Abstract:
Gene expression in normal unexercised hearts; chronically exercised, minimimally hypertrophied hearts; and hearts with hypertrophy due to renovascular hypertension is described. Methods of screening compounds for potential hypertrophic effects on cardiac muscle are provided.

Description:
RELATED APPLICATION  
       [0001]    This application claims priority to U.S. provisional application 60/280,048 filed Mar. 30, 2001. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The present invention relates to gene expression in normal unexercised hearts; chronically exercised, minimimally hypertrophied hearts; and hearts with hypertrophy due to renovascular hypertension.  
         BACKGROUND OF THE INVENTION  
         [0003]    Cardiac hypertrophy is the compensatory response of the myocardium to increased work. Cardiac hypertrophy causes an increase in the overall mass and size of the heart due to an increase in the size, not the number, of individual cardiac cells. (Since adult cardiac myocytes cannot divide, changes in cardiac myocyte number cannot occur in the adult heart.) Cardiac hypertrophy can occur in several ways. Physiologic hypertrophy is induced by exercise and seems to have no deleterious effect on the heart. Pathologic hypertrophy can be caused by pressure-overload (hypertension or aortic stenosis) or volume overload (mitral regurgitation), and this can lead to myocardial contractile failure (congestive heart failure and arrhythmia). Cardiac hypertrophy is a common clinical occurrence.  
         SUMMARY OF THE INVENTION  
         [0004]    According to one embodiment of the present invention there is provided a panel of genes that are differentially expressed in cardiac hypertrophic states; such genes are useful to identify and/or distinguish “good” (exercised-induced) cardiac hypertrophy from “bad” (hypertensive-induced) cardiac hypertrophy. 
       
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0005]    The present invention will be further described by way of example and with reference to the following figures:  
         [0006]    [0006]FIG. 1 shows genes differentially expressed in both RVhtn-induced cardiac hypertrophy hearts and Exercise-induced cardiac hypertrophy hearts.  
         [0007]    [0007]FIG. 2 shows genes differentially expressed in RVhtn-induced cardiac hypertrophy hearts and not in Exercise-induced cardiac hypertrophy hearts.  
         [0008]    [0008]FIG. 3 shows genes differentially expressed in Exercise-induced cardiac hypertrophy hearts vs. RVhtn-induced cardiac hypertrophy hearts. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0009]    The experimental model utilized by the present inventors compared gene expression in hearts from normal unexercised animals to both animals with chronically exercised, minimally hypertrophied hearts and animals with renovascular hypertensive cardiac hypertrophy produced by the one clip/one kidney model. Delineation of gene expression patterns derived from “good” (exercised-induced) and “bad” (hypertensive-induced) cardiac hypertrophy animal models were ascertained for use as disease markers. These markers are useful in the pre-clinical evaluation and optimization of novel lead compounds.  
         [0010]    The goal of the experiments was to determine the differential expression profile of mRNAs, using the GeneCalling technology, in hearts from normal unexercised rats to rats with chronically exercised, minimally hypertrophied hearts and rats with renovascular hypertensive cardiac hypertrophy produced by the one clip/one kidney model. Delineation of the differentially expressed genes in these two cardiac hypertrophy states identified genes that could be used as disease markers to separate “good” (exercised-induced) cardiac hypertrophy from “bad” (hypertensive-induced) cardiac hypertrophy during pre-clinical evaluation and optimization of novel lead compounds. Three sample groups were used in this study: 3 normal control rats (no cardiac hypertrophy), 3 rats with exercise cardiac hypertrophy induced through swimming in temperature-controlled water for 6 hrs/day for 6 weeks (3 hrs of swimming 2× a day with a 2 hr rest period in between), and 1 rat with renovascular hypertensive hypertrophy surgically induced using the one clip/one kidney model. Hypertrophy was assessed by heart wt/body wt ratios: Controls—0.24, 0.25, 0.25, Exercise—0.33, 0.30, 0.29 and RVhtn—0.32 (2 rats were not statistically different from controls and thus were not included in the GeneCalling study). All of the rats in the study were of the Han Wistar strain.  
         [0011]    The three sample groups in this study were compared for analysis in 2 different ways: Exercise-induced hypertrophy vs. control (Jobs 9838 and 34878) and Renovascular hypertensive hypertrophy vs. control (Jobs 9846 and 34877). All of these job comparisons were performed at thresholds of +/−1.5-fold expression difference and a statistical significance of 85%. However, the differences in jobs 9838 and 9846 were analyzed at different times; during which time there were several improvements to the difference finding software which explains, in part, why the number of differences that passed the thresholds and the n-fold modulations are slightly different. The data from the later jobs is the most accurate.  
         [0012]    In addition to improvements in the difference finding software, there was a sample mix-up error found in 4 of the subsequences (h0a0, i0a0, i0q0, l0h0) in the data from the original jobs 9838 and 9846. This sample mix-up problem centered on the single RVhtn sample being replaced by one of the exercise samples. This caused the misidentification of 5 genes as differentially expressed in the RVhtn sample. These genes were thrombin (m81397), C-reactive protein (m83176), 4-hydroxyphenolpyruvate (af082834), the rat ortholog of human very long-chain acyl-CoA synthetase homolog 2 (pg_rn_gbh af064255) and atrial natriuretic factor (m15868). There are two reasons that the problem was not identified earlier in the study: 1) There was only a single RVhtn sample and therefore no other samples for comparison, and 2) the exercise sample that was swapped in had its own set of differences that were not present in the other 2 exercise hypertrophy samples. The sample mix-up problem was corrected in all of the jobs, and four of the incorrectly identified genes, (thrombin, C-reactive protein, 4-hydroxyphenolpyruvate, rat VLCACS) were removed from the list of genes up-regulated in the RVhtn rat. (Once the incorrect data for ANP was removed, it was found that ANP was actually up-regulated +1.5-fold in the RVhtn sample, corresponding to previous literature reports.) A list of the genes seen to be modulated differently in the swapped Exercise hypertrophied rat (sample 72968) is shown in Table 1. This gene list indicates that this rat may have mounted an immunological response, although there is no retrospective pathology data to support this theory.  
                                                                       TABLE 1                       Genes Differentially Expressed in Exercise Sample 72968                                    Confirmed Genes                m83176   C-reactive Protein   +9.5           m81397   Thrombin   +5.8           af082834   4-hydroxyphenolpyruvate   +5.4           m15868   Atrial natriuretic factor (ANP)   −7.6                Not Confirmed Genes (with good GeneCalls)                k01933   Haptoglobin   +50.3           m29866   Complement component C3   +13.6           d17370   Cystathionine gamma-lyase   +7.6           x96721   Pregnenolone 16-alpha-   +4.4               carbonitrile-inducible cypP450           x02299   T-kininogen   +3.8           k00136   Glutathione S-transferase   +2.7                      
 
         [0013]    Over 35,200 gene fragments were assayed for expression level from an average of 85 subsequences in each job comparison. Overall, the exercise-induced hypertrophy rats showed fewer differences when compared to normal rats than the RVhtn rats did. The exercise-induced hypertrophy vs. control job comparison identified 103(0.3%)/82(0.2%) differences while the Renovascular hypertensive hypertrophy vs. control Job comparison identified 803(2.3%)/705(2.1%) differences. The greater than 7-fold increase in the number of differences in the RVhtn job vs. the Exercise job can, in part, be attributed to the smaller number of samples used in the RVhtn job (using a single RVhtn rat heart instead of triplicate samples allows for more background noise in the job comparison that would have been dampened by using triplicate samples). The larger number of differences can also be explained by the speculation that since RVhtn induced hypertrophy leads to a disease phenotype (progression toward heart failure) while exercise induced hypertrophy does not, the gene expression changes in the RVhtn cardiac hypertrophy are likely to be larger and greater in number.  
         [0014]    Of the total number of gene expression changes identified in all of the jobs, 160 were submitted to confirmation by GeneCall/Poisoning and 27 by Isolation/Poisoning, as described herein. A total of 46 GeneCall/Poisonings (29%) were completed with a successful poisoning, and a total of 23 differentially expressed bands submitted to Isolation/Poisoning (85%) were successfully isolated and sequenced. These confirmed bands identified fragments from 26 genes known in rat, 3 genes that could be defined by their similarity to a gene with known function in another species, 3 rat ESTs, and 2 novel sequences.  
         [0015]    A list of genes surmised, based on the literature, to possibly have an association with cardiac hypertrophy was prepared. Of these genes, 5 were identified to be differentially expressed in this study (ANP, alpha cardiac myosin heavy chain, SERCA-2, skeletal muscle actin and cyclin G). In addition, after the differentially expressed genes in these studies were determined, another 6 of the genes found to be differentially expressed were also found to have been previously reported to be associated with hypertrophy in the literature (cyclin D2, p27kip1 and the mitochondrial beta oxidation enzymes: HAD, ECH1, ECHB and MCAD). Of these 11 genes previously reported to be associated with hypertrophy, only cyclin D2 was found by the present study to be modulated in the opposite direction from the direction reported in the literature. Also, of the 11 genes previously reported, all of them were found to be differentially expressed in the renovascular hypertension-induced cardiac hypertrophy (RVhtn) samples and not changed in the exercise-induced cardiac hypertrophy samples (See FIG. 2). This may be a reflection of the lack of studies that have been reported describing gene expression changes in cardiac hypertrophy caused by exercise.  
         [0016]    Differentially Expressed Genes  
         [0017]    The goal of this study was to determine whether physiologic cardiac hypertrophy and pathologic hypertrophy could be differentiated by gene expression patterns and if so, could these genes be identified for use as a panel of markers to differentiate these two hypertrophy states. We have shown that physiologic and pathologic hypertrophy can be delineated at the level of gene expression patterns. Firstly, there are far fewer genes changing in the physiologic hypertrophy model compared to the pathologic hypertrophy model (0.2% of assayed bands changed in the exercise samples vs. 2.0% in the RVhtn samples). In the exercise job comparison (34878), there were 82 differences +/−1.5-fold or greater and 24 of these differences were +/−2.0-fold or greater. In the RVhtn job comparison (34877), there were 705 differences +/−1.5-fold or greater and 263 of these differences were +/−2.0-fold or greater.  
         [0018]    Secondly, of the 34 genes identified to be differentially expressed in this study, we only confirmed 7 that were differentially expressed in the same direction in both of the cardiac hypertrophy models (See FIG. 1; up-regulated—telethonin and skeletal muscle actin; down-regulated—cyclin D2, calsequestrin isoform, novel gene fragment, MOA and UCP2). There were an additional 7 bands down-regulated in both exercise and RVhtn and 6 bands up-regulated. However, since this pattern of expression was not of interest in determining gene expression patterns that could be used to differentiate physiological and pathological hypertrophy, these differences were not pursued further.  
         [0019]    A total of 17 genes were confirmed that were differentially expressed only in the RVhtn samples, 9 genes were confirmed that are only differentially expressed in the exercise samples and 1 gene was identified that was modulated in opposite directions in the RVhtn samples vs. the exercise samples. From this group, a panel of 20 cardiac hypertrophy marker genes has been compiled (See Table 2). These genes can be examined in experiments where cardiac hypertrophy is induced by lead compounds or drug candidates and they will be predictive in determining whether the hypertrophy induced by the compound aligns with the physiologic cardiac hypertrophy model (induced by exercise) or the pathologic cardiac hypertrophy (induced by renovascular hypertension).  
                                                                       TABLE 2                       20 Cardiac Hypertrophy Marker Genes                                    “Good” Hypertrophy Markers                u17254   NGFI-B   +4           j03179   D-binding protein   +4           s58745   TEF   +2           y00979   Beta beta enolase   +2           isolated   Rat nocturnin   +2           af106658   UBP45   +2           aa819672   rat EST (458 bp)   +2           ai407719   rat EST (640 bp)   +2           ai103318   rat EST sim. to Sop2-like gene   −2                “Bad” Hypertrophy Markers                x03894   UCP1   +4           m30596   Cytosolic malic enzyme   +2           m15868   ANP   +1.5           X62908   Cofilin   −10           d86924   p27kipl   −4           u06713   SM-20   −3           x15938   MYH6   −3           d16479   Mitochondrial long-chain   −3               3-ketoacyl-CoA thiolase           j02791   Medium chain acyl-CoA   −2               dehydrogenase           af043106   SERCA-2   −2           x70871   Cyclin G   −2                      
 
