The feature that most clearly separates eukaryotes from prokaryotes is the presence of eukaryotic cells.
The nucleus.
In fluorescence microscopy, the immediate source of the light detected is light that has been _____ the sample.
Emitted by.
Transmission electron microscopy is used to
Observe subcellular organelles & macromolecules.
Which of the following are the most commonly used mammals for genetic studies?
Mice
The original cell was thought to have arisen from the self-replicating _____ by a phospholipid membrane.
RNA
The genome of eukaryotes consists of genes derived from
Both archaebacteria & eubacteria.
Bacteriophages are
Viruses that infect bacteria.
In visualizing protein localization within a cell, what are the relative advantages & disadvantages of tagging proteins with green fluorescent protein (GFP) versus using a fluorescent antibody specific to the protein of interest (immunofluorescence)?
The main advantage of using GFP-tagged protein is that fixing, which kills cells & cause artifactual results, is not required. This makes it possible to observe subcellular localization in live cells. The localization of GFP-tagged proteins can be observe
The most abundant molecule in cell is
Water
Lipids with unsaturated fatty acids
Increase the fluidity of membranes
What is the effect of a beta-barrel on the permeability of a membrane?
It increases permeability.
Whereas small uncharged molecules can diffuse through the hydrophobic core of a phospholipid bilayer, a larger polar substance such as glucose must enter cells by
Binding to carrier proteins that facilitate the passage of specific molecules across membranes.
Passive transport across a membrane refers to
Transport in the energetically favorable direction.
In the most complete sense, "proteomics" refers to the large-scale analysis of
Cell proteins, protein localization, & protein interaction networks.
Which of the following is not an analytical instrument approach that can be applied to meet the goals of proteomics?
Atomic absorption analysis of the metal content of plant tissues.
Analytical instruments include: high-throughput microscope analysis of the distribution of GFP fusion proteins in cells, mass spectrometry, two-dimensional gel electrophoresis.
Proteins must have more than one ____ to have a quaternary structure.
Polypeptide chain.
The alpha helix is an example of which level of protein structure?
Secondary
Which of the following is not one of the four major classes of organic molecules in the cell?
Water
What is the major carbohydrate-storage molecule in plants?
Starch
Introducing a double bond into a fatty acid has the effect of putting a(n) _____ into the conformation of the molecule.
Kink.
Molecules that are partly water-soluble and partly water-insoluble are
Amphipathic
Which of the following bases is not found in DNA?
Uracil
Which of the following classes of amino acids is buried within the folded structure of the protein?
Nonpolar
Mass spectrometry is currently the major tool used in proteomics because of its ability to assign very precise
Mass-to-charge ratios to peptides, which can then be matched to predicted values for peptides in a complete protein database.
Cell Theory
The cell is the basic unit of life.
What is a cell?
Aqueous solution of organic molecules (proteins, nucleic acids, lipids, & carbohydrates).
Enclosed by a cell membrane.
Capacity for reproduction (mitosis & meiosis).
Carries out metabolism.
Nothing less than a cell is living.
Human body cells.
10^13 cells in the human body.
>200 different types of cells in the human body
Cell unity & diversity
Unity: cells share certain features -- common fundamental properties = DNA, plasma membrane, energy (ATP), metabolism.
Diversity: variation in appearance & function.
First self-replicating molecule
RNA was most likely the first self-replicating molecule because it is single-stranded, making it able to act as a template to a double-strand.
RNA world.
Evolution of cells.
Common prokaryotic ancestor gave rise to archaea & eubacteria.
Eukaryotic cell = fusion of archaebacteria & eubacteria.
Endosymbiosis
Mitochondria & chloroplasts are originated in eukaryotic cells via this process.
Model Organisms
Escherichia coli (E. coli), Saccharomyces cerevisiae (yeast), Arabidopsis thaliana (plant), Drosophila melanogaster (fruit fly), Caenorhabditis elegant (round worm), Xenopus (African frog -eggs), Zebra fish, Mus musculus (mice).
2 types of cell cultures
Primary cell lines: cells straight from the tissue with no passage. Because it is straight from the tissue it is a heterogenous mixture of cell types, but they're in their physiological conditions.
Cell lines: have at least one pass (usually 50-60 times).
HeLa cells
Cancer cell line for biomedical purposes.
