Cellular Biology Exam #1

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 linked to polar heads that contain carbohydrates (amphipathic). * Cholesterol: 4 hydrocarbon rings - NOT LINEAR (amphipathic).

Human body cells.

10^13 cells in the human body. >200 different types of cells in the human body

Missense mutation

A point mutation where a sequence of DNA that result in a change in amino acids.

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 variants). Allows us to see distribution of a specific molecule. Has two filters -- before light reaches the specimen - to excite the dye and filter that illuminates light (emits longer wavelengths - less energy).

Molecules that are partly water-soluble and partly water-insoluble are

Amphipathic

Phospholipid

Amphipathic

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 large aggregates of nonfunctional proteins. How does the cell prevent this from occurring?

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 entire chain is synthesized and the completed protein can fold appropriately.

How to visualize proteins in cells

Antibodies (about 3 months & expensive) GFP tagged proteins (quicker & cheaper)

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.

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.

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.

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.

The genome of eukaryotes consists of genes derived from

Both archaebacteria & eubacteria.

HeLa cells

Cancer cell line for biomedical purposes.

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.

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.

In the most complete sense, "proteomics" refers to the large-scale analysis of

Cell proteins, protein localization, & protein interaction networks.

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

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

How to identify proteins

Column chromatography Gel electrophoresis - SDS page - two dimensional gel electrophoresis ^^ separation Mass spectrometry ^^ identification

Evolution of cells.

Common prokaryotic ancestor gave rise to archaea & eubacteria. Eukaryotic cell = fusion of archaebacteria & eubacteria.

Confocal Microscopy

Contrast is even better than light microscopy because it can focus on thinner tissues & put the images together to form 3D picture.

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).

Robert Hooke

Developed microscope & was the first to name a 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.

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.

In fluorescence microscopy, the immediate source of the light detected is light that has been _____ the sample.

Emitted by.

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).

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 & detected by fluorescence microscopy, without the need to fix and stain the cell as would be required for the detection of proteins with antibodies.

Which of the following is an example of posttranslational modification?

Glycosylation Proteolysis Palmitoylation

Electron Microscopy

Higher resolution than light microscopy. Used to resolve the fine structure of the cell. Two types: Transmission EM and Scanning EM.

Mass spectrometry

ID proteins based on mass:charge ratio. Read by experts.

Lipids with unsaturated fatty acids

Increase the fluidity of membranes

Polymerization of nucleotides

Involves the formation of phosphodiester bonds between 5' phosphate of one nucleotide & 3' hydroxyl of another.

What is the effect of a beta-barrel on the permeability of a membrane?

It increases permeability.

Introducing a double bond into a fatty acid has the effect of putting a(n) _____ into the conformation of the molecule.

Kink.

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 plasmid and transform E. coli with the new recombinant vector. You find no expression. What might be occurring?

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 be no expression of the gene of interest.

Proteomics

Large scale analysis of - cell proteins - protein localization - protein interaction networks

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-ray crystallography - 3D protein structure (individual atoms in a polypeptide).

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.

Polypeptides

Linear chains containing two distinct ends - one terminating in amino group (N terminus) and one in a carboxyl group (C terminus).

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.

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 need membrane transport proteins. Charged molecules, despite size, cannot cross freely.

Which of the following are the most commonly used mammals for genetic studies?

Mice

Techniques to study cells

Microscopy Subcellular fractionation -- velocity centrifugation (density gradient)

Endosymbiosis

Mitochondria & chloroplasts are originated in eukaryotic cells via this process.

Which of the following classes of amino acids is buried within the folded structure of the protein?

Nonpolar

What technique would you use to determine if the level of a specific mRNA had been increased in response to an inducer?

Northern blotting

Transmission electron microscopy is used to

Observe subcellular organelles & macromolecules.

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. Follow the protein expression by locating GFP tag.

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.

Nonsense mutation

Point mutation where a sequence of DNA that results in a premature stop codon

Proteins must have more than one ____ to have a quaternary structure.

Polypeptide chain.

