Expression patterns and promoter activity analysis of UGP2 in pigs.

The function of the UDP-glucose pyrophosphorylase 2 gene (UGP2) in pig is not clear. In the present study, we used RNA isolated from Large White pigs and Chinese indigenous MeiShan pigs to examine the temporal coordination of changes in gene expression within muscle tissues. We cloned both the complete genomic DNA sequence and 2077-bp 5ꞌ-flanking sequence of porcine UGP2, to determine the genomic sequence. Real-time RT-PCR revealed that UGP2 was highly expressed in liver and skeletal muscle of MeiShan pigs. Among different types of muscle fibers, the UGP2 had the highest expression in both soleus muscle and longissimus dorsi in Large White pigs. In the progression of muscle fibers at different growth stages, UGP2 plays a role in the early days after birth in Large White pigs, while in MeiShan pigs it is important later. Furthermore, the 5ꞌ-flanking sequence we cloned exhibited the promoter activity of UGP2, and the sequence 588 bp upstream from the transcriptional site had the greatest activity.

Differential expression and effect of the porcine ANGPTL4 gene on intramuscular fat.

In a previous study, we investigated differences in gene expression in backfat between Meishan and Large White pigs and their F1 hybrids, Large White x Meishan, and Meishan x Large White pigs. One potential differentially expressed sequence tag from the mRNA differential display was a homolog of the human angiopoietin-like 4 (ANGPTL4) gene, which encodes a protein that is secreted by both liver and white adipose tissues and can inhibit lipoprotein lipase activity and stimulate white adipose tissue lipolysis. Here, ANGPTL4 mRNA was found to be upregulated in the backfat of Large White compared with that in the Meishan pigs and the F1 hybrids, Meishan x Large White and Large White x Meishan, whereas expression was lowest both in the longissimus dorsi and the heart, as shown by the tissue distribution profile. Only one mutation, a G/A transition located in the third intron, was found. The ANGPTL4 G/A polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) showed a significant effect on intramuscular fat (IMF), water moisture of the longissimus dorsi, meat marbling of the longissimus dorsi, and pH of the longissimus dorsi (P < 0.05). This site seemed to be significantly (P < 0.05) additive in its actions on IMF, water moisture, and pH, whereas it showed significant dominance in its action on meat marbling (P < 0.05). This locus can be potentially considered as a marker for IMF improvement.

Real-time reverse transcription-PCR expression profiling of porcine troponin I family in three different types of muscles during development.

In this study, the expression profiling of three troponin I isoforms (TNNI1, TNNI2 and TNNI3) was investigated in two pig breeds differing in muscularity (Yorkshire and Meishan) at six stages (fetal 60 days and postnatal 3, 35, 60, 120, and 180 days) and three types of muscles (longissimus dorsi muscle, LD; semitendinosus, ST; cardiac muscle, CM) using relative real-time quantitative PCR. Significant differences of troponin I expression in three muscles were found between Yorkshire and Meishan breeds at some stages. The expression peak of TNNI1 and TNNI2 in LD and ST was at postnatal 35 or 60 days in Yorkshire and at postnatal 120 or 180 days in Meishan pigs, while it occurred in CM at postnatal 3 days in two pig breeds. The relative expression values of TNNI1 and TNNI2 were significantly higher in LD than ST at most of stages after birth. The expression ratio of TNNI2 versus TNNI1 favoured TNNI2 expression in ST and LD, but on the contrary in CM. The expression peak of TNNI3 occurred at postnatal 60 and 120 days in Yorkshire and Meishan pigs, respectively. TNNI1 and TNNI3 were co-expressed in CM during the fetal and earlier stages after birth.

Molecular characterization, expression pattern and association analysis of the porcine BTG2 gene.

B-cell translocation gene 2 (BTG2), a member of the B-cell translocation gene family with anti-proliferative properties, have been characterized to be involved in cell growth, differentiation and survival. In this study, we cloned the full length sequences of cDNA and genomic DNA of BTG2 gene from the porcine skeletal muscle. Spatial expression analysis showed that the porcine BTG2 gene is expressed predominantly in muscle. Temporal expression analysis in longissimus dorsi muscle demonstrated that the expression of BTG2 gene has the highest expression at 60 days old in Large White while with a peak expression at 120 days old in Meishan. Temporal analysis also revealed that the expression of BTG2 gene is generally higher in Large White than in Meishan at all the developmental stages tested (65 days of conception and 3, 35, 60, 120, and 180 days of postnatal). A single nucleotide polymorphism (G417C) in the intron of BTG2 gene was then detected by PCR-RFLP in Large White × Meishan F2 resource population and association analysis suggested that this polymorphic site had significant association (P < 0.05) with the buttock fat thickness, fat percentage, lean muscle percentage, ratio of lean to fat and carcass length.

