Genetics Exam 3 Study Guide: Gene Regulation, Genetic Technologies, Genomics, and Cancer Genetics

Gene Expression and Regulation in Bacteria

Constitutive vs. Regulated Expression

Gene expression in bacteria can be either constitutive (always active) or regulated (controlled in response to environmental or cellular signals).

• Constitutive expression: Genes are transcribed continuously, regardless of environmental conditions. Example: housekeeping genes.

• Regulated expression: Genes are transcribed only under specific conditions, allowing cells to adapt to changes.

Negative vs. Positive Control

Gene regulation involves mechanisms that either inhibit (negative control) or promote (positive control) transcription.

• Negative control: Regulatory proteins (repressors) bind to DNA and prevent transcription.

• Positive control: Regulatory proteins (activators) enhance transcription by facilitating RNA polymerase binding.

Operon Architecture

Operons are clusters of genes under a single promoter, allowing coordinated regulation.

• Promoter: DNA sequence where RNA polymerase binds to initiate transcription.

• Operator: DNA region where regulatory proteins bind to control gene expression.

• Structural genes: Genes encoding proteins with related functions.

Allosteric vs. DNA-Binding Domains

Regulatory proteins often have distinct domains for function.

• Allosteric domain: Binds effector molecules, causing conformational changes.

• DNA-binding domain: Interacts with specific DNA sequences to regulate transcription.

Structural Motifs of DNA Regulatory Proteins

Common motifs include helix-turn-helix, zinc finger, and leucine zipper, which facilitate DNA binding.

• Helix-turn-helix: Found in many bacterial repressors.

• Zinc finger: Utilizes zinc ions for structural stability.

• Leucine zipper: Dimerizes proteins for DNA interaction.

lac Operon: Role and Mechanisms of Control

The lac operon regulates lactose metabolism in Escherichia coli.

• Inducible system: Activated in presence of lactose.

• Negative control: Lac repressor binds operator, blocking transcription.

• Positive control: CAP-cAMP complex enhances transcription when glucose is low.

trp Operon: Role and Mechanisms of Control

The trp operon regulates tryptophan biosynthesis.

• Repressible system: Turned off when tryptophan is abundant.

• Feedback inhibition: Tryptophan acts as a corepressor, enabling repressor binding to operator.

Regulatory Mutations

Mutations in regulatory regions or proteins can alter gene expression patterns.

• Operator mutations: May prevent repressor binding, leading to constitutive expression.

• Promoter mutations: Can reduce or enhance transcription.

Gene Expression and Regulation in Eukaryotes

cis-acting vs. trans-acting Elements

Gene regulation in eukaryotes involves both local (cis) and distant (trans) factors.

• cis-acting elements: DNA sequences (e.g., promoters, enhancers, silencers) that regulate nearby genes.

• trans-acting factors: Proteins (e.g., transcription factors) that bind to cis-elements to modulate expression.

Enhancer and Silencer Regulatory Regions

Enhancers increase, while silencers decrease, transcription rates.

• Enhancers: Can act at a distance, often looping DNA to interact with promoters.

• Silencers: Suppress gene expression by recruiting repressive proteins.

Transcription Factors

Proteins that bind DNA and regulate transcription initiation.

• General transcription factors: Required for all genes.

• Specific transcription factors: Regulate subsets of genes.

Epigenetic Modifications

Heritable changes in gene expression without altering DNA sequence.

• Methylation: Addition of methyl groups to DNA, often silencing genes.

• Acetylation: Addition of acetyl groups to histones, generally activating genes.

Open vs. Closed Chromatin

Chromatin structure influences gene accessibility.

• Open chromatin (euchromatin): Accessible, transcriptionally active.

• Closed chromatin (heterochromatin): Condensed, transcriptionally silent.

Genomic Imprinting

Parent-of-origin-specific gene expression due to epigenetic marks.

• Imprinted genes: Expressed from only one parental allele.

Topologically Acting Domains

Chromatin is organized into domains that regulate gene expression.

• TADs (Topologically Associating Domains): Regions of the genome that interact more frequently with themselves than with other regions.

RNAi Mechanisms

RNA interference (RNAi) silences gene expression post-transcriptionally.

• siRNA and miRNA: Small RNAs that guide silencing complexes to target mRNAs.

Post-Translational Modifications

Protein function can be regulated after translation.

• Phosphorylation, ubiquitination, methylation: Modify protein activity, stability, or localization.

Forward and Reverse Genetics, Recombinant DNA Technologies

Forward Genetics

Identifies genes responsible for a phenotype by mutagenesis and screening.

• Strategy: Randomly mutate organisms, screen for altered traits, map and identify causative genes.

• Model organisms: Drosophila, Arabidopsis, Mus musculus are commonly used.

• Insertional inactivation: Disrupts gene function by inserting DNA elements.

Reverse Genetics

Starts with a gene and investigates its function by targeted disruption or modification.

• Strategy: Knockout, knockdown, or modify specific genes, then observe phenotypic effects.

• Gain-of-function vs. loss-of-function mutations: Overexpression or inactivation of genes to study effects.

