Genetics😊

Wobble Hypothesis

• Wobble bases
• 3rd base of codon on mRNA
• 1st base of anticodon on tRNA (5' → 3' direction)
• These positions = wobble positions

• Wobble hypothesis
• Binding between these bases allows imprecision
• One anticodon base can bind with various codon bases
• Wobble bases pair via non-Watson Crick base pairing
• Watson Crick pairing: A–T, G–C
• Wobble pairing occurs in RNA only
• Does not follow Watson-Crick rule

Table:

Codons / AA possible

• 4 rise to number of BPs

Point Mutations

• Detection
• PCR → RFLP
• PCR → Sequencing.

• Example:

UCA (Serine) to	Mutation
UCU (serine)	Silent
CCA (proline)	Missense
UAA (stop)	Non sense

Frameshift Mutation

• DMD, Tay-Sach’s

ㅤ	DMD	BMD
Mutation	Frameshift / Non-sense	In-frame mutation
Protein	Truncated dystrophin protein	Dystrophin protein quality affected
BMD	More severe	Less severe

• This frame is non sense

Genetic Code

1. 16

2. 64

3. 128

4. 256
ANS
• 256
• 4 raise to (no. nucleotides)
• ie, 4 x 4 x 4 x 4

BLUE-WHITE ASSAY

• Principle
• Insertional inactivation of lacZα gene

Complementation

• Host
• Mutated E. coli → defective Lacα gene

• Vector
• Contains intact Lacα gene region
• site for DNA insertion

• Insertion
• DNA of interest ligated into LacZα region → disrupts Lacα in Vector

• Mechanism
• Non-recombinant plasmid → Intact Lacα → α-complementation
• functional β-galactosidase → cleaves X-gal →
• Blue colony
• Recombinant plasmid present → Disrupted Lacα → no complementation
• inactive β-galactosidase → X-gal not cleaved →
• White colony

LACTOSE OPERON IN E. COLI

• Operon model = operating unit

• Function: switch on/off genes

• Normal state
• Glucose present
• Lac Z, Y, A kept switched off to save energy
• No need to use lactose
• No need to make lactose-utilizing proteins

Mechanism of switching off Z, Y, A

• Operon → OP One
• Operator, Promoter, One
• (in that order)

• Lac I gene = inhibitory

• Housekeeping / constitutive (always active)

• Forms repressor tetramer (protein) continuously

• Repressor tetramer binds operator site

• Promoter upstream to operator

• RNA Polymerase bound to promoter

• Repressor tetramer binding to operator inhibits RNA Polymerase and transcription

Mechanism of switching on Z, Y, A

• Removes Repressor Tetramer
• Occurs if:
• ↓ Glucose in environment
• ↓ Glucose → ↑ cAMP → Form cAMP-CAP complex
• bind to CAP site
• CAP → Positive promoter
• Its presence is required for gene expression
• Presence of lactose in environment

Genetics

Pyrimidine and Purine Ring Synthesis

• Nitrogenous bases are fundamental to DNA and RNA.

Mnemonic:

• Glycine is necessary for Purine
• not Pyrimidine, ring formation.

Disorders of Purine Synthesis

Lesch-Nyhan Syndrome

• Cladribine, Pentostatin
• ⛔ ADA
• NOTE: SCID
• ADA deficiency

• Lesch Neyhan syndrome
• ⛔ HGPRT

• Defect: HGPRTase → Purine salvage pathway blocked.
• Pathway:
• Hypoxanthine ⇏ IMP
• Guanine ⇏ GMP

• PRPP accumulates → activates De novo purine synthesis → ↑ Purine production.

• Excess purines → undergo catabolism → ↑ Uric acid.

Kelley-Seegmiller Syndrome

• Defect:
• Partial defect of HGPRTase

Disorders of Purine Catabolism

• End product of purine catabolism: uric acid

Enzyme deficiency vs Features

DNA Structure and Organisation

Nucleoside:

• Nitrogenous base + ribose/deoxyribose sugar.

• Linked by a thermolabile Beta-N-Glycosidic linkage.

Nucleotide:

• Nucleoside + phosphate group at 5' hydroxyl group.

• Linked by a Phospho ester linkage.