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.

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). Cell population becomes more homogenous -- immortalized cell lines, usually tumors or cancel cells. These are "modified" cell lines are not in their physiological conditions.

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; associated with membrane INDIRECTLY.

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.

The original cell was thought to have arisen from the self-replicating _____ by a phospholipid membrane.

RNA

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.

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 elongation because it is required for the phosphodiester bond that forms between it and 5′ phosphate group of the next nucleotide. Dideoxynucleotides have hydrogens only at both the 2′ and 3′ positions (i.e., both hydroxyl groups are gone at these positions and therefore when incorporated into growing strands of DNA, elongation ceases because there is no 3′ hydroxyl for the subsequent phosphodiester bond to form).

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.

Molecule techniques

Recombinant DNA - cloning Detection of nucleic acids: south and northern blots Detection of protein: western blot

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.

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.

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.

The alpha helix is an example of which level of protein structure?

Secondary

Column chromatography

Separates based on size, hydrophobicity, affinity for other molecules, & charge Ion exchange charge Gel filtration size Affinity (specific interaction - highest purification)

two dimensional gel electrophoresis

Separation of proteins based on isoelectric point (the pH at which a particular molecule carriers NO net electric charge)

SDS page

Separation of proteins only based on size. Function / make proteins negatively charged. Proteins migrate baed on MW.

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.

RNA viruses

Some viruses have an RNA core rather than DNA. This RNA serves as genetic material.

What is the major carbohydrate-storage molecule in plants?

Starch

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 cell wall; primarily glucose. Beta (1-->4)

Cell Theory

The cell is the basic unit of life.

Semi conservative Replication

The complementarity of DNA strands allows each strand to serve as a template for synthesis of the other.

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 observed in real time in response to various inducers by infusing them into the sample while on the microscope. One potential disadvantage of using GFP-tagged proteins is that the presence of the GFP tag (which has a size of approximately 30 kilodaltons) could alter the structure of the protein and give artifactual results. In fact, that sometimes occurs.

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

The feature that most clearly separates eukaryotes from prokaryotes is the presence of eukaryotic cells.

The nucleus.

Polymerization of amino acids

Through peptide bond btwn the alpha amino group of one amino acid & the alpha carboxyl group of a second.

Transmission Electron Microscopy

Transmits a beam of electrons THROUGH the sample. Generates a 2D image.

Passive transport across a membrane refers to

Transport in the energetically favorable direction.

Cell unity & diversity

Unity: cells share certain features -- common fundamental properties = DNA, plasma membrane, energy (ATP), metabolism. Diversity: variation in appearance & function.

Which of the following bases is not found in DNA?

Uracil

Bacteriophages are

Viruses that infect bacteria.

The most abundant molecule in cell is

Water

Which of the following is not one of the four major classes of organic molecules in the cell?

Water

The composition of the cell

Water 70% Inorganic ions Organic molecules - Carbohydrates - Lipids - Proteins - Nucleotides

Signal sequences are sequences of hydrophobic amino acids that target membrane translocation. Signal sequences are found

at the amino terminus of the secreted protein.

cAMP activates cAMP-dependent protein kinase by

binding regulatory subunits and inducing their release from the catalytic subunits.

Three-dimensional structure of myoglobin is revealed..

by X-ray crystallography.

Protein phosphatases

catalyze the removal of phosphate residues from proteins.

The proteins shaped like a "double chamber" that are involved in protein folding are called

chaperonin proteins.

The function of aminoacyl tRNA synthetases is to

covalently attach amino acids to their corresponding tRNA molecules.

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.

The two strands of DNA in the double helix are held together by

hydrogen bonds between the bases of each strand.

Transgenic mice carry a foreign gene

in all of their cells.

A proteasome is a

multisubunit protease complex that degrades proteins marked for destruction.

Cholesterol & steroid hormones

steroid hormones derived from cholesterol.

The primary function of rRNAs in the ribosome is

to catalyze peptide bond formation.

The process by which proteins are made from RNA templates is called

translation.