Identification of three novel SNPs and association with carcass traits in porcine TNNI1 and TNNI2.

In this study, two novel SNPs (EU743939:g.5174T>C in intron 4 and EU743939:g.8350C>A in intron 7) in TNNI1 and one SNP (EU696779:g.1167C>T in intron 3) in TNNI2 were identified by PCR-RFLP (PCR restriction fragment length polymorphism) using XbaI, MspI and SmaI restriction enzyme, respectively. The allele frequencies of three novel SNPs were determined in the genetically diverse pig breeds including ten Chinese indigenous pigs and three Western commercial pig breeds. Association analysis of the SNPs with the carcass traits were conducted in a Large White × Meishan F(2) pig population. The linkage of two SNPs (g.5174T>C and g.8350C>A) in TNNI1 gene had significant effect on fat percentage. Besides these, the g.5174T>C polymorphism was also significantly associated with skin percentage (P < 0.05), shoulder fat thickness (P < 0.05) and backfat thickness between sixth and seventh ribs (P < 0.05). The significant effects of g.1167C>T polymorphism in TNNI2 gene on fat percentage (P < 0.01), lean meat percentage (P < 0.05), lion eye area (P < 0.05), thorax-waist backfat thickness (P < 0.01) and average backfat thickness (P < 0.05) were also found.

Association of 3 polymorphisms in porcine troponin I genes (TNNI1 and TNNI2) with meat quality traits.

The contractile protein troponin I (TnI), a constituent protein of the troponin complex located on the thin filaments of striated muscle, is involved in inhibition of calcium-induced myosin ATPase activity (and thus contraction). TnI-slow (slow-twitch skeletal muscle isoform, named TNNI1) and TnI-fast (fast-twitch skeletal muscle isoform, named TNNI2) are muscle-fiber-type-specific proteins, and expression of their genes may affect the composition of muscle fiber, thereby influencing the meat quality traits. Thus, the TnI genes are potential candidate genes for traits related to meat quality in animals. Association of 2 SNPs (EU743939:g.5174T>C in intron 4, and EU743939:g.8350C>A in intron 7) of the TNNI1 gene and a SNP (EU696779:g.1167C>T in intron 3) of the TNNI2 gene with 11 meat quality traits were studied on 334 Large White x Meishan F(2) pigs. In the TNNI1 gene, g.5174T>C and g.8350C>A were found to be significantly associated with intramuscular fat content and meat color value of biceps femoris. The g.5174T>C also showed significant effects on meat color value and marbling score of longissimus dorsi, as well as pH of longissimus dorsi and semispinalis capitis. The g.1167C>T polymorphism in the TNNI2 gene affected significantly the pH of longissimus dorsi, meat color value of longissimus dorsi and semispinalis capitis, marbling score of longissimus dorsi, and intramuscular fat.

Genetic polymorphisms and preliminary association analysis with production traits of the porcine SLC27A4 gene.

Solute carrier family 27 (fatty acid transporter), member 4 (SLC27A4) is a fatty acyl-CoA synthetase producing very long chain fatty acid-CoA for lipid metabolic pathways, suggesting that the SLC27A4 gene is a potential candidate gene for traits related to fat deposition in animals. This study was conducted to sequence the genomic region from exon 6 to 12 of porcine SLC27A4 and detect polymorphisms by comparative sequencing. In silico mapping assigned SLC27A4 gene between gene COQ4 (coenzyme Q4 homolog) and URM1 (ubiquitin related modifier 1 homolog) on pig chromosome 1q24-q2.12 where significant QTL affecting backfat depth had previously been identified. Thirty six putative sites of variation were detected, of which 31 polymorphisms including 28 SNPs and 3 indels were located in the intronic region, and 5 in the exonic regions. The g.1777G>A (EU703769) in intron 8 was confirmed by PCR-RFLP using HpaII restriction enzyme and further genotyped in four Chinese native pig breeds (Meishan, Erhualian, Tongcheng and Qingping) and three western meat-type pig breeds (Duroc, Large White and Landrace). Allele G was exclusively present in Tongcheng and Qingping pigs and predominant in the other pig populations analyzed. Significant differences of backfat at rump, body weight at birth and average daily gain on weaning between the AG and GG genotype were observed in Landrace pig population (P < 0.05).

