Ablation of pituitary pro-opiomelanocortin (POMC) cells produces alterations in hypothalamic POMC mRNA levels and midbrain mu opioid receptor binding in a conditional transgenic mouse model.

The hypothalamic-pituitary-adrenal (HPA) axis is regulated by stress-related excitatory inputs, and various inhibitory and negative-feedback controls by glucocorticoids and opioids, including pro-opiomelanocortin (POMC)-derived peptides. The role of POMC-derived peptides of pituitary origin in the modulation of brain POMC mRNA expression and opioid receptor binding was investigated using a line of transgenic mice that express a fusion gene composed of the pituitary expression-specific promoter region of the POMC gene driving the herpes simplex viral-1 thymidine kinase (TK). Male adult mice were treated with the antiherpes agent ganciclovir that selectively ablates cells expressing TK. Following treatment, POMC mRNA levels, measured by quantitative solution hybridization/RNase protection assays, were decreased by 48% in the pituitary of the TK+/+ mice, reflecting an expected loss of the pituitary corticotrope POMC cells. This treatment also significantly lowered pituitary beta-endorphin immunoreactivity content and plasma concentrations of corticosterone. In contrast, POMC mRNA levels were increased by 79% in the hypothalamus of the TK+/+ mice with pituitary POMC cell ablation. Binding of [(3)H]DAMGO to mu opioid receptors, as measured by quantitative autoradiography, was significantly reduced in several brain regions including the central grey, median raphe and superficial grey layer of the superior colliculus. These regions are innervated by hypothalamic POMC neurones. No significant differences in binding to either kappa or delta opioid receptors were found in the brain regions studied. These results suggest that POMC-derived peptides of pituitary origin may exert a tonic negative-feedback effect on hypothalamic POMC neurones. In turn, the downregulation of central mu opioid receptors in this model may be mediated through a mechanism related to hypothalamic POMC overexpression.

Novel and previously reported single-nucleotide polymorphisms in the human 5-HT(1B) receptor gene: no association with cocaine or alcohol abuse or dependence.

Evidence from animal self-administration and human genetics studies suggests that the serotonin(1B) (5-HT(1B)) receptor may be involved in modulating responses to cocaine or alcohol. We hypothesize that polymorphisms, including single-nucleotide polymorphisms (SNPs), in the human 5-HT(1B) receptor gene, may be associated with individual differences in vulnerability to cocaine or alcohol abuse or dependence. A total of 210 subjects were studied, including individuals with a primary diagnosis (DSM-IV criteria) of cocaine abuse or dependence, alcohol abuse or dependence, and controls with no history of previous or current illicit drug or alcohol abuse or dependence. Genomic DNA samples were isolated from each individual. For 157 of the subjects, polymerase chain reaction (PCR) was used to amplify the entire coding region of the 5-HT(1B) receptor gene as well as parts of the 5' and 3' untranslated regions. PCR products were sequenced in forward and reverse directions on an automated sequencer. Amplified DNA from an additional 53 subjects was sequenced in the 5' untranslated region to gain additional data on the frequency of one identified SNP. Seven polymorphisms were identified: one novel SNP in the 5' untranslated region (UTR) of the gene (A-161T); one SNP not reported in any published scientific communication (but found to be recorded in GenBank) in the 3' UTR (A1180G); two novel dinucleotide deletions at positions - 184/- 183 and - 182/- 181; and three previously identified SNPs (T-261G, C129T, G861C). Data were stratified by ethnicity and pooled Relative Risk was calculated for combined alcohol abuse and dependence cases and controls, and also for combined cocaine abuse and dependence cases and controls. No significant differences between cases and controls were found.

Elevation of guinea pig brain preprodynorphin mRNA expression and hypothalamic-pituitary-adrenal axis activity by "binge" pattern cocaine administration.