Techniques to study cells
Microscopy
Subcellular fractionation
-- velocity centrifugation (density gradient)
How to visualize proteins in cells
Antibodies (about 3 months & expensive)
GFP tagged proteins (quicker & cheaper)
Antibody Expression Process
PCR: clone genes - amplify proteins.
Inserted into expression vector (usually plasmids).
Inject protein into an animal -- immune system responds with anti-body.
Remove blood serum to obtain antibody.
Tag antibody with a secondary antibody, then color with
GFP Tagged Protein
GFP-A fused protein to GFP tag.
Transfection.
Sometimes GFP disrupts protein localization.
From jellyfish. Can be fused to any protein of interest using standards methods of recombinant DNA, and the GFP-tagged protein can then be expressed in cells & dete
Light Microscopy
Light source must be focused on specimen.
Light must pass through sample.
Lenses must be arranged to focus an image of the specimen in the eye.
Robert Hooke
Developed microscope & was the first to name a cell.
Fluorescence Microscopy
Advantage is INCREASED SENSITIVITY.
Selective staining of different components.
How are molecules visualized?
- Dyes that bind to particular structures or molecules.
- Dyes coupled to antibodies (immunofluorescence).
- Fluorescent protein tags (GFP and va
Confocal Microscopy
Contrast is even better than light microscopy because it can focus on thinner tissues & put the images together to form 3D picture.
Electron Microscopy
Higher resolution than light microscopy. Used to resolve the fine structure of the cell.
Two types: Transmission EM and Scanning EM.
Transmission Electron Microscopy
Transmits a beam of electrons THROUGH the sample.
Generates a 2D image.
Scanning Electron Microscopy (SEM)
Electrons scattered to look at the surface details of a specimen (specimen is coated with metal that reflect electrons to produce the image). Produces a 3D image.
X-ray Crystallography
Beam of x-rays directed at crystals of protein. Pattern (protein structure) is detected on x-ray film. A high resolution technique that can determine the arrangement of individuals atoms.
Ways of seeing cell structure
Light Microscopy
- gross cell structures (organelles, cytoskeleton, nucleus)
Transmission Electron Microscopy
- Ultrastructure (ribosomes, golgi stacks, DNA strands)
Scanning Electron Microscopy
- 3D surface view of an object (red blood cells, cilia)
X-ra
Differential Centrifugation
Repeated centrifugation at higher speeds. Separates on size & density. Components must differ greatly to achieve separation.
Decreasing density in supernatant with increased speeds of centrifugation.
The composition of the cell
Water 70%
Inorganic ions
Organic molecules
- Carbohydrates
- Lipids
- Proteins
- Nucleotides
Carbohydrates
Simple sugars & polysaccarides
Polysaccaride: monosaccharides join together by dehydration reaction in which H2O is removed & the sugars are linked together by a glycosidic bond between two of their carbons.
Different glycosidic linkages give rise to different polysaccharides.
Storage - amylopectin (starch) & glycogen.
* Store carbs for energy; composed entirely of a glucose configuration; PRIMARY LINKAGE = alpha (1-->4)
Occasionally link alpha (1-->4) alpha (1-->6).
Not edible by us - Structural - Cellulose & chitin.
* Plant c
Lipids
* Fatty acids (simplest)
* Phospholipids: glycerol phospholipids (two fatty acids bound to C atoms in glycerol, 3rd bound to phosphate group) and sphingomyelin (only non-glycerol phospholipids in cell membranes).
* Glycolipids: two hydrocarbon chains link
Cholesterol & steroid hormones
steroid hormones derived from cholesterol.
Polymerization of nucleotides
Involves the formation of phosphodiester bonds between 5' phosphate of one nucleotide & 3' hydroxyl of another.
Polymerization of amino acids
Through peptide bond btwn the alpha amino group of one amino acid & the alpha carboxyl group of a second.
Polypeptides
Linear chains containing two distinct ends - one terminating in amino group (N terminus) and one in a carboxyl group (C terminus).
Three-dimensional structure of myoglobin is revealed..
by X-ray crystallography.
Phospholipid
Amphipathic
Fluid Mosaic Model
Proteins are inserted into a lipid bilayer. Phospholipids provide basic structure; membrane proteins carry out specific functions.
Transmembrane or integral membrane proteins = embedded directly into bilayer.
Peripheral membrane proteins = not inserted; a
Transport
Membranes are impermeable to solutes & ions.
Membrane transport proteins are necessary to take up ions, amino acids, & sugars.
