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Genetic polymorphism of human parotid salivary amylase detected by isoelectric focusing electrophoresis and silver staining
159
Forensic Science International, 35 (198’7)159 - 163 Elsevier Scientific Publishers Ireland Ltd.
GENETIC POLYMORPHISM OF HUMAN PAROTID AMYLASE DETECTED BY ISOELECTRIC FOCUSING ELECTROPHORESIS AND SILVER STAINING
SALIVARY
S. TSUCHIDA and S. IKEMOTO Laboratory of Human Biology and Department 329-04 (Japanl
of Legal Medicine,
Jichi Medical School, Tochigi
(Received February 5th, 1987) (Accepted April 27th, 1987)
Summary In 332 samples of human parotid saliva collected at random from a Japanese population, the genetic polymorphism of salivary a-amylase was detected by isoelectric focusing electrophoresis in a pH range of 5.2-7.2 polyacrylamide gel followed by silver staining. This polymorphism, that was tentatively designated Amy, S, consisted of extra three isozymes of a normal pattern (Amy, N) and isoelectric points of these three isozymes were 5.5, 5.8 and 6.1, respectively. The inheritance was controlled by a dominant allele at an autosomal locus. The frequency of the genes determining these phenotypes were studied as follows: Amy,S = 0.014 f 0.004, Amy,N = 0.986 + 0.004.
Key words: Personal identification: Salivary a-amylase polymorphism: Isoelectric focusing electrophoresis
Introduction
Isozymes of human salivary a-amylase (Amy,) have been separated by various electrophoretic techniques and the inherited variants have been described by Kamaryt and Laxova [l], Boettcher and de la Lande [2], Wolf et al [3], Skude [4], Merritt et al. [5] and Ikemoto et al. [6]. However, the frequency of the individual variants described has been low. Tbe introduction of isoelectric focusing electrophoresis has increased the resolution of Amy, isozymes and the polymorphism with higher frequency have been reported by De Soyza [7], Pronk and Frants [8], Kuhnel and Tischberger [9], Eckersall and Beeley [lo] and Pronk et al. [11,12]. We studied the salivary a-amylase in human parotid saliva using isoelectric focusing electrophoresis with a pH gradient of 5.2-7.2 and silver staining. In the present study, a new phenotype of Amy, has been described. Material and Methods
Samples of human parotid saliva were collected in glass test tubes using a 0379-0'7381871$03.50
0 198’7Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
160
double-chamber cup of the Curby type and stored frozen at - 20°C until use. The sample solution was prepared as previously described [13]. Isoelectric focusing electrophoresis was carried out as follows: Reagent A: 29.1% acrylamide and 0.9% N,N’-methylenebisacrylamide, Reagent B: 87% glycerol. Reagent C: 0.004% riboflavin. Reagent A (6.7 ml), Reagent B (2.3 ml) and Reagent C (0.4 ml) were mixed and 1 ml of Ampholine pH 6-8 (LKB, Bromma, Sweden) was added to it. Then, the combined solution was made up to 20 ml with distilled water and was poured into a 105 mm x 160 mm x 1.0 mm glass plate mould and photopolymerized. Sample solutions (5 ~1) were dipped on rectangular pieces of filter paper and applied 2 cm from the cathode side on the gel plate. Isoelectric focusing was done on the LKB 2117 multiphor apparatus at 4OC. The anode wick contained 2% Ampholine pH 4 -6 and the cathode wick 1 M NaOH. The isoelectric focusing was started with 100 V and the voltage was gradually increased to 1200 V over a 60-min period. Then, the same voltage was kept for 3.5 h until the end of the electrophoresis. After electrophoresis, the gels were stained using the BIORAD (Richmond, California) silver staining reagent. The a-amylase activity of the salivary samples was determined by the Neo Amylase test DAIICHI (DAIICHI Pure Chemicals Co., Ltd., Japan) and the units per litre (IU/ll with samples were diluted to 0.2 x lo6 international distilled water so that an even activity of enzyme was loaded onto each track of the gel. Diluted salivary samples (5 ~11 were applied. After electrophoresis, gels were stained by the starch-iodine method for a-amylase described by Ward et al. [14]. Results
Isoelectric focusing electrophoresis in a pH gradient of 5.2-7.2 followed by silver staining of human parotid saliva yielded about 25 bands and three polymorphic proteins were found (Fig. 1). The phenotypes of this polymorphism were determined by the presence or absence of all of these three bands. When the gel was stained for amylase, these three bands had the activity of amylase. The isoelectric points (~1) of these three variant isozymes were estimated at pH 6.5, 5.8 and 6.1, respectively, when compared with p1 marker proteins. This variant was tentatively designated Amy, S, which was detected by silver staining and the common phenotype was named Amy, N. The saliva collected from type Amy, N individuals at different hours during a day and on different days never revealed these bands. On the other hand, the saliva collected from type Amy, S individuals at different hours during a day invariably revealed these three bands. As a confirmation test, the electrophoresis of type Amy, S saliva using half volume of the original amount and type Amy, N saliva using twice the original amount was performed. These isoelectric focusing patterns showed despite some difference in the no essential change in characteristics, intensity of staining.
