Mapping of 21 genetic markers to a QTL region for meat quality on pig chromosome 17.
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Animals↗
Biomedical subjects
D Rocha
Publications and source records attributed to D Rocha.
Comparative mapping reveals multiple rearrangements between pig chromosome 6 and human 19q13.
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Animals↗
Prospecting for pig single nucleotide polymorphisms in the human genome: have we struck gold?
Gene-to-gene variation in the frequency of single nucleotide polymorphisms (SNPs) has been observed in humans, mice, rats, primates and pigs, but a relationship across species in this variation has not been described. Here, the frequency of porcine coding SNPs (cSNPs) identified by in silico methods, and the frequency of murine cSNPs, were compared with the frequency of human cSNPs across homologous genes. From 150,000 porcine expressed sequence tag (EST) sequences, a total of 452 SNP-containing sequence clusters were found, totalling 1394 putative SNPs. All the clustered porcine EST annotations and SNP data have been made publicly available at http://sputnik.btk.fi/project?name=swine. Human and murine cSNPs were identified from dbSNP and were characterized as either validated or total number of cSNPs (validated plus non-validated) for comparison purposes. The correlation between in silico pig cSNP and validated human cSNP densities was found to be 0.77 (p < 0.00001) for a set of 25 homologous genes, while a correlation of 0.48 (p < 0.0005) was found for a primarily random sample of 50 homologous human and mouse genes. This is the first evidence of conserved gene-to-gene variability in cSNP frequency across species and indicates that site-directed screening of porcine genes that are homologous to cSNP-rich human genes may rapidly advance cSNP discovery in pigs.
Animals↗
Single nucleotide polymorphism identification, linkage and radiation hybrid mapping of the porcine pituitary adenylate cyclase-activating polypeptide type I receptor gene to chromosome 18.
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with diverse biological actions. Type I PACAP receptors (PACAPR) are specific for PACAP, whereas type II and III PACAPRs are less restricted. To localize and analyse the variation of this gene, a 559-bp long intronic fragment of the porcine PACAPR gene was amplified by polymerase chain reaction and sequenced in samples from five different pig breeds. One single nucleotide polymorphism was identified and its allele frequency was determined in all five breeds. Linkage analysis in a Berkshire x Yorkshire reference family placed the PACAPR gene on chromosome 18, between SW787 and S0062 (SW787- 8.1 cM -PACAPR- 3.0 cM -S0062). Radiation hybrid mapping confirmed that the PACAPR gene was linked to SW1682 on chromosome 18 (28.8 cR(3000); LOD = 10.4).
Animals↗
Characterization of the porcine FSCN3 gene: cDNA cloning, genomic structure, mapping and polymorphisms.
Fascin 3 (FSCN3)is a testis-specific actin-bundling protein involved in spermatid development. Here we describe the molecular characterisation of the porcine FSCN3 gene. The 1,800-bp cDNA sequence contains a 1,497-bp open reading frame encoding a protein of 498 amino acids with a calculated molecular mass of 56.2 kDa and an isoelectric point of 6.82. The porcine FSCN3 protein shares high identity with other mammalian FSCN3. The FSCN3 gene contains seven exons, spans approximately 9 kb, and maps to pig chromosome 18. We also identified 24 DNA polymorphisms.
Amino Acid Sequence↗
Mapping of the porcine serine carboxypeptidase vitellogenic-like gene (CPVL) to chromosome 18.
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Animals↗
Confirmation of the mapping of the porcine calumenin gene (CALU ) to chromosome 18.
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Animals↗
Analysis of public single nucleotide polymorphisms in commercial pig populations.
More than 5500 pig single nucleotide polymorphisms (SNPs) were recently identified and deposited in the public domain. To test the usefulness of these public SNPs, 109 SNPs were analysed for polymorphism within six commercial pig populations. A functional polymerase chain reaction (PCR) assay was obtained for 103 SNPs and it was possible to validate c. 59% by PCR-restriction fragment length polymorphism. Furthermore, polymorphism was found using a relatively limited number of genomic DNA samples, indicating that these polymorphisms are segregating at a useful frequency in these populations. The high percentage of validated markers demonstrates the utility of these public pig SNPs to identify loci responsible for economically important traits in commercial pig populations.
Animals↗
The effect of RU486 on the gene expression profile in an endometrial explant model.
