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(Botany) : Molecular Basis of Inheritance
(Botany) : Molecular Basis of Inheritance
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Molecular Basis of Inheritance 2
Nucleic acids :
* Friedrich Meisher discovered Nucleic acid in Nucleus of Pus cells (1869) and named it ‘Nuclein’
• Altmann found its acidic nature and termed ‘ Nucleic acid’
• DNA term was given by Zarich
* 2types of nucleic acid- 1. DeoxyRibonucleic Acid (DNA) - Genetic material in most organisms 2.
Ribonucleic Acid (RNA)-
* Genetic material in some viruses * Messenger molecule * Adapter molecule
* Catalytic (Ribozyme) * Structural component
DNA
DNA is a long polymer of deoxyribonucleotides.
• The length of DNA is usually defined as number of nucleotides (or a pair of nucleotide referred to as
base pairs , bp) present in it. This also is the characteristic of an organism.
i). Bacteriophage 174 -5386 nucleotides . ssDNA
ii). Bacteriophage lambda - 48502 base pairs (bp) - dsDNA
iii). Escherichia coli - 4.6 × 106 bp- dsDNA
iv). Haploid content (Genome) of human DNA . 3.3 × 109 bp. - dsDNA
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Molecular Basis of Inheritance 4
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Molecular Basis of Inheritance 5
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Molecular Basis of Inheritance 6
SALIENT FEATURES OF THE DOUBLE-HELIX STRUCTURE OF DNA
(i) It is made of two polynucleotide chains, where the backbone is constituted by sugar-phosphate, and the
bases project inside.
(ii) The two chains have anti-parallel polarity. It means, if one chain has the polarity 5’3', the other has
3' 5'
(iv) The bases in two strands are paired through hydrogen bond (H-bonds) forming base pairs (bp).
* This generates approximately uniform distance between the two strands of the helix.
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Molecular Basis of Inheritance 7
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Molecular Basis of Inheritance 8
Types of DNA :
1) Circular and Linear DNA
2) Repititive DNA – Sequence of N-bases is repeated more than once inDNA , present in centromere and
satellite region of chromosome
3) Palindromic DNA- Segment of DNA which reads the same when orientation of reading is kept same. ,
Restriction site for RE
C-Value of DNA :
* Total amount of DNA in genome
* Measured in pigogram (pg)
* Bacteria which was infected with viruses that had radioactive DNA were radioactive, indicating that
DNA was the material that passed from the virus to the bacteria.
* Bacteria that were infected with viruses that had radioactive proteins were not radioactive.
* This indicates that proteins did not enter the bacteria from the viruses.
* DNA is therefore the genetic material that is passed from virus to bacteria
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Molecular Basis of Inheritance 10
PROPERTIES OF AS A GENETIC MATERIAL RNA WORLD
A molecule that can act as a genetic material RNA was the first genetic material.
must fulfill the following criteria: RNA used to act as a genetic material as well as a
(i) It should be able to generate its replica (Replica- catalyst (there are some important biochemical
tion). reactions in living systems that are catalysed by
(ii) It should chemically and structurally be stable. RNA catalysts and not by protein enzymes).
(iii) It should provide the scope for slow changes
(mutation) that are required for evolution. But, RNA being a catalyst was reactive and
(iv) It should be able to express itself in the form of hence unstable.
'Mendelian Characters’. * Therefore, DNA has evolved from RNA with
chemical modifications that make it more stable.
The two strands of DNA being complementary if * DNA being double stranded and having comple-
separated by heating come together, when mentary strand further resists changes by
appropriate conditions are provided. evolving a process of repair.
* Further, 2'-OH group present at every nucleotide
in RNA is a reactive group and makes RNA DNA REPLICATION
labile and easily degradable. It is Synthesis of new DNA strand from parent
* RNA is also now known to be catalytic, hence DNA or on existing DNA strand.
reactive. * It occurs in S-phase of cell cycle
* It occurs in cytoplasm in prokaryotes , nucleus/
Therefore, DNA chemically is less reactive and Mt/chloroplast in Eukaryotes.
structurally more stable when compared to RNA. * While giving double helical structure of B-DNA ,
* Therefore, among the two nucleic acids, the Watson & Crick gave the scheme for DNA
DNA is a better genetic material. Replication.