161
1
2
3
4
5
6
7
Fig. 1. Isoelectric focusing patterns of parotid saliva in a pH range of 5.2-7.2 polyacrylamide gel followed by silver staining. (1) Amy, S; (2)Amy, N; (3) Amy, S, using half of the original amount; (4) Amy, N, using twice the original amount; (5) mixture of Amy, S and Amy, S; (6) mixture of Amy, S and Amy, N; (7) mixture of Amy, N and Amy, N.
Salivary samples collected at random from Japanese population were studied for the phenotype frequencies of Amy, S and Amy, N. Nine (2.7%) out of 332 samples were of the type Amy, S and 323 (97.3Ohl were of the type Amy, N, respectively. Family studies including 30 families and 55 offspring were done to test genetic hypothesis. Of one mating where both parents had the Amy, S phenotype, there was one Amy, S offspring. Of 26 matings where both parents were Amy, N phenotype, all 44 offspring had the expected Amy, N phenotype. Of 3 matings where one parent was Amy, S and the other Amy, N, there were 6 Amy, S and 1 Amy, N offspring. These findings suggested that the inheritance of the phenotype of Amy, S isozyme was governed by an autosomal dominant allele. From this hypothesis, the estimated gene frequencies that determined these phenotypes in Japanese population were, for AmyIS, 0.014 and for AmyIN, 0.986. Discussion
Isoelectric focusing electrophoresis in a pH gradient of 5.2-7.2 followed by silver staining of Amy, has offered more marked resolution than previously described and has revealed that the normal pattern (type Amy,
162
Nl consist of approximately 20 isozymes, together with the variant pattern (type Amy, Sl which has three extra isozymes with the phenotype frequency of 2.7% in 332 Japanese population within the p1 distribution of the type Amy, N. The genetic variants of Amy, have previously been detected by polyacrylamide gel electrophoresis [4,5,14]. Ikemoto et al. [S] observed the variant Amy, phenotypes by polyacrylamide gel electrophoresis among 150 Japanese and described that the phenotype and gene frequencies of Amy, variant were 0.02 and 0.010 ( f 0.0061,respectively. The relationship between the variant phenotype detected in this study and that previously observed by polyacrylamide gel electrophoresis was not found (Table Il. Using isoelectric focusing electrophoresis, Amy, has been investigated by De Soyza [7], Pronk and Frants [8], Eckersall and Beeley [9,15] and Pronk et al. [lo] and the frequency of variant phenotypes was found to be higher than that previously described. The relationship between the Amy, variant in this study and those previously observed by isoelectric focusing electrophoresis is unclear. It appears that Amy, variants previously described are major isozymes, while the three isozymes of type Amy, S are all minor. Karn et al. [16] have demonstrated the evidence for posttranscriptional modification of Amy, isozymes in human whole saliva. They suggested that salivary amylase modifier was an enzyme contributed by oral bacteria. This phenomenon was not observed in clean parotid saliva. In this study, we investigated Amy, isozyme in parotid saliva in order to minimize the possible contamination by bacteria in oral cavity. Therefore, this variant could not be attributed to post secretory modification caused by the oral environment. The family data in the present study suggested autosomal dominant inheritance. However, the previously reported inheritance of Amy, system which was detected by isoelectric focusing electrophoresis was that of autosomal codominant alleles. It is not impossible that this variant phenotype was determined by autosomal codominant alleles both AmyIN and TABLE
1
