Mutation

Genomics
MUTATION
General properties of
mutation
 Happens at any time, in any cell, could be good or bad
 Mutation generates new genes  increase variability  increase
the adaptation
 Mutation can be detrimental
 Can be classified in a variety of ways
 Location: Somatic mutation, germ-line mutation
 Conditional mutation: temperature-sensitive mutation ….
 Kind of DNA alteration: change base, continuous or not …

Ordinary Siamese cat. Biochemical

pathway leading to black pigmentation
is temperature-sensitive and
inactivated at normal body
temperature. The tips of
the legs, ears, and tail are cooler than
the rest of the body, so the pigment is
deposited in the hair in these areas.
The molecular basis of
mutation
(gene

mutation)
Base substitution: replace one base with another base

 Insertion/Deletion

 Expanding tri-nucleotide repeat (or longer repeat)

 Suppressor mutation: the second mutation occur to suppress the first
mutation, sometimes turn the first mutation to the original condition.
 Intragenic suppressor: Occur in
the same gene
 Intergenic
suppressor:
occur in the
different genes
 Phenotypic effect of mutation
 Missense mutation: base substitution changes one amino acid
 Nonsense mutation: base substitution changes one amino acid codon
to stop codon
 Silence mutation: changes codon but still code for the same amino
acid
 Neutral mutation: change amino acid of protein but does not change
the function.
 Loss-of-function mutation
 Gain-of-function mutation
 Conditional mutation: expressed under specific conditions
 Lethal mutation
 Three important mutations (among many mutations) in Indian
SARS-CoV2 strain(B.1.617) found to have association with
increased spreading of virus by increasing the binding of virus
with hACE2 receptor in many human cells (specifically epithelial
cells).
 E484Q (Glu-Gln)
 L452R (Leu- Arg)
 P681R (Pro-Arg)
 Mutation rate
 Mutation rate: number of mutations per biological unit
 4 mutations in 100,000 gametes of achondroplasia
 Affected by frequency of primary mutation in DNA (spontaneous or
induced mutation), the mutation repair, method of calculation.
 Vary among organisms and among genes
 Mutation frequency: incidence of the specific type of mutation
within the specific group of organism
 1 in 20,000 persons in the US carries mutation gene of
achondroplasia
 Mutation rate varies among
organisms and among genes within
organisms
 Prokaryote and virus: 1*10-8 – 1*10-10
(per cell), eukaryote : 1*10-5 -1*10-6
(per gamete)
 The ability of repair mutation
Cause of mutation

 Spontaneous mutation: natural change of DNA structure

 Induced mutation: change in DNA due to the environmental
radiation or chemicals
1. Spontaneous replication error
 Replication error is rare but do occur
 The primary cause of error is tautomeric shift (positions
of protons in the DNA bases change)  Purine and
pyrimidine bases exist in different chemical forms called
tautomers
 Mispairing due to wobble, the flexibility of helical
structure of DNA allows protonated and other forms of
bases can pair
2. Spontaneous chemical change
 Depurination:
 the loss of a purine base from a nucleotide
 results
when the covalent bond connecting the purine to
the 1-carbon atom of the deoxyribose sugar breaks
 Common cause of spontaneous mutation
 Deamination:
 the loss of an amino group (NH ) from a base.
2

