Measurement of [Ca2+] using the Fluorometric Imaging Plate Reader (FLIPR).
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Calcium↗
Biomedical subjects
E M Tucker
Publications and source records attributed to E M Tucker.
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.
Animals↗
A new reciprocal translocation (7q+;15q-) in the domestic pig.
A reciprocal translocation was identified in a phenotypically normal Large White boar. Chromosome preparations from the carrier were studied by flow sorting, chromosome painting and G-banding. The flow karyotype displayed one additional clearly distinguishable peak, while in situ hybridization and G-banding showed two abnormal chromosomes involved in the translocation. All the results suggested that the translocation involved chromosomes 7 and 15 and the karyotype investigated was 38,XY,rcp(7;15)(q24;q12). The parents and three full sibs of the carrier had normal karyotypes. It would seem that the translocation had arisen de novo.
Animals↗
Identification of pig chromosomes in pig-mouse somatic cell hybrid bivariate flow karyotypes.
To identify pig chromosomes in pig-mouse somatic cell hybrids, dual-laser flow karyotypes and GTG-banded metaphase spreads of pig, mouse, and 7 pig-mouse hybrid cell lines were compared. Pig chromosomes no. 1, 2, 5, 6, 10, 11, 13, 14, 16, 18, X and Y were tentatively assigned to individual peaks in the pig flow karyotype on the basis of DNA content vs. relative chromosome length. In the 7 hybrid cell lines, 7 out of 8 peaks distinct from those of the mouse cell line could be correlated with the presence of pig chromosomes no. 5, 9, 10, 11 or 16, 14, 15, and 18, whereas 1 peak appeared to correspond to the presence of 1 middle-size chromosome (3, 4, or 7). Other pig chromosomes present in the hybrids could not be detected with certainty due to superposition with mouse peaks and mouse chromosome rearrangements.
Animals↗
Synteny mapping in the horse using horse-mouse heterohybridomas.
In a study of 35 horse-mouse heterohybridoma cell lines, synteny in the horse was found between LDHB, PEPB and IGF1 and between NP, MPI and IDH2. A synteny between ADA and PEPC was also indicated. The loci for horse immunoglobulin light chain (IgL) genes and for LDHA were independent.
Adenosine Deaminase↗
Preparation of chromosome-specific paints and complete assignment of chromosomes in the pig flow karyotype.
Sorted chromosomes from each of the 20 clusters of the male porcine bivariate flow karyotype were amplified and biotinylated using DOP-PCR. The chromosomes comprising each cluster were identified by fluorescence in situ hybridization (FISH) of the 20 probes to R-banded male pig metaphase spreads. A standard flow karyotype for the pig is presented.
Animals↗
The production of equine monoclonal immunoglobulins by horse-mouse heterohybridomas.
Studies were carried out to determine the optimum conditions for the production of equine monoclonal antibodies (MAbs). Lymphocytes from ponies immunised with influenza A equine 2 virus, isolate A/Equine/Newmarket/79 (H3N8) were fused with mouse myeloma (NSO) cells and with horse-mouse heterohybridomas made aminopterin-sensitive by selective growth in 8-azaguanine. Although all fusions initially resulted in heterohybridoma colonies that secreted equine immunoglobulin, many of these were unable to maintain secretion for longer than a few weeks. Increasing the time between immunisation and the booster injection of Newmarket/79 virus, the inclusion of Freund's incomplete adjuvant and the use of an aminopterin-sensitive primary heterohybridoma as the fusion partner, improved the production of HIg-secreting heterohybridomas. After two clonings eight cell lines were established which maintained anti-Newmarket/79 antibody secretion for over a year. FACS analysis of the cell lines provided a useful means of predicting breakdown of MAb secretion by the cell lines, thus enabling re-cloning to be carried out in time.
Animals↗
Bivariate flow cytometry of farm animal chromosomes: a potential tool for gene mapping.
Bivariate flow karyotypes of chromosomes from sheep, cattle and pig lymphocytes and from a cattle-mouse somatic cell hybrid line were obtained using a dual laser fluorescence-activated cell sorter (FACS). Pig chromosomes were resolved into 19-20 peaks, indicating that most, if not all, pig chromosomes could be separated by this technique. Sheep chromosomes showed incomplete separation but three clear peaks, presumably representing the three large metacentric chromosomes, plus five other clusters were obtained. Cattle chromosomes showed poor separation but about four peaks could be distinguished, indicating that certain chromosomes could be sorted in this species. The use of cattle-mouse hybrids may enable other individual cattle chromosomes to be obtained. It is concluded that FACS separation will be a useful additional tool for gene mapping.
Animals↗
A chromosome 1-specific DNA library from the domestic pig (Sus scrofa domestica).
Chromosomes were prepared from lymphocytes of a male domestic pig and flow-sorted on a dual-laser FACS. Twenty spots were observed, corresponding to the known pig karyotype of 18 pairs of autosomes plus the X and Y. DNA was isolated from 10,000 copies of the presumed chromosome 1 spot, restricted with Sau3A, ligated into the vector pGEM4z, and PCR amplified using universal primers; the products were then re-ligated into pUC18. After transformation into Escherichia coli, 210,000 independent colonies were obtained, 5% of which contained only vector DNA. The average insert size of the library was 405 bp. Southern blotting revealed that 36% of the clones contained single-copy DNA and that the remainder contained moderately or highly repetitive DNA. Screening with a (CA)n probe revealed that roughly 1% of the clones contained microsatellite sequences. A bulk insert of the library was biotinylated by PCR and used as a probe for chromosomal in situ suppression hybridization to pig chromosomes, which confirmed that the library is specific for chromosome 1. However, sequences from the centromeric and telomeric regions seem to be underrepresented in the library.
