Hum Genet (1992) 89 : 105-106

�9 Springer-Verlag 1992

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

1 Duncan Guthrie Institute of Medical Genetics, Yorkhill, Glasgow G3 8S J, UK 2 Department of Biochemistry, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK

Received June 15, 1991

Summary. A cDNA for human platelet 6-phosphofruc- tokinase (PFKP) has been isolated from a human Raji cell line cDNA library. Using this cDNA as a probe, the gene for human PFKP, previously mapped to chromo- some 10p te r -p l l . 1 , has been further localized to 10p15 by non-isotopic in situ hybridization.

Introduction

Mammalian 6-phosphofructokinase (PFK; ATP: D-fruc- tose-6-phosphate 1-phosphotransferase, EC 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) phospho- fructokinase genes. Variable expression of the L, M and P genes leads to the formation of homo- and heterotet- rameric forms of the enzyme with different catalytic and regulatory properties.

PFKM has been assigned to chromosome lcen-q32 (Vora et al. 1982), PFKL to chromosome 21q22.3 (van Keuren et al. 1986) and PFKP to chromosome 10pter- p l l . 1 (Vora et al. 1983). We have recently cloned a PFKP cDNA (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 C- terminus 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 lym- phocyte cultures from 3 human males of normal karyotype. Chro- mosome spreads from 2 synchronized lymphocyte cultures were also prepared for the confirmation and refinement of the initial re- sult (Yunis et al. 1978).

In situ hybridization

The probe was labelled with biotin-11-dUTP and used at a concen- tration of 0.5 ng/gl for in situ hybridization according to the meth- od described by Garson et al. (1987). The distribution of hybrid- ization 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 hybrid- ization; 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 confirma- tion 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 10p15.2- p15.3 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 hy- brids to assign the human PFKP gene to human chromo- some 10p. The work described here supports this assign- ment 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

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