• DNA:
• dTMP only in DNA

• RNA:
• UMP only in RNA

Polynucleotide Chain:

• Nucleotides link via 3' 5' phosphodiester linkages.

• Synthesis is in the 5' to 3' direction.

DNA and RNA Polymerases:

• Synthesize → 5' to 3' direction.

• Read template → 3' to 5' direction.

• Require deoxynucleotide triphosphates and Mg or Mn.

• Most DNA polymerases have:
• 5'->3' Polymerase activity.
• 3'->5' exonuclease activity (proofreading).

• DNA Polymerase I
• Has 5'->3' exonuclease activity.

• Klenow fragment:
• Product of DNA Polymerase I without 5'->3' exonuclease activity.
• Used in PCR.

Watson and Crick DNA Model

• Chargaff’s Rule: A + G = C + T (Purines = Pyrimidines).

• Breaking bonds
• Hydrogen bond → Unwinding → 2 single strands
• β N Glycosidic linkage → Base excision error
• 3’ - 5’ phosphodiester linkage on both strands → DsDNA break

• Describes a double helix.

• Two strands are complementary and anti-parallel.

• Backbone:
• 3' 5' phosphodiester linkages + deoxyribose.

• "Steps":
• Nitrogenous bases linked by hydrogen bonds.
• A-T:
• 2 hydrogen bonds.
• G-C:
• 3 hydrogen bonds.
• Mnemonic: C-G bonds are like Crazy Glue.

Types of DNA

B Type DNA

• Most common, physiological form

• Right-handed helix

• 10 base pairs per turn

• Rise per base pair: 3.4 Å

• One full turn: 34 Å

• Width: 20 Å

• One major groove + one minor groove per turn

A Type DNA

• Forms after DNA dehydration during extraction

• Seen in DNA-RNA hybrids or RNA-RNA hybrids

• Right-handed helix

• 11 base pairs per turn

• Grooves: same dimension

Z Type DNA

• Seen in GC-rich sequences

• Only left-handed helix

• 12 base pairs per turn

Chromosomes

• Chromosomes are DNA condensed with histones.
• Histones are basic, positively charged proteins.
• Positive charge (from lysine and arginine).

What does the division of a chromosome perpendicular to the normal axis of division lead to?

1. Ring chromosome

2. Isochromosome

3. Acrocentric chromosome

4. Subtelocentric chromosome
ANS

Histone Types:

• Dimers of H2A, H2B, H3, H4
• form an octamer core for nucleosomes.
• String of bead appearance of nucleosome

• H1 is a linker histone, absent in nucleosome
• Several nucleosomes connected by linker fragment

• 10 nm fibril = one nucleosome fibril (width 10 nm)

• First level of compactness: 10 nm fibril folds



• Nucleosome

• Second level compactness: 30 nm fibril



• Refolding like β pleated sheets

Chromosome Scaffold

• 30 nm fibrils form loops on chromosome scaffold

• Chromosome scaffold = tubular proteins in center

• Multiple condensations → highly condensed chromosome
• Contains centromere, short arm, long arm

Functions of Chromosomes

• Replication

• Transcription

For these, chromosomes undergo:

1. 1st → Uncondensation

2. f/b → Unbinding

3. then replication or transcription

Types of Chromatin

• Euchromatin:
• Transcriptionally active.
• Uncondensed
• Less densely stained with Giemsa stain.

• Heterochromatin:
• Transcriptionally inactive
• Condensed
• More densely stained
• Constitutive:
• In centromeres and telomeres
• Facultative:
• Example is the Barr body

Constitutive heterochromatin:

• Found in Centromere and Telomeric ends.

• Centromere:
• A region subjected to repeated breakage during anaphase.
• Breakage is due to mitotic spindle contraction and longitudinal breaking.
• Provided with non-coding sequences to prevent detrimental gene duplication or deletion.
• Marked as a dark dot.

• Telomeric ends:
• Undergo progressive shortening with each cell division or replication.
• If these ends contained coding sequences, gene deletion would occur.

Facultative heterochromatin:

• Lyonisation of X chromosome
• Random inactivation of one X chromosome
• In every female somatic cell:
• One X chromosome is transcriptionally active.
• The other X chromosome is transcriptionally inactive.
• NOTE: In germ cells, both X chromosomes are active.