 Deaminationmay occur spontaneously or be induced by

mutagenic chemicals.
 Alter the pairing properties of bases
3. Chemically induced mutation
a number of environmental agents are capable of
damaging DNA, including certain chemicals and
radiation.
 Any environmental agent that significantly
increases the rate of mutation above the
spontaneous rate is called a mutagen.
 Base analogs: chemicals with structures similar to that of any of the four
standard bases of DNA
 Alkylating agent
 Chemicals that donate alkyl groups.
 These agents include methyl (CH3) and ethyl (CH3–CH2) groups, which
are added to nucleotide bases by some chemicals.
 Deamination
 Bases are deaminated by chemical agents
 Hydroxylamine
 Add hydroxyl group to cytosine
 Oxidative reaction
 Reactive forms of oxygen (including superoxide radicals, hydrogen peroxide, and
hydroxyl radicals)
 Are produced in the course of normal aerobic metabolism, radiation, ozone, peroxides,
and certain drugs.
 Induce chemical change in DNA
 Intercalating agents
 such as proflavin, acridine orange, ethidium bromide, and dioxin
 Same size of nucleotide
 Cause mutatin by sandwiching themselves between adjuscent bases of DNA
Chemicals may alter
DNA bases.
(a) The alkylating agent
ethylmethanesulfonate
(EMS) adds an ethyl group
to guanine, producing 6-
ethylguanine, which pairs
with thymine, producing a
CG:TA transition mutation.
(b) Nitrous acid
deaminates cytosine to
produce uracil, which
pairs with adenine,
producing a CG:TA
transition mutation.
(c) Hydroxylamine
converts cytosine into
hydroxylaminocytosine,
which
frequently pairs with
adenine, leading to a
4. Radiation
 high energies of X-rays, gamma rays, and cosmic rays
are all capable of penetrating tissue and damaging
DNA
 These forms of radiation, called ionizing radiation,
dislodge electrons from the atoms that they
encounter, changing stable molecules into free
radicals and reactive ions, which then alter the
structures of bases and break phosphodiester
Study of mutation
 Analysis of reverse mutation
 Detecting mutation with Ames test
 Old test
 testing the cancer-causing potential of chemicals
 administer them to laboratory animals (rats or mice) and
compare the incidence of cancer in treated animals
 Time consuming and expensive
 Ames test
 Based on the principles that both cancer and mutations result
from damage of DNA
 Found more than 90% carcinogen are mutation
 Mutagen in bacteria could serve as indicator
DNA REPAIR
 Most DNA repair mechanisms require two nucleotide strands of
DNA because most replace whole nucleotides, and a template
strand is needed to specify the base sequence.
 Many types of DNA damage can be corrected by more than one
pathway of repair.
 ensures that almost all mistakes are corrected. If a mistake escapes
one repair system, it’s likely to be repaired by another system.
 Four general mechanisms of DNA repair: mismatch repair, direct
repair, base-excision repair, and nucleotide-excision repair
Mismatch repair

 Mismatch is first recognized and fixed by proofreading of DNA

polymerase enzymes
 If mismatch escapes proofreading, then it will be corrected by
mismatch repair proteins
 Proteins for mismatch repair in E.coli differentiate between old
and new synthesized strand by methylated nucleotide of specific
sequence on the old (template) strand.
 Exonuclease degrades the mismatch strand and then DNA
polymerase and DNA ligase synthesize the correct sequence
match complementary with the old strand.
Direct repair

 Change modified nucleotides directly into the correct structures by chemical

conversion
 For ex., photolyase in E.coli , O6-methylguanine- DNA methyltransferase..
Base excision repair

 Modified base is excised by DNA glycosylases and the nucleotide

is replaced
 Each enzyme catalyzes the excision of one specific nucleotide
 After removal of nucleotide, DNA polymerase synthesize the
correct nucleotide at nick position
Nucleotide excision repair

 Remove the DNA lesion that distort the DNA double helix: dimer,
large hydrocarbons …
 can repair many different types of DNA damage
 found in cells of all organisms from bacteria to humans and is
one of the most important of all repair mechanisms.
Reading

 Other mechanisms of DNA repair

 Genetic diseases and faulty DNA repair
GENOMICS
 Field of genetics that attempts to understand the
content, organization, function, and evolution of
genetic information contained in whole genomes.
 Genomics consists of three fields:
 Structural genomics determines the organization and
sequence of the genetic information contained within a
genome,
 Functional genomics characterizes the function of
sequences elucidated by structural genomics.
 Comparative genomics, compares the gene content,
function, and organization of genomes of different organisms
Structural genomics

