Quantitative founder-effect analysis of French Canadian families identifies specific loci contributing to metabolic phenotypes of hypertension.

The Saguenay-Lac St-Jean population of Quebec is relatively isolated and has genealogical records dating to the 17th-century French founders. In 120 extended families with at least one sib pair affected with early-onset hypertension and/or dyslipidemia, we analyzed the genetic determinants of hypertension and related cardiovascular and metabolic conditions. Variance-components linkage analysis revealed 46 loci after 100,000 permutations. The most prominent clusters of overlapping quantitative-trait loci were on chromosomes 1 and 3, a finding supported by principal-components and bivariate analyses. These genetic determinants were further tested by classifying families by use of LOD score density analysis for each measured phenotype at every 5 cM. Our study showed the founder effect over several generations and classes of living individuals. This quantitative genealogical approach supports the notion of the ancestral causality of traits uniquely present and inherited in distinct family classes. With the founder effect, traits determined within population subsets are measurably and quantitatively transmitted through generational lineage, with a precise component contributing to phenotypic variance. These methods should accelerate the uncovering of causal haplotypes in complex diseases such as hypertension and metabolic syndrome.

Chromosomal mapping of HCaRG, a novel hypertension-related, calcium-regulated gene.

We recently identified a novel gene that is negatively regulated by extracellular calcium concentration with higher levels of transcripts in hypertensive animals (SHR). We named this gene HCaRG (Hypertension-related, Calcium-Regulated Gene). In this work we report the chromosomal localization of the HCaRG gene among different species. We identified a BglII RFLP between BN.lx and SHR rats. We then analysed the strain distribution pattern of this RFLP in 31 RIS, originating from BN.lx and SHR rats, and compared it to the segregation of 475 markers localized in the rat genetic map. Hcarg localizes to the rat chromosome 7 between the markers Mit3 and Mit4. This region is homologous to human chromosome 8q21-24. We identified three clones in Genbank that contain the sequence of HCaRG. It was therefore possible to narrow down the localization of human HCaRG to chromosome 8q24.3. Furthermore, a suggestive localization of mouse Hcarg based on conservation of linkage between human and mouse is on chromosome 15. We previously identified a putative calcium-binding motif (EF-Hand) and a nuclear receptor-binding domain (LxxLL) in the rat sequence of the HCaRG protein. Sequence comparison between five different species showed that these domains are highly conserved. Furthermore, a search of ESTs in Genbank for homologous sequences showed that HCaRG is expressed only in eukaryotes, particularly in mammals.

Predictors of target organ damage in hypertensive blacks and whites.

The purpose of this study was to evaluate the association of the insulin resistance syndrome with both blood pressure and target organ damage in blacks and whites with essential hypertension. Eighty-two black and 63 white French Canadian patients were studied. None had diabetes, and antihypertensive medications had been discontinued for >/=1 week. Measurements included 24-hour blood pressure monitoring, fasting plasma lipids, insulin sensitivity determined with the Bergman minimal model, echocardiogram, microalbumin excretion, and inulin and lithium clearances. Compared with the white French Canadians, black patients had an attenuated nighttime reduction in blood pressure (P<0.02), increased cardiac dimensions (P<0.001), greater microalbumin excretion (P<0.05), increased inulin clearance (indicative of glomerular hyperfiltration; P<0.001), and decreased lithium clearance (indicative of increased sodium reabsorption in the proximal tubule; P<0.001). Blood pressure levels were not related to insulin resistance; although in blacks, the nighttime reduction in systolic blood pressure was inversely related to fasting plasma insulin (r=-0.18, P<0.04). In a stepwise multivariate analysis (including blood pressure levels and components of the insulin resistance syndrome as independent variables), race was the strongest predictor of left ventricular mass (r=0.53, P<0.000), relative wall thickness (r=0.49, P<0.000), and both inulin (r=0.53, P<0.000) and lithium (r=0.41, P<0.000) clearances. Nighttime systolic blood pressure was also a significant determinant of concentric left ventricular hypertrophy (r=0.37, P<0.000). In blacks, microalbumin excretion was related to insulin resistance. These observations are consistent with the hypothesis that there is a genetic contribution to cardiac hypertrophy, glomerular hyperfiltration, and sodium retention in blacks with essential hypertension.

