Term
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Insulin consists of an alpha and beta polypeptide chain
Proinsulin precurser is post-translationally modified where the C chain is removed and S-S bonds between cysteines are formed
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Definition
•The structure and function determined by its sugar monomers and the positions of glycosidic linkages.
Storage
•Starch, a storage polysaccharide of plants, consists entirely of glucose monomers.
-Plants store surplus starch as granules within chloroplasts and other plastids.
•Glycogen is a storage polysaccharide in animals.
•Humans and other vertebrates store glycogen mainly in liver and muscle cells.
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Term
nProkaryote – Cells without defined nuclear envelope, i.e. - Bacterial world
nEukaryote – Cells with defined nucleus separated from the cytoplasm by a membrane structure – Everything except Bacteria and viruses
nViruses – Submicroscopic particles which can not replicate without a host. Viruses consists of genetic material (RNA or DNA) and an encapsulating protein envelop (caspid). |
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Definition
nSimilarities:
nPlasma Membrane
nGenetic info in DNA code, same code (circular vs linear)
nShared homology and pathways
nChemical energy as ATP
nSimilar mechanisms for photosynthesis
nSimilar mechanism for inserting proteins into membranes
nSimilar Proteosome structure |
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Term
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Leeuwenhoek – use of microscope to view unicellular organisms
Hooke – sent by the Royal Society to investigate early microscopy and described observations as “cells”
Matthias Schleiden & Theodor Schwann- Co-founders of cell theory
Organisms are comprised of one or more cells
Cells are the structural units of life
Rudolf Virchow- Cells arise from the division of living cells
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Definition
•dehydration reaction occurs when two monomers bond together through the loss of a water molecule.
•Enzymes- macromolecules that speed up the dehydration process.
•Polymers are disassembled to monomers by hydrolysis, a reaction that is essentially the reverse of the dehydration reaction. |
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Term
Western analysis of DNA Mismatch Repair proteins separated on a denaturing PAGE gel. Published 2007.
Denaturing gels contain SDS (detergent) and remove tertiary and most secondary structure. Proteins migrate based on relative size. |
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Definition
nMolecular technique where a primary antibody (Ab) is raised to a particular substance under study (a protein for example). The secondary Ab is raised against the heavy chain of the primary antibody and it contains a chemical capable of causing luminescence. The 10Ab binds to the protein and the 20Ab binds to the 10Ab. The 20Ab contains a peroxidase which can excite luminol and the signal (luminescence) is detected on an film. |
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Term
Genetic information:
Encoded in nucleic acids.
Utilized based upon the needs of the cell or the organism as a whole
Conserved across life forms: different species carry similar genes. |
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Definition
•formed when a dehydration reaction joins two monosaccharides .
•This covalent bond is called a glycosidic linkage. |
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Term
•A slight change in primary structure can affect a protein’s structure and ability to function.
•Sickle-cell disease, an inherited blood disorder, results from a single amino acid substitution in the protein hemoglobin. |
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Definition
•Nucleic acids are polymers called polynucleotides.
•Each polynucleotide is made of monomers called nucleotides.
–nitrogenous base, a pentose sugar, and a phosphate group.
•The portion of a nucleotide without the phosphate group is called a nucleoside. |
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Term
•determined by interactions between R groups
–These interactions between R groups include hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals interactions.
•Strong covalent bonds called disulfide bridges may reinforce the protein’s structure. |
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Definition
•two or more polypeptide chains form one macromolecule.
•Collagen is a fibrous protein consisting of three polypeptides coiled like a rope.
•Hemoglobin is a globular protein consisting of four polypeptides: two alpha and two beta chains. |
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Term
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Organization and complexity
Genetic program
Capable of replication
Acquire and utilize energy
Utilize chemical reactions
Transport and movement
Intra- and Inter-Cellular Communication
Self regulation – maintain homeostasis
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Definition
•general formula CH2O.
•Glucose (C6H12O6)= most common
•classified by:
–location of the carbonyl group (as aldose or ketose)
–number of carbons in the carbon skeleton
•in aqueous solutions many sugars form rings.
•major fuel for cells
•raw material for building molecules.
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Term
Structure
•cellulose is a major component of the tough wall of plant cells.
•two ring forms for glucose: alpha (a) and beta (b).
alpha glucose are helical.
beta glucose are straight.
•H atoms on one strand can bond with OH groups on other strands.
•Parallel cellulose molecules held together this way are grouped into microfibrils, which form strong building materials for plants. Human's can't hydrolyze
Chitin, found in the exoskeleton of arthropods and cell walls of many fungi.
