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At least 1,315 records · Page 73Linked to original sources
PubMed2002

Identification of N-myc regulatory regions involved in embryonic expression.

Our knowledge on the regulation of the N-myc proto-oncogene expression comes mostly from in vitro studies. Very few in vivo analyses have been performed to identify the regulatory elements involved in N-myc developmental expression. In the present study, we defined DNA regions required for the regulated expression of N-myc during early embryogenesis. We showed that the expression of N-myc driven by the human N-myc sequences previously described to control N-myc expression in appropriate cell types in vitro cannot rescue the mouse N-myc mutant phenotype, suggesting that regulatory elements necessary for N-myc embryonic expression were missing. To identify the regulatory DNA regions involved in N-myc expression, transgenic mouse lines carrying N-myc/lacZ reporter constructs were generated. Beta-galactosidase staining analysis at different stages of gestation revealed that >16 kb of mouse N-myc genomic sequences are required to recapitulate the entire spatiotemporal expression pattern of the endogenous N-myc gene between embryonic d 8.5 and 11.5. This observation supported the notion that the sequences previously identified by in vitro assays were not sufficient to reproduce the N-myc embryonic expression pattern. However, regulatory elements that can direct specific expression in the visceral arches, the limb buds, the CNS, and the dorsal root ganglia are included into the mouse N-myc genomic sequences tested. Altogether, these findings indicated that the regulation of the spatiotemporal expression pattern of N-myc during development necessitates multiple regulatory DNA elements.

Animals↗
PubMed2001

PROP1 gene screening in patients with multiple pituitary hormone deficiency reveals two sites of hypermutability and a high incidence of corticotroph deficiency.

Alterations of the gene encoding the pituitary transcription factor PROP1 were associated with congenital forms of multiple pituitary hormone deficiencies in several families. Among 23 patients with multiple pituitary hormone deficiencies screened for a PROP1 gene abnormality, nine belonging to eight unrelated families had homozygous PROP1 gene defects. All mutations were located in exon 2 and affected only two different sites: a homozygous AG deletion at codons 99/100/101 (n = 5); homozygous point mutations affecting codon 73: R73C (n = 2) or R73H (n = 1), and a R73C/R99X double-heterozygous mutation (n = 1). R73H and R99X were never described. All patients were born to unaffected parents, and consanguinity was documented in two patients. They had complete GH, LH-FSH, and TSH deficiencies and normal basal levels of PRL. Delayed ACTH deficiency was diagnosed in four of nine patients. At magnetic resonance imaging the anterior pituitary was hypoplastic in seven patients and hyperplastic in two. This study found two novel mutations (R73H and R99X) and underlines the high incidence of PROP1 gene alterations in patients with multiple pituitary hormone deficiencies. A corticotroph deficiency was frequently observed in association with GH, TSH, and gonadotropin deficiencies and should be carefully sought during follow-up.

Adolescent↗
PubMed2002

A novel form of autosomal recessive pure hereditary spastic paraplegia maps to chromosome 13q14.

BACKGROUND: Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous disorder characterized by a progressive weakening and spasticity of the lower limbs. HSP is classified according to the presence or absence of accompanying neurologic problems and by the mode of inheritance. Currently, 17 loci have been linked to the various forms of HSP. OBJECTIVE: To determine the chromosomal location of a gene causing pure autosomal recessive spastic paraplegia. METHODS: Genotyping using fluorescently labeled microsatellite markers was performed on three affected individuals and three unaffected individuals from a family displaying pure autosomal recessive HSP (ARHSP) and sensorineural deafness. All family members were then included in the analysis to narrow the genetic interval. Candidate genes were screened for the presence of mutations by heteroduplex analysis. RESULTS: The paraplegic trait linked to a 1.8-Mb region of chromosome 13q14 flanked by the FLJ11712 gene and the microsatellite marker D13S270. The deafness did not link to this region and did not cosegregate with the paraplegic trait. CONCLUSION: The HSP that this family had represents a novel genetic form of pure ARHSP as no other form of HSP (autosomal dominant or recessive) has been linked to chromosome 13.

Abnormalities, Multiple↗
PubMed1993

Cleavage of potato virus Y polyprotein in Escherichia coli depending on the proteolytic activity of viral protease.

