MSC Second year : Semester 1: Molecular biology

 Credit 1: Genomics 

1 mark

b) Explain an example of SNP in humans.

- 

SNP Definition: A SNP (Single Nucleotide Polymorphism) is a tiny difference in our genetic code, specifically in a single "letter" of our DNA.

A Single Nucleotide Polymorphism (SNP) refers to a minor genetic variation in the DNA sequence of individuals, involving the substitution of a single nucleotide (genetic "letter") at a specific location within the genome. This variation can influence traits, disease susceptibilities, and responses to treatments, albeit typically in combination with other genetic and environmental factors.

Example SNP: One well-known SNP is called rs1800497. It's like a tiny spelling change in a gene called IL-6.

Impact on Immune System: This SNP can affect how much a protein called IL-6 is made. IL-6 helps our body's defense system and how it responds to inflammation (swelling).

Health Connection: Depending on our SNP version (CC, CG, or GG), we might have different levels of IL-6. This can slightly change our chances of getting certain diseases or how we respond to treatments. But it's just one part of a bigger puzzle involving genes and the environment.

a) Enlist the techniques used in gene sequencing

- Sanger Sequencing: A traditional method that reads DNA by creating fragments that stop at specific points, revealing the sequence.

Illumina Sequencing: Uses fluorescently labeled nucleotides to read DNA and is highly efficient for large-scale sequencing.

Ion Torrent Sequencing: Detects hydrogen ions released during DNA replication to determine the sequence.

454 Pyrosequencing: Measures light emission as nucleotides are added to a DNA strand, providing sequence information.

PacBio Sequencing: Monitors real-time DNA polymerization to directly read DNA sequences.

Nanopore Sequencing: Passes DNA through nanopores, measuring changes in electrical current to determine the sequence.

SMRT Sequencing: Measures real-time DNA replication speed to determine sequence information.

Hybrid Sequencing (e.g., PromethION): Combines different technologies to improve accuracy and read lengths.

Shotgun Sequencing: Breaks DNA into fragments, sequences them, and assembles them to reveal the full sequence.

Metagenomic Sequencing: Studies DNA of whole microorganism communities to understand genetic diversity.

Targeted Sequencing: Focuses on specific regions of interest within the genome, reducing costs and analysis efforts.

7 mark

a) How do telomeres play a central role in gene aging?

-Protective Caps: Telomeres are protective caps at the ends of chromosomes, made up of repetitive DNA sequences.

Shortening with Division: During cell division, telomeres naturally shorten due to incomplete DNA replication.

Mitotic Clock: Telomere shortening acts as a "mitotic clock," counting the number of cell divisions a cell can undergo.

Replicative Senescence: As telomeres shorten, cells reach a critical point where they can no longer divide. This state is called replicative senescence.

Cellular Dysfunction: Cells with short telomeres experience reduced DNA repair and replication efficiency, leading to cellular dysfunction.

Accumulation of Damaged Cells: Cells unable to divide accumulate over time, contributing to tissue aging and decline.

Limited Tissue Repair: Stem cells, crucial for tissue repair, also have limited divisions due to telomere shortening.

Stem Cell Exhaustion: Shortened telomeres in stem cells lead to their decreased ability to regenerate tissues.

Age-Related Diseases: Short telomeres are linked to age-related diseases like heart disease, cancer, and neurodegenerative disorders.

Genetic Instability: Telomere shortening can cause genetic instability, increasing the risk of mutations and dysfunctional cells.

Cellular Aging: Cellular functions decline due to shortened telomeres, contributing to overall cellular aging.

Epigenetic Changes: Telomere shortening triggers epigenetic alterations, influencing gene expression and cellular functions.

Telomerase Enzyme: Telomerase can lengthen telomeres by adding back lost DNA sequences.

Selective Telomerase Activity: Telomerase is active in stem cells and immune cells, aiding in their longevity and function.

Balancing Act: While telomerase activation can extend cell lifespan, excessive activation might lead to uncontrolled cell growth, promoting cancer.

In essence, telomeres serve as cellular clocks, influencing the number of divisions cells can undergo and contributing to age-related changes, diseases, and cellular decline.

b) “Histone modification is an example of epigenetic change” explain

-

Histones and DNA Packaging: Histones are proteins that help package DNA into a compact structure known as chromatin.

Epigenetic Changes: Epigenetic changes refer to modifications that affect gene expression without altering the DNA sequence.

Histone Modification Definition: Histone modification involves chemical alterations to the histone proteins within chromatin.

Chemical Groups: These modifications include adding or removing chemical groups like acetyl, methyl, or phosphate groups.