* In fact, the presence of thymine at the place of “The parent DNA strands break and each strand
uracil also confers additional stability to DNA. acts as template for synthesis of new DNA
* RNA being unstable, mutate at a faster rate strand’
CENTRAL DOGMA OF MOLECULAR * 3 modes of DNA replication :
BIOLOGY 1. Semi-conservative – accepted mode , experimen-
* Francis Crick proposed the Central dogma in tally proven by Messelson and Stahl
molecular biology, which states that the genetic 2.Conservative 3.Dispersive
information flows from DNA RNA Protein.
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Molecular Basis of Inheritance 11
DNA REPLICATION
Messelson and Stahl’s Experiment (1958)
* Experimental model-E.coli * He used -
1. Heavy isotope of N2- N15 2. Light isotope of N2- N14
* He used Density gradient centrifugation by CsCl
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Molecular Basis of Inheritance 12
1) ORI = origin of replication 2 types of new strands :
* DNA replication starts at ori 1.Continuous/Leading strand :
* 1 ori (prokaryotes) , >1 ori (eukaryotes) * Synthesized continuously
* If desired DNA is to be propagated through * Single primer required
recombinant DNA technology , then vector is * Okazaki DNA fragments absent
required because vector has Ori. * Faster formation
2) Activation of Nucleotides (DEEP) : * Formed on parent / Template strand 3' 5'
* Deoxyribonucleotide,Phosphorylase Enzyme ,
Energy, two Pi required. 2.Lagging strand :
* Deoxyribonucleoside triphosphates serve dual * Synthesized discontinuously
purposes - * >1 primer required
1. In addition to acting as substrates * Okazaki fragments present (joined by DNA
2. they provide energy for polymerisation reaction ligase)
(the two terminal phosphates in a * Slower
deoxynucleoside triphosphates are high-energy * Formed on parent / Template strand 5'3'
phosphates, same as in case of ATP).
c)Topoisomerase -
* It release tension due to super coiling at opposite
end of replication fork.
* It nicks , release tension &then re-seals.
* In prokaryotes , this function is performed by
DNA Gyrase. * In eukaryotes, the replication of DNA takes
place at S-phase of the cell-cycle.
4) Primer formation : * The replication of DNA and cell division cycle
* DNA-P cannot initiate replication on its own. should be highly coordinated.
* So , RNA Primer (small sequence of ss RNA , * A failure in cell division after DNA replication
complimentary to parent DNA strand) is formed results into polyploidy(a chromosomal anomaly).
at 3’ end of parent strand Proof reading :
* using enzyme DNA dependent RNA Poly- * Done mainly by DNA-P I
merase/RNA Primase * In 3'5' direction
* After completion of new strand , primer is * Exonuclease activity
removed(5'÷3') & gap is filled by DNA-P I
DNA replication is
5) Elongation of DNA strand * Fast
* After primer addition , DNA-P III attaches * Semi-conservative
¡¥activated nucleotides¡¦ to the primer two * Semi-continuous
terminal pyrophosphate high energy are removed * Bidirectional
releasing energy which is used in forming * Almost accurate
phospho-di-ester bonds * Energy consuming
* DNA-P III adds about 2000 bps/second
* DNA-P forms new strand in 5' 3' direction
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Molecular Basis of Inheritance 13
RNA :
* RIBONUCLEIC ACID
* Single stranded polynucleotide * Parts of RNA:
1.Ribose sugar 2.Phosphate 3.Nitrogen bases- A , G, C, U
TYPES OF RNA :
1.mRNA 2.tRNA 3.rRNA
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Molecular Basis of Inheritance 14
t-RNA : 2 models 4.sc RNA – small cytoplasmic RNA
1.Inverted L model – * 7-s scRNA + Proteins signal recognition
* by Kim & Klug peptide (SRP)*
* 3-D , Difficult to study * They help in binding ribosome & nascent peptide
2.Clover leaf model - to ER for formation of regulatory protein.
* By Holley * All 3 types of RNA are required in protein
* 2-D / Secondary structure , easy to study synthesis i.e mRNA, tRNA and rRNA
* Second, the two RNA molecules if produced simultaneously would be complementary to each other,
* hence would form a double stranded RNA.