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Definition
•Fats are constructed from two types of smaller molecules: glycerol and fatty acids.
•Glycerol is a three-carbon alcohol with a hydroxyl group attached to each carbon.
•A fatty acid consists of a carboxyl group attached to a long carbon skeleton.
•water molecules form hydrogen bonds with each other and exclude the fats.
•three fatty acids are joined to glycerol by an ester linkage, creating a triacylglycerol, or triglyceride.
Animal fats= saturated
plant/fish fats= unsaturated
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Term
•Nucleoside = nitrogenous base + sugar.
•There are two families of nitrogenous bases:
–pyrimidines (cytosine, thymine, and uracil) have a single six-membered ring
–purines (adenine and guanine) have a six-membered ring fused to a five-membered ring
•DNA, sugar is deoxyribose
•RNA, sugar is ribose.
•Nucleotide = nucleoside + phosphate group. |
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Definition
•Nucleotide polymers are linked together to build a polynucleotide.
•Adjacent nucleotides are joined by covalent bonds that form between the –OH group on the 3' carbon of one nucleotide and the phosphate on the 5' carbon on the next.
•These links create a backbone of sugar-phosphate units with nitrogenous bases as appendages.
•The sequence of bases along a DNA or mRNA polymer is unique for each gene. |
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Term
•two polynucleotides spiraling around an imaginary axis, forming a double helix.
•In the DNA double helix, the two backbones run in opposite 5¢ → 3¢ directions from each other, an arrangement referred to as antiparallel.
•One DNA molecule includes many genes.
•The nitrogenous bases in DNA pair up and form hydrogen bonds:
–adenine (A) always with thymine (T)
–guanine (G) always with cytosine (C) |
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Definition
n“What?”: what are the function(s) of a gene in vivo?
n“Why?” and “how?”: we create a loss of function mutation of the gene of your interest in mouse and observe the phenotype.
nLimitations: time consuming and expensive. Mice in the lab and mice in the wild are different. Quite often when you do not observe a phenotype, it does not mean that the gene is not important (in the wild or in human). |
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Term
nInsulin: an ancient gene (found in worms, fly).
nMore ancient: closer homology—importance
nClose homology of proteins allow for treatment of disease. Patients with diabetes mellitus can be treated with bovine or porcine insulin. Allergic reactions are usually few.
nWhy are there allergic reactions?
nAmino acid substitutions change the structure of the protein. |
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Definition
nData base managed by National Center for Biotechnology Information
nTool used to search homology of protein or nucleotide sequences
nAvailable to the public.
nDetermine if the gene or protein you discovered is novel.
nUsed as an aid to identify function of previously unknown genes, proteins. |
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Term
nSpeciation - Speciation is the origin of a new species capable of making a living in a new way from the species from which it arose. As part of this process it has also acquired some barrier to genetic exchange with the parent species.
nHomolog - A gene related to a second gene by descent from a common ancestral DNA sequence. The term, homolog, may apply to the relationship between genes separated by the event of speciation (see ortholog) or to the relationship between gene separated by the event of genetic duplication (see paralog)
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Definition
nOrtholog - Orthologs are genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Identification of orthologs is critical for reliable prediction of gene function in newly sequenced genomes.
nParalog - Paralogs are genes related by duplication within a genome. Orthologs retain a similar function in the course of evolution, whereas paralogs may evolve new functions, even if these are related to the original one. |
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Term
nCells are capable of replicating themselves through division.
nReplication involves:
nError free synthesis of new DNA
nSeparation of replicated DNA
nPartitioning of cell contents
nReforming membranes
nSome cells types are replicated frequently, others not frequently (epithelial lining vs cardiac cells)
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Definition
nThe first example of cultured cells capable of replicating and sustaining growth were isolated by George Gey (Johns Hopkins) from Henrietta Lacks hence - HeLa cells.
nHeLa Cells:
nDerived from human tumor cells
nKept alive through nutrients (growth media)
nStill used today – we use them in the labs
nProvide a method to experiment in an in-vitro system that contains human cells
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Term
nCells use energy to maintain a charge across their membranes. A difference between a live cell and dead cell is the membrane charge potential.