Histone Tails: Modifications occur on specific amino acids within the "tails" of histone proteins.

Chromatin Structure: Modifications change the structure of chromatin, affecting how tightly DNA is wound around histones.

Gene Accessibility: Modified histones can open up or condense chromatin, influencing the accessibility of genes to cellular machinery.

Gene Expression Regulation: The accessibility of genes determines whether they are actively transcribed (expressed) or silenced.

Acetylation Example: Acetyl groups can be added to histones, relaxing chromatin and promoting gene expression.

Methylation Example: Methylation of histones can lead to compact chromatin, inhibiting gene expression.

Gene Activation and Repression: Histone modifications can activate or repress specific genes.

Cell Identity and Development: Histone modifications help establish and maintain cell identity during development.

Environmental Response: Cells can modify histones in response to environmental cues, influencing gene expression.

Cell Division Inheritance: Histone modifications can be passed on during cell division, potentially impacting gene expression in daughter cells.

Epigenetic Memory: Histone modifications contribute to epigenetic memory, allowing cells to remember past experiences and adapt accordingly.

In summary, histone modification is an example of epigenetic change as it involves altering the structure of chromatin through chemical modifications to histone proteins. This process controls gene accessibility and influences gene expression patterns without changing the DNA sequence, impacting various aspects of cell behavior, development, and adaptation.

b) What is alternative gene expression? Give examples

- 

Alternative gene expression refers to the phenomenon where different forms of a gene are transcribed and translated to produce distinct protein isoforms or RNA products. This diversity in gene expression arises from variations in splicing, transcription start sites, or other regulatory mechanisms. Here are 15 points and examples to elaborate:

1. Definition: Alternative gene expression occurs when different versions of a gene are expressed, resulting in the production of diverse RNA transcripts or protein isoforms.

2. Splicing Variants: One common mechanism is alternative splicing, where exons (coding regions) of a gene are combined in different ways during RNA processing.

3. Tissue-Specific Expression: Different tissues express specific isoforms of a gene to fulfill specialized functions.

Example: Troponin T gene in muscle cells vs. heart cells.

4. Developmental Changes: Expression patterns can change during development to support different stages of growth.

Example: Immunoglobulin genes during B-cell maturation.

5. Cellular Responses: Cells can switch gene isoforms in response to signals or stressors.

Example: Bcl-x gene in response to apoptosis signals.

6. Disease Implications: Altered gene isoforms can lead to disease.

Example: Tau protein isoforms in neurodegenerative disorders like Alzheimer's.

7. Variations in Exons: Alternative exons can be included or excluded, leading to different protein functions.

Example: CD44 gene variants in cancer metastasis.

8. Transcription Start Sites: Different transcription start sites can result in varied mRNA sequences.

Example: Heterogeneous nuclear ribonucleoprotein (hnRNP) genes.

9. Promoter Usage: Different promoters control gene expression in specific contexts.

Example: Estrogen receptor alpha with tissue-specific promoters.

10. RNA Editing: Nucleotide modifications can lead to alternative RNA sequences.

- Example: APOBEC enzymes editing apolipoprotein B mRNA.

11. MicroRNA Regulation: MicroRNAs can bind to target mRNAs, affecting their stability and translation.

- Example: miR-34 regulation of p53 pathway genes.

12. Noncoding RNA Variants: Different noncoding RNA isoforms can regulate gene expression.

- Example: Long noncoding RNA (lncRNA) variants in X-chromosome inactivation.

13. Immune Response Diversity: Antigen receptor genes diversify during immune responses.

- Example: Immunoglobulin and T cell receptor gene rearrangements.

14. Hormonal Control: Different hormone levels can trigger alternative expression.

- Example: Glucocorticoid receptor isoforms in response to stress.

15. Environmental Adaptation: Alternative expression can help organisms adapt to environmental changes.

- Example: Heat shock proteins responding to elevated temperatures.

In summary, alternative gene expression introduces diversity in RNA and protein products through mechanisms like splicing variants, transcription start site variations, and regulatory changes. This diversity enables cells to fine-tune their functions, respond to dynamic environments, and carry out specialized roles in development and disease.

Credit 2: Genetically modified plants and animals

5 mark

a) Social and ethical concerns of genetically modified organism

Credit 3: Mobile DNA Elements

1 mark

f) Give 5 characteristics of Drosophila transposons.

-

1) Abundance: Drosophila genomes contain a diverse array of transposons, making up a substantial portion of their DNA.

