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cell lecture 3
DNA & the Nucleus
Introduction to DNA and Nucleus
Overview of DNA's role and the structure of the nucleus as its storage site.
Milestones in Genetics
The Science of Genetics
Scientific methods evolve based on problem-solving and knowledge acquisition.
Knowledge from various fields interconnects, highlighting the non-static nature of scientific views.
Key Discoveries
1865: Gregor Mendel
Established patterns of inheritance; developed the laws of segregation and independent assortment.
1869: Friedrich Miescher
Isolated phosphorus-rich substances from white blood cells, termed them "nuclein"; later identified as nucleic acids.
Thomas Hunt Morgan (1866–1945)
Developed the chromosome theory, linking chromosomes to inheritance using Drosophila melanogaster.
1928: Frederick Griffith
Discovered transformation in Streptococcus pneumoniae.
1944: Avery, MacLeod, and McCarty
Identified DNA as the transforming substance, refuting the dominance of proteins as genetic material.
Barbara McClintock (1902–1992)
Developed chromosomal staining techniques and discovered transposons (jumping genes).
1950: Erwin Chargaff
Formulated Chargaff’s rules:
Base composition varies by species.
A=T and G=C in any given species.
1953: Watson and Crick, Wilkins & Franklin
Developed the double-helical model of DNA, synthesizing prior findings.
DNA Sequencing Developments
1961-1966: Nirenberg’s Codon Discoveries
Identified the triplet coding system for amino acids.
1985: Polymerase Chain Reaction (PCR)
Revolutionized DNA amplification, enabling detailed study from small samples.
1990-2003: Human Genome Project
Aimed to map all human genes, enhancing understanding of genetic contributions to diseases.
Bioinformatics
Emerged from the Human Genome Project, linking genomic data with practical applications (e.g., genetic testing).
Structural Biology of DNA
DNA Structure and Function
Deoxyribonucleic Acid (DNA) Characteristics
Antiparallel double helix structure.
Length measured in base pairs (bp) or kilobase pairs (kbp).
Nucleic Acids Overview
Key Components
Comprised of nucleotides with deoxyribose sugar in DNA; ribose sugar in RNA.
DNA features include different bases, backbones, and groupings.
Chromosome Structure
Prokaryotic Chromosomes
Characteristics
Circular, double-stranded DNA associated with proteins, existing in nucleoid. Also carry circular DNA plasmids.
Maintains access for transcription and replication.
Eukaryotic Chromosomes
Structure
Larger, linear DNA molecules characterized by 2 telomeres, centromeres, and gene regions.
Unique and Repeated Sequences
Unique Sequences
Include protein-coding exons and regulatory elements.
Repeated Sequences
Simple repeats, segmental duplications, and mobile elements like transposons.
Tandem repeats: back to back
Interspersed repeats: scattered around the genome
Many kinds of transposons: LINEs, SINEs, retroviral, and DNA
LINEs: long interspersed nuclear elements, retrotransposons, can copy themselves
SINEs: short interspersed nuclear elements, can not move on their own
Sanger Sequencing
Sanger sequencing, also known as the chain termination method, was developed in the 1970s by Frederick Sanger. It involves the synthesis of DNA strands using labeled dideoxynucleotides, which lead to the termination of the nucleotide chain during replication.
Basic Steps:
The DNA sample is mixed with a primer, DNA polymerase, and four dNTPs.
Dideoxynucleotides (ddNTPs), which lack a 3' hydroxyl group, are also included in small amounts.
When a ddNTP is incorporated, it prevents the addition of further nucleotides, resulting in fragments of varying lengths.
Gel Electrophoresis:
The resulting DNA fragments are separated by size using gel electrophoresis, allowing the identification of the terminating ddNTP by fluorescence.
Reading the Sequence:
The sequence of the DNA can be read based on the color of the fluorescence emitted from the terminating ddNTPs.
Applications:
Sanger sequencing was widely used for genome sequencing and remains important for sequencing small DNA fragments and verifying results from next-generation sequencing.
This technique laid the foundation for later advancements in DNA sequencing methodologies.
