GENETIC VARIATION IN AMOUNT OF SALIVARY AMYLASE IN THE BANK VOLE, CLETHRZONOMYS GLAREOLA

J. PETER HJORTH1, MIRIAM MEISLERZ AND J. TBNNES NIELSENl

'Instilute of Ecology and Genetics, Un:versity of Aarhus, DK-8000 Arhus, Denmark 2Department of Human Genetics, University of Michigan, Ann Arbor, Michigan 48109

Manuscript received January 2,1979 Revised copy received March 26,1979

ABSTRACT

Several investigated bank vole populations are polymorphic for the number of salivary amylase loci, and individual chromosomes may carry one, two or three linked amylase structural genes. In the present study, we have used bank vole stocks homozygous for different chromosomes to investigate the relation- ship between amylase production and gene number. By measuring the amy- lase activity in parotid glands and the percentage of amylase protein in saliva, we have been able to demonstrate that the amount of salivary amylase is directly proportional to the proposed gene number. The paper also describes the allele, Am?", which codes for a heat-labile salivary amylase. The relative amounts of the heat-labile isozyme have been determined in different hetero- zygotes containing this allele, and these results also support the multiple locus model. Finally, a stock devoid of salivary amylase activity was established. Animils from this strain have, however, a protein in the parotid glands and in saliva that is very similar to amylase in molecular weight, amino acid com- position and in its binding to glycogen and cyclohepta-amylose. In genetic crosses, the protein segregates as an amylase allele. Therefore, this protein, encoded by the functionally null allele AmyN, may represent an incorrectly processed amylase precursor.

I N rodents and some other mammals including man, large quantities of amylase are produced in both the salivary glands and the pancreas. In several species,

genetic studies of electrophoretic variants have demonstrated that salivary and pancreatic amylases are determined by closely linked genes (KARN and MALA- CINSKI 1978). These related structural genes may have arisen by duplication of a common ancestral gene. In the bank vole, Ctethrionomys g-lareola, the salivary amylase is determined by more than one structural locus (NIELSEN 1969, 1977a,b), and in the house mouse, Mus musculus, several genes code for the pancreatic amylase (NIELSEN 1974; HJORTH et at., in preparation), so that in these two species at least, additional duplications may have occurred during evolution.

In bank vole populations, four salivary amylase alleles, represected by the bands S, A, H and B, have been demonstrated by means of agar gel electro- phoresis (NIELSEN 1969, 1977a,b). A number of animals carried chromosomes Genetics 92: 915-930 July, 1979.

916 J. P. HJORTH. M. MEISLER AND J. T. IVIELSEN

with closely linked genes for two amylase isozymes, and we have established homozygous stock with double-banded patterns. In addition to the electro- phoretic (qualitative) variation, we have found quantitative variation, as for instance in the relative amounts of the two isozyme forms in some of the double- banded stocks. To indicate both the qualitative and the quantitative phenotype of the stocks, they were designated, for example, “S”, “A”, “SA” and “SA”, where A and S refer to the electrophoretic form of the amylase, while upper and lower case letters are used to indicate the relative amounts of the isozyme forms in double-banded animals. In the given example the “S” and “A” stocks are single-banded, whereas “SA” and ‘-SA” are double-banded. In the “SA” stock the amounts of the two amylase forms are the same, while the “SA” stock shows a pattern with relatively more A than S amylase.

Genetic analysis of the quantitative variation was carried out by measuring the relative amounts of different salivary amylase isozymes from individual heterozygotes. Salivary gland homogenates were electrophoresed, the slides stained for protein and the intensity of the bands determined by means of an integrating scanner. An outline of the analysis is given by the following example: We studied offspring from crosses among wild “A” and laboratory stock “B” animals with two structural salivary loci, i.e., with the genotype Amy” AmyB/ AmyB AmyB. The ratio of A:B amylase in these double-banded offspring fell into three groups, namely, 1:2, 2:2 or 3:2. This result was interpreted to mean that the wild-derived chromosomes carried either one, two or three salivary amylase genes. Animals homozygous for these different types of chromosomes would then carry two, four o r six amylase structural genes. The model is consistent with our results from a number of other crosses (NIELSEN 1977a,b). The genes must be close together on the chromosome, because no recombination has been scored among more than a thousand offspring of informative matings. Thus, the genetic basis for the quantitative variation in bank vole salivary amylase is probably a polymorphism for the number of structural genes on the chromosomes.