THE RELATIONSHIP BETWEEN THE PHENOTYPES OF SALIVARY AMYLASE DETECTED BY THE METHOD OF WARD ET AL. [14] WITH HORIZONTAL POLYACRYLAMIDE GEL ELECTROPHORESIS IN pH 8.5 AND THAT DETECTED BY THE METHOD OF PRESENT STUDY PhQnOtypQS by
present method
PhQnotypQS by the method of ward Qt aL 11.41
Total
Amy, normal
Amy, vatint
AmY, S
70 12
3 0
73 12
Total
82
3
85
Amy, N
163
AmyIS. According to this hypothesis, the phenotype of Amy, N would be the homozygous expression of the normal genotype and that of variant isozyme would be the heterozygous expression of the normal cross with a variant genotype. The homozygous expression of variant genotype would be another phenotype which has not been detected. It is likely that the family data is insufficient for analysis of the inheritance. At the moment, no other phenotypes except two, which are normal (Amy, NJ and variant (Amy, S) phenotypes, are detected in the family data. Therefore, we frame a hypothesis that the variant phenotype of Amy, is determined by an autosomal dominant allele. Acknowledgements The authors are indebted to Dr. S. Tomita for drawing the attention to this problem and wish to thank E. Nishiumi for providing human parotid saliva. References
6
10 11 12
13 14 15 16
J. Kamaryt and R. Laxova, Amylase heterogeneity; Some genetik and clinical aspects. Humangenetik, 10965) 579 - 586. B. Boetteher and F.A. De La Lande, Electrophoresis of human saliva and identification of inherited variants of amylase isozymes. Au&. J. Ezp. BioL &fed Sci, 47 (1969) 97- 103. R.O. Wolf, L.L. Taylor, J.D. Niswander and J.T. Schwartz, The heritability of human salivary isoamylase. Arch. Oral BioL, 16 (1971) 1357 - 1359. G. Skude, Isozyme variation of human salivary amylase. Humangenetik, 12 (1971) 255-256. A.D. Merritt, M.L. Rivas, D. Bixler and R. Newell, Salivary and pancreatic amylase: Electropboretic characterization and genetic studies. Am. J. Hum. Genet., 25 (1973) 510522. S. Ikemoto, K. Minaguchi and H. Hinohara, Genetic polymorphisms of human parotid salivary proteins (Pa, Pb, Pr, Db and Pm) and salivary amylase isozyme in Japanese population. Hum. Hered. 27 (1977) 328-331. K. De Soyza, Polymorphism of human salivary amylase. Hum. Genet., 45 (1978) 189- 192. J.C. Pronk and R.R. Frants, New genetic variant of parotid salivary amylase. Hum. Hered. 29 (1979) 181- 186. P. Kuhnel and H. Tischberger, Amylase, polymorphism of human parotid saliva: Detection of a new allele, Amy,5 by isoelectric focusing and Amy, population data from Germany. Ekctrophoresis, 1 (1980) 186 - 190. P.D. Eckersall, R.J. Mairs and J.A. Beeley, An improved procedure for isoelectric focusing of human salivary proteins. Arch. Oral BioL, 26 (1981) 727 - 733. J.C. Pronk, R.R. Frants, W. Jansen, A.W. Eriksson and G.J.M. Tonino, Evidence for duplication of the human salivary amylase gene. Hum. Genet, 60 (1982) 32-35. J.C. Pronk, W.J. Jansen, A. Pronk, C.F.A.M. v.d. Pol, R.R. Frants and A.W. Eriksson, Salivary protein polymorphism in Kenya: Evidence for a new AMY1 allele. Hum. Hered. 34 (1984) 212-216. S. Ikemoto, H. Hinohara, S. Tsuchida and K. Tomita, Phenotype and gene frequencies of acid phosphatase(s-AcP) in the human parotid saliva. Hum. Genet., 71 (1985) 30-32. J.C. Ward, A.D. Merritt and A.D. Bixler, Human salivary amylase: Genetics of electrophoretic variants. Am. J. Hum. Gene&, 23 (1971) 403-409. P.D. Eckersall and J.A. Beeley, Genetic analysis of human salivary a-amylase isozymes by isoelectric focusing. B&hem. Genet., 19 (1981) 1055- 1062. R.C. Karn, J.D. Shulkin, A.D. Merritt and R.C. Newell, Evidence for post-transcriptional modification of human salivary amylase (Amy,) isozymes. Biochem. Genet., 10 (1973) 341350.