Chromatin Structure and Function
Chromatin Dynamics
chromatin = DNA + all protein (found in nucleus of eukaryotic cells)
—> at interphase, chromatin is organized by being packed but this dense packing makes it difficult to express genetic information coded in these regions
histones = DNA binding proteins
nucleosomes = DNA wound around a histone
Types
Euchromatin: Active transcription area. Contains transcriptionally active genes.
Heterochromatin: Dense packing inhibits transcription. Loops of chromatin fibres are formed by binding to cohesin protein.
Constitutive Heterochromatin: Centromeres and telomeres are sections of heterochromatin that have specific roles.
Centromeres and Telomeres:
Centromeres: appear as distinct constrictions of chromosomes. Maintain cohesion between sister chromatids. Assembly site for kinetochores. Defined by repetitive sequence (CEN) that varies between species.
Telomeres: repetitive sequence found at tips of chromosomes. Protect the ends of chromosomes from degradation. Sequence of repeats (TTAGGG).
Histones and Transcription Regulation
Nucleosome Forming
DNA wraps around 8 histones, forming nucleosomes pivotal for gene regulation.
Histones:
Acetylation “opens” chromatin to allow transcription and methylation of a histone tail tightens chromatin packing which causes gene slicing
Methylation and Acetylation create a histone code that can modify transcription activity
Epigenetics and DNA Methylation
Epigenetics refers to heritable changes in gene expression that do not involve alterations to the underlying DNA sequence. One of the key mechanisms of epigenetic regulation is DNA methylation, which involves the addition of methyl groups to the DNA molecule, typically at cytosine bases in a CpG dinucleotide context.
Effects of DNA Methylation:
DNA methylation can repress gene transcription, effectively turning off genes without changing their sequence.
This process is crucial for normal development, cellular differentiation, and maintenance of genome stability.
Epigenetic Reprogramming:
DNA methylation patterns can be dynamically regulated in response to environmental signals and during cell division, allowing cells to adapt and modify their gene expression profiles according to external demands.
Nuclear Structure
Nucleus Functions The nucleus serves as the genetic information storage and regulation centre in eukaryotic cells.
Nuclear Envelope and Pores Structure The nucleus is surrounded by a double-membraned nuclear envelope, which contains nuclear pores that allow for the transport of molecules between the nucleus and the cytoplasm.
Protein Transport Mechanisms Nuclear Localization Signals (NLS) are signal sequences that facilitate the import of larger proteins into the nucleus, ensuring the necessary proteins are available for processes occurring within the nucleus.
Nucleolus
The nucleolus is a dense, spherical structure located within the nucleus of eukaryotic cells. It is primarily involved in the synthesis and assembly of ribosomal RNA (rRNA) and the formation of ribosome subunits.
Functions:
Ribosome Biogenesis: The nucleolus is the site where rRNA is transcribed and combined with ribosomal proteins to form the large and small subunits of ribosomes.
Regulation of Cellular Processes: The nucleolus also plays a role in regulating various cellular processes, including the cell cycle, stress responses, and apoptosis.
Structure:
The nucleolus is not membrane-bound and is composed of three main regions: the fibrillar center (where rRNA transcription occurs), the dense fibrillar component (where early processing of rRNA takes place), and the granular component (where assembly of ribosomal subunits occurs).
Significance:
The size and number of nucleoli within a cell can vary based on the cell's activity level, especially during times of high protein synthesis. Overactive nucleoli can be indicative of cancerous cells due to their increased demand for ribosome production.
DNA Organization
Levels of DNA Organization:
Nucleosomes:
DNA wraps around histone proteins to form nucleosomes, the fundamental units of chromatin, which helps condense DNA.
Chromatin Structure:
Chromatin is classified as euchromatin (loosely packed, transcriptionally active) and heterochromatin (tightly packed, transcriptionally inactive).
Chromosomes:
During cell division, chromatin condenses into visible chromosomes, with each chromosome containing a single continuous molecule of DNA.
FISH (Fluorescence In Situ Hybridization)
FISH is a cytogenetic technique used to detect and localize specific DNA sequences on chromosomes. It employs fluorescent probes that bind to complementary DNA sequences in the sample.