Small lipid soluble and small, uncharged polar molecules can diffuse freely through bilayer.
Larger uncharged polar molecules ne
The proteins shaped like a "double chamber" that are involved in protein folding are called
chaperonin proteins.
Signal sequences are sequences of hydrophobic amino acids that target membrane translocation. Signal sequences are found
at the amino terminus of the secreted protein.
Which of the following is an example of posttranslational modification?
Glycosylation
Proteolysis
Palmitoylation
Protein phosphatases
catalyze the removal of phosphate residues from proteins.
cAMP activates cAMP-dependent protein kinase by
binding regulatory subunits and inducing their release from the catalytic subunits.
The function of aminoacyl tRNA synthetases is to
covalently attach amino acids to their corresponding tRNA molecules.
The primary function of rRNAs in the ribosome is
to catalyze peptide bond formation.
A proteasome is a
multisubunit protease complex that degrades proteins marked for destruction.
The growing polypeptide chain coming off the ribosomal complex is fairly unstable. It has a tendency to fold back on itself and can aggregate with adjacent polypeptides, and these processes, if allowed to occur, would yield degraded, improperly folded, or
An entire class of proteins, called chaperones, prevents these potential problems. As polypeptides come off the ribosomes, these chaperones quickly bind to and stabilize the growing chain. This activity prevents improper or premature folding until the ent
What technique would you use to determine if the level of a specific mRNA had been increased in response to an inducer?
Northern blotting
The SDS in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of a protein is used to
denature the protein and give it an overall negative charge.
Transgenic mice carry a foreign gene
in all of their cells.
The two strands of DNA in the double helix are held together by
hydrogen bonds between the bases of each strand.
The process by which proteins are made from RNA templates is called
translation.
The characterization of restriction endonucleases was a key step in the development of recombinant DNA technology. What is the function of these enzymes?
Cleavage of DNA at specific sequences
Which of the following correctly outlines the process of RNA interference, beginning with a double-stranded RNA molecule?
Cleavage by Dicer; association with RISC; unwinding of siRNA; pairing with target mRNA; mRNA cleavage
A common method of DNA sequencing is based on premature termination of DNA synthesis using dideoxynucleotides in the DNA polymerase reactions. Why does termination of a growing strand cease when a dideoxynucleotide is incorporated?
RNA, or ribonucleic acid, uses ribose and contains hydroxyl groups at both the 2? and 3? positions. DNA is deoxynucleic acid, with deoxy referring to one less hydroxyl group at the 2? position. Both molecules are dependent on the 3? hydroxyl for elongatio
Suppose you are studying a mammalian transcription factor that you would like to express in bacteria so that you can purify large quantities of it to use in biochemical studies. You introduce the cDNA encoding the transcription factor into an expression p
Lack of expression could result from a number of factors. Remember the plasmid must have appropriate unique restriction sites, an origin of replication, and a selective antibiotic resistance marker. If any of these are perturbed during cloning, there will
Two classes of membrane transport proteins
Carrier protein: acts like enzymes - solute must fit - great specificity.
Active transport.
Channel proteins "ion channels": discriminate on sis & charge. Can exist in open or closed state.
Passive transport.
How to identify proteins
Column chromatography
Gel electrophoresis
- SDS page
- two dimensional gel electrophoresis
^^ separation
Mass spectrometry
^^ identification
Column chromatography
Separates based on size, hydrophobicity, affinity for other molecules, & charge
Ion exchange charge
Gel filtration size
Affinity (specific interaction - highest purification)
Gel electrophoresis
Proteins migrate based on size & charge.
Migrates until it reaches a pH at which the charge of the protein is neutralized.
Then separated second time based on size. Lower MW = faster movement through gel.
SDS page
Separation of proteins only based on size.
Function / make proteins negatively charged. Proteins migrate baed on MW.
two dimensional gel electrophoresis
Separation of proteins based on isoelectric point (the pH at which a particular molecule carriers NO net electric charge)
Mass spectrometry
ID proteins based on mass:charge ratio.
Read by experts.
Proteomics
Large scale analysis of
- cell proteins
- protein localization
- protein interaction networks
Semi conservative Replication
The complementarity of DNA strands allows each strand to serve as a template for synthesis of the other.
RNA viruses
Some viruses have an RNA core rather than DNA.
This RNA serves as genetic material.
Retroviruses
Replicate in an unusual way - the RNA serves as template for synthesis of a complementary DNA by the RNA dependent DNA polymerase reverse transcriptase, this process is called reverse transcription.
