Exam 2

I. Seminal Experiments in DNA History

1. Frederick Griffith’s Experiment (1928)

   • Demonstrated transformation in bacteria.

   • Showed that a "transforming principle" could transfer genetic information.

   • Question: Does this prove DNA is the genetic material? (No, other possibilities exist.)

2. Avery, MacLeod, and McCarty (1944)

   • Identified DNA as the "transforming principle."

   • Used enzyme treatments to eliminate proteins, RNA, and DNA, proving that DNA alone caused transformation.

3. Hershey-Chase Experiment (1952)

   • Used bacteriophages labeled with radioactive phosphorus (DNA) and sulfur (protein).

   • Showed that DNA, not protein, is the genetic material transferred to bacteria.

4. Meselson-Stahl Experiment (1958)

   • Used nitrogen isotopes (15N & 14N) to show DNA replicates semiconservatively.

   • After one replication cycle, all DNA had an intermediate density (hybrid of heavy and light nitrogen).

   • After two cycles, DNA was either hybrid or fully light.

II. DNA Structure

1. Components of DNA

   • Nucleotides consist of:

     • Nitrogenous base (A, T, C, G)

     • Deoxyribose sugar

     • Phosphate group

   • Purines (Adenine & Guanine) vs. Pyrimidines (Cytosine & Thymine)

   • Chargaff’s Rules: A = T, C = G

2. Double Helix Model

   • Rosalind Franklin’s X-ray diffraction data used by Watson & Crick.

   • DNA is antiparallel and held together by hydrogen bonds:

     • A-T (2 bonds)

     • C-G (3 bonds)

III. DNA Replication

1. Key Enzymes and Their Roles

   • Helicase: Unzips DNA strands.

   • Single-Strand Binding Proteins (SSBPs): Stabilize unwound DNA.

   • Primase: Synthesizes RNA primers.

   • DNA Polymerase III: Synthesizes new DNA strands (5’ → 3’ direction).

   • DNA Polymerase I: Replaces RNA primers with DNA.

   • Ligase: Seals Okazaki fragments on lagging strand.

   • Topoisomerase: Relieves supercoiling.

2. Leading vs. Lagging Strand

   • Leading strand: Continuous synthesis.

   • Lagging strand: Discontinuous synthesis via Okazaki fragments.

3. Origin of Replication

   • Prokaryotes: Single origin.

   • Eukaryotes: Multiple origins.

4. End-Replication Problem & Telomerase

   • Linear chromosomes lose DNA at ends with each replication.

   • Telomerase extends telomeres to prevent loss.

   • Cancer cells reactivate telomerase for immortality.

IV. PCR (Polymerase Chain Reaction)

1. Steps of PCR

   • Denaturation (95°C): DNA strands separate.

   • Annealing (45-68°C): Primers bind to target DNA.

   • Extension (72°C): Taq polymerase synthesizes new DNA.

2. PCR Components

   • Template DNA, primers, DNA polymerase (Taq), dNTPs, buffer.

3. Applications of PCR

   • Cloning, forensic analysis, medical diagnostics, evolutionary studies.

V. DNA Sequencing

1. Sanger Sequencing

   • Uses dideoxynucleotides (ddNTPs) to terminate replication.

   • DNA fragments are separated by size using gel electrophoresis.

2. Next-Generation Sequencing (NGS)

   • Faster, parallel sequencing of millions of DNA fragments.

   • Used for whole-genome sequencing.

Review Questions

1. How did Griffith’s experiment demonstrate transformation? Griffith's experiment demonstrated transformation by showing that harmless strains of bacteria could acquire virulence when exposed to heat-killed virulent strains, indicating that some genetic material was transferred between the bacteria.

2. Why did Hershey and Chase use radioactive phosphorus and sulfur?

   They used radioactive phosphorus to label DNA and radioactive sulfur to label proteins, allowing them to trace which component entered the bacterial cells during infection.

3. What are Chargaff’s rules, and how do they support complementary base pairing? Chargaff's rules state that in a given DNA molecule, the amount of adenine (A) is equal to the amount of thymine (T), and the amount of cytosine (C) is equal to the amount of guanine (G). This observation supports complementary base pairing by confirming that A pairs with T and C pairs with G, which is essential for the structure and function of DNA.