nEnergy is initially derived from sunlight and trapped in carbohydrates through photosynthesis
nCells release energy from carbohydrates and other molecules through metabolism
nCells use this energy to maintain a charge potential across its membrane
nCells use energy to drive cellular functions within the cell
nATP, NAD, FAD, and GTP are the main energy currencies in the cell |
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Definition
nCells carry out various chemical reactions to maintain homeostasis
nEnzymes are used to achieve the activation energy to enable chemical reactions to occur rapidly
nMetabolism is the term used to represent the total of a cell’s chemical reactions |
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Term
nChemical reactions occur within the cell by forming or breaking covalent bonds between bio-molecules
nCell’s Chemical Reactions
nSynthesis of and modification of nucleic acids
nSynthesis and modification of proteins
nConversion of lipid derivatives
nSynthesis of molecules such as steroids, ATP, hormones
nCatabolism of sugars, fatty acids, proteins
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Definition
nSome cells move through diapedesis, or with structural attachments
nSpecialized structures allow motility within the cell and motility of the cell
nCells work in combination with other cells to allow the organism to move
nCells transport materials into their cytoplasm, organelles and nucleus.
nMany specialized events occur in certain organelles. Since proteins are made in the cytoplasm they need to be conveyed to areas where they are needed.
nChannels allow the transport of ions, while specialized protein complexes allow for other materials to cross the membrane
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Term
nCells receive messages from their environment and respond to this stimuli:
nChemicals
nTemperature
npH
nHost-pathogen response
nCells communicate with other cells
nCell density (Quorum Sensing)
nChecking homeostasis of adjoining cells
nCells send signals from the exterior through the cell
nSignal transduction pathways
nResponse to hormones
nRegulate gene transcription |
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Definition
nCells adapt to changes in the environment to maintain homeostasis.
nCells adapt and regulate themselves in response to problems in gene function
nCells which become transformed (become cancerous) alter their regulation to favor cell survival, growth and division
nBased upon the signals received from the environment or other cells, cells may change their functions to compensate |
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Term
nEukaryotes Have:
nNuclear envelope with membrane
nLinear Chromosomes, Complex chromatin formation
nSpecialized cytoplasmic organelles with membranes: Golgi, lysosomes, peroxisomes, etc.
nComplex cytoskeletal system
nComplex flagella and cillia
nEndocytosis and phagocytosis
nUnique cell division w/ microtubule/mitotic spindle apparatus
nTwo gene copies per cell
nSexual reproduction requiring meiosis |
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Definition
nOrganelles are defined cellular structures which have some function related to cell survival.
nNucleus – Contains genetic code information to program cell activities
nMitochondria – responsible for oxidative metabolism, energy generation and maintaining voltage differential across the membrane
nLysosomes and peroxisomes – provide metabolic compartments for breakdown of macromolecules and invading pathogens.
nEndoplasmic Recticulum and Golgi Apparatus – responsible for protein sorting and transport
nCytoskeleton – provides structural framework, coordinates internal movement and diapedesis
nPeroxisomes -breakdown of long chain fatty acids through beta-oxidation
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Term
nDifferent types of cells are used commonly in the field of molecular biology. Common organismal and cellular models include:
nEscherichia coli (bacteria)
nSaccharomyces cerevisiae (yeast)
nCaenorhabditis elegans (nematode)
nDrosophila (fruit fly)
nArabidopsis thaliana (mustard plant)
nMus musculus (mouse)
nHomo sapiens (human cells, e.g. Hela cells) |
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Definition
nEasy to manipulate in culture
nWell understood organism
nSmall, Hardy
nEasy to introduce new DNA material
nRapid growth (colonies double every half hour)
nRapid results (hours days)
nAlpha complementation (positive selection for gene introduction)
nGenome completely mapped
nLittle Ethical consideration |
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Term
nEukaryotic organism – better human model than bacteria
nHaploid and diploid forms
nWell understood organism
nSmall, Hardy
nEasy to introduce new DNA material
nRapid growth (hours, days)
nRapid results
nGenome completely mapped
nLittle Ethical consideration |
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Definition
nEukaryotic complex organism – better human model
nWell understood organism
nSmall, Hardy
nEasy to introduce new DNA material
nRapid predictable growth (days, weeks)
nRapid results
nGenome completely mapped
nEvery cell and its differentiation has been mapped and investigated (apoptosis 131 cells die during formation to adult, total of 959 cells result)
nSome Ethical consideration |
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Term
nEukaryotic complex organism – best? human model
nWell understood organism
nSmall, Hardy?
nHarder to introduce new DNA material
nRapid predictable growth
nFairly Rapid results (weeks, months)
nGenome completely mapped
nModerate Ethical considerations
nGood in-vivo disease model |
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Definition
nBest human model at cell level
nNot Well understood at organism level
nEasy to culture – but most are transformed
nHard to introduce new DNA material
nRapid predictable growth
nFairly Rapid results (weeks, months)
nGenome completely mapped
nLow Ethical considerations – except stem cells
nNot good in-vivo disease model at organism level |
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Term
n“What?”: northern blot can determine the expression activity of a single gene. But it takes forever to determine the expression of all genes by northern blot.
