Construction of a new porcine whole-genome framework map using a radiation hybrid panel

We have constructed a radiation hybrid (RH) map of the porcine genome using an RH panel generated by an irradiation dose of 5000-rad (Sus scrofa radiation hybrid map, SSRH map). Normal porcine aortic endothelial cells were irradiated and fused with a thymidine kinase-deficient mouse cell line, L-M (TK-). A total of 110 cell lines were selected and used for further analysis. Among 1091 microsatellite (MS) markers selected for mapping, 842 markers (77%) could be typed on the panel. The framework map comprised 342 MS markers and an additional 247 MS markers were then added to generate the whole-genome map. The average retention frequency for the data set was 30.6%. The total map length was 5596.2 centiRay (cR). Using an estimated physical length of 2718 Mbp, the average ratio between cR and physical distance over the porcine genome was estimated to be 0.49 Mb/cR.     

Characterisation of a bovine/murine hybrid cell panel informative for all bovine autosomes

A bovine/murine hybrid cell panel consisting of 57 cell lines was typed with 124 markers by PCR, Southern hybridisation and isozyme analysis in order to establish its utility as a resource for genome mapping. All bovine chromosomes, including the sex chromosomes were represented in the panel. Computerised analysis of syntenies indicated that there are no cell lines containing only a single bovine chromosome. The panel was used to map 10 new bovine microsatellite markers, and the MYL6 and CPE genes. This panel is informative for all bovine chromosomes other than the sex-specific region of the X chromosome and can be used in synteny mapping studies. At present, due to the relatively small number of markers typed, the resolution of the panel does not go beyond the chromosomal level.     

Mapping of 1400 expressed sequence tags in the bovine genome using a somatic cell hybrid panel

A bovine/hamster hybrid cell panel consisting of 30 independent hybrids was developed to locate genes. Polymerase chain reaction analysis of 279 microsatellites on the cattle linkage map in this panel revealed the presence of all chromosomes in either entire or fragmented form. Among primer pairs prepared from bovine 3'-expressed sequence tags (ESTs), 1400 ESTs were assigned to specific chromosomes, of which 1303 were newly assigned in this study, and mapped 854 (61%) to 1 of 192 chromosomal segments using this panel. The regional mapping of new genes to cattle chromosomes can be rapidly achieved using this panel.     

A radiation hybrid map of bovine chromosome 24 and comparative mapping with human chromosome 18

We present herein a bovine chromosome 24 (BTA24) radiation hybrid (RH) map using 40 markers scored on a panel of 90 RHs. Of these markers, 29 loci were ordered with odds of at least 1000:1 in a framework map. An average retention frequency of 17.4% was observed, with relatively higher frequencies near the centromere. The length of the comprehensive map was 640 centiray5000 (cR5000) with an average marker interval of approximately 17.3 cR5000. The observed locus order is generally consistent with currently published bovine linkage and physical maps. Nineteen markers were either Type I loci or closely associated with expressed sequences and thus could be used to compare the BTA24 RH map with human mapping information. All genes located on BTA24 were located on human chromosome 18, and previously reported regions of conserved synteny were extended. The comparative data revealed the presence of at least six conserved regions between these chromosomes.     

ChickRH6: a chicken whole-genome radiation hybrid panel

As a first step towards the development of radiation hybrid maps, we have produced a radiation hybrid panel in the chicken by fusing female embryonic diploid fibroblasts irradiated at 6 000 rads with HPRT-deficient hamster Wg3hCl₂ cells. Due to the low retention frequency of the chicken fragments, a high number of clones was produced from which the best ones were selected. Thus, 452 fusion clones were tested for retention frequencies with a panel of 46 markers. Based on these results, 103 clones with a mean marker retention of 23.8% were selected for large scale culture to produce DNA in sufficient quantities for the genotyping of numerous markers. Retention frequency was tested again with the same 46 markers and the 90 best clones, with a final mean retention frequency of 21.9%, were selected for the final panel. This panel will be a valuable resource for fine mapping of markers and genes in the chicken, and will also help in building BAC contigs.     

A high-resolution radiation hybrid map of bovine chromosome 5q1.3-q2.5 compared with human chromosome 12q

In this study we present a comprehensive 3000-rad radiation hybrid map on bovine chromosome 5 (BTA5) of a region between 12.8 and 74.0 cM according to the linkage map, which contains a quantitative trait loci for ovulation rate. We mapped 28 gene-associated sequence tagged site markers derived from sequences of bovine BAC clones and 10 microsatellite markers to the BTA5 region. In comparison with HSA12q, four blocks of conserved synteny were apparent showing three chromosomal breakpoints and two inversions in this segment of BTA5. Therefore, we have improved breakpoint resolution in the human-bovine comparative map, which enhances the determination of candidate genes underlying traits of interest mapped to BTA5.     

