ABSTRACT
Haemophilus parasuis is the causative agent of polyserositis in pigs, a mostly fatal disease on the rise especially in early-weaned pigs and in pig herds with a high-health status. The mechanisms by which H. parasuis propagates through the body and colonizes the serous membranes are unknown. We have used an H. parasuis microarray to identify virulence genes involved in host adaptation. H. parasuis gene expression was analysed under in vitro growth conditions mimicking the environmental conditions encountered during an infection. These included iron-limitation, acidic and temperature stress and growth under microaerobic conditions. A kinetic impression of the gene regulation was obtained by analysing the transcription 10, 30 and 60 min after induction of the altered growth conditions. A total of 75 regulated H. parasuis genes were identified, most of which coded for transporters of iron and sugar metabolites, metabolic enzymes, DNA metabolism and hypothetical proteins with unknown functions. Furthermore, H. parasuis genes were identified that have homology to known virulence factors in other pathogenic bacteria. Homologues of some of the identified H. parasuis genes are known to be expressed during natural and experimental infections in pathogens of the Pasteurellaceae family.
Subject(s)
Bacterial Proteins/genetics , Environment , Gene Expression Profiling , Gene Expression Regulation, Bacterial/genetics , Haemophilus parasuis/growth & development , Haemophilus parasuis/genetics , Oligonucleotide Array Sequence Analysis , Virulence Factors/geneticsABSTRACT
BACKGROUND: Microarray profiling has the potential to illuminate the molecular processes that govern the phenotypic characteristics of porcine skeletal muscles, such as hypertrophy or atrophy, and the expression of specific fibre types. This information is not only important for understanding basic muscle biology but also provides underpinning knowledge for enhancing the efficiency of livestock production. RESULTS: We report on the de novo development of a composite skeletal muscle cDNA microarray, comprising 5500 clones from two developmentally distinct cDNA libraries (longissimus dorsi of a 50-day porcine foetus and the gastrocnemius of a 3-day-old pig). Clones selected for the microarray assembly were of low to moderate abundance, as indicated by colony hybridisation. We profiled the differential expression of genes between the psoas (red muscle) and the longissimus dorsi (white muscle), by co-hybridisation of Cy3 and Cy5 labelled cDNA derived from these two muscles. Results from seven microarray slides (replicates) correctly identified genes that were expected to be differentially expressed, as well as a number of novel candidate regulatory genes. Quantitative real-time RT-PCR on selected genes was used to confirm the results from the microarray. CONCLUSION: We have developed a porcine skeletal muscle cDNA microarray and have identified a number of candidate genes that could be involved in muscle phenotype determination, including several members of the casein kinase 2 signalling pathway.
Subject(s)
DNA, Complementary/genetics , Gene Expression Profiling/methods , Muscle, Skeletal/chemistry , Muscle, Skeletal/metabolism , Oligonucleotide Array Sequence Analysis/methods , Swine/genetics , Transcription, Genetic/genetics , Animals , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Casein Kinase II , Cloning, Molecular/methods , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Gene Expression Regulation/genetics , Genes, Tumor Suppressor , Muscle Fibers, Fast-Twitch/chemistry , Muscle Fibers, Fast-Twitch/metabolism , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Phenotype , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/genetics , Psoas Muscles/chemistry , Psoas Muscles/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Tumor Suppressor Proteins/biosynthesis , Tumor Suppressor Proteins/geneticsABSTRACT
Among genes involved in sex determination and differentiation, DMRT1 is the only one characterized to date containing a domain (the DM domain) that is conserved between phyla. To study DMRT1 transcriptional regulation within mammalian phyla, we generated transgenic mice that express green fluorescent protein (GFP) or Cre-recombinase (Cre) under the control of 2.6 kb of pig DMRT1 5' flanking sequences (pDMRT1p-GFP and pDMRT1p-Cre, respectively). Within the pDMRT1p-GFP positive mice, GFP expression was observed in the XY genital ridge by embryonic day 11.5 (e11.5) and remained detectable during testis embryonic development to birth. GFP expression was restricted within testis cords as soon as cords were detectable. No fluorescence was observed in developing ovaries, although more sensitive RT-PCR analysis revealed transgene expression in embryonic ovaries from e13.5 to e15.5. RT-PCR performed on fluorescent activated cell sorter (FACS)-purified GFP cells from e14.5, e17.5, and e19.5 developing testis showed that GFP expression was restricted to cells expressing the endogenous mouse Dmrt1. GFP cells also expressed Mis and Oct4, showing that the transgene is expressed in both Sertoli cell and germ cell compartments. In postnatal testis, transgene expression was detectable by GFP fluorescence from P0 to P21 in mice heterozygous for the transgene and through adulthood in mice homozygous for the transgene. In pDMRT1p-Cre positive mice, Cre expression was detected within the genital ridges of both XY and XX embryos. We conclude that DMRT1 regulatory mechanisms during sexual differentiation are functionally conserved across mammalian evolution. The transgenic mouse lines described should provide useful marker systems for studies involving Dmrt1 gene expression during sex differentiation.
Subject(s)
Gene Expression Regulation, Developmental , Regulatory Sequences, Nucleic Acid/genetics , Sex Differentiation/genetics , Testis/embryology , Testis/metabolism , Transcription Factors/genetics , 5' Flanking Region/genetics , Animals , Base Sequence , Cloning, Molecular , Conserved Sequence/genetics , Humans , Male , Mice , Mice, Transgenic , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Swine/genetics , Testis/cytology , Transgenes/geneticsABSTRACT
An investigation into the role of CD45 isoforms in T cell antigen receptor signal transduction was carried out by transfecting CD45-negative CD4(+)CD8(+) HPB-ALL T cells with the CD45R0, CD45RBC, and CD45RABC isoforms. Fluorescence resonance energy transfer analysis showed that the CD45R0 isoform, but not the CD45RBC or CD45RABC isoforms, was found as homodimers and also preferentially associated with CD4 and CD8 at the cell-surface. A comparison was therefore made of T cell antigen receptor signaling between sub-clones expressing either CD45R0 or CD45RBC. Under basal conditions CD4-associated p56(lck) tyrosine kinase activity and cellular protein tyrosine phosphorylation levels were higher in the CD45R0(+) than in the CD45RBC(+) sub-clones. Upon CD3-CD4 ligation, TCR-zeta phosphorylation, ZAP-70 recruitment to the p21/p23 TCR-zeta phosphoisomers, ZAP-70 phosphorylation, as well as p56(lck), c-Cbl and Slp-76 phosphorylation, were all markedly increased in CD45R0(+) compared with CD45RBC(+) cells. T cell antigen receptor (TCR) stimulation alone also promoted c-Cbl phosphorylation in CD45R0(+) but not in CD45RBC(+) cells. Our results are consistent with a model in which association of CD45R0 with CD4 generates a more active pool of CD4-associated p56(lck) kinase molecules. Upon CD3-CD4 co-ligation, the active p56(lck) increases the intensity of T cell antigen receptor signal transduction coupling by promoting TCR-zeta chain phosphorylation and ZAP-70 recruitment.