Balancer Chromosomes

Special chromosomes used in genetic screens to maintain mutations and prevent recombination.

• Example: Used in Drosophila genetics.

Complementation Tests

Determine if mutations affect the same gene or different genes.

• Modifier screens: Identify genes that enhance or suppress a phenotype.

CRISPR and RNAi Mechanisms

Modern tools for genome editing and gene silencing.

• CRISPR: Uses Cas9 nuclease and guide RNA to create targeted DNA breaks.

• RNAi: Silences gene expression via small RNAs.

Transgenes and Enhancer Trapping

Introduce foreign genes or reporter constructs to study gene regulation.

• Enhancer trapping: Identifies regulatory elements by linking them to reporter genes.

Restriction Enzymes, Plasmid Engineering, and Shuttle Vectors

Tools for manipulating DNA in recombinant technology.

• Restriction enzymes: Cut DNA at specific sequences.

• Plasmid engineering: Design plasmids for gene cloning and expression.

• Shuttle vectors: Function in multiple host species.

Transformation, Plant and Animal Cloning

Methods for introducing DNA into cells and generating genetically identical organisms.

• Transformation: Uptake of foreign DNA by cells.

• Cloning: Producing genetically identical plants or animals.

Genomics

Primer Walking vs. Shotgun Sequencing

Methods for sequencing DNA.

• Primer walking: Sequentially extends sequencing from known regions.

• Shotgun sequencing: Randomly fragments DNA, sequences pieces, and assembles them computationally.

Human Genome Sequencing and Next-Generation Platforms

Technologies for large-scale DNA sequencing.

• Human genome sequencing: Completed using Sanger and next-generation methods.

• Next-generation platforms: Illumina (short reads), PacBio (long reads), Nanopore (real-time, long reads).

Open Reading Frames (ORFs), Gene Duplication, Horizontal Gene Transfer

Key concepts in genome analysis.

• ORFs: DNA sequences that can be translated into proteins.

• Gene duplication: Source of genetic innovation.

• Horizontal gene transfer: Movement of genes between species.

Transcriptome Sequencing and Microarray Methodology

Methods for studying gene expression.

• Transcriptome sequencing: RNA-seq quantifies all transcripts in a cell.

• Microarrays: Measure expression of thousands of genes simultaneously.

Synteny

Conservation of gene order across species.

• Example: Human and mouse genomes share syntenic blocks.

Genetics of Cancer

Definition and Hallmarks of Cancer

Cancer is a disease characterized by uncontrolled cell growth and division.

• Hallmarks: Include sustained proliferative signaling, evasion of growth suppressors, resistance to cell death, etc.

Cancer Progression

Cancer develops from normal cells to benign tumors, then to malignant tumors capable of invasion and metastasis.

• Benign: Non-invasive, localized growth.

• Malignant: Invasive, can spread to other tissues.

Sporadic vs. Familial vs. Hereditary Cancer

Cancers can arise from random mutations, familial predisposition, or inherited genetic defects.

• Sporadic: No family history, random mutations.

• Familial: Multiple cases in a family, but not strictly inherited.

• Hereditary: Caused by inherited mutations (e.g., BRCA1/2).

Paired vs. Non-Paired Genome Sequencing Approaches

Methods for identifying cancer mutations.

• Paired: Compare tumor and normal tissue from the same patient.

• Non-paired: Sequence only tumor tissue.

Driver Genes: Proto-Oncogenes, Oncogenes, Tumor Suppressor Genes

Genes that contribute to cancer development when mutated.

• Proto-oncogenes: Normal genes that promote cell growth; become oncogenes when mutated.

• Oncogenes: Mutated, overactive proto-oncogenes driving cancer.

• Tumor suppressor genes: Inhibit cell growth; loss leads to cancer.

Gene Amplification, Point Mutations, SMRs, SVs

Types of genetic alterations in cancer.

• Gene amplification: Increased copies of oncogenes.

• Point mutations: Single nucleotide changes.

• SMRs (Small Mutational Rearrangements): Small-scale DNA changes.

• SVs (Structural Variants): Large-scale rearrangements (deletions, duplications, translocations).

Tumor Genetic Heterogeneity/Homogeneity

Tumors may contain genetically diverse (heterogeneous) or similar (homogeneous) cell populations.

• Heterogeneity: Complicates treatment and diagnosis.

TP53, BRCA1, and BRCA2

Key genes in cancer susceptibility and progression.

• TP53: Tumor suppressor, "guardian of the genome."

• BRCA1/2: DNA repair genes; mutations increase breast and ovarian cancer risk.

Chromothripsis/Chromoplexy, MMBIR

Complex genomic rearrangements in cancer.

• Chromothripsis: Massive chromosome shattering and reassembly.

• Chromoplexy: Multiple, complex rearrangements.

• MMBIR: Micro-homology-mediated break-induced replication, a DNA repair mechanism.

Gene Therapy: Gene Augmentation, Suppression, Genome Editing

Therapeutic approaches to treat genetic diseases, including cancer.

• Gene augmentation: Add functional copies of genes.

• Gene suppression: Silence harmful genes.

• Genome editing: Directly correct mutations (e.g., CRISPR).