaaaggtat aaagaaatgc attcagagaa agatcatata ttagccagtt gaaaattaaa  11400 

cacaaaatga gtgcatatta cattacttaa tcttgcagtc aaaggtaaaa agtcaaccta  11460 

aaggtatact acctgctttc ttatcgcact gcaaatagaa attaccacaa attttatttt  11520 

ggaaataatc tcagaaaaca taatttttta tgtactatta aaacatttac tttccaaata  11580 

ttctgtcatt caggagtatg gaagtatcga tggcttcttt aaaatgaagc aggagggtct  11640 

ggcagagagt atctatgaaa taagttcctc tgaccttcac gcttaatttt ctgaatggag  11700 

tggagcaaat tacttcaagc ttcacttaac ttgcatatga aatgaaccgt acaaaaatac  11760 

aagagtgtca ggagaaagtt atgctctggt aaatattttg caaaacagat aaaagataat  11820 

actagagctc tgtcctcaaa gagttaagca gctaatctaa ggaggtaaac tctatgtcag  11880 

caggatgaac tgctcttccc tttcctcctc aataaattgc aaatcatcta gtccaacatc  11940 

tttaccacca gtgcctgagg ctccagagga gccattgcct tctcaaggtc acataggtgg  12000 

tgggtgagtt aggaccaaat ctagaattcc tgactccagt aacttctgaa gtcattttgt  12060 

tttttatttt tatggtttta ttataagaat acttgctaag cacacttacc ccctgcattg  12120 

attaataact ctaggatctc aggtggatcc agcacataga aatatgaatt cgtttctatt  12180 

tggacttcat gatatattta cattatcacc ttggaatcac cctaacattc aggattgtat  12240 

cttgttataa tcaaaaagga tgttgcatcc cctgaacagt catcagtcag ggaagcagag  12300 

gagggaaagt aatcttgcga ggaagagaaa atactattta agggacagtc agagaacata  12360 

atggaattca aactttctgg gaaaacctac atacataaat gtattagtgg ccatcctaaa  12420 

tgtctttata tctttgaggc tttattttcc ctactccaaa tagacacatt tagttattca  12480 

tttcttttaa aatggtattt ctctttttaa actatttctt gactttttta ataaaaagag  12540 

atgcaagcaa gaggatattt aataaaaagt aagagagttg agcttaaggc ttattaaaag  12600 

accccctttt tctagttagt caggagctct aatgtgccct ggctacctat taaatggtgg  12660 

caataaactg gaagctcagt gatgactcta gcctgcttct cctaatagct gttaagcctc  12720 

aaatgccctt tagagtgtgt atgtccttta aagtagctat taagaaggaa agcagcagca  12780 

gcagatattg tctagaaaga agccccaaga agctgaggtt tcagcttggg catttgtttt  12840 

cgccatccca tgctccattt ccctctgctg gaactgtgca cctcagtgta ttctccctct  12900 

atacctcaca gcaggaactg cttgcccccc cccccccccc ccaacataca tggctggaac  12960 

tgaatagact tttactttcc cgaggtgctt ctacagttcc ctctgccagc aggggaacag  13020 

atggaaatag caatcacctg ccagaaggtg gcgtgcagca aggatgtgca tcttttgccg  13080 

ctactgcttt ctgattccta aaaattactc agagatcact catgtgttca gtgattcagg  13140 

ttctgttgaa gataccaaag atattcggtt ggtcaaaatg acgggcatat aaaggcttct  13200 

caggtttctg aggtaaactg aagggtcaga attccagttg tggatgaagg aaatggtgtt  13260 

atgactgcct caaggttttg tagcaagtca tagggaacca agaggaatct tgttttcctc  13320 

agaggtcatg ccaactccaa ctcccgttcc ctaaactgtc tctgagccat agactagtaa  13380 

tggactcttc aagctctacc attaggtatc ttttaaagaa agctggttat tactatttat  13440 

tcattttttt ctcttctgtg cagtgcaaaa gatatgaaac atcggctagg tttcctgctg  13500 

caaaaatctg attcctgtga acacaattct tcccacaaca agaaggacaa agtggttatt  13560 

tgccagaggt aagagaaaag gccttggtga agatgtactt agtattaact atctgatgat  13620 