Heritability estimates of obesity measures in siblings with and without hypertension.

The goal of the present study was to evaluate mean values and heritability estimates of 3 global and 11 regional obesity measures in siblings with (HPT, n=209) or without (non-HPT, n=91) early-onset (age

Arterial pressure, left ventricular mass, and aldosterone in essential hypertension.

The purpose of the present study was to evaluate the relationship of aldosterone to blood pressure and left ventricular size in black American (n=109) and white French Canadian (n=73) patients with essential hypertension. Measurements were obtained with patients off antihypertensive medications and included 24-hour blood pressure monitoring, plasma renin activity and aldosterone, and an echocardiogram. Compared with the French Canadians, the black Americans had higher body mass indexes, higher systolic blood pressures, attenuated nighttime reduction of blood pressure, and lower serum potassium concentrations (P:<0.01 for each). Left ventricular mass index, posterior wall thickness, interventricular septal thickness, and relative wall thickness were also greater (P:<0.01 for each) in the black American patients. Supine and standing plasma renin activity was lower (P:<0.01 and P:<0.05, respectively) in the black Americans, whereas supine plasma aldosterone concentrations did not differ, and standing plasma aldosterone was greater (P:<0.05) in the black Americans (9.2+/-0.7 ng/dL) than in the French Canadians (7.3+/-0.6 ng/dL). In the black Americans, supine plasma aldosterone was positively correlated with nighttime systolic (r=0.30; P:<0.01) and diastolic (r=0.39; P:<0.001) blood pressures and inversely correlated with the nocturnal decline of systolic (r=-0.29; P:<0.01) and diastolic (r=-0.37; P:<0.001) blood pressures. In the black Americans, standing plasma aldosterone was positively correlated with left ventricular mass index (r=0.36; P:<0.001), posterior wall thickness (r=0.33; P:<0.01), and interventricular septal thickness (r=0.26; P:<0.05). When the black American patients were divided into obese and nonobese groups, significant correlations between plasma aldosterone and both blood pressure and cardiac mass were observed only in the obese. In the French Canadians, overall, plasma aldosterone did not correlate with either blood pressure or any measures of heart size. However, among obese French Canadians, supine plasma aldosterone correlated with nighttime diastolic (r=0.53, P:<0.02) and systolic (r=0.44, P:<0.01) blood pressures but not with cardiac mass. These results are consistent with the hypothesis that aldosterone contributes to elevated arterial pressure in obese black American and obese white French Canadian patients with essential hypertension and to the attenuated nocturnal decline of blood pressure and left ventricular hypertrophy in obese, hypertensive black Americans.

HCaRG, a novel calcium-regulated gene coding for a nuclear protein, is potentially involved in the regulation of cell proliferation.

Since a negative calcium balance is present in spontaneously hypertensive rats, we searched for the gene(s) involved in this dysregulation. A cDNA library was constructed from the spontaneously hypertensive rat parathyroid gland, which is a key regulator of serum-ionized calcium. From seven overlapping DNA fragments, a 1100-base pair novel cDNA containing an open reading frame of 224 codons was reconstituted. This novel gene, named HCaRG (hypertension-related, calcium-regulated gene), was negatively regulated by extracellular calcium concentration, and its basal mRNA levels were higher in hypertensive animals. The deduced protein showed no transmembrane domain, 67% alpha-helix content, a mutated calcium-binding site (EF-hand motif), four putative "leucine zipper" motifs, and a nuclear receptor-binding domain. At the subcellular level, HCaRG had a nuclear localization. We cloned the human homolog of this gene. Sequence comparison revealed 80% homology between rats and humans at the nucleotide and amino acid sequences. Tissue distribution showed a preponderance in the heart, stomach, jejunum, kidney (tubular fraction), liver, and adrenal gland (mainly in the medulla). HCaRG mRNA was significantly more expressed in adult than in fetal organs, and its levels were decreased in tumors and cancerous cell lines. We observed that after 60-min ischemia followed by reperfusion, HCaRG mRNA declined rapidly in contrast with an increase in c-myc mRNA. Its levels then rose steadily to exceed base line at 48 h of reperfusion. HEK293 cells stably transfected with HCaRG exhibited much lower proliferation, as shown by cell count and [(3)H]thymidine incorporation. Taken together, our results suggest that HCaRG is a nuclear protein potentially involved in the control of cell proliferation.

