Association analysis of polymorphisms of porcine LMP2 and LMP7 genes with haematological traits

Low molecular weight polypeptides 2 (LMP2) and low molecular weight polypeptides 7 (LMP7) are located within the major histocompatibility complex and have been associated with autoimmune disease. In this study, polymorphisms of porcine LMP2 and LMP7 genes were analyzed by PCR-SSCP and DNA sequencing methods. Four SNPs (DQ659151:g.2115T>C; DQ659151:g.4343A>G; DQ872631:g.1232C>G; DQ872631:g.2847C>T) were identified. Four SNPs of genes were analyzed for association with 22 haematological traits in Large White (n = 195), Landrace (n = 84) and Songliao Black (n = 86) pig population. Of all the 22 traits, seven were significant associated with the SNPs of LMP2/LMP7 gene (P < 0.05). They included white blood cell count (WBC) (P = 0.028), neutrophilic granulocyte count (GRAN) (P = 0.037), monocytes percentage (MO%) (P = 0.015), red blood cell (RBC) (P = 0.004), red blood cell volume distribution width (RDW) (P = 0.004), mean platelet volume (MPV) (P = 0.016) and CD4(+)CD8(+)% (P = 0.045). These results suggest LMP2/LMP7 gene should be regarded as molecular marker to estimate animal's immune status for their effects on hematological traits.     

Molecular characterization, expression profiles of the porcine SDC2 and HSPG2 genes and their association with hematologic parameters

Heparan sulfate proteoglycans (HSPGs) located at the cell surface and in the extracellular matrix of most animal tissues are proteoglycan coreceptors that carry heparan sulfate chains, and play a vital role in infections of many diseases. HSPGs are classified as glypican, syndecan, perlecan and agrin according to different core proteins. Syndecan-2 (SDC2) is one of the four coding genes of syndecan, while heparan sulfate proteoglycan 2 (HSPG2) is for perlecan. In this study, we cloned the cDNA of porcine SDC2 and analyzed its genomic structure. The porcine SDC2 and HSPG2 were mapped to SSC4p12-13 and SSC6q24-25 by the SCHP panel respectively, further IMpRH panel analysis showed that they were most closely linked to the marker SWR362 and SW709. One special domain named the 4.1 m domain (putative band 4.1 homologues’ binding motif) was found in the prediction amino acid sequence of porcine SDC2. RT-PCR showed that both of porcine SDC2 and HSPG2 were expressed widely in detected tissues: heart, liver, spleen, lung, kidney, stomach, muscle, fat and lymph. Upon stimulation in healthy Tongcheng piglets with PRRSV, SDC2 mRNA did not induce a prominent change in the PAMs, while HSPG2 mRNA displayed a dramatic decline. In addition, synonymous mutation g.32A>G of the SDC2 gene was detected and confirmed to be significantly associated with hematocrit, mean corpuscular volume and hemoglobin concentration in the peripheral blood (p < 0.05). A single nucleotide polymorphism g.83.A>G was found in the HSPG2 gene and the association analysis showed that it was significantly associated with mean corpuscular hemoglobin (p < 0.05). Our results confirmed the relation of porcine SDC2 and HSPG2 to the immunity in pigs, and these two genes could be used as candidate genes for improving immune traits in industrial pig breeding.     

Molecular characterization of porcine hyaluronidase genes 1, 2, and 3 clustered on SSC13q21

