Abstract
The purpose of the investigation was to examine the correlation of enzyme production in response to the evolutionary impact of AMY1 gene copy numbers in individuals. Based upon research obtained in previous studies, there was an assumed positive correlation between gene copy numbers and the production of salivary amylase protein levels in mg/mL. Variances in copy numbers can be explained by individual ancestral history. This is primarily dependent on the specific geographical locations where gene copy numbers within a sample population presented similar numbers of the AMY1 gene, due to similar levels of starch consumption within individual’s diets. This provided evidence towards ancestral starch consumption affecting current gene copy numbers. Individually, it was hypothesized that a subject with a moderate level of ancestral starch consumption would produce values of AMY1 diploid gene copies and consequent amylase production close to the mean values of others with a similar diet (Tracey 2019). Testing of this hypothesis was conducted through a series of investigations including methods of spectrometry of amylase absorbance for both a controlled and collected sample of salivary amylase which provided the basis for forming calibration curves to demonstrate protein levels, determined to be 1.61 mg/mL, compared to copy numbers, determined to be two (2), in terms of absorbance and comparative starch consumption. Moreover, the data collected proved the hypothesis by representing the positive correlation between salivary amylase gene copy numbers and enzyme expression, consequent to the predetermined mean values for the respective ancestral diet type.
Introduction
The metabolism of starch in Homo sapiens is initiated by salivary alpha amylase; an enzyme utilized for the hydrolyzation of starch into its core compounds (Tracey, 2019). Amylases are secreted proteins that hydrolyze 1,4-alpha-glucosidic bonds in oligosaccharides and polysaccharides, and therefore are required for catalyzation in the initial step of digestion of dietary starches and glycogen (Meisler and Ting, 1993). The human genome has an agglomeration of several amylase genes that are expressed at high levels in either the salivary gland or pancreas. Provided that the level of expression of this gene is hypothesized to be higher in individuals with ancestral heritage where there were high levels of starch consumed, it can be concluded that the quantity of this enzyme and its activity varies dependently on such variables and will have variances between individuals.
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The regulation of starch digestion and the correspondence to AMY1 gene copies have broad implications in the field of epigenetics. Clinical researchers, such as the study published by Poole et al. (2019) “Human Salivary Amylase Gene Copy Number Impacts Oral and Gut Microbiomes” focuses on the aspect of microbiome variances and connects this to gene and enzyme regulation. Provided an individual’s AMY1 gene copy numbers are dependent upon ancestral and individual starch consumption levels; this therefore represents the cumulative result of ancestral microbiome adaptations throughout generations. An example of implications of this currently being observed are regarding copy number variants (CNVs) of the AMY1 locus encoding for salivary amylase. CNVs explain how entire genes can be deleted or replicated, and in humans, the salivary α-amylase gene locus (AMY1) shows immense variation in number of copies between individuals (Perry et al, 2007). However, for the CNVs to become common in the population, there needs to be an advantage to having a large number of copies of the salivary amylase gene. The variation indicates the importance of the salivary amylase in human physiology and evolution. Therefore, this study established AMY1 copy numbers as a genetic factor associated with microbiome composition and function.
It is the AMY1 gene which determines levels at which the enzyme is present, and the differences in number of gene copy numbers can be determined while following chromosome 1 on individual genomes (Morris et al. 2015). Individuals have varying levels of the active enzyme, ranging anywhere from 2-20 copies respectively (Tracey 2019). Background research has provided evidence that the expression of the enzyme is related to the starch diet both modernly and ancestrally, with specific insight on individuals with higher ancestral levels of starch consumption containing a higher level of the enzyme in modern offspring (Mandel et al. 2010). In this context, CNVs can be tracked as the gene copy varies for each individual along chromosome 1 due to tandem repeat copies along the chromosome (Morris, 2019). Each individual has two copies of each tandem repeat with the location having various alleles all differing in copy number such that an individual’s genotype at six to eight different regions can be used to uniquely identify an individual (Morris 2019). This property of amylase allows the use of a PCR and gel electrophoresis techniques by employing tandem repeat sites as targeted and amplified regions by sequence specific primers that target flanking regions of the variable repeats. The resulting amplified DNA fragments can then be separated and detected using gel electrophoresis (Tracey 2019). This information obtained from previous studies provided a baseline for forming a hypothesis for this investigation, hypothesizing that due to ancestral migration patterns of in habitation in urban areas where food sources are not solely dependent on agricultural (predominantly starch based) resources, there will be a moderate level of amylase production on an individual basis.
