BIOL 2153 EXAM 3

Chromosomal Rearrangements

1. Types of Chromosomal Rearrangements

  • Deletions:

    • Homozygous Deletions: Often lethal or harmful, depending on the size of the deletion and the genes affected.

    • Heterozygous Deletions: Can cause mutant phenotypes due to gene dosage effects (haploinsufficiency), increased risk of mutations in the other gene copy, or uncovering recessive mutant alleles.

    • Recombination: Recombination can occur between homologous chromosomes only at regions of similarity. No recombination occurs in a deletion loop, and genetic map distances in deletion heterozygotes may not be accurate.

  • Duplications:

    • Phenotypic Effects: Duplications can be harmful, especially when they involve dosage-sensitive genes. The increased gene copy number can lead to novel phenotypes.

    • Homozygous or Heterozygous Duplication: Depending on size, can be lethal or harmful.

    • Unequal Crossing Over: Unequal crossing over between duplicated regions leads to changes in copy number.

    • Types of Duplications:

      • Tandem Duplications: In the same order or reverse order.

      • Nontandem (Dispersed) Duplications: Can be in the same order or reverse order.

  • Inversions:

    • Phenotypic Effects: Most inversions do not result in abnormal phenotypes unless they disrupt genes or place genes near regulatory sequences or heterochromatin.

    • Pericentric Inversion: Involves the centromere within the inverted segment.

    • Paracentric Inversion: Involves no centromere within the inverted segment.

    • Recombination:

      • Inversion Loop: Forms during recombination to align homologous regions.

      • Pericentric Inversion: Crossing over within the inversion loop leads to genetically unbalanced recombinant chromatids.

      • Paracentric Inversion: Crossing over can result in chromatids with two centromeres or no centromere, leading to nonviable gametes.

  • Translocations:

    • Reciprocal Translocations: No loss of genetic material, but can lead to phenotypic changes if the breakpoints affect genes.

    • Meiosis and Segregation: 3 chromosome segregation patterns are possible in a translocation heterozygote. Only alternate segregation leads to viable progeny.

    • Robertsonian Translocation: Affects acrocentric chromosomes, often resulting in the loss of a small chromosome.

    • Down Syndrome: May arise from a Robertsonian translocation between chromosomes 21 and 14.

2. Mechanisms Causing Chromosomal Rearrangements

  • Non-homologous End Joining (NHEJ): A repair mechanism for double-strand breaks that leads to incorrect joining of DNA ends, resulting in deletions, duplications, inversions, or translocations.

  • Homologous Recombination: Can lead to duplications or deletions during misalignment in meiosis or DNA repair.

3. Experimental Methods to Detect Chromosomal Rearrangements

  • Fluorescence in Situ Hybridization (FISH): Uses fluorescent probes to detect structural changes in chromosomes.

  • Karyotyping: Chromosome staining to identify large-scale rearrangements.

  • Genome Sequencing: Identifies rearrangements through read count analysis, revealing duplications and deletions.

4. Effects on Crossing Over During Meiosis

  • Deletions: Gene dosage effects and deletion loops.

  • Duplications: Unequal crossing over due to misalignment (gene dosage effects).

  • Inversions: Pericentric and paracentric inversion loops lead to semi-sterility.

  • Translocations: Alternate segregation pattern is essential for viable gametes, leading to semi-sterility.



Transposable Elements

1. Types of Transposable Elements

  • Retrotransposons:

    • Mechanism: "Copy and Paste" through an RNA intermediate.

  • DNA Transposons:

    • Mechanism: "Cut and Paste" without an RNA intermediate.

2. Structural Features

  • Inverted Repeats (IRs): Sequences of 10-200 base pairs at the ends.

  • Transposase Gene: Encodes the enzyme responsible for recognizing IRs and cutting DNA at their borders.



Genetic Code

1. Understanding the Genetic Code

  • Reading the Code: Codons are triplets of nucleotides that encode amino acids.

  • Degeneracy: Multiple codons can code for the same amino acid.

  • Key Features of Codons:

    • Triplicate Bases: Each codon consists of 3 nucleotides.

    • Non-overlapping: Codons are read sequentially, with no overlap.

