G. E. Sulimova

A Genome Scan for Positive Selection in Thoroughbred Horses

Thoroughbred horses have been selected for exceptional racing performance resulting in system-wide structural and functional adaptations contributing to elite athletic phenotypes. Because selection has been recent and intense in a closed population that stems from a small number of founder animals Thoroughbreds represent a unique population within which to identify genomic contributions to exercise-related traits. Employing a population genetics-based hitchhiking mapping approach we performed a genome scan using 394 autosomal and X chromosome microsatellite loci and identified positively selected loci in the extreme tail-ends of the empirical distributions for (1) deviations from expected heterozygosity (Ewens-Watterson test) in Thoroughbred (n = 112) and (2) global differentiation among four geographically diverse horse populations (FST). We found positively selected genomic regions in Thoroughbred enriched for phosphoinositide-mediated signalling (3.2-fold enrichment; P<0.01), insulin receptor signalling (5.0-fold enrichment; P<0.01) and lipid transport (2.2-fold enrichment; P<0.05) genes. We found a significant overrepresentation of sarcoglycan complex (11.1-fold enrichment; P<0.05) and focal adhesion pathway (1.9-fold enrichment; P<0.01) genes highlighting the role for muscle strength and integrity in the Thoroughbred athletic phenotype. We report for the first time candidate athletic-performance genes within regions targeted by selection in Thoroughbred horses that are principally responsible for fatty acid oxidation, increased insulin sensitivity and muscle strength: ACSS1 (acyl-CoA synthetase short-chain family member 1), ACTA1 (actin, alpha 1, skeletal muscle), ACTN2 (actinin, alpha 2), ADHFE1 (alcohol dehydrogenase, iron containing, 1), MTFR1 (mitochondrial fission regulator 1), PDK4 (pyruvate dehydrogenase kinase, isozyme 4) and TNC (tenascin C). Understanding the genetic basis for exercise adaptation will be crucial for the identification of genes within the complex molecular networks underlying obesity and its consequential pathologies, such as type 2 diabetes. Therefore, we propose Thoroughbred as a novel in vivo large animal model for understanding molecular protection against metabolic disease.

The genetic origin and history of speed in the Thoroughbred racehorse

Selective breeding for speed in the racehorse has resulted in an unusually high frequency of the C-variant (g.66493737C/T) at the myostatin gene (MSTN) in cohorts of the Thoroughbred horse population that are best suited to sprint racing. Here we show using a combination of molecular- and pedigree-based approaches in 593 horses from 22 Eurasian and North-American horse populations, museum specimens from 12 historically important Thoroughbred stallions (b.1764-1930), 330 elite-performing modern Thoroughbreds and 42 samples from three other equid species that the T-allele was ancestral and there was a single introduction of the C-allele at the foundation stages of the Thoroughbred from a British-native mare. Furthermore, we show that although the C-allele was rare among the celebrated racehorses of the 18th and 19th centuries, it has proliferated recently in the population via the stallion Nearctic (b.1954), the sire of the most influential stallion of modern time, Northern Dancer (b.1961).

Association of DNA polymorphisms of the growth hormone and prolactin genes with milk productivity in Yaroslavl and Black-and-White cattle