Forensic Science International, 35 (198’7)159 - 163 Elsevier Scientific Publishers Ireland Ltd.
GENETIC POLYMORPHISM OF HUMAN PAROTID AMYLASE DETECTED BY ISOELECTRIC FOCUSING ELECTROPHORESIS AND SILVER STAINING
SALIVARY
S. TSUCHIDA and S. IKEMOTO Laboratory of Human Biology and Department 329-04 (Japanl
of Legal Medicine,
Jichi Medical School, Tochigi
(Received February 5th, 1987) (Accepted April 27th, 1987)
Summary In 332 samples of human parotid saliva collected at random from a Japanese population, the genetic polymorphism of salivary a-amylase was detected by isoelectric focusing electrophoresis in a pH range of 5.2-7.2 polyacrylamide gel followed by silver staining. This polymorphism, that was tentatively designated Amy, S, consisted of extra three isozymes of a normal pattern (Amy, N) and isoelectric points of these three isozymes were 5.5, 5.8 and 6.1, respectively. The inheritance was controlled by a dominant allele at an autosomal locus. The frequency of the genes determining these phenotypes were studied as follows: Amy,S = 0.014 f 0.004, Amy,N = 0.986 + 0.004.
Key words: Personal identification: Salivary a-amylase polymorphism: Isoelectric focusing electrophoresis
Introduction
Isozymes of human salivary a-amylase (Amy,) have been separated by various electrophoretic techniques and the inherited variants have been described by Kamaryt and Laxova [l], Boettcher and de la Lande [2], Wolf et al [3], Skude [4], Merritt et al. [5] and Ikemoto et al. [6]. However, the frequency of the individual variants described has been low. Tbe introduction of isoelectric focusing electrophoresis has increased the resolution of Amy, isozymes and the polymorphism with higher frequency have been reported by De Soyza [7], Pronk and Frants [8], Kuhnel and Tischberger [9], Eckersall and Beeley [lo] and Pronk et al. [11,12]. We studied the salivary a-amylase in human parotid saliva using isoelectric focusing electrophoresis with a pH gradient of 5.2-7.2 and silver staining. In the present study, a new phenotype of Amy, has been described. Material and Methods
Samples of human parotid saliva were collected in glass test tubes using a 0379-0'7381871$03.50
0 198’7Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
160
double-chamber cup of the Curby type and stored frozen at - 20°C until use. The sample solution was prepared as previously described [13]. Isoelectric focusing electrophoresis was carried out as follows: Reagent A: 29.1% acrylamide and 0.9% N,N’-methylenebisacrylamide, Reagent B: 87% glycerol. Reagent C: 0.004% riboflavin. Reagent A (6.7 ml), Reagent B (2.3 ml) and Reagent C (0.4 ml) were mixed and 1 ml of Ampholine pH 6-8 (LKB, Bromma, Sweden) was added to it. Then, the combined solution was made up to 20 ml with distilled water and was poured into a 105 mm x 160 mm x 1.0 mm glass plate mould and photopolymerized. Sample solutions (5 ~1) were dipped on rectangular pieces of filter paper and applied 2 cm from the cathode side on the gel plate. Isoelectric focusing was done on the LKB 2117 multiphor apparatus at 4OC. The anode wick contained 2% Ampholine pH 4 -6 and the cathode wick 1 M NaOH. The isoelectric focusing was started with 100 V and the voltage was gradually increased to 1200 V over a 60-min period. Then, the same voltage was kept for 3.5 h until the end of the electrophoresis. After electrophoresis, the gels were stained using the BIORAD (Richmond, California) silver staining reagent. The a-amylase activity of the salivary samples was determined by the Neo Amylase test DAIICHI (DAIICHI Pure Chemicals Co., Ltd., Japan) and the units per litre (IU/ll with samples were diluted to 0.2 x lo6 international distilled water so that an even activity of enzyme was loaded onto each track of the gel. Diluted salivary samples (5 ~11 were applied. After electrophoresis, gels were stained by the starch-iodine method for a-amylase described by Ward et al. [14]. Results
Isoelectric focusing electrophoresis in a pH gradient of 5.2-7.2 followed by silver staining of human parotid saliva yielded about 25 bands and three polymorphic proteins were found (Fig. 1). The phenotypes of this polymorphism were determined by the presence or absence of all of these three bands. When the gel was stained for amylase, these three bands had the activity of amylase. The isoelectric points (~1) of these three variant isozymes were estimated at pH 6.5, 5.8 and 6.1, respectively, when compared with p1 marker proteins. This variant was tentatively designated Amy, S, which was detected by silver staining and the common phenotype was named Amy, N. The saliva collected from type Amy, N individuals at different hours during a day and on different days never revealed these bands. On the other hand, the saliva collected from type Amy, S individuals at different hours during a day invariably revealed these three bands. As a confirmation test, the electrophoresis of type Amy, S saliva using half volume of the original amount and type Amy, N saliva using twice the original amount was performed. These isoelectric focusing patterns showed despite some difference in the no essential change in characteristics, intensity of staining.
