BIOL4341: Chapters 9-12 Notes

• Study notes for BIOL4341 Chapters 9, 10, 11 and 12
• Notes written by Scott Buckel, PhD

Types of Restriction Endonucleases

• Type I and III restriction endonucleases are large, multisubunit complexes with endonuclease and methylase activities
• Type II restriction endonucleases are simpler, not requiring ATP, and catalyze hydrolytic cleavage of DNA phosphodiester bonds in the recognition sequence

DNA Cloning via PCR

• Restriction endonuclease cleavage sites allow following of amplified DNA cloning.

Constructed E. coli Plasmid pBR322 Key Features

• The origin of replication sequence is where replication initiates
• Resistance genes provide means for selection
• Recognition sequences are for restriction endonucleases

Selectable and Screenable Markers

• Selectable markers identify cells that uptake plasmid DNA
• A selectable marker permits cell growth (positive selection) or kills cells (negative selection) under specific conditions
• A screenable marker uses a gene encoding for a protein, causing cells to produce a colored or fluorescent molecule

Terminal Tags for Affinity Purification

• Tags are peptides or proteins binding simple, stable ligands with high affinity and specificity
• Used for purifying target proteins by affinity chromatography
• Fusing a gene encoding the target protein with a tag allows affinity purification

cDNA Library Construction from mRNA

• Complementary DNAs (cDNAs) are double-stranded DNA fragments from mRNA templates
• Construction relies on reverse transcriptase
• A cDNA library is a population of clones made by inserting cDNA fragments into vectors for cloning

Three Levels of Protein Function

• Phenotypic function describes a protein's effects on the entire organism
• Cellular function describes a protein's interaction network at the cellular level
• Molecular function describes a protein's precise biochemical activity

Protein Function and Sequence/Structural Relationships

• Comparative genomics uses genome databases to assign gene functions and perform genome comparisons
• BLAST (Basic Local Alignment Search Tool) is an algorithm allowing rapid genome database searching
• Genome annotation converts residue sequences into useful information about gene location and function and critical sequences

Fusion Proteins and Immunofluorescence

• Green fluorescent protein (GFP) from jellyfish marks location
• Fusing a target gene to GFP creates a highly fluorescent fusion protein
• Variants exist with other colors/characteristics

Immunofluorescence

• Visualize endogenous protein using fixation
• Epitope tags are short protein sequences bound by an antibody

Effects of Protein Deletion

• Mutating or deleting a gene provides a way to understand its function
• CRISPR/Cas systems have now replaced the need for traditional approaches

CRISPR/Cas Systems Defined

• CRISPR stands for clustered, regularly interspaced short palindromic repeats
• CRISPR sequences are regularly spaced short repeats in the bacterial genome surrounding sequences from phage pathogens
• Cas protein is a nuclease

Current CRISPR Technology

• Only needs two components being a single Cas protein (Cas9) along with a single guide RNA (sgRNA)
• sgRNA consists of gRNA and tracrRNA fused into a single RNA
• Cas9 has two separate nuclease domains that cleave one DNA strand each
• The sgRNA guide sequence can be altered to target any genomic sequence
• This must be paired with the target DNA sequence to activate the nuclease domains

Fatty Acids

• Fatty acids are hydrocarbon derivatives
• Oxidation of fatty acids to CO2 and H2O is highly exergonic

Nomenclature: Unbranched Fatty Acids

• Chain length and number of bonds are separated by a colon
• Numbering begins at the carboxyl carbon
• Double bond positions are indicated by Δ and a superscript number

Polyunsaturated Fatty Acids

• Polyunsaturated fatty acids (PUFAs) have more than one double bond in their backbone
• Omega-3 (ω-3) fatty acids has a double bond between C-3 and C-4 relative to the most distant carbon (ω)
• Omega-6 (ω-6) fatty acids has a double bond between C-6 and C-7 relative to ω

Triacylglycerols Composition and Properties

• Triacylglycerols are the simplest lipids constructed from fatty acids
• They consist of three fatty acids, each with an ester linkage from a single glycerol
• They are nonpolar and hydrophobic
• It can be simple, with one kind of attached fatty acid, or mixed, with two or three different fatty acids