• Barr body







• Used for sex determination

• Inactivation / Lyonisation of X chromosome

• Mechanism: DNA Methylation

• Gene: XIST gene

• Time: 6th day of gestation

• Facultative Heterochromatin
• “Heterochromatin” → highly condensed and stains densely
• "facultative" → because random.

• Appear as Davidson bodies in WBCs
• Female with absent Barr body = Turner
• Normal males: 1-1 = 0 Barr body
• Normal females: 2-1 = 1 Barr body
• Turner syndrome females: 1-1=0

Question Discussion: Barr Body

• A. Euchromatin

• B. Constitutive heterochromatin

• C. Facultative heterochromatin

• D. Hypersensitive heterochromatin
Explanation:
• C
• Barr body
• Inactivated X chromosome.
• Seen in females (used for sex determination).
• Example of facultative heterochromatin

DNA Replication

1. Leading Strand Synthesis:
• Template: 3’ → 5’ strand.
• Process: RNA primase adds primer → DNA polymerase III (prokaryotes) or ε (eukaryotes) synthesizes continuously → DNA polymerase I removes primer.

2. Lagging Strand Synthesis:
• Template: 5’ → 3’ strand.
• Process: Multiple RNA primers → DNA polymerase III synthesizes Okazaki fragments → DNA polymerase I removes primers → DNA ligase seals gaps.

DNA Polymerases

DNA Proofreading:

• 3’ → 5’ exonuclease ability

• Performed by
• DNAP I, II, III
• ε, δ

Requirements of DNA Polymerase

• Template strand
• Must be 3' → 5' direction.
• New strand synthesized complementary and antiparallel (5' → 3').

• Primer
• Essential to initiate elongation.

• dNTPs (deoxynucleotide triphosphates)
• Incorporated as monophosphate nucleotides in the chain.
• Triphosphates provide energy.
• Energy from breaking last two phosphate bonds.
• Drives 3' → 5' phosphodiester bond formation.

• Cofactors
• Requires Magnesium or Manganese ions.

• Buffer system
• Maintains optimal pH for activity.

Question Discussion:

• Q. DNA Polymerase requires all except?
• A. RNA Primer
• B. 3' to 5' strand to act as a template
• C. dNTP
• D. 5' to 3' strand act as a template

• Explanation:
• The template strand for DNA Polymerase must be 3' to 5'.

Properties of DNA Polymerases

• Proofreading
• Requires a 3' to 5' exonuclease activity.

• repair activity.
• If a defect is found, they remove defective strands from the other end.

• All DNA Polymerases have:
• 5' → 3' Polymerase activity.
• 3' → 5' exonuclease activity.

• Exception:
• DNA Polymerase 1 has both
• 5' → 3' Polymerase activity.
• 3' → 5' exonuclease activity.
• 5' → 3' exonuclease activity.

Klenow Fragment

Definition

• A fragment of Taq DNA Polymerase I (E. coli)
• Retains:
• 5' → 3' polymerase activity
• 3' → 5' exonuclease (proofreading) activity
• 5' → 3' exonuclease activity.

• Treatment with Subtilisin
• removes the 5' → 3' exonuclease subunit
• It disrupts precise DNA work.
• Klenow fragment avoids this

Uses

1. Used in PCR
• Convert ssDNA → dsDNA
• Used before discovery of thermostable Taq polymerase

2. Blunt-End Formation
• Removes 3' overhangs
• via 3' → 5' exonuclease
• Fills 5' overhangs
• via 5' → 3' polymerase
• Converts sticky ends
• blunt ends (for cloning).

3. Labelling DNA Probes
• Incorporates radioactive/modified nucleotides for hybridization assays.

Exo-Klenow Fragment

• Lacks both exonuclease activities,
• retain only the 5’ → 3’ polymerase function.

• Applications:
• Used in microarrays
• to make fluorescent probes.
• Performs dA/dT tailing
• adding adenine or thymine residues to ends of DNA

Question Discussion: Klenow Fragment

• Q. All of the following are true about Klenow fragment except:
• A. It is a product of DNA polymerase I.
• B. It has 5'3' polymerase activity.
• C. It has 5'3' exonuclease activity.
• D. It has 3'5' exonuclease activity.

Difference between Replication and Transcription

Replication

• The parent double-stranded DNA unwinds.

• Each of the two strands acts as a template.