         [0020]    Gene expression changes caused by pathological cardiac hypertrophy has been extensively studied. It is known that pathologic hypertrophy induces large changes in energy metabolism in cardiomyocytes and modified contractile properties in the myocardium. There is a decrease in the rate of calcium uptake into the sarcoplasmic reticulum and an alteration in the speed of relaxation of the hypertrophied heart muscle. An increase in the ratio of beta myosin heavy chain isoform to the alpha myosin heavy chain isoform results in a slower rate of ATP cycling and ultimately a slower velocity of contraction and relaxation and an improved economy of cardiac pumping function. Most of the 17 genes identified that are only differentially expressed in the RVhtn sample and not changed in the exercise samples fit into this model.  
         [0021]    Genes Specific to Exercise-induced Hypertrophy  
         [0022]    Nine genes were found to be differentially expressed specifically in response to Exercise-induced hypertrophy and not changed in response to Renovascular hypertension-induced hypertrophy. Expression of eight of the genes was increased while expression of one of the genes was decreased. In addition, there was one gene that was modulated in opposite directions in the exercise-induced hypertrophy rats and the RVhtn-induced hypertrophy rats. Together, these ten genes give a profile of expressed genes specific to exercise-induced hypertrophy that can be used as markers to distinguish exercise-induced or “good” hypertrophy from renovascular hypertension-induced or “bad” hypertrophy”. Some of the more interesting of these genes are discussed below.  
         [0023]    Rat Immediate Early Gene Transcription Factor NGFI-B (GenBank u17254)  
         [0024]    NGFI-B (nerve growth factor-induced-B, also called Nur77 or TIS-1) is an immediate early gene originally identified due to rapid, transient induction in the rat pheochromocytoma cell line PC12 by nerve growth factor (NGF) (Milbrandt, 1988). NGFI-B has structural features of a ligand-activated transcriptional regulator and is a member of the NGFI-B subfamily of nuclear receptors. Other NGFI-B subfamily members are Nur-related factor 1 (Nurr1) and Neuron derived orphan receptor (NOR-1).  
         [0025]    NGFI-B (1692 bp) encodes a 564aa, 61 kD protein encoding an orphan nuclear receptor that is constitutively expressed in adult rat tissues with highest levels of mRNA found in the pituitary and high levels found in the cerebral cortex, muscle, ventral prostate, thymus and adrenal glands (Bandoh et al., 1997). NGFI-B mRNA is expressed in heart at low but detectable levels (approx. 100 attomoles/mg total RNA). NGFI-B expression can be induced by a variety of stimuli including stressors, cAMP, phorbol ester, growth factors, peptide hormones, neurotransmitters as well as physical stimulation such as membrane depolarization, mechanical agitation and a magnetic field (Maruyama et al., 1998). NGFI-B expression in the brain has also been shown to be induced in vivo by various treatments and insults [for example, NGFI-B is induced in the cerebral cortex, midbrain, and cerebellum of animals that experienced a convulsant-induced seizure (Watson and Milbrandt, 1989)]. NGFI-B has also been reported to be induced by light in the suprachiasmatic nucleus of the hypothalamus (Rusak et al., 1992; Morris et al., 1998), expressed at high levels and involved in induction of apoptosis in T-cells and T-cell hybrids (Woronicz et al, 1994; Liu et al., 1994), induced during the early stage of glomerulonephritis (Hayashi et al., 1996) and in small-cell lung cancer tumors (Ueda et al., 1999). However, NGFI-B knockout mice thrive and reproduce normally (Crawford et al., 1995). NGFI-B has been reported to regulate steroid hydroxylase transcription (no changes seen in this study; rat 21-hydroxylase mRNA, GenBank u56853, GeneCall: 0 of 9), corticotropin releasing hormone (CRH) gene (no changes seen in this study; rat corticotropin releasing hormone, GenBank m54987, GeneCall: 0 of 10) and human brain fructose-biphosphate aldolase C (no changes seen in this study; rat brain mRNA for aldolase C, GenBank x06984, GeneCall: 0 of 1). Previous studies have shown that NGFI-B expression can be induced in skeletal muscle cell lines (Lim et al., 1995) but is not induced in mouse gastrocnemius muscle by electrical stimulation of the sciatic nerve in a pattern of brisk intermittent exercise (Abu-Shakra, 1993).  
         [0026]    NGFI-B response element (NBRE; 5′-AAAGGTCA-3′ or 5′-GAATGCCA-3′) NGFI-B can bind to the NBRE as a monomer or heterodimerize with RXR and bind retinoic acid response elements to activate transcription (no changes seen in this study; rat retinoid X receptor alpha, GenBank I06482, GeneCall: 0 of 8). Also, homodimers of NGFI-B have been reported to bind to a novel response element (NurRE), within the pro-opiomelanocortin gene (POMC) promoter (no changes seen in this study; rat pro-opiomelanocortin gene, GenBank k01877, GeneCall: 0 of 6). Both a truncated form of NGFI-B lacking the putative ligand binding domain and full-length NGFI-B could activate transcription from a reporter plasmid in experiments in COS cells (Wilson et al., 1991). It was postulated that the NGFI-B ligand may be synthesized by COS cells, that a ligand may not be required by NGFI-B in order to activate transcription or the activation may have been weak compared to activation with a ligand.  
         [0027]    Identification of NGFI-B as the gene with the highest-fold induction in the exercise hypertrophied rats is a very interesting finding and has not been previously reported. Induction was only seen after 6 weeks of exercise training and subsequent cardiac hypertrophy and not in the RVhtn sample. (Transcriptional activation has previously been reported to have an immediate-early component, which can occur in the absence of de novo protein synthesis, and a delayed-early component, which is dependent on de novo protein synthesis—Williams and Lau, 1993).  
         [0028]    There was no full-length mRNA sequence available for rat NGF, only genomic sequence containing the second (coding) exon. This exon was not detected using any of the CuraGen subsequences. It would be of interest to assay the exercise hypertrophy-induced cardiac tissue to determine whether there is increased levels of NGF protein that is contributing to the upregulation of NGFI-B.  
         [0029]    Rat D-binding Protein (DBP) (GenBank j03179)  
         [0030]    DBP is a member of the PAR subfamily of basic/leucine zipper (bZip) transcription factors (the two other family members are Thyrotroph Embryonic Factor, TEF—also identified as up-regulated approximately 2-fold in this study, and Hepatic Leukemia Factor, HLF). The PAR domain is a conserved proline- and acidic-rich domain to the immediate amino-terminal side of the basic domain. Significant protein expression of DBP is confined to the liver, however DBP mRNA is present in most tissues (excluding testis). DBP was cloned due to its ability to bind to the D-site in the albumin promoter and activate transcription in the adult liver (Mueller et al., 1990). Expression of DBP is down-regulated upon induction of regenerative growth and dedifferentiation. DBP has also been reported to bind the promoters and transactivate several other genes expressed in the liver including cholesterol 7 alpha hydroxylase, cytochrome P450 CYP2C6, alcohol dehydrogenase, serpin, aldolase B and phosphoenolpyruvate carboxykinase (PEPCK) (Roesler et al., 1992). The ability of DBP to bind and activate the PEPCK promoter is especially interesting since PEPCK catalyzes the formation of phosphoenolpyruvate from oxaloacetate in the gluconeogenesis pathway showing that DBP can transcriptionally regulate an enzyme in a metabolic pathway. Transcription of phosphoenolpyruvate carboxykinase (PEPCK) has also been reported to be stimulated by retinoic acid and RAR, cAMP or thyroid hormone tri-iodothyronine with CCAAT-enhancer-binding protein (C/EBP) (Park et al., 1997) showing cooperativity of a transcription factor and nuclear receptors at affecting transcription of an enzyme. However, the ability of DBP to affect transcription of PEPCK has been used to explain the highest expression levels of PEPCK in the liver (contradicts our finding of induction of expression of DBP in the exercise-hypertrophied heart). Expression of liver-enriched transcription factors, including DBP, has been seen in cardiac mesoderm during fetal development (Van den Hoff et al., 1994) indicating that increased DBP expression during exercise-induced cardiac hypertrophy may be due to a reversion to a fetal expression pattern. DBP has been shown to have circadian expression patterns (Fonjallaz et al., 1996). Several of the genes identified to be modulated by exercise-induced cardiac hypertrophy have been shown to play a role in circadian rhythm (nocturnin, DBP and TEF). This may indicate a disruption of normal circadian rhythm gene expression in the exercise-induced hypertrophied heart.  
         [0031]    Rat Muscle-specific Beta-enolase (Beta Beta Enolase) (GenBank y00979 &amp; aa851223)  
         [0032]    There are 3 isozymes of enolase (2-phospho-D-glycerate hydrolase) that occur in mature mammalian tissues: alpha alpha—called non-neuronal enolase; gamma gamma—neuron-specific enolase; and beta beta—muscle-specific enolase. There has been a muscle-specific enhancer identified within the first intron of the human beta enolase gene (Feo et al., 1995). Enolase is an enzyme involved in the last stage of the glycolytic pathway that catalyzes the dehydration of 2-phosphoglycerate to phosphoenolpyruvate (one step prior to the formation of pyruvate and generation of the second ATP molecule). AA851223 is a 521 bp EST with 98% identity to beta beta enolase (y00979). It contains a point mutation difference from y00979 that creates a RE site and fragment m1n0-181.2, not seen in y00979. Beta beta enolase is most highly expressed in adult skeletal muscle. In a simplistic view, a switch from alpha alpha (non-neuronal) enolase to beta beta (muscle-specific) enolase expression occurs during the final stage of cell differentiation and beta beta enolase expression increases with the functional maturation of myotubes. However, the hybrid alpha beta enolase still contributes approximately 30% to total enolase activity in the adult heart. It has been reported that during pathological cardiac growth following aortic stenosis, there is a downregulation of the beta enolase gene leading to a less mature cardiac phenotype-increased alpha to beta enolase subunit ratio (Keller et al., 1995). This is exactly the opposite effect that was seen in the exercise-induced hypertrophied hearts. No change in expression of any of the enolase isozyme genes were seen in the RVhtn-hypertrophied hearts. It has been reported that enolase activity is low in adult rat liver and only the alpha alpha enolase isozyme is expressed (Keller et al., 1995).  
         [0033]    The set of 20 Cardiac Hypertrophy Marker Genes (Table 2), or a subset thereof, will be useful to evaluate drug compounds that cause hypertrophy to determine whether the hypertrophy resembles exercise-induced hypertrophy and would therefore be considered “Good” or if it resembles Renovascular-hypertension-hypertrophy and would therefore be considered “Bad”. These 20 genes would most likely be used in a screening panel to examine their expression in heart tissue from rats or other mammals treated with the compounds under investigation.  
         [0034]    The set of 9 genes that were differentially expressed in the exercise-hypertrophy samples (see Table 2), or a subset thereof, are useful to determine whether cardiac hypertrophy is similar to exercise-induced cardia hypertrophy. Although there have been many studies outlining the changes in gene expression in the heart due to volume or pressure-overload-induced hypertrophy, there are few studies that address the gene changes in exercise-induced cardiac hypertrophy, and none of the genes in the present study have been previously associated with “Good” hypertrophy. In particular, NGFI-B is an orphan nuclear receptor with activity that could be modulated by a small molecule compound. There are two novel sequences in this group, cgrny0n0162.9 — 9838-115 and cgrnw0c0282 — 9838-301, that extend rat ESTs but at this point only represent gene fragments and not full-length genes.  
       EXPERIMENTAL  
     Example 1  
     Materials and Methods  
       [0035]    Organism Source: rattus norvegicus  
         [0036]    Tissue Source: heart  
         [0037]    Sample Groups Submitted:  
                                                                         Sample Group   ID#&#39;s   Strain   Organ   Treatment                                1.   Control   72965/72966/   Han Wistar   Heart   Control               72967   rats       2.   Exercise   72968/72969/   Hans Wistar   Heart   Exercise               72970   rats       3.   RVhtn   72971   Hans Wistar   Heart   Renovascular                   rats       hypertrophy                  
 