2) Classified Types: Drosophila transposons are mainly classified into two major classes: DNA transposons and retrotransposons.

3) Retrotransposons: Retrotransposons in Drosophila are similar to retroviruses, involving a reverse transcription step in their replication.

4) Regulation: Some Drosophila transposons are regulated by piwi-interacting RNAs (piRNAs) that suppress their activity to maintain genome stability.

5) Genomic Impact: Transposons have played a significant role in shaping the genome structure and evolution of Drosophila species by contributing to genetic diversity and rearrangements.

f) Give 5 characteristics of maize transposons.

-

1) Ubiquity: Transposons are a major component of the maize genome, contributing significantly to its size and complexity.

2) Class Variation: Maize transposons are classified into two major classes: Class I (retrotransposons) and Class II (DNA transposons).

3) Ac-Ds System: The Ac-Ds transposon system in maize is a well-studied example of DNA transposons that can cause color variation in kernel tissues.

4) Mutagenesis: Maize transposons have been harnessed as mutagenic agents in genetic studies, facilitating the discovery of new traits and genes.

5) Genome Structure Impact: The movement and insertion of transposons have played a significant role in maize genome evolution, including gene regulation and genome size variation.

5 mark

b) Write the characteristics of Tn5 and Tn10

-

Tn5 Transposon:

1) Discovery: Tn5 was one of the first transposons extensively studied and was discovered in Escherichia coli (E. coli) bacteria.

2) Structure: Tn5 is a DNA transposon composed of about 5.3 kilobase pairs (kb) and has terminal inverted repeats (IR) at its ends.

3) Transposition Mechanism: Tn5 uses a "cut-and-paste" transposition mechanism. It excises from its original location and inserts itself at a new site.

4) Transposase Enzyme: Tn5 encodes its transposase enzyme, which recognizes the IR sequences and catalyzes the transposition process.

5) Insertion Sequences: Tn5 contains two insertion sequences (IS) within it, IS50L and IS50R, which are involved in transposition and regulate the transposase activity.

Tn10 Transposon:

1) Type and Origin: Tn10 is also a DNA transposon found in E. coli. It belongs to the IS3 family of transposons.

2) Structure: Tn10 is approximately 9.4 kb in size and contains terminal inverted repeats (IR) at its ends.

3) Transposition Mechanism: Similar to Tn5, Tn10 employs a "cut-and-paste" transposition mechanism, excising from one location and inserting into another.

4) Transposase Enzyme: Tn10 encodes its transposase enzyme, which recognizes the IR sequences and facilitates the transposition process.

5) Insertion Effects: Tn10 can disrupt genes upon insertion, leading to genetic rearrangements and changes in bacterial phenotypes. It has been extensively used in genetic studies and mutagenesis.

a) Add a relative description of the replicative and non-replicative transposons.

-

Replicative Transposons:

• Mechanism: Replicative transposons use a "copy and paste" mechanism to move within the genome.
• Duplication: Upon transposition, a copy of the transposon is created and inserted at a new site while the original copy remains at its original location.
• Impact: Replicative transposition increases the number of transposons in the genome, leading to an expansion of transposon sequences.
• Example: The bacterial IS3 family transposons, like Tn5, often utilize a replicative mechanism.
• Evolutionary Impact: This mechanism can contribute to genetic diversity and potentially drive evolutionary changes.

Non-Replicative Transposons:

• Mechanism: Non-replicative transposons use a "cut and paste" mechanism to move within the genome.
• Movement: The transposon is excised from its original location and inserted at a new site, leaving no duplicate.
• Impact: Non-replicative transposition maintains the number of transposon copies in the genome.
• Example: Tn10 is an example of a non-replicative transposon found in bacteria.
• Precision: This mechanism is more direct and efficient, as it involves the direct movement of the transposon without creating a new copy.
• 
  

b) Explain auto splicing of group I introns.

-

Auto-splicing of Group I introns is a remarkable process that involves the removal of intron sequences from RNA molecules without the assistance of external factors like spliceosomes. Here are 15 points explained in an easy-to-remember manner:

1. RNA Self-Catalysis: Group I introns are RNA molecules with the ability to catalyze their own removal from precursor RNA molecules.
2. Intron Structure: Group I introns have a specific folded structure that includes characteristic domains.
3. Self-Splicing Steps: Auto-splicing occurs in two steps: an initial cleavage and then a ligation.
4. 3D Structure: The intron's 3D structure allows it to bring the reactive sites close together.
5. GTP Cofactor: GTP molecule can act as a cofactor, aiding in the self-splicing process.
6. Exon Formation: After splicing, exons are joined, creating a mature RNA molecule.
7. Multistep Reaction: Self-splicing involves multiple chemical reactions within the intron.
8. Ribozyme Activity: The intron acts as a ribozyme, a catalytic RNA molecule.
9. RNA World Hypothesis: Auto-splicing supports the RNA World hypothesis suggesting early life depended on RNA-based processes.
10. Ancient Mechanism: Auto-splicing is thought to be an ancient mechanism before protein enzymes evolved.
11. RNA Folding: Proper RNA folding is crucial for group I introns to perform self-splicing.
12. Catalytic Core: The intron's catalytic core is responsible for cleavage and ligation steps.
13. Substrate Binding: The intron substrate binds to the catalytic core through specific interactions.
14. Conserved Sequences: Group I introns share conserved sequences critical for their self-splicing ability.
15. In Vivo Significance: Group I introns can impact gene expression, RNA processing, and mobility of genetic elements in various organisms.
16. 
    

b) Comment on Controlling of Tn 10 transposition.

-

1. Transposon Disruption: Tn10 transposition can disrupt genes, affecting bacterial functions.
2. Transposase Control: Transposase enzyme guides Tn10 movement, preventing random jumps.
3. DNA Recognition: Transposase identifies specific DNA sequences for insertion.
4. Preventing Damage: Control prevents harmful gene disruptions by regulating transposition.
5. Cointegrate Formation: Transposition can form cointegrates, inhibiting movement.
6. Host Factors: Bacterial proteins influence transposition rates.
7. Environmental Signals: Environmental cues affect transposase activity.
8. Genetic Diversity: Controlled transposition aids adaptation by generating diversity.
9. Transposition Fidelity: Regulation minimizes errors and mutations.
10. Biotech Use: Controlled transposition aids genetic studies and analysis.
11. Evolution Impact: Regulated movement influences bacterial evolution.
12. Stability Maintenance: Control prevents genomic instability.
13. Gene Expression Impact: Transposition can influence gene expression.
14. Mutation Prevention: Regulation avoids harmful mutations.
15. Balancing Change: Control balances innovation and genetic stability.

c) Differentiate between LINES and SINES.

LINES vs. SINES Comparison

Characteristic	LINES (Long Interspersed Nuclear Elements)	SINES (Short Interspersed Nuclear Elements)
Size	Larger (6,000 to 8,000 base pairs)	Smaller (100 to 300 base pairs)
Autonomous	Encode own transposition machinery	Non-autonomous, lack own machinery
Transposition	Can transpose independently	Rely on LINES machinery for transposition
Transposition Mechanism	Reverse-transcribe RNA to DNA for insertion	RNA transcribed and reverse-transcribed by LINES
Examples	Human L1 elements	Human Alu elements

Credit 4: Proteomics

1 mark

d) Give 2 examples of amino acids with aromatic R groups.

e) Give 2 applications of proteomics.

7 mark

a) Write the steps involved in separation and purification of proteins in a crude extract of microbial cells.

b) What are the essential steps involved in the study of metabolomics?

c) Methods used in Protein structure analysis.

b) Explain protein foot printing as a tool in molecular biology

a) What alterations are made in DNA by transposons

c) Write a note on metabolomics.

b) Explain the factors affecting the expression of proteins.

a) What is MALDI? How is it used in proteomics.

b) Comment on 2D electrophoresis as a tool in characterization of protein.

a) Justify the retrovirus life cycle involves transposition like events

a) DMS foot printing.

 b) Auto regulation of ara C. 

c) tRNA splicing in eukaryotes.

c) Explain transesterification reaction in mRNA splicing

a) Explain is RNA and its applications.

b) Explain role of riboswitches with examples

c) Explain positive control of ara operon.

a) Comment on role of cAMP-CAP in regulation of lac operon.

c) Explain DNA helicase assay.

a) Explain method used for measuring transcription rates with suitable example.

a) What are the important parts of gene casettes involved in integrons and also mention their roles?

a) What is trade - off mechanism? Give an example.

b) What is genetic trade-off mechanism? Write its significance.

Q. The short DNA shown below is to be sequenced, using your knowledge

of how the Sanger method works. Dideoxynucleotides (dd NTPs) are

added in relatively small amounts. Asteris R represents radioactive label.

What length of strands will be synthesized on the 3 5    template below

*5 ---3 OH  

3 ---ACGACGCAGGACATTAGA3 5   

Nucleotide mixtures added in below,

dGTP, dATP, dTTP, dCTP, ddTTP

b) Regulation of E. coli. lac promoter.