ggggatgttc tgtgagaagg aacttgtgct cctagttaag ccagatttgg atcaagatag  13680 

cctccatttt catggagatc ataactacat ttgaaatttc tatacattta gtgaaaaact  13740 

gccctcatca ataacatatt ttgtcataac gatggaaaat aaaatctttg ccttcattca  13800 

ggatcttaga tttcttgccc caattttttt accatggcat tccaattatt ctgtttctct  13860 

ctattttttc tagagtgagc caagaggaag tcaagaaatg ggctgaatca ctggaaaacc  13920 

tgattagtca tgaatgtaag tctgacagca acctgggatg aggtactctg gataagacaa  13980 

gttatattat gctggtctaa tagaaactgc agcaaggcct ggcttctttc tgatgttcag  14040 

actcaggaga ctctttaggt cttaaattca gtctgtttaa aattttaata tgccctagag  14100 

ctttgtgata tacaatgaaa agtttatgca ggaaccatgt ggaaaaccat ctctctcatc  14160 

acaaggaaaa acggaagaga gaaaaaaaat gataaatatc aataccttct tgcaaaatca  14220 

atctcagttt ctctttccca aattgacctt ggtaattgat agctgcatag gcatttcaga  14280 

agcaaaatac ttccttgaaa gaggcttcca acttgagtaa gaatcattag gtagaactgg  14340 

gaaccactgg atatcaaaca cagattaggg ttacctgact ccaggtgact tgaaaaaagc  14400 

aggggaaaaa gggattgctt gaatccatgc tttatccccc aagtacctca gctttatgtg  14460 

aaatagcata tccaagaggc caaccagtgt gatgacaact gtggtccttt ctcctgtatc  14520 

ataggtgggc tggcagcttt caaagctttc ttgaagtctg aatatagtga ggagaatatt  14580 

gacttctgga tcagctgtga agagtacaag aaaatcaaat caccatctaa actaagtccc  14640 

aaggccaaaa agatctataa tgaattcatc tcagtccagg caaccaaaga ggtaggtttt  14700 

ttatggatac ataaaaattg tacgtattta tggagtatgt gtgatatttt gatacatgca  14760 

tacaatgtga taacaatcaa atcagggcaa ttgctatata catatctcaa acatttatta  14820 

tttctacgtg ttgagaacat tccaaatctc ctcttctagc tatcttaaaa tatacaataa  14880 

actattgata actatatcac cctaatgtgc tatcaaacac tagaacctat tccctctacc  14940 

caactttcta tctattcctt ctacccatta gccaacctga ccaaaaaggt aagcttttat  15000 

ggcagagaac tctctggatc ttagtgaagg ttcctagaat agtggagctg actatcataa  15060 

tcttgacaac cccaaataaa tcagtttttt aaaaaatctc ttttatccat gtggcttacc  15120 

ataacctccc tgcatgaatt tttctgatga atctccccaa tttgttagac agaacagaag  15180 

atcttgccct gctctctcta aagcagaaag gttcattctg aacctttcat actctctcac  15240 

atgtgccaag gaggacccca atgtcacttt tgttttttgc ttctgaaata cagagggtgc  15300 

actgccactt acaagtcact acaaagcata caggcttgca tcctcaacag ggatataggt  15360 

ctaatgaagc cttggccttt gcccctcagg tgaacctgga ttcttgcacc agggaagaga  15420 

caagccggaa catgctagag cctacaataa cctgctttga tgaggcccag aagaagattt  15480 

tcaacctgat ggagaaggat tcctaccgcc gcttcctcaa gtctcgattc tatcttgatt  15540 

tggtcaaccc gtccagctgt ggggcagaaa agcagaaagg agccaagagt tcagcagact  15600 

gtgcttccct ggtccctcag tgtgcctaat tctcacctga aggcagaggg atgaaatgcc  15660 

aagactctat gctctggaaa acctgaggcc aaatattgat ctgtattaag ctccagtgct  15720 

ttatccacat tgtagcctaa tattcatgct gcctgccatg tgtgagtcac ttctacgcat  15780 

aaactagata tagcttttgg tgtttgagtg ttcatcaggg tgggacccca ttccagtcca  15840 

