DAT Biology

Ionic bonds

Transfer of e- from one atom to another where both atoms have different electronegativities

Electronegativity

Attraction an atom has for elections

Covalent bonds

* e- shared b/w atoms of similar electronegativities

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* Can have single, double or triple bonds

Non-polar covalent bonds

Equal sharing of e- b/w two atoms of similar electronegativity

Polar covalent bonds

Unequal sharing of e- b/w two atoms of different electronegativities → forms a dipole

H-bonding

Weak bond b/w hydrogen attached to a highly electronegative atom and a negatively charged atom on another molecule (F, O or N)

5 Properties of H2O

* Excellent solvent
* High heat capacity
* Ice floats due to low density (water expands due to H-bonding that forms a lattice)
* Cohesion / surface tension: attracted to like substances
* Adhesion: attracted to unlike substances

Macromolecules

Polymers formed from monomers (1 unit) e.g., polysaccharide

How are macromolecules formed?

==**Dehydration rxn:**== forms bonds; H2O by-product (monomers → polymers)

How are macromolecules broken down?

==**Hydrolysis:**== breaks bonds using H2O (polymers → monomers)

Examples of monosaccharides

* Glucose and fructose
* OH down = alpha
* OH up = beta

Examples of covalent bonds

* Peptide bonds
* Glycosidic linkage

How is the glycosidic linkage formed?

dehydration / condensation rxn

How many H2O molecules are lost for every glycosidic linkage formed?

1 H2O

Disaccharide

2 sugars joined by **glycosidic linkage**

What is **sucrose** made of?

glucose + fructose

What is **lactose** made of?

glucose + galactose

What is **maltose** made of?

glucose + glucose

Examples of 𝜶-glucose polysaccharides

* **Starch:** stores energy in plants
* **Glycogen:** stores energy in animals

Examples of β-glucose polysaccharides

* **Cellulose:** walls of plant cells
* **Chitin:** β-glucose w/ nitrogen groups

Lipids

* ==**Functions:**== insulation, energy storage, form cholesterol and phospholipids in membranes, participate in endocrine signalling

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* Covalent C-C bonds

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* ==**Triglycerides / triacylglycerols:**== 3 FA + glycerol backbone
* __Saturated:__ no double bonds; straight chains; unhealthy cuz chains stack densely(can form fat plaques)
* __Unsaturated:__ contains double bonds w/ kinks in chains; healthier cuz chains stack less densely; cis or trans
* ==**Phospholipids / diacylglycerols:**== 2 FA + phosphate group + glycerol backbone
* Amphipathic: both hydrophobic and hydrophilic
* ==**Steroids:**== three 6-membered rings + one 5-membered ring
* Hormones and cholesterol

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* ==**Cell membrane fluidity:**== cell membranes change membrane FA composition to maintain steady degree of fluidity
* **Cold weather (rigid membranes):** __cholesterol & mono + polyunsaturated FA__ added into membrane to avoid cell membrane rigidity → increased fluidity
* **Warm weather (fluid & flexible membranes):** __cholesterol__ added into membrane to restrict movement/flexibility; __saturated FA__ tails become straight and pack tightly → decreased fluidity

Lipid derivatives

* **Waxes:** esters of FA + alcohol for protective coating on skin (lanolin)

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* **Carotenoids:** FA chains w/ conjugated double bonds; pigment producing colours in plants & animals

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* **Adipocytes:** specialized fat cells
* __White fat cells:__ primarily contain triglycerides w/ thin layer of cytoplasm around it
* __Brown fat cells:__ have lots of cytoplasm, lipid droplets scattered throughout and lots of mitochondria

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* **Glycolipids**: 2 FA + carbohydrate group + glycerol backbone

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* **Lipoproteins:** lipid cores surrounded by phospholipids and apolipoproteins

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* **Porphyrins / tetrapyrroles:** 4 joined pyrrole rings w/ a metal center atom
* Chlorophyll and hemoglobin

Proteins

* Monomer: amino acid

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* Polymer: peptide

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* Linkage: ==**peptide bonds (covalent bonds)**==

