Muscle ESTs II: Cloning, Sequencing, and Mapping the Pig Gene for the Intermediate Filament Protein Desmin

The results of sequencing large numbers of random cDNAs in the human genome project has clearly shown value in gene discovery and mapping. We previously demonstrated a simple approach to rapidly identify musclespecific pig cDNAs for sequencing by strong hybridization to muscle cDNA probes. In this report, we sequenced additional muscle cDNAs, emphasizing clones that were not strongly expressed in muscle. Fewer clones matched known muscle-specific genes in the pig or other species, indicating selection by hybridization is most accurate for strongly hybridizing clones showing weak signals with non-muscle probes. Some muscle-specific clones, however, also were identified by sequencing the weakly hybridizing class of cDNAs. One of these was M239, encoding muscle-specific desmin, which is an intermediate filament protein involved in holding muscle fibers together. As desmin may be an interesting candidate gene for muscle tenderness, the desmin gene (DES) was fully sequenced and mapped both physically and genetically. Pig desmin is highly conserved, being more than 97% identical at the amino acid level to human and mouse desmin. Linkage of DES was observed for three loci already mapped to pig chromosome 15 (SSC15), and physical mapping placed DES on SSC15q23-26. This mapping information will be useful in further studies on the role of DES in muscle biology and muscle tenderness traits

Quantitative Measurement of PIT1, GH, and PRL mRNA and Circulating Hormone Levels in Pig Families Segregating PIT1 Genotypes

PIT1 is a member of the POU-domain family gene and is a positive regulator for growth hormone (GH), prolactin (PRL), and thyrotroph-stimulating hormone β(TSHB) in several mammalian species. Previous studies in pigs have shown an association of PIT1 polymorphisms with growth and carcass traits in which the primarily Chinese alleles were associated with heavier birth weight and greater backfat. To further investigate the role of PIT1 in controlling pig growth traits, Chinese Meishan (MS) pigs segregating PIT1 polymorphisms were used to study the differences of GH and PRL at both mRNA and circulating hormone levels. A total of 60 animals from nine litters was used to collect both pituitary and blood samples at day 1, 15, and 30 after birth. A novel procedure that involves standard curve quantification was used to quantify mRNA amount for GH, PRL, PIT1-α, and PIT1-β. Conventional hormone assays were done to measure the circulating amount of GH and PRL from the blood. Statistical analyses of PIT1 genotypes, sex, and days on either mRNA (PIT1 alternative transcripts, GH, and PRL) or circulating hormone (GH and PRL) were done by using the leastsquares procedure. This study investigates GH and PRL at both mRNA and circulating hormone levels in MS pigs segregating PIT1 polymorphisms. Results from this study will provide useful information on the role of PIT1 in controlling pig growth traits

Heat Stress during Pig Oocyte In Vitro Maturation Impacts Embryonic Development and Gene Expression

Gene expression of heat stressed oocytes matured in vitro was evaluated for potential markers which could be used to characterize the effects of heat stress on developing oocytes in the pig. Three heat stress scenarios were administered during in vitro oocyte maturation. Heat stressed oocytes had reduced maturation rates, decreased developmental competency, and altered expression of heat stress and developmental competency markers

Development of New Placental and Fetal Expressed Sequence Tags (EST) for Gene Discovery in Pig Reproduction

One major problem that has high economic impact on pig reproduction is the unexplained loss of potential porcine conceptuses during the first month of gestation. To better understand when and how these losses occur, it is imperative to investigate the underlying genetic regulatory mechanisms. We have recently initiated a large-scale cDNA sequencing project to provide molecular information regarding the genes expressed in female reproductive tissues. cDNA libraries are planned for ovary, hypothalamus, pituitary, placenta, uterus, and several stages of embryonic development. Sequence information will also be highly useful in developing sequence-tagged sites for physical mapping and developing comparative links between the human, mouse, and pig genome maps. We have previously reported the creation of two cDNA libraries, porcine fetal (day 20), and conceptus (day 17). Sequencing of these libraries produced 220 Expressed Sequence Tags (ESTs), with 180 sequences analyzed by clustering algorithms, and 139 clusters identified within these sequences. We now report the creation of two more libraries from porcine fetal (day 45) and placental tissues. The day 45 fetal library has 971,150 independent clones (average insert: 1.4 kb), whereas the placental library has 1,320,000 independent clones. Initial sequencing of the fetal library has produced 119 ESTs (81 clusters), whereas we have obtained 1411 ESTs (1056 clusters) from the placental library. After clustering all sequences thus far obtained, we have identified 1,233 unique clusters. Sequences obtained in this project will be deposited into Genbank dbEST, and all comparative homolog

Addition of Thirteen Genes to the Porcine Comparative Gene Map Reveals New Regions of Conserved Synteny