The endogenous opioid system and the hypothalamic-pituitary-adrenal (HPA) axis have been implicated in many of the neurobiological effects of cocaine. Previous studies in our laboratory showed that "binge" pattern cocaine administration increases preprodynorphin (ppDyn) mRNA levels in the caudate putamen and circulating levels of corticosterone in the rat. The present study extended these findings to guinea pigs, a species known to have a kappa opioid receptor profile similar to that of humans. Male guinea pigs were treated with: (a) "binge" pattern cocaine for 7 days (subchronic) (3 x 15 mg/kg/day, hourly, intraperitoneal); (b) "binge" pattern saline for 5 days followed by "binge" pattern cocaine for 2 days (subacute); or (c) "binge" pattern saline for 7 days. Thirty minutes after the final injection, levels of ppDyn mRNA were quantitated in the nucleus accumbens, caudate putamen, frontal cortex, amygdala, hippocampus, and hypothalamus using a solution hybridization RNase protection assay. Regional distribution of ppDyn mRNA levels in the guinea pig brain was similar to that found in rat, with highest levels in the nucleus accumbens and caudate putamen. In the caudate putamen, ppDyn mRNA was significantly increased following either 2 days (38% increase) or 7 days (32% increase) of "binge" pattern cocaine administration as compared to saline-treated controls. No significant changes in ppDyn mRNA levels were found in any other brain region. Both subacute and subchronic "binge" cocaine administration significantly elevated plasma levels of adrenocorticotropin hormone (ACTH) and cortisol. However, the ACTH and cortisol increases were significantly blunted following 7 days of "binge" cocaine administration as compared to 2 days of drug treatment, reflecting the development of HPA tolerance or adaptation to repeated cocaine administration. Thus, the ppDyn mRNA and HPA responses to cocaine in guinea pigs are similar to those observed in rats.

Detection of single nucleotide polymorphisms of the human mu opioid receptor gene by hybridization or single nucleotide extension on custom oligonucleotide gelpad microchips: potential in studies of addiction.

The human mu opioid receptor (MOR) plays a central role in mediating the effects of opioids, both endogenous and exogenous. Epidemiological studies have shown that addiction in general, and especially opiate addiction, has a heritable component. Clinical and laboratory studies suggest that the MOR gene may contribute to the heritable component of vulnerability to develop opiate addiction. Naturally occurring single nucleotide polymorphisms (SNPs) have been identified in the MOR gene by conventional methods. Two coding region SNPs, the A118G and C17T substitutions, occur at high allelic frequencies (10.5% and 6.6%, respectively, in our previous studies). These common SNPs cause amino acid changes in the receptor, and may have implications for differences in individual responses to opioids, as well as decreased or increased vulnerability to opiate addiction. The A118G substitution encodes a variant receptor with binding and signal transduction differences in response to beta-endorphin in cellular assays. Recent innovations in microchip technology offer new potential methods for SNP detection. We report here on the development of two separate approaches using custom oligonucleotide gelpad microarrays for detection of these two common SNPs of the MOR gene in human DNA samples. First, PCR-amplified genomic DNA samples were used to produce target sequences, which were labeled with fluorescent dye and hybridized to custom microchips. Oligonucleotides on these reusable microchips were designed to query nucleotide substitutions at positions 17 and 118 of the MOR gene. Thirty-six human DNA samples were assayed both on these custom microchips and by conventional automated gel sequencing, with highly concordant identification of both heterozygous and homozygous substitutions. A second approach was developed for the C17T SNP utilizing single nucleotide extension on custom microchips. These custom gelpad microchips have potential for the rapid and inexpensive detection of specific SNPs for genetic and genomic studies.

Opioid receptor and peptide gene polymorphisms: potential implications for addictions.

Addictions to drugs of abuse and alcohol have been shown by studies of genetic epidemiology to have both a heritable and an environmental basis, with these factors influencing addiction to different substances to a different extent. In the search for specific alleles of specific genes that may contribute to the development of the addictions, many researchers have focused on the endogenous opioid system, which mediates a diverse array of neurological, physiological, and behavioral functions. The endogenous opioid system is also centrally important in mediating the effects of drugs of abuse and alcohol. Polymorphisms, including single nucleotide polymorphisms, have been identified in genes of the endogenous opioid receptors and peptides. A number of recent genetic association studies and a few studies of potential function provide clues as to which genes and which alleles may have implications for human physiology and pathophysiology, including the addictions.

