Analgesic (omega)-conotoxins CVIE and CVIF selectively and voltage-dependently block recombinant and native N-type calcium channels.

Neuronal (N)-type Ca(2+) channel-selective omega-conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new omega-conotoxins, CVIE and CVIF, were discovered from a Conus catus cDNA library. Both conopeptides potently displaced (125)I-GVIA binding to rat brain membranes. In Xenopus laevis oocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba(2+) currents through recombinant N-type (alpha1(B-b)/alpha(2)delta1/beta(3)) Ca(2+) channels. Recovery from block increased with membrane hyperpolarization, indicating that CVIE and CVIF have a higher affinity for channels in the inactivated state. The link between inactivation and the reversibility of omega-conotoxin action was investigated by creating molecular diversity in beta subunits: N-type channels with beta(2a) subunits almost completely recovered from CVIE or CVIF block, whereas those with beta(3) subunits exhibited weak recovery, suggesting that reversibility of the omega-conotoxin block may depend on the type of beta-subunit isoform. In rat dorsal root ganglion sensory neurons, neither peptide had an effect on low-voltage-activated T-type channels but potently and selectively inhibited high voltage-activated N-type Ca(2+) channels in a voltage-dependent manner. In rat spinal cord slices, both peptides reversibly inhibited excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. Homology models of CVIE and CVIF suggest that omega-conotoxin/voltage-gated Ca(2+) channel interaction is dominated by ionic/electrostatic interactions. In the rat partial sciatic nerve ligation model of neuropathic pain, CVIE and CVIF (1 nM) significantly reduced allodynic behavior. These N-type Ca(2+) channel-selective omega-conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.

QTL for meat tenderness in the M. longissimus lumborum of cattle.

Meat tenderness has been difficult to improve using standard genetic selection. Marker assisted selection holds great promise if markers for meat tenderness can be identified. Here, we report quantitative trait loci (QTL) for beef tenderness identified in 598 animals of three Charolais-Brahman x Belmont Red pedigrees after screening the whole genome using 183 DNA markers. In addition to the usual Warner-Bratzler peak force measurements, tenderness was also measured using compression, adhesion and pressure-heat-treated peak force. Three QTL for meat tenderness in the M. longissimus lumborum muscle were found, two of which have not been reported before. One is located in the HEL9-CSSM47 interval on bovine chromosome 8 with a LOD of 3.1 and an effect of 1.02 phenotypic standard deviations for tensile strength of cooked muscle as measured by adhesion. A second QTL is located near CSRM60 on bovine chromosome 10 with a LOD of 2.4 and an effect of 0.48 phenotypic standard deviations for compression. The third QTL is in a region of bovine chromosome 7 that has previously been reported to have a QTL affecting peak force. This region also shows effects on compression and a combined tenderness index. These QTL are all for the myofibrillar component of meat tenderness. No QTL were found for pressure-treated peak force, which is an estimate of the connective tissue component muscle of meat tenderness.

muO-conotoxin MrVIB selectively blocks Nav1.8 sensory neuron specific sodium channels and chronic pain behavior without motor deficits.

The tetrodotoxin-resistant voltage-gated sodium channel (VGSC) Na(v)1.8 is expressed predominantly by damage-sensing primary afferent nerves and is important for the development and maintenance of persistent pain states. Here we demonstrate that muO-conotoxin MrVIB from Conus marmoreus displays substantial selectivity for Na(v)1.8 and inhibits pain behavior in models of persistent pain. In rat sensory neurons, submicromolar concentrations of MrVIB blocked tetrodotoxin-resistant current characteristic of Na(v)1.8 but not Na(v)1.9 or tetrodotoxin-sensitive VGSC currents. MrVIB blocked human Na(v)1.8 expressed in Xenopus oocytes with selectivity at least 10-fold greater than other VGSCs. In neuropathic and chronic inflammatory pain models, allodynia and hyperalgesia were both reduced by intrathecal infusion of MrVIB (0.03-3 nmol), whereas motor side effects occurred only at 30-fold higher doses. In contrast, the nonselective VGSC blocker lignocaine displayed no selectivity for allodynia and hyperalgesia versus motor side effects. The actions of MrVIB reveal that VGSC antagonists displaying selectivity toward Na(v)1.8 can alleviate chronic pain behavior with a greater therapeutic index than nonselective antagonists.

