How does ancestral starch consumption affect amylase gene copy numbers and salivary

amylase enzyme concentration in Homo sapiens?

Dazhong Duo (400381968, Lab Section 11)

Instructors: Lovaye Kajiura, Rosa da Silva, Lauren Tiller, and Mahrukh Fatima

Department of Biology, McMaster University, Hamilton, Ontario, Canada

 Abstract

This investigation aimed to find linkages between the ancestral diets of Homo sapiens,

amylase gene copy numbers, and salivary amylase concentrations. It was hypothesized that

individuals with starch abundant ancestral diets possessed greater amylase gene copy numbers

and higher amylase concentrations in their saliva. These experiments determined the AMY1A

gene copy number and salivary amylase concentration of Unknown Students A and B. These

values were then substituted into a larger group of sample data to observe trends and

relationships between variables. An amylase enzyme assay was created by reacting starch-iodine

solutions with increasing concentrations of amylase. Moreover, data conveying the AMY1A

gene copy numbers in a sample group of students was found using polymerase chain reactions

and gel electrophoresis. Notably, the results showed a negative correlation between amylase

concentration and the absorbance of the starch-iodine solutions with a high R2value of 0.9532.

Further, the average salivary amylase concentrations of 0.400 mg/ml, 0.600 mg/ml, and 1.649

mg/ml, for low, moderate, and high starch diets, respectively, indicate a direct relationship

between starch levels and the concentration of salivary amylase produced. Finally, a direct

correlation with an r-value of 0.664 was found between amylase gene copy numbers and the

starch levels in the diets of individuals. Ultimately, it was concluded that the findings of this

investigation strongly support the hypothesis that ancestral diets are directly proportional to the

AMY1A copy numbers and salivary amylase concentrations in Homo sapiens.

Introduction

The amylase enzyme is produced by both the salivary glands and pancreas of Homo

sapiens. Amylase is crucial for the digestion of starch because it possesses the ability to
hydrolyze the alpha-1, 4-glycosidic linkages in polysaccharides. The polysaccharides are broken

down into maltose, glucose, and larger oligosaccharides which are then metabolized by the body

(Tracey et al. 2021). The AMY1A gene in Homo sapiens DNA is located on both copies of

chromosomes and, specifically, codes for salivary amylase which is produced by epithelial cheek

cells (Tracey et al. 2021).

Humans can possess between 2 and 20 copies of the AMY1A gene, the exact copy

number may be affected by a range of variables such as random duplication mutations or

environmental factors (Tracey et al. 2021). Moreover, an individual’s relative copy number is

suggested to have been influenced by their ancestral starch consumption. Literature has shown

that populations that evolved high-starch diets had a higher average copy number of the AMY1A

gene compared to those that evolved with low-starch diets. (Santos et al. 2012). Furthermore,

direct correlative evidence was found between variations in AMY1A gene duplications and the

concentration of amylase expressed in saliva (Pajic et al. 2019). This suggests that humans and

perhaps other species were under selective pressure to produce varying amounts of salivary

amylase in response to the starch content in their diets. For example, characteristically,

agricultural societies and hunter-gatherers in arid environments had exceptionally high starch

content in their diets. Conversely, pastoralists and hunter-gatherers living in rainforests or

circum-arctic regions consumed much less starch because of its scarcity in their environment

(Perry et al. 2007). Therefore, through natural selection, populations in areas plentiful in starch

had the evolutionary need to have higher salivary amylase concentrations for digestion.

Consequently, individuals that had more tandem repeats of the AMY1A gene had better chances

of survival and therefore passed on more of their genetic information to future generations

(Tracey et al. 2021). Ultimately, it is strongly suggested that evolutionary pressures have led to a
positive correlation between ancestral starch consumption, AMY1A gene duplication, and

salivary amylase concentration in Homo sapiens populations.

