Comparative mapping of sheep inhibin subunit βb to chromosome 2 in sheep and cattle by fluorescence in situ hybridization

A cosmid clone containing the complete sheep inhibin subunit β_B gene (INHBB) was assigned to sheep and cattle homologous chromosome bands 2q31-q33 by fluorescence in situ hybridization. The assignment of INHBB in sheep excludes another candidate gene as the site of the Fec^B mutation.     

Enumeration of anaerobic oxalate-degrading bacteria in the ruminal contents of sheep

Abstract Concentrations of oxalate-degrading anaerobes in ruminal contents of sheep were determined from counts of colonies producing clear zones on a calcium oxalate medium (D agar with 7 mM CaCl₂). Viable counts of oxalate degraders from a 55-kg sheep fed a diet containing 32% halogeton (4.6% oxalate) averaged 2.6 × 10⁶/ g (dry weight). When the halogeton concentration in the diet was reduced to 16%, counts of oxalate degraders decreased nearly 300-fold. Oxalate-degrading isolates from this sheep were similar to OxB, the type strain of Oxalobacter formigenes . When a 45-kg sheep was fed diets containing 2.2, 1.5, and 0.8% oxalate, viable counts of oxalate degraders (enumerated on D agar with 14 mM CaCl₂ and 20% filter-sterilized ruminal fluid) represented 0.85, 0.52, and 0.06% of the total viable population, respectively; total viable counts were essentially unchanges by these concentrations of dietary oxalate. Similar percentages of oxalate degraders were also observed when a 23-kg sheep was fed diets containing 1.5 or 0.8% oxalate. This report presents the first direct measurements of the concentrations of oxalate-degrading bacteria in the rumen and supports the concept that the availability of oxalate in the diet influences the proportion of oxalate-degrading bacteria in the rumen     

Assignment of five polymorphic ovine microsatellites to bovine syntenic groups

A panel of bovine somatic cell hybrids was used to map ovine microsatellites. Five of seven microsatellites were assigned to five bovine syntenic groups. These microsatellites were designated D5S10 (MAF23), D1S4 (MAF46), D13S1 (MAF18), D4S3 (MAF50), and DXS2 (MAF45), mapped to syntenic groups U3 (chromosome 5), U10 (chromosome 1), U11, U13, and the X chromosome, respectively. Two remaining sheep microsatellites amplified rodent DNA in the hybrid somatic cell panel, and were not assigned to bovine syntenic groups. Assignment of ovine-derived microsatellites to bovine syntenic groups provides additional evidence of the usefulness of microsatellites for mapping closely related species. The use of ovine and bovine microsatellites will aid in development of comparative genomic maps for these two species.     

Cloning, sequencing and linkage mapping of the NRAMP1 gene of sheep and deer

The ovine NRAMP1 and cervine NRAMP1 cDNAs were cloned by RT PCR of RNA derived from macrophage enriched leukocyte preparations. The complete coding and 3' regions were sequenced. Both sheep and deer NRAMP1 proteins are 548 amino acids long. There are 77 and 73 amino acid differences, respectively, compared to the mouse Nramp1 sequence. Dinucleotide repeats were found in both the ovine and cervine 3' non-coding sequence. Amplification of these regions in individual sheep and deer showed them to be polymorphic micro-satellites. They have polymorphism information content values of 0·76 and 0·84 in sheep and deer, respectively. Using these microsatellites, the ovine NRAMP1 gene was mapped in a linkage group on ovine chromosome 2q and cervine NRAMP1 was mapped in a linkage group syntenic with human chromosome 2, mouse chromosome 1 and sheep chromosome 2.     