The assignment by linkage mapping of four genes from human chromosome 22 to bovine chromosome 5 and 17.

Polymerase chain reaction oligonucleotides were designed to amplify bovine specific sequences for four genes that are located on human chromosome 22 (HSA22): crystallin beta A4 (CRY B A4), parvalbumin (PVALB), tissue inhibitor of metalloproteinase 3 (TIMP3) and matrix metalloproteinase 11 (MMP11). Single strand conformation analysis of these bovine gene fragments defined polymorphisms within a population of three large half-sib families of three F1 Charolais x Brahman sires and a composite herd comprising an equal proportion of Africander, Brahman, Hereford and Shorthorn breeds (CSIRO pedigree). The DNA marker genotypes were used to define linkage associations to other DNA markers already placed on the CSIRO linkage map. The genes TIMP3 and PVALB were assigned to BTA5 and CRYbetaA4 and MMP11 to BTA17.

An enhanced linkage map of the sheep genome comprising more than 1000 loci.

A medium-density linkage map of the ovine genome has been developed. Marker data for 550 new loci were generated and merged with the previous sheep linkage map. The new map comprises 1093 markers representing 1062 unique loci (941 anonymous loci, 121 genes) and spans 3500 cM (sex-averaged) for the autosomes and 132 cM (female) on the X chromosome. There is an average spacing of 3.4 cM between autosomal loci and 8.3 cM between highly polymorphic [polymorphic information content (PIC) > or = 0.7] autosomal loci. The largest gap between markers is 32.5 cM, and the number of gaps of > 20 cM between loci, or regions where loci are missing from chromosome ends, has been reduced from 40 in the previous map to 6. Five hundred and seventy-three of the loci can be ordered on a framework map with odds of > 1000 : 1. The sheep linkage map contains strong links to both the cattle and goat maps. Five hundred and seventy-two of the loci positioned on the sheep linkage map have also been mapped by linkage analysis in cattle, and 209 of the loci mapped on the sheep linkage map have also been placed on the goat linkage map. Inspection of ruminant linkage maps indicates that the genomic coverage by the current sheep linkage map is comparable to that of the available cattle maps. The sheep map provides a valuable resource to the international sheep, cattle, and goat gene mapping community.

Novel omega-conotoxins from Conus catus discriminate among neuronal calcium channel subtypes.

omega-Conotoxins selective for N-type calcium channels are useful in the management of severe pain. In an attempt to expand the therapeutic potential of this class, four new omega-conotoxins (CVIA-D) have been discovered in the venom of the piscivorous cone snail, Conus catus, using assay-guided fractionation and gene cloning. Compared with other omega-conotoxins, CVID has a novel loop 4 sequence and the highest selectivity for N-type over P/Q-type calcium channels in radioligand binding assays. CVIA-D also inhibited contractions of electrically stimulated rat vas deferens. In electrophysiological studies, omega-conotoxins CVID and MVIIA had similar potencies to inhibit current through central (alpha(1B-d)) and peripheral (alpha(1B-b)) splice variants of the rat N-type calcium channels when coexpressed with rat beta(3) in Xenopus oocytes. However, the potency of CVID and MVIIA increased when alpha(1B-d) and alpha(1B-b) were expressed in the absence of rat beta(3), an effect most pronounced for CVID at alpha(1B-d) (up to 540-fold) and least pronounced for MVIIA at alpha(1B-d) (3-fold). The novel selectivity of CVID may have therapeutic implications. (1)H NMR studies reveal that CVID possesses a combination of unique structural features, including two hydrogen bonds that stabilize loop 2 and place loop 2 proximal to loop 4, creating a globular surface that is rigid and well defined.

Molecular cloning and expression in Escherichia coli of an aquaporin-like gene from adult buffalo fly (Haematobia irritans exigua).

