Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 13 de 13
Filter
1.
Proc Natl Acad Sci U S A ; 101(13): 4695-700, 2004 Mar 30.
Article in English | MEDLINE | ID: mdl-15070780

ABSTRACT

Inactivating mutations of the pro-opiomelanocortin (POMC) gene in both mice and humans leads to hyperphagia and obesity. To further examine the mechanisms whereby POMC-deficiency leads to disordered energy homeostasis, we have generated mice lacking all POMC-derived peptides. Consistent with a previously reported model, Pomc(-/-) mice were obese and hyperphagic. They also showed reduced resting oxygen consumption associated with lowered serum levels of thyroxine. Hypothalami from Pomc(-/-) mice showed markedly increased expression of melanin-concentrating hormone mRNA in the lateral hypothalamus, but expression of neuropeptide Y mRNA in the arcuate nucleus was not altered. Provision of a 45% fat diet increased energy intake and body weight in both Pomc(-/-) and Pomc(+/-) mice. The effects of leptin on food intake and body weight were blunted in obese Pomc(-/-) mice whereas nonobese Pomc(-/-) mice were sensitive to leptin. Surprisingly, we found that Pomc(-/-) mice maintained their acute anorectic response to peptide-YY(3-36) (PYY(3-36)). However, 7 days of PYY(3-36) administration had no effect on cumulative food intake or body weight in wild-type or Pomc(-/-) mice. Thus, POMC peptides seem to be necessary for the normal response of energy balance to high-fat feeding, but not for the acute anorectic effect of PYY(3-36) or full effects of leptin on feeding. The finding that the loss of only one copy of the Pomc gene is sufficient to render mice susceptible to the effects of high fat feeding emphasizes the potential importance of this locus as a site for gene-environment interactions predisposing to obesity.


Subject(s)
Appetite Depressants/pharmacology , Dietary Fats/pharmacology , Leptin/pharmacology , Peptide YY/pharmacology , Pro-Opiomelanocortin/deficiency , Pro-Opiomelanocortin/genetics , Animals , Base Sequence , Body Weight/drug effects , DNA Primers , Energy Intake , Hypothalamic Hormones/genetics , Hypothalamus/physiology , Kinetics , Melanins/genetics , Mice , Mice, Knockout , Mutagenesis, Site-Directed , Neuropeptide Y/genetics , Obesity/genetics , Peptide Fragments , Phenotype , Pituitary Hormones/genetics , Polymerase Chain Reaction , Transcription, Genetic
2.
J Invest Dermatol ; 121(4): 674-80, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14632181

ABSTRACT

Reduced coat 3 (Rco3) is a new spontaneous autosomal recessive mutation with defects in hair structure and progressive alopecia. Here we describe chromosomal mapping and molecular identification of the Rco3 mutation. The murine Rco3 locus maps to a 2-Mb interval on chromosome 15 encompassing the keratin type II gene cluster. Recently, mK6irs1 was described as a type II keratin expressed in Henle's and Huxley's layer of the murine inner root sheath. Genomic sequencing revealed a 10-bp deletion in exon 1 of mK6irs1 resulting in a frameshift after 58 amino acid residues and, therefore, the absence of 422 carboxy-terminal amino acid residues containing the complete alpha-helical rod domain. Henle's and Huxley's layers show no immunoreactivity with mK6irs1-specific antibodies and the absence of intermediate filament formation in electron microscopic images. These results indicate that the expression of functional mK6irs1 is indispensable for intermediate filament formation in the inner root sheath and highlights the importance of the keratinization of the inner root sheath in the normal formation of the hair shaft.


Subject(s)
Alopecia/genetics , Frameshift Mutation , Keratins/genetics , Alopecia/physiopathology , Amino Acid Sequence , Animals , Base Sequence , Chromosomes, Mammalian , Cloning, Molecular , Disease Models, Animal , Keratins/deficiency , Mice , Mice, Inbred AKR , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Molecular Sequence Data , Phenotype
4.
Nature ; 404(6773): 95-9, 2000 Mar 02.
Article in English | MEDLINE | ID: mdl-10716450

ABSTRACT

The earliest cell fate decision in the mammalian embryo separates the extra-embryonic trophoblast lineage, which forms the fetal portion of the placenta, from the embryonic cell lineages. The body plan of the embryo proper is established only later at gastrulation, when the pluripotent epiblast gives rise to the germ layers ectoderm, mesoderm and endoderm. Here we show that the T-box gene Eomesodermin performs essential functions in both trophoblast development and gastrulation. Mouse embryos lacking Eomesodermin arrest at the blastocyst stage. Mutant trophoectoderm does not differentiate into trophoblast, indicating that Eomesodermin may be required for the development of trophoblast stem cells. In the embryo proper, Eomesodermin is essential for mesoderm formation. Although the specification of the anterior-posterior axis and the initial response to mesoderm-inducing signals is intact in mutant epiblasts, the prospective mesodermal cells are not recruited into the primitive streak. Our results indicate that Eomesodermin defines a conserved molecular pathway controlling the morphogenetic movements of germ layer formation and has acquired a new function in mammals in the differentiation of trophoblast.