Materials and Methods
Refer to the McMaster University Biology 1A03 Fall laboratory manual for detailed accounts of the procedures followed throughout the investigation. Changes made to procedures listed are in investigation 4, in which there was no ice bucket used for the dilutions of the solutions. Additionally, during investigation 7, 5ul of ladder was used opposed to 10ul in the first well of the gel electrophoresis.
Results
Through collection of experimental data, it was determined that the level of AMY1 protein concentration present in saliva is connected through a linear relationship to the number of diploid AMY1 gene copies. Correlational analysis was used to investigate whether or not there was a linear relationship between two variables. There was a strong positive correlation between the data points; however, correlational analysis cannot be used to make a conclusion about a cause and effect relationship provided this does not account for other experimental variables that could be affecting the relationship between the two variables examined.
From this, a calibration curve was generated based on data represented from a starch iodine solution. The quantitative measure of how much light is absorbed by a substance and how much light is being transmitted at any given time was determined using a spectrophotometer. Readings are always taken by comparison with a standard or blank; containing all the ingredients of the test solution except the material being tested. From within the machine, light photons are transmitted through a filter which can be adjusted to regulate the colour of the beam dependent on its wavelength prior to passing through a sample. After it has passed through the sample, the light strikes a light-sensitive phototube producing an electric current proportional to the light energy received. (Tracey, 2019). The spectrophotometer, which was set to a 620 nm wavelength to measure absorbance level values, plotted the respective absorbance levels against amylase concentration to produce a calibration curve using a linear equation and value of R2= 0.77. As amylase concentration increased, there was a corresponding increase in the absorbance values (Appendix Table 1).
The saliva samples obtained during investigation 4 presented a trend alike to that within the values from the initial table, by a basis of a series of 10-fold dilutions (Appendix Table 2). The serial dilutions corresponded to concentrations relative to the initial sample by 10-1, 10-2, 10-3 respectively. Based upon the data collected from the previous calibration curve, the known absorbance of salivary amylase connected to the calculated value of 1.61 mg/mL amylase concentration in saliva.
To model the relationship between ancestral diet types (low, moderate, or high) compared to the determined concentration of salivary amylase, standard deviation bars and range error bars were generated.
The DNA extraction obtained from a buccal swab provided the ability to isolate and denature the proteins to be used in a series of polymerase chain reactions (PCR). DNA extraction and isolation occurred through cell lysis via disruption of the cell walls and cell membranes, as well as denaturation of proteins. The process of the PCR amplified the sample of DNA at the target flanking region of the AMY1 gene as the sample underwent 35 cycles which produced 3.4 x 1010 copies of the target sequence. Applying techniques of gel electrophoresis, a DNA image was created. Using the gel imaging software Bio-Rad to process the image, a semi-log graph plotting the base pair lengths of DNA fragments obtained from the PCR process was produced and used as a template for the measurement of the migration of DNA from their original position in the gel. The original length (Appendix Table 4) of each fragment of replicated DNA was found by the ratio of adjusted actin and amylase volume levels. Provided DNA is a negatively charged molecule, gel electrophoresis can be used to separate DNA fragments according to size. Agarose has the ability to create a gelatinous matrix that contains pores through which the molecules can move. The smaller fragments move faster through the pores of the gel than the larger ones, and with time the fragments become separated into distinct fragments. Fragments of DNA will migrate in the electrical field at rates which are inversely proportional to the log10 of their molecular weight, provided the use and creation of a semi-log graph. This was concluded from a loaded 100bp ladder sample into the first well of the gel. The results of the gel imaging representing a subject with a moderate level of ancestral starch consumption demonstrated a travelled distance of 36mm for 510 bp of actin, and a distance of 39mm for 400 bp of amylase. The imaged bars of DNA produced and adj. volume (int) of 433,242 for amylase, and 710,698 for actin; representing values for one AMY1 gene copy. This data was used to produce a scatter-plot graph demonstrating values collected from all subjects comparing salivary amylase concentrations to numbers of amylase gene copies; representing the positive correlation between the variables and therefore supporting the hypothesis.
Discussion
The premise of this investigation was to examine the correlation between enzyme production, gene copy numbers, and evolutionary influences of ancestral diet types pertaining to the consumption of starch. The hypothesis assumed a strong positive correlation between the salivary AMY1 gene, respective copy numbers of the gene, and ancestral starch intake. Regarding a subject with a moderate ancestral starch diet, individuals in a population with a similar diet types are assumed to demonstrate values close to the average for gene copy numbers of the AMY1 diploid gene, and consequently the amount of amylase produced is estimated to be a similar quantity to the mean average of others with similar dietary habits (Tracey 2019).