Sibling resemblance of erythrocyte ion transporters in French-Canadian sibling-pairs affected with essential hypertension.

OBJECTIVES: Erythrocyte Na+/Li+ countertransport and Na+,K+ cotransport are increased in some Caucasians with essential hypertension. This study examines the relative contributions of genetic and shared environmental factors to the activity of these ion carriers in French-Canadian sibling-pairs affected with essential hypertension. DESIGN: The activity of Na+/Li+ countertransport and Na+,K+ cotransport (rate of Na+ o-dependent Li+ efflux and bumetanide-sensitive 86Rb influx, respectively) was measured in 122 French-Canadian siblings with essential hypertension, including 36 brother/brother and 48 sister/sister pairs. Sibling/sibling correlations were estimated using the FCOR program of the S.A.G.E. package. RESULTS: Na+/Li+ countertransport and Na+,K+ cotransport were respectively higher by 27% (P = 0.002) and 42% (P = 0.0009) in erythrocytes from men compared with women. Intra-individual correlation analysis did not reveal a significant effect of age on the activity of these ion transporters in both males and females, and an influence of plasma lipids (triglycerides, cholesterol, low-density lipoprotein, high-density lipoprotein) in females. In males, Na+,K+ cotransport was correlated with the level of serum triglycerides only (P = 0.01). Familial correlation analysis showed that sibling resemblance of Na+/Li+ countertransport and Na+,K+ cotransport was higher in men (r = 0.26 and 0.39) than in women (r = 0.01 and 0.03, respectively). CONCLUSION: The present data indicate that different factors contribute to the regulation of monovalent ion carriers in erythrocytes from Caucasian men and women with essential hypertension. The activity of erythrocyte Na+/Li+ countertransport and Na+,K+ cotransport appears to be more strongly determined by inheritable factors in men than in women.

Localization of mRNA coding for the three subtypes of atrial natriuretic factor (ANF) receptors in rat anterior pituitary gland cells.

Atrial Natriuretic Factor (ANF) action is mediated by highly selective and specific receptors. Three subtypes have been characterized and cloned: ANF receptor-A, -B and -C. These subtypes are all expressed in the anterior pituitary of the rat. In the present study, the mRNA for each subtype was detected by in situ hybridization. The amounts of ANFR-A and -B mRNA were found to be similar, and to be twice that of ANFR-C mRNA. At the ultrastructural level, the three types of ANFR mRNA were expressed in three anterior pituitary cell types, namely lactotrophs, corticotrophs, and gonadotrophs, identified by their hormonal content. No signal was revealed in somatotrophs or thyrotrophs. The different forms of mRNA were similar in terms of subcellular localization: in the cytoplasmic matrix and the nuclear euchromatin. These data indicate that the anterior pituitary is an important target tissue for ANF action.

Apoptosis in target organs of hypertension.