• Two new strands are synthesized.

Transcription

• Only the transcription unit unwinds.

• It is a chromosomal segment coding for protein or RNA.

• Aim: formation of a single-stranded RNA.

• Template vs Coding Strand
• Template strand
• Only the 3'→5' strand
• Serves as template for RNA synthesis.
• Synthesizes new RNA in 5'→3' direction.
• Coding strand
• 5'→3' strand
• Does not participate.
• Same polarity and sequence as RNA.
• Difference: T replaced by U in RNA.

Steps to determine RNA sequence

• When template strand sequence is provided:
• Step 1: Check polarity.
• Template strand should be 3' → 5'.
• If polarity is not mentioned, assume 5' → 3'
• and reverse it to 3' → 5'.
• Step 2: Synthesize a complementary sequence.
• This will be the RNA product in the 5'3' direction.

• When coding strand sequence is provided:
• Maintain the same polarity and sequence.
• Replace all Thymine (T) with Uracil (U).
• The gene sequence is the coding strand sequence.

Question Discussion: RNA Sequence Determination

• Q. The strand of DNA used as the template for transcription has the base sequence GATCTAC. What is the base sequence of RNA product?
• A. CTAGATG
• B. GTAGATC
• C. GAUCUAC
• D. GUAGAUC
Explanation:
• Given template: GATCTAC.
• Assume it's 5'GATCTAC3'.
• Actual template must be 3'CATCTAG5'.
• Complementary RNA is 5'GUAGAUC3'.

Genes and Gene Expression

• A gene is a chromosome segment coding for protein or RNA.
• Contain
• Exons (coding)
• Make 2% of total genome
• Introns
• They are also non coding

• Transcription:
• Initial product is Heteronuclear RNA (hnRNA) or Primary Transcript

Transcription

• DNA → RNA

• Strand transcribed = Template/Minus/Antisense strand

• Other strand = Coding/Plus/Sense strand
• Not involved in transcription
• Same sequence as RNA (T → U)
• Mnemonic: CoPy Sense (Coding, Plus, Sense) → (Replace T → U)

RNA Polymerase (RNAP)

1. Prokaryotic RNAP (multisubunit)

Subunit	Function
β subunit	Catalytic, binds Mg²⁺
σ subunit	Binds promoter

2. Eukaryotic RNAP

• 2 → 3 → 1

Post-transcriptional modifications:

Types of Nucleases

• Endonucleases → cleave within RNA (site-specific)

• Exonucleases → degrade RNA from the ends
• 5′→3′ exonucleases:
• act from 5′ end
• 3′→5′ exonucleases:
• act from 3′ end

Reason for Cap and Tail

• Nucleus is rich in exonucleases

• To protect endogenous RNA from degradation, modifications are added:
• 5′ cap at 5′ end
• Poly-A tail at 3′ end

5’ Capping

• 7-Methyl guanosine cap added at 5’ end.

• Enzymes:
• Guanyl transferase – Nucleus
• Methyl transferase – Cytoplasm

• Methyl group donor:
• SAM (S-adenosyl methionine).

• Functions:
• Stabilizes mRNA (blocks 5’→3’ exonuclease).
• Helps initiation of translation.
• Binding of mRNA to 43S pre-initiation complex.

Splicing:

1. Introns removed
• By endonucleases/Spliceosomes
1. SnRNP/Snurps
• SnRNA (Ribozyme) : Rich in uracil +
• 60 proteins : RR, SR motif +
• SnrnP → SnRNA + Protein + Primary transcript
2. Primary transcript

2. Exons joined.

3’ Poly A Tailing

• Poly-A tail added.

• Addition of 40–200 adenosine residues at 3’ end.

• Enzyme: Polyadenylate polymerase.

• Functions:
• Stabilizes mRNA (blocks 3’→5’ exonuclease).
• Facilitates exit of mRNA from nucleus → cytoplasm.
• Recruitment of 40S ribosome.
• Poly A tail is translated into the polylysine tail
• AAA is the codon for lysine amino acid.

