Phosphoglucomutase 1 (PGM1) deficiency is a stable characteristic
of the
erythroleukaemic cell
line, K562, whereas the activity of the isozymes of the other two PGM loci
(PGM2 and PGM3) is
slightly elevated. In this study the molecular basis of PGM1 deficiency
was
investigated by a
combined approach utilising protein electrophoresis, immunodetection,
cytogenetic techniques, and
DNA and RNA analysis. Isoelectric focusing and activity staining confirmed
that K562 has no
detectable PGM1 activity. Immunoblot analysis of extracts, separated by
isoelectric focusing, starch
gel and SDS gel electrophoresis, using monospecific anti-PGM1 antibodies
showed that K562
contained no detectable immunoreactive material. Karyotype analysis revealed
the presence of two
intact chromosomes 1 and a derivative chromosome 1,
der(1)t(1; 11), each of which carried a copy
of the PGM1 gene as demonstrated by fluorescence in situ
hybridization using a PGM1 cosmid as
probe. Southern blot analysis using a PGM1 cDNA clone as probe
suggested
that the PGM1 genes
had not been subject to any gross structural rearrangements. We were also
able to determine that K562 is type PGM1 2+1+
by restriction endonuclease analysis of genomic DNA. Very low levels
of PGM1 mRNA which appeared to be full length transcripts were detected
in
K562 using a reverse
transcriptase PCR technique. We conclude that the most likely cause of
PGM1
enzyme deficiency in K562 is abnormal regulation of transcription.