    • Linear: Read from 5’ to 3’ without interruption.

2. Classes of Mutations

  • Missense Mutations: Change one amino acid to another; can be conservative (similar properties) or non-conservative (different properties).

  • Nonsense Mutations: Convert a codon into a stop codon, leading to premature termination.

  • Frameshift Mutations: Insertions or deletions of nucleotides (not in multiples of 3) cause a shift in the reading frame, often disrupting protein function.

  • Silent Mutations: Do not change the amino acid sequence due to codon redundancy.



Gene Expression: Transcription

1. Central Dogma

  • DNA → RNA → Protein

  • Transcription: Process by which RNA is synthesized from a DNA template.

2. Key Differences Between DNA and RNA

  • DNA: Double-stranded, deoxyribose, A, T, G, C.

  • RNA: Single-stranded, ribose, A, U, G, C.

3. Steps of Transcription

  • Initiation: RNA polymerase binds to the promoter and starts transcription.

  • Elongation: RNA polymerase adds nucleotides to the growing RNA strand.

  • Termination: Transcription ends when a terminator sequence is encountered.

4. Alternative Splicing

  • Produces different protein isoforms from the same pre-mRNA by including or excluding exons.



Gene Expression: Translation

1. Steps of Translation

  • Initiation: Small ribosomal subunit binds to mRNA; the initiator tRNA binds to the start codon.

  • Elongation: tRNA molecules add amino acids to the growing polypeptide chain.

  • Termination: Release factors bind to the stop codon, releasing the polypeptide chain.

2. Key Features of tRNA

  • Anticodon: Complementary to the mRNA codon.

  • Amino Acid Attachment: Each tRNA carries a specific amino acid.

3. Wobble Base

  • The 5’ base in the tRNA anticodon can form non-standard pairings with mRNA codons, allowing a single tRNA to recognize multiple codons.



Gene Regulation

1. Cis-Acting Elements

  • Promoters: Directly upstream of genes, where RNA polymerase binds.

  • Enhancers: Can be far from the gene, increasing or decreasing transcription.

  • Silencers: Decrease transcription.

  • Insulators: Block enhancer-promoter interactions.

2. Trans-Acting Factors

  • Transcription Factors: Proteins that bind to cis-elements (e.g., promoters, enhancers) to regulate transcription.

3. Reporter Assays

  • Used to identify cis and trans elements by observing changes in gene expression linked to a reporter gene.

4. Histone Modifications

  • Histone Acetylation: Typically increases gene expression by loosening chromatin.

  • DNA Methylation: Typically represses gene expression by preventing transcription factor binding.



Epigenetics

1. Mechanisms of Epigenetic Changes

  • DNA Methylation: Addition of methyl groups to cytosine bases, typically repressing gene expression.

  • Histone Modifications: Acetylation or methylation of histones can alter chromatin structure and gene expression.

2. Inheritance of Epigenetic Marks

  • Epigenetic modifications can be passed down during DNA replication, influencing gene expression without altering the DNA sequence.

3. CpG Islands

  • CpG Islands: Regions rich in CpG sites where DNA methylation typically occurs, repressing transcription.

Genomic Imprinting

Key Concepts:

  • Imprinting = Silenced: Genomic imprinting refers to the silencing of certain genes based on their parental origin (either maternal or paternal).

  • Inheritance Pattern of Imprinted Genes:

    • In genomic imprinting, specific genes are expressed in a parent-of-origin manner, meaning that the expression of a gene depends on whether it was inherited from the mother or the father.

    • Imprinting: The gene from one parent is silenced (turned off), and only the allele from the other parent is expressed.

    • Resetting of Imprints: Genetic imprints are reset during gamete formation and early embryonic development to ensure that the imprinting marks are correctly established for the next generation.

Major Concepts:

  • Why doesn't methylation at all DNA locations cause imprinting?

    • Methylation in imprinting specifically occurs at particular regions of the genome (imprinted loci), not at all DNA locations. These imprinted regions undergo specific methylation patterns during gamete formation to mark them for silencing based on parental origin.

  • When do epigenetic marks get reset?