Apoptosis or programmed cell death frequently parallels abnormalities in cell proliferation and differentiation. As hypertrophy/hyperplasia or remodeling occurs in organs affected by hypertension, we evaluated the degree of apoptosis in the heart, kidney, and brain in situ in genetically hypertensive mice and rats as well as in cultured vascular smooth muscle cells. Apoptosis was characterized by morphological features, DNA fragmentation, and laddering as well as by terminal deoxynucleotidyl transferase labeling of the 3' OH ends of both extracted DNA and tissue sections. The present report provides the first evidence of increased apoptosis in whole organs of genetically hypertensive rat and mouse strains: in the heart of spontaneously hypertensive rats (SHR) and in the heart (ventricular cardiomyocytes), kidney (inner cortex and medulla), and brain (cortex, striatum, hippocampus, and thalamus) of spontaneously hypertensive mice, with a higher effect of apoptotic inducers in cultured aortic smooth muscle cells derived from SHR. Both types of known apoptotic processes, oligonucleosomal cleavage and large DNA fragmentation, were observed in vascular smooth muscle cells, but only the former appeared to be increased in SHR. This study underlines the importance of cell death dysregulation in hypertension, reveals a new route for investigation of the pathogenesis of hypertension, and suggests novel targets of therapeutic intervention.

Alternative splicing of natriuretic peptide A and B receptor transcripts in the rat brain.

1. In the present study we searched for variants of alternative splicing of guanylyl cyclase A and B mRNA in rats in vivo. 2. Guanylyl cyclase A2 and guanylyl cyclase B2 isoforms of guanylyl cyclase produced by alternative splicing leading to the deletion of exon 9 of both transcripts were quantified in several rat organs. 3. Only one alternative splicing was found in the regulatory domain, encoded by exons 8-15. 4. Quantification of the guanylyl cyclase B2 isoform in different rat organs and in cultured aortic smooth muscle cells showed that this alternative splicing was tissue-specific and occurred predominantly in the central nervous system where the alternatively spliced variant represented more than 50% of the guanylyl cyclase B mRNA. 5. The same alternative splicing existed for guanylyl cyclase A mRNA but at very low levels in the organs studied. 6. Alternative splicing of guanylyl cyclase B exon 9 in the brain may play an important role in signal transduction, since the expressed protein possesses a constitutionally active guanylyl cyclase acting independently of C-type natriuretic peptide regulation.

Regulation of natriuretic peptide receptor A and B expression by transforming growth factor-beta 1 in cultured aortic smooth muscle cells.

Two types of natriuretic peptide receptors (NPR-A and NPR-B) are membrane guanylate cyclases whose relative expression varies in different tissues. Because natriuretic peptides have been shown to inhibit aortic smooth muscle proliferation, we investigated the regulation of NPR-A and NPR-B in these cells under different proliferative conditions. NPR subtype mRNA levels were measured by our newly developed quantitative reverse transcription-polymerase chain reaction assay using mutated NPR-A and NPR-B cRNA as internal standards. The functional impact of their expression was determined by atrial natriuretic peptide (ANP)- and C-type natriuretic peptide (CNP)-induced stimulation of cyclic GMP production. In the intact aorta, NPR-B mRNA levels were found to be 10-fold higher than those of NPR-A. This dominance was further amplified (1000-fold) in long-term cultures (10 to 15 passages) of aortic smooth muscle cells (ASMC). Higher cyclic GMP production with CNP than with ANP was observed in cultured ASMC from Wistar-Kyoto (WKY) rats. Similar stimulation by the two agonists was noted in spontaneously hypertensive rat (SHR) cells, paralleled by a 10-fold increase in NPR-A mRNA levels and ANP stimulation of cyclic GMP in hypertensive cells. The present study also evaluated NPR-A and NPR-B mRNA control by transforming growth factor-beta 1 (TGF-beta 1), an important regulator of cell proliferation that is overexpressed in SHR ASMC. TGF-beta 1 decreased both NPR-A and NPR-B mRNA levels with a predominant effect in SHR cells at high cell density.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth hormone receptor binding protein in rat anterior pituitary.