attttcctaa gtttctttga gggttccatg ggagcaaata tctaaataat ggcctggtag  15900 

gtctggattt tcaaagattg ttggcagttt cctcctccca acagttttac ctcgggatgg  15960 

ttggttagtg catgtcacat gacatccaca tgcacatgta ttctgttggc cagcacgttc  16020 

tccagactct agatgtttag atgaggttga gctatgatat gtgcttgtgt gtatgtctat  16080 

gtgtatatat tatatataca ttagacacac atatacatta tttctgtata tagatgtctg  16140 

tgtatacata tgtatgtgtg agtgtatgta tacacacaca cacacacaca cacacacact  16200 

tttgcaagag tgatgggaaa gaccctaggt gctcataact agagtatgtg tatgtactta  16260 

catgggtgtt ttgatctctg ttctttcata ctacatttga acagggcaaa atgaactaac  16320 

tgccatgtag gctaagaaag aaatgctaac ctgtggaaag ttggttttgt aaaattccat  16380 

ggatcttgct ggagaagcat ccaaggaact tcatgcttga tttgaccact gacagcctcc  16440 

accttgagca ctattctaag gagcaaatac cttagctccc ttgagctggt tttctctgat  16500 

ggcacttttg agctcctaag ctgccagcct tcccttcttt tcctgggtgc tcagggcatg  16560 

cttattagca gctgggttgg tatggagttg gcagacagga tgttcaactt aatgaagaaa  16620 

tacagctaag gccttgccag caacacctgc cgtaagttac tggctgagtg agggcataga  16680 

agttaaaggt tactgttttt atcctctatc cttttttcct ttcctgatca aggtgctctt  16740 

ctcatttttt cctgagaacc ttagccatca gatgaggctc cttagtttat tgtggttggt  16800 

tgttttttct ttataatggc tctgggctat atgcctatat ttataaacca gcagcagggg  16860 

aaagattata ttttataaga gggaacaaat tttcacaatt tgaaaagccc acataagttt  16920 

tctcttttaa ggtagaatct tgttaatttc attccaaaca tcggggctaa cagagactgg  16980 

aggcatttct ttttaggctc tgagactaaa tgagaggaaa agaaaagaaa aaaaaaatga  17040 

ttgtctaacc aattgtgaga attactgttt gaaacttttc aaggcacatt gaaatacttg  17100 

aaaacttctc atttatgtta tttatgatgt tattttgtac gtgttattat tattatattg  17160 

ttttataaat ggaggtacag gatatcacct gaattattaa tgaatgccca ggaagtaatt  17220 

ttcttctcat tcttctaaaa ctactgcctt tcaaagtgca cacacacgcg tccacataca  17280 

ctgcattcgt tgctccagta taaattacat gcatgagcac ctttctggct tttaagccaa  17340 

tataatgggc tgcaaaatga agacaccaga gtgtatgcat acaaatctca ctgtattaaa  17400 

gatgcaggtt ttctaattgt acccttcttg tctctctggc aatcttgccc ttaatatccc  17460 

tggagttcct catcagtgtc attttctgtt atacacagtt ccacaatttt gtctctagtt  17520 

gacttcaaat gtgtaacttt attggtcttg ccctattata attgtcatga ctttcagatt  17580 

gtatctgaac tcacagactg ctgtcttact aataggtctg gaaggtcacg ctgaatgaga  17640 

agtaaattat tttatgtaat acatttttga gtgtgttttt cagttgtatt tccctgttat  17700 

ttcatcacta tttccaatgg tgagcttgcc tgctcatgct ccctggacag aatactcctt  17760 

ccttttgcat gcctgtttct atcatgtgct tgataggcct caaagctaat gcttccagtg  17820 

aaacacacgc atcttaataa taagggtaaa taaacgctcc atatgaaact atttgcttgg  17880 

aaacacatta atgatccaga gacatgctat gagaaacatc agggtgtagg gtgactttag  17940 

aaaaatactc atactgagtc tttaatccct cctgtgccag tgaactctgg gaaagaaagt  18000 

acaaactgaa tattgtttat tctttagttc atgccactgc tctgcttggc tctactcata  18060 

gaaccaaggc aatcttagct tcagagactg caaaacagat taagtgattt gcttgcagat  18120 

