Gene Expression and Genetic Disorders Lecture Notes
GENE EXPRESSION
MicroRNAs (miRNAs)
Transcribed from DNA but not translated into proteins.
Class of post-transcriptional regulators.
Mechanism: Can inhibit gene expression by binding to specific locations on mRNA, preventing translation.
Biological Implications: MiRNAs are implicated in cancer and genetic translocations (mutations).
Types of mRNA
Polycistronic mRNA:
Found in prokaryotes.
Codes for more than one protein.
Contains multiple initiation and termination codons.
Monocistronic mRNA:
Present in eukaryotes.
Codes for a single protein.
Contains one initiation and one termination codon.
Gene Expression in Prokaryotes
Operon:
Functional arrangement of clustered genes in prokaryotes, includes regulatory and structural genes.
Trp Operon:
A repressible operon where genes are expressed in the absence of tryptophan.
When tryptophan is present, it activates the repressor, shutting down expression.
Lac Operon:
An inducible operon where genes are expressed in the presence of lactose.
Gene Expression in Eukaryotes
Enhancers:
Short DNA regions that bind with proteins to increase gene expression either distally or proximally.
Promoters:
Proximal DNA sequences that bind to RNA polymerase, initiating transcription.
Located near the transcription site on the same strand, upstream (towards the 3' region of the antisense strand).
TATA Box:
Binds transcription factors, enabling the positioning of RNA polymerase.
Typically found within 30 base pairs of the transcription start site.
Transcriptional Start Site:
The specific nucleotide within the promoter where transcription begins, located upstream of the ATG start codon.
5' Untranslated Region (UTR):
A segment of mRNA between the transcriptional start site and the translational start site.
Controls the rate of translation initiation.
Open Reading Frame (ORF):
The segment of a gene that codes for a protein, beginning with an ATG start codon and ending with a stop codon.
3' Untranslated Region (UTR):
The segment of mRNA following the stop codon that can also be regulatory.
Genetic Mosaicism and Chimerism
Genetic Mosaicism:
The presence of two or more genetically different populations of cells in one individual derived from the same fertilized egg.
Chimerism:
The fusion of more than one fertilized zygote during early embryonic development.
MUTATIONS
Mutagenesis:
The process of creating mutations, which can be caused by radiation, chemicals, excess heat, and viruses.
Forward Genetics:
The method of introducing random mutations and screening for the desired phenotype, followed by mapping the mutations to identify affected genes.
Reverse Genetics:
The process of mutating a specific gene of interest to determine resulting phenotypes, for example through targeted transposon insertion, deletion, or CRISPR-Cas modifications.
Types of Mutations
Frameshift Mutation:
The addition or deletion of nucleotides that causes a shift in the reading frame.
Nonsense Mutation:
Introduces a premature stop codon into the sequence, leading to dysfunctional proteins.
Repeat Expansion:
A mutation where the number of times a short DNA sequence is duplicated increases.
Transversion:
A mutation where a purine is replaced with a pyrimidine or vice versa.
Silent Mutation:
A mutation where the nucleotide change does not affect the amino acid encoded.
Missense Mutation:
A mutation that results in a codon change leading to a different amino acid and polypeptide sequence.
GENETIC DISORDERS
Genetic disorders can be categorized as dominant, recessive, sex-linked, epistatic, or variably expressed.
Nondisjunction:
Improper separation of chromatids during meiosis, leading to gametes with extra or missing chromosomes (e.g., Down's syndrome).
Trinucleotide Repeats:
Repeated sequences of nucleotides can lead to conditions such as Huntington's disease and fragile X syndrome.
Defective Genes:
Genes that fail to produce the correct protein, exemplified by conditions like sickle cell anemia and hemophilia (due to gene mutations).
Crossover Frequency:
Frequency of crossing over during meiosis, influencing genetic variability.
DNA REPLICATION
Overview:
DNA replication is semi-conservative, resulting in daughter cells containing one old strand and one new strand of DNA.