We have cloned a 5-kb cDNA corresponding to the 3'-half of genomic RNA of potato virus Y (PVY), the type member of plant potyvirus. Based on its nucleotide sequence, the cDNA was presumed to encode PVY protease responsible for the self-processing of polyprotein precursor expressed from PVY genome. To test its proteolytic activity, the cDNA was modified so as to express the protease-polymerase-coat protein (CP) portion of PVY polyprotein in Escherichia coli. By Western blotting analysis of the cell extract of E. coli expressing the polyprotein, mature CP was detected. A large deletion in the polymerase-coding region did not affect the emergence of mature CP, but a small deletion in the putative protease region resulted in disappearance of mature CP and appearance of the intact polyprotein. These results indicated that the polyprotein expressed in E. coli was cleaved depending on the proteolytic activity of PVY protease.

Amino Acid Sequence↗
PubMed2005

New methods for researching accessory proteins.

Receptor activity-modifying proteins (RAMPs) control the pharmacology of the receptors for the calcitonin family of peptide hormones. There are five of these peptides: calcitonin, calcitonix/calcitixin gene-related peptide (CGRP), adrenomedullin, amylin, and now adrenomedullin 2. The calcitonin receptor is specific for calcitonin when expressed alone but it can function as an amylin or CGRP receptor when co-expressed with a RAMP. The calcitonin receptor-like receptor (CRLR) will not reach the cell surface without any one of the three RAMP proteins to function as either a CGRP or adrenomedullin receptor. This system was discovered more than 6 yr ago. At the time, it was reasonable to think that nature would employ accessory proteins, such as the RAMPs, to enable flexible signaling systems for other ligand families and that these would be discovered in time. In reality, many more new peptide ligands have been discovered than accessory proteins. Why is this? Developments in bioinformatics facilitate the discovery of both seven transmembrane ligands and accessory proteins. Proteomics and transcriptomics can be used together to define likely accessory proteins that can be experimentally tested. Comparative genomics was used in the discovery of adrenomedullin 2. The existence of multiple RAMPs within several species of fish suggests an alternative endocrinology. Finally, genetics offers a direct view of receptors, ligands, and accessory proteins in human disease--either as causative or contributing factors.

Adrenomedullin↗
PubMed1990

cDNA cloning of androgen-stimulated mRNAs in rat seminal vesicles: partial characterization of newly isolated cDNA clones, pSv-1 and pSv-2.

As the first step in surveying the molecular mechanism of androgen-responsive gene expression in rat seminal vesicles, the effect of androgen on the mRNAs was examined by in vitro translation assay. When the in vitro translation products of mRNAs from castrated animals (48 h) were compared with those from castrated and testosterone-treated animals (48 h) by SDS-PAGE, several discrete bands which were stimulated or repressed in response to androgen were observed in addition to major peptide bands of SVS IV and SVS V. From these findings, we constructed a partial cDNA library from the seminal vesicle poly(A +)RNAs of androgen-treated rats and screened by differential colony hybridization. Two distinct cDNA clones, pSv-1 and pSv-2, whose mRNAs were differentially stimulated in response to androgen and seemed to be expressed specifically in the seminal vesicles, were isolated. pSv-1 and pSv-2 hybridized to mRNAs of 1,600 and 3,500 nucleotides in length, respectively. These cDNA sequences, newly isolated in the present study, may provide useful probes for the study of molecular mechanism of androgen-responsive gene expression.

Androgens↗
PubMed2006

Molecular and functional analysis of new members of the wheat PR4 gene family.

Five new genes belonging to the pathogenesis-related (PR) 4 family have been cloned and characterised in Triticum aestivum. Two full-length genes, named wPR4e and wPR4f-b, were isolated by library screening, demonstrating the presence of a small intron only in wPR4f-b. Two other PR4 genes (wPR4f-a and wPR4f-c) were isolated by PCR, showing very high sequence identity with wPR4f-b and constituting a new sub-family. Transcription start analysis was performed by RLM-RACE, leading to the isolation of a fifth gene, named wPR4g, that is highly homologous to wPR4e; both encode putative vacuolar PR4 proteins (Wheatwin7 and Wheatwin5, respectively). wPR4e and wPR4f sub-family genes are induced by F. culmorum infection, by chemicals that lead to systemic acquired resistance and by wounding, showing different spatial and temporal induction pathways. In silico analysis of the 5' untranslated regions of wPR4e and wPR4f-b revealed the presence of several abiotic and biotic stress-responsive elements. wPR4e and wPR4f-b putative promoters were fused to the beta-glucuronidase (GUS) reporter gene, and transient and stable expression assays demonstrated that both are able to drive expression of GUS. Characterisation of these new PR4 genes and particularly of their 5' untranslated regions, as well as the determination of their expression patterns, will contribute to our understanding of the responsiveness of this gene family to various stress conditions and of its role in plant defence.