In this paper, we describe a salivary amylase allele with no amylase activity, a functionally null allele, Amy“. We also demonstrate that the allele A m y , giving the electrophoretic phenotype “S”: codes for a heat-labile form of the enzyme. By measuring the total amount of amylase in parotid glands from animals of different genotypes, by determining the relative amounts of heat- labile amylase in saliva from heterozygotes containing the A m p allele and by determining the percentage of amylase protein in saliva, we have obtained three independent types of data that support the multiple amylase loci model.

MATERIALS AND METHODS

Animals: As the bank \role is not commonly used as a laboratory animal, our source of gznetic variation has been animals trapped in the wild. We have established 12 pure-breeding lines with different amylase types. These lines vary with respect to both electrophoretic alleles and the number of salivary amylase genes on their chromosomes (Table 1). Eleven of these chromosomes have been described previously (NIELSEN 1969, 1977a,b). The AmyN allele was

GENETIC VARIATION IN AMOUNT O F AMYLASE

TABLE 1

List of bank uole strains

91 7

Phenotype Genes on chromosomes

“S” “A” “H” ‘3” “SA” “A” “AH’ “AB” “B’ “SA”

“Ab” “N” (no activity)

Am ys” Amy* AmyH AmyB Amy8 A m y 4 Amy* Amy” Amy4 Amy” Amy* Amy€: AmyB Amy€: Am+ Amy* AmyA Amy* Amy* AmyB AmyN

found in a heterozygous animal from a sample of bank voles kindly provided by E. PINCENT, Edinburgh. In crosses, the allele behaves as a null allele, and animals from the homozygous AmyN/AmyLv stock show very little salivary amylase activity. In spite of this, the viability of the strain in the laboratory is not impaired.

Sample Preparation: Saliva samples for agar gel electrophoresis were obtained by flushing the mouth of etherized animals with a drop of distilled water.

Tissues were homogenized in 1 ml of 0.05 M phosphate buffer, pH 6.9, containing 7 mM sodium chloride (buffer A). Homogenates were centrifuged for ten min at 12,000 X g. The supernatant was stored at -20” and assayed within a few days Amylase in these preparations is stable €or several months at -20”.

Saliva for measurement of heat stability and for glycogen precipitation was obtained from animals anesthetized by intraperitoneal injection of 1.0 to 1.5 mg of nembutal. To stimulate salivation, 0.1 mg of isoproterenol was injected. Parotid glands were removed from animals killed by cervical dislocation. The glands were cleaned using a stereo microscope with low magnification. For dissection of the different salivary glands, the animals were anesthetized with nembutal and killed by bleeding.

Electrophoresis: Electrophoresis of amylase in 0.9% agar gel at pH 7.3 has been described previously (SICK and NIELSEN 1964; SICK 1965; NIELSEN 1969). Electrophoresis in polyacrylam- ide gels under nondenaturing conditions was performed in 7% gels with the TRIS-glycine buffer system described by CLARKE (1964). Electrophoresis in the presence of sodium dodecyl sulfate was carried out with the buffer system described by LAEMivLr (1970). The acrylamide con- centration was 10% for tube gels and 9% for slab gels. All polyacrylamide gels were strained for protein with 2.8% Coomassie brilliant blue R in a solution of methanol, water and acetic acid, 5:5:1.

Amylase determination: Amylase activity was assayed by measurement of the free reducing sugar groups produced whcn amylase acts on starch. We have employed the procedure of DAHLQVIST (1962), with the following modifications: The final reaction volume was 1 ml, the final concentration of starch (soluble starch after Zulkowsky, Merck) was 2% and the reaction time was only two min. Enzyme samples were diluted in buffer A, so that the final reaction mixture contained less than 2 pU. One unit (U) of amylase activity is defined as the amount of amylase that liberates one mole of free reducing groups per minute at 30”.

Heat inact uution: Inactivation was carried out in buffer A containing 0.01 % bovine serum albumin. The heat treatment was initiated by adding 100 pl cold enzyme solution to 400 pl pre- heated buffer, and was terminated by transferring the mixture to an ice bath. The residual enzyme activity was assayed within 20 min.

918 J. P. HJORTH, M. MEISLER A N D J. T. NIELSEN

Glycogen precipitation: Amylase was precipitated from saliva by procedure I of SCHRAMM and LOYTER (1966). High molecular weight oyster glycogen was obtained from ICN Pharma- ceuticals Inc., New York, and precipitated from 40% ethanol before use in the precipitation of amylase.

