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PubMed1999

Hypertension associated with decreased testosterone levels in natriuretic peptide receptor-A gene-knockout and gene-duplicated mutant mouse models.

Mice lacking the gene (Npr1) encoding the natriuretic peptide receptor A (NPRA) have hypertension with elevated blood pressure and cardiac hypertrophy. In particular, Npr1 gene-deficient male mice exhibit lethal vascular events similar to those seen in untreated human hypertensive patients. Serum testosterone levels tend to be lower in hypertensive male humans than in normal males without hypertension, but the genetic basis for this tendency remains unknown. To determine whether Npr1 gene function affects the testosterone level, we measured serum testosterone in male hypertensive mice lacking a functional Npr1 gene, wild-type animals with two copies, and the gene-duplicated littermates expressing four copies of the gene. In the Npr1 gene-knockout (zero-copy) mice, the serum testosterone level was 62% lower than that in the two-copy control mice (80+/-10 ts. 120+/-14 ng/ml, respectively; P < 0.005). Serum testosterone in the four-copy mice was 144% (P < 0.005) of that in the two-copy wild-type control mice. To investigate the role of NPRA in testicular steroidogenesis, we analyzed atrial natriuretic peptide (ANP)-dependent guanylyl cyclase activation, accumulation of intracellular cGMP, and testosterone production in purified primary Leydig cells from animals with zero, two, or four copies of the Npr1 gene. Leydig cells lacking the Npr1 gene did not show ANP-stimulated guanylyl cyclase activation or cGMP accumulation and had no ANP-dependent testosterone production. ANP stimulation of Leydig cells from the four-copy males elicited a 2-fold greater production of cGMP compared to that in the two-copy wild-type counterparts (260+/-12 vs. 126+/-7 pmol/l x 10(6) cells; P < 0.001). Similarly, ANP-dependent testosterone production in Leydig cells was nearly twice as high in four-copy mice as in two-copy wild-type controls (561+/-18 vs. 325+/-11 ng/l x 10(6) cells; P < 0.001). ANP-dependent guanylyl cyclase activation and production of cGMP in Leydig cells increased progressively with the number of Npr1 gene copies. Our results establish the existence of an alternate mechanism for testicular steroidogenesis that is stimulated by NPRA-dependent cGMP signaling, in addition to that mediated by gonadotropins, via a cAMP pathway. These findings demonstrate the role of Npr1 gene function in the maintenance of serum testosterone levels and testicular steroidogenesis and provide a genetic link between hypertension associated with decreased NPRA and low testosterone levels.

Animals↗
PubMed1996

Heterologous expression of the cloned guinea pig alpha 2A, alpha 2B, and alpha 2C adrenoceptor subtypes. Radioligand binding and functional coupling to a CAMP-responsive reporter gene.

Functional studies have shown that 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4,5-tetrahydro-3- benzazepine (SKF 104078) has very low affinity for prejunctional alpha 2-adrenoceptors (alpha 2-AR) in the guinea pig atrium. In this study, we have cloned guinea pig homologues of the human alpha 2-C10, alpha 2-C4 AR subtypes and have studied them in isolation by heterologous expression in cultured mammalian cells. Oligonucleotide primers, designed from conserved areas of the human alpha 2-ARs were used in a polymerase chain reaction (PCR) with template cDNA synthesized from guinea pig atrial mRNA. Three PCR products were obtained that shared identity with the three human alpha 2-AR subtypes. A guinea pig (gp) genomic library was screened with a cDNA clone encoding a portion of the gp-alpha 2A, and genes containing the complete coding sequences of the guinea pig alpha 2A, alpha 2B, and alpha 2C AR subtypes were obtained. These guinea pig genes were subcloned into a eukaryotic expression plasmid and were expressed transiently in COS-7 cells. The binding of the alpha 2-selective antagonist [3H]MK-912 to membranes prepared from these cells was specific and of high affinity with Kd values of 810 pM for gp-alpha 2A, 2700 pM for gp-alpha 2B and 110 pM for gp-alpha 2C. Competition for the binding of [3H]MK-912 by SKF 104078 indicated that it was of moderately high affinity (approximately 100 nM) but that it was not selective for any of the guinea pig alpha 2-AR subtypes. Co-expression of guinea pig alpha 2-AR subtypes with a cyclicAMP-responsive chloramphenicol acetyltransferase (CAT) reporter gene resulted in agonist-dependent modulation of CAT activity. For the gp-alpha 2 A, a biphasic response was obtained with low concentrations of noradrenaline (NE) decreasing forskolin-stimulated CAT activity and high concentrations causing a reversal. For the gp-alpha 2B, NE produced mostly potentiation of forskolin-stimulated activity, and for the gp-alpha 2C, NE caused mainly inhibition. Overall, the pharmacology of the cloned guinea pig alpha 2-AR subtypes was in agreement with data obtained for the native guinea pig receptors and was functionally similar to that of the cloned human alpha 2-AR subtypes.

