Assignment No.

4
Advance Molecular Genetics

Submitted by: Hajra Qayyum (MBI173001)

Submitted to: Dr. Shaukat Iqbal Malik
Dated: 26th December, 2017

Department of Bioinformatics and Biosciences

Capital University of Science and Technology, Islamabad
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1. Single Amino Acid Polymorphism (SAP)

Single-nucleotide polymorphisms (SNPs) are the main form of genomic sequence variation and
the major genetic basis of physiological or pathological variants for individuals.

1.1. Types

There are two types of SNPs: within coding regions and within non-coding regions. Due to
degeneracy of the genetic codes, SNPs within coding regions may not change the amino acid
sequence of the corresponding protein. Therefore, SNPs within coding regions should be further
divided into two categories: synonymous and non-synonymous. The non-synonymous
polymorphisms result in variation of amino acid sequences and often have significant impact on
functions of genes. Recently, an exome sequencing analysis identified more than 53000 SNPs
located in coding regions from 200 human exomes, among which less than half of SNPs were
synonymous and the rest were non-synonymous. This suggests that non-synonymous
polymorphisms are widely distributed in human genome.

1.2. Genetic Structures

More importantly, for human and other diploid-genome organisms, SNPs imply two possible
genetic structures in a single allele: either homozygous state or heterozygous state. It was
reported that the different genetic structures of coding SNPs were tightly associated with
physiological and pathological traits of individuals. In addition, for heterozygous state, the ratio
of allele-specific gene expressions is also associated with various traits of individuals. Therefore,
researchers should collect, both qualitatively and quantitatively, the genetic structural SNPs'
information of individuals. Theoretically, only the qualitative genetic structural information of
SNPs is able to be detected at the genome level by sequencing or microarray approaches,
whereas both qualitative and quantitative information of coding SNPs can be collected at the
transcriptome level (Figure 1A). Several methods have been developed for detecting variable
allele-specific expressions at mRNA level. On the other hand, only the non-synonymous
polymorphisms on amino acids of proteins can be detected both qualitatively and quantitatively
at the proteome level.

1.3. SAP Mutations Associated Diseases

The SNP analysis at the genome level has been extensively studied to reveal their associations
with particular traits or diseases, which is indirect and non-quantitative. On the other hand, the
SAPs play more important roles in affecting functions of genes and present the direct association
with phenotypes. Furthermore, the quantitative traits should be ultimately determined by the
expressed proteins. For example, di Fede et al found a β-amyloid precursor protein mutation
(APP_A673V) that caused familial Alzheimer's disease only in the homozygous state, while
heterozygous carriers were unaffected since the wild-type peptide could interact with the mutant
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peptide. In the present work, we combined non-targeted and targeted proteomics strategy, both
qualitatively and quantitatively detected SAP peptides in population level.

1.4. SAP Databases

a) SAAPdap

Available freely at http://www.bioinf.org.uk/saap/dap/

b) dpSAP

Online database available freely at http://www.megabionet.org/dbSAP/index.html

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2. Protein Sequencing
2.1. Definition

Protein sequencing refers to the detection of proteins’ primary structure, which contains the
number of polypeptide chains in proteins. Polypeptides and proteins can be used equally in many
cases. Amino acid sequence of polypeptides is the biological function of proteins.

2.2. Principle

Protein sequencing is mainly relying on chemical or enzymatic digestion methods to separate

peptides and detect the amount and composition of amino acid residues. This post will introduce
the principles and experimental steps of protein sequencing.

2.3. Sequencing Methods

a) Edman degradation

Before sequencing process is initiated, it is necessary to break all non-covalent interaction by

denaturants (like high concentration of urea or GuHCl. This process will also separate subunits,
in case of oligomeric proteins. Occasionally, subunits of a oligomeric protein are connected by
covalent interactions. In that case special treatments are required to separate subunits. The
protein is treated with Edman’s reagent (phenyl isothiocyanate) which reacts with the N-terminal
amino acid and under mild acidic condition forms a cyclic compound Phenyl thiohydantoin
derivative (PTH–amino acid) of N-terminal amino acid is released. Amino acid of PTH –amino
acid derivative is identified by chromatographic Proteomics & Genomics property of the PTH –
amino acid derivative. In this process N-terminal aminos acid is identified after first cycle. Since
this method proceeds from the N terminal residue, the reaction will not work if that N-terminal
of a protein is blocked (generally due to posttranslational modification). After first cycle of the
reaction, amino group of the second amino acid is free for reaction with Edman’s reagent and at
the end of reaction PTH derivative of second amino acid from N-terminal is released. The
process continues till end of sequence or a disulfide bond is encountered in the sequence. PTH-
cysteine derivative will remain attached with polypeptide and PTH-cystein will not be released.
Thus, reduction of disulfide bond in the polypeptide sequnce needed before sequencing process
can be initiated. Reduction of free cysteine can be done by use of - marcaptoethanol.
Further, the accuracy of each cycle is 98%. So after 60 steps the accuracy is less than 30%. Thus,
this method cannot be used for sequencing of proteins larger than 50 amino acids. In case of
larger proteins it has to be broken down to short peptide fragments using cleavage proteases such
as trypsin (cleaves a protein at carboxyl side of lysine and arginine residues) or chymotrypsin
(cleaves at carboxyl side of tyrosine, tryptophan and phenylalanine). Specific cleavage can also
be achieved by chemical methods like cynogen bromide, which always cleaves at carboxyl side
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of methionine residue (a protein with 12 methionine will yield 13 fragment polypeptide on

