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Molecular
biology
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N-Base Nucleoside Nucleotide
Adenine Adenosine AMP,ADP,ATP
Guanine Guanosine GMP,GDP,GTP
Cytosine Cytidine CMP,CDP,CTP
Uracil Uridine UMP, UDP,UTP
Thymine Thymidine TMP,TDP,TTP
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Cytoplasm
4
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OTC deficiency Orotic aciduria
• Increased Orotic acid in blood & • Increased Orotic acid in blood &
urine urine
• No Megaloblastic anemia • Megaloblastic anemia
• Hyperammonemia • No hyperammonemia
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Q. A 55-year-old woman is diagnosed with liver cancer and is put on
treatment using 5-FU. Which of the following enzyme reaction is
affected by this therapy?
a) Purine nucleoside phosphorylase
b)Thymidylate synthase
c) PRPP synthase
d)Adenosine deaminase
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Rasburicase in tumor lysis syndrome
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8
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Molecule Absorption of Due to • Below 400 nm is
light at conjugated UV light
double bonds • 400-800 nm is
in the rings Visible spectrum
DNA 260 nm (UV) Nitrogenous • The absorption
bases band of
Proteins 280 nm (UV) Aromatic porphyrins is
amino acids known as Soret
(maximum by band)
tryptophan)
NAD/NADP 340 nm (UV) Adenine
Porphyrins 400 nm Pyrrole rings
(Visible)
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DNA
Replication
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ENZYMES:
1. Helicase
2. Topoisomerase
3. SSBs
4. Primase
5. DNA Polymerase III
6. DNA Polymerase I
7. Ligase
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Q. A 42 year old male presents with premature aging and is
diagnosed with Werner syndrome. The most likely mechanism
is:
a) Increased telomere length
b) Increased advanced glycation end products
c) Decreased lipid peroxidation
d) Short telomere with damaged DNA and loss of Helicase
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So, DNA Polymerase I has :
• 5’ →3’ exonuclease (primer removal)
• 5’→3’ polymerase (synthesis)
• 3’→5’ exonuclease (proofreading)
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Single strand break repair
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Coding & Non-Coding RNAs
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RNA Polymerases
RNA Effect of α-
Type Location
transcribed amanitin
18, 5.8, 28
I Nucleolus Insensitive
rRNA
mRNA, Highly
II Nucleoplasm
miRNA, snRNA sensitive
tRNA, 5s rRNA
III Nucleoplasm Less sensitive
, U6 snRNA
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Q. A scientist is using an in-vitro transcription and
translation system to study a gene function. When he used a
base sequence GATCTAC as DNA template during his
experiment. What will be the base sequence of his RNA
product?
a. CTAGATG b. GTAGATC
c. CUAGAUG d. GUAGAUC
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RIBOZYMES
rRNA Ribozyme
having Ribonuclease P
23 s rRNA role in (cleaves the
28 s rRNA SPLICING
(Prokaryotes tRNA)
(Eukaryotes) (removal of
Introns )
TELOMERASE is not a RIBOZYME
RNase H is not a RIBOZYME
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OPERON MODEL
31
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Lac operon Tryptophan operon
Inducible (Lactose is the inducer) Repressible (Trp is co-repressor)
Catabolic system Anabolic system
Usually off Usually on (inactive when high trp
is present)
3 structural genes- Z, Y & A 5 structural genes (A,B,C,D & E)
Repressor protein is active Repressor protein is inactive
Turned on when glucose cAMP is not necessary here
decreased, cAMP increased &
Lactose present
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CODON AMINO ACID
AAA Lysine
UUU Phenyl-alanine
CCC Proline
GGG Glycine
AUA Isoleucine
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Translation
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Factors in translation in prokaryotes &
eukaryotes
Prokaryotes Eukaryotes
Initiation IF 1,2 , 3 eIF 1, 2, 3, 4F
Elongation EF- Tu, Ts, G eEF 1α,1β ,1γ ,
eEF 2
Termination RF 1, 2, 3 eRF
Translocation – EF-G & eEf-2
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Blotting Sample to Probe KNOWN AS
be (labelled)
detected
Southern DNA DNA DNA-DNA
Hybridizati
on
Northern RNA DNA DNA-RNA
Hybridizati
on
Western Protein Antibody
(antigen)
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PCR steps
1. Denaturation
2. Annealing/ hybridization of primer to the parent strand
3. Extension/ elongation/amplification/polymerization
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Methylation has role in :
DNA :
• DNA Replication & Transcription
• Mismatch repair
• X-chromosome inactivation
• Chromatin remodelling or chromosome segregation (heterochromatin
formation)
RNA:
• Splicing, Alternate splicing and regulation of splicing
• 7-methyl guanosine cap in MRNA
• Gene regulation or regulation of gene expression - Gene silencing & Genome
imprinting (Epigenetics)
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Methylation has role in :
Others:
• Embryonic development
• Maintenance of genome stability
• Carcinogenesis
• Atherosclerosis,
• ageing ,
• exercise
• B-cell differentiation
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DNA Methylation found in :
1. Prokaryotes
2. Eukaryotes: some fungi, plants, vertebrates
• But not found in single celled- Eukaryotes
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4 methods of gene/DNA sequencing
1. Sanger’s or chain termination method – dideoxy nucleotide method
2. Maxam & gilbert – cut dsDNA at specific positions
3. Pyro sequencing method- sequencing by synthesis , addition of
deoxynucleotide is accompanied by release of flash light
4. NGS – for massive production – fast & cheap
• Sanger's method is used for DNA sequencing using
didexoynucleotides
• Sanger reagent (1-fluoro 2,4 dinitrobenzene -FDNB) is a reagent used
for protein sequencing.
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• Edman reagent- Phenyl Iso Thio Cyanate – PITC
• Sanger’s reagent – (1-Fluoro 2,4 Di Nitro Benzene (FDNB) or Di Nitro
Fluoro Benzene) is a reagent used for protein sequencing
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Gene transfer methods:
1. Transformation – naked DNA from env. or dead bacteria to a living
bacteria – transformers or robots
2. Transfection – infection – injection – transfer of DNA into
eukaryotic cell deliberately
3. Transduction – duck & virus – transfer of DNA & RNA through
virus, called bacteriophage
4. Conjugation – by cell to cell contact , formation of pilus in donor
bacteria to recepient bacteria
5. Lipofection – if liposome used (liposome can carry DNA or drugs)
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CRISPR – Cas 9 system
• Causes double strand break
• CRISPR- Clustered Regularly Interspersed Short Pallindromic Repeats
• Cas-9 → CRISPR associated endonuclease
• It is an immune system in bacteria against bacteriophages
• Transmitted to progeny
• So many generations protected from viruses
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