Adrenergic alpha-Antagonists↗
PubMed2004

Cre/loxP system for generating tissue-specific knockout mouse models.

Alteration of the mouse genome by conventional transgenic and gene-targeted approaches has greatly facilitated studies of gene function. However, a gene alteration expressed in the germ line may cause an embryonic lethal phenotype resulting in no viable mouse to study gene function. Similarly, a gene alteration may exert its effect in multiple different cell and tissue types, creating a complex phenotype in which it is difficult to distinguish direct function in a particular tissue from secondary effects resulting from altered gene function in other tissues. Therefore, methods have been developed to control conditions such as the timing, cell-type, and tissue specificity of gene activation or repression. This brief review provides an overview of the Cre/LoxP system for generating tissue-specific knockout mouse models.

Animals↗
PubMed1999

Statistical methods for testing functional divergence after gene duplication.

Functional innovations after gene duplication may result in altered functional constraints between member gene clusters of a gene family. This type (type I) of functional divergence is measured by the coefficient of functional divergence (theta lambda), which can be interpreted as the decrease in rate correlation between gene clusters, or the probability that the evolutionary rate at a site is statistically independent between two gene clusters. A simple stochastic model has been developed for estimating theta lambda and testing its statistical significance. The current model includes the model of rate variation among sites as a special case when theta lambda = 0. Moreover, we have developed a site-specific profile based on the hidden Markov model to identify critical amino acid residues that are responsible for these functional differences between two gene clusters, which may have great potential in functional genomics.

Gene Duplication↗
PubMed1984

Chromosomal organization of the human dihydrofolate reductase genes: dispersion, selective amplification, and a novel form of polymorphism.

The human dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) gene family includes a functional gene (hDHFR) and at least four intronless genes. Three intronless genes (hDHFR-psi 2, hDHFR-psi 3, and hDHFR-psi 4) are identifiable as pseudogenes because of DNA sequence divergence from the functional gene with introns, while one intronless gene (hDHFR-psi 1) is completely homologous to the coding sequences of the functional gene. Analysis of genomic DNA from two panels of somatic human-rodent cell hybrids with specific molecular probes provide insight into the chromosomal organization and assignment of these genes. The five genes are dispersed in that each one is found on a different chromosome. The functional gene hDHFR has been assigned to chromosome 5, and one pseudogene (hDHFR-psi 4), to chromosome 3. In a human cell line (HeLa) that was selected for methotrexate resistance, the functional locus became amplified, while there was no amplification of the four intronless pseudogenes. hDHFR-psi 1 was found to be present in DNA of some individuals and absent from DNA of others, consistent with a recent evolutionary origin of this gene originally suggested by its sequence identity to the coding portions of the functional gene. The presence or absence of this intronless pseudogene represents a previously unreported form of DNA polymorphism.

Animals↗
PubMed1997

Retinoic acid-responsive enhancers located 3' of the Hox A and Hox B homeobox gene clusters. Functional analysis.