Isoproterenol-stimulated saliva from single animals was diluted to approximately 1.3 mg protein per ml. Two 600 pl samples of diluted saliva from each animal were precipitated with 4 and 5 mg of glycogen, respectively. The glycogen-precipitated protein was recovered by cen- trifugation for six min at 10,000 x g, washed once in 500 pl of 40% ethanol in buffer A, and finally dissolved in buffer A. Autodigestion was performed at 30" for 20 min. Amylase activity and protein concentration were measured within a few hours. The protein determination was performed according to the micro-method described by LOWRY et al. (1951), using bovine serum albumin as a standard. The recovery of amylase activity was always greater than 80%. We obtained closely similar values for the specific activity of duplicate preparations from a single saliva sample. However, the specific activity of preparations from different saliva samples varied considerably (from 700 to 1450 p U per mg protein). This variation was independent of the geno- type of the strains and appears to be caused by variable quantities of an inactive protein that is co-precipitated by glycogen (see RESULTS).

Zmmunotitrat on: Immunoprecipitation studies were carried out with gamma globulin pre- pared from rabbit antiserum against mouse salivary amylase, kindly provided by D. OWERBACH. One p unit of amylase from tissue extracts was incubated with gamma globulin in a total volume of 0.6 ml of 0.05 M phosphate buffer, pH 6.8, containing 0.14 M sodium chloride. Samples were incubated for 30 min at 37" and then for 16 hr at 25". After centrifugation to remove immuno- precipitated protein, an 0.5 ml aliquot of the supernatant was assayed for nmprecipitated amy- lase activity.

Amino acid analysis: Amino acid analysis was carried out with the assistance of T. ELLEBAEK PETWSEN and K. K. THOMSEN, Institute of Molecular Biology, University tof Aarhus, using the method of MOORE and STEIN (1963). Samples containing about 200 pg of protein were dialyzed against water and hydrolyzed in 6~ HC1 for 24 hours at 110" in vacuo, and the amino acid composition was determined by using a Beckman amino acid analyzer. Values were not cor- rected for amino acid destruction.

RESULTS

Tissue distribution of amylase: Salivary amylase activity in bank voles is normally present in high concentrations in the parotid glands, whereas the other salivary glands, the submandibularis and the sublingualis, produce other proteins found in the saliva. In Table 2, we have compared the amylase content of differ-

TABLE 2

Amylase activity in tissues from a BB/N and N / N individual

SU amylase per m g gland Tissue BBiN N M

Parotid gland Submandibular gland Sublingual gland Pancreas Brain Kidney Liver Spleen Serum

29.5 0.03 0.04 5.90

<O.Ol <O.Ol <0.01 <0.01 <0.01

0.95 0.01 0.01 4.90

<O.Ol <O.Ol <O.Ol <0.01 <0.01

GENETIC VARIATION IN AMOUNT O F AMYLASE

TABLE 3

Amylase activity in parotid glands from bank voles of various genotypes

919

Number Mean value Genotype of individuals in pU/mg gland

N/N 8 0.4

A/A A/B B/B H/H N/BB

AA/AA AA/BB BB/BB ABjAB H/SAA

S AA/S AA

AAB/AAB

8.5 21.8

8 34.5 9

3 4

18 1 42 30.7

16 40.9

The individual values are given in Figure 1.

ent tissues from two animals of the genotypes AmyB AmyB/AmyN and AmyN/ AmyN, respectively. The animal homozygous for the AmyN allele showed a very low level of amylase activity in the parotid glands, while the activity level in the pancreas was normal. The nature of this mutant allele will be discussed in the final section of this paper.

Total salivary amylase activity: Table 3 shows the results of a determination of amylase activity in parotid glands from animals of various genotypes. The specimens were grouped in accordance with the proposed number of structural genes that code for active amylase. The amylase content of the glands varies considerably during the feeding cycle. In “resting” periods, part of the synthe- sized amylase is stored in the glands as zymogen granules, but large amounts of the enzyme are released from the store and secreted when the animals are eating. In this study, no attempt was made to control this variation, and we did observe a considerable range of values among animals of the same genotype. However, when the data from specimens with the same number of salivary amylase genes were pooled, a clear pattern became evident: the maximum values of activity increased with the gene number (Figure 1) as did the mean values for the differ- ent groups. These mean values for animals with two, four or six amylase genes are 14.9, 30.7 and 45.0, respectively, closely resembling the proportions of 1:2:3. The original classification of strains was based on their relative contribution to the total amylase in heterozygotes. We have shown here that this classification also groups strains with respect to their absolute amylase activities.