* This would prevent RNA from being translated into protein
* and the exercise of transcription would become a futile one.
* Template/Non coding/Anti-sense strand of DNA –
* Strand with polarity - 3'5'
* RNA is synthesized on this strand in 5'3'as RNA Polymerase works in 5'3'¦
* Coding /Non template/ Sense strand of DNA –
* Strand with polarity - 5'3'
* RNA formed is similar to coding strand , but formed on template strand
Transcription Unit
* It is segment of DNA involved in transcription.
* A transcription unit in DNA is defined primarily by the three regions in the DNA: (i) A Promoter (ii) The
Structural gene (iii) A Terminator
Structural gene is flanked on both sides by promoter (5' of coding strand) and terminator( 3' of coding
strand)
* Promoter –
* provides binding site for RNA-Polymerase
* present at 5' of coding strand (upstream) or 3' of template strand
* The presence of promoter defines coding and template strand
*By switching promoter with terminator , the definition of coding and template strand reverses.
Recognition/conserve sequences –
* present in promoter region , recognised by RNA-Polymerase (sigma factor)
* may be present further upstream or downstream to the promoter
* In prokaryotes-
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Molecular Basis of Inheritance 16
* at -10 bp( 10 bp before/upstream to start point) TRANSCRIPTION IN PROKARYOTES
* TATAAT (Pribnow box) * There is single DNA-dependent RNA poly-
* In eukaryotes – merase that catalyse transcription of all types of
* at -20 bp (20 bp before/upstream to start point) RNA in bacteria.
* TATAAAT / TATATAT (Hogness/TATA box) * It occurs in cytoplasm.
* -70 to -80 bp CAAT box * 3 steps –
1.Initiation 2.Elongation 3.Termination
RNA polymerase
* Forms RNA in 5'3' on template strand 1.Initiation –
* In prokaryotes – only one type which forms all * Sigma factor of RNA Polymerase recognise
types of RNA conserved sequences in promoter region.
* In eukaryotes – 3 types of RNA Polymerase * Now RNA polymerase binds to promoter and
1. RNA polymerase I (nucloelus) transcribes initiates transcription.
rRNAs (28S, 18S, and 5.8S)
2. RNA polymerase II (nucleoplasm)transcribes 2. Elongation –
mRNA – the heterogeneous RNA (hnRNA). * RNA Polymerase uses nucleoside triphosphates
3. RNA polymerase III (nucleoplasm) is transcribes as substrate and polymerises in a template
tRNA , 5s rRNA, scRNAs and snRNAs depended fashion following the rule of
complementarity.
RNA Polymerase has 5 subunits : * It somehow also facilitates opening of the helix
* Core enzyme = 2 + 1+1 + 1 = role in and continues elongation till it reaches terminator
elongation of RNA site.
* Sigma factor (£m ) = role in initiation of tran- * Only a short stretch of RNA remains bound to
scription the enzyme.
* Rho factor (£l )= role in termination of transcrip-
tion 3. Termination –
* Holoenzyme = core enzyme + Sigma factor * Once the polymerases reaches the terminator
region , Rho factor helps in release of RNA
Terminator – Polymerase,
* Defines end of transcription * the nascent RNA falls off, so also the RNA
* present at 3' of coding strand (downstream) or 5' of polymerase.
template strand * This results in termination of transcription.
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Molecular Basis of Inheritance 17
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Molecular Basis of Inheritance 18
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Molecular Basis of Inheritance 20
* Insertion or deletion of three or its multiple bases TRANSLATION
insert or delete one or multiple codon hence one * The process of polymerisation of amino acids to
or multiple amino acids, and reading frame form a polypeptide.
remains unaltered from that point onwards * It occurs in cytoplasm in both prokaryotes and
eukaryotes.
RAM HAS RED CAP * Cellular factory for protein synthesis is Ribo-
RAM HAS BRE DCA P some.
RAM HAS BIR EDC AP * The order and sequence of amino acids are
RAM HAS BIG RED CAP defined by the sequence of bases in the mRNA.