Molecular characterization and association with carcass traits of the porcine SLC39A7 gene.

In this study, the molecular characterization and potential association of SLC39A7 gene with carcass traits were investigated in pigs. The sequence of SLC39A7 cDNA was obtained by in silico cloning and RT-polymerase chain reaction (PCR). Two transcripts, variant 1 (2398 bp) and variant 2 (2088 bp), of the SLC39A7 gene were identified. Expression analysis of SLC39A7 in 10 different tissues by RT-PCR showed that variant 1 was ubiquitously expressed in all tissues analysed, but variant 2 was not found in fat tissue. The cDNA regions of variant 1 and 2 were organized in seven and eight exons respectively. A c.205G>A substitution in exon 3, which changes a codon for glycine into a codon for arginine, (p.Gly69Arg) and a c.1138-216T>C substitution in intron 6 were detected by PCR-HpaII-restriction fragment length polymorphisms (RFLP) and PCR-cofI-RFLP respectively. Significant differences were found in the allele frequencies of c.205G>A among six Chinese indigenous pig breeds and two commercial pig breeds. Linkage analysis showed that the c.205G>A polymorphism within the SLC39A7 gene was closely linked to the marker Sw1856 on pig chromosome 7 in a Large White x Meishan F(2) resource population. The QTL and association studies between polymorphisms of the SLC39A7 gene and carcass traits were carried out. Significant associations of the SLC39A7 polymorphisms with backfat thickness at thorax-waist (p < 0.05), average backfat thickness (p < 0.05) and leaf fat weight (p < 0.01) were found. Additional F-drop test or marker assisted association analyses also supported the association of the mutation in SLC39A7 with the above traits. Together, the present study provided the useful information for the characterization of SLC39A7 gene and potential association with carcass traits in pigs.

Identification of a differential gene HUMMLC2B between F1 hybrids Landrace x Yorkshire and their female parents Yorkshire.

In order to investigate heterosis on a molecular basis, suppression subtractive hybridization was used to analyze the differences in gene expression between porcine F1 hybrids Landrace x Yorkshire and their female parents Yorkshire. From two specific subtractive cDNA libraries, the clones screened out by reverse Northern high-density blots screening were chosen to clone full-length cDNA by RACE. An expression-upregulated gene for Yorkshire skeletal muscle, designated as HUMMLC2B, was identified. Porcine HUMMLC2B contains an open reading frame (ORF) encoding 169 amino acids residues with 59 and 115 nucleotides in the 5' and 3' untranslated regions (UTRs), respectively. In the porcine genome, it contains seven exons separated by six introns. High allelic variations and four SINEs were detected in it. Comparison of derived amino acid sequence of HUMMLC2B with database sequences revealed highly conserved 12 amino acid residues in a putative calcium-binding region. RT-PCR analysis showed a tissue-specific pattern of expression in skeletal muscle and a similar level of expression during skeletal muscle development. The possible role of HUMMLC2B and its relation to porcine heterosis are discussed.

Lipopolysaccharide receptors and signal transduction pathways in mononuclear phagocytes.

There is little question but that bacterial lipopolysaccharides (LPS) remain one of the most potent stimuli which can affect macrophage activation. Although the precise biochemical mechanisms responsible for this remain to be fully defined, there is now evidence accumulating from a number of laboratories that functional receptors for these bacterial products do exist and may contribute to the initial triggering event. Unfortunately, there is currently no consensus as to which of the candidate receptors identified to date serves as the primary binding target for LPS, and it is possible that the difference in macrophage cell types, LPS probes, and detection systems will all influence the nature of the binding. At the present time, therefore, macromolecules of 96-kDa, 95-kDa (adhesion beta chain), 80-kDa, 65-kDa, and 55-kDa may be considered as possible LPS targets. With the exception of the 96-kDa protein identified by Hampton and his co-workers, there exists some experimental evidence for a functional role for each of the molecules so far identified. It is apparent that the molecular cloning and sequencing and subsequent biochemical characterization of these LPS receptors will be required to determine unequivocally their role in LPS-mediated triggering events. Such information will be invaluable in sorting out the relevant biochemical second signals involved in macrophage activation. Although much new information has recently been accumulated on potential signaling pathways for LPS, the definitive events remain far from unequivocally established. In view of the obvious importance of LPS-macrophage interactions in the overall capacity of the mammalian host to respond appropriately to the potentially hostile prokaryotic environment, a precise delineation of LPS-mediated macrophage activation is critical to our understanding of this important inflammatory mediator cell.