The results obtained throughout this investigation were collected through sequential experiments which utilized various chemical processes and biological principles. The findings of the study present a clear association between the concentration of amylase and ancestral diet type with regards to enzyme production, gene copy numbers, and genetic evolution. The results obtained from the investigations produced a value of 1.61 mg/mL of amylase concentration in saliva for a moderate level of ancestral dietary patterns; being within a close approximation to the mean concentration of 2.64 mg/mL with a standard deviation of plus or minus 1.8 and a range of values from 0.0 to 7.5 mg/mL as obtained in the study conducted by Perry et al (2007). This demonstrates that the collected value of 1.61mg/mL is within one standard deviation from the mean, and therefore correlates to the mean value of moderate starch from the respective population (Appendix Table 3). Furthermore, the reviewed literature by Perry et al. hypothesized individuals with moderate ancestral starch consumption to have 2 copies of the AMY1 gene, which corresponded to the determined value of 2 gene copies (Appendix Table 5). Within the entire population of the study, however, there is a lack of correlation between variables with outliers of gene copy numbers up to 11. Therefore, there is not a strong correlation between individuals within the study, however, the scatterplot does represent a correlation between amylase concentration and gene copy numbers as salivary amylase concentration increases in response to an increase to gene copy numbers. This relationship suggests that the hypothesis proposed by Perry et al. (2007) was correct in the correlation between amylase concentration and AMY1 gene copy numbers based upon ancestral diet, however, the proportionality of the variables measured in this investigation are not as apparent as the original study, demonstrating a somewhat weaker correlation between variables.
The discrepancies in the collected data may reflect various genetic influences on the AMY1 expression such as CNVs or regulatory region single nucleotide polymorphisms (SNPs) in which one of two or more alleles at a locus that differ by either a single nucleotide base or have variable numbers of tandem nucleotide repeats (Madsen 2010). Tandem repeats are widely distributed in the human genome and differences observed between genes can be attributed to variances in short sequences of DNA repeated in tandem. Regarding SNPs, it is known that the AMY1 gene is 10kb in length, and has 11 exons and 10 introns, generates a product of 511 amino acids, and is located at locus 1p21. It can be divided into three genes: AMY1A, AMY1B, and AMY1C. Restriction maps of these three genes reveal that their sequences are identical over a 27 kb region and hence, they are thought to be the product of recent gene duplication.
Further, discrepancies may be a result of non-genetic factors that may include individual hydration status, stress level, and short-term dietary habits (Perry et al. 2007). As noted that stress is a factor which may affect amylase expression, the sample population of this investigation being solely students may have altered results due to the comparative high-stress between the general population studied by Perry et al. (2007) and the sample in this study.
A potential source of error within this investigation includes sample size, which was a lab group of 30 subjects whereas in the study by Perry et al. there were 226 test subjects which provided more accurate results and a better representation of an entire population group. Subjects within this investigation group formed hypothesis’ based on ancestral diet types, however, much of this was an estimation and there was no verification of this data for accuracy. Secondly, there were external variables such as such as hydration status, psychosocial stress level, and short-term dietary habits which may alter amylase expression (Sonestedt, 2018). Provided subjects within this experiment were not in a controlled environment to alleviate external influences, this is an extraneous variable which may have altered results. Additionally, the process of PCR to amplify the target DNA may have produced errors during enzymatic copying or denaturing due to thermal influences in the thermocycler if not entirely protected by the heat resistant Go-Taq used (Tracey 2019). This error in PCR would correspondingly result in an inaccurate measurement of amylase and actin gene expression, which would consequently alter all corresponding data and figures. To reduce potential margins of error in future research, external stressors and variances in individuals should be considered and a controlled subject group should be analyzed.
The premise behind the hypothesis stated that the higher number of gene copies present, the more mRNA made in a given amount of time leading to an overall increase in the secretion of necessary proteins. The results supported the hypothesis, as there was a strong correlation between gene copy numbers and the amount of amylase present. The conclusion in the data was determined from comparison to the study published by Perry et al. (2007). The results identified a moderate positive correlation between variables; provided a gene copy number of 2 was determined, as well as a value of amylase concentration of 1.61 mg/mL, which is within one standard deviation of the value found in the study by Perry et al (2007).
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