Hum Genet (1992) 89 : 105-106
9 Springer-Verlag1992
Short communications
Regional chromosomal assignment of the human platelet phosphofructokinase gene to 10p15 Norma Morrison 1, Craig Simpson 2, Linda Fothergill-Gilmore 2, Elizabeth Boyd 1, and J. Michael Connor 1 1Duncan Guthrie Institute of Medical Genetics, Yorkhill, Glasgow G3 8SJ, UK 2Department of Biochemistry, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK Received June 15, 1991
Summary. A c D N A for human platelet 6-phosphofructokinase (PFKP) has been isolated from a human Raji cell line c D N A library. Using this c D N A as a probe, the gene for human PFKP, previously mapped to chromosome 1 0 p t e r - p l l . 1 , has been further localized to 10p15 by non-isotopic in situ hybridization.
Introduction Mammalian 6-phosphofructokinase (PFK; ATP: D-fructose-6-phosphate 1-phosphotransferase, E C 2.7.1.11) is a key regulatory enzyme in glycolysis. There are three types of subunit of the tetrameric protein PFK that are differentially expressed during development (Vora 1982) and that display distinct tissue specificities (Dunaway et al. 1988). These subunits are encoded by the muscle (PFKM), liver (PFKL) and platelet (PFKP) phosphofructokinase genes. Variable expression of the L, M and P genes leads to the formation of homo- and heterotetrameric forms of the enzyme with different catalytic and regulatory properties. P F K M has been assigned to chromosome l c e n - q 3 2 (Vora et al. 1982), P F K L to chromosome 21q22.3 (van Keuren et al. 1986) and PFKP to chromosome 10pterp l l . 1 (Vora et al. 1983). We have recently cloned a PFKP c D N A (Simpson and Fothergill-Gilmore 1991) and have used this as a probe to define more precisely the regional chromosomal assignment of the human PFKP gene.
Materials and methods DNA probe The cDNA clone pCS16 (Simpson and Fothergill-Gilmore 1991) was used as a probe for the PFKP gene. This clone consists of 238 bp of the downstream untranslated region and 900 bp of the Cterminus coding sequence inserted into Bluescript KS.
Offprint requests to."J. M. Connor
Chromosome preparation Using standard cytogenetic methods, metaphase chromosome spreads were obtained from phytohaemagglutinin-stimulated lymphocyte cultures from 3 human males of normal karyotype. Chromosome spreads from 2 synchronized lymphocyte cultures were also prepared for the confirmation and refinement of the initial result (Yunis et al. 1978).
In situ hybridization The probe was labelled with biotin-11-dUTP and used at a concentration of 0.5 ng/gl for in situ hybridization according to the method described by Garson et al. (1987). The distribution of hybridization signals in chromosome spreads from non-synchronized preparations was analysed statistically using the chi-square test.
Results and discussion Eighty-eight metaphases were scored following hybridization; the positions of 336 hybridization signals were recorded. Of these, a highly significant (P < 0.005) 39 signals (11.6%) were located on chromosome 10, with 17 (5%) comprising a signal peak on 10p15. For confirmation and refinement, the probe was hybridized to longer chromosomes obtained from synchronized lymphocyte cultures. The positions of 36 signals fell within 10p15 and could be assigned with confidence to a single sub-band. Thirty-three signals (91.7%) were located at 10p15.2p15.3 with 24 (66.7%) at 10p15.3. Vora et al. (1983) used an immunochemical method in the study of a series of rodent-human somatic cell hybrids to assign the human PFKP gene to human chromosome 10p. The work described here supports this assignment and indicates a regional localization to 10p15. Sequence data for pCS16 has been entered into the E M B L / G E N B A N K database under accession number M64784.
Acknowledgements. This work was partly funded by the WHO (Grant 08/181/230) and was carried out with support from the MRC as part of the Human Genome Mapping Project.
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References Dunaway GA, Kasten TP, Sebo T, Trapp R (1988) Analysis of the phosphofructokinase subunits and isoenzymes in human tissues. Biochem J 251 : 677-683 Garson JA, Berghe JA van den, Kemshead JT (1987) Novel nonisotopic in situ hybridization technique detects small (1 kb) unique sequences in routinely G-banded human chromosomes: fine mapping of N-myc and beta-NGF genes. Nucleic Acids Res 15 : 4761-4770 Keuren M van, Drabkin H, Hart I, Harker D, Patterson D, Vora S (1986) Regional assignment of human liver-type 6-phosphofructokinase to chromosome 21q22.3 by using somatic cell hybrids and a monoclonal anti-L antibody. Hum Genet 74: 34-40 Simpson CJ, Fothergill-Gilmore L (1991) Isolation and sequence of a cDNA encoding human platelet phosphofructokinase. Biochem Biophys Res Commun 180 : 197-203
Vora S (1982) Isozymes of phosphofructokinase. In: Rattazzi MC, Scandalios JG, Whitt GS (eds) Isozymes: current topics in biological and medical research, vol. 6. Liss, New York, pp 119167 Vora S, Durham S, Martinville B de, George DL, Francke U (1982) Assignment of the human gene for muscle type phosphofructokinase (PFKM) to chromosome 1 (region cen-q32) using somatic cell hybrids and monoclonal anti-M antibody. Somatic Cell Genet 8 : 95-104 Vora S, Miranda AF, Hernandez E, Francke U (1983) Regional assignment of the human gene for platelet-type phosphofructokinase (PFKP) to chromosome 10p: novel use of polyspecific rodent antisera to localize human enzyme genes. Hum Genet 63 : 374-379 Yunis J J, Sawyer HR, Ball DW (1978) The characterization of high-resolution G-banded chromosome of man. Chromosoma 67 : 293-307
9 Springer-Verlag1992
Short communications
Regional chromosomal assignment of the human platelet phosphofructokinase gene to 10p15 Norma Morrison 1, Craig Simpson 2, Linda Fothergill-Gilmore 2, Elizabeth Boyd 1, and J. Michael Connor 1 1Duncan Guthrie Institute of Medical Genetics, Yorkhill, Glasgow G3 8SJ, UK 2Department of Biochemistry, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK Received June 15, 1991
Summary. A c D N A for human platelet 6-phosphofructokinase (PFKP) has been isolated from a human Raji cell line c D N A library. Using this c D N A as a probe, the gene for human PFKP, previously mapped to chromosome 1 0 p t e r - p l l . 1 , has been further localized to 10p15 by non-isotopic in situ hybridization.
