Review Flashcards

• Meselson and Stahl cultured E Coli with 2 N isotopes to show that DNA replication is semi conservative 

• Hershey and Chase’s experiment: DNA not protein is the genetic material of bacteriophage

• Reverse Transcription: DNA strand is made complementary to RNA molecule 

• Post-transcriptional processing: splicing out introns 

• Bacteria does not acquire genetic diversity through binary fission 

• A particular cell does not make the same protein for their entire lifespan 

• Different cell types contain different proteins, all DNA containing cells in 1 organism is basically the same

• Promoters help regulate gene activity

• Anticodons form units of genetic code in tRNA that correspond to complementary codons in mRNA.

• tRNA transfers amino acids to ribosome while mRNA codes instructions for building polypeptide

• In eukaryotes translation takes place in cytoplasm, rough ER surface, or ribosome
  In prokaryote it takes place in ribosome 

DNA Replication 

When DNA is copied – interphase, S phase of cell cycle

Recognition of origin site on DNA, concept of unwinding enzyme Helicase 

RNA primer

DNA polymerase – functional definition

Concept of complementary relationship among bases – semiconservative

Discontinuous/continuous or lagging /leading or Okazaki fragments (due to

antiparallel backbones and 5’ to 3’ generation of new segments)

DNA ligase – functional definition

Mitosis 

concept of chromatid pairs or ‘doubled chromosomes’

prophase – condensation, spindle formation

metaphase – alignment of chromosomes

anaphase – separation of chromatids or equivalent statement

telophase or origin of cytokinesis – nuclear membrane reforms, cell plate or cell furrow

Transcription (Occurs in the nucleus):

• RNA polymerase binds to the promoter region on the DNA to initiate transcription.

• RNA polymerase synthesizes a complementary RNA strand using one DNA strand as a template; uracil (U)replaces thymine (T) in RNA.

• The new mRNA strand grows in the 5’ to 3’ direction.

• After transcription, introns (noncoding regions) are removed, and exons (coding regions) are joined together by spliceosomes.

• A 5’ cap is added to protect mRNA and assist with ribosome binding; a poly-A tail is added to prevent degradation and aid in mRNA export.

• The mature mRNA exits the nucleus and enters the cytoplasm.

Translation (Occurs in the cytoplasm):

• The small ribosomal subunit binds to the mRNA, followed by the first tRNA (carrying methionine) and the large ribosomal subunit to form the initiation complex.

• tRNA molecules bring specific amino acids to the ribosome, matching anticodons to codons on the mRNA.

• The ribosome has three sites: A (aminoacyl), P (peptidyl), and E (exit); peptide bonds form between amino acids in the P site.

• During translocation, the ribosome moves along the mRNA, shifting tRNAs from the A to P to E sites, elongating the polypeptide chain.

• Translation ends when a stop codon is reached; a release factor binds, causing the release of the completed polypeptide and disassembly of the ribosome.

• The ribosome reaches a stop codon (UAA, UAG, or UGA).

• A release factor binds, causing the release of the polypeptide.

• The ribosome disassembles.

 1. DNA Replication

• Purpose: To copy the DNA before cell division.

• When: During the S phase of interphase in the cell cycle.

• Result: Two identical DNA molecules.

2. Transcription

• Purpose: To convert a gene (DNA sequence) into an mRNA molecule.

• Where: In the nucleus of eukaryotic cells.

• Result: A single-stranded mRNA complementary to the DNA template.

3. Translation

• Purpose: To use the mRNA sequence to build a polypeptide (protein).

• Where: In the cytoplasm, on ribosomes.

• Result: A functional protein that performs work in the cell.

• Nucleotide made of sugar, phosphate, nitrogen base 

• transcription : initiation, elongation, termination 

Initiation: RNA polymerase binds to DNA promoter region, DNA strand unwind and RNA polymerase starts synthesising 

Elongation: RNA polymerase moves along DNA template strand adding RNA nucleotides in 5’ to 3’ direction 

Termination: RNA polymerase reaches termination sequence, DNA rewinds into double helix  

Operon: cluster of genes under control of promoter

Key components: promoter, operator, and structural genes

Regulation:

• Off when lactose is absent: A repressor binds to the operator, blocking transcription.

• On when lactose is present: Lactose (or allolactose) binds the repressor, removing it from the operator, allowing transcription.

Transcription Factors in Eukaryotes

Definition: Proteins that bind to specific DNA sequences to regulate transcription (either promoting or blocking RNA polymerase binding).
Types:

• General transcription factors: Needed for all transcription.

• Specific transcription factors: Regulate particular genes by binding to control elements like enhancers.

Enhancers and Repressors

• Enhancers: DNA sequences far from the gene that increase transcription when bound by activator proteins (a type of transcription factor). They can loop the DNA to interact with the promoter.

• Repressors: Proteins or DNA elements that decrease transcription, often by blocking activator binding or recruiting proteins that compact chromatin.