Administration of RU486 in vivo during the receptive phase rapidly renders the endometrium non-receptive to the implanting embryo. In order to identify key pathways responsible for endometrial receptivity we have used cDNA arrays to monitor gene expression changes in short-term endometrial explants in response to RU486. Endometrial biopsies from five normal fertile women at mid-secretory phase were cultured in the presence of estradiol and progesterone with or without RU486 for 12 h. cDNA arrays were produced containing approximately 1000 sequence-verified clones which included genes known to be important in angiogenesis, apoptosis, cell signalling, extracellular matrix remodelling and cell cycle regulation. cDNA probes from the paired endometrial samples were hybridized to the arrays and hybridization signals were quantified. A total of 12 genes displayed significant changes in expression; six were up-regulated and six down-regulated following RU486 treatment. For five of these genes this is the first report suggesting that they are regulated by steroids in the endometrium. JAK1 and JNK1 were two of the genes shown by the arrays to be down-regulated in RU486-treated endometrial explants. This was confirmed by real time RT-PCR. JAK1 immunoreactivity was localized to both glandular epithelium and the stroma of normal endometrium and staining was much stronger in the luteal phase of the cycle. These results show that components of two important signalling pathways in endometrium-the JAK/STAT pathway, and the JNK pathway-are altered by RU486. Genes whose expression is controlled by these pathways are likely to be involved in the mechanism by which steroids render the endometrium receptive to the implanting embryo.
Adult↗
Insights into the molecular basis of male infertility.
The study of the genetic basis of human male infertility is complicated by genetic heterogeneity and because linkage analysis studies are difficult. The study has been limited so far to the analysis of genes located on the Y chromosome. Several genes and gene families have been discovered and mutation analysis of these candidate genes in infertile patients is ongoing. In recent years, several mouse models with impaired spermatogenesis or fertility have also been analysed, expanding our knowledge about the molecular basis of spermatogenesis and male fertility.
Animals↗
Differential gene expression in CD3epsilon- and RAG1-deficient thymuses: definition of a set of genes potentially involved in thymocyte maturation.
A set of 3000 mouse thymus cDNAs was analyzed by extensive measurement of expression using complex-probe hybridization of DNA arrays ("quantitative differential screening"). The complex probes were initially prepared using total thymus RNA isolated from C57BL/6 wild-type (WT), CD3epsilon- and RAG1-deficient mice. Over 100 clones displaying over- or under-expression by at least a factor of two between WT and knockout (KO) thymuses were further analyzed by measuring hybridization signatures with probes from a wide range of KO thymuses, cell types, organs, and embryonic thymuses. A restricted set of clones was selected by virtue of their expression spectra (modulation in KO thymuses and thymocytes, lymphoid cell specificity, and differential expression during embryonic thymus development), sequenced at one extremity, and compared to sequences in databases. Clones corresponding to previously identified genes (e.g., Tcrbeta, Tcf1 or CD25) showed expression patterns that were consistent with existing data. Ten distinct clones corresponding to new genes were subjected to further study: Northern blot hybridization, in situ hybridization on thymus sections, and partial or complete mRNA sequence determination. Among these genes, we report a new serine peptidase highly expressed in cortical epithelial cells that we have named thymus-specific serine peptidase (TSSP), and an acidic protein expressed in thymocytes and of unknown function that we have named thymus-expressed acidic protein (TEAP). This approach identifies new molecules likely to be involved in thymocyte differentiation and function.
Alternative Splicing↗
Mitochondrial DNA variation of Triatoma infestans populations and its implication on the specific status of T. melanosoma.
DNA sequence comparison of 412 base-pairs fragments of the mitochondrial cytochrome B gene was used to infer the genetic structure of nine geographical Triatoma infestans populations and their phylogenetic relationship with T. melanosoma and T. brasiliensis. T. infestans and T. melanosoma were compared by morphometry, allozyme and cytogenetic analyses, as well as subjected to reciprocal crosses, in order to clarify the taxonomic status of the latter. No differences were found to distinguish the two species and the crosses between them yielded progeny. T. infestans populations presented four haplotypes that could be separated in two clusters: one formed by the samples from Bolivia (Andes and Chaco) and the other formed by samples from Argentina and Brazil. Silvatic and domestic T. infestans populations from Bolivia (Andes) were genetically identical.
Animals↗
Molecular cloning, sequencing and expression of the mRNA encoding human Cdx1 and Cdx2 homeobox. Down-regulation of Cdx1 and Cdx2 mRNA expression during colorectal carcinogenesis.