Physical mapping confirms that sheep chromosome 10 has extensive conserved synteny with cattle chromosome 12 and human chromosome 13

The following loci, on human chromosome 13, have been newly assigned to sheep chromosome 10 using chromosomally characterized sheep-hamster cell hybrids: gap junction protein, beta 2, 26 kDa (connexin 26) (GJB2); gap junction protein, alpha 3, 46 kDa (connexin 46) (GJA3), and esterase D/formylglutathione hydrolase (ESD). This assignment of ESD is consistent with comparative mapping evidence, but not with an earlier report of it on sheep chromosome 3p26-p24. Cell hybrid analysis confirmed the location of another human chromosome 13 locus, retinoblastoma 1 (including osteosar-coma) (RBI), and the anonymous ovine genomic sequence RP11 on sheep chromosome 10. Isotopic in situ hybridization was used to regionally localize RP11 on to sheep 10q15-q22. The location of microsatellites AGLA226, OarDB3, OarHH41, OarVH58, and TGLA441, previously assigned to sheep chromosome 10 by linkage analysis, was confirmed by polymerase chain reaction using the cell hybrid panel. These mapping data provide further evidence that sheep chromosome 10 is the equivalent of cattle chromosome 12, and that these chromosomes show extensive conserved synteny with human chromosome 13.     

Pig microsatellites isolated from cosmids revealing polymorphism and localized on chromosomes

One of the most widely studied simple sequences in the mammalian genome is the (TG)_n dinucleotide sequence. Because these microsatellites are highly polymorphic, we chose to study microsatellites from cosmids to provide genetic markers for the porcine genome. After screening a porcine cosmid library with a (CA)₁₀ probe, 20 cosmids containing microsatellites were subcloned and 17 microsatellites identified by sequencing. Oligonucleotide primers flanking the repeat were designed for seven (TG)_n microsatellites with n > 14. These seven microsatellites revealed polymorphism and were regionally assigned to chromosomes by fluorescent in situ hybridization of initial cosmids. These seven loci will be useful for both the construction of the genetic map and as landmark loci on the physical map of the porcine genome.     

Chromosomal distribution of endogenous Jaagsiekte sheep retrovirus proviral sequences in the sheep genome

A family of endogenous retroviruses (enJSRV) closely related to Jaagsiekte sheep retrovirus (JSRV) is ubiquitous in domestic and wild sheep and goats. Southern blot hybridization studies indicate that there is little active replication or movement of the enJSRV proviruses in these species. Two approaches were used to investigate the distribution of proviral loci in the sheep genome. Fluorescence in situ hybridization (FISH) to metaphase chromosome spreads using viral DNA probes was used to detect loci on chromosomes. Hybridization signals were reproducibly detected on seven sheep chromosomes and eight goat chromosomes in seven cell lines. In addition, a panel of 30 sheep-hamster hybrid cell lines, each of which carries one or more sheep chromosomes and which collectively contain the whole sheep genome, was examined for enJSRV sequences. DNA from each of the lines was used as a template for PCR with JSRV gag-specific primers. A PCR product was amplified from 27 of the hybrid lines, indicating that JSRV gag sequences are found on at least 15 of the 28 sheep chromosomes, including those identified by FISH. Thus, enJSRV proviruses are essentially randomly distributed among the chromosomes of sheep and goats. FISH and/or Southern blot hybridization on DNA from several of the sheep-hamster hybrid cell lines suggests that loci containing multiple copies of enJSRV are present on chromosomes 6 and 9. The origin and functional significance of these arrays is not known.     

Two new sheep transferrin variants and the effect of neuraminidase

Transferrin phenotypes were determined in six breeds of sheep by starch gel electrophoresis. Two new variants, Tf H_Czech and Tf K_Czech, were found and some evidence of their genetic control was obtained. Tf H_Czeeh was detected only in Sumava sheep; it has an intermediate mobility between Tf A and Tf B. Tf K_Czech was found only in Tsigais; it was localized between Tf B and Tf C. The frequencies of corresponding alleles were very low.
Individual transferrin variants (I, A, H_Czech, B, K_czech, C, D, E, and P) were treated with neuraminidase. Electrophoretic mobility of the strong band was decreased by two steps in each case. It suggests that in the strong Tf band two sialic acid residues are accessible to the enzyme.     