A gene fragment encoding a putative member of the aquaporin gene family was amplified using cDNA prepared from unfed adult buffalo fly poly(A)+ RNA and degenerate PCR primers designed from highly conserved regions of amino acids found in all members of the aquaporin gene family. This PCR product was labelled with digoxigenin-dUTP and used as a probe to screen a lambdagt-11 cDNA library constructed from unfed adult buffalo fly. One positively hybridizing clone (AqpBF1), contained an insert of 1878 bp, and DNA sequence analysis revealed an open reading frame of 753 bp encoding a polypeptide of predicted Mr = 26 163 Da. Comparison of the AqpBF1 deduced protein sequence with the GenBank database revealed significant homology to many aquaporin genes, including 72% identity with a partial DNA sequence encoding a member (DRIP) of the MIP protein family isolated from Drosophila melanogaster. The most closely related, full-length, GenBank sequence was an aquaporin gene isolated from the digestive tract of the sap-sucking insect Cicadella viridis, which was 53% identical to the buffalo fly AqpBF1 protein sequence. The full-length coding sequence of AqpBF1 was cloned into the (His)6-fusion vector, pQE10, and the recombinant protein was expressed in Escherichia coli following induction by IPTG. The recombinant (His)6-fusion protein was localized predominantly in the membrane fraction of E. coli. The protein was solubilized from E. coli membranes with n-octyl beta-D-glucopyranoside and purified by affinity chromatography on a Ni++-sepharose column in the presence of detergent.

Genetic diversity of Asian water buffalo (Bubalus bubalis): mitochondrial DNA D-loop and cytochrome b sequence variation.

Swamp and river buffalo mitochondrial DNA (mtDNA) was sequenced for 303 bp of the cytochrome b gene for 54 animals from 14 populations, and for 158 bp of the D-loop region for 80 animals from 11 populations. Only one cytochrome b haplotype was found in river buffalo. Of the four haplotypes identified in swamp buffalo, one found in all populations is apparently ancestral both to the other swamp haplotypes and to the river haplotype. The phylogenetic relationships among the 33 D-loop haplotypes, with a cluster of 11 found in swamp buffalo only, also support the evolution of domesticated swamp and river buffalo from an ancestral swamp-like animal, most likely represented today by the wild Asian buffalo (Bubalus arnee). The time of divergence of the swamp and river types, estimated from the D-loop data, is 28,000 to 87,000 years ago. We hypothesise that the species originated in mainland south-east Asia, and that it spread north to China and west to the Indian subcontinent, where the rive type evolved and was domesticated. Following domestication in China, the domesticated swamp buffalo spread through two separate routes, through Taiwan and the Philippines to the eastern islands of Borneo and Sulawesi, and south through mainland south-east Asia and then to the western islands of Indonesia.

Classification of the southern African sanga and east African shorthorned zebu.

Humped African cattle, which are differentiated into zebu and sanga types, have traditionally been classified as Bos indicus. This paper discusses existing evidence and presents new evidence supporting the classification of southern African sangas as Bos taurus and East African zebus as 'taurindicus'. Classification is based on karyotype, frequencies of DNA markers and protein polymorphisms. The Boran, an East African zebu, has an acrocentric Y chromosome typical of Bos indicus. The southern African sanga breeds have a submetacentric Y chromosome typical of Bos taurus. Frequencies of four DNA markers support the hypothesis that the Tuli, a southern African sanga, had taurine ancestors and the Boran had both taurine and indicine ancestors. Frequencies for several protein polymorphisms strongly suggest that southern African sangas have more in common with taurine than with indicine breeds, while East African zebus are an admixture of African taurine and Asian indicine breeds.

A medium-density genetic linkage map of the bovine genome.