Amino Acid Sequence↗
PubMed2005

An essential Drosophila glutamate receptor subunit that functions in both central neuropil and neuromuscular junction.

A Drosophila forward genetic screen for mutants with defective synaptic development identified bad reception (brec). Homozygous brec mutants are embryonic lethal, paralyzed, and show no detectable synaptic transmission at the glutamatergic neuromuscular junction (NMJ). Genetic mapping, complementation tests, and genomic sequencing show that brec mutations disrupt a previously uncharacterized ionotropic glutamate receptor subunit, named here "GluRIID." GluRIID is expressed in the postsynaptic domain of the NMJ, as well as widely throughout the synaptic neuropil of the CNS. In the NMJ of null brec mutants, all known glutamate receptor subunits are undetectable by immunocytochemistry, and all functional glutamate receptors are eliminated. Thus, we conclude that GluRIID is essential for the assembly and/or stabilization of glutamate receptors in the NMJ. In null brec mutant embryos, the frequency of periodic excitatory currents in motor neurons is significantly reduced, demonstrating that CNS motor pattern activity is regulated by GluRIID. Although synaptic development and molecular differentiation appear otherwise unperturbed in null mutants, viable hypomorphic brec mutants display dramatically undergrown NMJs by the end of larval development, suggesting that GluRIID-dependent central pattern activity regulates peripheral synaptic growth. These studies reveal GluRIID as a newly identified glutamate receptor subunit that is essential for glutamate receptor assembly/stabilization in the peripheral NMJ and required for properly patterned motor output in the CNS.

Animals↗
PubMed2006

Site-specific transformation of Drosophila via phiC31 integrase-mediated cassette exchange.

Position effects can complicate transgene analyses. This is especially true when comparing transgenes that have inserted randomly into different genomic positions and are therefore subject to varying position effects. Here, we introduce a method for the precise targeting of transgenic constructs to predetermined genomic sites in Drosophila using the C31 integrase system in conjunction with recombinase-mediated cassette exchange (RMCE). We demonstrate the feasibility of this system using two donor cassettes, one carrying the yellow gene and the other carrying GFP. At all four genomic sites tested, we observed exchange of donor cassettes with an integrated target cassette carrying the mini-white gene. Furthermore, because RMCE-mediated integration of the donor cassette is necessarily accompanied by loss of the target cassette, we were able to identify integrants simply by the loss of mini-white eye color. Importantly, this feature of the technology will permit integration of unmarked constructs into Drosophila, even those lacking functional genes. Thus, C31 integrase-mediated RMCE should greatly facilitate transgene analysis as well as permit new experimental designs.

Animals↗
PubMed2006

Quantitative trait loci for locomotor behavior in Drosophila melanogaster.

Locomotion is an integral component of most animal behaviors and many human diseases and disorders are associated with locomotor deficits, but little is known about the genetic basis of natural variation in locomotor behavior. Locomotion is a complex trait, with variation attributable to the joint segregation of multiple interacting quantitative trait loci (QTL), with effects that are sensitive to the environment. We assessed variation in a component of locomotor behavior (locomotor reactivity) in a population of 98 recombinant inbred lines of Drosophila melanogaster and mapped four QTL affecting locomotor reactivity by linkage to polymorphic roo transposable element insertion sites. We used complementation tests of deficiencies to fine map these QTL to 12 chromosomal regions and complementation tests of mutations to identify 13 positional candidate genes affecting locomotor reactivity, including Dopa decarboxylase (Ddc), which catalyzes the final step in the synthesis of serotonin and dopamine. Linkage disequilibrium mapping in a population of 164 second chromosome substitution lines derived from a single natural population showed that polymorphisms at Ddc were associated with naturally occurring genetic variation in locomotor behavior. These data implicate variation in the synthesis of bioamines as a factor contributing to natural variation in locomotor reactivity.

Animals↗
PubMed2002

Genetic screening for maternal uniparental disomy of chromosome 7 in prenatal and postnatal growth retardation of unknown cause.