Solubility of Fatty Acids

• Poor solubility in water from the nonpolar hydrocarbon chain
• As chain length rises, the solubility starts to diminish along wit a decreased double bond number
• The carboxylic acid group is polar and ionized at neutral pH

Fatty Acids and Melting Points

• Saturated fatty acids have a waxy consistency at room temperature
• Unsaturated fatty acids are oily liquids at room temperature
• Extent of packing depends on the degree of saturation

Advantageous Use of Triacylglycerols

• Carbon atoms of fatty acids are more reduced than those of sugars, therefore oxidation yields more energy
• Triacylglycerols are hydrophobic and unhydrated, so the organism doesn't need to carry the added weight of water hydration that is normally associated with stored polysaccharides

Biological Membranes

• Biological membranes are comprised of a double layer of lipids and act as a barrier to both polar molecules and ions
• The one end of the molecules is amphipathic, hydrophobic with the other hydrophilic
• Hydrophobic regions associate with each other
• Hydrophilic regions interact with water

Glycerophospholipids Properties

• Glycerophospholipids, known also as phosphoglycerides, are a type of membrane lipid where two carbon atoms are attached to a fatty acyl through an ester linkage
• The first and second carbons, glycerol, and a highly polar group to the third carbon

Sphingolipids Properties

• Sphingolipids make up a large class of membrane phospholipids and glycolipids
• They contain a polar head group and two nonpolar tails, but contains no glycerol
• They also contains one molecule of the long-chain amino alcohol sphingosine, or one of its derivatives

Ceramides

• C-1, C-2, and C-3 of sphingosine are structurally analogous to the three carbons of glycerol in glycerophospholipids
• Ceramide is a compound when a fatty acid is attached in amide linkage to the -NH2 on C-2 and is structurally similar to diacylglycerol

Glycosphingolipids Composition

• Glycosphingolipids have head groups with 1+ sugars directly connected to the -OH at C-1 of the ceramide
• There is no phosphate, but typically occurs largely in the outer face of plasma membranes.

Glycolipids

• Sphingolipids are prominent in plasma membranes of neurons
• Human blood groups (O, A, B) are determined by the oligosaccharide head groups of glycosphingolipids

Phospholipid and Sphingolid Degradation

• Phospholipases of the A type remove one Ffa from another
• Lysophospholipases remove remaining fatty acids
• Lysosomal enzymes catalyze the stepwise removal of sugar units of gangliosides

Cholesterol Properties

• CHoolesterol is a major sterol in animal tissues
• It is amphipathic, containing a polar head group and a nonpolar hydrocarbon body
• Cholesterol is also a membrane constituent
• It also is similar to stigmasteol in plants and ergosterol in fungi.

Bisphosphate Importance

• Phosphatidylinositol 4,5-bisphosphate (PIP2) is found in the cytoplasmic face of plasma membranes, where it serves as a reservoir of messenger molecules for reactions
• Phospholipase C hydrolyzes PIP2 to IP3, or inositol 1,4,5-trisphosphate and diacylglycerol, intracellular messengers)

Nucleation

• Signaling proteins bind specifically to phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the plasma membrane
• Initiates formation of multienzyme complexes at the membrane's cytosolic surface creating a signal transduction pathway.

Eicosanoids and Their Four Classes

• Four major classes of eicosanoids include prostaglandins, thromboxanes, leukotrienes, & lipoxins

Prostaglandins and Their Functions

• Prostaglandins (PG) are a class of eiconasoids that contain a five carbon ring
• They stimulate contraction of the smooth muscles of the uterus
• Also affect blood flow to specific organs and the wake-sleep cycle, while also affecting the responsiveness of certain tissues of hormones
• Finally elevate body temperature to cause inflammation and pain

Lipid Quinones Characteristics

• Vitamin E is a collective name for group of lipids known as tocopherols
• Tocopherols are hydrophobic compounds that contain a substituted aromatic ring and a long isoprenoid side chain
• They also contain lipid deposits as with those of lipoproteins
• Being biological antioxidents, they are key to their function