RAM HAS EDC AP (R removed)
RAM HAS DCA P (RE removed) Machinery for translation :
RAM HAS CAP (RED removed) 1. r RNA ( as structural component of ribosome)
* This forms genetic basis of proof that codon is a 2. t RNA
triplet & it is read in a contiguous manner 3. m RNA
4.Ribosome
2. Substitution / Point mutation complete 5.Mg2+ ions
reading of frame is not altered 6.Amino acids (raw material)
* THE FAT CAT ATE THE RAT (BAT) 7.Aminoacyl t RNA synthetase enzyme
8.Initiation factors , elongation factors , release
a)Transition – factors
* If Pu is mutated to Pu or Py to Py When 2 subunits of ribosome combines ,
1.AUG (methionine) GUG(valine) there is formation of 3 sites
2.AUG (methionine) ACG (threonine) 1.P site (Peptidyl site)
2.A site ( Aminoacyl site)
b)Transversion – 3.E site ( Exit site)
* If Pu is mutated to Py or Py is mutated to Pu
1.AUG (methionine) CUG(Leucine) Process of translation :
2.AUG (methionine) AAG(Lysine) 1.Activation of amino acid :
* In the first phase itself amino acids are activated
1. Mis-sense mutation –if by change of 1 nitrogen in the presence of ATP
base , aa is changed , may be lethal, A min o acyl
* Sickel cell anemia * Amino acid + ATP t RNA synthetase
* AUG (methionine) GUG(valine)
2. Same-Sense / Silent mutation – if by change of 1 Amino acyl AMP- enzyme complex + PP
nitrogen base , aa is not changed , not lethal , role
in evolution AA + ATP + Aminoacyl synthetase enzyme (E) +
* CCC (proline) CCG (proline) Mg2+
3. Non -Sense mutation - if by change of 1 nitrogen AA-AMP-E Complex + Ppi
base , there is appearance of any stop codon , There is a separate gAmino acyl t-RNA
may be lethal , the protein synthesis stops after synthetase enzyme for each kind of amino acid
this point.
* UGG (Tryptophan) UGA (Stop codon) 2. Charging of tRNA or Aminoacylation of tRNA
Q) Assume there are – :
a) 5 types of Nitrogen bases in RNA and there are * Activated Amino acids linked to their cognate
2 bases in each codon , then calculate no. of tRNA AA-AMP-E Complex + specific t RNA
codons possible – tRNA –VAA (Charged tRNA) + AMP + Enzyme
b) 7 types of Nitrogen bases in RNA and there are
2500 amino acids, then calculate i) No. of 3. Formation of polypeptide chain :
nitrogen bases in codon ii) No. of codons possible * The amino acids are joined by a bond which is
known as a peptide bond.
* Formation of a peptide bond requires energy.
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Molecular Basis of Inheritance 21
* The Ribosome also acts as a catalyst (23S rRNA * This t-RNA of P site released from ribosome via
in bacteria is enzyme- Ribozyme) for the forma- E-site and dipeptide attaches with A site.
tion of peptide * Now t-RNA of A-site is transferred to P-site and
A) Binding of mRNA with smaller subunit of hence A-site becomes empty.
ribosome
B) Binding of charged tRNA with m RNA-Ribosome ii) Translocation – moving of ribosome on m RNA ,
complex from one codon to other.
C) Chain elongation – * Now , process of translocation and peptide bond
i) Peptide bond formation formation will continue till at A-site , stop codon
* After formation of initiation complex , A-site is appears.
established next to P-site (where 1st charged * The amino acids are getting added one after the
tRNA is attached) other , leading to polypeptide sequence , dictated
by DNA
Now next charged t RNA binds to A-site (codon-
anticodon binding) iii ) Chain – Termination :-
* When 2 charged t RNA are brought in close * Due to sliding of ribosome over m-RNA when
contact, then peptide bond formation is favoured any Non-sense codon (UAA, UAG, UGA)
energetically available at A site of ribosome , then polypeptide
* Catalyst Peptidyl transferase (Ribozyme) helps in chain terminates
this bond formation * The linkage between the last tRNA and the
* Peptide bond is formed between COOH of one polypeptide chain is broken by three release
amino acid and –NH2 of next amino acid. facctor called RF1, RF2, RF3 with the help of
GTP.