Animals↗
Chromosome painting using chromosome-specific probes from flow-sorted pig chromosomes.
Biotinylated chromosome-specific probes were prepared from flow-sorted pig chromosomes 1, 13, 18, X, and Y using the degenerate oligonucleotide-primed polymerase chain reaction. Probes prepared in this way can be used to confirm the identity of chromosomes in the bivariate pig flow karyotype and in pig x mouse somatic cell hybrids.
Animals↗
Synteny mapping of the bovine IGHG2, CRC and IGF1 genes.
A panel of bovine-murine hybrid cell lines was analysed for 10 loci, including three (IGF1, IGHG2 and the calcium release channel gene [CRC]) that have previously been mapped in man, but not in cattle. The IGF and CRC genes were indirectly mapped to chromosomes 5 and 18 respectively and the syntenies of the HOX2 and GH genes and of the NP and FOS genes were confirmed. The results also show that the IGHG2 locus, which is linked to NP and FOS on human chromosome 14, is separated from these genes in cattle. By showing synteny of the IGHG2 and MPI loci, the IGHG2 locus has been indirectly mapped to chromosome 21.
Animals↗
Identification of a 25 kDa polypeptide associated with the L antigen in low potassium-type sheep red cells.
Antisera to the L blood group antigen have been used, following radioiodination of low potassium-type sheep red cells and subsequent immunoprecipitation, to identify a polypeptide of the L antigen. Only LK, and not HK, cells express this 25 kDa component which is present in very low copy number.
Animals↗
Amino acid transport systems in sheep reticulocytes.
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Alanine↗
Two new sheep haemoglobins, one of which is replaced by haemoglobin C in anaemia.
Two new haemoglobin variants, provisionally named Hb G and Hb H, were found during a survey of 295 Welsh Mountain cross-bred sheep. Both haemoglobins appear to be beta chain variants controlled by genes allelic to those for the common forms Hb A and Hb B. Studies on an anaemic Hb AH and an Hb AG type sheep showed that Hb G, like Hb A, is replaced by Hb C in anaemia whereas Hb H, like Hb B, is not replaced.
Anemia↗
A bovine monoclonal antibody detecting a class I BoLA antigen.
The bovine IgG1 monoclonal antibody (mAb) ILA70 was made by immunizing a calf with peripheral blood mononuclear cells (PBM) from a BoLA-w10 homozygous heifer and subsequently fusing lymphocytes from the local lymph-node with the heterohybridoma 53B3. Family and population studies, antibody binding inhibition and immunoprecipitation of the target antigen all indicate that ILA70 detects a polymorphic epitope on a bovine class I major histocompatibility complex (MHC) molecule. The antibody is complement fixing and so may be used in a standard cytotoxicity assay. Ascitic fluid with antibody activity many times greater than that of the tissue-culture supernatant has been prepared in nude mice. The antibody-producing heterohybridoma has been subcloned three times and appears to be stable. Such heterohybridomas may prove to be a valuable source of particularly discriminating and informative mAbs for the serological analysis of the products of the bovine MHC.
Animals↗
The production and application of non-rodent monoclonal antibodies in veterinary science.
The requirement for monoclonal antibodies derived from species other than rats and mice is becoming increasingly realised in veterinary, as well as human, medicine. This paper reviews current knowledge of the production of inter-species hybridomas (heterohybridomas) by the fusion of rodent myeloma cell lines with lymphocytes from species of veterinary importance. To date a number of monoclonal immunoglobulins derived from sheep, cattle, pig, rabbit, mink and primate species have been produced to a variety of different bacterial, viral and nematode pathogens as well as to blood group and MHC determinants and to hormones. The technique opens up a number of possibilities for the future; some of these applications are discussed in relation to the antibodies produced thus far.
Animals↗
Blood genetic marker studies of a sheep-goat hybrid and its back-cross offspring.
Blood samples from a female sheep-goat hybrid and its back-cross male offspring were tested for electrophoretic variants of plasma albumin, transferrin and esterase, and of red cell carbonic anhydrase, nucleoside phosphorylase, NADH-diaphorase, 'X'-protein, superoxide dismutase, malic enzyme and haemoglobin. Red cells were also tested for blood group antigens. Both animals showed variants that could not be attributed to either sheep or goat alone, thus confirming previous chromosomal data that the female was a genuine sheep-goat hybrid.
Animals↗
Blood groups and biochemical polymorphism in the Namaqua sheep breed.
The Namaqua is an indigenous fat-tailed African breed of sheep which has remained relatively isolated and which at one time dwindled to near extinction. Frequency data are given for blood group antigens, red cell glutathione and potassium types, for electrophoretic variants of red cell haemoglobin, 'X' protein, nucleoside phosphorylase, NADH-diaphorase, lysine and carbonic anhydrase and of plasma esterase, transferrin and albumin. Of particular interest was the occurrence of the i blood group, a bimodal distribution in red cell glutathione concentrations and red cell potassium concentrations of around 57 mmol/l cells, i.e. neither typically LK nor HK type.
Animals↗