Subject(s)
Embryonic and Fetal Development/physiology , T-Box Domain Proteins/physiology , Trophoblasts/physiology , Xenopus Proteins , Amino Acid Sequence , Animals , Body Patterning/physiology , Chimera/genetics , Culture Techniques , Gastrula/physiology , Mesoderm/physiology , Mice , Molecular Sequence Data , Mutagenesis , Sequence Homology, Amino Acid , Signal Transduction , T-Box Domain Proteins/genetics , Xenopus
5.
Am Heart J ; 137(4 Pt 1): 698-705, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10097233

ABSTRACT

BACKGROUND: Genes encoding components of the renin-angiotensin system have been associated with elevated blood pressure (BP) and an increased risk of coronary artery disease. To explore the role of the angiotensinogen (AGT) gene in coronary atherosclerosis and thrombosis, we studied the effect of the AGT M235T gene variant on plasma AGT levels and BP in patients with coronary artery disease and in the subgroup of survivors of myocardial infarction as compared with angiographically defined control subjects. METHODS AND RESULTS: This was a case-control study of 301 white male subjects examined at Frankfurt University medical center. Plasma AGT levels increased stepwise according to the number of T235 alleles present (no T235 allele, 14.8 +/- 3.9 nmol/L; 1 allele, 15.7 +/- 5.1 nmol/L; 2 alleles, 17.3 +/- 4.7 nmol/L; P =.006). In a multivariate model, circulating AGT emerged as the most important predictor of diastolic pressure (P =.001). In addition, AGT M235T gene polymorphism remained a significant predictor of diastolic BP in a multivariate model adjusted for age, body mass index, fasting glucose, apolipoprotein B, presence of coronary artery disease, and treatment with antihypertensive agents ( P <.05). Finally, homozygosity for T235 was associated with increased univariate risk of coronary artery disease and myocardial infarction (odds ratio estimates 1.5; 95% confidence intervals 1.1 to 2.1, P =.03, and 1.0 to 2.1, P =.05, respectively). CONCLUSIONS: The significant relations observed between the AGT M235T variant, its protein product, and the cardiovascular disease phenotypes provide evidence for a possible role of elevated circulating AGT in the pathogenesis of coronary artery disease.


Subject(s)
Angiotensinogen/blood , Angiotensinogen/genetics , Cardiovascular Diseases/genetics , Polymorphism, Genetic , Renin-Angiotensin System/genetics , Alleles , Blood Pressure/genetics , Case-Control Studies , Coronary Artery Disease/genetics , Coronary Thrombosis/genetics , Gene Frequency , Genotype , Humans , Male , Middle Aged , Multivariate Analysis , Myocardial Infarction/genetics , Odds Ratio , Peptidyl-Dipeptidase A/blood , Risk Factors
6.
Mol Cell Biol ; 18(5): 3081-8, 1998 May.
Article in English | MEDLINE | ID: mdl-9566926

ABSTRACT

A strategy employing gene trap mutagenesis and site-specific recombination (Cre/loxP) has been used to identify genes that are transiently expressed during early mouse development. Embryonic stem cells expressing a reporter plasmid that codes for neomycin phosphotransferase and Escherichia coli LacZ were infected with a retroviral gene trap vector (U3Cre) carrying coding sequences for Cre recombinase (Cre) in the U3 region. Activation of Cre expression from integrations into active genes resulted in a permanent switching between the two selectable marker genes and consequently the expression of beta-galactosidase (beta-Gal). As a result, clones in which U3Cre had disrupted genes that were only transiently expressed could be selected. Moreover, U3Cre-activating cells acquired a cell autonomous marker that could be traced to cells and tissues of the developing embryo. Thus, when two of the clones with inducible U3Cre integrations were passaged in the germ line, they generated spatial patterns of beta-Gal expression.


Subject(s)
Embryonic and Fetal Development/genetics , Gene Expression Regulation, Developmental , Mice/genetics , Molecular Biology/methods , Viral Proteins , Animals , Body Patterning/genetics , Cell Differentiation/genetics , Clone Cells , Genes, Reporter , Integrases , Lac Operon , Mice, Transgenic , Mutagenesis , Recombination, Genetic , Stem Cells , Tissue Distribution , beta-Galactosidase/biosynthesis
7.
Trends Genet ; 13(9): 370-4, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9287493

ABSTRACT

Classical genetics depends upon investigation of function by random destruction with little information on structure. Modern mapping using random polymorphisms, cloning and sequencing investigates structure without function. The genome projects with their rapid gene discovery are, however, redefining classical genetic approaches. The efficient translation of this wealth of new information into insights in biological function at molecular, cellular and organismal levels requires large-scale approaches to the generation of mutants. Gene trapping in embryonic stem (ES) cells allows an efficient approach to the functional analysis of the murine genome. The usually separate processes of gene discovery, mapping, the observation of the expression pattern and the mutant phenotype in vivo, can be integrated by the use of an indexed library of insertionally mutated ES cell clones. It should be possible to generate mutants for a large proportion of the genes of the mammalian genome.