GH is synthesized by the somatotrophs in the pituitary, where it may have paracrine actions. In order to identify the GH target cells in rat pituitary, the cellular distributions of rat GH receptor binding protein messenger ribonucleic acid (rGH-RBP mRNA) and protein were investigated at the electronmicroscopic level, using in situ hybridization and immunocytology, respectively, on ultrathin frozen sections of rat pituitary. Ultrastructural distribution of 125I-bGH, 30 min after intracardiac administration was also performed in order to determine the pituitary cell types that bind the labeled hormone. In situ hybridization was performed using digoxigenin-labeled oligonucleotide probe revealed by indirect immunogold reaction. rGH-RBP mRNA was readily identified in the cytoplasmic matrix, associated with the endoplasmic reticulum and the nucleus of the somatotrophs, the lactotrophs and the gonadotrophs. No significant signal was detected in the corticotrophs or thyrotrophs. The number of gold particles in each pituitary cell type was estimated by direct counting, and was compared to the results of hybridization performed on rat liver sections as a control. The results showed that the level of rGH-RBP mRNA was higher in hepatocytes than in the pituitary cells, and was higher in the somatotrophs and lactotrophs than in the gonadotrophs. Immunocytological detection of rGH-RBP was performed using two monoclonal antibodies (mAbs 43 and 263) directed against independent epitopes of the extracellular domain of the rGH-R. Indirect immunocytological detection showed regionalization of rGH-RBP; it was present in the cytoplasmic matrix and the nucleus of the hepatocytes and in discrete pituitary cells: somatotrophs, lactotrophs and gonadotrophs, but not in thyrotrophs or corticotrophs. Gold particle number was also higher in somatotrophs and lactotrophs than in gonadotrophs and higher in the nucleus compared to the cytoplasmic matrix. Radioiodinated GH was uptaken 30 min after injection by the same three pituitary cell types, showing evidence for the functional role of the GH receptor. In conclusion, we find that the cellular localization of rGH-RBP mRNA and protein is similar in discrete cell subpopulations of the pituitary, suggesting a direct effect of GH on somatotrophs, lactotrophs and gonadotrophs, through paracrine, autocrine or intracrine mechanisms.

Increased cyclic guanosine monophosphate production and overexpression of atrial natriuretic peptide A-receptor mRNA in spontaneously hypertensive rats.

Atrial natriuretic peptide (ANP) specifically stimulates particulate guanylate cyclase, and cyclic guanosine monophosphate (cGMP) has been recognized as its second messenger. Spontaneously hypertensive rats (SHR) have elevated plasma ANP levels, but manifest an exaggerated natriuretic and diuretic response to exogenous ANP when compared to normotensive strains. In isolated glomeruli, the maximal cGMP response to ANP corresponds to a 12- to 14-fold increase over basal levels in normotensive strains (Wistar 13 +/- 2; Wistar-Kyoto 12 +/- 2; Sprague-Dawley 14 +/- 2) while a maximal 33 +/- 3-fold elevation occurs in SHR (P < 0.001). This hyperresponsiveness of cGMP is reproducible in intact glomeruli from SHR from various commercial sources. Furthermore, this abnormality develops early in life, even before hypertension is clearly established, and persists despite pharmacological modulation of blood pressure, indicating that it is a primary event in hypertension. In vitro studies have revealed a higher particulate guanylate cyclase activity in membranes from glomeruli and other tissues from SHR. This increase is not accounted for by different patterns of ANP binding to its receptor subtypes between normotensive and hypertensive strains, as assessed by competitive displacement with C-ANP102-121, an analog which selectively binds to one ANP receptor subtype. The hyperactivity of particulate guanylate cyclase in SHR and its behavior under basal, ligand (ANP), and detergent-enhanced conditions could be attributed either to increased expression or augmented sensitivity of the enzyme. Radiation-inactivation analysis does not evoke a disturbance in the size of regulatory elements normally repressing enzymatic activity, while the expression of particulate guanylate cyclase gene using mutated standard of A- and B-receptors partial cDNAs, quantified by polymerase chain reaction (PCR) transcript titration assay, manifests a selective increase of one guanylate cyclase subtype. Our data suggest that in hypertension, genetic overexpression of the ANP A-receptor subtype is related to the exaggerated biological response to ANP in this disease.