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Molecular Basis of Inheritance 22
Regulation of Gene Expression :
A translational unit in mRNA * Regulation over the functioning of genes is called
* The sequence of RNA that is flanked by the start regulation of gene expression. It can be exerted
codon (AUG) and the stop codon at four levels.
* and codes for a polypeptide (i) Transcriptional level during formation of primary
transcript.
Untranslated regions (UTR) – (ii) Processing level (regulation of splicing)
* An mRNA also have some additional sequences (iii)Transport of mRNAs from nucleus to cytoplasm
that are not translated (iv) Translational level
* The UTRs are present at both 5' end (before
start codon) and at 3' end (after stop codon). Operon Model :
* The UTR (untranslated regions) present on •An operon is a segment of DNA that functions as
mRNA are required for efficient translation single regulated unit comprising
process (by recognising the smaller subunit of 1.a regulator gene
ribosome by mRNA) 2.a promoter gene
3.an operator gene
* For initiation, the ribosome binds to the mRNA at 4.one or more structural genes
the start codon (AUG) that is recognised only by
the initiator tRNA. Operons involve two types –
* The ribosome proceeds to the elongation phase (1) Inducible operon model
of protein synthesis. (2) Repressible operon model.
* During this stage, complexes composed of an First operon lac–Operon was
amino acid linked to tRNA, sequentially bind to * discovered by Jacob and Monod (1961) in E.coli.
the appropriate codon in mRNA by forming
complementary base pairs with the tRNA The genes in a cell are expressed to perform a
anticodon. particular function or a set of functions.
* The ribosome moves from codon to codon along * For example, if an enzyme called beta-galactosi-
the mRNA. dase is synthesised by E. coli, it is used to
* Amino acids are added one by one, translated catalyse the hydrolysis of a disaccharide, lactose
into Polypeptide sequences into galactose and glucose;
* dictated by DNA and represented by mRNA. * the bacteria use them as a source of energy.
* At the end, a release factor binds to the stop * Hence, if the bacteria do not have lactose around
codon, terminating translation and releasing the them to be utilised for energy source, they would
complete polypeptide from the ribosome. no longer require the synthesis of the enzyme
Summary of Translation – beta-galactosidase.
I.Amino acid activation – 1 ATP required * In simple terms, it is metabolic, physiological or
II.Charging of t-RNA – No ATP required (X) environmental conditions that regulate expression
III.Polypeptide chain formation - of genes.
* The development and differentiation of embryo
1.Chain initiation :- into adult organisms are also a result of the
a)m-RNA ribosome (smaller subunit) complex- (X) coordinated regulation of expression of several
b)Addition of charged t-RNA – 1 GTP required sets of genes.
c)Larger subunit of ribosome attaches 2. Chain – * In prokaryotes, control of the rate of transcrip-
elongation :- tional initiation is the predominant site for control
a)Peptide bond formation –X of gene expression.
b)Translocation- 1 GTP required 3. Chain termination * In a transcription unit, the activity of RNA
- 1 GTP required polymerase at a given promoter is in turn regu-
lated by interaction with accessory proteins,
which affect its ability to recognise start sites.
* These regulatory proteins can act both positively
(activators) and negatively (repressors).
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Molecular Basis of Inheritance 23
* The accessibility of promoter regions of prokaryotic DNA is in many cases regulated by the interaction of
proteins with sequences termed operators.
* The operator region is adjacent to the promoter elements in most operons and in most cases the se-
quences of the operator bind a repressor protein.
* Each operon has its specific operator and specific repressor.
* For example, lac operator is present only in the lac operon and it interacts specifically with lac repressor
only.
Inducible Operon Model :
• It is found in catabolic pathway
• Ex: Lactose operon or Lac operon.
The Lac operon
* Geneticist , Francois Jacob and a biochemist, Jacque Monod
* a transcriptionally regulated system
* In lac operon (here lac referes to lactose),
* a polycistronic structural gene is regulated by a common promoter and regulatory genes.
* Such arrangement is very common in bacteria and is referred to as operon.