161
1
2
3
4
5
6
7
Fig. 1. Isoelectric focusing patterns of parotid saliva in a pH range of 5.2-7.2 polyacrylamide gel followed by silver staining. (1) Amy, S; (2)Amy, N; (3) Amy, S, using half of the original amount; (4) Amy, N, using twice the original amount; (5) mixture of Amy, S and Amy, S; (6) mixture of Amy, S and Amy, N; (7) mixture of Amy, N and Amy, N.
Salivary samples collected at random from Japanese population were studied for the phenotype frequencies of Amy, S and Amy, N. Nine (2.7%) out of 332 samples were of the type Amy, S and 323 (97.3Ohl were of the type Amy, N, respectively. Family studies including 30 families and 55 offspring were done to test genetic hypothesis. Of one mating where both parents had the Amy, S phenotype, there was one Amy, S offspring. Of 26 matings where both parents were Amy, N phenotype, all 44 offspring had the expected Amy, N phenotype. Of 3 matings where one parent was Amy, S and the other Amy, N, there were 6 Amy, S and 1 Amy, N offspring. These findings suggested that the inheritance of the phenotype of Amy, S isozyme was governed by an autosomal dominant allele. From this hypothesis, the estimated gene frequencies that determined these phenotypes in Japanese population were, for AmyIS, 0.014 and for AmyIN, 0.986. Discussion
Isoelectric focusing electrophoresis in a pH gradient of 5.2-7.2 followed by silver staining of Amy, has offered more marked resolution than previously described and has revealed that the normal pattern (type Amy,
162
Nl consist of approximately 20 isozymes, together with the variant pattern (type Amy, Sl which has three extra isozymes with the phenotype frequency of 2.7% in 332 Japanese population within the p1 distribution of the type Amy, N. The genetic variants of Amy, have previously been detected by polyacrylamide gel electrophoresis [4,5,14]. Ikemoto et al. [S] observed the variant Amy, phenotypes by polyacrylamide gel electrophoresis among 150 Japanese and described that the phenotype and gene frequencies of Amy, variant were 0.02 and 0.010 ( f 0.0061,respectively. The relationship between the variant phenotype detected in this study and that previously observed by polyacrylamide gel electrophoresis was not found (Table Il. Using isoelectric focusing electrophoresis, Amy, has been investigated by De Soyza [7], Pronk and Frants [8], Eckersall and Beeley [9,15] and Pronk et al. [lo] and the frequency of variant phenotypes was found to be higher than that previously described. The relationship between the Amy, variant in this study and those previously observed by isoelectric focusing electrophoresis is unclear. It appears that Amy, variants previously described are major isozymes, while the three isozymes of type Amy, S are all minor. Karn et al. [16] have demonstrated the evidence for posttranscriptional modification of Amy, isozymes in human whole saliva. They suggested that salivary amylase modifier was an enzyme contributed by oral bacteria. This phenomenon was not observed in clean parotid saliva. In this study, we investigated Amy, isozyme in parotid saliva in order to minimize the possible contamination by bacteria in oral cavity. Therefore, this variant could not be attributed to post secretory modification caused by the oral environment. The family data in the present study suggested autosomal dominant inheritance. However, the previously reported inheritance of Amy, system which was detected by isoelectric focusing electrophoresis was that of autosomal codominant alleles. It is not impossible that this variant phenotype was determined by autosomal codominant alleles both AmyIN and TABLE
1