Single-nucleotide polymorphism in the human mu opioid receptor gene alters beta-endorphin binding and activity: possible implications for opiate addiction.

Opioid drugs play important roles in the clinical management of pain, as well as in the development and treatment of drug abuse. The mu opioid receptor is the primary site of action for the most commonly used opioids, including morphine, heroin, fentanyl, and methadone. By sequencing DNA from 113 former heroin addicts in methadone maintenance and 39 individuals with no history of drug or alcohol abuse or dependence, we have identified five different single-nucleotide polymorphisms (SNPs) in the coding region of the mu opioid receptor gene. The most prevalent SNP is a nucleotide substitution at position 118 (A118G), predicting an amino acid change at a putative N-glycosylation site. This SNP displays an allelic frequency of approximately 10% in our study population. Significant differences in allele distribution were observed among ethnic groups studied. The variant receptor resulting from the A118G SNP did not show altered binding affinities for most opioid peptides and alkaloids tested. However, the A118G variant receptor binds beta-endorphin, an endogenous opioid that activates the mu opioid receptor, approximately three times more tightly than the most common allelic form of the receptor. Furthermore, beta-endorphin is approximately three times more potent at the A118G variant receptor than at the most common allelic form in agonist-induced activation of G protein-coupled potassium channels. These results show that SNPs in the mu opioid receptor gene can alter binding and signal transduction in the resulting receptor and may have implications for normal physiology, therapeutics, and vulnerability to develop or protection from diverse diseases including the addictive diseases.

Regional quantitation of preprodynorphin mRNA in guinea pig gastrointestinal tract.

The endogenous opioid peptide dynorphin has been shown by immunochemical studies to be widely distributed in the gastrointestinal tract. The aim of this study was to determine basal levels of preprodynorphin (ppDyn) mRNA in different regions of the gastrointestinal tract of the guinea pig. A modified sensitive and specific solution hybridization RNase protection assay was used to quantitate ppDyn mRNA, with confirmation by gel analysis of the RNase protected hybrids and PCR amplified cDNA. This method combines high sensitivity and sufficient throughput to analyze large number of samples in a single assay. Low but measurable amounts of ppDyn mRNA were detected in fundus, duodenum, jejunum, ileum, cecum, and rectum. The rectum contained significantly more ppDyn mRNA than the stomach, small bowel, and cecum. The muscularis/myenteric plexus layer of both ileum and rectum contained a higher concentration of ppDyn mRNA per microg total RNA compared to the mucosa/submucosa/submucosal plexus. However, a greater absolute amount of ppDyn mRNA (80-85%) localized to the mucosal layer. The greater absolute amount of ppDyn mRNA in the mucosal layer may indicate the presence of dynorphin in the endocrine cells of the mucosa.

Distribution of nociceptin/orphanin FQ receptor transcript in human central nervous system and immune cells.

We have examined the distribution of the opioid receptor-like-1 (ORL-1) transcript in the human CNS as well as human immune cells by RT-PCR and RNAse protection. The hORL-1 mRNA was distributed throughout the brain and particularly abundant in cortical areas, striatum, thalamus and hypothalamus. In the immune system, gene transcription was observed in normal circulating lymphocytes and monocytes as well as in T, B and monocytic cell lines. A splice variant, lacking 15 nucleotides at the junction between exon 1 and exon 2, showed a distribution similar to the already known ORL-1 transcript. Altogether these results show comparable expression levels of the hORL-1 gene in both nervous and immune systems, suggesting that the ORL-1-encoded receptor may participate to neuronal and non-neuronal physiological functions in humans.

Distribution and polysome association of full-length and 3' truncated preproenkephalin mRNA in the guinea-pig brain.