Defining the molecular mechanisms involved in cancer formation and progression is still a major challenge in colorectal-cancer research. Our strategy was to characterize genes whose expression is altered during colorectal carcinogenesis. To this end, the phenotype of a colorectal tumour was previously established by partial sequencing of a large number of its transcripts and the genes of interest were selected by differential screening on high-density filters with mRNA of colorectal cancer and normal adjacent mucosa. Fifty-one clones were found over-expressed and 23 were underexpressed in the colorectal-cancer tissues of the 5 analyzed patients. Among the latter, clones 6G2 and 32D6 were found of particular interest, since they had significant homology with several homeodomain-containing genes. The highest degree of similarity was with the murine Cdx1 for 6G2, and with the murine Cdx2 and hamster Cdx3 for 32D6. Using a RT-PCR approach, complete sequence of both types of homeobox-containing cDNA was obtained. The amino-acid sequence of the human Cdx1 is 85% identical to the mouse protein, and human Cdx2 has 94% identity with the mouse Cdx2 and hamster Cdx3. Tissue-distribution analysis of Cdx1 and Cdx2 mRNA showed that both transcripts were specifically expressed in small intestine, in colon and rectum. Twelve tissue samples from colorectal adenocarcinomas and the corresponding normal mucosa were analyzed by Northern blot. Expression of the 2 types of mRNA was either reduced or absent in 10 of them. Several colon-cancer cell lines were also analyzed. Cdx2 mRNA was absent from LS174T cells and Cdx1 mRNA was absent in PF11, TC7 and SW480 cells; none was detected in HT29 cells. It was concluded that decrease in human Cdx1 and/or Cdx2 expression is associated with colorectal tumorigenesis.
Adenocarcinoma↗
Differential mRNA expression in untreated and TNF-alpha elicited murine dendritic cells precursors.
We have compared the pattern of gene expression in long term cultured precursor dendritic cells (DC), either untreated (immature) or cultured for two days in the presence of recombinant murine (rm)-TNF alpha (mature). The hybridization signature of complex cDNA probes prepared from total RNA extracted from immature and mature DC were analyzed using a mouse thymic cDNA library, gridded on high density filters. For each clone spotted on the filters, we have measured using an imaging plate device the hybridization signals of the complex probe obtained from immature or mature DC. Comparative analysis of these values allowed us to identify differentially expressed gene products. Our goal is to identify a new set of genes induced or repressed during DC maturation elicited by rmTNF alpha treatment.
Animals↗
Modulation of mRNA levels in the presence of thymocytes and genome mapping for a set of genes expressed in mouse thymic epithelial cells.
Modulation of gene expression in mouse thymic epithelium upon culture in the presence of thymocytes (coculture) was studied by comparison of hybridization signatures on a set of nearly 5000 mouse thymus cDNA clones. Forty-nine differentially expressed clones (usually down-regulated in coculture) were characterized by tag sequencing. Many of them corresponded to entities that had not been described previously in the mouse, and were further characterized by genome mapping. This set of genes appears to be involved in growth regulation and differentiation within the thymus.
Animals↗
From hybridization image to numerical values: a practical, high throughput quantification system for high density filter hybridizations.
Hybridization to sets of bacterial colonies or PCR products arrayed on high density filters is used in a number of experimental schemes. In many cases it is desirable to collect quantitative information ('hybridization signatures') rather than indications on 'positive' and 'negative' colonies. We present a practical system, based on an imaging plate analyser and a customized version of commercial software, that makes such quantification feasible, and define its performance in terms of reproducibility and linearity. The system is far superior to methods based on autoradiography and should be useful in many projects that involve the increasingly popular high density filter format.
Animals↗
Differential gene expression in the murine thymus assayed by quantitative hybridization of arrayed cDNA clones.
High-throughput measurement of hybridization signatures obtained using complex probes prepared from poly(A)+ RNA and high-density cDNA colony filters is described. The performance of the system, elimination of artifacts, and verification of the validity of the data are discussed. cDNAs corresponding to sequences present at levels of approximately 0.01% in the complex probe can be detected. Good correlation is observed between expression profiles determined by this method and by Northern blotting. The method is applied to a preliminary investigation of differential expression in three cell types present in the murine thymus.
Animals↗
Overexpression of YAP2, coding for a new yAP protein, and YAP1 in Saccharomyces cerevisiae alleviates growth inhibition caused by 1,10-phenanthroline.
A new member of the yeast yAP family, designated YAP2, has been isolated and characterized. The protein displays a high homology with the DNA-binding domain of yAP-1, which contains a basic DNA-binding domain and leucine zipper motif and binds in vitro to the same cis-element. Growth arrest of yeast caused by low concentrations of 1,10-phenanthroline, resulting in zinc and/or iron deprivation, is overcome by over-expression of YAP1 or YAP2. In fact, yeast cells over-expressing YAP1 or YAP2 display pleiotropic drug resistance. On the other hand, a yap2 null mutant has an increased thermotolerance under starvation conditions caused by 1,10-phenanthroline. The latter mutant might become an excellent tool in the study of pathways leading toward thermotolerance acquisition.
Amino Acid Sequence↗