cleavage with cynogen bromide (CNBr). Protein fragments after a protease (for example
trypsine) will be separated and sequenced. Let us assume that the following two peptide
sequences are obtained.

b) Protein sequencing using Sanger’s reagent and dansyl chloride

Here, the N terminal amino acid of the protein is labeled by dyes like Sanger’s reagent (fluoro-
dinitrobenzene) or dansyl chloride. The labeled protein is then hydrolyzed by 6M HCl at 110 ºC
by the above mentioned method and loaded in Dowex 50 column and the elution profile is
matched with the standard profile obtained from FNB or DNSCl derivative of all the amino
acids, to obtain the N terminal amino acid. The reagents produce coloured derivatives which can
be easily detected by absorbance
 Disadvantages of this method include
1. Once we get the N terminal amino acid, the protein is already hydrolyzed in constituent
amino acids. Thus we cannot repeat the cycle with same sample. For second amino acid
sequencing we require new stock of protein sample and the N-terminal residue need to be
cleaved from the protein using an appropriate protease such as amino peptidase. This makes
the process very tedious and complicated.
2. These dyes selectively labels the amine groups present in the protein and therefore can label
the amine groups present in the side chains as well, which may give erroneous results.

c) Protein sequencing using Molecular Biology techniques

If first few N-terminal amino acid of a protein is known, complete amino acid sequence can be
derived using Molecular Biology techniques. A simple example is as follow: The genome
sequence of Calotropis procures, a plant, or the sequence of porcelain B, a novel cystein protease
from the plant, gene is not yet known. Thus, the only information for cloning of cDNA we have
is the fifteen N-terminal amino acid residues. The double stranded cDNA can be amplified with
help of degenerate primer (based of N-terminal amino acid sequence) and oligo dT primer. Total
RNA can be isolated from young leaf or latex of the plant and first strand of cDNA can be
synthesized with oligo dT primer by reverse transcription. The second strand of cDNA can be
synthesized and the subsequent amplification of double stranded cDNA can be achieved by PCR
with degenerate primer as forward and oligo dT primer as reverse primmer. The amplified
double stranded cDNA of expected size can be subjected to TA cloning and confirmed by
sequencing. Once sequence of cDNA is available, it can be translated in protein sequence.

2.4. Applications

There are several applications of protein sequencing, which are:-

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a) Identification of the protein family to which a particular protein belongs and finding the
evolutionary history of that protein. Function prediction.
b) Prediction of the cellular localization of the protein based on its target sequence (sequence of
amino acids at the N terminal end of the protein which determines the location of the protein
inside the cell).
c) Prediction of the sequence of the gene encoding the particular protein.
d) Discovering the structure and function of a protein through various computational methods
and experimental methods.

3. Pleiotropy versus Polygenic Inheritance

3.1. Pleiotropy

In pleiotropy, a single gene affects many traits. That means the gene product is used in many
types of cells in different tissues. Sometimes, the gene product may act as a signaling molecule,
affecting the functions of many tissues. The gene, which is responsible for the coat color of mice
is pleiotropic. The dominant allele Y produces the yellow coat color and the recessive allele y
produces the agouti color in the mice. The genotype yy produces agouti color mice. The
genotype Yy produces yellow color mice. The genotype YY produces two traits, the coat color
and lethality. Therefore, the mice embryo with the genotype YY will terminate prematurely.
Since one gene is involved in the determination of the trait in pleiotropy, only three different
genotypic outcomes can be observed in the offspring. The mechanism of pleiotropy is shown in
figure 1.

Figure 1: One gene controlling multiple effects

When a mutation occurs in this gene, many symptoms may arise since the gene affects many
traits. Phenylketonuria is a disease caused by a mutation in the gene which is coded for the
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enzyme, phenylalanine hydroxylase. The symptoms of phenylketonuria are mental retardation,

reduced hair, and skin pigmentation. A gene carrying a mixture of both beneficial and harmful
traits with the same gene is referred to as an antagonistic pleiotropy. The p53 gene suppresses the
undifferentiated cell proliferation, preventing cancer. At the same time, it suppresses stem cell
proliferation, preventing the tissue regeneration in the old. Aging is another example of
antagonistic pleiotropy, which increases the fitness in youth but decreases the fitness as one
grows old. Albinism occurs when the melanin production of melanin is altered by a mutation. It
affects the organism’s skin, hair, and eye. Autism, schizophrenia, sickle cell anemia, and Marfan
syndrome are examples of diseases caused by pleiotropy.

