ABSTRACT
A large number of putative single nucleotide polymorphisms (SNPs) have been identified from the bovine genome-sequencing project. However, few of these have been validated and many will turn out to be sequencing artefacts or have low minor allele frequencies. In addition, there is little information available on SNPs within coding regions, which are likely to be responsible for phenotypic variation. Therefore, additional SNP discovery is necessary to identify and validate polymorphisms both in specific genes and genome-wide. Sequence-tagged sites within 286 genes were resequenced from a panel of animals representing a wide range of European cattle breeds. For 80 genes, no polymorphisms were identified, and 672 putative SNPs were identified within 206 genes. Fifteen European cattle breeds (436 individuals plus available parents) were genotyped with these putative SNPs, and 389 SNPs were confirmed to have minor allele frequencies above 10%. The genes containing SNPs were localized on chromosomes by radiation hybrid mapping and on the bovine genome sequence by Blast. Flanking microsatellite loci were identified, to facilitate the alignment of the genes containing the SNPs in relation to mapped quantitative trait loci. Of the 672 putative SNPs discovered in this work, only 11 were found among the validated SNPs and 100 were found among the approximately 2.3 million putative SNPs currently in dbSNP. The genes studied in this work could be considered as candidates for traits associated with beef production and the SNPs reported will help to assess the role of the genes in the genetic control of muscle development and meat quality. The allele frequency data presented allows the general utility of the SNPs to be assessed.
Subject(s)
Body Composition/genetics , Cattle/genetics , Polymorphism, Single Nucleotide , Animals , Cattle/anatomy & histology , Cattle/growth & development , Chromosomes, Mammalian , Gene Frequency , Phenotype , Radiation Hybrid Mapping , Sequence Analysis, DNAABSTRACT
DNA damage and other forms of stress are believed to be important factors in reducing the efficiency of in vitro embryo transfer techniques in farm animals. The expression of mRNAs from stress-responsive genes such as gadd153 (CHOP-10, ddit3) may provide a means of assessing the quality of embryos produced in vitro. Treatment of bovine granulosa cell cultures with the DNA-damaging agents, methyl methane-sulphonate (MMS) or sodium arsenite, induced the expression of an mRNA, which hybridized with the hamster gadd153 cDNA. Part of the corresponding bovine cDNA was amplified by nested polymerase chain reaction (PCR), cloned, and sequenced. Using a sensitive reverse transcriptase-PCR assay we have investigated the expression of gadd153 and beta-actin in blastocyst-stage bovine embryos treated with MMS or sodium arsenite. Both agents produced an increase in the ratio of gadd153 mRNA relative to beta-actin. These results show that there are changes in gene expression in blastocyst-stage bovine embryos in response to genotoxic stress, suggesting that an increase in gadd153 mRNA is a useful marker of DNA damage and metabolic stress in preimplantation embryos.
Subject(s)
Blastocyst/metabolism , CCAAT-Enhancer-Binding Proteins/genetics , DNA Damage/drug effects , Gene Expression/drug effects , Mutagens/pharmacology , RNA, Messenger/analysis , Transcription Factors/genetics , Actins/genetics , Animals , Arsenites/pharmacology , Base Sequence , Cattle , Cells, Cultured , Cricetinae , DNA, Complementary/chemistry , Female , Granulosa Cells/drug effects , Granulosa Cells/metabolism , Methyl Methanesulfonate/pharmacology , Molecular Sequence Data , Oxidative Stress , Reverse Transcriptase Polymerase Chain Reaction , Sodium Compounds/pharmacology , Transcription Factor CHOPABSTRACT
CHOP-10 (also known as gadd153 or Ddit3) is one of the genes overexpressed by mammalian cells exposed to cytotoxic agents or to nutrient stress. The response of this gene to stress was studied in the mouse blastocyst and in F9 embryonal carcinoma cells. When mouse blastocysts were exposed to the alkylating agent MMS, the metabolic inhibitor sodium arsenite or an inhibitor of protein glycosylation tunicamycin, levels of the CHOP-10 mRNA were increased by two- to threefold relative to the mRNA for beta-actin. There was no increase in gene expression when blastocysts were treated with the inhibitor of nucleotide synthesis PALA. These results show that the response of CHOP-10 is dependent on the type of stress applied to the embryo. When F9 embryonal carcinoma cells were treated with MMS or sodium arsenite, CHOP-10 expression was induced by fourfold within 4 hr of treatment. The induction following tunicamycin treatment was slower requiring at least 24 hr. The response to tunicamycin was greater in cells treated with retinoic acid to induce differentiation. The results suggest that there is a link between the extent of glycoprotein synthesis and the sensitivity of CHOP-10 to tunicamycin. The inhibitor PALA did not change CHOP-10 expression in the presence or absence of retinoic acid. In F9 cells an increase in the expression of CHOP-10 was followed by cell death due to apoptosis. The overexpression of CHOP-10 may be a marker for one of the pathways that lead to apoptosis in the blastocyst. These results suggest that there is more than one control system regulating growth arrest in the blastocyst and the fetal outcome may differ depending on the type of stress encountered in culture.
Subject(s)
Blastocyst/metabolism , CCAAT-Enhancer-Binding Proteins , DNA-Binding Proteins/genetics , Stress, Physiological/physiopathology , Transcription Factors/genetics , Actins/genetics , Animals , Apoptosis , Arsenates/pharmacology , Aspartic Acid/analogs & derivatives , Aspartic Acid/pharmacology , Blastocyst/drug effects , Blotting, Northern , DNA-Binding Proteins/drug effects , DNA-Binding Proteins/metabolism , Enzyme Inhibitors/pharmacology , Female , Methyl Methanesulfonate/pharmacology , Mice , Mice, Inbred C57BL , Phosphonoacetic Acid/analogs & derivatives , Phosphonoacetic Acid/pharmacology , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Stress, Physiological/chemically induced , Transcription Factor CHOP , Transcription Factors/drug effects , Transcription Factors/metabolism , Tumor Cells, Cultured , Tunicamycin/pharmacology , Up-RegulationABSTRACT
Mammalian cells mount an active response to nutrient limitation by overexpressing the growth arrest specific (GAS) and the growth arrest and DNA damage (GADD) genes. During embryogenesis in rats, there are quantitative and temporal differences in GAS and GADD gene expression during the development of the placenta, heart and kidney. Genes associated with the inhibition of DNA synthesis (p53 and GAS1) were predominantly expressed during the early stages of development, whereas those genes associated with inhibition of protein synthesis [GADD153 (also known as CHOP-10 or Ddit3) and C/EBP-beta] were more highly expressed during the later stages. The GADD45 gene was expressed throughout development. There were distinct periods of GAS3 and GAS6 gene expression during the development of the placenta, heart and kidneys, which is consistent with the proposed roles of these genes in cell interactions. These results show that there is a change in the expression of genes associated with the negative regulation of growth as the fetus develops.
Subject(s)
Apoptosis/genetics , DNA Damage/genetics , Embryonic and Fetal Development/genetics , Fetal Growth Retardation/genetics , Genes, p53 , Heart/embryology , Kidney/embryology , Placenta/embryology , Analysis of Variance , Animals , Blotting, Northern , Female , Pregnancy , RNA/genetics , RatsABSTRACT
Although institutional outbreaks of pneumococcal infection have been reported, secondary cases of pneumococcal meningitis do not seem to have been described. We report two cases of pneumococcal meningitis involving the same serotype occurring in individuals with direct contact.