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* Amino group + carboxyl groups + 𝜶 - Carbon bound to R chain (AA)
* Functions: storage, transport, defense (antibodies), enzymes **(exception: RNA e.g., ribosomes can act as an enzyme)**

Factors affecting enzymatic activity

* Substrate and enzyme concentration
* Temperature
* pH
* Presence/absence of inhibitors

Cofactor

Non-protein molecule assisting enzymes → donate or accept electrons or fxnl groups

Holoenzyme

Union of cofactor + enzyme

Apoenzyme / apoprotein

When an enzyme is **not** combined w/ a cofactor

Inorganic cofactor

Metal ions (Fe2+ or Mg2+)

Coenzyme

Organic cofactor e.g., vitamins

Protein classification

* **Simple:** formed only of AA
* Albumins and Globulins: fxnl proteins
* Scleroprotein: structural protein
* **Conjugated:** simple protein + non-protein
* Lipoprotein: protein bound to lipid

Protein Structure

* **Primary:** linear chain of AA connected by ==**peptide bonds**==

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* **Secondary:** 3D shape due to **H-bonding** b/w amino and carboxyl groups of adjacent AA into ==𝜶-helices or β-sheets== / H-bonding of peptide backbone

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* **Tertiary:** 3D structure due to ==**non-covalent interactions**== b/w AA side chains
* Also has **disulfide bonds:** covalent bond b/w cysteines

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* **Quaternary:** 3D shape due to grouping of 2 or more separate peptide chains (e.g., 𝜶-helix + β-sheets)

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* All proteins have a 1º structure, most have 2º & large proteins have 3º or 4º structures

Non-covalent bonds

H-bonds, ionic bonds, hydrophobic interactions, Van Der Waals forces

Protein types

* **Globular**

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* **Fibrous/structural proteins**

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* **Membrane proteins:** membrane pumps, channels or receptors

Fibrous / Structural proteins

* Water insoluble
* Dominantly 2º structure
* Long polymers
* Maintain and add strength to cellular and matrix structure (collagen or keratin)

Globular proteins

* Somewhat water soluble
* Dominantly 3º structure
* Functions: enzymes, hormones, storage, antibodies, osmotic regulation

Protein denaturation

* Protein reversed back to its 1º structure
* Usually irreversible but can be reversed w/ removal of denaturing agent
* All folding information is encoded in primary structure

Protein digestion

Eliminates **all** protein structure, including 1º structure

Nucleic acids

* ==**Functions:**== encode, express and store genetic information

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* **Monomer:** ==nucleotides== (nitrogen base + 5-carbon sugar + phosphate group)
* ==Nucleosides:== sugar + nitrogen base

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* **Polymers:** ==nucleic acid== (DNA and RNA)

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* ==**Linkage:**== phosphodiester bonds

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### Nitrogenous bases

* DNA: adenine, thymine, guanine, cytosine
* A - T: 2 H-bonds
* G - C: 3 H-bonds
* RNA: adenine, uracil, guanine, cytosine
* A - U: 2 H-bonds
* G - C: 3 H-bonds
* ==**Chargaff’s Rule:**== A & T and G & C are always present in equal amounts

Purines

* 2 rings
* Adenine and Guanine (**Pur**e **A**s **G**old)\*\*

Pyrimidines

* 1 ring
* Cytosine, Uracil, Thymine **(CUT**)**

DNA

Deoxyribose sugar + 2 antiparallel strands of a double helix

RNA

* Ribose sugar
* Single stranded

Cell Theory

* All living organisms are composed of one or more cells
* Cell is the basic unit of structure, fxn, and organization in all organisms
* All cells come from pre-existing, living cells
* Cells carry hereditary info

RNA World Hypothesis

* Self-replicating RNA molecules were precursors to current life
* RNA stores genetic info like DNA and catalyzes rxns like an enzyme
* RNA played a major role in evol. of cellular life
* Evidence: RNA is unstable relative to DNA due to its extra OH group → more likely to participate in chemical rxns