         [0038]    RNA was isolated from the samples by grinding frozen tissue in liquid Nitrogen, followed by extraction of 2 grams of powder and purification of the total RNA using a standard Trizol protocol. Yields of total RNA for each sample passed the usual QC evaluations. The OD260/280 ratio was in the range of 1.6-1.8 and denaturing gel electrophoresis indicated that the ratio of the cytoplasmic ribosomal RNAs was correct. mRNA was purified from 50 micrograms of total RNA using oligo-dT magnetic beads, with yields ranging from 1832 to 2878 nanograms.  
                                                 Genomics Facility Processing Specifications:            Sample Group   Sample #s   ng cDNA   # Subsequences               Control   72965/72966/72967   982/951/1134   87       Exercise   72968/72969/72970   950/1107/953   86       RVhtn   72971   777   85                  
 
         [0039]    GeneCalling chemistry was performed on 1 ng of cDNA per subsequence pair. The GeneCalling method has 3 main steps: restriction endonuclease digestion, adapter ligation and PCR amplification. Briefly, the cDNA was digested with a standard set of 96 different pairs of restriction enzymes with 6 base-pair recognition sites (subsequence pairs). Complementary adapters were ligated to the digested cDNA and adapter-specific primers were used to direct 20 cycles of PCR, amplifying fragments containing sites for the pairs of restriction enzymes used. One adapter-specific primer is biotin-labeled while the other is labeled with the fluorescent dye FAM. Following PCR amplification, the biotin labeled DNA was purified on immobilized streptavidin. Denatured single-stranded DNA fragments were resolved by capillary electrophoresis on MegaBace instruments, and FAM-labeled fragments were detected upon laser excitation. Since the biotin label is necessary for purification and the FAM label is necessary for detection, all detected fragments result from restriction digestion with both enzymes. The output of the electrophoresis instruments was processed using the Java-based internet-ready Open Genome Initiative (OGI) software suite. Three independent reactions from the same cDNA sample were compared for quality of electrophoretic peak resolution and reproducibility of peak patterns. Composite traces from each sample were generated and then compared between three independent samples for peak quality and reproducibility. The resulting traces, placed in an Oracle database, represent the total gene expression profile for the tissue sampled and treatment in question. The databases for each sample can be compared to identify differences in gene expression resulting from the different cardiac hypertrophy treatments. The composite traces calculated for each sample group, based on average peak height and variance, were compared among sample groups using software designed to identify peaks representing differential expression.  
       Example 2  
     Analysis Strategy  
       [0040]    Goal: To identify genes that can be used as markers for “good” hypertrophy (i.e. exercise-induced, in this study) vs. “bad” hypertrophy (renovascular hypertension -induced).  
         [0041]    Jobs:  
         [0042]    Exercise-induced hypertrophy vs. control, # diffs=103 (0.3%)  
         [0043]    Exercise-induced hypertrophy vs. control, # diffs=82 (0.2%)  
         [0044]    Renovascular hypertensive hypertrophy vs. control, diffs=803 (2.3%)  
         [0045]    Renovascular hypertensive hypertrophy vs. control, # diffs=705 (2.1%)  
         [0046]    Analysis Strategy:  
         [0047]    a. Focused on known genes that were increased or decreased in expression in exercise vs. control and not changed or modulated in the other direction in the renovascular hypertension vs. control job.  
         [0048]    b. Focused on known genes that were increased or decreased in expression in renovascular hypertension vs. control and not changed or modulated in the other direction in the exercise vs. control job.  
         [0049]    In the four job comparisons, at least 34,000 bands per comparison were assayed. Jobs were run at stringency settings of N-fold difference cut-off greater than 1.5-fold in either direction and band significance of 0.85 or greater. All differences were visually inspected to ensure that the difference-finding algorithm had not identified questionable differences, shoulder peaks or occasional noise. The average number of subsequences used in the comparison was 85 (out of a total of 96).  
         [0050]    Confirmation and gene identification of differentially expressed bands can occur by two routes. The most efficient method is GeneCalling/Poisoning. In this case, cDNA fragments representing differentially expressed genes can be identified by database searching with the 6 base-pair restriction enzyme recognition sequences at the fragment ends and the exact length of each fragment (determined electrophoretically, subtracting linker length). Database searching for genes predicted to have restriction fragments of matching lengths enables the immediate identification of all of the genes whose sequences reside in that database and “flags” fragments derived from novel genes by virtue of their absence from the database. Given a three nucleotide size window, database lookup can provide a unique assignment of gene identity. This single hit is often referred to as a “GeneCall”. The detection of multiple fragments derived from the same gene which show differential expression of the same directional modulation increases the likelihood that the prediction of the gene identity is correct.  
         [0051]    The differentially expressed gene fragment and the gene, cDNA or EST sequence identified by the database lookup, are unequivocally linked through a positive poisoning reaction. In this process, the reaction containing the fragment of interest is performed a second time using the same end primers, but in the presence or absence of an excess of an unlabeled oligonucleotide whose sequence is derived from the predicted gene fragment. If the identity of the fragment was predicted correctly, the unlabeled oligo will out-compete the universal oligo for priming that fragment and appear in the chromatogram to ablate that peak specifically without affecting the amplification of the other fragments. The efficiency and success of the GeneCalling/Poisoning method relies predominantly on the quality of the database used. A database with large contigs assembled from EST sequences or a high proportion of full length sequences increase the probability that a gene is correctly identified and confirmed using the poisoning reaction. In this study, differentially expressed gene fragments were GeneCalled against the CuraGen SeqCalling Rat Assemblies (SCDB3) database, as well as the GenBank Rat and GenBank Rat Patent databases.  
         [0052]    An alternative but more time consuming method for gene identification and confirmation, Isolation/Poisoning, relies on the isolation of the differentially expressed fragment from the re-amplified GeneCalling chemistry reaction from a preparative gel. The gel-purified fragment is re-amplified and cloned in a standard PCR product cloning vector. The insert is sized and fragment. The Poisoning reaction is performed and analyzed as described above. Successful ablation of the peak using the unlabeled oligonucleotide based on the cloned sequence unambiguously identifies the sequence as corresponding to the original differentially expressed gene fragment. Subsequently, the gene identification is obtained by standard BLASTN or BLASTX analysis of the poisoning cloned sequence, against the CuraGen SeqCalling Rat Assemblies (SCDB3) database, as well as GenBank non-redundant DNA and protein databases. The BLAST analysis, and additional sequence analysis discussed herein, is conveniently performed within the GeneScape environment. Hits to genes from a different species are recognized with a “Similar to” (Sim.) identity assignment. The assignment of a “NOVEL” identification to a gene fragment is the result of either the complete absence of any BLAST hits or a BLASTN result with p&gt;1e-5.  
       Example 3  
     Results  
       [0053]    10.1 Job Array 34889: Exercise vs. RVhtn  
         [0054]    Jobs  
         [0055]    Job 9838: Exercise-induced hypertrophy vs. control  
         [0056]    Job 34878: Exercise-induced hypertrophy vs. control  
         [0057]    Job 9846: Renovascular hypertensive hypertrophy vs. control  
         [0058]    Job 34877: Renovascular hypertensive hypertrophy vs. control  
                                                                                                                   Band Statistics for Array 34889                        N-fold       Total   Differentially   Locked   Bands   Genes       Job   Set A   Set B   Thresholds   Subsequences   Bands   Expressed Bands   Bands   Confirmed   Confirmed                    9838   Rat hyper   rat hyper   +1.5/−1.5   86   35611   103   9   27   19           exer   ctrl       34878   Rat hyper   rat hyper   +1.5/−1.5   86   35599   82   0   0   0           exer   ctrl       9846   Rat hyper   rat hyper   +1.5/−1.5   85   35201   804   38   39   26           reno   ctrl       34877   Rat hyper   rat hyper   +1.5/−1.5   83   34405   705   0   1   1           reno   ctrl                  
 
