Further characterization of a somatic cell hybrid panel: ten new assignments to the bovine genome

Thirty-six partially characterized hamster-bovine hybrid cell lines were used for the determination of synteny groups. Sixteen additional reference loci, selected for their coverage of the bovine genome, were analysed on these hybrid cells. This increases to 25 the number of synteny groups detected. This panel was then used to make synteny assignments for 10 additional loci, eight by Southern blotting (COL1A1, COL1A2, FAS, CTSB, CTSL, CHRNG, HEXB and HTR1A) and two by polymerase chain reaction (PCR) amplification (HRH1 and ETH1112), These loci were assigned to international synteny groups U12 (HRH1), U13 (COL1A2), U17 (CHRNG), U21 (COL1A1, FAS), U29 (ETHI1112), to chromosome 20 (U14 or U25) for HEXB and HTR1A, and to the same local synteny group (A), which is probably U18, for CTSB and CTSL. For three loci already mapped in humans (COL1A1, COL1A2 and CHRNG), the present results are in accordance with the predictions based on comparative mapping between the human and bovine species.     

Assignment of bovine synteny group U2 to chromosome 9

One cosmid containing a microsatellite (INRA144, D9S14) was assigned to bovine synteny group U2 by somatic cell genetics and localized to bovine chromosome 9q25 by fluorescent in situ hybridization. These results permitted the assignment of one more synteny group to a bovine chromosome. There are now 22 out of 31 bovine synteny groups which are related to a chromosome. The mapping data have been entered in the BovMap database, Jouy-en-Josas, France.     

Sheep gene mapping: synteny between COL2AI and the LDHB-PEPB-TPI-GAPD-LALBA-IGFl group

DNA extracted from 21 hamster-sheep hybrid cell lines was subjected, after Southern blotting, to hybridization with a type-II alpha 1 collagen genomic probe (COL2A1). The corresponding locus was found to be syntenic with the LDHB-PEPB-TPI-GAPD-LALBA-IGF1 group in sheep.     

Direct characterization of bovine microsatellites from cosmids: polymorphism and synteny mapping

A (TG)₈ oligonucleotide probe was used to screen 186 cosmids from a commercial bovine cosmid library. Of the 56 positive discovered, 7 were sequenced in the region of the microsatellite and analysed for polymorphism. These microsatellites, IDVGA-2, -3, -7, -8, -9, -10, -11 showed the following number of alleles and polymorphism information content (PIC) values (7/0.616, 8/0.693, 6/0.641, 5/0.643, 2/0.239, 10/0.844, 6/0.720). The microsatellites were also assigned to synteny groups as follows: IDVGA-2/U17, IDVGA-3/U16, IDVGA-7/U7, IDVGA-8/U29, IDVGA-9/U3, IDVGA-10/U19, IDVGA-11/A (probably U18).     

Regional assignments of NP and MPI on chromosome 7 in pig, Sus scrofa

Summary. Fibroblasts from a pig with a spontaneous reciprocal translocation involving chromosome 7, were used to prepare a set of pig-mouse somatic cell hybrids. The isozyme analysis strongly indicated that in pigs, the NP (nucleoside phosphorylase) gene is located on the distal part of the long arm of chromosome 7, (q21-qter), while the MPI (mannose phosphate isomerase) gene is in the region q21-pter. This confirmed previously reported chromosomal assignment of these genes in pigs and that this synteny has been evolutionarily conserved in several different animal species.     

Further data on chromosomal assignments of pig enzyme loci LDHA, LDHB, MPI, PEPB and PGM1, using somatic cell hybrids

Summary. Clear interspecies differentiation between the chromosomes in pig-mouse somatic cell hybrids was achieved by using the THA-technique for the cytogenetic analysis. The assignments of LDHB and MPI to pig chromosomes nos 5 and 7 respectively, reported previously, were confirmed by analysis of 34 hybrid clones. The LDHA, PEPB and PGM1 genes were assigned to pig chromosomes nos 2, 5 and 6 respectively. Both LDHB and PEPB were indicated to be located on the long arm, except the most proximal part, of pig chromosome no. 5. The proposed synteny between LDHB and PEPB in pigs is in accordance with the synteny observed between these two loci in several other mammalian species.     