Rat granulosa cells express the gonadotrophin-releasing hormone gene: evidence from in-situ hybridization histochemistry.

There is still debate as to whether natural sequence gonadotrophin-releasing hormone (GnRH) is produced in the mammalian gonads and concerning its potential role as a paracrine modulator of gonadal function. To address this question, we have used in-situ hybridization histochemistry with an oligonucleotide probe complementary to the GnRH decapeptide coding sequence, to determine the cellular site(s) of expression of the GnRH gene in rodent ovaries. GnRH mRNA was detected in granulosa and thecal cells from ovarian follicles at all stages of development (primary-->Graafian), with no significant change in grain density during follicular development. The granulosa cell compartment always contained more mRNA than the thecal cell compartment. Corpora lutea expressed the GnRH gene to the same extent as thecal cells. These results indicate that preproGnRH mRNA is detectable under physiological conditions in the mammalian ovary, though whether this produces authentic GnRH decapeptide or an alternative protein product is not known. The physiological significance of these findings remains to be determined.

Ultrastructural non-radioactive in situ hybridization of GH mRNA in rat pituitary gland: pre-embedding vs ultra-thin frozen sections vs post-embedding.

In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultra-thin frozen sections, or on ultra-thin sections of tissues embedded in hydrophilic resin such as Lowicryl. With the purpose of comparing the sensitivity, resolution, and ultrastructural preservation of these three methods, we examined the expression of the growth hormone (GH) gene in anterior pituitary cells by in situ hybridization at the ultrastructural level, using a synthetic oligonucleotide complementary to the codons of the mRNA from Gln 45 to Ser 54 labeled at the 3' end of biotin-21dUTP. All these methods gave similar results: mRNA was located on the lamellar endoplasmic reticulum of somatotrophs. The pre-embedding method gave the best ultrastructural preservation, with low resolution with the enzymatic detection system and an intermediate sensitivity. A probe concentration of 10 pmol/ml was sufficient to obtain a signal. With this method gold particles could not be used without pre-treatment. The frozen section method gave the best sensitivity (a signal was observed with 4 pmol/ml of probe) but the lowest ultrastructural preservation. On ultra-thin Lowicryl sections, resolution was as high as with the frozen-section method, ultrastructural conservation was intermediate, and sensitivity was low. These results indicate that the last method seems to be a good compromise between sensitivity and ultrastructural preservation.

The yeast KEX-2-processing endoprotease is active in the Golgi apparatus of transfected NIH 3T3 fibroblasts.

Proteolytic processing of polyprotein precursors at pairs of basic amino acids is a prerequisite for the generation of bioactive peptide hormones. While the mammalian endoproteases responsible for these cleavages are yet to be identified, this function has been unequivocally assigned in yeast to the product of the KEX-2 gene. To study the molecular mechanisms involved in polyprotein processing, we have transfected the yeast KEX-2 gene into mouse NIH 3T3 fibroblasts and established a new cell line (called 2N-DK) where the KEX-2 endoprotease is permanently expressed. Immunofluorescence studies show that the KEX-2 enzyme is retained within the Golgi of the 2N-DK cells. The evidence for this cellular location is supported by measurement of intracellular and extracellular KEX-2 enzyme activity. In this permanently transfected cell line, KEX-2 activity is exclusively intracellular, in contrast to the situation previously described in transiently infected cell lines, where extracellular KEX-2 activity was detected. Furthermore, infection of 2N-DK cells with a recombinant retrovirus expressing a cDNA coding for porcine proopiomelanocortin (POMC) resulted in the synthesis of POMC and its efficient processing into beta-lipotropin and beta-endorphin, two of its physiologically authentic maturation products. These results suggest that in the fibroblast cell line 2N-DK, proteolytic processing of POMC by KEX-2 endoprotease occurs in the Golgi apparatus.

Ultrastructural distribution of somatostatin-14 and -28 in rat adrenal cells.