Subject(s)
Cloning, Molecular/methods , Gene Targeting/methods , Genetic Techniques , Animals , Base Sequence , Chromosome Mapping , DNA, Complementary , Gene Expression Regulation, Developmental , Gene Library , Genome , Humans , Mice , Molecular Sequence Data , Mutagenesis , Mutation , Stem Cells
8.
Proc Natl Acad Sci U S A ; 93(26): 15279-84, 1996 Dec 24.
Article in English | MEDLINE | ID: mdl-8986802

ABSTRACT

A strategy employing gene-trap mutagenesis and site-specific recombination (Cre/loxP) has been developed to isolate genes that are transcriptionally activated during programmed cell death. Interleukin-3 (IL-3)-dependent hematopoietic precursor cells (FDCP1) expressing a reporter plasmid that codes for herpes simplex virus-thymidine kinase, neomycin phosphotransferase, and murine IL-3 were transduced with a retroviral gene-trap vector carrying coding sequences for Cre-recombinase (Cre) in the U3 region. Activation of Cre expression from integrations into active genes resulted in a permanent switching between the selectable marker genes that converted the FDCP1 cells to factor independence. Selection for autonomous growth yielded recombinants in which Cre sequences in the U3 region were expressed from upstream cellular promoters. Because the expression of the marker genes is independent of the trapped cellular promoter, genes could be identified that were transiently induced by IL-3 withdrawal.


Subject(s)
Apoptosis/genetics , Hematopoietic Stem Cells/physiology , Mutagenesis , Recombination, Genetic , Viral Proteins , Animals , Cell Line , Cell Survival/drug effects , Cloning, Molecular , DNA Primers , Gene Expression Regulation, Enzymologic , Genes, Reporter , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Integrases/biosynthesis , Integrases/genetics , Interleukin-3/genetics , Interleukin-3/pharmacology , Interleukin-3/physiology , Kanamycin Kinase , Kinetics , Mice , Phosphotransferases (Alcohol Group Acceptor)/biosynthesis , Phosphotransferases (Alcohol Group Acceptor)/genetics , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Simplexvirus/genetics , Thymidine Kinase/biosynthesis , Thymidine Kinase/genetics , Transfection
9.
J Virol ; 70(8): 4927-32, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8763996

ABSTRACT

A new generation of retrovirus vectors for gene therapy has been developed. The vectors have the ability to excise themselves after inserting a gene into the genome, thereby avoiding problems encountered with conventional retrovirus vectors, such as recombination with helper viruses or transcriptional repression of transduced genes. The strategy exploited (i) the natural life cycle of retroviruses, involving duplication of terminal control regions U5 and U3 to generate long terminal repeats (LTRs) and (ii) the ability of the P1 phage site-specific recombinase (Cre) to excise any sequences positioned between two loxP target sequences from the mammalian genome. Thus, an independently expressed selectable marker gene flanked by a loxP target sequence was cloned into the U3 region of a Moloney murine leukemia virus vector. A separate cassette expressing the Cre recombinase was inserted between the LTRs into the body of the virus. LTR-mediated duplication placed vector sequences, including Cre, between loxP sites in the integrated provirus. This enabled Cre to excise from the provirus most of the viral and nonviral sequences unrelated to transcription of the U3 gene.


Subject(s)
Genetic Therapy , Genetic Vectors , Retroviridae/genetics , Animals , Humans , Plasmids
10.
Ann Intern Med ; 125(1): 19-25, 1996 Jul 01.
Article in English | MEDLINE | ID: mdl-8644984