Introduction Mammalian 6-phosphofructokinase (PFK; ATP: D-fructose-6-phosphate 1-phosphotransferase, E C 2.7.1.11) is a key regulatory enzyme in glycolysis. There are three types of subunit of the tetrameric protein PFK that are differentially expressed during development (Vora 1982) and that display distinct tissue specificities (Dunaway et al. 1988). These subunits are encoded by the muscle (PFKM), liver (PFKL) and platelet (PFKP) phosphofructokinase genes. Variable expression of the L, M and P genes leads to the formation of homo- and heterotetrameric forms of the enzyme with different catalytic and regulatory properties. P F K M has been assigned to chromosome l c e n - q 3 2 (Vora et al. 1982), P F K L to chromosome 21q22.3 (van Keuren et al. 1986) and PFKP to chromosome 10pterp l l . 1 (Vora et al. 1983). We have recently cloned a PFKP c D N A (Simpson and Fothergill-Gilmore 1991) and have used this as a probe to define more precisely the regional chromosomal assignment of the human PFKP gene.
Materials and methods DNA probe The cDNA clone pCS16 (Simpson and Fothergill-Gilmore 1991) was used as a probe for the PFKP gene. This clone consists of 238 bp of the downstream untranslated region and 900 bp of the Cterminus coding sequence inserted into Bluescript KS.
Offprint requests to."J. M. Connor
Chromosome preparation Using standard cytogenetic methods, metaphase chromosome spreads were obtained from phytohaemagglutinin-stimulated lymphocyte cultures from 3 human males of normal karyotype. Chromosome spreads from 2 synchronized lymphocyte cultures were also prepared for the confirmation and refinement of the initial result (Yunis et al. 1978).
In situ hybridization The probe was labelled with biotin-11-dUTP and used at a concentration of 0.5 ng/gl for in situ hybridization according to the method described by Garson et al. (1987). The distribution of hybridization signals in chromosome spreads from non-synchronized preparations was analysed statistically using the chi-square test.
Results and discussion Eighty-eight metaphases were scored following hybridization; the positions of 336 hybridization signals were recorded. Of these, a highly significant (P < 0.005) 39 signals (11.6%) were located on chromosome 10, with 17 (5%) comprising a signal peak on 10p15. For confirmation and refinement, the probe was hybridized to longer chromosomes obtained from synchronized lymphocyte cultures. The positions of 36 signals fell within 10p15 and could be assigned with confidence to a single sub-band. Thirty-three signals (91.7%) were located at 10p15.2p15.3 with 24 (66.7%) at 10p15.3. Vora et al. (1983) used an immunochemical method in the study of a series of rodent-human somatic cell hybrids to assign the human PFKP gene to human chromosome 10p. The work described here supports this assignment and indicates a regional localization to 10p15. Sequence data for pCS16 has been entered into the E M B L / G E N B A N K database under accession number M64784.
Acknowledgements. This work was partly funded by the WHO (Grant 08/181/230) and was carried out with support from the MRC as part of the Human Genome Mapping Project.
106
References Dunaway GA, Kasten TP, Sebo T, Trapp R (1988) Analysis of the phosphofructokinase subunits and isoenzymes in human tissues. Biochem J 251 : 677-683 Garson JA, Berghe JA van den, Kemshead JT (1987) Novel nonisotopic in situ hybridization technique detects small (1 kb) unique sequences in routinely G-banded human chromosomes: fine mapping of N-myc and beta-NGF genes. Nucleic Acids Res 15 : 4761-4770 Keuren M van, Drabkin H, Hart I, Harker D, Patterson D, Vora S (1986) Regional assignment of human liver-type 6-phosphofructokinase to chromosome 21q22.3 by using somatic cell hybrids and a monoclonal anti-L antibody. Hum Genet 74: 34-40 Simpson CJ, Fothergill-Gilmore L (1991) Isolation and sequence of a cDNA encoding human platelet phosphofructokinase. Biochem Biophys Res Commun 180 : 197-203
Vora S (1982) Isozymes of phosphofructokinase. In: Rattazzi MC, Scandalios JG, Whitt GS (eds) Isozymes: current topics in biological and medical research, vol. 6. Liss, New York, pp 119167 Vora S, Durham S, Martinville B de, George DL, Francke U (1982) Assignment of the human gene for muscle type phosphofructokinase (PFKM) to chromosome 1 (region cen-q32) using somatic cell hybrids and monoclonal anti-M antibody. Somatic Cell Genet 8 : 95-104 Vora S, Miranda AF, Hernandez E, Francke U (1983) Regional assignment of the human gene for platelet-type phosphofructokinase (PFKP) to chromosome 10p: novel use of polyspecific rodent antisera to localize human enzyme genes. Hum Genet 63 : 374-379 Yunis J J, Sawyer HR, Ball DW (1978) The characterization of high-resolution G-banded chromosome of man. Chromosoma 67 : 293-307