Thermostability studies: When the thermal stability of salivary amylase from

920 J. P. H J O R T H , M. MEISLER AND J. T. NIELSEN

I

CLASS I

a.

CLASS II 0

CLASS IU

I

0 20 40 60 80 100 pUNlTS OF AMYLASE ACTIVITY PER mg GLAND

F i G U R E 1.-Distribution of amylase activity of individual bank voles (from Table 3) . Class 0 are animals from the “ N ’ strain. Animals in Class I, I1 and I11 have two, four or six genes for salivary amylase, respectively.

a number of our stocks was investigated, we found that the enzyme was stable a t 48”, with a single exception (Figure 2). In the “S” strain, with genotype AmySU/ AmySu, an unstable amylase isozyme was identified. Two other stocks, namely “SA” ( A m f AmyA/AmyS AmyA) and L L ~ A ” ( A m y AmyA AmyA/Amf AmyA AmyA) carry alleles with the same electrophoretic mobility, but they do not con- tain a thermolabile component (Figure 2). Since amylase is a monomeric enzyme, it is unlikely that subunit interactions can be responsible for the sta- bility of the A m y allele in the “SA” and “SA” stocks. Therefore, we have desig- nated the allele that codes for the thermolabile isozyme as AmpU and the geno- type of strain “S” as AmyS“/AmySU.

The percentage of thermolabile amylase was measured in four types of hetero- zygotes, all having the AmySU allele (Figure 3 ) . In the AmySU/AmyH animals, approximately 56% of the amylase is stable. If this value is corrected for the residual activity found irr homozygous AmySu individuals (Figure 2), the stable amylase (from AmyH) is 52% o€ the total enzyme activity. Using the same correction, the AmySU/AmyA AmyA and AmySU/AmyA AmyB heterozygotes both had 69% of the stable amylase, while the corrected value for AmyS”/AmyA AmyA AmyB individuals is 78%. Thus, the proportion of heat-labile enzyme in

GENETIC VARIATION IN AMOUNT O F AMYLASE 92 1

0 5 IO 15 MINUTES AT 4 8 * C

FIGURE 2.-Heat inactivation at 48” of salivary amylase from homozygotes. 0: genotype Su/Su, A: SA/SA, .: SAMSAA, : H/H, v: BB/BB, A: ABJ‘AB, 7: AH/AH, 0: AABJ AAB.

these heterozygotes is close to one-half, one-third and one-fourth, respectively; a result which again indicates that the amylase production is directly proportional to the proposed number of structural genes on the chromosomes.

Quantitation of amylase protein in saliva: Amylase can be purified from mix- tures of protein by precipitation with glycogen (SCHRAMM and LOYTER 1966), and we have utilized this method to quantitate the amount of amylase protein in saliva from the bank vole stocks. Aliquots of saliva contining 0.3 to 0.6 mg of total protein from animals with two, four or six amylase genes, and from animals of the “N” strain, were treated with increasing amounts of glycogen (Figure 4). The filled symbols in the figure represent the percentages of precipitated amylase, and it can be seen that 1 mg of glycogen was sufficient to precipitate almost all (>95%) of the amylase in these samples. The open symbols in Figure 4 show the precipitated protein as a percentage of the total salivary pro- tein. The values for animals with two, four or six genes are 26%, 42% and 58%, respectively, so that it is apparent that the concentration of amylase in secreted saliva increases with the proposed number of genes. The data for the “N’ stock will be discussed in the last paragraph in this section.

922

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J. P. H J O R T H , M. MEISLER A N D J. T. NIELSEN

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Saliva from ten strains was analyzed by this method using saturating amounts of glycogen to precipitate the amylase. For each saliva sample, we calculated the amount of glycogen-precipitable protein per mg of nonprecipitable protein. The values for the individual specimens are given in Figure 5. The “N” strains are shown separately, and the results will be discussed later. The remaining animals are grouped in accordance with their gene number (2,4 or 6). The mean values for the three groups are 0.44, 0.84 and 1.32 mg, which gives the same 1 :2:3 ratio as that found for the amylase activity in the parotid glands.