         [0059]    [0059]                                                                                                                           Confirmation Status for Array 34889                    Isolations   Isolations   Isolations   Isolations   Poisons   Poisons   Poisons   Poisons   Poisons       Job   Job Title   Requested   Completed   In Process   Dropped   Requested   Passed   Failed   Dropped   In Process                    9838   Exercise-   16   14   0   2   39   13   22   4   0           induced           hypertrophy           vs. control       34878   Exercise-   0   0   0   0   0   0   0   0   0           induced           hypertrophy           vs. control       9846   Renovascular   11   9   0   2   118   30   75   13   0           hyper-tensive           hypertrophy           vs. control       34877   Renovascular   0   0   0   0   3   1   0   0   2           hyper-tensive           hypertrophy           vs. control                    
         [0060]    [0060]                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             Gene List for Array 34889            Gene Name   Accno   Bands   9838   34878   9846   34877   Description                    01.05.02 Ubiquination                UBP45: Rat   af106658   i0c0-   + 2.3     +2.1   —   —   Rat UBP45 is a deubiquiti-           deubiquitinating       220.1                   nating enzyme that was orig-       Enzyme (ubp45)       i010-                   inally cloned from skeletal               166.5                   muscle. Deubiquitinating               m0r0-                   enzymes are cysteine pro-               61.4                   teases that cleave ubiquitin                                   from ubiquitin-conjugated                                   protein substrates. There are                                   more than 90 identified                                   deubiquitinating enzymes with                                   significant sequence diver-                                   sity indicating a broad range                                   of substrate specificities.                                   Medline 10603300,                                   TrEMBL AAF14189,                    Job 9838           Comment 1: Gene fragments are actually identical to both rat UBP45 (af106658 &amp; af202454) and rat UBP69       (af106659 &amp; af202453). UBP45 and UBP69 are identical over the 3′ end of their sequence (about 1080 bp       out of 2095 bp total for UBP69). Both were cloned from skeletal muscle. There is also a very high       identity over the 3′ half of the sequence to mouse, human and chicken UBP41.               02.01.01 Peptide Hormones                ANF:Rat   m15868   i0a0-   —   1     −3     +1.7   ANF is a potent vasoactive           hypothalamic       135.5                   peptide synthesized and       atrial natriuretic                           secreted by mammalian heart       factor (ANF, ANP                           atria in response to volume       or atriopeptin) [ C ]                           expansion. ANF inhibits                                   sodium reabsorption in the                                   distal tubules of the kidney                                   and causes vasodilation                                   playing a key role in                                   cardiovascular homeo-                                   stasis. ANF has a CGMP-                                   stimulating activity.                                   Medline 2951736,                                   Swiss-Prot P01161.                    Job 9846           Comment 1: Original down regulation of ANP in this sample has been attributed to a sample mix-up. The       differences were failed.       Comment 2: In most reported studies, increased ventricular mass has been associated with high       ventricular expression of ANF. PMID 10381898               02.12.04 Cyclic Nucleotide Phosphodiesterases                PDE4B3: Rat   u95748   g1k0-   —   —     −1.9     −1.8   Cyclic AMP, a second mes-           cAMP-specific       370.4                   senger in the action of many       phosphodiesterase                           hormones, is formed from ATP       PDE4B                           by adenylate cyclase and de-       (PDE4B3)                           graded by a specific phospho-                                   diesterase. At least seven                                   different cyclic nucleotide                                   phosphodiesterases are known                                   to exist in mammalian                                   tissues. The PDE4 gene                                   family is one of the largest                                   with isoforms expressed in                                   many tissues including                                   skeletal muscle, airway                                   smooth muscle cells, brain,                                   lymphocytes, liver and                                   kidney. PDE4 is the primary                                   membrane-bound phospho-                                   diesterase found in rat                                   cardiac muscle. PDE4B3 is a                                   721aa “long form” PDE4B                                   splice variant with a                                   unique 79aa N-terminal                                   region.                                   Medline 1848733, 7480160,                                   9371714,                    Job 9846           Comment 1: There are no literature reports associating changes in PDE4 gene expression with cardiac       hypertrophy.               02.14.01 Transcription Factors                TEF: Rat gene   Cgrnk0n0319_9838-   k0n0-     +2     +2.1   —   —   Novel rat gene fragment (319           fragment   420   319                   bp) that overlaps the 3′-most       containing the 3′-                           12 nucleotides of rat thyro-       end of thyrotroph                           troph embryonic factor (TEF;       embryonic factor                           GenBank entry S58745 which       (S58745) [ N ]                           contains the sequence for rat                                   TEF CDS but no 3′UTR                                   sequence). TEF is a member of                                   the PAR (proline and acidic                                   amino acid-rich) subfamily of                                   basic leucine zipper (bZip)                                   transcription factors. TEF is                                   expressed exclusively in the                                   anterior pituitary during                                   embryogenesis but is found in                                   several tissues in juvenile                                   and adult rats. TEF can bind                                   DNA as a homodimer or as a                                   heterodimer with DBP. TEF and                                   DBP have both been implicated                                   to play roles in circadian                                   rhythms.                                   Medline 1916262, 8617210,                                   Swiss-Prot P41224,                    Job 9838           Comment 1: A contig can be made spanning this gene fragment and the rat TEF CDS (S58745) by using       2 mouse ESTs (Al892971 and AA138848) that are 96% identical at the nucleotide level to rat TEF       (S58745). No other bands were predicted to be detected with the standard 96 subsequence analysis.                    DBP: Rat D-   j03179   m0v0-     +4.3     +4.1   —   —   Rat D-binding protein (DBP)           binding protein       193.4                   is a member of the PAR sub-       (DBP)                           family of basic leucine                                   zipper (bZip) transcription                                   factors. Significant expres-                                   sion of DBP is confined to                                   the liver, however DBP mRNA                                   is present in most tissues                                   (excluding testis). DBP has                                   been reported to bind the                                   promoters and transactivate                                   several genes expressed in                                   the liver including albumin,                                   cholesterol 7 alpha                                   hydroxylase, cytochrome P450                                   CYP2C6, phosphoenolpyruvate                                   carboxykinase and angio-                                   tensinogen. DBP has been                                   shown to have circadian                                   expression patterns. PMID:                                   1059357 Medline 91249397,                     Job 9838           Comment 1: DBP has been reported to be expressed in embryonic cardiac mesoderm (prior to hepatic       endoderm formation). Histochem J 26:20-31, 1994               02.14.02 Nuclear Hormone/Orphan Receptors                NGFI-B: Rat   u17254   k0n0-     +4.4     +3   —   —   NGFI-B (nerve growth factor-           immediate early       78.6                   induced-B) is an immediate       gene transcription       l0w0-                   early gene originally       factor NGFI-B       97.7                   identified due to rapid,               w0c0-                   transient induction by nerve               108.1                   growth factor (NGF). NGFI-B                                   (1692 bp) encodes a 564 aa,                                   61 kd protein encoding an                                   orphan nuclear receptor that                                   can be induced by a variety                                   of stimuli, including                                   stressors. NGFI-B has been                                   reported to regulate steroid                                   hydroxylase transcription.                    03.03.02 Cyclins                Cyclin D2: Rat   d16308   l0e1-   —   +1.5     −1.6     −1.6   Cyclin D2 is normally ex-           cyclin D2       134.3                   pressed in the G1 phase of                                   the cell division cycle and                                   promotes progression through                                   G1 of the cell cycle. D-type                                   cyclins assemble with cyclin-                                   dependent kinases (CDK4 and                                   CDK6) to form holoenzymes                                   that facilitate exit from G1                                   by phosphorylating key sub-                                   strates (including Rb).                    Job 9846           Comment 1: Previous report associated an upregulation of cyclin D2 with LV hypertrophy in rats (days       3 to 21 post aortic constriction). Am J Physiol 275(3 Pt 2):H814-22, 1998                    Cyclin D2: Rat   I09752   g0y0-     +1.7     +1.5     −2.7     −2.3   Cyclin D2 is normally ex-           cyclin D2 (VIN1)       152                   pressed in the G1 phase of       [ C ]       l0w0-                   the cell division cycle and               97.7                   promotes progression through                                   G1 of the cell cycle. D-type                                   cyclins assemble with cyclin-                                   dependent kinases (CDK4 and                                   CDK6) to form holoenzymes                                   that facilitate exit from G1                                   by phosphorylating key sub-                                   strates (including Rb).                    Job 9838           Comment 1: Rat cyclin D2 (VIN1) is actually down regulated −3.2 fold in this job. This difference is       masked by an upregulated band from the immediate early gene transcription factor NGFI-B at this size       also.       Job 9846       Comment 1: Previous report associated an upregulation of cyclin D2 with LV hypertrophy in rats (days 3       to 21 post aortic constriction). Am J Physiol 275(3 Pt 2):H814-22, 1998                    Cyclin G: Rat   x70871   l0e1-   —   —     −1.8     −2.1   Cyclin G was identified as a           cyclin G.       343.3                   target of the p53 tumor sup-                                   pressor protein (levels of                                   cyclin G are increased after                                   induction of p53 by DNA                                   damage.) The function of                                   cyclin G has not yet been                                   established but it may be                                   associated with growth                                   stimulation. Cyclin G has                                   homology to  S. pombe  Cig1, a                                   B-type cyclin. Overexpression                                   of cyclin G may play a role                                   in facilitating apoptosis.                                   Medline 10467405, Swiss-Prot                                   p39950,                    03.03.03 Cyclin Dependent Kinase Inhibitors                p27kip1: Rat   d86924   d0v0-   —   —     −5.1     −4   The p27kip1 protein binds to           cyclin-dependent       129.3                   and inhibits complexes formed       kinase inhibitor                           by cyclin D1-CDK4, cyclin       p27 (p27kip1)                           A-CDK2 and cyclin E-CDK2.                                   Overexpression causes G1                                   arrest in cell cycle. p27kip1                                   has a region of sequence                                   similarity to the p21 cyclin-                                   Cdk inhibitor (p21Cip1/WAF1)                                   and may mediate TGF-b-induced                                   G1 arrest.                                   