A radiation hybrid map of bovine chromosome one

Radiation hybrid (RH) mapping has proven to be an extremely powerful approach to constructing high density maps of human chromosomes and is experiencing increased use in other animals, including cattle. A 5000 rad bovine whole-genome radiation hybrid panel was recently constructed in order to integrate existing cattle linkage maps with evolutionarily conserved genes and provide high resolution comparative maps relative to humans and mice. We utilized this panel to construct a 19 marker framework map of bovine chromosome 1 (BTA1), which included 8 Type I loci and 11 Type II loci ordered with at least 1000:1 odds. A 35 marker comprehensive map including 15 Type I loci and 20 Type II loci was also produced. Of the 15 Type I loci ordered on the comprehensive map, three are ordered on HSA3 and five are ordered in three blocks on HSA21 on the human cytogenetic maps.     

A 12 000-rad whole-genome radiation hybrid panel in sheep: application to the study of the ovine chromosome 18 region containing a QTL for scrapie susceptibility

Whole-genome radiation hybrid (RH) panels have been constructed for several species, including cattle. RH panels have proven to be an extremely powerful tool to construct high-density maps, which is an essential step in the identification of genes controlling important traits, and they can be used to establish high-resolution comparative maps. Although bovine RH panels can be used with ovine markers to construct sheep RH maps based on bovine genome organization, only some (c. 50%) of the markers available in sheep can be successfully mapped in the bovine genome. So, with the development of genomics and genome sequencing projects, there is a need for a high-resolution RH panel in sheep to map ovine markers. Consequently, we have constructed a 12 000-rad ovine whole-genome RH panel. Two hundred and eight hybrid clones were produced, of which 90 were selected based on their retention frequency. The final panel had an average marker retention frequency of 31.8%. The resolution of this 12 000-rad panel (SheepRH) was estimated by constructing an RH framework map for a 23-Mb region of sheep chromosome 18 (OAR18) that contains a QTL for scrapie susceptibility.     

Construction of a river buffalo (Bubalus bubalis) whole-genome radiation hybrid panel and preliminary RH mapping of chromosomes 3 and 10

The buffalo (Bubalus bubalis) is a source of milk and meat, and also serves as a draft animal. In this study, a 5000-rad whole-genome radiation hybrid (RH) panel for river buffalo was constructed and used to build preliminary RH maps for BBU3 and BBU10 chromosomes. The preliminary maps contain 66 markers, including coding genes, cattle expressed sequence tags (ESTs) and microsatellite loci. The RH maps presented here are the starting point for mapping additional loci that will allow detailed comparative maps between buffalo, cattle and other species whose genomes may be mapped in the future. A large quantity of DNA has been prepared from the cell lines forming the river buffalo RH panel and will be made publicly available to the international community both for the study of chromosome evolution and for the improvement of traits important to the role of buffalo in animal agriculture.     

A higher resolution radiation hybrid map of bovine chromosome 13

In this paper, we present a radiation hybrid framework map of BTA13 composed of nine microsatellite loci, six genes and one EST. The map has been developed using a recently constructed 12''000 rad bovine-hamster whole-genome radiation hybrid panel. Moreover, we present a comprehensive map of BTA13 comprising 72 loci, of which 45 are microsatellites, 20 are genes and seven are ESTs. The map has an estimated length of 2694.7 cR_12''000. The proposed order is in general agreement with published maps of BTA13. Our results only partially support previously published information of five blocks of conserved gene order between cattle and man. We found no evidence for the existence of an HSA20 homologous segment of coding DNA on BTA13 located centromeric of a confirmed HSA10 homologous region. The present map increases the marker density and the marker resolution on BTA13 and enables further insight into the evolutionary development of the chromosome as compared to man.     

New gene assignments using a complete, characterized sheep-hamster somatic cell hybrid panel

The generation and characterization of new sheep-hamster cell hybrids is reported from the fusion of sheep white blood cells with six different hamster auxotrophs. Selection from these and previously generated cell hybrids has led to the production of a panel of 30 hybrids covering the complete sheep genome of 28 chromosomes. Over half of the cell hybrids in this panel contain single sheep chromosomes. By complementation, the following new assignments have been made using the panel: phosphoribosyl N-formylglycinamide amidotransferase (PRFGA) to sheep chromosome (chr) 11; adenylosuccinate synthetase (ADSS) to sheep chr 12; adenylosuccinate lyase (ADSL) to sheep chr 3q; 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS) to sheep chr 16; dihydrofolate reductase (DHFR) to sheep chr 5; and adenine phosphoribosyltransferase (APRT) to sheep chr 14. The gene phosphoribosylaminoinidazole-carboxamide formyltransferase/Inosinicase (PRACFT) has now been regionally assigned to chr 2q. By isozyme analysis, phosphogluconate dehydrogenase (PGD) was assigned to sheep chr 12, anchoring the sheep syntenic group U1 to this chromosome, and mannose phosphate isomerase (MPI) was assigned to sheep chr 18. Furthermore, the chromosomal assignment of 110 microsatellites was confirmed using this cell panel.     