3.2. Polygenic Inheritance

In polygenic inheritance, a particular trait is determined by more than one gene. Thus, the effect
of one gene on the trait is small. Here, the contributing genes exhibit incomplete dominance.
Thus, the trait in the offspring is a mixture of parental traits. The external environmental factors
also have an effect on polygenic inheritance. Most of the metric and meristic traits are under the
influence of polygenic inheritance. The polygenic traits exhibit a continuous distribution in a
population. Thus, the distribution curve of the polygenic inheritance is bell-shaped. A great
variability of genotypes can be observed within a population in polygenic traits. The organisms
at the middle of the distribution curve consist of a combination of both dominant and recessive
alleles. The individuals with many of the dominant alleles or recessive alleles may appear at the
end of the curve. The distribution curve of the polygenic inheritance of height in humans is
shown in figure 2.

Figure 2: Polygenic inheritance of height

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3.3. Pleiotropy vs Polygenic Inheritance

The color of the human eye is controlled by 16 different genes. The eye color is determined by
the amount of melanin produced in front of the iris. The color can be either black, brown, green,
hazel or blue. The skin color of humans is another example of polygenic inheritance. The color
of the skin is determined by the amount of melanin produced in the skin. When the number of
dark alleles present in the skin is high, the color of the skin becomes darker.

3.3. Similarities between Pleiotropy and Polygenic Inheritance

a) Pleiotropy and polygenic inheritance describe the relationship between genes and their traits.
b) Both pleiotropy and polygenic inheritance can occur in all living organisms.

3.4. Difference between Pleiotropy and Polygenic Inheritance

Pleiotropy Polygenic Inheritance

Definition Pleiotropy is the controlling of Polygenic inheritance is the
multiple traits by a single controlling of a single trait by
gene. multiple genes.

Outcome Pleiotropy has only three Polygenic inheritance has

genotypic outcomes many genotypic outcomes.
Trait In pleiotropy, a particular trait
In polygenic inheritance, a
is influenced by one gene. particular trait is influenced by
many genes.
Effect of One Gene on the The effect of one gene on its The effect of one gene on the
Trait trait is 100%. trait is small.

Mendelian Inheritance The pleiotropy follows Polygenic inheritance is a non-

Mendelian inheritance Mendelian inheritance pattern.
patterns.

Effect of the Environmental Typically, pleiotropy is not The traits of the polygenic
Factors affected by the environmental inheritance are highly affected
factors. by the environmental factors.

Examples Albinism, phenylketonuria, Height, weight, body shape,

autism, schizophrenia, sickle eye color, skin color, and hair
cell anemia, and Marfan color of humans are controlled
syndrome are examples of by polygenic inheritance.
pleiotropy.
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3.5. Conclusion

Pleiotropy and polygenic inheritance describe the influence of genes on their phenotypes. In
pleiotropy, a single gene controls a particular character, obeying Mendelian inheritance patterns.
In polygenic inheritance, a single trait is controlled by many genes. The main difference between
pleiotropy and polygenic inheritance is the patterns of influences of genes on their traits.

4. Reference

1. Zhi-Duan Su, Liang Sun, Dan-Xia Yu, Rong-Xia Li, Huai-Xing Li, Zhi-Jie Yu, Quan-Hu
Sheng, Xu Lin, Rong Zeng, Jia-Rui Wu; Quantitative detection of single amino acid
polymorphisms by targeted proteomics, Journal of Molecular Cell Biology, Volume 3, Issue
5, 1 October 2011, Pages 309–315.

2. Carles Ferrer-Costa, Modesto Orozco, Xavier de la Cruz, Characterization of disease-

associated single amino acid polymorphisms in terms of sequence and structure
properties11Edited by J. Thornton, In Journal of Molecular Biology, Volume 315, Issue 4,
2002, Pages 771-786, ISSN 0022-2836, https://doi.org/10.1006/jmbi.2001.5255.

3.  Morgan, F.J. "AAS Biographical Memoirs - Pehr Victor Edman 1916 - 1977." 8 April 1998.
University of Melbourne. Accessed 30 March 2008.

4. Biemann K., Cone C., Webster B.R., Arsenault G.P. Determination of the amino acid
sequence in oligopeptides by computer interpretation of their high-resolution mass spectra J.
Am. Chem. Soc., 1966, 88(23), p.5598-606. 

5. Henzel W.J., Watanabe C., Stults J.T. Protein Identification: The Origins of Peptide Mass
Fingerprinting. Journal of the American Society for Mass Spectrometry, 2003, 14(), p.931.

6. “Pleiotropy.” Genetics-notes. N.p., n.d. Web. Available here. 13 July 2017.

7. Bailey, Regina. “What Is Polygenic Inheritance?” ThoughtCo. N.p., n.d. Web. Available
here. 13 July 2017.