tctcaatcaa ttttcaaggg atagagttca ccttccagag ccattctttt atttccagtt  18180 

acccgcctgt ttgagagatg atagagcagt gggaaattga gagagttgaa aggagctata  18240 

gattcttacc caaacttcaa aaatccttcc ctcccttttg ttaattctct ttcctggaaa  18300 

agaggtcata aaatgttcac atcctcagta ataggccctg tgctgtgtct attatgtcat  18360 

gagactccca tttcctgacc cttctttccc attgtaagag tagtagttac aaggtgttaa  18420 

ggatagatga tcttcaacac ttttgagaaa tagatccatt tacggatctg gtaaaaacta  18480 

tggaccgaac catcttttaa gaaaaaaatt cagagaggaa tctaaatttt gtgtgctttg  18540 

aggggaaact ctcagaatct cccctcaaaa ctatcattct tctcttatac tatagatgtg  18600 

tcagactctc actgggactg tatagttgct gctccctgta tttgataata tctatcaaga  18660 

actgcagggt aattcaaagt cacgctatta gcagcaagtg tgagcagtgt tggtttcccc  18720 

agtctctaca tccctcatcc tttctttctt ctttatggtt gtctattaaa gaaataaaaa  18780 

aaaatattgg ctgaccgttt ttctgaagat aatgtatatc aaggaccacc ttttgaaaaa  18840 

cactcattat tcgagaacaa agacacaaca tacgagaatc tctgggatac attcaaagca  18900 

gtgtgtagag ggaaatttat agcactaaat gcccacaaga gaaagcagga aagatctaaa  18960 

attgataccc taacatcaca attaaaagaa ctagaaaagc aagagcaaac acattcaaaa  19020 

gctagcagaa gacaagaaat aactaagatc agagcagaac tgaaggaaat agagacacaa  19080 

aaaacccttc aaaaaattaa tgaatccagg agctggtttt ttgaaaagat taacaaaatt  19140 

gatagactgc tagcaagact aataaagaag aaaagagaga agaatcaaat agacacaata  19200 

aaaaatgata aaggggatat caccaccgat cccacagaaa tacaaactac catcagagaa  19260 

tactataaac acctctacgc aaataaacta gaaaatctag aagaaatgga taaattcctc  19320 

gatacataca ccctcccaag accaaaccag gaagaagttg aatctctgaa tagaccaata  19380 

acaggctctg aaattgaggc aataatcaat agcttaccaa ccaaaaaaag tccaggacca  19440 

gatggattca cagctgaatt ctaccagacg tacaaagagg agctggtacc attccttctg  19500 

aaactattcc aatcaataga aaaagaggga atcctcccta actcatttta tgaggccagc  19560 

atcatcctga taccaaagcc tggcagagac acaaccaaaa aagagaattt tagaccaata  19620 

tccttgatga acattgatgc aaaaatcctc aataaaatac tggcaaaccg aatccagcag  19680 

cacatcaaaa agcttatcca ccatgatcaa gtgggtttca tccctgggat gcaaggctgg  19740 

ttcaacatac gcaaatcaat aaatgtaatc cagcatataa acagaaacaa agacaaaaac  19800 

cacatgatta tctcaataga tgcagaaaag gcatttgaca aaatttaaca actcttcatg  19860 

ctaaaaactc tcaatcaatt aggtattgat gggacgtatc tcaaaataat aagcactatc  19920 

tatgacaaac tcacagccaa tatcatactg aatgggcaaa aactggaagc attccctttg  19980 

aaaacgggca caagacaggg atgccctctc tcaccactcc tattcaacat agtgttggaa  20040 

gctctggcca gggcaattag gcaggagaag gaaataaagg gtattcaatt aggagaagag  20100 

gaagtcaaat tgtccctgtt tgcagatgac atgattgtat atctagaaaa ccccatcgtc  20160 

tcagcccaaa atctccttaa gctgataagc aacttcagca aagtctcagg atacaaaatc  20220 

aatgtacaaa aatcacaagc actcttatac atcaataaca gacaaacaga gagccaaatc  20280 

atgagtgaac tcccattcac                                              20300 

 
           