Process:
Unwinding: DNA helicase unwinds the DNA by breaking hydrogen bonds, forming a replication fork.
Prokaryotic DNA Replication:
One origin of replication and two replication forks formed bi-directionally.
Single-Strand Binding Proteins (SSB):
Stabilize unwound strands that serve as templates.
RNA Primase:
Synthesizes a primer (5-10 base pairs) complementary to the template strand.
DNA Polymerase III:
Carries out DNA replication, adding nucleotides in a 5' to 3' direction.
Leading and Lagging Strands:
Leading strand runs 5' to 3', whereas lagging strand runs 3' to 5' and is synthesized in Okazaki fragments.
Gap Filling:
DNA polymerase I fills gaps and removes RNA primers.
Topoisomerase:
Initiates unwinding of supercoiled DNA during replication.
Proofreading:
DNA polymerase has activities to ensure the accuracy of DNA replication.
EUKARYOTIC DNA REPLICATION
Initiation: Occurs at multiple origins of replication, creating replication bubbles.
Elongation:
DNA polymerases add nucleotides at the 3' ends; DNA polymerase requires an RNA primer for synthesis.
Termination: The meeting of leading strands of different replication bubbles halts replication; gaps are filled by DNA polymerases and nicks are joined by DNA ligase.
TRANSCRIPTION
Definition: The process of synthesizing RNA from a DNA template using RNA polymerase.
Modification in Eukaryotes:
Introns are removed, a poly-A tail is added to the 3' end, and a 5' cap is added before export from the nucleus.
Exons: The coding segments for protein synthesis.
TRANSLATION (PROTEIN SYNTHESIS)
Process Overview: The mRNA sequence guides the assembly of amino acids into a protein.
Differences in Translation:
Prokaryotic translation occurs simultaneously with transcription, while eukaryotic translation is a separate process.
Phases:
Initiation: mRNA associates with ribosomes and tRNAs bind to mRNA codons.
Elongation: tRNA adds amino acids to the growing peptide chain.
Termination: Occurs when the ribosome reaches a stop codon (UAA, UGA, UAG); release of the newly synthesized protein.
mRNA can be reused multiple times before degradation.
MITOCHONDRIAL DNA
Definition: Small circular DNA inherited maternally, crucial for energy production pathways.
Mutations: Mitochondrial mutations can lead to mitochondrial diseases affecting energy production in organs like muscle and brain.
AUTOSOMAL DOMINANT DISORDERS
Examples:
Huntington's Disease: Brain disorder; loss of abilities with adult onset.
Marfan Syndrome: Connective tissue disorder affecting cardiovascular systems.
Polycystic Kidney Disease: Cysts formation on kidneys.
Others include achondroplasia, porphyria, and neurofibromatosis.
AUTOSOMAL RECESSIVE DISORDERS
Examples:
Tay-Sachs Disease: Degenerative disorder, leading to death by age 5.
Sickle Cell Anemia: Red blood cells adopt a sickle shape causing blockage.
Others include cystic fibrosis, galactosemia, and phenylketonuria (PKU).
SEX-LINKED DISORDERS
X-Linked Dominant Disorders:
Ichthyosis Simplex: Severe dry skin disorder.
Hypertrichosis: Generalized hairiness.
X-Linked Recessive Disorders:
Hemophilia: Blood-coagulation defect;
Duchenne's Muscular Dystrophy: Affects muscle strength and function, typically diagnosed in early childhood.
Fragile X Syndrome: Genetic disorder linked to cognitive impairments.
Lesch-Nyhan Disease: Enzyme deficiency leading to self-destructive behavior.
Y-LINKED DOMINANT DISORDERS
Examples:
Hairy ears: Excessive hair growth on the ears.
SEX CHROMOSOME ANOMALIES
Conditions:
Jacob's Syndrome (XYY): Extra Y chromosome.
Turner Syndrome (X): Missing X chromosome.
Edwards (Trisomy 13) and Patau Syndrome (Trisomy 18).