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Molecular Basis of Inheritance 24
1. z gene codes for beta-galactosidase (â-gal), Types of Genes :
which is primarily responsible for the hydrolysis (1) House Keeping Genes (Constitutive Genes)
of the disaccharide, lactose into its monomeric :
units, galactose and glucose. * These genes are constantly expressing them-
2. y gene codes for permease, which increases selves in a cell because their products are
permeability of the cell to â-galactosides. required for normal cellular activities
3. a gene encodes a transacetylase * Ex: genes for glycolysis , regulator genes
Hence, all the three gene products in lac operon (2) Non-constitutive Genes (Luxury / Smart
are required for metabolism of lactose. Genes)
* In most other operons as well, the genes present * The genes are not always expressing themselves
in the operon are needed together to function in in a cell.
the same or related metabolic pathway. * They are switched on or off according to the
* Lactose is the substrate for the enzyme beta- requirement of cellular activities
galactosidase and it regulates switching on and * lactose system in Escherichia coli (structural
off of the operon. Hence, it is termed as genes)
inducer(Lactose). If lactose absent , Switch Off
* In the absence of a preferred carbon source such * If lactose present , Switch On
as glucose, * Repressor protein (i-gene) + Operator gene =
* if lactose is provided in the growth medium of the Switch Off
bacteria, * Repressor protein (i-gene) + Inducer (Lactose/
* the lactose is transported into the cells Allolactose) = Switch On
* through the action of permease Human Genome project (HGP) :
(Remember, a very low level of expression of lac * HGP is a mega project
operon has to be present in the cell all the time, * Started by U.S. Department of Energy and
otherwise lactose cannot enter the cells). National Institute of Health
* Lactose then induces the operon in the following * For sequencing human genome
manner. * In 1990
* The repressor of the operon is synthesised (all- * Welcome Trust (UK) joined project as a major
the-time – constitutively) from i gene. partner
* Repressor protein binds to the operator region of * Later on Japan, France, Germany, China & some
the operon & prevents RNA polymerase from other countries also joined it.
transcribing operon.
* In presence of an inducer, like lactose or * Expected scheduled completion – 2005
allolactose, * But completed in 2003 , in 13 years
* repressor is inactivated by interaction with * Till 2003 , all 23 (21 A + X +Y) chromosomes
inducer except chromosome no. 1
* This allows RNA polymerase access to the * Last chromosome to be sequenced is chromo-
promoter and transcription proceeds some 1 (May 2006)
* Essentially, regulation of lac operon can also be * Why called Mega project ???
visualised as regulation of enzyme synthesis by 1. Human genome is said to have approximately
its substrate. 3 x 109 bp,
* Glucose or galactose cannot act as inducers for * and if the cost of sequencing required is US $ 3
lac operon per bp (the estimated cost in the beginning),
* Regulation of lac operon by repressor is referred * total estimated cost of the project would be
to as negative regulation. approximately 9 billion US dollars
* Lac operon is under control of positive regulation
as well, but it is beyond the scope of discussion at 2. If obtained sequences were to be stored in
this level. typed form in books,
* and if each page of the book contained 1000
letters
* and each book contained 1000 pages,
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Molecular Basis of Inheritance 25
* then 3300 such books would be required to store * The cloning resulted into amplification of each
the information of DNA sequence from a single piece of DNA fragment so that it subsequently
human cell. could be sequenced with ease.
3. The enormous amount of data expected to be * Commonly used hosts- Bacteria & Yeast
generated also necessitated the use of high speed * Vectors-
computational devices for data storage and 1. BAC (bacterial artificial chromosomes)
retrieval, and analysis. 2. YAC (yeast artificial chromosomes
* HGP was closely associated with the rapid
development of a new area in biology called as 4. Fragments were sequenced using Auto-
Bioinformatics (use of high speed computational mated DNA sequencers:-
devices for data storage and retrieval, and * that worked on the principle of a method devel-
analysis) oped by Frederick Sanger.
Goals of HGP : * Sanger is also credited for developing method for
(i) Identify all approximately 20,000-25,000 Genes in determination of amino acid sequences in pro-
human DNA teins.
(ii) Determine the Sequences of the 3 billion chemical 5. These sequences were then arranged based on
base pairs that make up human DNA; some overlapping regions present in them.
(iiii) Store this information in databases; * This required generation of overlapping frag-
(iv) Improve tools for data analysis; ments for sequencing.