Hyaluronidase genes (HYAL) encode hyaluronidase enzymes required for hyaluronan degradation. Both in humans and in mouse, clustered hyaluronidase genes have been identified. Here, the porcine hyaluronidase cluster consisting of genes HYAL1, HYAL2 and HYAL3 was characterized. The porcine cDNA sequences and proteins share homologies to human orthologs of 85 and 81% for HYAL1, 87 and 89% for HYAL2 and 86 and 83% for HYAL3, respectively. The porcine hyaluronidase proteins approximately share a 40% homology with each other. Furthermore, genes FUS1 and FUS2 were found within this cluster, which was assigned to SSC13q21. A total of seven SNPs were detected in the genes (four in HYAL1, two in HYAL2 and one in HYAL3). Three of the four SNPs in HYAL1 led to amino acid exchanges (C622G --> Asp24 to Glu; C633T --> Pro28 to Leu, and G1298T --> Ala250 to Ser). The amino acid replacements induce putative changes in the extended strand at Asp24, in the extended strand and the random coil at Pro28, and finally in the random coil and the alpha helix at Ala250. Frequency estimations for four SNPs located in genes HYAL1 and HYAL3 using animals (n = 295) of nine European and six Chinese pig breeds indicated several significant deviations. For example, there were no significant differences in allele frequencies between pigs representing breeds Hampshire and Jiangquhai at SNP C633T (HYAL1), but between Hampshire respectively Jiangquhai animals and Rongchang pigs. Analysis of the same breeds at SNP C588T (HYAL3) indicates significant differences between Hampshire and Jiangquhai respectively Rongchang, but not between Jiangquhai and Rongchang. The breed G?ttingen Minipig displayed significant differences concerning two SNPs with respect to the other European pig breeds tested. For all three hyaluronidase genes, N-glycosylation sites are typical. For HYAL2 the lysosomal character was proven. The catalytic site responsible for HAase activity is conserved in the three enzymes. Expression of hyaluronidases was determined by RT-PCR and quantitative PCR. Broad gene expression was observed in different tissues for the three genes, respectively.     

Molecular characterization, expression profiles, and association analysis with hematologic parameters of the porcine HPSE and HPSE2 genes

Heparan sulfate (HS), which consists of repeating disaccharide units, plays an essential role in inflammation and viral infections. Heparanase (encoded by the HPSE gene) can cleave the HS chains of heparan sulfate proteoglycans (HSPGs), which are known to be important participants in immune responses. HPSE2 (heparanase 2) is a homologous gene of HPSE. To investigate the functions of HS, which is the primary receptor of the porcine reproductive and respiratory syndrome virus (PRRSV), the two genes involved in the metabolic process of HS were studied. Here, we present a study of tissue expression profiles, polymorphisms of the HPSE and HPSE2 genes, and the changes of their mRNA levels in porcine alveolar macrophages (PAMs) induced by PRRSV. Both genes are preferentially expressed in porcine immune or immune-related organs under normal conditions, e.g., in the lung, spleen, and lymph node. Moreover, a synonymous mutation c.750A>G located in exon5 of the HPSE gene was detected, and was significantly associated with the white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (HGB), and hematocrit (HCT) in the peripheral blood (p?<?0.05). A single nucleotide polymorphism (SNP) c.2073A>G was found in the HPSE2 gene and association analysis showed that it was significantly associated with the WBC content in the blood (p?<?0.05). Upon stimulation in healthy piglets with PRRSV, the HPSE mRNA was obviously up-regulated, while the HPSE2 mRNA did not induce a prominent change in PAMs.     

Molecular cloning, expression, and characterization of endoglucanase genes from Fibrobacter succinogenes AR1.

A cosmid gene library was constructed in Escherichia coli from genomic DNA isolated from the ruminal anaerobe Fibrobacter succinogenes AR1. Clones were screened on carboxymethyl cellulose, and 8 colonies that produced large clearing zones and 25 colonies that produced small clearing zones were identified. Southern blot hybridization revealed the existence of at least three separate genes encoding cellulase activity. pRC093, which is representative of cosmid clones that produce large clearing zones, was subcloned in pGem-1, and the resulting hybrid pRCEH directed synthesis of endoglucanase activity localized on a 2.1-kb EcoRI-HindIII insert. Activity was expressed from this fragment when it was cloned in both orientations in pGem-1 and pGem-2, indicating that F. succinogenes promoters functioned successfully in E. coli. A high level of endoglucanase activity was detected on acid-swollen cellulose, ball-milled cellulose, and carboxymethyl cellulose; and a moderate level was detected on filter paper, Avicel, lichenan, and xylan. Most activity (80%) was localized in the periplasm of E. coli, with low but significant levels (16%) being detected in the extracellular medium. The periplasmic endoglucanase had an estimated molecular weight of 46,500, had an optimum temperature of 39 degrees C, and exhibited activity over a broad pH range, with a maximum at pH 5.0.     