Isolation of subtelomeric DNA sequences labelling sheep and goat chromosome ends

Two techniques that make it possible to isolate telomere DNA are presented, using sheep as an example. The first technique is based upon the screening of a sheep BAC library with PCR amplified DNA segments preserved from high-power laser beam irradiation. Twenty-three BACs hybridising to 13 subtelomeric regions in sheep and goats were obtained (out of 27 in the sheep complement), of which 13 recognised more than one region, telomeric or not. Twenty-three microsatellites were isolated from these BACs and 22 were genetically mapped on the sheep international genetic map, always consistently with the cytogenetical localisation in 17 cases out of 22. These results are discussed. The second technique is based upon the selective cloning of subtelomeric enriched DNA. Preliminary results were obtained by this approach.     

Analysis of integration sites of Jaagsiekte sheep retrovirus in ovine pulmonary adenocarcinoma

Ovine pulmonary adenocarcinoma (OPA) is an infectious lung tumor of sheep caused by Jaagsiekte sheep retrovirus (JSRV). To test the hypothesis that JSRV insertional mutagenesis is involved in the oncogenesis of OPA, we cloned and characterized 70 independent integration sites from 23 cases of OPA. Multiple integration sites were identified in most tumors. BLAST analysis of the sequences did not disclose any potential oncogenic motifs or any identical integration sites in different tumors. Thirty-seven of the integration sites were mapped to individual chromosomes by PCR with a panel of sheep-hamster hybrid cell lines. Integration sites were found on 20 of the 28 sheep chromosomes, suggesting a random distribution. However, four integration sites from four different tumors mapped to chromosome 16. By Southern blot hybridization, probes derived from two of these sites mapped to within 5 kb of each other on normal sheep DNA. These sites were found within a single sheep bacterial artificial chromosome clone and were further mapped to only 2.5 kb apart, within an uncharacterized predicted gene and less than 200 kb from a mitogen-activated protein kinase-encoding gene. These findings suggest that there is at least one common integration site for JSRV in OPA and add weight to the hypothesis that insertional mutagenesis is involved in the development of this tumor.     

Evidence for altered cell-cycle traverse of the non-modal cells of the heteroploid MCa-11 line

Classic stem cell theory states that the growth of heteroploid cell populations is due to the proliferation of 'main stemline'cells with modal DNA content and chromosome number. Cells with non-modal DNA content and chromosome number are thought to be blocked and/or destroyed at mitosis. To test this, we studied two chromo-somally stable cell populations (mouse bone marrow and WCHE-5 cells) and one heteroploid, chromosomally diverse cell line (MCa-11). The heteroploid MCa-11 cells showed significant [³H]dT labelling for cells with DNA contents below the modal G_o/G₁ peak and above the modal G₂ peaks ( P <0.001). This was consistent with the presence of cells with the non-modal DNA content that were engaged in replicative DNA synthesis. A percentage labelled mitosis analysis showed that MCa-11 cells with non-modal DNA content and chromosome number were able to complete mitosis, although with prolonged pre-karyokinetic time. These results suggest that many non-modal cells present in heteroploid cell populations are capable of continued proliferation.     

Characteristics of some unclassifiable strains of staphylococci isolated from goats and sheep

A degoke , G.O. 1985. Characteristics of some unclassifiable strains of staphylococci isolated from goats and sheep. Journal of Applied Bacteriology 59 , 257–262.
Fourteen strains of catalase-positive, Gram-positive and coagulase-negative cocci that were sensitive to 20 μg/ml of furazolidone were isolated from goats and sheep. Two of the strains had glycerol with glucose teichoic acids whilst another possessed glycerol, glucose, glucosamine and acetylglucosamine teichoic acids. Six strains had peptidoglycan type L-Lys-Ala-Gly₄ peculiar to Staphylococcus sciuri and Staph. lentus but other phenotypic characters were different from those of Staph. sciuri and Staph. lentus . The guanine plus cytosine (G + C) content of the DNA determined for three of the strains examined ranged from 32.9–34.6 mol %. The coagulase-positive staphylococcal strain of caprine origin examined had glycerol and glucosarnine teichoic acids in addition to peptidogylcan type L-Lys-Gly_5–6. The characteristics of the strains of staphylococci described herein are different from those already described in the literature.     