Central Dogma of Genetics

* Biological info cannot be transferred backwards from protein to either protein or nucleic acid
* Info must travel from DNA → RNA → proteins

Elements that can be visualized w/ naked eye

==**100 µm - 1m**==

* Adult female
* Ostrich egg
* Chicken egg
* Frog egg
* Human egg

Elements that can be visualized w/ light microscope

==**100 nm - 1mm**==

* Mitochondria
* Bacteria
* Plant and animal cell
* Human egg
* Frog egg

Elements that can be visualized w/ electron microscope

==**0.1 nm - 10 µm**==

* Atom
* Lipids
* Protein
* Virus
* Bacteria
* Mitochondria
* Plant and animal cell

Microscopy techniques

* Stereomicroscope (light)
* Compound Microscope (light)
* Phase Contrast Microscope
* Confocal Laser Scanning Microscope and Fluorescence
* Scanning Electron Microscope (SEM)
* Cryo SEM
* Transmission Electron Microscope
* Electron Tomography

Stereomicroscope (light)

* Uses visible light to view surface of sample

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* **Pro:** can view living samples
* **Con: l**ow resolution relative to a compound microscope

Compound Microscope (light)

* Uses visible light to view thin section of sample

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* Uses multiple lenses

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* **Pro:** can view **some** living samples (single cell layer)
* **Con:** requires staining for good visibility

Phase Contrast Microscope

* Uses light phases and contrast for detailed observation of living organisms, including internal structures if thin

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* **Pro:** good resolution and contrast
* **Con:** not ideal for thick samples and produces a ‘halo effect’ around perimeter of samples

Confocal Laser Scanning Microscope / Fluorescence

* Used to observe thin slices while keeping a sample intact
* Common method for viewing chromosomes during mitosis
* Can be used w/o fluorescence → laser light is used to scan dyed specimen

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* **Pro:** can observe specific parts of a cell using fluorescent tagged antibodies
* **Con:** can cause artifacts

SEM

* **Pro:** view surface of 3D objects w/ high resolution


* **Cons:** cannot use on living samples; preparation is extensive as sample needs to be dried and coated; costly

Cryo SEM

* **Pro:** sample is not dehydrated co can observe in their ‘natural form’


* **Cons:** cannot use on living samples; samples must be frozen which can cause artifacts

TEM

* Used to view thin x-sections and internal structures within samples at very high magnification

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* **Pro:** high resolution


* **Cons:** cannot use on living samples; preparation of sample is extensive; costly

Electron Tomography

* Not a microscope but a technique used to build a 3D model of sample using TEM data

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* **Pro:** can view objects in 3D and see objects relative to one another


* **Cons:** cannot use on living samples; preparation of sample is extensive; costly

Centrifugation

* Used to separate a liquified sample into its different components by spinning it rapidly

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* Spins and separates liquified cell homogenates into layers based on density

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* Cells separate from most dense to lease dense

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* **Cell fractionation:** largest component of cells pellet first at the bottom and progressively spin faster: nuclei layer → mitochondria → larger macromolecules/viruses/ribosomes

Differential centrifugation

* Relies on density, shape, speed at which macromolecule travels

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* Forms continuous layers of sediments where insoluble proteins are found in the pellet while soluble proteins remain in the supernatant liquid above the pellet

Density centrifugation

Only relies on density

Rate of reaction in equilibrium

Rate of formation of reactants and products is equal

Enzymes

* Catalysts lowering the activation energy of a rxn → ↑ rate of rxn
* Substrate specific
* Remain unchanged during rxn
* Have an active site that binds substrates via induced fit
* Catalyze forward and reverse reactions
* Have varying fxn depending on pH and temperature

Prosthetic group

Cofactors that bind tightly or covalent to an enzyme

ATP

* Potential energy stored as chemical energy

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* Source of activation energy

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* Formed by phosphorylation of ADP using energy from glucose → ==**endergonic**==

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* Broken apart via hydrolysis → ==**exergonic**==

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* ATP stores energy generated from the ==exergonic reactions== in the electron transport chain, and can then be used to fuel ==endergonic reactions==

