RESUMO
UNLABELLED: Three-dimensional fluorescence in situ hybridization (3D-FISH) is used to study the organization and the positioning of chromosomes or specific sequences such as genes or RNA in cell nuclei. Many different programs (commercial or free) allow image analysis for 3D-FISH experiments. One of the more efficient open-source programs for automatically processing 3D-FISH microscopy images is Smart 3D-FISH, an ImageJ plug-in designed to automatically analyze distances between genes. One of the drawbacks of Smart 3D-FISH is that it has a rather basic user interface and produces its results in various text and image files thus making the data post-processing step time consuming. We developed a new Smart 3D-FISH graphical user interface, NEMO, which provides all information in the same place so that results can be checked and validated efficiently. NEMO gives users the ability to drive their experiments analysis in either automatic, semi-automatic or manual detection mode. We also tuned Smart 3D-FISH to better analyze chromosome territories. AVAILABILITY: NEMO is a stand-alone Java application available for Windows and Linux platforms. The program is distributed under the creative commons licence and can be freely downloaded from https://www-lgc.toulouse.inra.fr/nemo
Assuntos
Cromossomos/genética , Hibridização in Situ Fluorescente/métodos , Software , Núcleo Celular/genética , Núcleo Celular/metabolismo , Genes , Processamento de Imagem Assistida por Computador/métodos , Interface Usuário-ComputadorRESUMO
A high proportion of purebred Hampshire pigs carries the dominant RN- mutation, which causes high glycogen content in skeletal muscle. The mutation has beneficial effects on meat content but detrimental effects on processing yield. Here, it is shown that the mutation is a nonconservative substitution (R200Q) in the PRKAG3 gene, which encodes a muscle-specific isoform of the regulatory gamma subunit of adenosine monophosphate-activated protein kinase (AMPK). Loss-of-function mutations in the homologous gene in yeast (SNF4) cause defects in glucose metabolism, including glycogen storage. Further analysis of the PRKAG3 signaling pathway may provide insights into muscle physiology as well as the pathogenesis of noninsulin-dependent diabetes mellitus in humans, a metabolic disorder associated with impaired glycogen synthesis.
Assuntos
Glicogênio/metabolismo , Músculo Esquelético/enzimologia , Mutação Puntual , Proteínas Quinases/genética , Proteínas Quinases Ativadas por AMP , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Northern Blotting , Clonagem Molecular , DNA Complementar/isolamento & purificação , Regulação Enzimológica da Expressão Gênica , Homozigoto , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Especificidade de Órgãos/genética , Fenótipo , Proteínas Quinases/biossíntese , Proteínas Quinases/isolamento & purificação , Homologia de Sequência de Aminoácidos , SuínosRESUMO
Using in situ hybridization with a human probe, we have mapped the nucleoside phosphorylase gene on pig chromosome 7. These results are in agreement with those obtained by other groups, but give a more precise localization in the q2.1----q2.2 region of chromosome 7.
Assuntos
Pentosiltransferases/genética , Purina-Núcleosídeo Fosforilase/genética , Suínos/genética , Animais , Bandeamento Cromossômico , Mapeamento Cromossômico , Sondas de DNA , Masculino , Hibridização de Ácido NucleicoRESUMO
Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are members of the glycoprotein hormone family and play essential roles in gametogenesis and sexual development of mammals. Fragments of the porcine genes coding for the beta subunits of FSH and LH were amplified by PCR and used as probes for radioactive in situ hybridization in order to map these loci in the pig. Primers were chosen on the genomic DNA nucleotide sequences of FSHB and LHB as published in GenBank. Fragments of 1,127 bp (FSHB) and 1,239 bp (LHB) were cloned and verified by sequencing. FSHB was localized to pig chromosome bands 2p1.6-->p1.2 and LHB to pig chromosome band 6q2.1. The localization of LHB to the so-called halothane region of chromosome 6 could be expected from comparative mapping data. For FSHB, no conclusions can be drawn in this respect since, up to the present, too few genes are located on porcine chromosome 2.