Characterization of amylase protein in saliva: The procedure €or glycogen precipitation used in this study is generally considered to be specific for amylase and it has proved to be so in our laboratories when used on homogenates of mouse salivary or pancreatic glands. However, when glycogen-precipitated protein from vole saliva is analyzed by native polyacrylamide gel electrophoresis, a protein devoid of amylase activity appears in addition to the expected active amylase protein (Figure 6) . From polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, we find the apparent molecular weight of this inactive protein to be about 58,000, whereas that of active amylase is about 55,000 (Figure 7) . The inactive amylase protein is also obtained, together with active

GENETIC VARIATION IN AMOUNT O F AMYLASE 923

I oc

80

0 w G 5 60 - 0 w LL

I-

U Lz W

a

f, 40

a

20

0

0 1 2 3 mg GLYCOGEN

FIGURE 4.--The effect of increasing amounts of glycogen on the percent precipitated amy- lase (filled symbols) and protein (open symbols) in saliva from specimens of four strains. 0 and 0 : genotype B/B, A and A: AA/AA, 0 and M: AAB/AAB, V; IVJN. The initial amount of total salivary protein was between 0.3 and 0.6 mg in each experiment.

amylase, when vole saliva is subjected to affinity chromatography using sepha- rose coupled with cyclohepta-amylose (CHA), a method described to be specific for purification of a-amylase (SILVANOVICH and HILL 1976). The relative amount of inactive amylase protein is fairly constant from repeated glycogen precipitations of the same saliva sample, but it varies considerably among indi- vidual animals and may account for as much as 50% of the glycogen-precipitable protein. The specific amylase activity of glycogen precipitates from saliva varies almost two-fold among individual voles. This is most likely due to variable pro- portions of the inactive amylase protein. The variation in specific activity falls within the same range in animals having two, four or six amylase genes. There- fore, since the amount of active amylase increases with the proposed gene num- ber, the amount of inactive amylase must increase also to keep the specific activity of glycogen precipitates within the same range. Thus, the amounts of active and inactive amylase protein seem to be related. This quantitative rela- tionship, together with the amylase-like binding of the inactive protein to both

924 J. P. H J O R T H , M. MEISLER AND J. T. NIELSEN

I

0

: 0 0

1 I I I I

0 I II m FIGURE 5.-Glycogen-precipitable protein relative to other proteins in saliva from animals

of ten different strains. Class 0 individuals are of genotype N/N. Class I comprises four indi- viduals of type A/A, two BJB, three Su/Su and two H/H. Class I1 comprises four AAIAA, three BB/BB and two AH/AH, and class I11 three AAB/AAB and two SAA/SAA animals.

glycogen and CHA-sepharose and the slightly higher molecular weight of the inactive protein, may indicate that it is an amylase precursor.

The low-amylase strain “N”: The amylase activity in parotid slands from strain “N” is less than 5% of the value observed for stocks with one amylase locus (Table 1, Figure l), whereas the activity of the pancreas is within the normal range (Table 2).

In genetic crosses, this AmyN allele acts as a null allele, since heterozygotes, known to carry Amy” on one chromosome, exhibit the electrophoretic amylase pattern coded for by the other chromosome. It is inherited in a simple Mendelian fashion.

When saliva from “N” individuals was treated with increasing amounts of glycogen, salivary protein was precipitated following a curve similar to that for other vole strains (Figure 4). The amount of protein precipitated with saturating amounts of glycogen is close to that observed for strains having one amylase locus (Figure 5 ) .

Using native polyacrylamide gel electrophoresis, a major and a minor protein were resolved. The mobility of the major component (N) is more anodal than that of any other amylase isozyme, while the minor component shows the same

- N

+

GENETIC VAllIATION IN AMOUNT OF AMYLASE 925

O D 5 3 O .02 .04 .06 I“‘

I 2 3 FIGUIIE 6.-Native polyacrylamide gel electrophoresis of proteins from glycogen precipitates

of saliva showing the isozyme bands N, A, and B. Gel 1 : genotype MA, 2: WN, and 3: BIB. A gel similar to thres was sliced and assayed for amylase activity as indicated by the OD 530 ma profile. The arrow indicates the presumptive precursor.

mobility as the amylase-inactive protein discussed previously (Figure 6). Exami- nation of glycogen-precipitated protein from the “N” strain by polyacrylamide gels in the presence of sodium dodecyl sulfate revealed that the molecular weight of the N protein was slightly greater (approximately I000 daltons) than that of active amylase (Figure 7). In Amys/AmyB A m p heterozygotes, the molecular weight difference was also evident.