4. Why is telomerase important in aging and cancer? Telomerase is important in aging and cancer because it helps maintain the length of telomeres, which protect chromosome ends from deterioration. In normal somatic cells, telomerase activity is low, leading to progressive telomere shortening with each cell division, contributing to aging. However, in many cancer cells, telomerase is reactivated, allowing these cells to divide indefinitely and evade the normal aging process, thus promoting tumor growth.

5. What are the key differences between Sanger sequencing and NGS? Sanger sequencing is a method based on the selective incorporation of chain-terminating dideoxynucleotides during DNA replication, allowing for the determination of the DNA sequence of a single fragment, while Next-Generation Sequencing (NGS) utilizes massively parallel sequencing technology to simultaneously sequence millions of DNA fragments, resulting in a much higher throughput and allowing for comprehensive analysis of entire genomes.

6. How does PCR amplify DNA, and why is Taq polymerase used? PCR, or Polymerase Chain Reaction, amplifies DNA by repeatedly cycling through three main steps: denaturation, annealing, and extension. During denaturation, the double-stranded DNA is heated to separate it into two single strands. In the annealing step, short DNA primers bind to the target sequence at a lower temperature, and during extension, Taq polymerase synthesizes new DNA strands by adding nucleotides complementary to the template strand. Taq polymerase is used because it is heat-stable, allowing it to withstand the high temperatures needed for denaturation without denaturing itself, thus enabling the amplification process to be efficient and sustainable.

   III. Restriction Enzymes and Vector Construction

   • Restriction Endonucleases:

     • Cut DNA at specific palindromic sequences.

     • Create "sticky ends" or "blunt ends" for cloning.

     • Example: EcoRI produces sticky ends.

   • Restriction Mapping:

     • Determining the arrangement of restriction sites on DNA.

     • Used in plasmid vector construction.

   • Plasmid Features:

     • Origin of replication (ORI): Ensures DNA replication.

     • Selectable marker: E.g., antibiotic resistance gene.

     • Multiple Cloning Site (MCS): Region with many restriction sites.

   IV. Molecular Cloning Process

   1. Cut DNA and vector with the same restriction enzyme.

   2. Ligate foreign DNA into vector using DNA ligase.

   3. Transform bacteria with recombinant plasmid.

   4. Select for successfully transformed bacteria.

      • Antibiotic selection: Only bacteria with plasmid survive.

      • Blue/White Screening:

        • Blue colonies: Empty vector (functional lacZ gene).

        • White colonies: Recombinant plasmid (disrupted lacZ gene).

   V. Gene Editing Techniques

   • Transgenic Organisms:

     • Introduction of foreign genes into an organism.

     • Example: Knockout mice for studying gene function.

   • Gene Therapy:

     • Introduction of functional genes to treat genetic disorders.

     • Challenges:

       • Delivery of genes to correct cells.

       • Ensuring proper gene expression.

   VI. CRISPR-Cas9 Gene Editing

   • CRISPR System:

     • Uses guide RNA to target specific DNA sequences.

     • Cas9 endonuclease cuts DNA, allowing modifications.

   • Repair Mechanisms:

     • Non-Homologous End Joining (NHEJ): Error-prone, may cause mutations.

     • Homology-Directed Repair (HDR): Uses a template to introduce precise changes.

   • Applications:

     • Disease gene corrections (e.g., sickle cell anemia).

     • Potential for "designer babies" (ethical concerns).

   VII. Ethical Considerations in Genetic Engineering

   • Human Gene Editing:

     • Germline modifications are heritable and controversial.

     • First CRISPR babies: Edited CCR5 gene for HIV resistance.

   • Concerns:

     • Unintended mutations

     • Long-term ecological and health impacts

     • Ethical debates on genetic enhancement

   Review Questions

   1. How do restriction enzymes contribute to DNA cloning? Restriction enzymes, or endonucleases, cut DNA at specific palindromic sequences, creating 'sticky' or 'blunt' ends for cloning. They aid in determining the arrangement of restriction sites on DNA through restriction mapping, which is essential in plasmid vector construction. Plasmids, serving as cloning vectors, contain an origin of replication, selectable markers, and multiple cloning sites, facilitating the insertion and replication of foreign DNA.

   2. What features make plasmids useful as cloning vectors? Plasmids are useful cloning vectors due to their origin of replication, selectable markers (e.g., antibiotic resistance), and multiple cloning sites, which allow for the easy insertion and replication of foreign DNA.

   3. What is the role of selectable markers in molecular cloning? Selectable markers in molecular cloning, such as antibiotic resistance genes, allow for the identification of successfully transformed bacteria that contain the recombinant plasmid, facilitating the selection of cells that have incorporated foreign DNA.