Medline 8033212, 8033213,                                   9264403,                                   Swiss-Prot P46414,                    Job 9846           Comment 1: Downregulation of p27kip1 has been proposed to modulate the adaptive growth of       cardiomyocytes during pressure overload-induced LVH. Am J Physiol 273:H1358-67, 1997.               04.01.01 Fatty Acid Synthesis                Cytosolic Malic   m30596   g1i0-   —   —     +1.8     +2   Malic enzyme (also called           enzyme: Rat       192.8                   NADP+—linked malate enzyme)       cytosolic malic                           catalyzes the oxidative       enzyme                           decarboxylation of Malate to                                   Pyruvate and produces NADPH                                   from NADP+. NADPH is then                                   used as the reducing agent                                   and a source of hydrogens for                                   chain elongation in fatty                                   acid synthesis. This reac-                                   tion occurs in the cytosol                                   and is involved in the                                   process of bringing Acetyl                                   CoA from the mitochondria                                   to the cytosol for fatty                                   acid synthesis. In this                                   process, Oxaloacetate and                                   Acetyl CoA are condensed to                                   form Citrate which is trans-                                   ported into the cytosol. Once                                   in the cytosol, Citrate is                                   cleaved back into Acetyl CoA                                   and Oxaloacetate. The Oxalo-                                   acetate is then converted                                   into Malate and then Pyruvate                                   (by Malic enzyme) to be                                   transported back into the                                   mitochondria and recycled.                    04.01.02.01 Mitochondrial Beta Oxidation                HAD: Rat L-3-   af095449   s0c0-   —   —     −1.7     −1.8   Third step in mitochondrial           hydroxyacyl-CoA       214                   beta oxidation. Catalyzes the       dehydrogenase                           oxidation of the hydroxyl       precursor                           group at C-3 into a keto                                   group and generates NADPH.                                   This enzyme is specific for                                   the L-isomer of the hydroxy-                                   acyl substrate.               ECH1: Rat   d16478   m1n0-   —   —     −2.3     −7   Second step in mitochondrial       mitochondrial       385.5                   beta oxidation. Catalyzes the       long-chain enoyl-                           stereospecific hydration       CoA hydratase                           between C-2 and C-3 of Enoyl                                   CoA.                                   Swiss-Prot Q62651,               ECHB: Rat   d16479   d0g0-   —   —     −3.1     −2.3   Final step in mitochondrial       mitochondrial       101                   beta oxidation. Uses CoA-SH       long-chain 3-       g0m0-                   to cleave the 3-ketoacyl-CoA       ketoacyl-CoA       105.3                   freeing up acetyl-CoA to go       thiolase                           into the Krebs cycle and                                   adding a new CoA group onto                                   the now-exposed 3-keto group                                   to create an acyl-CoA n-2                                   shorter than the previous                                   acyl-CoA and this restarts                                   the beta oxidation cycle.                                   Swiss-Prot Q60587,               MCAD: Rat   j02791   m1n0-   —   —     −2.1     −2.2   Short-chain, medium-chain,       medium chain       202.4                   and long-chain acyl-CoA       acyl-CoA                           dehydrogenases catalyze the       dehydrogenase                           initial reaction in the beta-                                   oxidation of fatty acids.                                   Medium chain acyl-CoA                                   dehydrogenase covers the                                   initial dehydrogenation of                                   C4-C12 straight chain acyl-                                   CoA&#39;s in mitochondrial beta                                   oxidation. (EC1.3.99.3)                                   OMIM 201450,                                   Swiss-Prot P11310,                    04.03.01 Glycolysis/Gluconeogenesis                Beta beta   gber_aa851223   m1n0-     +1.9     +1.9   —   —   521 bp EST with 98% identity           enolase-seq var:       181.2                   to  rattus norvegicus  beta       EST encoding                           beta enolase (y00979).       beta beta       enolase-sequence       variant, cloned       from rat placenta.                    Job 9838           Comment 1: AA851223 is a 521 bp EST with 98% identity to beta beta enolase (y00979). Contains a point       mutation difference from y00979 that creates a RE site and fragment m1n0-181.2, not seen in y00979.                    Beta beta   y00979   m0r0-     +2.1     +1.5   —   —   Rat muscle-specific beta-           enolase: Rat       251.6                   enolase (beta beta enolase).       muscle-specific                           Enzyme involved in the last       beta-enolase                           stage of the glycolytic       (beta beta                           pathway, catalyzes the de-       enolase). [ C ]                           hydration of 2-phosphogly-                                   cerate to phosphoenolpyruvate                                   (one step prior to the                                   formation of pyruvate and                                   generation of the second ATP                                   molecule).                    04.04.03 ATP/Proton Motive Force Interconversion                UCP2: Rat UCP2   ab010743   g0m0-   −1.4   −1.7   −1.6   −1.8   UCP2 (uncoupling protein 2)                   120.7                   is 59% homologous to UPC1.                                   UPCs are transmembrane                                   proteins of the inner mito-                                   chondrial membrane that can                                   uncouple ATP production from                                   mitochondrial respiration,                                   allowing energy to be                                   released as heat and de-                                   creasing energy metabolism                                   efficiency. Unlike UCP1 and                                   UCP3, UCP2 has a wide tissue                                   distribution including                                   heart, kidney, lung,                                   placenta, lymphocytes and                                   white fat.                    Job 9838           Comment 1: Down-regulated in both exercise and RVhtn hypertrophy jobs.                    UCP1: Rat   x03894   l0n0-   —   —     +4     +3.7   UCP1 (uncoupling protein 1)           nuclear mRNA for       181.2                   is a transmembrane protein       mitochondrial       m0s0-                   found in the mitochondrial       uncoupling       91.7                   inner membrane. When       protein                           activated by cold, UPC1 can       (UCP1) (mapping                           allow hydrogen ions to pass       request made)                           through the inner mito-                                   chondrial membrane, abol-                                   ishing the hydrogen ion                                   gradient necessary to drive                                   ATP synthesis which raises                                   the body&#39;s metabolic rate                                   and generates heat. The                                   protein is normally kept in                                   an inactive state by nucleo-                                   tides that bind the protein.                                   UPC1 expression is                                   restricted to brown adipose                                   tissue and induced upon                                   birth, cold acclimation and                                   overfeeding.                    Job 9846           Comment 1: Poisoning is only a pass partial. Looks like modulation is about 30% of total modulation       for the band. (28% modulation was seen in the poisoning reaction done on the control samples.)               04.05.02 Aspartate Family                ASAT: Rat   d00252   l0m0-   —   —     −1.8     −1.7   During amino acid degrada-           cytosolic       403.3                   tion, the alpha-amino group       aspartate                           of many amino acids is       aminotransferase                           transferred to 2-oxoglutarate       (also called                           (alpha-ketoglutarate) by an       Transaminase A                           aminotransferase to form       or glutamate                           glutamate. Aspartate amino-       oxaloacetate                           transferase catalyzes the       transaminase-1)                           transfer of the amino group                                   of aspartate to 2oxoglutarate                                   using pyridoxal-phosphate as                                   a cofactor. L-ASPARTATE +                                   2-OXOGLUTARATE = OXALO-                                   ACETATE + L-GLUTAMATE. In                                   eukaryotes, there are two                                   isozymes: a cytoplasmic form                                   and a mitochondrial form.                                   Swiss-Prot P13221,                    04.08.03 Catecholamine Metabolism                MAO-A: Rat   d00688   f0i0-     −1.4     −1.8     −1.7     −2   Monoamine oxidase A (MAO-A)           monoamine       172.2                   belongs to the flavin mono-       oxidase A       g1n0-                   amine oxidase family and               106.9                   catalyzes the oxidative                                   deamination of biogenic and                                   xenobiotic amines and has                                   important functions in the                                   metabolism of neuroactive                                   and vasoactive amines in                                   the central nervous system                                   and peripheral tissues.                                   MAO-A preferentially                                   oxidizes biogenic amines                                   such as 5-hydroxytryptamine                                   (5-HT), norepinephrine and                                   epinephrine. It is localized                                   to the mitochondrial outer                                   membrane.                                   Swiss-Prot P21396,                    Job 9846           Comment 1: Catecholamines control myocardial contractibility by interactions with the beta adrenergic       receptors and adenylate cyclase system.               04.11.02.06 Water                AQP1:Rat   X67948   u0g1-   —   —   +1.9     +1.8     Aquaporin 1 is a six-trans-           channel integral       235.5                   membrane domain protein that       membrane protein                           is present in many fluid       28.                           secreting and absorbing                                   tissues including kidney,                                   brain, heart and eye. Forms                                   a water-specific channel that                                   provides the plasma membranes                                   of red cells and kidney                                   proximal tubules with high                                   permeability to water,                                   thereby permitting water to                                   move in the direction of an                                   osmotic gradient.                                   Pharmacologically inhibited                                   by submillimolar concentra-                                   tions of Hg2+.                                   Medline 97445992, Swiss-Prot                                   P29975,                    Job 34877           Comment 1: Aquaporin1/CHIP28 has also been identified to be expressed in freshly dispersed,       differentiated, cultured rat aortic vascular smooth muscle cells but not highly expressed in other       smooth muscle tissues. PMID: 8393626       Comment 2: There is some data to support the regulation of AQP1 by arginine vasopressin (AVP) and       atrial natriuretic peptide (ANP). PMID:9299519               05.01.01 Components                Sim to human   ai103318   g1g0-     −1.9     −1.8   —   —   Rat EST (536 bp) cloned from           Sop2-like: Rat       117.8                   normalized rat embryo.       