Systematic chromosomal deletion of bacterial ribosomal protein genes

Detailed studies of ribosomal proteins (RPs), essential components of the protein biosynthetic machinery, have been hampered by the lack of readily accessible chromosomal deletions of the corresponding genes. Here, we report the systematic genomic deletion of 41 individual RP genes in Escherichia coli, which are not included in the Keio collection. Chromosomal copies of these genes were replaced by an antibiotic resistance gene in the presence of an inducible, easy-to-exchange plasmid-born allele. Using this knockout collection, we found nine RPs (L15, L21, L24, L27, L29, L30, L34, S9, and S17) nonessential for survival under induction conditions at various temperatures. Taken together with previous results, this analysis revealed that 22 of the 54 E. coli RP genes can be individually deleted from the genome. These strains also allow expression of truncated protein variants to probe the importance of RNA-protein interactions in functional sites of the ribosome. This set of strains should enhance in vivo studies of ribosome assembly/function and may ultimately allow systematic substitution of RPs with RNA.     

A radiation hybrid map of chicken chromosome 15

We have constructed a radiation hybrid (RH) map of chicken chromosome (GGA) 15. This map can be used as a resource to efficiently map genes to this chromosome. The map has been developed using a 6000 rad chicken-hamster whole-genome radiation hybrid panel (ChickRH6). In total, six microsatellite loci, 18 sequence tagged sites (STSs) from BAC end sequences and 11 genes were typed on the panel. The initial framework map comprised eight markers, and an additional 23 markers were then added to generate the final map. The total map length was 334 centiRay6000 (cR6000). The estimated retention frequency for the data set was 18%. Using an estimated physical length of 21 Mb, the ratio between cR6000 and physical distance over GGA15 was estimated to be 0.063 Mb/cR6000. The present map increases the marker density and the marker resolution on GGA15 and enables fast mapping of new chicken genes homologous to genes from human chromosomes 12 and 22.     

Mapping of bovine prolactin and rhodopsin genes in hybrid somatic cells

The genes encoding bovine prolactin and rhodopsin were assigned to syntenic groups on the basis of hybridization of DNA from a panel of bovine-hamster hybrid somatic cell lines with cloned prolactin and rhodopsin gene probes. Prolactin was found to be syntenic with previously mapped glyoxalase, BoLA and 21-hydroxylase genes, establishing a syntenic conservation with human chromosome 6. The presence of bovine rhodopsin sequences among the various hybrid cell lines was not concordant with any gene previously assigned to one of the 23 defined autosomal syntenic groups. Thus, rhodopsin marks a new bovine syntenic group, U24, leaving only five cattle autosomes unmarked by at least one biochemical or molecular marker.     

Mapping and SNP analysis of bovine candidate genes for meat and carcass quality

The chromosomal localization of 13 bovine genes was determined using radiation hybrid (RH) mapping. The RH mapping data were in agreement with published data using either linkage, somatic cell hybrids or in situ hybridization. Mutation analysis using single-stranded conformational polymorphism, restriction fragment length polymorphism (RFLP) and sequencing revealed 13 SNPs in four different genes, namely carboxypeptidase E (CPE), uncoupling protein 2 (UCP2), single-minded (Drosophila) homologue 1 (SIM1) and methallothionein IIa (MT2A). With the exception of one mutation in CPE, all other mutations are either silent or are situated in an intron. The polymerase chain reaction RFLP was used on unrelated animals from different cattle breeds for determing allelic distribution.     

DNA sequence of the ribosomal protein genes rp12 and rps19 from a plant-pathogenic mycoplasma-like organism

A segment of a ribosomal protein operon from a plant-pathogenic mycoplasma-like organism (MLO) was cloned and sequenced, to provide supplemental molecular data pertinent to the question of MLO phylogeny. Comparisons of the deduced amino acid sequences indicate an ancient divergence of the MLOs from the animal-pathogenic mycoplasmas. Furthermore, although both the plant and animal pathogens have A-T rich genomes, a fundamental difference was apparent in their usage of the UGA codon.