Polymorphisms of the prolactin (bPRL) and growth hormone (bGH) genes were studied comparatively in the Russian and German Black-and-White and Yaroslavl cattle breeds. Two polymorphisms were studied for each gene. In the case of the bPRL gene, the polymorphism of the 5′-untranslated region was examined by microsatellite analysis and the RsaI polymorphism of exon 3, by RFLP analysis. In the case of the bGH gene, the MspI polymorphism of intron III and the AluI polymorphism of exon 5 were assessed by RFLP analysis. Differences in allele and genotype frequencies were observed both between and within breeds. The heterozygosity at the RsaI marker was low (9.4%) in the Russian Black-and-White breed; that at the microsatellite of the bPRL gene was low (3.2–24%) in all breeds examined. Homozygotes BB at the bPRL gene, which had not been reported earlier for European cattle breeds, were detected in the German Black-and-White and Yaroslavl breeds (at frequencies 0.16 and 0.13, respectively). The frequency of allele MspI(−) of the bGH gene in the Yaroslavl breed was extremely low (0.02), comparable only with that of the Holstein cattle (0.02). The heterozygosity at the AluI polymorphism was higher than at the MspI polymorphism of the bGH gene and reached 55% in the Yaroslavl breed. Genotype BB of the RsaI polymorphism of the bPRL gene tended to show a negative association with the fat content in milk. The genotypes of the AluI polymorphism of the bGH gene were associated with the fat content in milk in the Yaroslavl (F=4.56, P=0.013) and German Black-and-White (F=4.1, P=0.041) breeds: the highest fat content in milk was observed in the subsample of cows with heterozygous genotype VL.

Polymorphism of Bovine Prolactin Gene: Microsatellites, PCR-RFLP

In the samples of Russian Ayrshire and Gorbatov Red cattle breeds, distribution of frequencies of prolactin (PRL) gene alleles generated due to the presence of polymorphic RsaI site in exon 3 were studied. In the breeds, the frequencies of the Ballele of the PRLgene (with RsaI(+) site) detected by the PCR–RFLP method were 14.1 and 8.6%, respectively. In Black Pied, Ayrshire and Gorbatov Red cattle breeds, variation of the microsatellite dinucleotide repeat in the regulatory region of the gene PRLwas also studied. Gorbatov Red breed was monomorphic at the microsatellite locus with the only allele 164 bp in length. Two alleles (164 bp and 162 bp) were detected in the other breeds studied. The frequencies of 164-bp allele of the microsatellite locus were 93.7 and 90.0% in Black Pied and Ayrshire breeds, respectively. In Gorbatov Red breed of dairy type with good beef qualities and low milk-fat yield, lower level of heterozygosity for PRLgene was demonstrated compared to Ayrshire and Black Pied breeds that have high milk-fat yield. In three cattle breeds, higher mean estimate of polymorphism information content of PCR–RFLP in exon 3 (PIC = 0.21) was revealed compared with the same estimate (PIC = 0.09) for the microsatellite locus variability in the regulatory region of the PRLgene. Characteristics of allele Bdistribution of thePRLgene in the representatives of the Bovidae family are considered.

κ-casein gene (CSN3) allelic polymorphism in Russian cattle breeds and its information value as a genetic marker

The frequencies of the κ-casein gene (CSN3) alleles and genotypes have been determined in five Russian cattle breeds (Bestuzhev, Kalmyk, Russian Black Pied, Yaroslavl, and Yakut breeds) by means of PCR-RFLP analysis using two independent restriction nucleases (HinfI and TaqI) and by allele-specific PCR. Typing alleles A and B of CSN3 is of practical importance, because allele B is correlated with commercially valuable parameters of milk productivity (protein content and milk yield) and improves the cheese yielding capacity. The frequencies of the B allele of CSN3 in the breeds studied vary from 0.16 to 0.50; and those of the AB and BB genotypes, from 0.27 to 0.60 and from 0.02 to 0.23, respectively. The Yaroslavl breed had the highest frequencies of CSN3 allele B and genotype BB (0.50 and 0.23, respectively). The frequencies of the B allele and BB genotype in other breeds studied varied from 0.25 to 0.32 and from 0.03 to 0.09, respectively. In none of the breeds studied have the observed and expected heterozygosities been found to differ from each other significantly. However, the observed genotype distributions significantly differ from the expected one in some herds (in most such cases, an excess of heterozygotes is observed). Two herds of the Yaroslavl breed dramatically differ from each other in the heterozygosity level: a deficit (D = −0.14) and an excess (D = 0.20) of heterozygotes have been observed at the Mikhailovskoe and Gorshikha farms, respectively. In general, however, the heterozygosity of the Yaroslavl breed corresponds to the expected level (D = 0.04). Analysis of breeds for homogeneity with the use of Kulback’s test has shown that all cattle breeds studied are heterogeneous, the CSN3 diversity within breeds being higher than that among different breeds, which is confirmed by low Fst values (0.0025–0.0431). Thus, a DNA marker based on CSN3 gene polymorphism is extremely important for breeding practice as a marker of milk quality; however, it is inapplicable to marking differences between breeds or phylogenetic relationships between cattle breeds because of the high diversity with respect to this locus within breeds.