Homeobox genes control the spatial identity and differentiation of tissues in the developing vertebrate embryo. Retinoids are signaling molecules involved in the regulation of Hox genes. We previously identified a 3' enhancer called the RAIDR5, which contained a DR5 retinoic acid response element (RARE) and was responsible for the retinoic acid (RA)-associated expression of the murine Hoxa-1 gene in teratocarcinoma cells. We demonstrate that a similar enhancer, which contains a DR5 RARE, is located at a DNase I-hypersensitive site 3' of the murine Hoxb-1 gene. This enhancer, the Hoxb-1 RAIDR5, regulates the RA responsiveness of the Hoxb-1 gene and is different in location and sequence from the RA-regulated 3' Hoxb-1 enhancers previously described. Several DNA elements within the murine Hoxa-1 RA-inducible RAIDR5 enhancer, including the DR5 RARE, conserved element (CE) 1, and CE2, are conserved in the murine Hoxb-1 RAIDR5 enhancer, the human homolog of Hoxa-1, and in the chicken Hoxb-1 gene. Gel shifts show that the CE2 sequence TATTTACTCA binds an RA-inducible factor, while UV cross-linking indicates that a 170-kDa protein binds to this sequence. Thus, the Hoxa-1 and Hoxb-1 genes possess 3' enhancers with similar sequences through which their expression and responsiveness to endogenous retinoids are controlled.

Amino Acid Sequence↗
PubMed2003

Regulatory gene mutations affecting apolipoprotein gene expression: functions and regulatory behavior of known genes may guide future pharmacogenomic approaches to therapy.

A pharmacogenomic approach to therapy requires systematic knowledge of the regulatory regions of the genes, as well as basic understanding of transcriptional regulatory mechanisms of genes. Using the apolipoprotein (apo) A-I/CIII gene cluster as a model system, we have identified by in vitro and in vivo studies the regulatory elements and the factors which control its transcription. Studies in transgenic mice established that the hepatocyte nuclear factor (HNF-4) binding site of the apoCIII enhancer, which controls transcription of both genes, is required for the intestinal expression of apoA-I and apoCIII genes, and enhances synergistically their hepatic transcription in vivo. The three Sp1 sites of the enhancer are also required for the intestinal expression of apoA-land apoCIII genes in vivo, and for the enhancement of the hepatic transcription. The regulation of the apoE/apoCI/apoCIV/apoCII cluster is also cited. It is expected that identification of the regulatory regions of genes will be soon accelerated by the sequencing of several mammalian genomes. The functional analyses of the regulatory domains of genes involved in lipid homeostasis, combined with cross-species sequence comparisons in the near future, may identify natural regulatory gene polymorphisms in the general population that will permit rational pharmacogenomic approaches for treatment of dyslipidemias.

Animals↗
PubMed1998

10-Hydroxylation of nortriptyline in white persons with 0, 1, 2, 3, and 13 functional CYP2D6 genes.

OBJECTIVE: To investigate the disposition and effects of nortriptyline and its major metabolite 10-hydroxy-nortriptyline line in panels of white subjects with different CYP2D6 genotypes, including those with duplicated and multiduplicated CYP2D6*2 genes and to evaluate the contribution of the number of functional C gamma P2D6 alleles to the metabolism of nortriptyline, used here as a model drug for CYP2D6 substrates. METHODS: Oral single doses of 25 to 50 mg nortriptyline were given to five poor metabolizers of debrisoquin (INN; debrisoquine) with no functional CYP2D6 gene, five extensive metabolizers with one functional CY2D6 gene, five extensive metabolizers with two functional CYP2D6 genes, five ultrarapid metabolizers with duplicated CYP2D6*2 genes, and one ultrarapid metabolizer with 13 copies of the CYP2D6*2 gene. Plasma kinetics of nortriptyline and 10-hydroxynortriptyline were analyzed. Anticholinergic effects (inhibition of salivation and accommodation disturbances), sedation, blood pressure, and effect on supine and erect pulse rate were measured. RESULTS: There was a clear relation between the C gamma P2D6 genotype and the plasma kinetics of nortriptyline and 10-hydroxynortriptyline. The proportion between the apparent oral clearances of nortriptyline in the groups with 0, 1, 2, 3, and 13 functional genes was 1:1:4:5:17. The proportions between AUC(nortriptyline) to AUC(10-hydroxynortriptyline) ratios in the groups with 0, 1, 2, 3, and 13 functional genes were 36:25:10:4:1. Oral plasma clearance of nortriptyline and AUC(nortriptyline) to AUC(10-hydroxynortriptyline) ratio both correlated significantly with the debrisoquin metabolic ratio (rS = -0.89, p = 0.0001; rS = 0.92, p = 0.0001). Although ultrarapid metabolizer subjects were given double the nortriptyline dose (50 mg), inhibition of salivation was not more pronounced compared with the other genotype groups given 25 mg nortriptyline. CONCLUSION: The results of this study show the quantitative importance of the CYP2D6 genotype, especially the presence of multiple functional CYP2D6 genes for the pharmacokinetics of nortriptyline and 10-hydroxynortriptyline. Genotyping of subjects with multiple copies of functional genes may be of great value for differentiating ultrarapid metabolizers from patients who do not comply with the prescription and for assuring adequate drug choice and dosage for these patients.