Exception to one gene one protein theory

1. Differential RNA processing / RNA editing

• Occurs in few cells

• Exception to one gene one protein theory
• one gene gives more than 1 protein

Single ApoB gene give rise to

• ApoB100 in Liver

• ApoB48 in Intestine
• A chemical modification, C to U conversion
• gives rise to stop codon in intestinal mRNA
• causes shorter protein ApoB48 synthesis

2. Alternate splicing

• Also an exception to one gene one protein theory

• Gives rise different mature mRNAs from single type of transcript

Exceptions in Post-transcriptional Modifications

• Histone mRNA is an exception.
• Histone genes resemble prokaryotic genes.
• No introns → No splicing.
• No poly-A tail addition.
• 3' end protected by stem-loop structure.

Translation

Functional mRNA

tRNA (Transfer RNA)

Structure

• 75–90 nucleotides;

• 2° Structure: Cloverleaf shape

• 3° Structure: Inverted L shape

Four Arms:

Gene Expression

• Gene expression = A gene giving rise to RNA or protein.

Steps of Translation

• Ribosome reads mRNA from 5' → 3' direction.

Key differences

Total:

• 4 ATPs / per added amino acid
• 2 ATP → charging
• 2 GTP → elongation

No. of ATPs	Function
2 ATP	tRNA charging
1 GTP	Entry of aminoacyl-tRNA into A site
1 GTP	Translocation

• (Do not count → Additional costs)
• Initiation: 1 GTP
• Termination: 1 GTP

1. tRNA Charging (Aminoacylation)

• Aminoacyl-tRNA Synthetase
• Charges tRNA with correct AA
• uses ATP
• 1 unique enzyme / amino acid
• Amino acid + tRNA → Aminoacyl-tRNA
• Only point where proof reading of translation happens

2. Initiation

• Prokaryotes
• Ribosome: 70S (50S + 30S)
• Ribosome binds to Shine-Dalgarno sequence.
• Recognized by 16S rRNA
• Initiator tRNA delivers fMet to the P site.

• Eukaryotes
• Ribosome: 80S (60S + 40S).
• Initiator tRNA: Met-tRNAi^Met
• No formylation unlike prokaryotes
• Translation starts once Start Codon (AUG codon) is identified
• Kozak sequence
• AUG codes for
• Methionine (in eukaryotes)
• N-formylmethionine (fMet) (in prokaryotes)
• Sequence
• tRNA → Ternary complex + 40S → 43S → (+ mRNA) → 48S →(+ 60S) → 80S initiation complex
• Facilitated by eIF4F complex
• 4E, 4G, 4A
• eIF4E: Binds to 5' cap of mRNA.
• eIF4G: Acts as scaffold protein.
• eIF4A: RNA helicase that unwinds secondary structures in mRNA.
• FEGA (elF4F) = 4E + 4G + 4A

3. Elongation (Common)

• Ribosome translocates 3 nucleotides:
• Mnemonic: APE

Sites	Function
A site	Incoming Aminoacyl-tRNA ↳ (except initiator tRNA which binds P site).
P site	Holds growing Peptide
E site	Exit site for empty tRNA

• Peptidyl transferase
• Catalyze Peptide bond formation between amino acids
• Is a Ribozyme:
• 23S rRNA (of 50S) in prokaryotes
• 28S rRNA (of 60S) in eukaryotes
• Does not require ATP or GTP

• Translocation
• Movement of the ribosome along mRNA
• Shifts ribosome by one codon.
• Requires GTP
• Involves elongation factors:
• EF-G in prokaryotes
• eEF-2 in eukaryotes

4. Termination

• Stop codon (UAA, UAG, UGA) reaches A site.
• Mnemonics:
• UAA = U Are Away
• UGA = U Go Away
• UAG = U Are Gone

• Release factors → Signal termination.
• Prokaryotes: RF1/2/3.
• Eukaryotes: eRF1 + eRF3.

• Requires GTP.

