Environmental and Comparative Genomics Research Projects
Horses

Sepsis and Gene Expression in Foals
Very little is known about the molecular basis of septicemia in the neonatal foal, the most common cause of death in the first 7 days of life. Despite increased awareness of risk factors for sepsis and advances in medical management for critically ill foals, mortality of septic foals remains high. To begin to understand the molecular mechanisms associated with susceptibility to sepsis and the individual differences in response to treatment for sepsis, an analysis of the differential expression of expressed sequence tags (ESTs) previously isolated from a septic Thoroughbred foal was initiated. Blood samples were collected from foals presented to Tufts University Large Animal Hospital and used for extraction of total RNA. Foals were grouped as 0=healthy (n=3), 1=non septic/mildly ill (n=5), 2= septic/very ill (n=12). Differential mRNA expression of 7 ESTs (MHC Class II DQ alpha chain, granulocyte colony-stimulating factor receptor, complement C5a receptor, cell adhesion molecule GMP 140, LY6H protein-lymphocyte antigen, interleukin-1 receptor antagonist, and c-fms protooncogene) known to be involved in cell signaling/cell communication and cell/organism defense was examined in the RNA from septic and non-septic foals. Target gene expression was normalized to beta-2-microglobulin expression using real time RT-PCR. ANOVA results considering all 3 groups separately showed significant effect of gene (P<0.001) and health status (P<0.018) but there were no significant differences in the interaction of gene and health status. There was a significant correlation (P<0.05) between the expression of five of the genes (GCSF receptor, IL1ra, LY6H lymphocyte antigen, and complement C5a receptor) and the foal's white blood cell count. Preliminary results using the stallions of the international reference mapping family indicate that IL1ra, LY6H and c-fms protooncogene are polymorphic, work is underway to test polymorphism status of the other genes. Further work must be done with a larger sample size using RT-PCR analysis of genes suspected to be involved in the events that lead to the septic condition or the individual's positive or negative response to medical treatment. Knowledge of these molecular events in neonatal foals may one day help to prevent sepsis in the genetically predisposed and/or create individualized therapies for this condition.

M. Smith, M.R. Paradis ,and A. Alcivar-Warren. 2004. DIFFERENTIAL EXPRESSION OF HORSE ESTs IN NORMAL AND SEPTIC FOALS. Book of Abstracts. Plant and Animal Genome XII, January 10-14, 2004, Town & Country Convention Center, San Diego, CA. Abstr. P699, p. . (View Abstract)

Genetic Diversity of Horses
Three hundred and twenty cDNAs were isolated from a cDNA library originated from blood of a Thoroughbred septic foal. Single passage sequencing using M13 reverse primer provided 181 unique sequences. The remaining clones were either redundant, with very small insert (<50 bp), unreadable, or contained no insert. Out of the 181 unique sequences, 67 showed similarities to known genes, 56 were similar to unknown genes, and 58 were novel sequences. Seven of the 67 known genes were of mitochondrial-DNA origin (ATPase, cytochrome b, NADH1-3, COI, 16s rRNA) and 60 were of nuclear-DNA origin with different functions, including those involved in cell division (histone, cyclin I, cyclin dependent kinase2), cell signalling/cell communication (IL1-ra, G-protein receptors 1&2, immunoglobulin receptor, GCSF receptor, glia-derived neurite promoting factor, c-fms protooncogene, complement C5a receptor, IGF-II receptor, Burkitt's lymphoma receptor1, EGF-like EMR-2, cell adhesion GMP140, myeloid-associated differentiation protein, protein kinase C-delta13, cleft lip and palate transmembrane protein, myocardial vascular inhibitor factor), cell structure/motility (tropomyosin, lysosome-associated membrane protein), gene/protein expression (RNA polymeraseII, Not 56-like, RING zinc finger protein, polypirimidine tract-binding protein, ribosomal L10&L18, 45S pre rRNA), metabolism (vacuolar type H+ATPase, 75Kd iron sulphur protein, CAB1, NAD+-specific isocitrate dehydrogenase, ornithine decarboxylase antyzime, lysosomal pepstatin insensitive protease), and cell/organism defense (beta lactoglobulin II, transferrin, P2XM, zinedin, y-L aminoacid transporter1, MHC class II DQ-alpha, Ig lambda, LY6H protein, HIV-EP2/Shnurri2), among others. Ninety four out of 181 clones contained microsatellites of 3 or more repeats, 98% of which had flanking sequences suitable for allele amplification by PCR. These ESTs will be a valuable addition to the horse EST database and may be useful for gene mapping and other genetic analysis in horses.

Pascual, I., Dhar, A.K., Y. Fan, M.R. Paradis, M.V. Arruga, and A. Alcivar-Warren. 2001. Isolation of expressed sequence tags from a Thoroughbred horse (Equus caballus) 5'RACE cDNA library. Book of Abstracts, Plant & Animal Genome IX, The International Conference on the Status of Plant and Animal Genome Research. January 13-17, 2001, Town and Country Hotel., San Diego, CA., P63, p. 76.(View Abstract)

Horse EST database(HorseESTbase)
Horse ESTs classified according to function from FASTA searches. (View Horse ESTs)