Human papilloma viruses and cervical tumours: mapping of integration sites and analysis of adjacent cellular sequences

BackgroundIn cervical tumours the integration of human papilloma viruses (HPV) transcripts often results in the generation of transcripts that consist of hybrids of viral and cellular sequences. Mapping data using a variety of techniques has demonstrated that HPV integration occurred without obvious specificity into human genome. However, these techniques could not demonstrate whether integration resulted in the generation of transcripts encoding viral or viral-cellular sequences. The aim of this work was to map the integration sites of HPV DNA and to analyse the adjacent cellular sequences.MethodsAmplification of the INTs was done by the APOT technique. The APOT products were sequenced according to standard protocols. The analysis of the sequences was performed using BLASTN program and public databases. To localise the INTs PCR-based screening of GeneBridge4-RH-panel was used.ResultsTwelve cellular sequences adjacent to integrated HPV16 (INT markers) expressed in squamous cell cervical carcinomas were isolated. For 11 INT markers homologous human genomic sequences were readily identified and 9 of these showed significant homologies to known genes/ESTs. Using the known locations of homologous cDNAs and the RH-mapping techniques, mapping studies showed that the INTs are distributed among different human chromosomes for each tumour sample and are located in regions with the high levels of expression.ConclusionsIntegration of HPV genomes occurs into the different human chromosomes but into regions that contain highly transcribed genes. One interpretation of these studies is that integration of HPV occurs into decondensed regions, which are more accessible for integration of foreign DNA.

Identification of Bovine Leucocyte Adhesion Deficiency (BLAD) Carriers in Holstein and Brown Swiss AI Bulls in Iran

BLAD is a hereditary disease in Holstein dairy cattle. The defective allele of CD18 gene, which is responsible for this disease, has recessive inheritance. The recessive homozygous form (BL/BL) is lethal and since carrier animals have viability, BLAD frequency increases by use of carrier bulls in Artificial Insemination (AI). BLAD carriers can be detected easily by means of polymerase chain reaction followed by restriction analysis of the amplicons. In this study DNA samples from Holstein (n = 30) and Brown Swiss (n = 10) bulls from Abbas Abad AI center (Khorasan state of Iran) were analysed. A 101-bp fragment from the polymorphic region of CD18 gene located on chromosome 1 was amplified by PCR. Restriction enzymes TaqI and HaeIII were used to identify genotypes. Digestion products were screened by electrophoresis on 8% non-denaturing polyacrylamide gel and visualized by ethidium bromide staining. Frequencies of BL/TL (carrier) genotypes in Holstein and Brown Swiss bulls were 3.33% and 0%, respectively. Our pedigree studies of the carrier bull in this experiment revealed that the mutation was inherited by him from Hawkeye bull (CANM 369995, BL). Although the elimination of BLAD-carrier bulls from the Holstein world would be the most efficient method to control this genetic disorder, many BLAD-carrier bulls are still listed commercially for AI, and BLAD is still occurring in Iran. Monitoring the prevalence of BLAD carriers in random selected herds may be helpful in judging the effectiveness of the BLAD-control program.