The preproenkephalin gene is expressed in the mammalian central and peripheral nervous system as well as in other tissues, including the pituitary, adrenal medulla, lymphocytes, and reproductive and digestive systems. In the guinea-pig brain, preproenkephalin mRNA transcripts are cleaved at a specific site located in the 3' untranslated region. We used a solution hybridization RNAse protection assay to measure total levels of preproenkephalin mRNA and to determine the ratio of full-length to truncated forms in the pituitary and 12 brain regions of the guinea-pig. The overall distribution of preproenkephalin mRNA was found to be similar to that observed in other species in which it has been measured, with the highest levels of expression located in the caudate putamen and nucleus accumbens. The 3' truncated form was found throughout the brain in varying levels depending on the region. In nine regions examined, levels of the shorter form varied between 35 and 59% of total preproenkephalin mRNA content. The cerebellum and pons/medulla contained 17% each. In the pituitary, the only non-brain tissue studied, the truncated form constituted only 4% of total preproenkephalin mRNA. No correlation between absolute mRNA levels and the distribution between the two forms was observed. Both the full-length and 3' truncated mRNA forms were associated with polysomes isolated from the guinea-pig caudate putamen by sucrose density gradient centrifugation, suggesting that both mRNA forms may be actively translated. The distribution between the two forms, however, was different on the polysomal and dissociated monosomal gradients: the polysomal preproenkephalin mRNA contained a higher proportion of the full-length form whereas the monosomal fractions contained slightly more of the truncated form. This discontinuity in the amount of the two forms between polysomal and non-polysomal mRNA may suggest that the full-length form is more readily incorporated into polysomes.

Guinea pig preprodynorphin mRNA: primary structure and regional quantitation in the brain.

We isolated and sequenced genomic and cDNA clones of the guinea pig preprodynorphin (ppDyn) mRNA. The sequence of ppDyn mRNA was deduced from a combination of genomic and cDNA clones: The primary structure of two coding exons was derived from a genomic clone and 5' and 3' untranslated sequences were obtained using rapid amplification of cDNA ends (RACE). The predicted mRNA of 2,350 nucleotides coincides well with the size of transcripts in Northern blot analyses of RNA from different brain regions. The deduced amino acid sequence of guinea pig ppDyn shares 70%, 68%, and 61% identity to porcine, human, and rat ppDyn, respectively. The 5' untranslated sequences of guinea pig hippocampal and adrenal ppDyn mRNA are identical; both contain sequences of exon I and, like porcine mRNA, lack an exon (exon II) present in human and rat mRNA. Quantitative solution hybridization RNase protection analysis of total RNA from selected guinea pig brain regions was performed. The nucleus accumbens was found to have the greatest abundance of ppDyn mRNA, followed by caudate putamen, hippocampus, hypothalamus, amygdala, frontal cortex, olfactory bulb, and pons/medulla.

Steady-state methadone in rats does not change mRNA levels of corticotropin-releasing factor, its pituitary receptor or proopiomelanocortin.

Male Fischer rats received either methadone (a long-acting opioid agonist, 10 mg/kg/day) or saline (24 microliters/day) subcutaneously by osmotic minipumps for 7 days. Chronic steady-state methadone administration did not alter (a) corticotropin-releasing factor (CRF) mRNA in the hypothalamus, (b) proopiomelanocortin (POMC) and CRF type 1 receptor (CRF-R1) mRNAs in the anterior lobe and neurointermediate/posterior lobe of the pituitary, or (c) circulating levels of corticosterone. No change was found in levels of either POMC mRNA in the hypothalamus and amygdala, or CRF mRNA in the frontal cortex, olfactory bulb and amygdala. These results demonstrate that neither the activity of the hypothalamic-pituitary-adrenal axis, nor the beta-endorphin and CRF systems in the brain, are altered by steady-state occupancy of opioid receptors with the long-acting opioid agonist methadone.

Corticotropin-releasing factor and type 1 corticotropin-releasing factor receptor messenger RNAs in rat brain and pituitary during "binge"-pattern cocaine administration and chronic withdrawal.