BIOTECHNOLOGY
Sanger Sequencing
Description: Involves amplifying DNA, denaturing it, and sequencing the strands using ddNTPs.
Process: Employs a primer, then adds polymerase and dNTPs until a ddNTP halts further elongation. The length of the fragments indicates the sequence.
High Throughput Technologies
Transcriptomics (RNA-seq): Measures gene expression levels using Next-Generation Sequencing (NGS).
Advantages: Wider measurable range, no dependency on known genome, eliminates hybridization artifacts.
CRISPR-Cas9**
Description: A genome-editing technology employing gRNA and Cas protein to modify specific DNA sequences.
Utility: Allows for targeted modifications through specific gRNA design.
Other Techniques
Microarray Analysis: Utilized for cancer analysis by assessing mRNA from tumors & identifying gene activity.
Transposon Mutagenesis: Used to study gene function by inserting transposons and assessing fitness.
INHERITANCE PRINCIPLES
Law of Segregation: Alleles segregate independently during gamete formation (disproven by linked genes).
Pleiotropy: The impact of a single gene affecting multiple traits (e.g., sickle cell anemia).
Epistasis: Interactions between genes where one gene can mask the expression of another, exemplified by albinism.
Polyploidy: The condition of having more than two homologous sets of chromosomes in an organism or cell.
PROKARYOTIC GENE EXPRESSION
Generally polycistronic with multiple open reading frames (ORFs) per promoter.
Operons facilitate co-transcription of genes, with no introns present. Multiple ribosomal binding sites are present in the mRNA.
Bacterial Gene Transfer Methods
Conjugation: Direct transfer of genetic material between bacteria.
Transduction: Transfer of genetic material through bacteriophages.
Transformation: Uptake of free DNA from the environment by bacteria.
BLASTING TECHNIQUES
Northern Blotting: Transfers RNA to a membrane.
Southern Blotting: Transfers DNA to a membrane.
Western Blotting: Detects proteins on a membrane.
Eastern Blotting: Detects post-translational modifications of proteins.
STOP CODONS
UAG, UAA, UGA: Signaling the termination of protein synthesis.
DNA POLYMERASE
In prokaryotes: Types I, II, III perform various roles in DNA replication and repair.
Eukaryotic enzymes include polymerases alpha, beta, gamma involved in replication and repair mechanisms.
DNA TECHNOLOGY
Identifying desired DNA.
Cutting DNA using restriction enzymes.
Inserting DNA into vectors (like plasmids).
Connecting DNA pieces with ligase.
Transforming into host cells for cloning.
Storing clones.
Identifying cloned genes with radioactive probes.
Analyzing DNA fragments through electrophoresis.
CLONING
Eukaryotic Gene Cloning Process
Gene Selection: Eukaryotic genes (introns need removal for bacterial insertion).
Reverse Transcriptase: Used to create cDNA from mRNA, removing introns.
Plasmid Isolation: Use plasmids as vectors for genetic engineering.
Restriction Enzymes: Cut DNA for insertion into vectors.
Transformation into Host Cells: Clone the bacteria for amplification.
CHROMOSOME STRUCTURE
Kinetochore: Proteins associated with chromosome centromeres during division.
Chromatid: Each strand of a duplicated chromosome.
Telomeres: Repetitive sequences that protect chromosome ends from degradation.
QTL Analysis: Identifying chromosomes linked with complex traits in genetic crosses.
CODONS
Codon chart includes:
Amino Acids: List of codons and their corresponding amino acids.
RNA MICROASSAY
Analyzing gene interactions through droplet placement on glass slides, binding with complementary strands, and measuring activity
PCR Steps
Denaturation: Heating to unwind DNA.
Annealing: Cooling to allow primers to bind.
Extension: Adding complementary nucleotides and reheating.
RADIOACTIVE PROBES
Short strands that bind to complementary DNA, visualizable on X-ray film.
ELECTROPHORESIS
A technique for separating DNA, RNA, or protein fragments based on size and charge using an electric field.
Larger fragments migrate slower due to size constraints.