Distribution of BoLA-DRB3 allelic frequencies and identification of a new allele in the Iranian cattle breed Sistani (Bos indicus)

The distribution of the frequencies of BoLA-DRB3 gene alleles in the Iranian cattle breed Sistani was studied by the PCR-RFLP (“hemi-nested”) assay using restriction endonucleases RsaI, HaeIII and BstYI. In the examined cattle breed (65 animals) 32 alleles have been identified one of which being described for the first time (6.15% frequency). The nucleotide sequence of the polymorphic region of exon 2 of this allele has been determined and submitted in the GenBank database under accession number DQ486519. The submitted sequence has maximum homology (92%) with the previously described sequence DRB3-mRNA from Bos indicus (AccN X79346) and differs from it by 24 nucleotide substitutions which result in 16 amino acid substitutions. The peptide (on the basis of the reconstructed amino acid sequence) has 89% identity to the sequence encoded by the BIDRBF 188 locus (Bos indicus). The results obtained permit the sequence described by us to be considered as a new allele of the BoLA-DRB3 gene (DRB3.2*X). The total frequency of the main six alleles (DRB3.2*8, *10, *11, *20, *34 and *X) occurring with a frequency of over 5% is about 60% in Iranian Sistani cattle. Fifteen alleles have <1% frequency. The highest frequency was observed for DRB3.2*8 allele (21.54%) like in other previously described breeds of Bos indicus (up to 23.07%). The Iranian breed Sistani has a high level of similarity by the spectrum of BoLA-DRB3 alleles and their frequencies to other Bos indicus breeds and significantly differs by these criteria from the Bos Taurus breeds. The Iranian Sistani herd under study includes alleles associated with to resistance to leukemia (DRB3.2*11 and *23) and to different forms of mastitis (DRB3.2* 2, *7, *11, *23 and *24) although their frequencies are low (from 0.77 to 5.37%). On the whole, a high level of diversity of BoLA-DRB3 gene alleles and the availability of alleles associated with resistance to different diseases makes this breed of interest for breeding practice.

Altered Expression of the SEMA3B gene in Epithelial Tumors

Tumor-specific expression downregulation may be indicative of a gene’s involvement in tumor suppression. For instance, SEMA3B mRNA levels are decreased in many cell lines of small-cell and non-small cell lung cancer, and SEMA3B was shown to suppress the growth of the NSCLC cell line NCI-H1299 and tumor formation in immunodeficient mice. In this work, SEMA3B expression levels were determined in epithelial tumors of different localizations. In cell lines of renal, breast, and ovarian cancer, SEMA3B mRNA levels were frequently (4/11, 36%) decreased as much as 10–250-fold according to semiquantitative RT-PCR assay. SEMA3B expression levels were also determined in primary tumor extracts of kidney, lung, breast, ovarian, and colorectal cancer. In clear cell renal cell carcinoma, SEMA3B expression was decreased 5–1000-fold in 25 of 51 extracts (49%) compared to 5/51 (10%) extracts with increased mRNA levels; the result was highly significant: P < 0.0001 by Fisher’s exact test. SEMA3B was frequently downregulated in ovarian (5/16, 31% vs. 2/16, 12%) and colorectal cancer (6/11, 54% vs. 2/11, 18%). These results suggest that SEMA3B is involved in the suppression of kidney, ovarian, and colon tumor growth.

Activation of RHOA transcription in epithelial tumors may be caused by gene amplification and/or demethylation of the promoter region

RHOA is a small GTPase involved in morphogenesis, cell adhesion, and cell cycle control. RHOA (3p21.31) is a potential oncogene, causing cell malignant transformation. A study was made of the mechanisms activating RHOA transcription in several epithelial tumors. Semiquantitative RT-PCR revealed elevated transcription of RHOA in tumors (45 cases of breast cancer, renal cell carcinoma, and epithelial ovarian carcinoma; p < 10−4). For the first time transcriptional activation of RHOA in tumors was reliably associated with its amplification (p = 10−7). Digestion with four methyl-sensitive restriction enzymes (HpaII, HhaI, AciI, and Bsh1236I) and subsequent PCR revealed a change in methylation of the RHOA promoter region in 23 out of 45 tumors, hypomethylation being two times more frequent than hypermethylation. Demethylation of the RHOA promoter region was associated with a two-to tenfold increase in the transcription level of the gene. Thus, two mechanisms—amplification and demethylation of the promoter region—were shown to change the level of RHOA transcription in epithelial tumors.