Endogenous central corticotropin-releasing factor (CRF) may be involved in the neuroendocrine and behavioral responses to cocaine. In the present study, levels of CRF mRNA were measured in the hypothalamus and in several extrahypothalamic brain regions after different regimens of "binge"-pattern cocaine administration. Male Fischer rats were injected with saline or cocaine (15 mg/kg, 1 hr x 3/day) at the beginning of the light cycle, to approximate the manner in which cocaine is often abused by humans, both in terms of temporal pattern and in relation to circadian rhythm. Cocaine administered in this binge regimen produced time-dependent alterations of CRF mRNA levels in the hypothalamus. There was a significant increase in CRF mRNA levels on day 1, which returned to base-line levels on day 2, with elevated plasma corticosterone levels on both days. CRF receptor type 1 and prooplomeianocortin mRNA levels in the anterior lobe of the pituitary were not significantly altered after acute cocaine injections on day 1 or day 2. On day 14 of chronic binge-pattern cocaine administration, decreased hypothalamic CRF mRNA levels and an attenuated elevation in plasma corticosterone levels were found. After 10 days of withdrawal from 14-day binge cocaine, CRF mRNA returned to basal levels. CRF mRNA levels in the amygdala were also significantly increased on day 1 and returned to basal values on day 2. Chronic (14-day) binge cocaine administration did not alter CRF mRNA levels in the amygdala. These results suggest that the attenuated response in the hypothalamic-pituitary-adrenal axis to chronic binge cocaine administration is coupled to the cocaine-induced decreases in CRF gene expression in the hypothalamus and that activation of CRF gene expression in extrahypothalamic regions may have implications for a molecular understanding of the behavioral responses to cocaine.

Modulation of CRF-R1 mRNA in rat anterior pituitary by dexamethasone: correlation with POMC mRNA.

Glucocorticoids have been shown to decrease CRF receptor binding in the anterior pituitary. To determine whether glucocorticoids or CRF peptide modulate CRF-R1 gene expression, CRF-R1 mRNA levels in rat pituitary and brain were measured after administration of a synthetic glucocorticoid, dexamethasone (DEX) or rat/human CRF (r/hCRF), using a sensitive solution hybridization RNase protection assay. DEX (400 micrograms/day) or r/hCRF (100 micrograms/kg/day) was administered twice daily for 5 days to male rats. DEX treatment caused a significant decrease in CRF-R1 mRNA levels in the anterior pituitary. Also, a significant positive correlation was found between CRF-R1 and POMC mRNA levels in the anterior pituitary of the individual animals: levels of both mRNAs were reduced by DEX. However, r/hCRF treatment had no significant effect on CRF-R1 mRNA levels in the anterior pituitary. In extrahypothalamic brain regions, mRNA levels of CRF-R1 did not change following either DEX or r/hCRF administration. Our data suggest that in addition to POMC and CRF genes, CRF-R1 gene may also be subject to negative feedback control by glucocorticoids.

Structure and expression of the guinea pig preproenkephalin gene: site-specific cleavage in the 3' untranslated region yields truncated mRNA transcripts in specific brain regions.

We isolated the guinea pig preproenkephalin gene from a genomic library by hybridization to a rat cDNA probe. The entire nucleotide sequence of the gene was determined. Genomic Southern blot hybridization demonstrated that the gene exists in a single copy within the genome. On the basis of RNase protection transcript mapping and homology comparisons with known preproenkephalin sequences from other species and assuming a poly(A) tail length of 100 residues, we predicted an mRNA transcript of approximately 1,400 nucleotides encoded by three exons. Northern (RNA) blot analysis of total RNA from several brain regions showed high levels of preproenkephalin mRNA in the caudate putamen, nucleus accumbens, and hypothalamus, with detectable levels in the amygdala, ventral tegmental area, and central gray and also in the pituitary. Unexpectedly, in several brain regions, the mRNA appeared not only in the 1,400-nucleotide length but also in a shorter length of approximately 1,130 bases. Significant amounts of the shorter mRNA were found in the caudate putamen, nucleus accumbens, and amygdala. The longer, but not the shorter, transcripts from the caudate putamen were found to be polyadenylated, but the difference in size was not due solely to the presence of poly(A) tails. Northern gel analysis of total RNA from the caudate putamen with probes from each exon, together with RNase protection mapping of the 3' end of the mRNA demonstrated that the 1,400-base preproenkephalin mRNA transcripts are cleaved in a site-specific manner in some brain regions, yielding a 1,130-base transcript and a 165-base polyadenylated fragment derived from the terminal end of the 3' untranslated region of the mRNA. This cleavage may serve as a preliminary step in RNA degradation and provide a mechanism for control of preproenkephalin mRNA abundance through selective degradation.