DNA is then incorporated into host cell.
Genetic Code
Each amino acid codes is encoded by a set of 3 nucleotides (codon) on the mRNA.
The genetic code is degenerate with many amino acids specified by more than one codon.
Only tryptophan & methionine are encoded by a single codon.
Wobble hypothesis
Predicts that hydrogen bonding between the codon & anticodon at the third position is subject to modified base-pairing rules.
"I" in third position is the more relaxed position & modified base.
Frames gift mutation
Removal or addition of a nucleotide resulting in a shift of the entire following code unless it's addition/removal of a factor of 3.
Missense mutation
A point mutation where a sequence of DNA that result in a change in amino acids.
Nonsense mutation
Point mutation where a sequence of DNA that results in a premature stop codon
Molecule techniques
Recombinant DNA - cloning
Detection of nucleic acids: south and northern blots
Detection of protein: western blot
Enzyme restriction
Recognize specific sequences
DNA can be cut by restriction enzymes (molecular scissors). Cut DNA becomes vector DNS which can amplify the gene. After cloned, introduce recombinant DNA to host cell.
How to amplify a specific gene in vitro
PCR: polymerase chain reaction. Interested in a specific gene - know predicted sequence, but don't have DNA -- find the sequence & do PCR.
Two classes of membrane transport proteins
Carrier protein - soluble must "fit" - great specificity (acts like enzymes).
Channel protein "ion channels" - discriminate on size & charge - can exist in open or closed state.
Both form hydrophillic pathway across membrane.
Three steps of PCR
Denaturation (heat up to 95 to destory hydrogen bonds), primer annealing (designed complementary strand), & extension (using taq polymerase - heat stable enzyme - extend the primers).
DNA sequencing
The most common method of DNA sequencing is dideoxy chain termination sequencing, developed by Sanger. Template > dNTP > DNA polymerase (not Taq) > primers > ddNTP
Recombinant Libraries
Any genetic information. A genomic library that contains at least one copy of all sequences in the genome of interest. Constructed by cutting genomic DNA with a restriciton enzyme and ligating the fragments into vectors (choice of vector depends on the si
cDNA library
Contains complementary DNA copies made from the mRNA's present in a cell population & represents the genes that are transcriptionally active at the time the cells were collect for mRNA isolation. Genes are actually expressed.
cDNA is made from mRNA using
RNA interference
Control of gene expression (1/3 of genes regulated this way). Inhibit or degrade the target RNA by associating with complementary sequence.
Translation
The biological polymerization of amino acids into polypeptide chains. Requires amino acids, messenger RNA, ribosomes, & transfer RNA.
Peptide Bond
Linking amino acids
N terminus --> C terminus
rRNA
Catalytic activity.
Catalyze the formation of peptide bonds
Aminoacyl tRNA synthetases
The right amino acid must be attached to the right tRNA - individual enzymes that recognize a single amino acid & attach it to the correct tRNA
Three steps of translation
Initiation: assemble ribosomal formation
Elongation: move forward
Termination: until STOP codon
Different between prokaryotic &
Eukaryotic mRNA
Prokaryotic - multiple start sites, one mRNA codes multiple peptides (polycistronic) & initiated by SD sequence
Eukaryotic: single start site, one mRNA codes one peptide (monocistronic), initiated by Kozak sequence
Regulation of translation
Repressor protein binding to mRNA (ferritin - stores iron in cell or binding to 3' UTR); mRNA localization & length (in egg); RNA interference (micRNA)
^^ all gene specific
Global effect on overall translation
Initiation of translation - eukaryotic part I
GTP-elF2 escort tRNA to ribosome
Initiation factors recognize both 5' and 3' ends
Chaperones
Facilitate the folding of other proteins
Think of enzymes (not part of assembled protein)
Protein glycosylation
N-linked (serine)
O-linked (asparagine)
Add sugar to a protein which allows the protein to act like receptor cells
Synthesis of N-linked glycosylation
Occurs in ER
Hooked into a dolichol phosphate (lipid)carrier
O linked glycosylation
Occurs in Golgi
Much simpler
Linked to serine
Protein attachment to lipids
Cytosolic face =
n-myristoylation (n terminus)
Prenylation
(C terminus)
Palmitoylation
(Internal cysteine)
Extra cellular face = glycolipids important for signaling
(GPI anchor)