A synteny map of the horse genome comprised of 240 microsatellite and RAPD markers

To generate a domestic horse genome map we integrated synteny information for markers screened on a somatic cell hybrid (SCH) panel with published information for markers physically assigned to chromosomes. The mouse-horse SCH panel was established by fusing pSV2neo transformed primary horse fibroblasts to either RAG or LMTk mouse cells, followed by G418 antibiotic selection. For each of the 108 cell lines of the panel, we defined the presence or absence of 240 genetic markers by PCR, including 58 random amplified polymorphic DNA (RAPD) markers and 182 microsatellites. Thirty-three syntenic groups were defined, comprised of two to 26 markers with correlation coefficient (r) values ranging from 0.70 to 1.0. Based on significant correlation values with physically mapped microsatellite (type II) or gene (type I) markers, 22 syntenic groups were assigned to horse chromosomes (1, 2, 3, 4, 6, 9, 10, 11, 12, 13, 15, 18, 19, 20, 21, 22, 23, 24, 26, 30, X and Y). The other 11 syntenic groups were provisionally assigned to the remaining chromosomes based on information provided by heterologous species painting probes and work in progress with type I markers.     

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.     

Synteny-mapping horse microsatellite markers using a heterohybridoma panel

A panel of horse-mouse heterohybridoma cells was tested for genetic markers using biochemical and polymerase chain reaction-(PCR-) based tests. Biochemical markers included phospho-glucomutase ( PGM ), glucose phosphate isomerase ( GPI ) and 6-phosphogluconate dehydrogenase ( PGD ). Markers detected using PCR-based tests included microsatellite markers HTG2–15, HMS 1–3, 5–8, VHL20, ECA2 and genes for equine major histocompatibility gene ELA-DRA , tumour necrosis factor alpha (TNFA) and transferrin. The results were analysed for correlation and concordance. Based on the results, five synteny groups were identified, specifically between ELA-DRA, TNFA, HMS5 and HTG5 ; between HTG3 and HTG13 ; between HTG4, HTG8 and HMS3 ; between HTG6 and HMS1 ; and between HTG7, HTG9 and HMS6. Evidence was also found for synteny between HTG12, HMS7 and ECA2 , however, confirmation requires further testing. Cytogenetic evaluation of the cell lines making up the panel indicated that large metacentric chromosomes were preferentially lost or tended to break at the centromere. Consequently, the results from this analysis can be used to identify synteny, but not to exclude synteny.     

Regional localisations of VIM, HSD3b, ACTA1 and PGM1 in pigs

The comparative map between human and pig has progressed rapidly over the past 2 years. Nevertheless, some points still need to be clarified, particularly the correspondences between human chromosome 10 (HSA10) and porcine chromosome 10 (SSC10) and between human chromosome 1 (HSA1) and porcine chromosomes. The gene codings for vimentin (VIM) carried by HSA10 and three genes carried by HSA1 (hydroxy delta 5 steroid dehydrogenase 3 beta: HSD3B; alpha actin 1: ACTA1; and phosphoglucomutase 1: PGM1) were selected and the regional localisations on pig chromosomes were determined using a well-characterised somatic cell hybrid panel.     

An immunochemical method for the detection of the expression of human gene loci in human-rodent somatic cell hybrids with special reference to the GPI locus

Species-specific antibodies, prepared against unpurified human and Chinese hamster fibroblast extracts, were used to identify the parental origins of enzymes in human-hamster somatic cell hybrids. Results of the detection of the expression of the human glucosephosphate isomerase gene locus (GPI) by electrophoretic and immunochemical techniques were concordant in 17 instances. The human GPI synthesized by fibroblasts derived from skin explants and by somatic cell hybrids retaining the human GPI locus, regardless of whether the human parental cells were lymphocytes or fibroblasts, appeared to be antigenically identical.This work was supported by the Medical Research Council of Canada (Grant MA-4061). Personnel and operating support were provided by The Children's Hospital of Winnipeg Research Foundation, Inc.     