             4  
             480  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               A genomic sequence occurring upstream of RGS4  
             
           
            4 

ggattaatca tgacaaaagt aatctaaatc tcgttaagac tacttaatga tcaatctttc     60 

cctctgtttt ccctgactat agggaagtga attgccccaa tccttctcta tcacccccct    120 

gcagccatgc caatgcctta cctctgttat attcagccat aggggaagct tattctcata    180 

gaatcagggg ttggcatgta gtcactagct attcttggtg agactagtga agatgagtga    240 

aggaaaatat tgcataggtg aaatctcata ggcacaaata ggtgtttgtg agagtaacaa    300 

taaaagaaag tcattcccat actctagtag atgactcatt ttctcctcat tttttttttt    360 

tcaaggcgtt ctctacaacg gttaacctag taccaaaaat ccttctcttt tttcttggac    420 

aaatcctgtt caagttagca tggcatttac tacgtccaag acattgtcca gatgctgtgg    480 

 
           
             5  
             420  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               A genomic sequence occurring upstream of RGS4  
             
           
            5 

agagaaagaa aggcaggcag caaggagaaa aaacattttt taaaaaaaga aaattaaaat     60 

ccatgtaatg tctgatatct gttctgctgt atgtgtagat ctttccatat accaactcat    120 

tagccttatt ttacaggtga ggaaaatgag accgagagtc cttcttactt gaccaagttc    180 

acacagcaag atcacacatg gtagaaccaa tgttagaacc taggtgtata cttgctcatt    240 

caatatgtac aataattgca aaagtttcca taggtcttat tatatatcag gcactataaa    300 

tgctatgcat gtgtcaacta atttaaacct aagcaatatt ataaggaagg tactattata    360 

gaaatctcag ccttacaggt aagggaacag gaataaagag atgtgaggta atggcccaag    420 

 
           
             6  
             360  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               A genomic sequence occurring upstream of RGS4  
             
           
            6 

ataatctcct ttcaagtttt tatcctgtca cttgctagtt gtgtgatttg ggacaaatca     60 

tttaactcct tgtaaaggga gagaaggaag gctgtaaaaa aattaagtaa taaaaagata    120 

aactccttgt ggtatatttt gttattgttc aaaaatattt attgcccctc ttaggatgtc    180 

ttaggtcatt cttgcattgc tataaagaaa tacccaagtc tgggtaattt ataaagaata    240 

gaggttaaat tggctcacag ttctgcaggc tgcacaggaa gcatcccact ggcgtctact    300 

cacttctggt gaggactcag aaagcttttg cttatgacag caggctaagt gagagcaggt    360 

 
           
             7  
             420  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               A genomic sequence occurring downstream of 
      RGS4  
             
           
            7 

catggtattt ttactaccca ttgccttcta ggaaagggta taacaaatag gaaatattaa     60 

tatttttaat gcctttgagg gtgttaaaaa gcacaactct aaggactgtt tgtaaattcc    120 

aggtcaaatg ttgtttctcc ttctctattt cctaccttgg tgatggcctg atcttatatg    180 

gagtcactcc aactagaaac cacagaatca tccctagttc ctacttctga ctcactccat    240 

acactcaaaa gtcacctgac tctgcagaat ttctctagaa aaactctatg aaaacctatt    300 

cctgcctctc cacctgcata gatgtagctt catccaggct cttatggtgc atggcctcgg    360 

ttactgcctt atcctttcta ctggcctctc aatctcccat ctgataccca ttaatgtact    420 

 
           
             8  
             360  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               A genomic sequence occurring downstream of 
      RGS4  
             
           
            8 

ccaaatactt tttaggcaca ctgggaagtt acattgtttc ttgcaagtga caggttgtcc     60 

tttaattagt tctttctctc aaaaagagac tgctgactcc aaactgggaa gaaacccact    120 

caccagcaaa atgctgctga attcactctg atagttttct aatctctcat cagtagatga    180 

caataatgaa gccagtattg ttaccacaag actcagatat gtctatcacc caagatgatt    240 

tctctttaag acgcaataaa agggaacttt tctccccatt tattagcaac taagatgaaa    300 

tgagagccag agaaataaag tgaggaagga aagagaattt actaccttta caagctgaaa    360