THE RELATIONSHIP BETWEEN THE PHENOTYPES OF SALIVARY AMYLASE DETECTED BY THE METHOD OF WARD ET AL. [14] WITH HORIZONTAL POLYACRYLAMIDE GEL ELECTROPHORESIS IN pH 8.5 AND THAT DETECTED BY THE METHOD OF PRESENT STUDY PhQnOtypQS by
present method
PhQnotypQS by the method of ward Qt aL 11.41
Total
Amy, normal
Amy, vatint
AmY, S
70 12
3 0
73 12
Total
82
3
85
Amy, N
163
AmyIS. According to this hypothesis, the phenotype of Amy, N would be the homozygous expression of the normal genotype and that of variant isozyme would be the heterozygous expression of the normal cross with a variant genotype. The homozygous expression of variant genotype would be another phenotype which has not been detected. It is likely that the family data is insufficient for analysis of the inheritance. At the moment, no other phenotypes except two, which are normal (Amy, NJ and variant (Amy, S) phenotypes, are detected in the family data. Therefore, we frame a hypothesis that the variant phenotype of Amy, is determined by an autosomal dominant allele. Acknowledgements The authors are indebted to Dr. S. Tomita for drawing the attention to this problem and wish to thank E. Nishiumi for providing human parotid saliva. References
6
10 11 12
13 14 15 16
J. Kamaryt and R. Laxova, Amylase heterogeneity; Some genetik and clinical aspects. Humangenetik, 10965) 579 - 586. B. Boetteher and F.A. De La Lande, Electrophoresis of human saliva and identification of inherited variants of amylase isozymes. Au&. J. Ezp. BioL &fed Sci, 47 (1969) 97- 103. R.O. Wolf, L.L. Taylor, J.D. Niswander and J.T. Schwartz, The heritability of human salivary isoamylase. Arch. Oral BioL, 16 (1971) 1357 - 1359. G. Skude, Isozyme variation of human salivary amylase. Humangenetik, 12 (1971) 255-256. A.D. Merritt, M.L. Rivas, D. Bixler and R. Newell, Salivary and pancreatic amylase: Electropboretic characterization and genetic studies. Am. J. Hum. Genet., 25 (1973) 510522. S. Ikemoto, K. Minaguchi and H. Hinohara, Genetic polymorphisms of human parotid salivary proteins (Pa, Pb, Pr, Db and Pm) and salivary amylase isozyme in Japanese population. Hum. Hered. 27 (1977) 328-331. K. De Soyza, Polymorphism of human salivary amylase. Hum. Genet., 45 (1978) 189- 192. J.C. Pronk and R.R. Frants, New genetic variant of parotid salivary amylase. Hum. Hered. 29 (1979) 181- 186. P. Kuhnel and H. Tischberger, Amylase, polymorphism of human parotid saliva: Detection of a new allele, Amy,5 by isoelectric focusing and Amy, population data from Germany. Ekctrophoresis, 1 (1980) 186 - 190. P.D. Eckersall, R.J. Mairs and J.A. Beeley, An improved procedure for isoelectric focusing of human salivary proteins. Arch. Oral BioL, 26 (1981) 727 - 733. J.C. Pronk, R.R. Frants, W. Jansen, A.W. Eriksson and G.J.M. Tonino, Evidence for duplication of the human salivary amylase gene. Hum. Genet, 60 (1982) 32-35. J.C. Pronk, W.J. Jansen, A. Pronk, C.F.A.M. v.d. Pol, R.R. Frants and A.W. Eriksson, Salivary protein polymorphism in Kenya: Evidence for a new AMY1 allele. Hum. Hered. 34 (1984) 212-216. S. Ikemoto, H. Hinohara, S. Tsuchida and K. Tomita, Phenotype and gene frequencies of acid phosphatase(s-AcP) in the human parotid saliva. Hum. Genet., 71 (1985) 30-32. J.C. Ward, A.D. Merritt and A.D. Bixler, Human salivary amylase: Genetics of electrophoretic variants. Am. J. Hum. Gene&, 23 (1971) 403-409. P.D. Eckersall and J.A. Beeley, Genetic analysis of human salivary a-amylase isozymes by isoelectric focusing. B&hem. Genet., 19 (1981) 1055- 1062. R.C. Karn, J.D. Shulkin, A.D. Merritt and R.C. Newell, Evidence for post-transcriptional modification of human salivary amylase (Amy,) isozymes. Biochem. Genet., 10 (1973) 341350.