OBJECTIVE: Many short-statured children lack an etiologic explanation for their retarded growth. Recently, uniparental disomy (UPD), the inheritance of both chromosomes of a chromosome pair from only 1 parent, has been associated with short stature for many chromosomes. Silver-Russell syndrome (SRS) represents an extreme syndrome of intrauterine growth retardation (IUGR) and slight dysmorphic signs, and maternal UPD of human chromosome 7 (matUPD7) has been observed in approximately 10% of SRS cases. In addition, matUPD7 has been reported in patients with only slight dysmorphic features and prenatal or postnatal growth retardation. The objectives of this study were to study the role of matUPD7 in growth failure of unknown cause and in cases of SRS, and to evaluate the efficiency of genetic testing for matUPD7 as a diagnostic tool. METHODS: DNA samples were studied from 205 children, 92 girls and 113 boys, with short stature of unknown cause and their parents. The patient cohort included 39 cases of SRS, 91 patients with IUGR and subsequent postnatal short stature, and 75 patients with postnatal growth retardation only. MatUPD7 was screened for by genotyping DNA samples from the patient, mother, and father with 13 chromosome-7-specific polymorphic microsatellite markers. RESULTS: Six (3%) of 205 matUPD7 cases were observed exclusively among 39 (15%) SRS patients studied. Patients with IUGR and/or postnatal growth retardation and with dysmorphic features did not reveal cases of matUPD7. CONCLUSIONS: Our results indicate that matUPD7 cases are predominantly observed among patients meeting the criteria of SRS, and matUPD7 is not a common cause for growth retardation. Genetic screening for cases of matUPD7 among growth-retarded patients should be focused on patients with severe IUGR and features of SRS. In addition, matUPD7 screening is advisable in individuals with cystic fibrosis and other recessive disorders mapped to chromosome 7 who have unusually short stature.

Abnormalities, Multiple↗
PubMed2005

Selection of D2S3, an Aedes aegypti (Diptera: Culicidae) strain with high oral susceptibility to Dengue 2 virus and D2MEB, a strain with a midgut barrier to Dengue 2 escape.

Family-based phenotypic selection was used to breed two genetic strains of Aedes aegypti L. that differ in susceptibility to infection with dengue serotype 2 virus (DEN-2) strain JAM1409. A Dengue 2 Susceptible on 3 chromosomes (D2S3) strain was bred from Ae. aegypti aegypti and Ae. aegyptiformosus P1 parents to have a high midgut infection rate (MIR) and a high disseminated infection rate (DIR). A Dengue 2 Midgut Escape Barrier (D2MEB) strain was bred from D2S3 and Houston P1 parents to have a high MIR and a low DIR. After selection in the F2 generation, single strand conformation polymorphism genotypes were determined at cDNA marker loci throughout the genome to test for Mendelian ratios and thereby identify regions containing deleterious or lethal alleles. Both strains were orally challenged with two other DEN-2 genotypes, two DEN-1 genotypes, one DEN-3 genotype, and two DEN-4 genotypes. There were significant differences in MIR and DIR for the different virus strains in both the D2S3 and the D2MEB mosquito lines.

Aedes↗
PubMed2004

Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord.

The Wharton's jelly of the umbilical cord contains mucoid connective tissue and fibroblast-like cells. Using flow cytometric analysis, we found that mesenchymal cells isolated from the umbilical cord express matrix receptors (CD44, CD105) and integrin markers (CD29, CD51) but not hematopoietic lineage markers (CD34, CD45). Interestingly, these cells also express significant amounts of mesenchymal stem cell markers (SH2, SH3). We therefore investigated the potential of these cells to differentiate into cardiomyocytes by treating them with 5-azacytidine or by culturing them in cardiomyocyte-conditioned medium and found that both sets of conditions resulted in the expression of cardiomyocyte markers, namely N-cadherin and cardiac troponin I. We also showed that these cells have multilineage potential and that, under suitable culture conditions, are able to differentiate into cells of the adipogenic and osteogenic lineages. These findings may have a significant impact on studies of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering by helping to eliminate worrying ethical and technical issues.

Adipocytes↗
PubMedDate not supplied

Sry-negative XX sex reversal in a family of Norwegian Elkhounds.

Two related female Norwegian Elkhounds were evaluated at 6 and 8 months of age for enlarged clitori. Both had a 78 XX karyotype. Histology of their internal reproductive tracts demonstrated 1 to be an XX true hermaphrodite with bilateral ovotestes and the other to be an XX male with bilateral aspermatogenic testes. Polymerase chain reaction-based tests of genomic DNA showed that both dogs lacked Sry, the testis-determining gene. Pedigree analysis was consistent with an autosomal recessive mode of inheritance, as has been reported in the American Cocker Spaniel and the German Shorthaired Pointer. This is the 1st reported case of familial Sry-negative XX sex reversal in the Norwegian Elkhound. A summary of 34 previously unreported cases of dogs with masculinized external genitalia and a normal 78 XX karyotype seen from 1980 to 1997 is given.

Animals↗