EST similar to                           Similar to human Sop2-like       human Sop2-like                           protein (y08999)- 92% over       gene cloned from                           198 bp and 98% over 62aa.       normalized                           ORF with similarity to y08999       embryo                           is encoded by nucleotides 1                                   to 186 in frame −1 of EST                                   ai103318. Sop2 (suppressor of                                   profilin-2) was first cloned                                   in  S. pombe , is a member of                                   the WD-repeat containing                                   protein family and is                                   presumed to have a role in                                   cortical actin-requiring                                   processes (shown to interact                                   with actin-related protein                                   (Arp3), profilin and actin.)                                   EST could not be extended.                                   Medline 8978670,                    05.01.01.03 Structural Arm: Actins &amp; Short Filaments                ACTA: Rat   v01218   g0c0-     +1.5     +1.5     +2.1     +2.1   Actins are highly conserved           skeletal muscle       294.7                   proteins that are involved in       actin [ C ]                           various types of cell                                   motility and are ubiquitously                                   expressed in all eukaryotic                                   cells. In vertebrates, 3 main                                   groups of actin isoforms                                   (alpha, beta and gamma) have                                   been identified. Alpha actins                                   are found in muscle tissues                                   and are a major constituent                                   of the contractile apparatus                                   while the beta and gamma                                   actins are primarily                                   components of the cyto-                                   skeleton and mediate internal                                   cell motility. Alpha actins                                   bind to myosin in the muscle                                   myofibrils and facilitate                                   release of ADP from myosin                                   following ATP hydrolysis                                   during muscle contraction.                                   Swiss-Prot p02568,                    Job 9846           Comment 1: Skeletal alpha actin may account for up to half of the actin mRNA in an adult heart with       cardiac alpha actin MrNA making up the rest (Mol Cell Bid 3:1985-95, 1983). However, increased       expression of skeletal alpha actin has long been known to be a marker of cardiac hypertrophy (J Clin       Invest 80:1194-9, 1987; PNAS 88:2132-6, 1991; Circ Res 72:857-64, 1993.       Comment 2: Cardiac alpha actin is reported as a partial fragment for  rattus norvegicus  (x00306). It       contains 1 predicted band, k0n0-116, that was present in exercise, RVhtn and control animals. The       complete cardiac alpha actin is reported for  rattus rattus  (X80130), and it is 98% identical to the       partial fragment reported for  rattus norvegicus . This  rattus rattus  fragment contains the same       predicted band, k0n0-116, plus 3 additional bands: i0l0-217, l0e1-367 and m0v0-37. These additional       bands may be slightly decreased in the RVhtn animal (−1.5), but do not seem to be changed in the       exercise animals.               05.01.01.04 Structural Arm: Heavy Filaments                MYH6: Rat alpha   x15938   l0w0-   —   —     −3.6     −3   Muscle myosin is a hexameric           cardiac myosin       131.1                   protein that consists of 2       heavy chain                           heavy chain subunits (MHC),                                   2 light chain subunits (MLC),                                   and 2 regulatory light chain                                   subunits (MLC-2). Muscle                                   myosin is found in the thick                                   filaments of the myofibrils.                                   ATP hydrolysis drives muscle                                   contraction which is produced                                   by the sliding of actin                                   filaments against myosin                                   filaments. Myosin is an                                   actin-activated ATPase. There                                   are two cardiac myosin heavy                                   chain isoforms, alpha and                                   beta, encoded by two separate                                   genes that are 4kb apart in                                   the rat genome. The cardiac                                   alpha isoform is a ‘fast’                                   ATPase, while the beta                                   isoform is a ‘slow’ ATPase.                                   Swiss-Prot P02563,                    Job 9846           Comment 1: Decreased mechanical performance has been reported to be due to decreased myosin       ATPase activity in various models of hemodynamic load (alpha-MHC has the highest ATPase activity and       beta-MHC has the lowest). A switch from alpha- to beta-MHC isozyme in response to cardiac overload       has previously been reported. In this study, we are seeing a decrease in expression of alpha-MHC, but       no change in beta-MHC expression.               05.01.01.05 Binding Proteins                CFL1: Rat cofilin   x62908   l0k0-   —   —     −11.2     −9.5   Cofilin is an actin modulated                   326                   protein that is widely                                   distributed throughout muscle                                   and non-muscle cells. Cofilin                                   controls actin polymerization                                   and depolymerization in a pH-                                   sensitive manner and has the                                   ability to bind g-and f-actin                                   in a 1:1 ratio of cofilin to                                   actin. Cofilin is the major                                   component of intranuclear and                                   cytoplasmic actin rods. Two                                   cofilin isoforms exist in                                   mammals, muscle type and                                   non-muscle type. Muscle type                                   was detected predominantly in                                   heart, skeletal muscle, C2                                   myotubes and testis by                                   Northern blot while non-                                   muscle type was seen in a                                   variety of non-muscle                                   tissues. There is only a                                   single cofilin sequence                                   available for rat.                                   Medline 8195165,                                   Swiss-Prot P45592,                    Job 9846           Comment 1: Although non-muscle and muscle isoforms have been reported for mouse and human, only a       single cofilin sequence is publicly available for rat. The rat sequence is more similar to the general       mouse cofilin sequence (non-muscle) than to the muscle specific sequence. There are no reports in the       literature of modulation in cofilin expression in association with cardiac hypertrophy.               05.01.03.03 Contractile CA + 2 Regulators                CASQ2: Rat   af001334   r0w0-     −18.3     −11.6     −18.5     −14.2   Calsequestrin is a high-       cardiac       434.5                   capacity, moderate affinity       calsequestrin       r0w0-                   Ca(2+)-binding protein               434.5                   localized in the terminal                                   cisternae luminal spaces of                                   the sarcoplasmic reticulum of                                   skeletal and cardiac muscle                                   cells. Calsequestrin                                   functions as a Ca(2+)                                   storage protein in the lumen                                   of the sarcoplasmic                                   reticulum. The release of                                   calcium bound to calseques-                                   trin through a calcium                                   release channel triggers                                   muscle contraction. Two                                   isoforms of calsequestrin                                   have been identified, cardiac                                   and skeletal, which both have                                   high acidic amino acid                                   composition. The two isoforms                                   are very similar except for                                   the carboxy-terminus. The                                   cardiac isoform is expressed                                   in heart and slow skeletal                                   muscle while the skeletal                                   muscle isoform is expressed                                   in fast and slow skeletal                                   muscle. Medline 7816057,                                   Swiss-Prot P51868,                    Job 9838           Comment 1: Calsequestrin sequence poisoned two bands, r0w0-434.5 (found only in control samples)       and r0w0-430.0 (found in hypertrophy samples and control samples). Sequence is found in the 3′ UTR of       calsequestrin, and this band is the only band for this gene that can be identified using GeneCalling.       There have been reports of a reduced ability of the sarcoplasmic reticulum to accumulate Ca2+\in       heart failure, but no expression changes in calsequestrin have previously been identified in       hypertrophy models.               05.10 Others            Sim to mouse   gber_aa799471   u0f0-     +2.8     +2.5     +1.4     +1.5   Telethonin is a sarcomeric           telethonin: Rat       69.2                   protein expressed in heart       EST (656 bp)       u0f0-                   and skeletal muscle (very       similar to mouse       69.3                   abundant transcript). Shown       telethonin                           to interact with titin/       (AJ223854) (87%                           connectin in a confirmation       identical, 3″end of                           dependent manner in Y2H       CDS)                           analyses and may be phos-                                   phorylated by titin/                                   connectin, suggesting a role                                   in myofibrillogenesis.                                   Specific to heart and muscle                                   tissue. Previously cloned in                                   human and mouse but sequence                                   is not yet reported for rat.                                   Swiss-Prot 015273,                    Job 9838           Comment 1: 87% identical to mouse telethonin (aj223854) gene, 58% identical to mouse telethonin       protein (translated).               07.02.03 Ion Pumps            SERCA-2: Rat   af043106   d0v0-   —   —     −2.7     −1.8   The SERCA Ca(2+)-ATPases           sarco/endoplasmic       133.9                   are intracellular pumps       reticulum Ca2+-                           located in the sarcoplasmic       ATPase (SERCA-                           or endoplasmic reticula of       2)                           muscle cells (integral                                   membrane protein). SERCA2                                   belongs to the large family                                   of P-type cation pumps that                                   couple ATP hydrolysis with                                   cation transport across                                   membranes. SERCA pumps                                   specifically maintain low                                   cytosolic Ca(2+) concen-                                   trations by actively trans-                                   porting Ca(2+) from the                                   cytosol into the sarco/                                   endoplasmic reticulum lumen.                                   ATP2A2 encodes 2 alterna-                                   tively spliced transcripts,                                   encoding isoforms SERCA2a and                                   SERCA2b, respectively.                                   SERCA2a and SERCA2b differ                                   in their carboxy termini and                                   have distinct tissue-                                   expression patterns. SERCA2a                                   is located primarily in heart                                   and slow-twitch skeletal                                   muscle, whereas SERCA2b is                                   present in smooth muscle and                                   nonmuscle tissues.                                   OMIM 108740,                                   Swiss-Prot P11508,                    Job 9846           Comment 1: Decreases in SERCA2a have been reported in hypertrophied cardiac tissue due to aortic       stenosis. Circulation 98(22):2477-86, 1998.               