Cloning,chromosome mapping and expression pattern of porcine PLIN and M6PRBP1 genes

The PAT proteins, named after the three PLIN/ADRP/TIP47 (PAT) proteins, PLIN for perilipin, ADRP for adipose differentiation-related protein and TIP47 for tail-interacting protein of 47 kDa, now officially named M6PRBP1 for mannose-6-phosphate receptor binding protein 1, is a set of intracellular lipid droplet binding proteins. They are localized in the outer membrane monolayer enveloping lipid droplets and are involved in the metabolism of intracellular lipid. This work describes the cloning and sequencing of porcine PLIN and M6PRBP1 cDNAs, the chromosome mapping of these two genes, as well as the expression pattern of porcine PAT genes. Sequence analysis shows that the porcine PLIN cDNA contains an open reading frame of 1551 bp encoding 516 amino acids and that the porcine M6PRBP1 cDNA contains a coding region of 1320 bp encoding 439 amino acids. Comparison of PLIN and M6PRBP1 amino-acid sequences among various species reveals that porcine and bovine proteins are the most conserved. Porcine PLIN and M6PRBP1 genes have been mapped to pig chromosomes 7 and 2, respectively, by radiation hybrid analysis using the IMpRH panel. Expression analyses in pig showed a high expression of PLIN mRNA in adipose tissue, M6PRBP1 mRNA in small intestine, kidney and spleen and ADRP mRNA in adipose tissue, lung and spleen.     

Characterization and physical mapping of the porcine CDS1 and CDS2 genes

CDP-diacylglycerol synthase (CDS) catalyzes the conversion of phosphatidic acid to CDP-diacylglycerol, an important precursor for the synthesis of phosphatidylinositol, phosphatidylglycerol, and cardiolipin. We amplified and sequenced 2,053 bp of the pig CDS1 mRNA. The structure of the pig CDS1 gene was determined, being very similar to that of the human, rat, and mouse genes with respect size and organization of the 13 exons. In addition, we identified three polymorphic positions in exons 10 and 11. One of them, the A/C1006, was genotyped in samples belonging to Iberian, Landrace, Large White, Pietrain, and Meishan pig breeds. Expression of this gene was also analyzed by real-time polymerase chain reaction (PCR) in different tissues showing a high CDS1 expression in testis. Moreover, a 1240-bp fragment of the pig CDS2 mRNA was amplified and sequenced. Finally, the CDS1 and CDS2 genes were physically mapped to porcine chromosomes 8 and 17, respectively, by using the INRA, University of Minnesota porcine Radiation Hybrid panel (IMpRH).     

Molecular characterization of pdc2 and mapping of three pdc genes from rice

 The anaerobic fermentation pathway is thought to play an important role under flooding conditions. The pyruvate decarboxylase 2 (pdc2) gene that encodes the first enzyme of this pathway has been cloned and characterized from rice. This gene has an open reading frame that putatively encodes a 603 amino-acid-residue protein with a molecular mass of 64 kDa. pdc2 has five introns dispersed throughout the coding region, which is also true for rice pdc1. Although the length of these introns in rice pdc2 are different from those in rice pdc1, they are located in exactly the same positions based on the deduced amino-acid sequences. The temporal and spatial expression patterns of pdc1 and pdc2 show that pdc2 is induced to a higher level during the early period (1.5–12 h) of anoxia than pdc1, which is induced more after longer time periods (24–72 h) of anoxia in both shoots and roots. The map positions of the three pdc genes have also been determined. Rice pdc1 is located on chromosome 5 between BCD454A and RZ67, pdc2 is located on chromosome 3 between RZ329 and RZ313, and pdc3 is mapped on chromosome 7 distal to RG351. Received: 19 May 1998 / Accepted: 29 September 1998     

The new kallikrein-like gene, KLK-L2. Molecular characterization, mapping, tissue expression, and hormonal regulation

Since in rodents the kallikreins are represented by a large multi-gene family, the restriction of this family in humans to three genes is somewhat surprising. In an effort to identify new human kallikrein genes, we examined a genomic area of about 300 kilobases on chromosome 19q13.3-q13.4, a region that contains most of the currently known kallikreins. By using the positional candidate approach, we were able to identify a new gene named KLK-L2 (for kallikrein- like gene 2). Screening of human EST libraries allowed us to delineate the full genomic and cDNA structure of the new gene. KLK-L2 consists of 5 coding exons and 4 introns and has significant similarities to other members of the kallikrein multi-gene family. Homology studies suggest that the protein is likely secreted. KLK-L2 is expressed mainly in breast, brain, and testis and to a lesser extent in many other tissues. KLK-L2 is up-regulated by estrogens and progestins in the breast cancer cell line BT-474.     