Analysis of Variance↗
PubMed2006

Complex functionality of gene groups identified from high-throughput data.

Relating experimental data to biological knowledge is necessary to cope with the avalanches of new data emerging from recent developments in high-throughput technologies. Automatic functional profiling becomes the de facto standard approach for the secondary analysis of high-throughput data. A number of tools employing available gene functional annotations have been developed for this purpose. However, current annotations are derived mostly from traditional analysis of the individual gene function. The complex biological phenomena carried out by the concerted activity of many genes often requires the definition of new complex functionality (related to a group of genes), which is, in many cases, not available in current annotation vocabularies. Functional profiling with annotation terms related to the description of individual biological functions of a gene may fail to provide reasonable interpretation of biological relationships in a set of genes involved in complex biological phenomena. We introduce a novel procedure to profile a complex functionality of a gene set. Complex functionality is constructed as a combination of available annotation terms. By profiling ChIP-chip data from Saccharomyces cerevisiae we demonstrate that this technique produces deeper insights into the results of high-throughput experiments that are beyond the known facts described in the functional classifications.

Animals↗
PubMed1994

Nucleoside diphosphate kinase enzyme activity of NM23-H2/PuF is not required for its DNA binding and in vitro transcriptional functions.

nm23 genes encode proteins that participate in tumor metastasis regulation and in various fundamental cellular processes, although the mechanisms remain undefined. All Nm23 proteins contain nucleoside diphosphate kinase (NDPK) activity whose significance to these regulatory effects is not yet evident. The protein product of the human nm23-H2 gene functions in vitro both as a nucleoside diphosphate kinase enzyme (NDPK-B; Gilles, A.-M., Presecan, E., Vonica, A. and Lascu, I. (1991) J. Biol. Chem. 266, 8784-8789) and as a transcription factor (PuF; Postel, E. H., Berberich, S. J., Flint, S. J. and Ferrone, C. A. (1993) Science 261, 478-480). To understand the significance of these two biochemical activities to NM23-H2 function, we have investigated the relationship between the DNA binding and transcriptional activity of NM23-H2 and its NDPK function. Using site-directed mutagenesis of the cDNA encoding NM23-H2, we have created a mutant substituting for the amino acid histidine 118, the presumed site of phosphorylation in the formation of the phosphoenzyme intermediate, the nonphosphorylatable amino acid phenylalanine. The H118F mutant protein is shown to be catalytically inactive as measured both in a radioisotopic assay that detects formation of the phosphorylated enzyme intermediate and in a coupled enzyme assay that indicates nucleoside diphosphate formation. These results confirm that histidine 118 is the critical residue for NDPK-B activity. In addition, the H118F mutant protein lacking enzymatic activity displayed normal DNA binding affinity for the c-myc promoter in electrophoretic mobility shift assays, and retained full transcriptional activity using the c-myc gene in vitro. These results indicate a lack of correlation between nucleoside diphosphate kinase activity of nm23-H2 on the one hand, and its DNA binding and transcriptional activity on the other, suggesting that the nm23-H2 gene encodes a bifunctional protein molecule.

Base Sequence↗
PubMed1987

Influence of transposable elements on the structure and function of the A1 gene of Zea mays.