ABSTRACT

BACKGROUND: Previous research has shown that the insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) gene is a major determinant of plasma ACE activity. It has been suggested that persons with the DD genotype (those who express, on average, the highest levels of circulating ACE) have an increased risk for myocardial infarction and coronary artery disease, particularly if they are otherwise at low risk. Subsequent studies, however, have not confirmed that ACE I/D gene polymorphism is a risk factor for coronary artery disease and myocardial infarction. OBJECTIVE: To investigate the association between the I/D polymorphism of the ACE gene and the risk for coronary artery disease and myocardial infarction in patients in whom coronary artery disease status was documented by angiography. DESIGN: Cross-sectional study. SETTING: University medical center. PATIENTS: 209 male case-patients with coronary artery disease and 92 male controls without coronary artery disease, as documented by coronary angiography. MEASUREMENTS: Assessment of the cardiac risk profile by questionnaire; classification of patients by the degree of coronary artery stenosis; levels of lipoproteins, apolipoproteins, and fibrinogen; and ACE I/D gene polymorphism assessed by polymerase chain reaction amplification. RESULTS: Plasma ACE activity was significantly associated with ACE I/D gene polymorphism. The ACE genotype was not associated with the presence of coronary artery disease or myocardial infarction. If a recessive effect of the D allele was assumed (DD compared with DI and II), the relative risk was 1.00 (95% CI, 0.76 to 1.30) for coronary artery disease and 1.03 (CI, 0.77 to 1.38) for myocardial infarction. Results of analyses were also negative when a dominant effect of the D allele was assumed and when low-risk subgroups were examined. The established risk factors age and apolipoprotein B level emerged as the most important risk predictors in multivariate analyses, followed by diastolic blood pressure and fasting glucose levels. CONCLUSIONS: In an angiographically defined study sample, ACE I/D gene polymorphism was not associated with an increased risk for coronary artery disease or myocardial infarction, despite its effects on plasma ACE activity.


Subject(s)
Coronary Disease/enzymology , Genotype , Myocardial Infarction/enzymology , Peptidyl-Dipeptidase A/genetics , Polymorphism, Genetic , Aged , Coronary Disease/blood , Cross-Sectional Studies , Humans , Male , Middle Aged , Myocardial Infarction/blood , Odds Ratio , Peptidyl-Dipeptidase A/blood , Regression Analysis , Risk Factors , Surveys and Questionnaires
12.
Electrophoresis ; 14(3): 169-73, 1993 Mar.
Article in English | MEDLINE | ID: mdl-8486126

ABSTRACT

Apolipoprotein B-100 is the principal protein component of lipoproteins with very low, intermediate, and low density. The interaction of apoB-100 with low density lipoprotein (LDL) receptors is responsible for the uptake of LDL into cells. An AT-rich hypervariable region is located adjacent to the 3' end of the apoB gene. It consists of a variable number of tandemly repeated sequences (VNTR). Two approaches were used to analyze this polymorphism. In both, the region harboring the VNTR was amplified with the polymerase chain reaction (PCR). In the first method, fluorescently labeled primers were used in the PCR reactions and products were separated in agarose gels by means of an automated fluorescent fragment analyzer. In the second method, PCR products were analyzed in denaturing polyacrylamide gels and detected with silver staining. Even in the highly sophisticated automated system, agarose gel electrophoresis did not always enable unequivocal assignment of VNTR alleles. In contrast, denaturing polyacrylamide gel electrophoresis made it possible to distinguish the 15 bp differences between the VNTR alleles in a precise and simple manner. The VNTR polymorphism was typed in 234 individuals. Among these were 136 patients with coronary artery disease and 74 healthy controls. Thirteen alleles could be distinguished. The allele containing 49 repeats (VNTR-49) was found in 9.2% of the coronary artery disease patients and in 4.7% of the controls. Thus, the VNTR-49 allele increases relative coronary risk by about twofold. It is concluded that the apoB VNTR polymorphism is a potentially useful genetic marker. Since agarose gel electrophoresis may lead to ambiguous results, we prefer typing by denaturing polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Apolipoproteins B/genetics , DNA/genetics , DNA/isolation & purification , Electrophoresis, Agar Gel/methods , Electrophoresis, Polyacrylamide Gel/methods , Alleles , Base Sequence , Coronary Disease/genetics , Genotype , Humans , Molecular Sequence Data , Nucleic Acid Denaturation , Polymerase Chain Reaction , Polymorphism, Genetic , Repetitive Sequences, Nucleic Acid
13.
Biochim Biophys Acta ; 1132(3): 329-31, 1992 Oct 20.
Article in English | MEDLINE | ID: mdl-1358203

ABSTRACT

Cytosolic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase (E.C. 4.1.3.5) is a highly regulated enzyme involved in isoprenoid biosynthesis and therefore a potential target for cholesterol-lowering drugs. Up to now, primary structure data have only been available for chicken, rat and hamster HMG-CoA synthase. Using in vitro amplification and direct sequencing, we have determined the nucleotide sequence of the coding region of the human cytosolic 3-hydroxy-3-methylglutaryl CoA synthase cDNA.


Subject(s)
Hydroxymethylglutaryl-CoA Synthase/genetics , Amino Acid Sequence , Base Sequence , DNA , Humans , Molecular Sequence Data , Polymerase Chain Reaction
SELECTION OF CITATIONS
SEARCH DETAIL
...