Hum. Genet. 35, 219 223 (1977) © by Springer-Verlag1977
Evidence for Two Additional Common Alleles at the PGM1 Locus (Phosphoglucomutase--E.C.: 2.7.5.1) A Comparison by Three Different Techniques
P. KUhnl, U. Schmidtmann, and W. Spielmann Institut fur Immunh~matologie der Universit~it Frankfurt/Main, D-6000 Frankfurt/Main, Federal Republic of Germany
Summary. Lysates of erythrocytes, leukocytes, lymphocytes, and extracts of sperms were investigated for the PGM~ isozymes by three techniques: starch gel electrophoresis, high voltage thin-layer agarose gel electrophoresis, and thinlayer isoelectric focusing on polyacrylamide gel. On starch, only the well known c o m m o n phenotypes 1, 2-1, and 2 were demonstrable. On agarose, different distances of the two main cathodal bands (a, b) among the phenotypes 2-1 were noted. Furthermore, on agarose, some types considered as homozygous on starch gel had a single, sharp banded pattern while others were broad and blurred. Optimal separation was achieved by isoelectric focusing on polyacrylamide gel. In 291 leukolysates, 10 different phenotypes were identified. These are considered as gene products of 4 different common alleles at the PGM~ locus as suggested by preliminary family investigations. In a random population from Hessen these four alleles had the following frequencies: PGM~ 1 0.6186, PGM~ 2 0.1718, PGM~ 3 0.1426, and PGM~ 4 0.067. The preliminary designation al, a2, a3 and a4 was chosen as the assumed polymorphism was demonstrated on acrylamide and agarose. The sum of the frequencies PGM~ 1 and PGM~ 3 (the gene products of which have apparently the same electrophoretic mobility on starch) is similar to the frequency of the old P G M I allele (0.757) in Caucasoids, PGM~ 2 and PGM] 4have a frequency of 0.2388 corresponding with the frequency of the old allele P G M 2. Introduction The enzyme phosphoglucomutase (E.C.: 2.7.5.1) occurs in many human tissues (McAlpine et al., 1970; Renninger, 1969). Since Spencer et al. (1964) first detected the polymorphism of PGM, three loci, PGMI, PGM2, and P G M 3 have been identified and assigned to chromosomes 1, 4, and 6, respectively (Nguyen et al., 1971; Douglas et al., 1973; McAlpine et al., 1975; Jongsma et al., 1973). About 80--95% of the enzyme activity is attributable to PGM~ in most human tissues (Harris and Hopkinson, 1976), except erythrocytes which have a smaller amount
220
P. Ktihnl et al.
of PGM1, but a correspondingly higher content of PGM2 (approximately 50 %). The P G M 3 contribution is relatively small in all tissues and apparently not detectable in erythrocytes at all (Harris and Hopkinson, 1976; Quick et al., 1974). Bissbort et al. (1975), however, were able to demostrate the polymorphism of P G M 3 in erythrocyte hemolysateS by means of horizontal starch gel electrophoresis. The three c o m m o n phenotypes in Whites, detectable on PGM-stained starch gels are expressions of two c o m m o n alleles at PGM~ (PGM~ and P G M 2) and analogously at P G M 3 (PGM31 and PGM]). Five additional rare alleles and a deficient allele at PGM1 with a total frequency of less than 0.001 have been identified (Harris et al., 1973; Fiedler and Pettenkofer, 1969). This report gives the results of the separation of P G M 1 gene products by three techniques, starch gel electrophoresis (SGE), agarose gel electrophoresis (AGE), and isoelectric focusing on polyacrylamide gel (PAGIF).
Material and Methods
Hemolysates were prepared according to the method of Marti (1963), leukolysates as reported by Sinha et al. (1970), lympholysates after Otto and Schmid (1970), and sperm lysates as described by Renninger and Sina (1970). Starch gel electrophoresis was performed using the method described by Spencer et al. (1964). Prolonged agarose gel electrophoresis was carried out with the following system: Tris--L-Histidine monohydrochloride 0.25 M, adjusted to pH 6~0for bridge buffer; gel buffer 1: 30 dilution of bridge buffer. 5~zlsamples were applied 5 cm from the anodal side of a 1% agarose gel with the following dimensions: 400 × 200 x 1 mm according to a previously described method (Kiahnl et al., 1974). Electrophoresis was performed at 20 V/cm at ÷ 12° C for 41/2 h in a DESAGA TLE double chamber. Isoeleetric focusing was performed on commercially available polyacrylamide gels (Ampholine PAGplates, LKB, Bromma, Sweden, pH range 3.5--9.5, polyacrylamide gel concentration T=5%,degree of cross-linkage C=3%, ampholine concentration 2.4% (w/v), gel dimensions 245× 110× 1 mm) with a total focusing time of 31/2 h. Staining solutions for all three methods (SGE, AGE, PAGIF) were prepared according to Spencer et al. (1964) with modifications (Schmidtmann et al., 1977).