Applications:
Gene Mapping:
FISH can help identify the location of genes on chromosomes, which is useful for mapping genetic disorders.
Cancer Diagnosis:
It is frequently used to detect chromosomal abnormalities associated with cancer, such as translocations or aneuploidies.
Monosomy and Trisomy Detection:
FISH can assist in identifying chromosomal abnormalities such as monosomy (missing chromosome) or trisomy (extra chromosome).
Procedure:
Sample Preparation:
Cells from culture or tissue specimens are fixed and prepared on slides.
Probe Hybridization:
Fluorescently labeled probes are applied to the slides to hybridize with the target DNA sequences.
Visualization:
The slides are examined under a fluorescence microscope, allowing researchers to visualize the locations of the labeled sequences, appearing as bright spots.
Nuclear Pores
Nuclear pores are large protein complexes that cross the nuclear envelope, facilitating communication between the nucleus and the cytoplasm. They serve as gateways for the transport of molecules, allowing selective passage of proteins, RNA, and other substances.
Structure and Function
Composition: Composed of proteins known as nucleoporins that create a complex structure.
Size: Each pore has a diameter of approximately 120 nm, allowing passive diffusion of small molecules while regulating the passage of larger molecules.
Transport Mechanism: Molecules transport through nuclear pores can be passive or active. Small molecules and ions can passively diffuse, while larger molecules require specific transport mechanisms that often involve signaling sequences.
Nuclear Localization Signals (NLS)
Nuclear Localization Signals (NLS) are short peptide sequences that facilitate the import of proteins into the nucleus. They are essential for ensuring that proteins required for nuclear functions are available within the nuclear compartment.
Key Points
Composition: Typically consists of basic amino acids (lysine and arginine) that interact with the import machinery.
Recognition: Importin proteins recognize NLS sequences and bind to them.
Transport: The importin-NLS complex is transported through the nuclear pore complex into the nucleus, where the importin is released, allowing the protein to function within the nucleus.
Simplified RAN/Importin Transport Mechanism
The RAN/Importin transport system helps proteins move into the nucleus through nuclear pores.
Steps of the RAN/Importin Process
Importin Binding: Importins find and attach to proteins with Nuclear Localization Signals (NLS) in the cytoplasm, creating a transport complex.
Transport through Nuclear Pores: This complex travels through the nuclear pore into the nucleus.
RAN-GTP Binding: Inside the nucleus, it meets RAN, a protein that is linked to GTP (RAN-GTP), which is more abundant there than in the cytoplasm.
Dissociation of Complex: RAN-GTP binds to importin, causing it to release the cargo protein within the nucleus.
Export of Importin: The complex of RAN-GTP and importin exits back to the cytoplasm through the nuclear pore.
RAN-GAP Hydrolysis: In the cytoplasm, a protein called RAN-GAP helps convert RAN-GTP to RAN-GDP (without the phosphate), allowing importin to detach and be recycled for new transport.
Importance
This process makes sure proteins that need to be in the nucleus get there to carry out their important jobs, keeping the cell functioning properly.
Nuclear Matrix and Lamina
Nuclear Matrix
The nuclear matrix is a framework of fibers found within the nucleus of eukaryotic cells. It provides structural support and helps organize the interior of the nucleus.
Key Features:
Composition: Consists of proteins and nucleic acids, forming a network that helps maintain the shape of the nucleus.
Functions:
Organizes chromatin and gene expression.
Plays a role in DNA replication, transcription, and RNA processing by anchoring nuclear components in specific locations.
Nuclear Lamina
The nuclear lamina is a dense fibrillar network located just beneath the inner membrane of the nuclear envelope. It is primarily composed of intermediate filament proteins called lamins.
Key Features:
Structure: Composed of lamins A, B, and C, which are proteins forming a mesh-like structure.
Functions:
Provides mechanical support to the nuclear envelope, maintaining the shape of the nucleus.
Involved in organizing chromatin and regulating gene expression.
Plays a crucial role during cell division by disassembling and reassembling with the nuclear envelope.