Chromosomal assignment of porcine microsatellites by use of a somatic cell hybrid mapping panel

A well-established and characterized somatic cell hybrid panel was used to map three polymorphic microsatellites. Microsatellite S0072, representing the linkage group S0007-S0072, was assigned to porcine chromosome 14. Micro-satellite S0009, representing the unassigned linkage group EAM-S0009-S0071, was assigned tentatively to porcine chromosome 11. Finally, S0062 was tentatively mapped to chromosome 18. S0062 may represent the first marker for porcine chromosome 18.     

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.     

Global gene expression analysis of bovine blastocysts produced by multiple methods

Reproductive efficiency using somatic cell nuclear transfer (SCNT) technology remains suboptimal. Of the various efforts to improve the efficiency, chromatin transfer (CT) and clone-clone aggregation (NTagg) have been reported to produce live cloned animals. To better understand the molecular mechanisms of somatic cell reprogramming during SCNT and assess the various SCNT methods on the molecular level, we performed gene expression analysis on bovine blastocysts produced via standard nuclear transfer (NT), CT, NTagg, in vitro fertilization (IVF), and artificial insemination (AI), as well as on somatic donor cells, using bovine genome arrays. The expression profiles of SCNT (NT, CT, NTagg) embryos were compared with IVF and AI embryos as well as donor cells. NT and CT embryos have indistinguishable gene expression patterns. In comparison to IVF or AI embryos, the number of differentially expressed genes in NTagg embryos is significantly higher than in NT and CT embryos. Genes that were differentially expressed between all the SCNT embryos and IVF or AI embryos are identified. Compared to AI embryos, more than half of the genes found deregulated between SCNT and AI embryos appear to be the result of in vitro culture alone. The results indicate that although SCNT methods have altered differentiated somatic nuclei gene expression to more closely resemble that of embryonic nuclei, combination of insufficient reprogramming and in vitro culture condition compromise the developmental potential of SCNT embryos. This is the first set of comprehensive data for analyzing the molecular impact of various nuclear transfer methods on bovine pre-implantation embryos.     

The bovine butyrophilin gene maps to chromsome 23

Chromosomal assignment of the bovine butyrophilin gene ( BTN ) was performed by analysis of DNA from somatic hybrid cell lines using the polymerase chain reaction. The gene was assigned to bovine chromosome 23 using two sets of primers specific for bovine BTN.     

Expression of galactose-1-P-uridyltransferase in Chinese hamster × human galactosemia somatic cell hybrids

Lymphocytes from a patient with classic galactosemia (GALT deficiency) were hybridized with a Chinese hamster cell line. Electrophoretic evaluation of GALT in 31 independently derived interspecific hybrid clones failed to demonstrate expression of the human GALT gene even when human chromosome 9 was present. Possible mechanisms for this lack of expression are presented.This work was supported in part by Grants HD-04612 and HD-05615 from the National Institute of Child Health and Human Development.     

Bovine and ovine DNA microsatellites from the EMBL and GENBANK databases

Bovine and ovine microsatellite sequences were extracted from the EMBL and GENBANK databases. When analysed for number of alleles and degree of heterozygosity in the CSIRO cattle reference families, allele numbers range from 1 to 14 with heterozygosities, in the polymorphic systems ranging from 15.8% to 100%. Six (46%) of the 13 bovine systems tested gave specific and polymorphic products in sheep. Similarly 2 of the 4 ovine systems gave specific and polymorphic products in cattle. These data define 11 bovine and 8 ovine microsatellite systems which are associated with known genes and are thus useful for comparative mapping studies.     

Comparative and physical mapping of 111 previously reported and 105 new porcine microsatellites

Here we report radiation hybrid mapping of 105 new porcine microsatellite markers on the IMpRH₇₀₀₀ radiation hybrid panel. In addition, we searched flanking sequences of these markers, as well as 673 previously reported RH-mapped microsatellite markers, for orthology to human sequences. Eighty-seven new and 111 previously mapped sequences exhibited orthology to human sequences. Using a stringent sequence alignment, 25 microsatellite-flanking sequences were found to be highly similar to genic sequences, whereas 173 were similar to non-genic sequences in the human genome. Five markers were located near known breakpoints of synteny between human and swine.