Assuntos
Mapeamento Cromossômico , Hormônio Foliculoestimulante/genética , Hormônio Luteinizante/genética , Suínos/genética , Animais , Sequência de Bases , Primers do DNA , Subunidade beta do Hormônio Folículoestimulante , Hibridização In Situ , Linfócitos/citologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodosRESUMO
The nerve growth factor beta gene (NGFB) belongs to a conserved syntenic group on human chromosome 1 and mouse Chromosome 3. The objective of this study was the isolation of a part of the porcine NGFB gene and its use as a genetic and physical marker in the pig genome. On the basis of a nucleotide sequence comparison among different species, NGFB-specific primers were chosen to amplify the corresponding porcine sequence by PCR. A pig genomic DNA fragment of 763 bp was isolated, and its DNA sequence, containing the complete coding sequence of mature NGFB, was determined. It was demonstrated that pig NGFB is largely homologous with NGFB of other species (mouse, cattle, chicken) and especially with human NGFB; the isolated clone shows 91% nucleotide and 99% translated amino acid sequence identity to the human NGFB sequence. The porcine DNA clone allowed us to identify an RFLP marker for genetic mapping in pigs and was used to map the NGFB gene to pig chromosome region 4q1.6-->q2.3 by radioactive in situ hybridization. Previously, we had localized to the same chromosome another member of this syntenic group, the gene encoding alpha 1 Na+,K+ ATPase (ATP1A1). These results show that a portion of the homologous region between human chromosome 1 and mouse Chromosome 3 is also conserved on pig chromosome 4.
Assuntos
Sequência Consenso , Sequência Conservada , Marcadores Genéticos , Fatores de Crescimento Neural/genética , Suínos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Mapeamento Cromossômico , Clonagem Molecular , DNA , Humanos , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Polimorfismo Genético , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
The genes coding for the regulatory type I alpha subunit (PRKAR1A) and the catalytic beta subunit (PRKACB) of cAMP-dependent protein kinase and the genes for interleukin 1 alpha (IL1A) and interleukin 1 beta (IL1B) were localized in the pig by means of radioactive in situ hybridization. PRKAR1A was mapped to 12p1.4 and PRKARB to 6q3.1-->q3.3. The genes for IL1A and IL1B were both assigned to Chromosome (Chr) 3, in the region q1.2-->q1.3 and q1.1-->q1.4, respectively. The cDNA nucleotide sequences of these porcine genes were compared with those of human, mouse, and cattle. The location of the genes was discussed in relation to the position of their homologous loci in these mammalian species.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Interleucina-1/genética , Suínos/genética , Animais , Bovinos/genética , Mapeamento Cromossômico , Proteínas Quinases Dependentes de AMP Cíclico/química , DNA Complementar/genética , Genes , Hominidae/genética , Humanos , Camundongos/genética , Dados de Sequência Molecular , Especificidade da EspécieRESUMO
The genes coding for the insulin-like growth factor-1 receptor (IGF1R) and a porcine endothelium-derived 21-residue vasoconstrictor peptide (EDN) were localized in the pig by means of radioactive in situ hybridization. IGF1R was mapped to chromosome region 1q1.7-->q2.1 and EDN to 7p1.3-->p1.2. The results are discussed in relation to the position of their homologous loci in man.
Assuntos
Mapeamento Cromossômico , Endotelinas/genética , Precursores de Proteínas/genética , Receptor IGF Tipo 1/genética , Suínos/genética , Vasoconstrição/genética , Animais , Endotelina-1 , Humanos , Hibridização In SituRESUMO
The porcine gene for luteinizing hormone/choriogonadotropin receptor (LHCGR) was localized to chromosome 3q2.2----q2.3 using radioactive and nonradioactive in situ hybridization. A computer-assisted image-analysis system was developed which facilitated detection of the position of silver grains and fluorescent spots on the chromosomes after in situ hybridization. Compared with autoradiographic visualization, the nonisotopic procedure proved to be more rapid, precise, and highly specific; however, nonradiographic in situ hybridization was much less efficient than the autoradiographic technique for the detection of unique DNA sequences with small probes. From these results and published gene-mapping data, it was concluded that the synteny between LHCGR and MDH1 observed in man is conserved in the pig genome.
Assuntos
Mapeamento Cromossômico , Receptores do LH/genética , Suínos/genética , Animais , Bandeamento Cromossômico , Sondas de DNA/genética , Fluorescência , Hibridização de Ácido NucleicoRESUMO
Four homeobox genes that belong to the four homeobox gene clusters known in mammals have been regionally assigned to four distinct porcine chromosomes in conserved regions between human and pig. HOXA11, HOXB6, HOXC8, and HOXD4 genes were mapped by radioactive in situ hybridization to porcine Chromosomes (Chrs) 18q21-24 (with a secondary signal in 16q14-21), 12p11-12, 5p11-12, and 15q22-23 respectively. Besides, we have also revealed the presence of a porcine homeobox (pig Hbx24) which, although showing DNA sequence homology with a mouse gene of HOXB cluster, was located on porcine Chr 3 (3p14-13) outside the Hox clusters. To support the identity of the homeobox gene clusters analyzed and in the light of the high sequence similarity among homeobox genes, we also localized markers known to be mapped near each Hox cluster in human. In this way, four genes were also mapped in pig: GAPD (5q12-21), GAD1 (15q21-22), INHBA (18q24), and IGFBP3 (18q24). Mapping of HOXA11, INHBA, and IGFBP3 on pig Chr 18 constitutes the first assignments of genes on this small chromosome. These new localizations extend the information on the conservation of four human chromosomal regions in the pig genome.