Previous studies have shown that somatostatin modulates angiotensin-induced aldosterone secretion by adrenal glomerulosa cells. This effect is mediated through specific receptors which do not show any preference for somatostatin-14 (S14) or the N-extended form somatostatin-28 (S28). The study of the distribution of 125I-Tyr [Tyr0, DTrp8] S14- and 125I-Tyr [Leu8, DTrp22, Tyr25] S28-binding in frozen sections of the rat adrenal by autoradiography indicated that both peptides bind to similar loci. High concentrations of binding sites were observed in the zona glomerulosa, and low concentrations were detected in the medulla. At the ultrastructural level, immunocytochemistry after cryoultramicrotomy revealed endogenous S14- and S28-like immunoreactive material in zona glomerulosa and in medulla. In glomerulosa cells, immunoreactive material was localized at the plasma membrane level, in the cytoplasmic matrix, in the mitochondria, and in the nucleus. S14- and S28-like materials were detected in both epinephrine and norepinephrine-storing cells of the adrenal medulla. In these cells, the distribution of either immunoreactive product was similar; it was observed in cytoplasmic matrix, secretory granules and nucleus, but not at the plasma membrane level. In situ hybridization does not reveal somatostatin mRNA in zona glomerulosa or medulla. These results demonstrate that S14 and S28 bind to, and are taken up by zona glomerulosa and adrenal medullary cells, but are not produced by these cells.

Construction of cloning vectors using the Vibrio harveyi luminescence genes luxA and luxB as markers.

A synthetic oligodeoxyribonucleotide harboring four new restriction sites was inserted into the luxB gene of Vibrio harveyi. This insertion did not disrupt the reading frame. An active beta-subunit was synthesized since a plasmid with both the luxA and mutated luxB genes conferred upon Escherichia coli the bacterial luciferase (Lux) phenotype in the presence of an aldehyde. Ligation of a piece of foreign DNA at these new cloning sites in the vector extinguish the Lux phenotype of the transformed bacteria. Therefore, the plasmid was used as a cloning vector, and recombinant DNA-containing bacteria were detected by the loss of bioluminescence. To create more versatile plasmids, the intergenic region of phage f1 was inserted outside of the lux genes. The selection by loss of bioluminescence presents several advantages over the white/blue selection of the lacZ gene on indicator plates.

Expression and pharmacological characterization of the human M1 muscarinic receptor in Saccharomyces cerevisiae.

The yeast S. cerevisiae has been examined as a heterologous host for the expression of mammalian neurotransmitter receptors which couple to guanine nucleotide regulatory (G) proteins. A cloned gene encoding the M1 subtype of human muscarinic receptor (HM1) was transformed into S. cerevisiae on a high copy plasmid under the control of the promoter for the yeast alcohol dehydrogenase (ADH) gene. Northern blotting demonstrated the presence of HM1 transcripts in transformants, and crude membranes prepared from these cells showed saturable binding of the muscarinic antagonist [3H]N-methyl scopolamine with a Kd of 179 pM and Bmax of 20 fmol/mg protein. Competition binding studies revealed pharmacological properties for these sites which were comparable to those reported for the M1 site in mammalian tissues. Yeast expressing HM1 did not exhibit high affinity agonist binding or cell-cycle arrest in the presence of muscarinic agonists, indicating that the mammalian receptor did not couple to the endogenous yeast G protein.

Intracellular proteolytic processing of proopiomelanocortin in heterologous COS-1 cells by the yeast KEX2 endoprotease.

We have transiently expressed the yeast KEX2 gene together with the proopiomelanocortin (POMC) cDNA in COS-1 cells. Characterization of the POMC-related immunoreactive peptides by gel permeation and reversed-phase high pressure liquid chromatography showed that the KEX2 enzyme was active and capable of carrying out cleavage of POMC to release the authentic maturation product beta-endorphin(1-31). Peptides resembling beta-lipotropin, the amino terminal glycopeptide, and ACTH(1-39) were also detected as major products in the cell extracts. Our results indicate that the KEX2 enzyme can proteolytically release from POMC a set of peptides similar to that normally found in interior pituitary.