 Genetic maps: provide a rough approximation of the

locations of genes relative to the locations of other
known genes
 Based on the genetic function of recombination
 Constructedby on the recombination frequencies  shown
only genes that express to the visible phenotypes
 Low resolution
 May not present the exact physical distance between gene
 Physicalmap: based on the direct analysis of DNA,
and they place genes in relation to distances measured
in number of base pairs, kilobases, or megabases
 Higher resolution and are more accurate than genetic maps.
 Number of techniques are used to generate the physical maps
restriction mapping determines the positions of restriction
sites on DNA.
sequence-tagged site (STS) mapping locates the positions
of short unique sequences of DNA on a chromosome.
fluorescent in situ hybridization (FISH), by which markers
can be visually mapped to locations on chromosomes.
sequencing
DNA sequencing method

 Sanger sequencing method

 Use dideoxyribonucleotides which lack
hydroxyl group at c3 position
 Lead to the termination of DNA synthesis
when ddNTP is incorporated into DNA
molecules
 ddNTP incorporates randomly to the elongated
DNA strand  generate population of diversed
length molecules
 Can be automated with fluorescent dye labled
ddNTP and laser scanner
 Sequencing the entire genome
 Chromosomal DNA should be broken into smaller fragments that can
be sequenced
 The sequenced fragments are then put back together in the correct
order
 Approaches:
 Map-based sequencing: requires the initial creation of genetic and
physical map of genome which contain the marker information  used to
align short sequenced fragments together.


 whole genome shotgun sequencing: small-insert clones are prepared

directly from genomic DNA and sequenced. Computers are used to
assemble the entire genome by examining overlap among the small-insert
clones.
 Single nucleotide polymorphism (SNPs): which are single-base-
pair differences in DNA sequence between individual members of
a species
 Arising through mutation
 SNPs are inherited as allelic variants (just like alleles that produce
phenotypic differences, such as blood types), although SNPs do not
usually produce a phenotypic difference.
 present throughout genomes
 Used in study the inheritance of particular gene, family relationship
 Expressed sequence tags (ESTs): markers associated with DNA
sequences that are expressed as RNA
 obtained by isolating RNA from a cell and subjecting it to reverse
transcription, producing a set of cDNA fragments that correspond to
RNA molecules from the cell
 used to find active genes in a particular tissue or at a particular point
in development.
 Bioinformatics: field consisting of molecular biology and
computer science that centers on developing databases,
computer-search algorithms, gene prediction software, and other
analytical tools that are used to make sense of DNA, RNA, and
protein sequence data
 develops and applies these tools to “mine the data,” extracting the
useful information from sequencing projects.
Functional genomics

 probing genome sequences for meaning—identifying genes,

recognizing their organization, and understanding their function
 The goals of functional genomics:
 identifying all the RNA molecules transcribed from a genome (the
transcriptome)
 identifying all the proteins encoded by the genome (the proteome)
 exploits both bioinformatics and laboratory-based experimental
approaches in its search to define the function of DNA
sequences.
 Predicting the function from sequence
 From nucleotide sequence: to deduce protein sequence and function
 Develop the computational method to define gene function from DNA
sequence without doing laboratory work
 Homology searches: compare the DNA and protein sequences
from the similar or different organisms  homologous genes
 Ortholog: Homologous genes found in different species that evolved
from the same gene in a common ancestor
 Paralog: Homologous genes in the same organism (arising by
duplication of a single gene in the evolutionary past)
 The commonly used homologous search program is BLAST (Basic
Local Alignment Search Tool) from NCBI
 Gene expression and microarray (gene chip)
 rely on nucleic acid hybridization in which a known DNA fragment is
used as a probe to find complementary sequences
 Probes are fixed on solid support and may be labeled, sample nucleic
acids are prepared in solution.
 Complementary between probes and nucleic acids in samples shows
the presence of known sequence
 Used to see the expression of genes and level of expression
 Genomewide mutagenesis: the way to identify the function of
gene by examining the phenotype of individuals that have
mutations in gene.
 Random inducement mutation on genomewide basis and mapping
with molecular markers are coupled and automated on mutagenesis
screen  used to search for the specific genes encoding particular
functions
Comparative genomics
 Prokaryotic genomes
 Eukaryotic genomes