(v) Transfer related technologies to other sectors, * Alignment of these sequences was humanly not
such as industries; possible.
(vi) Address the ethical, legal, and social issues * Therefore, Specialised Computer Based Pro-
(ELSI) that may arise from the project. grams were developed . These sequences were
Many non-human model organisms have also subsequently annotated and were assigned to
been sequenced - each chromosome
1.Bacteria
2.Yeast
3.Caenorhabditis elegans (a free living non-patho-
genic nematode)
4.Drosophila (the fruit fly)
5.Plants (rice and Arabidopsis)
Methodology :
* Two approaches have been recognized for
analysing human genome
(i) ESTs or Expressed Sequence Tags :
* To identify all the genes that are expressed as
RNA.
(ii) Sequence annotation :
* Sequencing both coding and noncoding regions of
whole genome and later assigning different
segions in the sequence with functions * Another challenging task was assigning the
* Blind approach genetic and physical maps on the genome.
* For sequencing, * This was generated using information on poly-
1.Total DNA from a cell is isolated morphism of restriction endonuclease recognition
2.Then converted into random fragments of sites & some repetitive DNA sequences known
relatively smaller sizes (recall DNA is a very long as Microsatellites
polymer, and there are technical limitations in * One of applications of polymorphism in repetitive
sequencing very long pieces of DNA) DNA sequences - DNA fingerprinting).
3. Then , Cloned in suitable host using specialised
vectors.
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Molecular Basis of Inheritance 26
Salient features of Human Genome : * DNA fingerprinting involves identifying differ-
(i) The human genome contains 3164.7 million ences in some specific regions in DNA sequence
nucleotide bases.(3.1647 Billion) called as repetitive DNA, because in these
(ii) The average gene consists of 3000 bases, sequences, a small stretch of DNA is repeated
* but sizes vary greatly many times.
* largest known human gene being dystrophin at * These repetitive DNA are separated from bulk
2.4 million bases genomic DNA as different peaks during density
* Smallest human gene-TDF (Testis Determining gradient centrifugation.
Factor on Y-chromosome Y 14 bases) * Bulk DNA forms a major peak and the other
(iii) The total number of genes is estimated at small peaks are referred to as satellite DNA.
30,000– much lower than previous estimates of * Depending on base composition (A : T rich or
80,000 to 1,40,000 genes. Almost all (99.9 per G:C rich), length of segment & number of
cent) nucleotide bases are exactly the same in all repetitive units , satellite DNA is classified into
people. many categories, such as micro-satellites, mini-
(iv) The functions are unknown for over 50 % of satellites etc.
discovered genes * These sequences normally don¡¦t code for any
(v) < 2 % of genome codes for proteins. proteins, but they form a large portion of human
(vi) Repeated sequences make up very large portion genome.
of the human genome. * These sequence show high degree of polymor-
(vii) Repetitive sequences are stretches of DNA phism & form basis of DNA fingerprinting.
sequences that are repeated many times, some- * Since DNA from every tissue (such as blood,
times hundred to thousand times. hair -follicle, skin, bone, saliva, sperm etc.), from
* They are thought to have no direct coding an individual show the same degree of polymor-
functions, phism, they become very useful identification tool
* but they shed light on chromosome structure, in forensic applications.
dynamics and evolution. * Further, as polymorphisms are inheritable from
(viii) Chromosome 1 has most genes (2968), & the Y parents to children, DNA fingerprinting is the
has the fewest (231) basis of paternity testing, in case of disputes.
(ix) Scientists have identified about 1.4 million
locations where single- base DNA differences * Polymorphism in DNA sequence is the basis of
(SNPs – Single Nucleotide Polymorphism, genetic mapping of human genome as well as of
pronounced as “snips”) occur in humans DNA fingerprinting
* This information helps in processes of finding * Polymorphism (variation at genetic level) arises
chromosomal locations for disease-associated due to mutations.
sequences
* and Tracing human history. * New mutations may arise in an individual either
* DNAFingerprinting (DNA Profiling/ Typing/ in somatic cells or in the germ cells (cells that
Imprinting) Historical Aspect : generate gametes in sexually reproducing
* Sir Alec jeffreys (1984) developed the DNA organisms).
fingerprinting technique. * If a germ cell mutation does not seriously impair
* Dr.K.kashyap and Dr. Lalji Singh started the individual's ability to have offspring who can
fingerprinting technology in India. transmit the mutation, it can spread to the other
members of population (through sexual reproduc-
* What is DNA-fingerprinting : tion).