This investigation collected data on salivary amylase concentration and AMY1A gene

copy numbers from Unknown Students A and B and compared it to their reported ancestral

starch diets. The purpose of these experiments was to find if there is an association between gene

evolution, gene copy number, and amylase enzyme production through comparison to larger data

sets. Ultimately, if Unknown Students A and B reported higher or lower starch content in their

ancestral diets, then their respective AMY1A gene copy numbers and salivary amylase

concentration should be directly proportional to the starch levels because of adaptive pressures

imposed on their ancestors to digest starch more effectively.

Methods

It must be noted that the complete experimental methods can be found in the BIO1A03

Fall 2021 lab manual (Tracey et al. 2021).

In Lab 3, multiple reactions between amylase, at varying concentrations, and starch were

conducted in order to create an amylase enzyme assay and absorbance spectroscopy. A gradient,

representative of a standard curve was created using iodine to visualize the amount of starch that

had been broken down. Iodine and starch produce a deep blue-coloured solution, as a result, the

colour of the produced solution became decreasingly blue as higher concentrations of amylase

were used. The absorbance of the solutions was then determined using a spectrophotometer,

subsequently, a standard curve was used to identify the salivary amylase concentrations of

Unknown Students A and B. Using sample data on students with low, moderate, and high starch

diets, the mean amylase concentrations were found for each group. Due to errors made in the
procedure, the absorbance values were taken from the Lab 3B sample data provided by Mahrukh

Fatima.

In Lab 4, polymerase chain reactions and gel electrophoresis were utilized to determine

the AMY1A gene copy numbers of a group of students. First, DNA was extracted from samples

of inner cheek cells, a thermocycler was then used to perform PCR. Subsequently, the amplified

DNA fragments were separated through gel electrophoresis. The approximate base pair lengths

of the amylase and actin genes were determined using a standard curve on Microsoft Excel.

ImageJ software was then used to find the band intensities which in turn allowed the calculation

of the amylase gene number of Unknown Students A and B (Tracey et al. 2021).

In the Formal Lab, the results of Labs 3 and 4 were compared to determine and calculate

correlations between diploid gene copy number, salivary amylase concentration, and ancestral

starch consumption for Unknown Students A and B and students in a larger sample size.

Results

Figure 1A shows a strong negative correlation between salivary amylase concentration

and the intensity of the colour blue in the solution. Similarly, the spectroscopy graphed in Figure

1B shows a downward trend as a negative correlation can be observed between amylase

concentration and the absorbance of the starch-iodine solutions. The value R2=0.9532 indicates

a very strong negative correlation between the dependant and independent variables. In other

words, salivary amylase concentration is inversely proportional to the absorbance of the starch-

iodine solutions. Moreover, the salivary amylase concentrations of Unknown Students A and B

(Table 1B) were calculated to be 0.503 mg/ml and 2.004 mg/ml respectively, using the standard

curve in Figure 1B (see Calculation 1A). These results followed the general upward trend for
moderate and high starch diets, respectively, higher starch diets were positively correlated with

high salivary amylase concentrations. Next, the average concentrations of salivary amylase were

calculated for students with low, moderate, and high starch diets. There was an upward trend for

low, moderate, and high starch diets with the average salivary amylase concentrations being

0.400 mg/ml, 0.600 mg/ml, and 1.649 mg/ml, respectively (Figure 1C, 1D, and Table 1C).

The base-pair length for the actin and amylase genes in Unknown Students A and B were

found to be approximately 327 and 138 base pairs respectively (Figure, 2B, 2C, and Calculation

2A). Using ImageJ analysis, the number of diploid amylase gene copies for Unkown Students A

and B were found to be 3 and 6 copies, respectively. After the values for gene copy numbers of

Unknown Students A and B were substituted back into the sample group (Table 2B), Figure 2A

was constructed. There is an observable upward trend in Figure 2A, as gene copy number

increased, the salivary amylase concentration increased as well. This is supported by the

moderately strong correlation coefficient (r) of approximately 0.664 (Figure 2A).