Disease	Repeat	ㅤ	Mnemonic / Key Fact
Huntington disease	CAG CHromosome 4	Exon Extrovert → talks/codes	Hunting in a CAGE CAG+E Exon; CAG repeat cage has 4 corners
Fragile X syndrome	CGG	Intron (Introvert → Non coding)	Jiji fragile
Myotonic Dystrophy	CTG Chromosome 19	Intron	myoTonic dysTrophy has a T
Friedreich's Ataxia	GAA Chromosome 9	Intron	Friend → sings → Gana → GAA Awesom heart (HOCM) and big toes (Hallux Valgus)

Mnemonic:

• CAG → CGG → CTG → GAA

• Hunting () fragile () muscle () to Fry ()

Epigenetics

• Transmissible (Hereditary), reversible chemical modification of DNA

• No change in DNA sequence

Functions

• Regulation gene expression

• X chromosome inactivation

• Genomic imprinting

• Aging process

• Mismatch Repair
• But Not in DNA excision and repair

• Chromatin Remodelling

• Cancer → When dysregulated

• RNA Splicing

• Suppression of repetitive element transcription and transposition

Common modifications

• DNA methylation
• Definitions
• CpG site: Cytosine > Guanine (5' → 3' DNA orientation).
• CpG islands: Genomic areas with high CpG site frequency.
• Process
• Cytosine in CpG → 5-methylcytosine.
• Alters gene expression.
• Effect:
• Mutes / Silencing (Decreased gene expression)
• Not involved in
• RNA splicing
• Base pair excision
• DNA replication

• DNA acetylation
• Histone acetylation → Euchromatin formation → Gene activation
• Histone deacetylation → Heterochromatin formation → Gene silencing

• ADP ribosylation, Phosphorylation, Partial proteolysis

Detection of epigenetic modification

• Methylation specific PCR

• DNA chromatin immunoprecipitation (ChIP)

• Bisulphite sequencing

• Methylation sensitive restriction endonuclease digestion

Genomic Imprinting (Gene Silencing)

• Epigenetic process → gene expression depends on parent of origin

• One allele is imprinted/marked during gamete formation
• Not expressed / Silenced

• Expression from either maternal or paternal allele only.

 

• Mechanism: DNA methylation (M for Mute).

• Normal Imprinting: One parental gene is silenced.

Syndrome	Chromosome	Clinical Features
Prader-Willi	Chr 15 → SNORP (P) Paternal deletion,  + Maternal imprinting ("PRADE") OR Maternal disomy of Chr 15	Mental retardation; Chubby, short, obese (↑ ghrelin) During Neonatal period ➔ Hypotonia (Floppy baby) / Difficult to feed (thin upper lip and downturned mouth) / short extremities / almond-shaped eyes. During Childhood Excessive eating (hyperphagia) / Obese and Short / learning difficulties / growth abnormalities / self-injurious behaviour. GORE (GHERLIN) → SNORT (SNORP)
Angelman	Chr 15 → UB3A (A) Maternal deletion,  Paternal imprinting OR Paternal disomy of Chr 15	Happy puppets, microcephaly Mental retardation;  Seizures;  Inappropriate laughter Ubekuttan → Ubequitin ligase 3A

• Prader Willi
• Mnemonic:
• Proud william - he is a prince with blue eyes.. he is just 15... but he is obese and arrogant
• Arrogant → Father → paternal deletion → also maternal disomy
• He is obese → so snores → SNORP gene
• Ghur nnu purath povilla (Gherlin)

• Angelman
• Mnemonic:
• Happy puppey → chirich chirich seizure avum (seizures)
• Angel 5 → Pathinanj (15)
• She is Happy → tell “you be you” → UB3A

---

Beckwith-Wiedemann syndrome.

• BWS = 11p15 imprinting defect → Overgrowth + Macroglossia + Omphalocele + Tumor risk (Wilms, Hepatoblastoma).

• Cause: ↑ copies of imprinted genes (placental overgrowth).

• LGA baby with hemihypertrophy.

• Macroglossia / Protruding tongue

• Omphalocele.

• Organomegaly (liver/spleen).

• Horseshoe kidney

• Double → Ear lobe creases.

• Increased risk of embryonal Tumors:
• Wilms tumor (nephroblastoma)
• Hepatoblastoma
• Neuroblastoma, rhabdomyosarcoma (less common)

---

William Syndrome

• Chromosome 7 micromutation

• Elastin Mutation
• Results in Williams syndrome

• Overfriendly

• HyperCalcemia

• Elfian facies

• Leads to Supravalvular aortic stenosis
• Differential BP
• Right arm BP > Left arm Bp > Lower Limb

---

ㅤ	Williams syndrome	Marfan syndrome
Mutations	Elastin Mutation	Fibrillin Mutation
Leads to	Supravalvular aortic stenosis	Dilatation of aortic root ↳ Rupture → Death

ㅤ	ㅤ
Supravalvular AS	• Vitamin D toxicity • William syndrome
Supravalvular PS	• Noonan syndrome

ㅤ	Seen in
GNAS	• Mccune Albright • Cardiac Myxoma
GNAS 1	• Pseudohypoparathyroid/ Albright Hereditary Osteodystrophy
GNAQ	• Sturge Weber (Sporadic)

MCQs

Q1. Which of the following is NOT associated with post-transcriptional modification?