The amino acid composition of glycogen-precipitated protein from the “N” stock is very similar to that from strain “B” (Table 4). However, the N protein is immnuologically distinguishable from active amylase, as can be seen from the immunotitration experiments. If the same amount of antiserum is added to saliva

526 J. P. HJORTH. M. MEISLER AND J. T. NIELSEN

Gal

Alb

o v

1 2 3 4 5

FIGURE 7.-Polyacryamide gel electrophoresis in the presence of SDS of proteins from gly- cogen precipitates of saliva. Genotypes of the individuals are, 1: BB/BB, 2: BB/N, 3 and 4: N/N, 5: BB/ mixed with the standards bacterial P-galactosidase (Gal), bovine serum albumin (Alb), and ovalbumin (Ov). B indicates active amylase, N the inactive amylase, and the arrow the presumptive precursor.

samples from animals of the two genotypes, AmyB AmyB/AmyB AmyB and AmyN/AmyB AmyB, the resulting precipitates are equivalent in terms of amylase activity (Table 5 ) . Since the N protein is virtually devoid of amylase activity, the result shows that this protein does not compete for antibody-binding sites. The nonreactivity was confirmed by immunoelectrophoresis against three differ- ent antisera to mouse salivary amylase.

Finally, the allelism between the electrophoretic N band and active amylase was tested by crossing a heterozygous Amyw/AmyB Amy” male to a homozygous Amy” AmyA/AmyA AmyA female. The resulting 25 offspring segregated into two groups: 13 with the A and B bands and 12 with the A and N bands.

The genetic information together with the character and quantity of the glycogen precipitable protein from saliva of the “ N y strain indicates that the AmyN allele is a mutant within the amylase structural gene, resulting in the production of a catalytically inactive gene product. The slightly greater molecu- lar weight of the abnormal N protein may be the result of incorrect precursor

GENETIC VARIATION IN AMOUNT O F AMYLASE

TABLE 4

Amino acid composiiion of glycogen-precipitated protein from saliurr of the strains BB/BB and N / N

927

Number of residues per 1,000 residues Strain BB/BB Strain N / N Res’due

Aspartic acid 134 1 43 Threonine 50 48 Serine 68 57 Glutamic acid 91 92 Proline 51 58 Glycine 129 119 Alanine 74 68 Valine 71 77 Methionine 18 17 Isoleucine 47 49 Leucine 52 49 Tyrosine 38 35 Phenylalanine 48 50 Lysine 50 50 Histidine 27 26 Arginine 56 58

processing that also causes the lack of amylase activity and the altered immuno- logical properties.

DISCUSSION

Previous studies of the quantitative variation in bank vole salivary amylase led to a model involving polymorphism for chromosomes with different number of amylase structural genes (NIELSEN 1977a,b). The model was based on data obtained by measuring the proportions of protein in amylase isozymes in hetero- zygous double-banded animals.

In the present paper, we have demonstrated the existence of a heat-labile amylase in the homozygous stock AmyS”/AmyS”. The amounts of stable amylase in different heterozygotes carrying this AmyS” allele were approximately one- half, two-thirds and three-fourths of the total amylase. This method of measur-

TABLE 5

Immunoprecipitniion of amylase aciiuity in saliva from a BB/BB and BB/N individual

pU amylase precipitated p l Antiserum BB/BB BB/N

5 0.16 0.18 10 0.29 0.33 20 0.69 0.68

Nonprecipitated amylase was measured and the amount of enzyme in the precipitate was calculated.

928 J. P. ITJOitTH, M. MEISLER A N D J. T. NIELSEN

ing the relative proportions of amylase isozymes in heterozygotes supports the earlier evidence for chromosomes that encode isozyme production in the ratio of 1 :2:3. Although the experiment did not disclose new categories of chromosomes within our stocks, the heat-inactivation technique can be useful for characteriz- ing new variant chromosomes in natural populations.

When the amylase activity in parotid glands was determined from stocks homozygous for chromosomes with gene numbers of one. two or three, we found that the enzyme content increased with the proposed gene number. A similar result was obtained by glycogen-precipitated saliva from the same stocks. Thus, there is a difference in the total amylase production of the three chromosome types and a similar difference in contribution of the enzyme in heterozygotes. The results taken together indicate a chromosome-specific regulation of amylase production. The regulation is likely to occur at the level of amylase synthesis, as has been observed for mouse salivary amylase (HJORTH, in preparation) and also for mouse pancreatic amylase (BLOOR, MEISLER and NIELSEN 1978; BLOOR et al., in preparation).