   4. Compare and contrast NHEJ and HDR in CRISPR gene editing. NHEJ (Non-Homologous End Joining) is an error-prone DNA repair mechanism in CRISPR editing that may cause mutations, while HDR (Homology-Directed Repair) uses a template for precise changes, leading to accurate gene modifications.

   I. Differences Between DNA and RNA

   • Sugar: RNA contains ribose instead of deoxyribose.

   • Bases: RNA uses uracil (U) instead of thymine (T).

   • Strand Structure: RNA is usually single-stranded and can form secondary structures.

   II. Types of RNA and Their Functions

   1. Messenger RNA (mRNA): Carries genetic instructions from DNA to ribosomes.

   2. Ribosomal RNA (rRNA): Structural and catalytic component of ribosomes.

   3. Transfer RNA (tRNA): Brings amino acids to ribosomes during translation.

   4. Small Nuclear RNA (snRNA): Involved in splicing of pre-mRNA.

   5. Micro RNA (miRNA) & Small Interfering RNA (siRNA): Regulate gene expression.

   6. Piwi-Interacting RNA (piRNA): Protects the genome from transposable elements.

   7. Long Noncoding RNA (lncRNA): Involved in gene regulation.

   III. Transcription Process

   1. Initiation:

      • Prokaryotes: Sigma factor binds to -10 and -35 promoter regions to recruit RNA polymerase.

      • Eukaryotes: Transcription Factor IID (TFIID) binds to the TATA box, recruiting RNA polymerase II.

   2. Elongation:

      • RNA polymerase synthesizes RNA 5’ → 3’.

      • The transcription bubble forms as RNA polymerase moves along the DNA.

      • Eukaryotic RNA polymerase II has a C-terminal domain (CTD) that helps process mRNA.

   3. Termination:

      • Prokaryotes:

        • Rho-dependent termination: Rho protein disrupts RNA-DNA interaction.

        • Intrinsic termination: Secondary structures (hairpins) destabilize RNA polymerase.

      • Eukaryotes:

        • Torpedo Model: Exonuclease degrades excess RNA, leading to polymerase release.

   IV. RNA Processing in Eukaryotes

   1. 5’ Capping:

      • A modified guanine is added to the 5’ end for stability and ribosome recognition.

   2. Splicing:

      • Introns are removed, and exons are joined by the spliceosome.

      • Consensus splicing sites: 5' GU------AG 3'.

      • Splicing increases genetic diversity (alternative splicing).

   3. Polyadenylation (3' Poly-A Tail):

      • Added to protect mRNA from degradation and enhance translation.

   V. Alternative Transcription and RNA Editing

   1. Alternative Splicing:

      • Produces multiple proteins from one gene (~70% of human genes are alternatively spliced).

   2. Alternative Polyadenylation:

      • Different poly-A sites create different mRNA isoforms.

   3. RNA Editing:

      • Guide RNAs (gRNAs) direct nucleotide modifications, like uracil insertions or deletions.

   VI. Transcription Differences Between Prokaryotes and Eukaryotes

   Feature	Prokaryotes	Eukaryotes
   Polymerase	Single RNA polymerase	RNA Pol I, II, III
   Initiation	Sigma factor at -10 and -35	Transcription factors at TATA box
   Processing	No mRNA modifications	Capping, splicing, polyadenylation
   Termination	Rho-dependent or intrinsic	Torpedo model

   Review Questions

   1. How does RNA differ from DNA structurally and functionally? RNA differs from DNA in structure and function: RNA contains ribose sugar, uses uracil instead of thymine, and is usually single-stranded. Functionally, RNA plays roles in protein synthesis (mRNA), forms part of ribosomes (rRNA), and transports amino acids (tRNA).

   2. How do splicing and polyadenylation contribute to mRNA function? Splicing removes introns and joins exons in mRNA, increasing genetic diversity. Polyadenylation adds a tail to mRNA for stability and enhanced translation.

   3. What are alternative splicing and RNA editing, and why are they important? Alternative splicing produces multiple protein forms from one gene, while RNA editing modifies RNA sequences. Both processes are important for increasing genetic diversity and allowing for gene regulation.

      I. Overview of Translation

      • Definition: Translation is the process of synthesizing proteins from mRNA.

      • Key Players:

        • mRNA: Carries the genetic code.