09 Unknown Function            Sim to mouse   cgrnl0y0157.2_9838-   l0y0-     +2.1     +2.1   —   —   Novel rat gene fragment (157           Nocturnin: 92%   73   157.2                   bp) with similarity to  mus         Sim. to Mus                           musculus probable nocturnin       musculus probable                           gene (u70139), 92% identity       nocturnin (U70139)                           at the nucleotide level and       [ N ]                           100% identity at the amino                                   acid level. This novel rat                                   gene fragment also has 77%                                   identity at the nucleotide                                   level and 80% identity at the                                   amino acid level to Xenopus                                   laevis nocturnin (u74761),                                   identified as a retina mRNA                                   that is expressed in peak                                   abundance at night. Nocturnin                                   has strong sequence                                   similarity to the C-terminal                                   domain of the yeast tran-                                   scription factor CCR4 as well                                   as a leucine zipper-like                                   dimerization motif and is                                   presumed to be a component of                                   the circadian clock or down-                                   stream effector of clock                                   function.                                   Medline 8962150, 9038221,                    Job 9838           Comment 1: Sequence could not be further extended                    Cgrnw0c0282_9838-   cgrnw0c0282_9838-   w0c0-     +2.6     +2.5   —   —   Novel 281 bp fragment that           301: Partially   301   282                   extends EST Al407719 (630 bp)       novel, extends rat                           by 23 bp to form a 653 bp       EST Al407719 [ N ]                           contig. No significant                                   homologies to genes of known                                   function at the DNA or                                   protein level. ORFs are found                                   in 5 reading frames with the                                   longest in frame +                                   3 (101aa).                    Job 9838           Comment 1: Novel 281 bp fragment that extends EST Al407719 by 23 bp to form a 653 bp contig:            GTCTAATGTCAGGGCGAAATCAAGCCCACGGCAAAGAATTATGAGACATCCCCAGGCACCAGG   (SEQ ID NO.1)           CTCACACTCCCAGGGCAGGACCAAAGACTGATGCCTAGAGCGGGTAAGGGGTGTCGTGGGTGTCCCTGAG       AAGCTCAGTC       CAGAGGGCCTTTGTCTAAGAGACTCTGAGAAAGGGATGGGTGGCAGGAAGCTTGGGGAATAAGGGTATTAA       GAAGAGAAT       AAATTAAAGGGGGGGCTTGAGGGACAAGGGGCCTGTGCTGTCCTTCAAACAGCTGGGAGCAGACCAGGGG       TGGGAAAGAG       GGTGGCGGGAAGAGCTTGATACACTATCTTAAGAAACACCGTTTACCCACTTCCCTCTTAACCACTGCAGTG       CACAACGA       GCCAGGGCACAGGGCAGGAGCCCACATGCCCCAGTGGCTTTCAACATGGCACGGGTATAAAGGGAAGCAG       CTGAGGGATA       TCTCAGGAAGGGGAAGTTATCCCCTGGTCCCCAAATGCTATAAGGCACAATTCTTGGAGGCAACTAGATTCC       ATCCAAAA       TATTAAAGGAAAAAAAAAAACAACTTCAAAACAGAAAACTTTAAATCCCAGGTCTACTGTGACTTCGCTTGGGC       CTGGTC AACACTCACCTAGCATCACAGGGGGCTAGC                    Cgrny0k0141.7_9838-   cgrny0k0141.7_9838-   y0k0-     +1.9     +1.5     −1.7     −1.7   141 bp gene fragment from rat           126: Partially   126   141.6                   that overlaps with rat EST       novel, extends rat       y0k0-                   H34237(310 bp) and extends       EST gber_h34237       141.7                   the EST by 57 bp creating a       [N]                           367 bp contig. Contig encodes                                   a 122aa ORF in frame-1 that                                   has 84% identity at the                                   nucleotide level and                                   approximately 70% identity                                   at the amino acid level to                                   human protein KlAA0025-                                   392aa(D14695), a predicted                                   transmembrane protein with                                   unknown function.                    Job 9838           Comment 1: 141 bp isolated fragment overlaps with rat EST H34237, creating a 367 bp contig:            CGGCTGCATTCTGAATTGGCCGGCTGGTTTTCTGCCGGAAACTGGTTGTGTAATAGGGNCGGGAGCCGGTG   (SEQ ID NO.2)           TAGAGACCA       CAGGTATTTCTTGTGCACTTGGTGTAGGGCCAAAAGCTCCTGAAGCAGCAGTGGCAGCCAAGTATTGCATGT       AGTACTNT       CTTGCATAGATCTGCTGGAACCAGGAGAGCTGCAGGCCACCCGTAGGTGGTGTAGCCAAAGAANCCGGGC       CCGAGGCCTT       GGAAAGTCTGCTGGACGGCTTCAGGCCTAGAGACGTTCTCCCATCCAGAGGGAGGAAGGTTCCGAAGGACT       TCCCTTTCC CGTAAGCCATCGCTTGAGGAATCCCCAGGATACTGTGCCTGACTAGT                    Cgrny0n0162.9_9838-   cgrny0n0.162.9 13  9839-   y0n0-     +2.6     −2.5   —   —   Novel 163 bp fragment that           115: Partially   115   162.9                   extends EST AA819672 (485 bp)       novel, extends rat                           53 bp to form a 538 bp       EST AA819672 [N]                           contig. Contig contains a                                   53aa ORF in frame − 1 starting                                   at nuc 1. No significant                                   homologies to genes of known                                   function at the DNA or                                   protein level. Does contain                                   strong homology to a 10aa                                   motif found in glutamine                                   amidotransferase class-II                                   proteins (esp. rat amino-                                   phosphoribosyltransferase-                                   D10853).                    Job 9838           Comment 1: Novel 163 bp fragment that extends EST AA819672 53 bp to form a 538 bp contig:            TTTTTTTTTTTTTTTTTAGACCCATATTAGGTTTATTTAATAACAGAGCACTCGCTTCTTTAAATAAAATATCTCA   (SEQ ID NO.3)           AAGT       TCTAGCTTTGCCTCAAACACAATGTTGCACCCAAACAGAAAAGCACAAATCAAACCAACAGAAAGATAGTTTT       TTTTAAA       AAATTATCTCCTTAGGCCTCTGTCTTTAACTTCCCCTTGTTCCTATTTCTATGAGAGAGACCGTAACGCACAGG       CTGAGG       AGACACACTGCCAACAAGGCTAATGTGCACCAGACCGAAGAGGGACAGCTCGGCTTTGGCCAGCCCTCTTC       CTGCAGGAT       ACCAATCCTATGTTTGCGTCAATCCTGACCTGCTCAGATGAAGCGGCACTCAGGCACTAGTCAGCCGTTGAC       CATACAAG       AACAGAGAACACTGGAGTAGACAGAGCTTTCTCCAGGAATGCTGACAGGCGTCCCTCCCTTTTGAGAAGTCC       TTTGCTTT CCTGACCCCTGTGCTTCAGGCACCCTGGCAAGGCCAACCAACTTCCTTCAGCTGTACA                    SM-20: Rat SM-20   u06713   f0k0-66   —   —     −4.1     −2.9   Identified by differential                                       screening of a rat aortic                                   smooth muscle cell cDNA                                   library to be induced                                   following treatment with                                   growth agonists (serum, PDGF,                                   angiotensin II). Expressed at                                   high levels in muscle cells                                   (smooth, skeletal and cardiac                                   as well as the brain - not                                   found in fibroblasts.) The                                   protein localizes to                                   filaments in the cytoplasm of                                   smooth muscle cells. In the                                   brain, see increased                                   expression in sympathetic                                   neurons deprived of nerve                                   growth factor (NGF)-proposed                                   role in regulation of                                   neuronal cell death. Text                                   article,                09.04 Novel       Cgrng0y0369_9846-   cgrng0y03969_9846-   g0y0-   —   —     −4     −2   Novel rat gene fragment (369       106: Novel -   106   369                   bp). No similarity to any       Sim. to mouse EST                           known genes at the nucleotide       AA286474 [ N ]                           or amino acid levels. ORFs                                   all 6 reading frames.                                   Sequence could not be                                   extended.                    Job 9846           Comment 1: Novel 369 bp fragment:            ggatccagtttgagacagtagctgttatgagatcactgcttctcctcatcttgctcctgagcacaggctgggagaagaca   (SEQ ID NO. 4)           tggggtgcactggggtatccttgtagaatagcagcctttattccttcctcaccttacctgtctctggaagggcagcaatt       gcatgggacaattaacatctggacttagcctctgatcatagctcagaaccttctggaatagcgggttagaggacagaatt       ttcagagcagggctgtttggtcaagaggtggacttaagccttccttgggcttcacttggccaactgaggcactcacaatg       tcccctcacgtctctgtcaatccaggttgacccccatctttggactagt                    Cgrng1k0329.8_9838-   cgrng1k0329.8_9839-   g1k0-     −6.4     −4.8     −7.7     −5.2   Novel rat gene fragment (327           225:   225   329.8                   bp). No similarity to any       Novel [ N ]       g1k0-                   known genes at the nucleotide               329.8                   or amino acid levels. ORFs                                   all 6 reading frames.                                   Sequence could not be                                   extended.                    Job 9838           Comment 1: Novel 327 bp fragment:            tctagaacctgtgaagtccagggagaagggagcacagtggccgtgggtgccactggcctcccagggaagcccttagatat   (SEQ ID NO. 5)           caccagtgtgcacagcagagcagcacacgtgtgtacgtgtgtgtatgtgtgtgtgcatgtgtgtgtatgtatgcctgggt       ccatgccggtgactgggcattggagggtctagggagggcaggactacagggactcctgcttggactgagccttcctacag       cctaggtagcctgtgtggctccagagccaggtagtcgtggtctctgtattagctggtcaggggaggcagtgaggggtatg tgggccc                    
         [0061]    References  
         [0062]    Abu-Shakra, S. R., Cole, A. J. and Drachman, D. B. Nerve stimulation and denervation induce differential patterns of immediate early gene mRNA expression in skeletal muscle. Brain Res Mel Brain Res 18, 216-20 (1993).  
         [0063]    Bandoh, S., Tsukada, T., Maruyama, K., Ohkura, N. and Yamaguchi, K. Differential expression of NGFI-B and RNR-1 genes in various tissues and developing brain of the rat: comparative study by quantitative reverse transcription-polymerase chain reaction. J Neuroendocrinol 9, 3-8 (1997).  
         [0064]    Crawford, P. A., Sadovsky, Y., Woodson, K., Lee, S. L. and Milbrandt, J. Adrenocortical function and regulation of the steroid 21-hydroxylase gene NGFI-B-deficient mice. Mol Cell Biol 15, 4331-16 (1995).  
         [0065]    Feo, S., Antona, V., Barbieri, G., Passantino, R., Cali, L., and Giallongo, A. Transcription of the human beta enolase gene (ENO-3) is regulated by an intronic muscle-specific enhancer that binds myocyte-specific enhancer factor 2 proteins and ubiquitous G-rich-box binding factors. Mol Cell Biol 15, 5991-6002 (1995).  
         [0066]    Fonjallaz, P., Ossipow, V., Wanner, G. and Schibler, U. The two PAR leucine zipper proteins, TEF and DBP, display similar circadian and tissue-specific expression, but have different target promoter preferences. EMBO J 15, 351-62 (1996).  
         [0067]    Hayashi, K., Ohkura, N., Miki, K., Osada, S. and Tomino, Y. Early induction of the NGFI-B/Nur77 family genes in nephritis induced by anti-glomerular basement membrane antibody. Mol Cell Endocrinol 123, 205-9 (1996).  
         [0068]    Keller, A., Rouzeau, J.-D., Farhadian, F., Wisnewsky, C., Marotte, F., Lamande, N., Samuel, J.-L., Schwartz, K., Lazar, M. and Lucas, M. Differential expression of alpha- and beta-enolase genes during rat heart development and hypertrophy. Am J Physiol 269, H1843-51 (1995).  
         [0069]    Lim, R. W., Zhu, C. Y. and Stringer, B. Differential regulation of primary response gene expression in skeletal muscle cells through multiple signal transduction pathways. Biochim Biosphys Acta 1266, 91-100 (1995).  
         [0070]    Liu, Z. G., Smith, S. W., McLaughlin, K. A., Schwartz, L. M. and Osborne, B. A. Apoptotic signals delivered through the T-cell receptor of a T-cell hybrid require the immediate-early gene nur77. Nature 367, 281-4 (1994).  
         [0071]    Maruyama, K., Tsukada, T., Ohkura, N., Bandoh, S., Hosono, T. and Yamaguchi, K. The NGFI-B subfamily of the nuclear receptor superfamily. Int J Oncol 12, 1237-43 (1998).  
         [0072]    Milbrandt, J. Nerve growth factor induces a gene homologous to the glucocorticoid receptor gene. Neuron 1, 183-8 (1988).  
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         [0074]    Mueller, C. R., Maire, P. and Schibler, U. DBP, a liver-enriched transcriptional activator, is expressed late in ontogeny and its tissue specificity is determined posttranscriptionally. Cell 61, 279-291 (1990).  
         [0075]    Ohshima, Y., Mitsui, H., Takayama, Y., Kushiya, E., Sakimura, K. &amp; Takahashi, Y. cDNA Cloning and Nucleotide Sequence of Rat Muscle-Specific Enolase (Beta Beta Enolase). FEBS Lett 242, 425-30 (1989).  
         [0076]    Park, E. A., Song, S., Olive, M. and Roesler, W. J. CCAT-enhancer-binding protein alpha (C/EBP alpha) is required for the thyroid hormone but not the retinoic acid induction of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription. Biochem J 15, 343-9 (1997).  
         [0077]    Roesler, W. J., McFie, P. J. and Dauvin, C. The liver-enriched transcription factor D-site-binding protein activates the promoter of the phosphoenolpyruvate carboxykinase gene in hepatoma cells. J Biol Chem 267, 21235-43 (1992).  
         [0078]    Rusak, B., McNaughton, L., Robertson, H. A. and Hunt, S. P. Circadian variation in photic regulation of immediate-early gene mRNAs in rat suprachiasmatic nucleus cells. Brain Res Mol Brain Res 14, 124-30 (1992).  
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         [0080]    Ueda, Y., Bandoh, S., Fujita, J., Sato, M., Yamaji, Y. and Takahara, J. Expression of nerve growth factor-induced clone B subfamily and pro-opiomelanocortin gene in lung cancer cell lines. Am J Respir Cell Mol Biol 20, 1319-(1999).  
         [0081]    Van den Hoff, M. J., Vermeulen, J. L., De Boer, P. A., Lamers, W. H. and Moorman, A. F. Developmental changes in the expression of the liver-enriched transcription factors LF-B1, C/EBP, DBP and LAP/LIP in relation to the expression of albumin, alpha-fetoprotein, carbamoylphosphate synthase and lactase mRNA. Histochem J 26, 20-31 (1994).  
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         1 
         