Subtle population structuring within a highly vagile marine invertebrate, the veined squid Loligo forbesi, demonstrated with microsatellite DNA markers

Microsatellite DNA markers were applied for the first time in a population genetic study of a cephalopod and compared with previous estimates of genetic differentiation obtained using allozyme and mitochondrial DNA (mtDNA) markers. Levels of genetic variation detected with microsatellites were much higher than found with previous markers (mean number of alleles per locus=10.6, mean expected heterozygosity ( H _E)=0.79; allozyme H _E=0.08; mtDNA restriction fragment length polymorphism (RFLP) H _E=0.16). In agreement with previous studies, microsatellites demonstrated genetic uniformity across the population occupying the European shelf seas of the North East Atlantic, and extreme genetic differentiation of the Azores population ( R _ST/ F _ST=0.252/0.245; allozyme F _ST=0.536; mtDNA F _ST=0.789). In contrast to other markers, microsatellites detected more subtle, and significant, levels of differentiation between the populations of the North East Atlantic offshore banks (Rockall and Faroes) and the shelf population ( R _ST=0.048 and 0.057). Breakdown of extensive gene flow among these populations is indicated, with hydrographic (water depth) and hydrodynamic (isolating current regimes) factors suggested as possible barriers to migration. The demonstration of genetic subdivision in an abundant, highly mobile marine invertebrate has implications for the interpretation of dispersal and population dynamics, and consequent management, of such a commercially exploited species. Relative levels of differentiation indicated by the three different marker systems, and the use of measures of differentiation (assuming different mutation models), are discussed.     

Cytogenetic studies of four South American species of Paullinia L. (Sapindaceae)

Four South American species of Paullinia ( P. elegans , P. meliaefolia , P. pinnata , and P. rhomboidea ) were compared using conventional chromosome staining, C-Giemsa and C-chromomycin A₃/4',6-diamidino-2-phenylindole (C-CMA₃/DAPI) banding, and fluorescence in situ hybridization (FISH) with a 45S ribosomal DNA (rDNA) probe. All species showed a somatic complement of 2 n  = 24 chromosomes, agreeing with earlier records in some cases, and showing a tendency for the chromosome number to be conserved in this genus. The chromosome number of P. rhomboidea is a new report. The karyotypes differed in chromosome size and degree of karyotype asymmetry. The chromosomal band patterns and location of the 45S rDNA sites are reported for the first time in the genus. Terminal C-CMA₃ bands were associated with the 45S rDNA sites, but varied in number and size between the species. The occurrence of other C-Giemsa bands that were not revealed by CMA₃ suggests that more than one family of repetitive DNA may be involved in karyotype differentiation. The systematic implications of these results on the infrageneric relationships are discussed.   © 2007 The Linnean Society of London . Botanical Journal of the Linnean Society , 2007, 154 , 313–320.     

The application of AFLP fingerprinting to construct a YAC contig containing ADH2 and MTP on sheep chromosome 6.

The low density of genetic markers on livestock maps limits progress in positional cloning projects. We demonstrate a strategy of combining comparative mapping with AFLP fingerprinting to develop physical maps in a defined region of the sheep genome. Sequence tagged sites for alcohol dehydrogenase 2 (ADH2) and microsomal triglyceride transfer protein (MTP) were developed and used to screen a sheep yeast artificial chromosome (YAC) library. Nine YACs were identified containing the microsatellite marker BM1329 and either ADH2 or MTP. Additional markers in the region were not available, and AFLP analysis was developed to identify sheep-specific bands within the YACs to determine their degree of overlap. Fourteen bands common to more than one YAC were analysed and provided the markers necessary to develop a YAC contig containing the three STS markers. One YAC (yac260B5) containing all three markers (ADH2, MTP, and BM1329) was mapped to sheep chromosome 6q1.6-->q1.8 by FISH analysis.     