        • Ribosomes: Catalyze protein synthesis.

        • tRNA: Brings amino acids to the ribosome.

        • Aminoacyl-tRNA Synthetase: Charges tRNA with amino acids.

      II. The Genetic Code

      • Triplet Codons: Three-nucleotide sequences encode amino acids.

      • Reading Frame: Defined by the start codon (AUG).

      • Wobble Hypothesis: Some tRNAs recognize multiple codons due to flexible base pairing.

      • Frameshift Mutations: Insertions or deletions shift the reading frame, disrupting translation.

      III. Translation Stages

      1. Initiation

      • Prokaryotes:

        • Shine-Dalgarno sequence helps ribosome binding.

        • Initiation factors (IF1, IF2, IF3) assist in forming the initiation complex.

        • Small ribosomal subunit binds mRNA; fMet-tRNA binds the start codon.

        • Large ribosomal subunit joins, forming the full ribosome.

      • Eukaryotes:

        • 5' Cap recruits ribosome.

        • Initiation factors (eIF1, eIF3, eIF4, eIF5) guide assembly.

        • Ribosome scans for the start codon (AUG) within the Kozak sequence.

        • Large subunit joins, and translation begins.

      2. Elongation

      • Elongation Factors (EFs) assist in:

        • Bringing charged tRNAs to the A site.

        • Forming peptide bonds between amino acids.

        • Translocating the ribosome along the mRNA.

      • Peptide bonds form between amino acids in the P site and A site.

      • Ribosome moves 5' to 3' on mRNA, shifting tRNAs from A → P → E site.

      3. Termination

      • Stop codons (UAA, UAG, UGA) are recognized by release factors (RFs).

      • Release Factors (RF1, RF2, RF3 in prokaryotes; eRF1 in eukaryotes) break the peptide-tRNA bond.

      • The ribosome disassembles, releasing the completed protein.

      IV. Protein Structure and Function

      • Primary Structure: Amino acid sequence.

      • Secondary Structure: Alpha helices and beta sheets.

      • Tertiary Structure: Three-dimensional folding.

      • Quaternary Structure: Multiple polypeptide subunits interacting.

      V. Post-Translational Modifications

      1. Phosphorylation: Kinases add phosphate groups; regulates activity.

      2. Ubiquitination: Tags proteins for degradation by the proteasome.

      3. Glycosylation: Adds sugar molecules; affects stability and function.

      4. Signal Sequences: Direct proteins to specific locations (ER, mitochondria, etc.).

      VI. Effects of Mutations on Protein Function

      • Missense Mutation: Changes one amino acid.

      • Nonsense Mutation: Introduces a stop codon, truncating the protein.

      • Silent Mutation: No change in amino acid sequence.

      • Frameshift Mutation: Alters the reading frame, leading to a nonfunctional protein.

      VII. Ribosome Function and Translation Efficiency

      • Polyribosomes: Multiple ribosomes translating a single mRNA simultaneously.

      • Polycistronic mRNA (Prokaryotes): One mRNA encodes multiple proteins.

      • Monocistronic mRNA (Eukaryotes): One mRNA encodes a single protein.

      Review Questions

      1. What are the key differences between prokaryotic and eukaryotic translation? Prokaryotes have a single RNA polymerase, while eukaryotes have three (RNA Pol I, II, III)., Initiation in prokaryotes involves sigma factors, whereas eukaryotes use transcription factors at the TATA box., Prokaryotic translation lacks mRNA modifications; eukaryotes involve capping, splicing, and polyadenylation., Termination mechanisms differ, with prokaryotes using Rho-dependent or intrinsic termination, and eukaryotes employing the torpedo model.

      2. How does tRNA contribute to translation accuracy? tRNA contributes to translation accuracy by bringing the correct amino acids to the ribosome, matching them to the corresponding mRNA codons through complementary base pairing.

      3. How do mutations affect protein function and stability?
         Mutations can alter protein function and stability by causing missense mutations (changing one amino acid), nonsense mutations (introducing a stop codon), silent mutations (no change in amino acid sequence), or frameshift mutations (shifting the reading frame, potentially leading to nonfunctional proteins).

      4. How do signal sequences direct proteins to their proper cellular locations? Signal sequences direct proteins to their specific cellular locations, ensuring proper functioning by guiding them to the endoplasmic reticulum, mitochondria, or other destinations.

         Replace selection with thisInsert below selectionTry againCancel

   ---