Repressor to activator switch by mutations in the first Zn finger of the glucocorticoid receptor: is direct DNA binding necessary?

Transfection of HeLa cells with cDNA vectors expressing the wild-type human glucocorticoid receptor (GR) enabled dexamethasone to strongly repress cytokine- and second messenger-induced expression of cotransfected chimeric reporter genes containing transcription regulatory DNA elements from the human interleukin 6 (IL-6) promoter. Deletion of the DNA-binding domain or of the second Zn finger or a point mutation in the Zn catenation site in the second finger blocked the ability of GR to mediate repression of the IL-6 promoter. Unexpectedly, deletion of the first Zn finger, a point mutation in the Zn-catenation site in the first finger, or one in the steroid-specificity domain at the base of the first finger converted GR into a dexamethasone-responsive activator that enhanced basal and interleukin 1-induced IL-6 promoter function. These first-finger mutants of GR also mediated dexamethasone-responsive enhancement of expression of the herpesvirus thymidine kinase-chloramphenicol acetyltransferase (TK-105-CAT and TK-80-CAT) reporter genes but not of the murine mammary tumor virus long terminal repeat-CAT or the c-fos-CAT (pFC700) reporter genes. Wild-type GR was able to specifically bind to DNA fragments containing glucocorticoid response element sequences in both the murine mammary tumor virus and IL-6 promoters, albeit weakly to the latter, in a sequential DNA-binding immunoprecipitation assay. The first-finger mutants of GR, however, were inactive in this assay. Thus, mutations in the first Zn finger unmask unusual promoter-specific activation properties of GR that may not require direct high-affinity binding of the mutant GR to target DNA.

On the mechanism for efficient repression of the interleukin-6 promoter by glucocorticoids: enhancer, TATA box, and RNA start site (Inr motif) occlusion.

The feedback inhibition of interleukin-6 (IL-6) gene expression by glucocorticoids represents a regulatory link between the endocrine and immune systems. The mechanism of the efficient repression of the IL-6 promoter by dexamethasone (Dex) was investigated in HeLa cells transiently transfected with plasmid constructs containing different IL-6 promoter elements linked to the herpesvirus thymidine kinase gene (tk) promoter and the bacterial chloramphenicol acetyltransferase gene (cat) and cotransfected with cDNA vectors constitutively expressing either the active wild-type or inactive mutant human glucocorticoid receptor (GR). The induction by interleukin-1, tumor necrosis factor, phorbol ester, or forskolin of IL-6-tk-cat chimeric constructs containing a single copy of the IL-6 DNA segment from -173 to -151 (MRE I) or from -158 to -145 (MRE II), which derive from within the multiple cytokine- and second-messenger-responsive enhancer (MRE) region, was strongly repressed by Dex in a wild-type GR-dependent fashion irrespective of the inducer used. The induction by pseudorabies virus of an IL-6 construct containing the IL-6 TATA box and the RNA start site ("initiator" or Inr element) but not the MRE region was also repressed by Dex in the presence of wild-type GR. DNase I footprinting showed that the purified DNA-binding fragment of GR bound across the MRE, the TATA box, and the Inr site in the IL-6 promoter; this footprint overlapped that produced by proteins present in nuclear extracts from uninduced or induced HeLa cells. Imperfect palindromic nucleotide sequence motifs moderately related to the consensus GR-responsive element (GRE) motif were present at the Inr, the TATA box, and the MRE II site in the IL-6 promoter; although MRE I and a GR-binding site between -201 and -210 in IL-6 both lacked a discernible inverted repeat motif, their sequences showed considerable similarity with negative GRE sequences in other Dex-repressed genes. Surprisingly, chimeric genes containing MRE II, which lacks a recognizable GACGTCA cyclic AMP- and phorbol ester-responsive motif, were strongly induced by both phorbol ester and forskolin, suggesting that MRE II (ACATTGCACAATCT) may be the prototype of a novel cyclic AMP- and phorbol ester-responsive element. Taken together, these observations suggest that ligand-activated GR represses the IL-6 gene by occlusion not only of the inducible IL-6 MRE enhancer region but also of the basal IL-6 promoter elements.