Lipopolysaccharide (LPS) binding to 73-kDa and 38-kDa surface proteins on lymphoreticular cells: preferential inhibition of LPS binding to the former by Rhodopseudomonas sphaeroides lipid A.

Using a photoactivable, radioiodinated lipopolysaccharide probe, [125I]ASD-LPS (derivatized from purified E. coli 0111:B4 S-LPS), we earlier reported the presence of a 73-kDa (p73) predominant LPS-binding protein on mouse lymphocytes and macrophages with specificity for the lipid A region of LPS. Both Re-LPS from Salmonella minnesota and purified lipid A will inhibit the binding of LPS to the p73 LPS receptor. In the studies reported here, we have found that non-toxic diphosphoryl lipid A purified from Rhodo-pseudomonas sphaeroides has the capability to inhibit the binding of [125I]ASD-LPS to the p73 protein. However, using the same LPS probe and photoaffinity cross-linking techniques, our data suggest that a less dominant 38-kDa (p38) LPS-specific binding protein identified on mouse splenocytes, J774.1 macrophage-like cell line, and 70Z/3 pre B-cell line by SDS-PAGE is not inhibited by purified lipid A, even at a concentration in 50-fold excess of that of [125]ASD-LPS. The binding of the LPS probe to the 38 protein could be inhibited in a dose-dependent manner by underivatized native S. minnesota Re-LPS (composed only of Kdo and lipid A). We speculate that this p38 LPS-binding protein may manifest a specificity for inner core oligosaccharide determinants on LPS.

Lipopolysaccharide/lipid A receptors on lymphocytes and macrophages.

Significant advances have been realized during the past five years in the understanding of the mechanism(s) by which endotoxic LPS interactions with mammalian lymphoreticular cells leads to characteristic cellular responses. There is now strong experimental evidence to support the concept that specific receptors for the lipid A component of LPS do, in fact, exist and are functional on these cells. While the available data do not rule out a potential contribution of nonspecific hydrophobic interactions of lipid A with the membrane bilayer in the cellular activation process, it would appear that interaction with the LPS receptor alone is sufficient to initiate triggering. Whether there exist more than one molecular entity which might function on mammalian cell membranes as a specific receptor for LPS, or whether different cell types may manifest different LPS receptors remains as an interesting area for future research. Further, the concept that molecular complexes of LPS with mammalian host proteins, such as the acute phase LPS binding protein, might trigger additional novel pathways for cell activation is both exciting and of potential importance. The precise mechanism or mechanisms by which LPS-receptor ligand interactions translate into appropriate transmembrane signalling events is currently uncertain. Clearly there exists evidence for contribution of many of the traditional second signals, although at present, the data are incomplete and not always consistent between laboratories. Of potential concern in this respect are the sometimes rather striking differences noted between lipid A and intact polysaccharide containing S-LPS. While such differences may be significant and important, it should be remembered that S-LPS itself is a potent stimulus for many lymphoreticular cell subpopulations, and any postulated pathways must encompass S-LPS as well as lipid A. In any case, it is likely that the further molecular-biochemical characterization of LPS receptors will yield crucial information for the eventual elucidation of the precise pathways for LPS transmembrane signalling. Such information will be invaluable in the future harnessing of the immunostimulatory potential of LPS as well as the abrogation of its profound deleterious pathophysiological effects in endotoxin shock.

Endotoxin receptors on mammalian cells.