Assuntos
Mapeamento Cromossômico , Genes Homeobox/genética , Família Multigênica/genética , Suínos/genética , Animais , Marcadores Genéticos , Humanos , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Polimorfismo Conformacional de Fita SimplesRESUMO
In the pig, the linkage group around the halothane gene (HAL), composed of S-GPI-HAL-H-A1BG-PGD, has been assigned to bands p1.2----q2.2 of chromosome 6. In man, ENO1-PGD and APOE-GPI constitute two syntenic groups situated on different chromosomes (1 and 19, respectively). Since GPI and PGD are linked in the pig, we have hybridized the human cDNA probes for ENO1 and APOE to pig chromosomes. These markers were assigned to pig chromosome 6, in the q2.2----q2.4 and cen----q2.1 regions, respectively, using in situ hybridization. Since GPI and APOE are situated in the same region, we combined the use of high resolution chromosome analysis and in situ hybridization to give a more precise localization in the q1.2 and q1.2----q2.1.2 regions of chromosome 6. A possible linear order of these genes is proposed.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 19 , Cromossomos Humanos Par 1 , Marcadores Genéticos , Halotano/farmacologia , Suínos/genética , Animais , Apolipoproteínas E/genética , Bandeamento Cromossômico , Ligação Genética , Glucose-6-Fosfato Isomerase/genética , Humanos , Hibridização de Ácido Nucleico , Fosfogluconato Desidrogenase/genética , Fosfopiruvato Hidratase/genéticaRESUMO
The TGF beta-1 and PGD loci have been localized by in situ hybridization to the C-greater than q2.1 and q2.2 -greater than q2.5 regions of pig chromosome 6. These assignments confirm that the conversation of syntenic groups around GPI and PGD extends to pigs where these two groups are uniquely found to be linked. Our data also support the hypothesis that the porcine and human inherited malignant hyperthermia syndromes are caused by mutations in homologous genes which map to human chromosome 19q, porcine chromosome 6q and murine chromosome 7.
Assuntos
Mapeamento Cromossômico , Fosfogluconato Desidrogenase/genética , Suínos/genética , Fator de Crescimento Transformador beta/genética , Animais , Bandeamento Cromossômico , Genes , Humanos , Hibridização de Ácido NucleicoRESUMO
For insight into the general organization of the swine leukocyte antigen (SLA) complex, the swine major histocompatibility complex (MHC), four sequences related to the heat-shock proteins HSP70 were characterized by screening of a pig genomic cosmid library with a swine cDNA HSP70 2.6-kb probe. This yielded three positive clones: HC2.2, HC3.2, and HC4.2. Restriction site maps revealed a large overlap of HC2.2 with HC3.2, whereas HC4.2 was independent. Southern blot hybridization with the 5' section, the central section, and the 3' section of the 2.6-kb probe and also with a swine 4.5-kb HSP70 genomic probe suggested the existence, within the overlapping clones, of three distinct HSP70 sequences encompassing a segment no longer than 22 kb. The HC4.2 clone, which hybridized with the same probes, displayed a single band of 7.3 kb, probably corresponding to one gene only. Fluorescent in situ hybridization on swine chromosome metaphases with the whole HC2.2 or HC4.2 cosmids allowed the assignment of HC2.2 to MHC region on Chromosome (Chr) 7 (Cen-p1.1), and of HC4.2 to Chr 14 (q2.4-2.5). Thus, as in humans, the swine MHC comprises three closely linked HSP70 loci. The presence of additional genes belonging to the same inducible HSP70 gene family can be expected from what is known in humans. The HSP70 gene found here on the pig Chr 14 may be one of these putative unidentified genes.