Discussion

The purpose of this investigation was to study the links between ancestral starch diet,

AMY1A gene copy number, and salivary amylase concentration in Homo sapiens. It was

hypothesized that higher proportions of starch in the ancestral diet of individuals are directly

proportional to their amylase gene copy numbers and salivary amylase concentrations.

From the results section, it was found that there was a very strong inverse relationship

between salivary amylase concentration and starch-iodine absorbance. Notably, this is supported

by the high R2value of 0.9532. This supports literature and background information which states

that the absorbance values decreased because the salivary amylase broke down the starch in the
solutions. Consequently, there was less amylose for the iodine to bind to, which resulted in

decreasingly blue solutions (Tracey et al. 2021). Evidently, salivary amylase plays a crucial role

in digesting starch into simple sugars that can be metabolized by the body (Pajic et al. 2019).

Furthermore, there was a directly proportional relationship between diet starch levels (low,

moderate, and high) and average salivary amylase concentrations which were 0.400 mg/ml,

0.600 mg/ml, and 1.649 mg/ml, respectively. This data supports the theory that the starch content

in the diet of individuals may directly influence the levels of salivary amylase produced by the

body (Pajic et al. 2019). Notably, the reported starch levels were from the current diets of

students rather than their ancestral diets, even still, academic literature has shown that salivary

amylase concentrations may be heavily influenced by environmental factors such as hydration

status, psychosocial stress level, and short-term dietary habits (Santos et al. 2012). Similarly, a

study on polymorphic patterns of human salivary amylase demonstrated evidence supporting the

idea that quantitative variations in amylase protein patterns do not necessarily reflect

polymorphisms in gene copy number (Bank et al. 1992). Finally, through PCR and gel

electerophoerisis, it was found that the base pair length of actin and amylase were the same for

both Unkown Students A and B (327 and 138 base pairs respectively). This coincides with the

theory that states the base pair length has no effect on the number of gene copies in an organism

(Farrelly et al. 1995).

In order to investigate the relation between amylase presence and ancestral starch diets,

copies of the AMY1A gene were observed in the DNA of individuals. A directly proportional

relationship was observed between amylase gene copy number and salivary amylase

concentration. This relationship had a moderately strong correlation coefficient (r) of

approximately 0.664. The general consensus across most literature on this subject states that
extensive copy number variation in the AMY1 genes is directly proportional to the salivary α-

amylase content in saliva (Santos et al, 2012). Subsequently, previous investigations have shown

that populations with more copies also eat more starch (Pajic et al. 2019). However, based on

this data, it cannot be concluded whether this correlation is because populations evolved in

response to high starch diets, or if the high starch diets simply developed because individuals

happened to have higher salivary amylase concentrations (Pajic et al. 2019). Nonetheless, the

former theory would be in accordance with substantial amounts of academic support; different

evolutionary pressures may have acted on amylase. For instance, higher AMY1 copy numbers

and protein levels likely improve the digestion of starchy foods and decrease the risk of intestinal

disease. Consequently, individuals from populations with high-starch diets evolved more AMY1

copies than those with traditionally low-starch diets through natural selection (Perry et al. 2007).

Overall, the diet, amylase gene copy number, and amylase concentrations of Unknown

students A and B support an association between gene evolution, gene copy number, and

amylase enzyme production because both students seem to have similar levels of starch in their

diet as their ancestors. Student A and their ancestors had lower levels of starch in their diet

because the gene copy number (3 copies) and amylase concentration (0.503 mg/ml) are lower.

Student B and their ancestors had higher levels of starch in their diet because the gene copy

number (6 copies) and amylase concentration are higher (2.004 mg/ml).

Ultimately the results of this investigation offer substantial support to the hypothesis that

the abundance of starch in the ancestral diets of Homo sapiens caused the adaptation of increased

amylase gene copy numbers and amylase concentrations in their saliva.