Results and Discussion
The patterns of nine different selected leukolysates, as obtained with the three methods, are given in Figure 1. In the lower part of the figure, the starch gel zymograms reveal only the three c o m m o n phenotypes P G M l l , PGM12-1, and PGMI2; similar patterns were observed in hemolysates and sperm lysates. Even after prolonged electrophoresis of 28 h no striking differences were observed. In respect to the distances of the two main cathodal bands, the patterns obtained after prolonged A G E (center of Fig. 1) were apparently heterogeneous in positions 3, 4, 7, and 8. Phenotypes considered as homozygous on starch (positions 1, 2, 5, 6, and 9) also differed slightly with respect to the sharpness of the main band. It was, however, not possible to resolve the broad and blurred bands of the phenotypes in positions 5 and 6 into two distinct isozymes in a photograph. The upper part of the figure gives the separation of the same 9 leukolysates obtained by P A G I F . Each allele apparently determines an additional characteristic minor band in the top(acidic) part of the P A A gel, which was omitted in the
Evidence for Two Additional Common Alleles
221
Fig. 1. Separation of PGM 1isozymes on starch, agarose, and polyacrylamide (SGE, AGE, and PAGIF) of nine selected leukolysates. The diagrammatic representation of the zymograms in the center part refers to nine phenotypes confirmed by family investigations. The tenth type a4 was found recently diagram. Their significance is still under investigation. Hemolysates were less suitable for P A G I F , due to their higher PGM2 activity, causing an unfavorable overlap with the PGM1 gene products. The results of the investigation of 291 leukolysates of non-related blood donors from Hessen and of 20 sperm extracts together with the findings of pedigree investigations in 15 families with 36 children--the propositus being preferably a3 or a4 heterozygous--confirm the assumed genetically determined polymorphism with 4 different c o m m o n genes at P G M 1. The gene frequencies are shown in Table 1. The sum of the frequencies of PGM~ 1 and PGM~ 3 is in good accord with the frequency of the "normal" P G M I allele (0.757, Harris and Hopkinson, 1976); correspondingly PGM~ 2 and P G M ] 4 have a frequency that fits closely to PGM~.
222
P. K0hnl et al.
Table 1. Distribution of PGMI phenotypes and gene frequencies of 291 non-related persons from Hessen (PAG1F results) Phenotype
al a2 a2 a3 a3 a3 a4 a4 a4 a4
- - al - - al - - a2 - - al - - a2 - - a3
n
Gene frequencies
(obs)
(exp)
111 59 9 55 14 5 24 9 4 1
111.35 61.85 8.59 51.34 14.25 5.92 24.12 6.70 5.56 1.31
PGM~ 1 0.6186 PGM~
0.1718
PGM~ 3 0.1426 PGM$
0.0670
~Z2=0.3985
0.80
Evidence for Two Additional Common Alleles at the PGM1 Locus (Phosphoglucomutase--E.C.: 2.7.5.1) A Comparison by Three Different Techniques
P. KUhnl, U. Schmidtmann, and W. Spielmann Institut fur Immunh~matologie der Universit~it Frankfurt/Main, D-6000 Frankfurt/Main, Federal Republic of Germany
Summary. Lysates of erythrocytes, leukocytes, lymphocytes, and extracts of sperms were investigated for the PGM~ isozymes by three techniques: starch gel electrophoresis, high voltage thin-layer agarose gel electrophoresis, and thinlayer isoelectric focusing on polyacrylamide gel. On starch, only the well known c o m m o n phenotypes 1, 2-1, and 2 were demonstrable. On agarose, different distances of the two main cathodal bands (a, b) among the phenotypes 2-1 were noted. Furthermore, on agarose, some types considered as homozygous on starch gel had a single, sharp banded pattern while others were broad and blurred. Optimal separation was achieved by isoelectric focusing on polyacrylamide gel. In 291 leukolysates, 10 different phenotypes were identified. These are considered as gene products of 4 different common alleles at the PGM~ locus as suggested by preliminary family investigations. In a random population from Hessen these four alleles had the following frequencies: PGM~ 1 0.6186, PGM~ 2 0.1718, PGM~ 3 0.1426, and PGM~ 4 0.067. The preliminary designation al, a2, a3 and a4 was chosen as the assumed polymorphism was demonstrated on acrylamide and agarose. The sum of the frequencies PGM~ 1 and PGM~ 3 (the gene products of which have apparently the same electrophoretic mobility on starch) is similar to the frequency of the old P G M I allele (0.757) in Caucasoids, PGM~ 2 and PGM] 4have a frequency of 0.2388 corresponding with the frequency of the old allele P G M 2. Introduction The enzyme phosphoglucomutase (E.C.: 2.7.5.1) occurs in many human tissues (McAlpine et al., 1970; Renninger, 1969). Since Spencer et al. (1964) first detected the polymorphism of PGM, three loci, PGMI, PGM2, and P G M 3 have been identified and assigned to chromosomes 1, 4, and 6, respectively (Nguyen et al., 1971; Douglas et al., 1973; McAlpine et al., 1975; Jongsma et al., 1973). About 80--95% of the enzyme activity is attributable to PGM~ in most human tissues (Harris and Hopkinson, 1976), except erythrocytes which have a smaller amount