           
             5  
           
           
             1  
             653  
             DNA  
             Rattus rattus  
           
            1 

gtctaatgtc agggcgaaat caagcccacg gcaaagaatt atgagacatc cccaggcacc     60 

aggctcacac tcccagggca ggaccaaaga ctgatgccta gagcgggtaa ggggtgtcgt    120 

gggtgtccct gagaagctca gtccagaggg cctttgtcta agagactctg agaaagggat    180 

gggtggcagg aagcttgggg aataagggta ttaagaagag aataaattaa agggggggct    240 

tgagggacaa ggggcctgtg ctgtccttca aacagctggg agcagaccag gggtgggaaa    300 

gagggtggcg ggaagagctt gatacactat cttaagaaac accgtttacc cacttccctc    360 

ttaaccactg cagtgcacaa cgagccaggg cacagggcag gagcccacat gccccagtgg    420 

ctttcaacat ggcacgggta taaagggaag cagctgaggg atatctcagg aaggggaagt    480 

tatcccctgg tccccaaatg ctataaggca caattcttgg aggcaactag attccatcca    540 

aaatattaaa ggaaaaaaaa aaacaacttc aaaacagaaa actttaaatc ccaggtctac    600 

tgtgacttcg cttgggcctg gtcaacactc acctagcatc acagggggct agc           653 

 
           
             2  
             367  
             DNA  
             Rattus rattus  
             
               misc_feature  
               59, 159, 224  
               n = A,T,C or G  
             
           
            2 

cggctgcatt ctgaattggc cggctggttt tctgccggaa actggttgtg taatagggnc     60 

gggagccggt gtagagacca caggtatttc ttgtgcactt ggtgtagggc caaaagctcc    120 

tgaagcagca gtggcagcca agtattgcat gtagtactnt cttgcataga tctgctggaa    180 

ccaggagagc tgcaggccac ccgtaggtgg tgtagccaaa gaanccgggc ccgaggcctt    240 

ggaaagtctg ctggacggct tcaggcctag agacgttctc ccatccagag ggaggaaggt    300 

tccgaaggac ttccctttcc cgtaagccat cgcttgagga atccccagga tactgtgcct    360 

gactagt                                                              367 

 
           
             3  
             538  
             DNA  
             Rattus rattus  
           
            3 

tttttttttt tttttttaga cccatattag gtttatttaa taacagagca ctcgcttctt     60 

taaataaaat atctcaaagt tctagctttg cctcaaacac aatgttgcac ccaaacagaa    120 

aagcacaaat caaaccaaca gaaagatagt tttttttaaa aaattatctc cttaggcctc    180 

tgtctttaac ttccccttgt tcctatttct atgagagaga ccgtaacgca caggctgagg    240 

agacacactg ccaacaaggc taatgtgcac cagaccgaag agggacagct cggctttggc    300 

cagccctctt cctgcaggat accaatccta tgtttgcgtc aatcctgacc tgctcagatg    360 

aagcggcact caggcactag tcagccgttg accatacaag aacagagaac actggagtag    420 

acagagcttt ctccaggaat gctgacaggc gtccctccct tttgagaagt cctttgcttt    480 

cctgacccct gtgcttcagg caccctggca aggccaacca acttccttca gctgtaca      538 

 
           
             4  
             369  
             DNA  
             Rattus rattus  
           
            4 

ggatccagtt tgagacagta gctgttatga gatcactgct tctcctcatc ttgctcctga     60 

gcacaggctg ggagaagaca tggggtgcac tggggtatcc ttgtagaata gcagccttta    120 

ttccttcctc accttacctg tctctggaag ggcagcaatt gcatgggaca attaacatct    180 

ggacttagcc tctgatcata gctcagaacc ttctggaata gcgggttaga ggacagaatt    240 

ttcagagcag ggctgtttgg tcaagaggtg gacttaagcc ttccttgggc ttcacttggc    300 

caactgaggc actcacaatg tcccctcacg tctctgtcaa tccaggttga cccccatctt    360 

tggactagt                                                            369 

 
           
             5  
             327  
             DNA  
             Rattus rattus  
           
            5 

tctagaacct gtgaagtcca gggagaaggg agcacagtgg ccgtgggtgc cactggcctc     60 

ccagggaagc ccttagatat caccagtgtg cacagcagag cagcacacgt gtgtacgtgt    120 

gtgtatgtgt gtgtgcatgt gtgtgtatgt atgcctgggt ccatgccggt gactgggcat    180 

tggagggtct agggagggca ggactacagg gactcctgct tggactgagc cttcctacag    240 

cctaggtagc ctgtgtggct ccagagccag gtagtcgtgg tctctgtatt agctggtcag    300 

gggaggcagt gaggggtatg tgggccc                                        327