The structure of the A1 gene of Zea mays was determined by sequencing cDNA and genomic clones. The gene is composed of four exons and three short introns. The 40.1-kd A1 protein is an NADPH-dependent reductase. Germinal derivatives of the mutable a1-m1 allele with either recessive or wild-type phenotype have been isolated. Sequence analysis of these revertant alleles indicates that frame-shift mutations abolish A1 gene function, whereas one additional amino acid within the protein sequence still allows wild-type gene expression. The presence of a second, promoter-like structure, upstream of the functional A1 gene promoter is discussed with respect to its possible involvement in differential expression of the A1 gene. The structure of the a1-m2 8004, 3456 and 4412 alleles, featuring distinguishable phenotypes in the presence of Spm(En), was also determined. In all alleles the 1080-bp-long inhibitor (I) element is located 15 bp upstream of the CAAT box of the A1 gene promoter. The unusual response of a1-m2 alleles to trans-active signals of the Spm(En) element is discussed with respect to the position of the I inserts. Also presented are data on the structure and insertion sites of transposable elements determined by cloning and sequencing of the mutable a1 alleles a1-mpapu, a1-mr 102 and a1-ml.

Alcohol Oxidoreductases↗
PubMed1987

Evidence for related functions of the RNA genes of Saccharomyces cerevisiae.

The yeast genes RNA2-RNA11 are necessary for splicing of nuclear intron-containing pre-mRNAs. We investigated the relationships among these genes by asking whether increased expression of one RNA gene leads to suppression of the temperature-sensitive lethality of a mutation in any other RNA gene. The presence of extra plasmid-borne copies of the RNA3 gene relieves the lethality of temperature-sensitive rna4 mutations. A region of the yeast genome (SRN2) is described that suppresses temperature-sensitive rna2 mutations when it is present on either medium or high-copy number plasmids. Neither suppression occurs via a bypass of RNA gene function since null alleles of rna2 and rna4 are not suppressed by elevated dosage of SRN2 and RNA3, respectively. These results suggest that the SRN2 and RNA2 gene products have related functions, as do the RNA3 and RNA4 gene products.

Genes, Fungal↗
PubMed1999

Adenovirus-mediated gene transfer of the beta2-adrenergic receptor to donor hearts enhances cardiac function.

Gene transfer to modify donor heart function during transplantation has significant therapeutic implications. Recent studies by our laboratory in transgenic mice have shown that overexpression of beta2-adrenergic receptors (beta2-ARs) leads to significantly enhanced cardiac function. Thus, we investigated the functional consequences of adenovirus-mediated gene transfer of the human beta2-AR in a rat heterotopic heart transplant model. Donor hearts received 1 ml of solution containing 1 x 1010 p.f.u. of adenovirus encoding the beta2-AR or an empty adenovirus as a control. Five days after transplantation, basal left ventricular (LV) pressure was measured using an isolated, isovolumic heart perfusion apparatus. A subset of hearts was stimulated with the beta2-AR agonist, zinterol. Treatment with the beta2-AR virus resulted in global myocardial gene transfer with a six-fold increase in mean beta-AR density which corresponded to a significant increase in basal contractility (LV + dP/dtmax, control: 3152.1 +/- 286 versus beta2-AR, 6250.6* +/- 432.5 mmHg/s; n = 10, *P < 0.02). beta2-AR overexpressing hearts also had higher contractility after zinterol administration compared with control hearts. Our results indicate that myocardial function of the transplanted heart can be enhanced by the adenovirus-mediated delivery of beta2-ARs. Thus, genetic manipulation may offer a novel therapeutic strategy to improve donor heart function in the post- operative setting.

Adenoviridae↗
PubMed2004

Functional divergence of duplicated genes formed by polyploidy during Arabidopsis evolution.

To study the evolutionary effects of polyploidy on plant gene functions, we analyzed functional genomics data for a large number of duplicated gene pairs formed by ancient polyploidy events in Arabidopsis thaliana. Genes retained in duplicate are not distributed evenly among Gene Ontology or Munich Information Center for Protein Sequences functional categories, which indicates a nonrandom process of gene loss. Genes involved in signal transduction and transcription have been preferentially retained, and those involved in DNA repair have been preferentially lost. Although the two members of each gene pair must originally have had identical transcription profiles, less than half of the pairs formed by the most recent polyploidy event still retain significantly correlated profiles. We identified several cases where groups of duplicated gene pairs have diverged in concert, forming two parallel networks, each containing one member of each gene pair. In these cases, the expression of each gene is strongly correlated with the other nonhomologous genes in its network but poorly correlated with its paralog in the other network. We also find that the rate of protein sequence evolution has been significantly asymmetric in >20% of duplicate pairs. Together, these results suggest that functional diversification of the surviving duplicated genes is a major feature of the long-term evolution of polyploids.