Enzyme regulation

* Km (Michaelis constant)
* Allosteric enzymes
* Competitive inhibition
* Noncompetitive inhibition
* Uncompetitive/anti-competitive inhibition

Km (Michaelis constant)

* Substrate \[ \] at which the rate of rxn = 1/2 max velocity of the enzyme (Vmax)
* Inversely represents binding affinity
* Small Km = less substrate needed to reach Vmax → higher binding affinity
* High Km = more substrate needed to reach Vmax → low binding affinity

Allosteric enzymes

* Have both an ==active site== for ==substrate binding== and an ==allosteric site== for an ==allosteric effector== (can be an activator or inhibitor

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* ==**Allosteric site:**== secondary location where an effector binds

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* ==**Active site:**== area of an enzyme where the substrate binds

Mechanism of enzyme rxn

1. Substrates (aka reactants) enter the active site of the enzyme

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2. Enzyme and substrate change shape slightly to better catalyze the reaction ==**(induced fit model)**==


1. When the substrate binds the enzyme → ==**Enzyme-Substrate Complex**==

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3. The enzyme facilitates the rxn by lowering the activation energy

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4. Products are released and the cycle repeats

Exergonic rxn

* Free energy is released
* Spontaneous with -𝛥G

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* Can be used to drive otherwise nonspontaneous rxns (ATP hydrolysis - exergonic - to facilitate endergonic rxns)

Endergonic rxn

* Free energy is absorbed
* Nonspontaneous with +𝛥G

PCR

* Creates a large amount of DNA by amplifying a DNA sample:

1\. Denaturation: High heat separates ds DNA

2\. Annealing: Sample is cooled so primers attach to separated strands

3\. Elongation: Polymerase synthesize new strands

* Cycle repeats to increase amount of DNA exponentially

Reverse Transcriptase

* Used to synthesize DNA from an RNA template
* Sometimes used to create complementary DNA (cDNA) off an mRNA template
* cDNA lacks introns
* Naturally used by viruses

DNA sequencing

* Used to determine the sequence of base pairs in a DNA or RNA molecule

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* Example: ==**Dideoxy Chain Termination**== is based on the principle that during DNA synthesis, addition of a nucleotide requires a free OH group on the 3ʹ carbon of the sugar of the last nucleotide of the growing DNA strand

Blotting techniques

* Used for identifying specific fragments of DNA, RNA or protein

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### Types:

* Southern: DNA
* Northern: RNA
* Western: Protein

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### Method:

1\. Electrophoresis: separates sample

2\. Sample is transferred to nitrocellulose gel

3\. Probe is added to hybridize and mark target fragment

Hybridization

Nucleic acids form complementary base pairs with nucleic acids of different strand during blotting

Gel electrophoresis

* Used to separate DNA molecules by size and charge: the smaller the molecule, the farther it travels down the gel
* After separating the DNA sample, it can be sequenced or probed to find location of specific sequence

Microarray Assays

Used to monitor the expression of large groups of genes across a genome

Recombinant DNA & Gene Libraries

* Recombinant DNA contains segments from multiple sources

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* Recombinant DNA is vital for creating ==**gene libraries**== (collections of DNA pieces from a genome)

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* **Process to make and use recombinant DNA:**


1. Using ==**restriction endonucleases**== (restriction enzymes) to cut specific segments of DNA called ==**restriction sites**==
* These enzymes create sticky ends, which allow new DNA pieces to bind
2. ==**DNA ligase**== connects the different fragments together
3. A vector can then be used to transfer foreign DNA into another cell

* ==**Vectors:**== plasmids and bacteriophages

Recombinant DNA in bacterial cloning

1. ==**Restriction enzyme**== is applied to __**both**__ the bacterial plasmid and the foreign DNA to create the same sticky ends
2. ==**DNA ligase**== attaches the fragments to create plasmid with new DNA
3. Plasmid is introduced into bacteria using ==**transformation**==
4. Bacteria can be grown to produce a product or form a colony