Q2. A patient with multiple colonic polyps and carcinoma, positive family history of HNPCC, has a defect in which repair mechanism? (NEET PG 2021)

Inhibitors of Nucleotide Synthesis

• Flu (Lefluomide, 5FU) → Pyramidine = Fire (Pyro = Fire)

• My Rib Azad Mercap → Purine

• Water, Fire, Tryme in Metha → Both

• inhibit deoxythymidine monophosphate → [dTMP] in
• humans (methotrexate),
• bacteria (trimethoprim)
• protozoa (pyrimethamine)

CPS Enzymes

Replication Steps

DNA Errors

1. Thymidine dimers repair

2. Base excision repair

3. Mismatch repair

4. Double-strand break repair
ANS
1

Base excision error

• This is the most common DNA error.

• Occurs because the Beta N-glycosidic linkage is weak

• Results in base excision.

• The backbone of the DNA remains intact.

Nucleotide Excision

• It is termed Nucleotide excision repair
• because a segment is excised

• UV light-mediated Pyrimidine dimerisation
• UV light induces dimerisation of adjacent pyrimidines.
• This creates an abnormal "kink" in the DNA.

• Repair
• UV specific endonuclease.
• AKA Excinuclease.
• Creates two nicks adjacent to the kink.
• Excises the entire affected nucleotide segment.

Mismatch error

• During DNA replication
• The parent double-stranded DNA unwinds.
• Each strand is a template for synthesis of new strands.
• Synthesis is done by DNA Polymerases.
• DNA Polymerases can introduce defects.

• Example:
• attaching a Thymine instead of Guanine is on the template.

• Cause Hereditary Non-Polyposis Colon cancer (HNPCC).

Double stranded DNA break

• Ionising radiation

• This is a severe error.

• The backbone of both DNA strands is broken.
• The backbone is made of 3' → 5' phosphodiester linkages.

• Occurs in the presence of Ionizing radiation.
• Example: Gamma rays.

• Ionizing radiation can cause ionisation of water.
• This can break ester and ether linkages in DNA.

Non-Homologous End Joining (NHEJ)

• More common than HDR.

• Mediated by Ku Helicase (a dimer)
• Each unit binds to both ends of the broken DNA.
• Unwinds and approximates the broken ends.
• Continues until base pairing is established.
• Excess strand material is removed.
• DNA ligase joins the ends.

• Disadvantage
• Loss of nucleotide sequences → detrimental mutations
• Results in gene knock-out.

• Causes
• Ataxia Telangiectasia
• Severe Combined Immunodeficiency (SCID)

• Used in CRISPR/Cas9 gene editing Normally
• But Nobel for using Homologous Repair for the same

NOTE: Different Fanconis

ㅤ	ㅤ
Fanconi disease/syndrome	• Proximal tubular reabsorption problem → Type 2 RTA • Glycosuria, aminoaciduria
Fanconi anemia (Not syndrome)	• Pancytopenia + radial ray
Fanconi Bickel syndrome	• Mutation in GLUT-2   • Bickel → Bi → 2 (GLUT 2) Defect in glucose sensing → ↓ insulin release • Postprandial Hyperglycemia. • Fasting Hypoglycemia • Glycogen accumulation disorder

Stranger things characters

• Dustin (Cleido cranial dysplasia)

• Robin (Cock robin position)

• Ray (Radial Ray) Hopper (Holt Oram ASD)

Homology-Directed Repair (HDR).