Promoter: start site for RNA polymerase, can bind there 

Operator: region where repressor protein can bind, next to or overlapping the promoter 

Structural genes: code for proteins with specific functions, the actual genes 

1. Mutations

   • Point mutations (silent, missense, nonsense)
     Missense: single base change leads to codon that codes for diff amino acid could alter protein structure + function
     Nonsense: changes codon into stop codon

   • Frameshift mutations (insertions, deletions)

   • Chromosomal mutations
     Can occur during DNA replication errors or during meiosis crossing over

Translocation: A segment from one chromosome breaks off and attaches to another chromosome.

Non-disjunction: Failure of chromosomes to separate properly during meiosis, leading to an abnormal number of chromosomes (e.g., Down syndrome, caused by trisomy 21).

2. Biotechnology (often included in this unit)

   • Gel electrophoresis

   • Polymerase Chain Reaction (PCR)

   • Recombinant DNA, plasmids, transformation

   • CRISPR and gene editing basics

Gel Electrophoresis

• Purpose: Used to separate DNA, RNA, or protein fragments based on size and charge.

• How it works:

  • DNA is loaded into a gel matrix and an electric field is applied.

  • Since DNA is negatively charged (due to phosphate groups), it moves toward the positive electrode.

  • Smaller fragments move faster through the gel, while larger fragments move more slowly.

• Applications:

  • DNA fingerprinting (e.g., for paternity testing or forensic analysis)

  • Genetic testing

Polymerase Chain Reaction (PCR)

• Purpose: A method used to amplify small amounts of DNA, creating millions of copies.

• Steps:

  • Denaturation: The double-stranded DNA is heated to separate the strands.

  • Annealing: Short primers bind to the target DNA sequence.

  • Extension: DNA polymerase adds nucleotides to extend the DNA strands.

• Applications:

  • Cloning genes for study

  • Diagnosing diseases (e.g., detecting viral infections like HIV)

  • Forensic analysis (e.g., identifying suspects or victims from tiny DNA samples)

Recombinant DNA, Plasmids, and Transformation

• Recombinant DNA refers to DNA molecules that are artificially created by combining DNA from different sources.

  • Plasmids: Small, circular DNA molecules often used in genetic engineering. They can carry genes of interest and be inserted into cells.

  • Transformation: The process of introducing recombinant DNA (such as plasmids) into a cell.

  • Process: For example, a plasmid carrying a gene of interest can be inserted into a bacterium, and the bacterium will express the gene, often producing proteins like insulin.

• Applications:

  • Producing genetically modified organisms (GMOs) for agriculture.

  • Creating recombinant proteins for medical use (e.g., insulin).

  • Gene therapy (replacing or repairing defective genes).

CRISPR and Gene Editing Basics

• CRISPR-Cas9: A gene-editing technology that allows scientists to make precise changes to an organism’s DNA.

  • How it works:

    • Cas9 is an enzyme that acts as molecular scissors, cutting DNA at a specific location.

    • Guide RNA directs Cas9 to the target DNA sequence, ensuring the cut is made in the correct location.

    • Once the DNA is cut, it can either be repaired (by introducing a new sequence) or disrupted (by causing a frameshift mutation).

• Applications:

  • Gene editing for disease treatment (e.g., sickle cell anemia or cystic fibrosis).

  • Creating genetically modified organisms in research or agriculture.

  • Potential applications in human gene therapy, although ethical concerns are involved.

• Alternative Splicing:

  • In eukaryotic cells, one gene can give rise to multiple proteins through alternative splicing. This occurs when different combinations of exons are joined together during RNA processing.

  • Significance: This increases protein diversity without requiring additional genes.

• Transcription in Prokaryotes vs Eukaryotes:

  • Prokaryotes: Transcription and translation are coupled (occur simultaneously in the cytoplasm).

  • Eukaryotes: Transcription occurs in the nucleus, and mRNA must be processed and transported to the cytoplasm before translation.

• DNA Repair Mechanisms:

  • Proofreading: DNA polymerase checks and corrects errors during replication.

  • Mismatch Repair: Fixes errors that escape proofreading during DNA replication.

  • Excision Repair: Involves the removal and replacement of damaged DNA, including damage caused by UV radiation (thymine dimers).

Viral Genetics and Reverse Transcription

• Retroviruses and Reverse Transcription:

  • Retroviruses, like HIV, use the enzyme reverse transcriptase to convert their RNA genome into DNA, which is then integrated into the host cell’s genome.

  • This reverse transcription process is important for understanding how RNA viruses replicate within host cells and cause diseases

DNA Repair Mechanisms

• Base Excision Repair: Repairs single damaged bases in the DNA.

• Nucleotide Excision Repair: Removes and replaces larger segments of damaged DNA, such as thymine dimers caused by UV light.

• Homologous Recombination: A repair mechanism that uses a homologous chromosome as a template to fix DNA double-strand breaks.