Thirteen genes were mapped to the porcine genome by using either linkage mapping of the PiGMaP families (eight genes) or typing of a porcine somatic cell hybrid panel (12 genes). The genes were chosen from interesting locations in the human genome. The physical gene assignments to pig chromosomes (SSC) with corresponding human chromosome (HSA) locations include the following: FGF7 (HSA15), MADH4 (HSA18), and MC4R (HSA18) to SSC1, RXRB (HSA6), and SSTR1 (HSA14) to SSC7, UCP1 (HSA4) to SSC8, PGR (HSA11) to SSC9, TTN (HSA2) and ANT1 (HSA4) to SSC15, GRIA1 (HSA5) to SSC16, AR (HSA-X), and GRIA3 (HSA-X) to SSC-X. Additionally, CD59 (HSA11) was linkage mapped to SSC2. The majority of the assignments confirm results from bidirectional chromosome painting (4). A rearrangement in gene order was detected within the region of correspondence between SSC1 and HSA15. Two assignments were made that were not expected from the painting results (MC4R and GRIA1) and one assignment of a gene from a region where the painting study was not informative (ANT1)

Acute Systemic Inflammatory Response to Lipopolysaccharide Stimulation in Pigs Divergently Selected for Residual Feed Intake

Background: It is unclear whether improving feed efficiency by selection for low residual feed intake (RFI) compromises pigs’ immunocompetence. Here, we aimed at investigating whether pig lines divergently selected for RFI had different inflammatory responses to lipopolysaccharide (LPS) exposure, regarding to clinical presentations and transcriptomic changes in peripheral blood cells. Results: LPS injection induced acute systemic inflammation in both the low-RFI and high-RFI line (n = 8 per line). At 4 h post injection (hpi), the low-RFI line had a significantly lower (p= 0.0075) mean rectal temperature compared to the high-RFI line. However, no significant differences in complete blood count or levels of several plasma cytokines were detected between the two lines. Profiling blood transcriptomes at 0, 2, 6, and 24 hpi by RNA-sequencing revealed that LPS induced dramatic transcriptional changes, with 6296 genes differentially expressed at at least one time point post injection relative to baseline in at least one line (n =4 per line) (|log2(fold change)| ≥ log2(1.2); q \u3c 0.05). Furthermore, applying the same cutoffs, we detected 334 genes differentially expressed between the two lines at at least one time point, including 33 genes differentially expressed between the two lines at baseline. But no significant line-by-time interaction effects were detected. Genes involved in protein translation, defense response, immune response, and signaling were enriched in different co-expression clusters of genes responsive to LPS stimulation. The two lines were largely similar in their peripheral blood transcriptomic responses to LPS stimulation at the pathway level, although the low-RFI line had a slightly lower level of inflammatory response than the high-RFI line from 2 to 6 hpi and a slightly higher level of inflammatory response than the high-RFI line at 24 hpi. Conclusions: The pig lines divergently selected for RFI had a largely similar response to LPS stimulation. However, the low-RFI line had a relatively lower-level, but longer-lasting, inflammatory response compared to the high-RFI line. Our results suggest selection for feed efficient pigs does not significantly compromise a pig’sacute systemic inflammatory response to LPS, although slight differences in intensity and duration may occur

Gene Discovery and Functional Genomics in the Pig

Advances in gene mapping and genomics in farm animals have been considerable over the past decade. Medium resolution linkage and physical maps have been reported, and specific chromosomal regions and genes associated with traits of biological and economic interest have been identified. We have reached an exciting stage in gene identification, mapping and quantitative trait locus discovery in pigs, as new molecular information is accumulating rapidly. Significant progress has been made by identifying candidate gene associations and low-resolution regions containing quantitative trail loci (QTL). However, we are still disadvantaged by the lack of tools available to efficiently use much of this new information. For example, current pig maps are neither of high enough resolution nor sufficiently informative at the comparative level for positional candidate gene cloning within QTL regions. As well, studying biological mechanisms underlying economically important traits such as reproduction is limited by the lack of molecular resources. This is especially important, as reproduction is very difficult to genetically improve by classical breeding methods due to the relatively low heritability and high expense in data collection. Thus, an improved understanding of porcine reproductive biology is of crucial economic importance, yet reproductive processes are poorly characterized at the molecular level. Recently, new methodologies have been brought to bear on a better understanding of pig molecular biology for accelerating genetic improvement in pigs. Several groups are developing molecular information in the pig, and the total Genbank sequence entries for porcine expressed genes have recently topped 100,000. Our Midwest EST Consortium has produced cDNA libraries containing the majority of genes expressed in major female reproductive tissues, and we have deposited nearly 15,000 gene sequences into public databases. These sequences represent over 8,900 different genes, based on sequence comparison among these data. Furthermore, we have developed computer software to automatically extract sequence similarity of these pig genes with their human counterparts, as well as the mapping information of these human homologues. Within our data set, we have identified nearly 1,500 pig genes with strong similarity to mapped human genes, and we are in the process of mapping 700 of these genes to improve the human-pig comparative map. This work and the complementary work of others can now be used to more rapidly understand and identify the genes controlling reproduction, so that genetic improvement of reproduction phenotypes can accelerate