• Best repair mechanism for double stranded DNA breaks

• Results in gene knock-in

• DNA Polymerase Epsilon or Beta become active

• Causes
• Fanconi's Anaemia
• HBOC (Human Breast and Ovarian Cancer Syndrome):
• BRCA1 mutations predispose to breast and ovarian cancer.
• Inherited in an autosomal dominant pattern.
• Nijmegen breakage syndrome (NBS)
• Werner syndrome
• Rothmund–Thomson syndrome

• A. B-DNA

• B. A-DNA

• C. Z-DNA

• D. C-DNA
Explanation
• B
• Freshly extracted DNA → B-DNA
• On dehydration → A-DNA

• A. Positive charge

• B. Negative charge

• C. Both

• D. No charge
Explanation
• B
• DNA has negatively charged phosphate groups at physiological pH.
• Allows interaction with positively charged histones.

Telomere - TAG

• Structure:
• Chromosome ends with TTAGGG tandem repeats at the 3’ end.

• Hayflick Limit:
• Limits cell division due to telomere shortening
• After 50 cell divisions → DNA replication stops → Leads to aging

• Steps
1. On removal of primer from 3’ end
2. The primer nucleotide sequence is not replicated in the daughter strand
3. End replication error
4. Telomere attrition (Shortening of ends of chromosomes)

Telomerase:

Function:

• "Immortality gene"

• RNA dependent DNA polymerase (RDDP).
• Reverse transcriptase activity.
• Mnemonic: Change 6 DP (RDP → RDNA Polymerase) to become immortal

• Contains intrinsic RNA template

• Adds DNA segments to 3’ end
• Prevents telomere shortening
• No Hayflick limit

Expression:

• Absent in somatic cells

• present in
• skin
• hematopoietic
• germline cells
• cancer cells
• lymphocytes

 

Gene Knock Down, Knock Out, and Knock In

Gene Knock Down

• Down-regulation of gene expression.

• Mediated by siRNA or miRNA

• Seen in DNA interference

• The gene sequence is not edited.

• Expression is suppressed at the translation level.

Gene Knock Out and Knock In

• Two methods of gene editing.

• CRISPR mediates gene editing by causing double-stranded DNA breaks.

• Repair mechanism proceeds in two ways:
1. Non-homologous end joining (NHEJ)
• Gene knock-out:
• Removal of a defective gene.
2. Homologous DNA repair (HDR)
• Gene knock-in:
• Replacement of a defective gene with a normal gene.
• Considered a better mechanism.

CRISPR-Cas9

• A genome editing tool.

• Trick: Cas9 acts as scissors.

• Mnemonic for CRISPR:
• C - Clustered
• R - Regularly
• I - Interspaced
• S - Short
• P - Palindromic
• R - Repeats

• Process:
• Uses Non Homologous → Gene knock-out
• (1) Virus invades the bacterial cell.
• (2) A new spacer is derived from the virus.
• Integrated into the CRISPR sequence (Adaptation).
• (3) Production of CRISPR RNA is formed.
• (4) CRISPR RNA guides molecular machinery.
• Targets and destroys the viral genome.
• Its memory is saved as interspersed spaces (Viral elemental memory).

• Important Information:
• CRISPR-Cas9 is a bacterial defence system against viruses.
• The FELUDA test, based on CRISPR-Cas9, is used for COVID.

miRNA /siRNA (MicroRNA / Small Interference RNA)

• Function:
• Interferes with gene expression at translational level.

• siRNAs: Have specific sequences.

• Mechanism:
• Hybridize with complementary sequences on mRNA
• usually in 3′ UTR
• UTR = Untranslated Regions
• Ribosome encounters dsRNA structure → recognizes as error → halts translation.
• Ribosome recruits endonuclease → nicks mRNA → fragmentation.
• Fragmented mRNA cannot be translated.

• Result:
• Gene expression down-regulation → called gene knockdown

• Q. miRNA is used for?
• A. Gene knock down
• B. Gene knock out
• C. Gene knock in
• D. RNA editing
Explanation:
• miRNA is a precursor for siRNA.
• Both bring about gene knock down.

• Q. miRNA binds to?
• A. 5' UTR
• B. 3' UTR
• C. Gene promotor
• D. Gene exons
ANS
3' UTR

• Q. Following CRISPR-mediated gene nicks, which of the following can result in gene knock in?
• A. Non-homologous end joining.
• B. Homologous DNA repair.
• C. Interference.
• D. Ku helicase mediated repair.
Explanation:
• B
• Interference leads to gene knock-down.
• NHEJ and Ku helicase-mediated repair result in gene knock-out.