    • Epigenetic marks (such as DNA methylation) are reset during germ cell formation (sperm and egg cells) and in the early stages of embryonic development to ensure the correct silencing of imprinted genes.



Genetic Screens and Complementation Tests

Key Concepts:

  • Complementation Groups:

    • Mutants are placed into complementation groups based on their ability to complement each other.

    • Same complementation group: If two mutants fail to complement, they likely affect the same gene, leading to similar phenotypic outcomes.

    • Larger complementation groups indicate larger genes because larger genes are more likely to be disrupted by mutations.

  • Biochemical Pathways:

    • By analyzing mutants’ ability to grow with different precursor substrates, we can assemble biochemical pathways.

      • If growth occurs with the substrate of a mutated gene → gene is involved in the pathway.

      • If growth occurs with the product of a mutated gene → gene is rescued, showing its role in the pathway.

Major Concepts:

  • Forward vs. Reverse Genetics:

    • Forward Genetics: Starts with phenotype and identifies the gene responsible by screening for mutations.

    • Reverse Genetics: Starts with a known gene and seeks to determine its function by observing the resulting phenotype when the gene is disrupted or knocked out.



Genetic Epistasis

Key Concepts:

  • Epistasis: The interaction between genes where one gene can mask the expression of another gene.

  • Double Mutant Phenotypes:

    • Linear Pathway: If two genes are in a linear pathway, the double mutant phenotype will resemble that of the downstream gene, as the upstream gene’s effect is masked.

    • Synergistic (Redundant) Pathway: If two genes are in a redundant parallel pathway, the double mutant will show a more severe phenotype than either single mutant.

    • Antagonistic Parallel Pathway: If two genes are in an antagonistic parallel pathway, the double mutant phenotype will be intermediate or less severe than the single mutants.



DNA Sequencing

Key Concepts:

  • Restriction Enzymes and DNA Shearing:

    • Restriction enzymes recognize specific DNA sequences and cut DNA into fragments, often producing blunt or sticky ends.

    • DNA shearing methods (sonication, hydrodynamic shearing, nebulization) are used to break DNA into smaller pieces for sequencing.

  • Genomic Libraries:

    • A genomic library is created by fragmenting genomic DNA, inserting the fragments into vectors (such as plasmids), and then introducing these vectors into host cells for replication and storage.

  • Molecular Cloning and Bacterial Transformation:

    • Molecular cloning involves extracting DNA, cutting it into fragments, ligating it into vectors, and introducing it into host cells.

    • Bacterial transformation involves introducing foreign DNA into bacteria to produce clones of the desired gene.

  • Sanger Sequencing (Chain Termination Method):

    • Sanger sequencing involves creating DNA fragments of different lengths using dideoxynucleotides (ddNTPs), which terminate chain elongation. These fragments are then separated by electrophoresis, and the sequence is determined based on the last nucleotide incorporated into each fragment.

  • Electrophoresis and DNA Fragment Separation:

    • Electrophoresis separates DNA fragments based on their size (smaller fragments move faster through the gel).

  • Shotgun Sequencing Strategy:

    • Shotgun sequencing involves randomly fragmenting the DNA and sequencing the fragments. Computational tools are then used to assemble the fragments into the complete genome sequence.

Major Concepts:

  • Why Fragment DNA Before Sequencing?

    • In shotgun sequencing, DNA is fragmented into smaller pieces to make sequencing manageable. These fragments are sequenced individually, and overlaps between sequences are used to assemble the full genome.

  • Why Use Plasmids for DNA Fragments?

    • Plasmids act as vectors to carry and replicate the DNA fragment of interest. This makes it easier to amplify and manipulate the DNA in the lab.

  • Why Transform DNA into Bacteria?

    • Bacteria can replicate the introduced foreign DNA, producing multiple copies for further study.

  • Importance of ddNTPs in Sanger Sequencing:

    • ddNTPs lack a 3' OH group, preventing the addition of further nucleotides and terminating chain elongation. Fluorescent labeling allows the detection of the last nucleotide in the sequence.

  • Shotgun Sequencing and Genome Assembly:

After DNA is fragmented and sequenced, computers use overlaps between the sequenced fragments to reconstruct the entire genome.