Overall, this investigation had some weaknesses and logical sources of error that may

have distorted results. Primarily, the large standard deviation and range (see Figures 1C, 1D)
indicate high variability in the salivary amylase concentrations for each diet group.

Consequently, the reliability of this data is hindered due to the low consistency. By addressing

some of the errors in the investigation, variability can be minimized. Mainly, the low

concentration iodine that was used was susceptible to degradation before use. Therefore, the

colours of the enzyme assay may have been affected. An improvement would be to use a higher

concentration of iodine or to ensure that materials have been prepared in a manner to reduce

degradation over time. An additional error was that the 100 bp ladder bands in the gel

electrophoresis image were not well separated (Figure 2B). This made have led to inaccuracies

when finding the bp length of the actin and amylase genes. By maintaining the amount of

electricity that ran through the pores of the 1.5% agarose gel, the 100 bp ladder bands would be

allowed to properly separate.

Literature Cited
Bank, R. A., Hettema, E. H., Muijs, M. A., Pals, G., Arwert, F., Boomsma, D. I., & Pronk, J. C.

(1992). Variation in gene copy number and polymorphism of the human salivary amylase

isoenzyme system in Caucasians. Human Genetics, 89(2).

https://doi.org/10.1007/bf00217126

Farrelly, V., Rainey, F. A., & Stackebrandt, E. (1995). Effect of genome size and RRN gene

copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial

species. Applied and Environmental Microbiology, 61(7), 2798–2801.

https://doi.org/10.1128/aem.61.7.2798-2801.1995

Pajic, P., Pavlidis, P., Dean, K., Neznanova, L., Romano, R.-A., Garneau, D., Daugherity, E.,

Globig, A., Ruhl, S., & Gokcumen, O. (2019). Independent amylase gene copy number

bursts correlate with dietary preferences in mammals.

https://doi.org/10.7554/elife.44628.018

Perry GH, Dominy NJ, Claw KG, Lee AS, Fiegler H, Redon R, Werner J, Villanea FA,

Mountain JL, Misra R, et al. (2007). Diet and the evolution of human amylase gene copy

number variation. Nat Genet. [accessed 2021 Dec 6]; 39(10):1256–1260.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2377015/doi:10.1038/ng2123

Santos, J. L., Saus, E., Smalley, S. V., Cataldo, L. R., Alberti, G., Parada, J., Gratacòs, M., &

Estivill, X. (2012). Copy number polymorphism of the salivary amylase gene:

Implications in human nutrition research. Journal of Nutrigenetics and Nutrigenomics,

5(3), 117–131. https://doi.org/10.1159/000339951

Tracey A, da Silva R, Mahalingam S, Rerecich T. (2021). Biology 1A03 Lab Manual Fall 2021

[accessed 2021 Sep 15]

 Appendices

Figure 1A: Amylase assay created in Lab 3A showing progressively increasing concentrations of

amylase reacting with starch. The samples are labelled with their amylase concentrations

(ug/mL).

Table 1A: Sample data for Lab 3B used to determine Amylase concentrations of UAI and UBI.
Figure 1B: Increasing Amylase Concentration compared to the Absorbance of Starch-Iodine

Solutions. Graphed using data from Table 1A, standard curve equation used to find salivary

amylase concentration for Unknown Students A and B

Table 1B: Sample Data showing a group of Students matched with the starch levels in their diet

as well as their salivary amylase concentration (mg/ml). Used to create Figure 1C.

Student Diet (High, Salivary

Name Moderate or Amylase
Low Starch) Concentration
(mg/ml)

Unknown Moderate 0.503

Student A

Unknown High 2.004

Student B

Student C Low 0.275

Student D Moderate 0.406

Student E High 3.262

 Student F High 1.544

Student G Low 0.654

Student H Low 0.135

Student I Moderate 0.66

Student J High 0.676

Student K Moderate 1.013

Student L High 1.37

Student M Moderate 0.821

Student N High 0.897

Student O Low 0.43

Student P High 2.865

Student Q Moderate 0.958

Student R High 2.39

Student S Low 0.59

Student T Moderate 0.345

Student U Low 0.314

Calculation 1A: Calculating salivary amylase concentration based on the standard curve in

Figure 1B. These numbers are inputted to complete Table 1B.