n“Why?” and “how?”: we perform “northern blot” on a chip that can potentially give us the information of all genes in the cell.
nLimitations: usually not very accurate. Need independent verification. |
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Definition
nConstruct radioactive probe (DNA with labeled nucleotides)
nMix “hot” DNA with protein mixture
nSeparate on a Non-denaturing gel by PAGE
nDry gel onto a membrane (filter paper)
nUse autoradiography to detect image of left by radiation on probe |
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Term
Band shows that DNA has been bound by a protein complex – non specific
Specificity demonstrated adding a specific antibody to the mix (lane 9). The antibody binds to the protein and makes the mass greater
Non-denaturing gels allow for protein interacts and structure |
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Definition
n“What?”: you want to observe the dynamic location(s) of your protein in living cells.
n“Why?” and “how?”: you create a fusion protein containing GFP (green fluorescence protein) and your protein, and express the fusion protein in the cell. The fusion protein should have the same dynamics as the WT protein and it is now fluorescent and can be observed directly with a fluorescence microscope.
nLimitations: sometimes GFP does affect the dynamics of a protein. |
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Term
n“What?”: you can determine the MW of a protein by SDS-PAGE. But correct MW is far from proving that it is the correct protein. So how to further confirm it is the right one?
n“Why?” and “how?”: western blot utilizes an antibody that has been shown to recognize the protein of your interest. So if you see a positive signal on the western blot, you are further assured that it should be the correct protein of interest.
nLimitations: antibodies quite often have off-targets, i.e., they recognize the wrong proteins too.
nWHY?? |
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Definition
n“What?”: you have a solution with lots of proteins. How to measure their MWs? Or, you have a protein solution, how do you know it is pure or contaminated with lots of other proteins?
n“Why?” and “how?”: SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). SDS is a detergent that will denature proteins to rod shapes and cover them with uniform negative charges. So the speed of migration in the gel is only determined by their MWs.
nLimitations: some proteins have charge densities when denatured by SDS and show incorrect MWs. |
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Term
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Leucine zippers are specialized Coiled coil motifs with DNA binding At N-terminal regions
Leucine appears every 7th position
Transcription factors
Zinc Finger Motif
Helix-loop-helix are found in transcription factors which have DNA binding and protein interaction domains.
Negative charge to attract Ca 2+
Coiled-coiled Motif- Two or more alpha helices can wrap around one another
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Definition
n“What?”: you have a mixture containing several components of different sizes, or densities. How do you separate them?
n“Why?” and “how?”: The starting suspension contains 3 components of 3 different sizes.
Analytical centrifugation, specifically sedimentation velocity, uses a special centrifuge (analytical centrifuge) with detection optics to measure the moving speeds of the components in a solution.
Its output numbers are in Svedberg units (S, = 10-13 second). S µ M/f (molecular mass/friction coefficient)
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Term
1. Crosslink Protein-DNA complexes in situ
2. Isolate nuclei and fragment DNA (sonication or digestion)
3. Immunoprecipitate with antibody against target nuclear proteinand reverse crosslinks
4a. Identify protein components ofisolated complexes
4b. Identify DNA sequence by PCR, cloning and sequencing |
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Definition
1. Prepare cell or tissue lysate. (Spin to precipitate insoluble components)
2. Add the primary antibody targeting protein of interest, to supernate
3.Add the secondary antibody conjugated with agarose resin
4.Wash the precipitated complexesand apply on SDS-PAGE |
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Term
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Molecular technique where an antibody (Ab) is raised to a particular substance under study (a protein for example).
The Ab is tagged with a fluorescent dye (fluorochrome).
The Ab binds to the protein and the fluorescence can be visualized by microscopy. |
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Definition
1. Resolve protein samples on Native PAGE
2. Electrophoretically transfer fractionated proteins from gel onto PVDF membrane
3. Block the membranewith neutral protein (BSA or milk casein)
4. Incubate the membranewith protein interacting with target protein(primary Ab)
5. Incubate the membranewith HRP-labeled antibody (secondary Ab)specific to primary Ab.
6. Incubate the blot withchemiluminescent HRPsubstrate and expose to film |
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Term
1. Apply protein/dye samplesinto polyacrylamide gel wells
2. Run the electrophoresis until dye reaches the end of the gel
3. Remove the gel from the apparatus and stain for proteins |
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Definition
Tailbleed mice to determine if a homozygous for the defect is present, possible that the defect was embryonically lethal and only exists as a heterozygote |
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