* It is a technique to identify a person on the basis * Allelic sequence variation has traditionally been
of his//her DNA specificity described as a DNA polymorphism if more than
* Technique of determining nucleotide sequences in one variant (allele) at a locus occurs in human
certain DNA segments , which is unique to an population with a frequency greater than 0.01.
individual. * In simple terms, if an inheritable mutation is
observed in a population at high frequency, it is
referred to as DNA polymorphism.
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Molecular Basis of Inheritance 27
* The probability of such variation to be observed * Consequently, DNA from a single cell is enough
in non- coding DNA sequence would be higher to perform DNA fingerprinting analysis.
as mutations in these sequences may not have * In addition to application in forensic science, it
any immediate effect/impact in an individual¡¦s has much wider application, such as in determin-
reproductive ability. ing population and genetic diversities.
* These mutations keep on accumulating genera- * Currently, many different probes are used to
tion after generation, and form one of the basis of generate DNA fingerprints.
variability/polymorphism. Technique of DNA fingerprinting :
* There is a variety of different types of polymor- (i) The DNA is isolated from the nuclei of white
phisms ranging from single nucleotide change to blood cells or spermatozoa or the hair follicle
very large scale changes. cells.
* For evolution and speciation, such polymorphisms (ii) Restriction endonuclease enzyme performs
play very important role. digestion of DNA molecules. The former cuts
* The technique of DNA Fingerprinting was DNA in to fragments. The fragments of DNA
initially developed by Alec Jeffreys. also contain the VNTRs.
* He used a Satellite DNA as probe that shows (iii) Gel electrophoresis is used to separate these
very high degree of polymorphism. fragments according to their size
* It was called as Variable Number of Tandem (iv) VNTRs are multiplied through PCR technique.
Repeats (VNTR 11-60 bp long)/Minisatellites The VNTRs are treated with alkaline chemicals
* The technique, as used earlier, involved Southern to split them into single stranded DNAs.
blot hybridisation using radiolabelled VNTR as a (v) SS DNA fragments of the gel are shifted onto a
probe. nylon paper/nitrocellulose membrane by Southern
(i) isolation of DNA blotting technique.
(ii) digestion of DNA by restriction endonucleases (vi) Radioactive SS-DNA-probes are used for
(iii) separation of DNA fragments by electrophoresis hybridization with VNTR on the nylon mem-
(iv) transferring (blotting) of separated DNA frag- brane.
ments to synthetic membranes, such as nitrocel- (vii) Now the nylon membrane is exposed at the front
lulose or nylon of X-ray film and mark the places where the
(v) hybridisation using labelled VNTR probe radioactive DNA probes have bound to the DNA
(vi) detection of hybridised DNA fragments by fragments. These places are marked as dark
autoradiography. bands when X-ray film is developed. This is
* The VNTR belongs to a class of satellite DNA known as Autoradiography.
eferred to as mini-satellite. (viii) The dark bands on X-ray film show the DNA
* A small DNA sequence is arranged tandemly in fingerprints.
many copy numbers. Applications of DNA Fingerprinting :
* The copy number varies from chromosome to (i) It is very useful in the detection of crime and
chromosome in an individual. illegal pursuits.
* The numbers of repeat show very high degree of (ii) Paternity maternity disputes can be solved by
polymorphism. DNA fingerprinting.
* As a result the size of VNTR varies in size from (iii) It can be used to study the breeding patterns of
0.1 to 20 kb. animals facing the danger of extinction.
* Consequently, after hybridisation with VNTR (iv) It provides information about relationship of man
probe, the autoradiogram gives many bands of with apes.
differing sizes. (v) It determining population and genetic diversities.
* These bands give a characteristic pattern for an
individual DNA
* It differs from individual to individual in a popula-
tion except in the case of monozygotic (identical)
twins.
* The sensitivity of the technique has been in-
creased by use of polymerase chain reaction.
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Molecular Basis of Inheritance 28
Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005