220
P. Ktihnl et al.
of PGM1, but a correspondingly higher content of PGM2 (approximately 50 %). The P G M 3 contribution is relatively small in all tissues and apparently not detectable in erythrocytes at all (Harris and Hopkinson, 1976; Quick et al., 1974). Bissbort et al. (1975), however, were able to demostrate the polymorphism of P G M 3 in erythrocyte hemolysateS by means of horizontal starch gel electrophoresis. The three c o m m o n phenotypes in Whites, detectable on PGM-stained starch gels are expressions of two c o m m o n alleles at PGM~ (PGM~ and P G M 2) and analogously at P G M 3 (PGM31 and PGM]). Five additional rare alleles and a deficient allele at PGM1 with a total frequency of less than 0.001 have been identified (Harris et al., 1973; Fiedler and Pettenkofer, 1969). This report gives the results of the separation of P G M 1 gene products by three techniques, starch gel electrophoresis (SGE), agarose gel electrophoresis (AGE), and isoelectric focusing on polyacrylamide gel (PAGIF).
Material and Methods
Hemolysates were prepared according to the method of Marti (1963), leukolysates as reported by Sinha et al. (1970), lympholysates after Otto and Schmid (1970), and sperm lysates as described by Renninger and Sina (1970). Starch gel electrophoresis was performed using the method described by Spencer et al. (1964). Prolonged agarose gel electrophoresis was carried out with the following system: Tris--L-Histidine monohydrochloride 0.25 M, adjusted to pH 6~0for bridge buffer; gel buffer 1: 30 dilution of bridge buffer. 5~zlsamples were applied 5 cm from the anodal side of a 1% agarose gel with the following dimensions: 400 × 200 x 1 mm according to a previously described method (Kiahnl et al., 1974). Electrophoresis was performed at 20 V/cm at ÷ 12° C for 41/2 h in a DESAGA TLE double chamber. Isoeleetric focusing was performed on commercially available polyacrylamide gels (Ampholine PAGplates, LKB, Bromma, Sweden, pH range 3.5--9.5, polyacrylamide gel concentration T=5%,degree of cross-linkage C=3%, ampholine concentration 2.4% (w/v), gel dimensions 245× 110× 1 mm) with a total focusing time of 31/2 h. Staining solutions for all three methods (SGE, AGE, PAGIF) were prepared according to Spencer et al. (1964) with modifications (Schmidtmann et al., 1977).
Results and Discussion
The patterns of nine different selected leukolysates, as obtained with the three methods, are given in Figure 1. In the lower part of the figure, the starch gel zymograms reveal only the three c o m m o n phenotypes P G M l l , PGM12-1, and PGMI2; similar patterns were observed in hemolysates and sperm lysates. Even after prolonged electrophoresis of 28 h no striking differences were observed. In respect to the distances of the two main cathodal bands, the patterns obtained after prolonged A G E (center of Fig. 1) were apparently heterogeneous in positions 3, 4, 7, and 8. Phenotypes considered as homozygous on starch (positions 1, 2, 5, 6, and 9) also differed slightly with respect to the sharpness of the main band. It was, however, not possible to resolve the broad and blurred bands of the phenotypes in positions 5 and 6 into two distinct isozymes in a photograph. The upper part of the figure gives the separation of the same 9 leukolysates obtained by P A G I F . Each allele apparently determines an additional characteristic minor band in the top(acidic) part of the P A A gel, which was omitted in the
Evidence for Two Additional Common Alleles
221
Fig. 1. Separation of PGM 1isozymes on starch, agarose, and polyacrylamide (SGE, AGE, and PAGIF) of nine selected leukolysates. The diagrammatic representation of the zymograms in the center part refers to nine phenotypes confirmed by family investigations. The tenth type a4 was found recently diagram. Their significance is still under investigation. Hemolysates were less suitable for P A G I F , due to their higher PGM2 activity, causing an unfavorable overlap with the PGM1 gene products. The results of the investigation of 291 leukolysates of non-related blood donors from Hessen and of 20 sperm extracts together with the findings of pedigree investigations in 15 families with 36 children--the propositus being preferably a3 or a4 heterozygous--confirm the assumed genetically determined polymorphism with 4 different c o m m o n genes at P G M 1. The gene frequencies are shown in Table 1. The sum of the frequencies of PGM~ 1 and PGM~ 3 is in good accord with the frequency of the "normal" P G M I allele (0.757, Harris and Hopkinson, 1976); correspondingly PGM~ 2 and P G M ] 4 have a frequency that fits closely to PGM~.
222
P. K0hnl et al.
Table 1. Distribution of PGMI phenotypes and gene frequencies of 291 non-related persons from Hessen (PAG1F results) Phenotype
al a2 a2 a3 a3 a3 a4 a4 a4 a4
- - al - - al - - a2 - - al - - a2 - - a3
n
Gene frequencies
(obs)
(exp)
111 59 9 55 14 5 24 9 4 1
111.35 61.85 8.59 51.34 14.25 5.92 24.12 6.70 5.56 1.31
PGM~ 1 0.6186 PGM~
0.1718
PGM~ 3 0.1426 PGM$
0.0670
~Z2=0.3985
0.80