Unknown Student A:

1.07= -0.0533x + 1.3381

-0.2681=-0.0533x

x≈5.03

x*100=503
 503/1000=0.503

Unknown Student B:

0.27= -0.0533x + 1.3381

-1.0681=-0.0533x

x≈20.040

x*100=2004.0

2004.0/1000=2.004

Table 1C: Data for Figure1C and 1D, displays figures for mean amylase concentration (mg/ml)

for students with low, moderate, and high amounts of starch in their diet. Displays standard

deviation minimum, and maximum values for these numbers.

Amount of Mean Amylase Standard Minimum Maximu Mean- Maximum

Starch in Diet Concentration (mg/ml) Deviation Value m Value Minimum -Mean
0.19736328 0.264666 0.2543333
Low 0.399666667 6 0.135 0.654 667 33
0.36907798 0.602905 0.4129842
Moderate 0.600015714 4 -0.00289 1.013 714 86
1.09355080
High 1.64875 2 0.186 3.262 1.46275 1.61325
Figure 1C: Salivary amylase concentration for students with low, moderate, and high starch in

their diet. Data bars represent the concentration of salivary amylase (mg/ml) for students with

low, moderate, and high amounts of starch in their diets. The error bars represent the standard

deviation for each category. The sample sizes for the low, moderate, and high groups are 6, 7,

and 8 respectively.
Figure 1D: Salivary amylase concentration for students with low, moderate, and high starch in

their diet. Data bars represent the concentration of salivary amylase (mg/ml) for students with

low, moderate, and high amounts of starch in their diets. The error bars represent the range for

each category. The sample sizes for the low, moderate, and high groups are 6, 7, and 8

respectively.

Table 2A: Gene copy number of Unkown Students A and B, found through analysis of data from

ImageJ software. The values for brightness and intensity for the amylase and actin band were

used to find the ratio between normalized amylase and actin light intensity. (Tracey et al.,

2021).

Table 2B: List of sample group students matched to their diploid amylase gene copy number as

well as salivary amylase concentration (mg/ml). Values used to create Figure 2A.

Student Amylase Gene Salivary

Name Copy Number Amylase
Concentration
(mg/ml)

Unknown 3 0.503
Student A

Unknown 6 2
Student B

Student C 2 0.275

Student D 5 0.406

Student E 9 3.262

Student F 12 1.544

Student G 4 0.654

Student H 1 0.135

Student I 6 0.66

Student J 6 0.676

Student K 7 1.013

Student L 8 1.37

Student M 5 0.821

Student N 4 0.897

Student O 3 0.43

Student P 5 2.865

Student Q 7 0.958

Student R 10 2.39

Student S 2 0.59

Student T 3 0.345

Student U 1 0.314
Figure 2A: The Relationship between Gene Copy Number and Amylase Enzyme Concentration.

The sample size is 21, the correlation coefficient (r) is approximately 0.664

Figure 2B: Gel electrophoresis results from Lab 4B, shows the amplified results of the actin and

salivary amylase genes for Unkown Students A and B. Lane 1: Control (no enzyme), Lane 2:

HindIII, Lane 3: EcoRI, Lane 4: BamHI, and Lane 5: Bg/II.

Figure 2C: The relationship between the DNA fragment size and the distance it migrated in 1.5%

agarose gel during gel electrophoresis. The standard curve was created by measuring the distance

travelled by each fragment and comparing it to the 100 bp ladder.

Calculation 2A: Calculations finding the distance migrated by the actin and amylase bands, the

standard curve from Figure 2C were used to determine the base pair length of the actin and

amylase genes.