Recent experiments from our laboratory, as well as those of several other investigators, have focused upon the identification and characterization of specific membrane-localized receptors for bacterial endotoxic lipopolysaccharides on mammalian cells. In this article, we have summarize the results of these studies with a primary emphasis upon experiments from our own laboratory. It would appear that some differences between laboratories in the identification of LPS receptor may be the result of different experimental techniques employed. In spite of these differences, however, there is an emerging picture which defines several LPS binding proteins as potentially dominant candidates for the functional LPS receptor on the cell membrane, including a protein of 70-80 kDa and perhaps a second protein of 30-40 kDa as well. Other membrane proteins with important functions in the cellular response to LPS include CD14, the CD11/18 family of adhesins and the 95 kDa scavenger receptor. Identification of specific cell membrane targets for LPS has important implications for immunotherapy of septic shock and perhaps cancer as well.

Molecular cloning and polymorphism of the porcine H2AFZ gene.

H2A histone family, member Z (H2A.Z) is required for early mammalian development. In the present study, the 932 bp of full-length cDNA encoding a 128 amino-acid protein and the sequences of intron 2 to 4 of the porcine H2A histone family, member Z (pH2AFZ) gene were obtained. By comparative sequencing of pH2AFZ gene in Large White and Meishan pigs, a 4 bp deletion/insertion in intron 2 was detected and a PCR-Bsu15I-RFLP was established to detect this variation. In DIV (4th Dam line of Chinese lean-type new lines) pigs, the first-parity females with AA genotype had fewer piglets born alive (-2.64 and -1.83 piglets per litter) than those with AB (P < 0.01) and BB (P < 0.05) genotype. The additive allelic and dominance effect were estimated to be 0.92 (P < 0.05) and -0.87 piglets per litter (P < 0.01) for number of piglets born alive, respectively. This result suggests that the pH2AFZ gene might be a good candidate gene of litter-size trait and provides some marker information for marker-assisted selection.

Identification of polymorphism and association analysis with reproductive traits in the porcine RNF4 gene.

The ring finger protein 4 gene (RNF4), which might play a role in fetal germ cell development as well as in oocyte and granulosa cell maturation, was one of the potential candidate genes for reproductive traits. In the present work, we isolated the complete coding sequence of porcine RNF4 gene, identified a single nucleotide polymorphism (SNP: T/C) in intron5, and developed a PCR-SacII-RFLP genotyping assay. Association of this SNP with reproductive traits was assessed in three populations with diverse genetic backgrounds. One was Chinese Qingping sows. Another was consisted of crossbred sows derived from Landrace, Large White, Chinese Tongcheng and/or Chinese Meishan (Line DIV). The third is Large White x Meishan (LW x M) F(2) slaughtered population. Statistical analysis demonstrated that, in the first parity, the difference between RNF4 genotypes and reproductive traits of both Qingping and Line DIV sows was not significant. In the second and subsequent litters, CC animals in Qingping population had more piglets born (+1.74 piglets) and piglets born alive (+2.02 piglets) than sows with the TT genotype (P<0.05). Line DIV sows inheriting the CC genotype had additional 0.69 piglets born compared to the TC animals (P<0.05) in second and subsequent litters. No significant difference was observed between genotypes and reproductive tracts components in F(2) animals. In addition, we found RNF4 gene has a significant additive effect on both piglet born and piglet born alive in Qingping animals (P<0.05). Results here suggested that the RNF4 SNP was significantly associated with litter size in two populations and could be useful in selection for increasing litter size in pigs. Further studies were needed to confirm these preliminary researches.

Differential proteome analysis of porcine skeletal muscles between Meishan and Large White.

Western and indigenous Chinese pig breeds show obvious differences in muscle growth and meat quality; however, the underlying molecular mechanism remains unclear. In this study, proteome analysis of LM between purebred Meishan and Large White pigs was performed by 2-dimensional gel electrophoresis and mass spectrometry. A total of 25 protein spots were differentially expressed in the 2 breeds. The 14 identified proteins could be divided into 4 groups: energy metabolism, defense and stress, myofibrillar filaments, and other unclassified proteins. Quantitative real-time PCR was used to analyze the partly differentially expressed proteins in mRNA level, which revealed a positive correlation between the content of the proteins and their mRNA levels. We also analyzed the mRNA levels of myosin heavy chain isoforms using quantitative real-time PCR. The results indicated that IIa and IIx fibers were elevated in Meishan pigs, whereas the IIb fiber was more highly expressed in Large White pigs. To the best of our knowledge, this was the first proteomics-based investigation of total skeletal muscle protein in different pig breeds, and these results may provide valuable information for understanding the molecular mechanism responsible for breed-specific differences in growth performance and meat quality.