Key Vitamin K Function

• Contains an aromatic ring to undergo a cycle of oxidation and reduction during the formation of active prothrombin, making it a blood plasma protien and therefore essential in blood clotting

Micelle Formation

• Micelles are spherical structures containing amphipathic molecules arranged with hydrophobic regions in the interior
• Hydrophilic head groups are on the exterior
• Formation is favored when the cross-sectional area of the head group is greater than that of the acyl side chain(s)

Vesicle Formation

• Vesicles or liposomes form spontaneously when a bilayer sheet folds back on itself to form a hollow sphere

Bilayer Formation

• Bilayers are a type of lipid aggregate where two lipid monolayers (leaflets) form a two demensional sheet
• Formation is favored when the cross-sectional areas of the head group and acyl side chain(s) are similar

Fluid Mosaic Model

• A pattern formed by individual lipid and protein units in a membrane
• Pattern can change while maintaining the permeability The Endomembrane System and its Importance Single membrane surrounds are made of endoplasmic reticulum (ER), Golgi apparatus, lysosomes, and various small vesicles Double membrane surrounds are made of the nucleus, mitochondrion, along with chloroplasts

Membrane Trafficking

• Membrane trafficking is the process where membrane lipids and protien are synthesized in ER and then transported to the destination organelles in the plasma membrane
• Lipids and protiens undergo covalent modifications in the Golgi Apparatus
• These modifications dictatate the locatation of the meture protiens

Proteins on the Membrane

• Membrane protiens can provide receotors for extracellular signals
• Transporters to carry specific polar or charged compounds
• Transporation across the plasma membrane or between organelles
• Enzymes

Different Aspects of Membrane protiens in Bilayers

• Integral Membrane protiens are firmly embedded within the lipid Bilayer
• Peripheral membrane protiens associate with the membrane with electrostatic actions and hydrogen bonding
• Amphitropic protiens associate reversibly with membranes

The different levels of Protiens in the membrane

• Monotopic use has small Hydrophobic domains that interact with only a single leaflet of the membrane
• Bitopic spans the Protien Bilayer to extend on either surface
• Contains a single hydrophobic sequence in this molecule
• Polytopic cross the membrane several times and have multiple transmembrane helices

Fluiditiy

• Acyl groups in the bilayer are ordered
• The liquid ordered version is gel like while the hydro carbons are not constrained
• The Liquid Disorderd is the state where Individual Hydrocarbon chains are in constant Motion when at the state ( Lateral to rotational )

Lipids and movement

• Lipids can move freely within their monolayer and therefore are mobile and can carry both rotation and diffusion,
• Transverse diffusion happens during proteins where they are called the translocators, or Flippases

Temperature can afffect membrane

• at Physiological Temperatures the Long chain Fatty acids tend to pack into a Ld state, Where as the kinks will interfere favoring ld
• Then shorter chains also favor the LD state

3 types of Active Transporters

• Flippases Catalyse translocation of the Amino acids (P) Sfrom extertnal to internal membrane
• These require 1 atp
• Floppases uses ATP and removes phosphorolopids and sterols from Cytoplasmic to Extracellular
• Scramblaces Move over all and are not atp dependent

Signalling

• General process of singal transduction into Animals with the following events from a LIGAND ( signal ) Interacts with a protien Receptor, which interacts with protien machinary , this then interacts within Celluar Metobolic activity which then ends

Protiens Transduced

• In the transduction events the G protiens are coupled by G protiens whuch are coupled to the receptors and then regualte enymes

Messages and Mesngers

• The first message is one thst intercats fron the outside
• While then the lower mw create a second one

Beta Addrenergenic Pathways

• Adrenegic proteins are ones bonded to epinephrine
• This is due to the GEF that stimulates proteins for the first time, while the stimuliatory will stimulat the protien in the s stae The Pathway is then the binding changes causing GTP Binding with Gs ALPHA releasing the beta Cyclic andenlylate forms the synthysis and is a cylic atp