Molecular cloning, expression, and characterization of endoglucanase genes from Fibrobacter succinogenes AR1

A cosmid gene library was constructed in Escherichia coli from genomic DNA isolated from the ruminal anaerobe Fibrobacter succinogenes AR1. Clones were screened on carboxymethyl cellulose, and 8 colonies that produced large clearing zones and 25 colonies that produced small clearing zones were identified. Southern blot hybridization revealed the existence of at least three separate genes encoding cellulase activity. pRC093, which is representative of cosmid clones that produce large clearing zones, was subcloned in pGem-1, and the resulting hybrid pRCEH directed synthesis of endoglucanase activity localized on a 2.1-kb EcoRI-HindIII insert. Activity was expressed from this fragment when it was cloned in both orientations in pGem-1 and pGem-2, indicating that F. succinogenes promoters functioned successfully in E. coli. A high level of endoglucanase activity was detected on acid-swollen cellulose, ball-milled cellulose, and carboxymethyl cellulose; and a moderate level was detected on filter paper, Avicel, lichenan, and xylan. Most activity (80%) was localized in the periplasm of E. coli, with low but significant levels (16%) being detected in the extracellular medium. The periplasmic endoglucanase had an estimated molecular weight of 46,500, had an optimum temperature of 39 degrees C, and exhibited activity over a broad pH range, with a maximum at pH 5.0.     

Molecular characterization, polymorphism and association of porcine MYST2 gene

MYST histone acetyltransferase 2 (MYST2) is an important reproduction related gene. In this study, we cloned the full-length cDNA sequence of porcine MYST2 gene through the rapid amplification of cDNA ends method. The porcine MYST2 gene encodes a protein of 611 amino acids which shares high homology with the MYST2 of six species: cattle (99%), rabbit (99%), human (99%), rat (99%), mouse (99%) and chicken (98%).The open reading frame of this gene is structured in 15 exons and 14 introns as revealed by computer-assisted analysis. The phylogenetic analysis revealed that the porcine MYST2 gene has a closer genetic distance with the MYST2 gene of cattle. PCR-RFLP was established to detect the GU373686:c.2872G > A substitution of porcine MYST2 gene mRNA and association of this mutation with litter size traits was assessed in Large White (n = 200) and Landrace (n = 200) pig populations. Results demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White sows and Landrace sows. These data serve as a foundation for further insight into this porcine gene.     

T cells lacking immunoproteasome subunits MECL-1 and LMP7 hyperproliferate in response to polyclonal mitogens

Immunoproteasomes comprise a specialized subset of proteasomes that is defined by the presence of three catalytic immunosubunits: LMP2, MECL-1 (LMP10), and LMP7. Proteasomes in general serve many cellular functions through protein degradation, whereas the specific function of immunoproteasomes has been thought to be largely, if not exclusively, optimization of MHC class I Ag processing. In this report, we demonstrate that T cells from double knockout mice lacking two of the immunosubunits, MECL-1 and LMP7, hyperproliferate in vitro in response to various polyclonal mitogens. We observe hyperproliferation of both CD4(+) and CD8(+) T cell subsets and demonstrate accelerated cell cycling. We do not observe hyperproliferation of T cells lacking only one of these subunits, and thus hyperproliferation is independent of either reduced MHC class I expression in LMP7(-/-) mice or reduced CD8(+) T cell numbers in MECL-1(-/-) mice. We observe both of these latter two phenotypes in MECL-1/LMP7(-/-) mice, which indicates that they also are independent of each other. Finally, we provide evidence of in vivo T cell dysfunction by demonstrating increased numbers of central memory phenotype CD8(+) T cells in MECL-1/LMP7(-/-) mice. In summary, this novel phenotype of hyperproliferation of T cells lacking both MECL-1 and LMP7 implicates a specific role for immunoproteasomes in T cell proliferation that is not obviously connected to MHC class I Ag processing.