Thirteen loci physically assigned to sheep Chromosome 2 by cell hybrid analysis and in situ hybridization

Sheep x hamster cell hybrids containing sheep metacentric Chromosome (Chr) 2 were produced by fusing blood leukocytes from normal sheep with hamster auxotrophic Ade F-minus mutants. Cell clones that were isocitrate dehydrogenase 1 (IDH1) positive were cytogenetically characterized, confirming that they contained sheep Chr 2. The following loci were newly assigned by Southern hybridization to sheep Chr 2: lipoprotein lipase (LPL), glycoprotein-4-beta galactosyltransferase 2 (GGTB2), neurofilament light polypeptide (68 kDa; NEFL), surfactant-associated protein 2 (SFTP2), lymphocyte-specific protein tyrosine kinase (LCK), and nebulin (NEB). These new assignments and the in situ localization of gelsolin (GSN) to sheep Chr 2pter-p24 are consistent with the predicted homology of cattle Chr 8 (U18) with sheep Chr 2p, and of cattle Chr 2 (U17) with sheep 2q. In addition, the assignment by cell hybrid analysis of loci previously mapped to Chr 2 in sheep, viz., cholinergic receptor, nicotinic, delta polypeptide (CHRND), collagen type III alpha 1 (COL3A1), fibronectin 1 (FN1), isocitrate dehydrogenase (IDH1), and villin 1 (VIL1), confirmed the localization of sheep syntenic group U11 to this chromosome. By nutritional selection and complementation of the hamster auxotrophic Ade F mutation, the multifunctional enzyme locus phosphoribosylaminoimidazolecarboxamide formyltransferase (AICAR transformylase)/IMP cyclohydrolase (inosinicase) (provisionally given the symbol PRACFT) has also been newly assigned to sheep Chr 2. This report significantly extends the number of loci physically mapped to sheep Chr 2 and confirms its close homology with cattle Chrs 2 and 8.     

Contribution to the physically anchored linkage map of the pig

Thirty-three microsatellites have been mapped on the PiGMaP porcine genetic map. By comparison with the previously published PiGMaP maps, the maps of chromosome 2 (140 cM/70 cM) and chromosome 3 (180 cM/110 cM) were extended and new markers were mapped on the p-arm extremity of chromosome 7 and on the centromeric extremity of chromosome 15. New orders are proposed for markers on chromosomes 3 and 17. Six microsatellites isolated from cosmids were also localized on the cytogenetic map by fluorescent in situ hybridization. We tested the subcloning ligation mixture–polymerase chain reaction (SLiM-PCR) method for isolating microsatellites from cosmids. Subcloning is more effective when the cosmid harbours several microsatellites whereas SLiM-PCR is more straightforward when the cosmid contains a single microsatellite. Fifteen anonymous microsatellites were regionally assigned by using a hybrid cell panel. For map integration, the determination of a regional assignment of anonymous microsatellites by using a hybrid cell panel offers an alternative to microsatellite isolation from cosmids and their localizations by in situ hybridization.     

Isolation and mapping of polymorphic microsatellites in cattle

Summary
A partial plasmid library with bovine genomic inserts of about 500 basepairs was screened with a (dC-dA)_n-(dG-dT)_n oligonucleotide probe for the repeated nucleotide motif (CA)_n. Eleven positive clones (0.3% of all colonies screened) were discovered and were subsequently isolated and sequenced. Eight microsatellite loci were analysed, one with eight alleles, one with seven alleles, three with six alleles, one with three alleles and two with two alleles. Six of these microsatellites were mapped by PCR-analysis of a panel of somatic hybrid lines.     

A comparative linkage and physical map of bovine chromosome 24 with human chromosome 18

Polymorphic microsatellites have been developed in the vicinity of nine genes on bovine chromosome (BTA) 24, all orthologous to genes on human chromosome (HSA) 18. The microsatellites have been isolated from bacterial and yeast artificial chromosome clones containing the genes. A linkage map was developed including these polymorphic markers and four anonymous, published microsatellites. Yeast artificial chromosomes containing six of these genes have also been mapped using fluorescent in situ hybridization (FISH), thereby tying the linkage map together with the physical map of BTA24. Comparing gene location on HSA18 and BTA24 identifies four regions of conserved gene order, each containing at least two genes. These genes identify six regions of conserved order between human and mouse, two more than in the human-bovine comparison. The breakpoints between regions of conserved order for human-bovine are also breakpoints in the human-mouse comparison. The centromere identifies a fifth conserved region if the BTA24 centromere is orthologous with the HSA18 centromere. Received: 17 September 1998 / Accepted: 4 December 1998