1. To ensure that the bacteria has included the plasmid, a gene for antibiotic resistance is added to the plasmid. Bacteria without the plasmid will perish under antibiotic conditions

Competitive inhibition

* Substance that mimics the substrate and inhibits the enzyme by binding at the active site
* Effect can be overcome by ↑ \[substrate\]
* Km ↑
* Vmax stays same

Vmax

Max velocity of a rxn at peak substrate saturation

Noncompetitive inhibition

* Substance inhibits enzyme by binding elsewhere than the active site → substrate still binds but the rxn is prevented from completing
* Vmax ↓
* Km stays same

Uncompetitive / anti-competitive inhibition

Occurs when an enzyme inhibitor binds only to the formed enzyme-substrate (ES) complex → prevents formation of product

Cooperativity

When an enzyme become more receptive to additional substrate molecules after one substrate molecule binds to the active site

Peripheral membrane proteins

* Loosely attached to the surface of one side of the membrane

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* Hydrophilic

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* Held in place by H-bonds and electrostatic interactions

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* Can disrupt/detach them by changing \[salt\] or pH

Integral membrane proteins

* Embedded in the cell membrane
* Hydrophobic
* Can be destroyed using detergent

Transmembrane proteins

Type of integral membrane that travels all the way through the cell membrane

Membrane proteins

* Channel proteins
* Ion channels
* Porins
* Recognition proteins
* Carrier proteins
* Transport proteins
* Adhesion proteins
* Receptor proteins

Channel proteins

Provide a passageway through the membrane for hydrophilic, polar and charged substances

Recognition proteins

* Glycoprotein (have an attached oligosaccharide) used to distinguish b/w self and foreign


* Example: MHC on macrophages

Ion channels

* Used to pass ions across the membrane
* Gated channels in nerve and muscle cells
* Voltage gated: respond to difference in membrane potential
* Ligand-gated: chemical binds to open channel
* Mechanically gated: respond to pressure or vibration

Porins

* Allow passage of certain ions and small polar molecules
* Increase rate of H2O passing in kidney and plant root cells
* Less specific

Carrier proteins

* Specific to mvt across membrane via integral membrane protein
* Changes shape after binding to specific mol. (glucose into cell)

Transport proteins

* Use ATP to transport materials across the membrane
* ==**Active transport (Na+/K+ pump):**== reqiures ATP
* ==**Facilitated diffusion:**== doesn’t require ATP

Adhesion proteins

Attach cells to neighbouring cells and provide anchors for stability via internal filaments and tubules

Receptor proteins

Serve as binding sites for hormones and other trigger molecules

Membrane properties

* ==**Phospholipid membrane permeability:**== allows small, uncharged, hydrophobic molecules to freely pass the membrane. Other molecules that are large, polar, or charged require a transporter
* Polar molecules can cross if they are small and uncharged

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* ==**Cholesterol:**== regulates fluidity of cell membrane (↑ temp = ↓ fluidity)
* Prokaryotes don’t have cholesterol in their membranes

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* ==**Glycocalyx**==: carbohydrate coat covering the outer face of the cell wall of bacteria and of the plasma membrane in animal cells
* **Fxns:** adhesive capabilities, barrier to infection, markers for cell-cell recognition

Nucleus

* Contains the cell’s DNA, and coordinates cell activities such as protein synthesis & reproduction

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* ==**Chromatin**==: general packaging structure of DNA around proteins in eukaryotes

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* ==**Chromosomes:**== tightly condensed chromatin when the cell is ready to divide

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* ==**Histones:**== organize DNA which coil around it into bundles called nucleosomes

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* ==**Nucleolus:**== inside the nucleus where ribosome is synthesized ==(using rRNA)==

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* Surrounded by double layer nuclear envelope w/ nuclear pores for transport

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* Contains nucleoplasm (no cytoplasm)

Nucleiod

Region w/n ==**prokaryotic cells**== containing genetic material

Nuclear lamina

* Dense fibrillar network inside of the nucleus of ==**eukaryotic cells**== that provides mechanical support

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* Helps regulate DNA replication, cell division and chromatin organization