We should like to discuss two types of genetic mechanisms as explanations for the three quantitative classes of bank vole salivary amylase. The animals may differ with respect to the number of amylase structural genes, or they could vary at regulatory sites. Duplication of the structural gene for salivary amylase in the bank vole was first suggested by the observation of pure-breeding lines with two isozyme forms (NIELSEN 1969). Regardless of the genotype of the other parent, off spring from such double-banded strains inherit both electrophoretic forms. Three lines of evidence strongly suggest that the two amylase isozymes in these double-banded homozygotes are the products of distinct structural genes, rather than the result of post-transcriptional modifications of single gene products. First, the relative amounts of the two isozymes do not vary among individuals from the same s tnin and are independent of physiological variables such as age and sex. Second, the relative amounts of the two forms in homozygous double-banded strains are close to the integral proportions of 1 : 1 or 1 :2. It seems unlikely that an enzymatic modification would produce such precise ratios. Third, in hetero- zygotes the integral ratio between isozymes from each chromosome is cis-deter- mined, and there are no effects from one chromosome on the product of the other. In summary, double-banded salivary amylase patterns in homozygotes appear to reflect the presence of at least two amylase structural genes.

The quantitative variation described in this report can adequately be explained by variation in gene number, and our results are, in fact, in striking agreement with the values predicted from earlier measurements.

It still remains possible, however, that part of the variation influencing the quantity of a single electrophoretic amylase isozyme may be produced by cis- acting regulatory sites Pinked to the structural gene. Such sites might regulate the amount of amylase mRNA transcribed from the structural gene and might act so as to fit the integral numbers 1 and 2. Alternatively, the level of amylase production might be determined by a site within the structural gene that influ- ences the efficiency of translation of amylase mRNA. The ultimate confirmation

GENETIC VARIATION IN AMOUNT O F AMYLASE 929

of the gene copy model could eventually be provided by hybridization of DNA and a reverse transcript of purified amylase mRNA.

In our preparations of glycogen-precipitated amylase, we have found signifi- cant quantities of an amylase-inactive protein whose molecular weight is approx- imately 3 to 4000 daltons greater than amylase. Since the protein binds to glycogen and also to cyclohepta-amyloze, it may represent an inactive amylase precursor, similar to that observed after in vitro synthesis of rat pancreatic amy- lase (MACDONALD, PRZYBYLA and RUTTER 1977). but further biochemical characterization of the protein is required before a final conclusion can be drawn.

In recent years, a variety of genetic system have been described in which segregation data indicate the existence of null alleles. Such alleles may result from deletion of the structural gene, resulting in the complete absence of the protein, or they may be caused by a mutation that alters the (enzymatic) proper- ties of the protein. The AmyN allele reported here belongs to the latter category. It behaves in crosses as a functionally null allele when the agar gel technique is used because no active salivary amylase is found in the homozygous AmyN/ AmyN stock. The homozygotes do, however, produce a protein that can be precipi- tated by both glycogen and cyclohepta-amylose. This protein has a total amino acid composition very similar to that of active amylase and segregates in crosses as an amylase allele. The slightly greater molecular weight of the inactive N amylase, as compared to normal salivary amylase, may be due to an incorrect processing of a precursor. The resulting protein is not only deficient in amylase activity, but also has altered immunological properties.

This research was supported by grants from the Danish Natural Science Research Council and by GM 24872 from the Public Health Service. Part of the study was made during a three- month visit of M. MEISLER in Arhus, which was supported by a grant from the University of Aarhus. We thank Ms. H. HBST, Ms. L. PEDERSEN, Ms. J. BRUUN and Ms. M. CLARK for their competent technical assistance.

LITERATURE CITED

BLOOR, J., M. MEISLER and J. T. NIELSEN, 1978 Genetic determination of rate of synthesis of

CLARKE, J. T., 1964 Simplified “disc” (polyacrylamide gel) electrophoresis. Ann. N.Y. Acad.

DAHLQVIST, A., 1962 A method for determination of amylase in intestinal content. Scan. J.

KARN, R. C. and G. M. MALACINSKI, 1978 The comparative biochemistry, physiology and ge-

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