Arabidopsis↗
PubMed1992

The lectin gene family of Ricinus communis: cloning of a functional ricin gene and three lectin pseudogenes.

Molecular hybridisation using a ricin cDNA probe has revealed that the ricin/Ricinus communis agglutinin (RCA) multigene family is composed of approximately eight members. Several genomic clones containing preproricin and preproricin-like sequences have been isolated. Partial analysis of three different genomic clones by DNA sequencing and ribonuclease protection has indicated that at least three members of the lectin gene family are non-functional. None of the original seventeen positive clones isolated appears to contain a Ricinus communis agglutinin (RCA) gene. One gene member analysed (pCBG3H1) represents a functional ricin gene similar in coding sequence to the published cDNA sequence and possesses typical eukaryotic consensus sequences and seed-specific elements within the flanking sequences. Investigation at the transcriptional level of the expression pattern of this gene revealed that mRNA accumulates during the post-testa stages of seed development. The pattern of accumulation of steady-state transcripts correlates closely with that previously observed at the protein and translatable RNA levels.

Amino Acid Sequence↗
PubMed1991

All internal promoter elements of Neurospora crassa 5 S rRNA and tRNA genes, including the A boxes, are functionally gene-specific.

The internal control elements of Neurospora crassa 5 S genes include an A box and a C box as in Xenopus and Saccharomyces cerevisiae, plus a novel element, the Ribo box at position +18 to +34. The Ribo box is also found in the 40 S rRNA promoter and a ribosomal protein gene but is absent from tRNA genes in N. crassa. The 5 S A box diverges from the tRNA A box consensus at two positions. We tested whether replacement of the 5 S A box with a tRNALeu A box sequence would increase 5 S gene transcription in vitro or would remove the requirement for the Ribo box. The 5 S gene with the tRNALeu A box was transcribed poorly, and the Ribo box and the C box are still required for transcription. We tested the function of the Ribo box and 5 S A box in a tRNA-like transcription unit by constructing hybrids between a 5 S gene and a tRNALeu gene. In the tRNA-like context, the 5 S A box supported a lower level of transcription than the tRNA A box, and the Ribo box was not required at all. Therefore, in N. crassa, all of the 5 S internal control elements are gene-specific. In particular, the 5 S and tRNA A box sequences are not functionally interchangeable and may bind different transcription factors. Transcription of the hybrids was initiated at the 5 S initiation site, suggesting that the mechanism of initiation site selection is the same in the 5 S and tRNA genes. Competition experiments with the tRNA B box suggested that the N. crassa 5 S and tRNA genes require at least one common transcription factor such as TFIIIC.

Base Sequence↗
PubMed1990

Functional characterization of the 5'-regulatory region of human factor IX gene.

Functional characterization of the 5'-regulatory region of the human factor IX gene was carried out by a series of gene dissection analyses. The region extending from -175 to -274 of the 5' end flanking sequence is required for the expression of this gene. Within this region, sequence elements AGCCACT at -238 and TCAAAT at -187 were assigned as the functional CAAT box and TATA box, respectively. The transcription initiation site was found to be at -150 for the factor IX chloramphenicol acetyltransferase chimeric gene. A negative regulatory (silencer) activity was located in the region spanning from 1.4 to 1.7 kilobases upstream of the promoter region. This region contains a short sequence element (ATCCTCTCC) known to have such activity. A strong promoter on the opposite strand was also located about 500 base pairs upstream of the promoter. The expression of factor IX gene was highly liver specific, as assessed by Northern blot analysis. Short sequence elements (TGGACC and CTTTGGACT) homologous to the known liver-specific elements were located in the vicinity of the defined promoter region.

Base Sequence↗