Isolation and sequence of the canine pancreatic phospholipase A2 gene.

A genomic library has been constructed in EMBL3 lambda phage using high molecular mass DNA isolated from canine spleen. A cDNA clone, shown to code for preprophospholipase A2 which is processed to the prosecretory form prior to release from secretory cells, was used to identify a lambda clone which contains the complete phospholipase A2 gene. Restriction enzyme and DNA sequence analysis indicate that the primary transcriptional unit for the phospholipase gene, approximately 9.0 kb, is organized into four exon sequences. Exon 1 encodes the 5' nontranslated sequence, the ATG initiation codon, and the hydrophobic core of the signal peptide. Exons 2-4 encode regions of the peptide of residues -11 to 43, 43 to 86 and 86 to 124, respectively. The 5' flanking region shows a TATA box at position -29 and multiple CAAT boxes at positions -279, -206, -183 and -159. Regions of the 5' flanking sequence in the canine sequence, from nucleotides -47 to -74 and -91 to -129, show high similarity to similar regions in the human gene. However, an analysis of 400 nucleotides of the 5' flanking sequence in transient expression studies was unable to identify tissue-specific promoter or enhancer sequences. Within 5' nontranslated regions the canine and human genes share a pyrimidine-rich sequence which may be involved in differential regulation of mRNA translation.

Structure of the canine pancreatic lipase gene.

Identification of three overlapping clones in a canine genomic lambda phage library allowed us to determine a detailed restriction enzyme map of the primary transcriptional unit of the pancreatic lipase gene (15.5 kilobase pairs) as well as 15 and 6 kilobase pairs of 5'- and 3'-flanking regions, respectively. DNA sequence analysis provided the primary structure of (a) 1,345 nucleotides (nt) of 5'-flanking sequence including CAAT and TATA boxes at positions -112 and -35, respectively, and a class 2 glucocorticoid receptor binding sequence at position -97, (b) 13,127 out of approximately 15,500 nt of the transcriptional unit which is organized into 13 exon sequences, and (c) 1,270 nt of 3'-flanking sequence. Exon 1 encodes the entire 5'-nontranslated mRNA sequence; exon 2, the ATG initiation codon and the hydrophobic portion of the signal peptide; and exon 6, Ser154 which shows homology to the active Ser152 in the porcine enzyme. Comparison of the amino acid sequences of human lipoprotein lipase, rat hepatic lipase, and Drosophila yolk proteins 1, 2, and 3 with canine pancreatic lipase shows that the central region of highest homology (encoded by exons 6-8 in the dog gene) contains four highly conserved subregions which may play a critical role in enzyme-substrate and protein-ligand binding for lipases and yolk proteins, respectively. Comparison of the sequences of 10 lipases from prokaryotes and eukaryotes identifies a 9-residue consensus sequence surrounding the active serine which includes the previously identified sequence Gly-X-Ser-X-Gly. The hydrophobic nature of this sequence in the 10 lipases contrasts with the hydrophilic nature of the corresponding sequences in serine proteases and thus defines an active site serine consensus sequence specific for lipases. An analysis of 5'- and 3'-flanking and intron 1-4 sequences in transient expression studies with AR4-2J and 266-6 cells was unable to reveal tissue-specific promoter or enhancer sequences.