A cattle genetic linkage map was constructed which covers more than 95 percent of the bovine genome at medium density. Seven hundred and forty six DNA polymorphisms were genotyped in cattle families which comprise 347 individuals in full sibling pedigrees. Seven hundred and three of the loci are linked to at least one other locus. All linkage groups are assigned to chromosomes, and all are orientated with regards to the centromere. There is little overall difference in the lengths of the bull and cow linkage maps although there are individual differences between maps of chromosomes. One hundred and sixty polymorphisms are in or near genes, and the resultant genome-wide comparative analyses indicate that while there is greater conservation of synteny between cattle and humans compared with mice, the conservation of gene order between cattle and humans is much less than would be expected from the conservation of synteny. This map provides a basis for high-resolution mapping of the bovine genome with physical resources such as Yeast and Bacterial Artificial Chromosomes as well as providing the underpinning for the interpolation of information from the Human Genome Project.

Denaturing gradient gel electrophoresis detection of polymorphism in a PCR fragment of sheep epidermal growth factor gene.

The ovine map is not yet well-developed, which represents a problem when looking for markers of a region of interest in sheep. A means of circumventing this is to use comparative mapping. In this study primers were determined using consensus sequences for the epidermal growth factor gene of humans, rats and mice, and an ovine epidermal growth factor gene fragment was amplified by polymerase chain reaction (PCR). A new set of specific ovine primers was chosen to study the polymorphism of this DNA fragment by denaturing gradient gel electrophoresis. Eighty-four individuals belonging to seven sheep breeds were studied with this technique and four alleles were detected. The heterozygosity rate was 0.57. Family analysis showed mendelian inheritance of the alleles. Usually, genetic analysis of type-I loci used in the comparative mapping is based on the detection of restriction fragment length polymorphisms in sheep DNA using cDNA probes from other species. Our work shows that another method, based on PCR and denaturing gradient gel electrophoresis techniques, can be efficiently used.

Genetic markers for the Booroola fecundity (Fec) gene in sheep.

Animals from the Booroola line of Australian Merino sheep are characterized by a high ovulation rate that can be attributed to the presence of a codominant allele (FecB). The specific function of the gene has not been identified. Effective use of the trait within the sheep breeding industry requires one or more genetic markers that can distinguish between alternative alleles at the locus Fec. With a combination of DNA minisatellite markers and polymorphic protein markers, a cluster of seven minisatellite fragments has been identified as being linked to the Fec gene and to the ovine A blood group locus. The minisatellite fragments have been derived from multilocus probes and hence cannot be used to define the chromosomal location of the Fec gene or to serve as diagnostic markers for Fec. The derivation of cloned single locus markers from the minisatellite fragments will enable finer scale mapping of the Fec and the A blood group locus in sheep.

Isolation and mapping of a single-locus minisatellite sequence marker to 1q36-qter and synteny group 10 in cattle and to 1q36-qter in sheep.

A minisatellite sequence (RD1613) was isolated from a bovine cosmid genomic library and its chromosomal location determined in cattle and sheep. In cattle, somatic cell hybrid panel analysis assigned RD1613 to the syntenic group U10 with a concordancy of 100%. In situ hybridization placed RD1613 onto bovine chromosome 1 in the region of bands q36-qter. This is the first in situ localization to chromosome 1 in cattle and allows the provisional assignment of syntenic group U10 to this chromosome. It was also found that RD1613 hybridized strongly to sheep genomic DNA. In situ hybridization localized RD1613 to sheep chromosome 1q36-qter, which is consistent with homology between cattle chromosome 1 and sheep chromosome 1q. The RD1613 probe detects a polymorphic single locus marker (designated D1S1) in both cattle and sheep and will be very useful in linkage studies.

Microsatellite mapping of a gene affecting horn development in Bos taurus.

The presence or absence of horns in Bos taurus is thought to be under the genetic control of the autosomal polled locus, characterized by two alleles: P dominant over p, and causing the polled or hornless phenotype. We have demonstrated genetic linkage between the polled locus and two microsatellite markers, GMPOLL-1 and GMPOLL-2, and have assigned the corresponding linkage group to bovine chromosome 1. This confirms the existence of the postulated polled locus and the hypothesized inheritance pattern. It will allow marker assisted selection for the polledness trait in breeding programs and is a first step towards positional cloning and molecular study of a gene that has been subjected to both natural and artificial selection.