Assuntos
Proteínas de Choque Térmico/genética , Suínos/genética , Animais , Mapeamento Cromossômico , Clonagem Molecular , Cosmídeos , Sondas de DNA , Hibridização In Situ , Complexo Principal de Histocompatibilidade , Mapeamento por RestriçãoRESUMO
We have developed a simple and efficient method to construct partial libraries of swine Chromosome (Chr) 11, starting with only 300 flow-sorted copies. DNA is amplified by PARM-PCR with primer containing at the 5'-end the sequence AGCU-. After amplification, digestion of PCR products with uracil DNA glycosylase generates cohesive ends corresponding to the SstI site. The amplified fragments can then be ligated in vector linearized with the SstI enzyme. Using five different primers, we PARM-PCR amplified and cloned swine Chr 11 DNA. These chromosome-specific libraries have been used to develop 14 different (TG)n microsatellites. Ten of these markers were assigned to Chr 11 by PCR analysis of a panel of Pig-Rodent somatic hybrids and by linkage analysis of the 171 individuals of the PiGMaP reference families. A complete linkage map of 147 cM of this chromosome was then realized by integrating existing markers.
Assuntos
Mapeamento Cromossômico , Repetições de Microssatélites , Suínos/genética , Animais , Sequência de Bases , Mapeamento Cromossômico/métodos , Primers do DNA , Citometria de Fluxo , Biblioteca Gênica , Genótipo , Dados de Sequência MolecularRESUMO
The genes coding for glucose regulated protein, 78kDal (GRP78), hormone-sensitive lipase (LIPE), plasminogen activator or urokinase (PLAU), and D-amino acid oxidase (DAO) were localized in the pig by radioactive in situ hybridization. GRP78 was mapped to 1q2.10-->q2.13 and LIPE was localized to chromosome 6cen-->q1.2. The genes for PLAU and DAO were both assigned to chromosome 14, in the region q2.4-->q2.6 and q2.1-->q2.3, respectively. The results are compared to mapping data in other mammalian species.
Assuntos
Mapeamento Cromossômico , Marcadores Genéticos , Proteínas de Choque Térmico , Chaperonas Moleculares , Suínos/genética , Animais , Proteínas de Transporte/genética , Bovinos , D-Aminoácido Oxidase/genética , Chaperona BiP do Retículo Endoplasmático , Humanos , Hibridização In Situ , Camundongos , Esterol Esterase/genética , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Using as a probe pig genomic DNA, including the complete interferon alpha gene, we have mapped the leukocyte interferon gene on pig chromosome 1 by in situ hybridization. A total of 196 silver grains were noted on the 106 metaphases scored: 31% of the grains were observed on chromosome 1, and 67% of these were localized in the region 1q2.2----q2.7.
Assuntos
Mapeamento Cromossômico , Interferon Tipo I/genética , Suínos/genética , Animais , Cariotipagem , Hibridização de Ácido NucleicoRESUMO
The gene encoding the porcine growth hormone (GH) has been localized to the q-arm of chromosome 12 using high-resolution R-banded chromosomes for in situ hybridization. We report here the localization of GH on the p-arm of this chromosome when using in situ hybridization on high-resolution G-banded chromosomes. Sequential Q- and R-banding show that this discrepancy is caused by a reversed orientation of chromosome 12 in the R-banded high-resolution karyotype published by Rønne et al. (1987) and the G-banded standard karyotype.
Assuntos
Hormônio do Crescimento/genética , Suínos/genética , Animais , Bandeamento Cromossômico/veterinária , Mapeamento Cromossômico/veterinária , Hibridização In Situ/veterinária , Masculino , MetáfaseRESUMO
Two genes coding for Na+,K(+)-ATPase alpha and beta subunits are localized on pig chromosome 4, to the q1.6-->q2.3 and 1.3-->q2.1 regions, respectively, by radioactive in situ hybridization. According to nucleotide and amino acid sequence comparisons with different human isoforms of Na+,K(+)-ATPase, these pig alpha beta ATPase genes show strong homologies with human alpha 1 and beta subunit ATPase genes, respectively. These results are discussed with respect to comparative mapping data of conserved genes in mammalian species. We showed that the pig cDNA probes encoding ATPase alpha and beta genes reveal DNA polymorphism in Meishan and Large White pigs.
Assuntos
Mapeamento Cromossômico/veterinária , ATPase Trocadora de Sódio-Potássio/genética , Suínos/genética , Animais , Células Cultivadas , Humanos , Hibridização In Situ/veterinária , Cariotipagem/veterinária , Metáfase , Camundongos , Polimorfismo Genético , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
The M13.13 minisatellite probe, consisting of a polymer of the M13 VNTR consensus sequence, cross-hybridized to ovine DNA and allowed detection of several polymorphic loci. Individual specific patterns were obtained in sheep using this probe. Pedigree analysis showed that individuals were heterozygous for most of the DNA fragments detected (88%). By studying the segregation of male's variable DNA fragments, a minimum of 10 loci were defined. The ovine DNA 'fingerprint' obtained with M13.13 is polymorphic enough to be used efficiently in animal identification, paternity testing, and possibly as a source of genetic markers for linkage analysis.