Molecular Cell- Test 1
Proteomics
Attempting to understand the functions and interactions of all of the proteins present in a particular cell. Proteomic studies aim to understand the structure and properties of every protein produced by a genome and to learn how these proteins interact with each other in biological networks to regulate cellular functions.
Centrl Dogma of Molecular Biology
DNA replication => DNA => *transcription* => RNA => *translation* => protein
Chromosome Theory of Heredity
Proposed that the hereditary factors responsible for Mendelian inheritance are located on the chromosomes within the nucleus.
DNA cloning
a process used to generate many copies of specific DNA sequences for detailed study and further manipulation
Independent variable
The condition that is changed
Wild type
an organism (E. coli, yeast, a mouse, or other model organism) with unaltered DNA The mutant strain is identical to the wild type except that it lacks one particular gene's functio
DNA sequencing
devised for rapidly determining the base sequences of DNA molecules.
Restriction Enzymes
have the ability to cleave DNA molecules at specific sequences
Bioinformatics
merges computer science and biology as a means of making sense of sequence data. Led to the recognition that the human genome contains approximately 20,000 protein-coding genes, about half of which were not characterized before genome sequencing.
Specialized Light Microscopes
-Developed to overcome distortions from slide preparation: -phase constant microscopy -interference contrast microscopy -fluorescence micoscopy -confocal microscopy
Escherichia coli advatages
-Easy to grow in the lab, divides rapidly (its generation time is 20-30 minutes), and is easily mutagenized for studies of gene function. -First to have genome sequenced. -readily takes up DNA by transformation from virtually any organism; has become the workhorse of cellular and molecular biology for isolating and cloning genes. -routinely used for the analysis and production of genes and proteins for research, industrial, and medical uses.
Biochemistry
-Past 75 years with roots going back one century prior. Especially important- Development of laboratory techniques like: -Ultracentrifugation -Chromatography -Radioactive labeling -Electrophoresis -Mass spectrometry All used for separating and identifying cellular components
Saccharomyces cerevisiae advatages
-Used o study processes unique to eukaryotic cells, such as chromosome pairing during cell division, organelle develop-ment, or cell signaling -unicellular, as well as easy to grow and mutagenize, and well-characterized mutant lines are available. -Used to isolate and characterize genetic mutants following mutagenesis by chemicals or radiation; can determine the normal cellular function of genes and proteins by studying mutant yeast strains that are deficient in particular genes and their protein products - Use of the yeast two-hybrid system which allows researchers to determine whether and how specific proteins interact within a living cell; contributed greatly to understanding of the complex molecular interactions involved in cellular function.
Mus musculus (mouse) advantages
-Used to study cellular and physiological processes specific to mammals -shares many cellular, anatomical, and physiological similarities with humans and is widely used for research in medicine, immunology, and aging. -subject to, and therefore useful for the study of, a variety of diseases that also affect humans, such as cancer, diabetes, and osteoporosis. -Numerous mouse strains have been bred or engineered in which particular genes have either been "knocked out" or introduced, making them extremely valuable in biomedical research.
Chlamydomonas reinhardtii (unicellular green algae) advatages
-Used to study processes in plants, such as photosynthesis and light perception, and some processes common to all organisms -easily grown in the lab on Petri plates and has been used to study photosynthesis, light perception, mating type, cellular motility, and DNA methylation. For flowering plant studies, Arabidopsis thaliana -one of the smallest genomes of any plant and a rapid (six-week) life cycle, facilitating genetic studies. -complete genome has been sequenced, and thousands of mutant strains have been created, enabling detailed studies of plant gene function
Drosophila melanogaster advatages
-Used to study processes such as communication between cells, differentiation of cells, or embryonic development -Flies are easy to grow and manipulate in the lab, have a short (two-week) generation time, produce numerous progeny, and have easily observable physical characteristics, such as eye color and wing shape. -Thousands of mutant strains are available, each defective in a particular gene; valuable for studies of embryogenesis, developmental biology, and cell signaling.
Other developments for cytology:
-dyes for staining and identifying cell structures (19th century) -improved optics -more sophisticated lenses -microtome -Pushed light microscopy to limit of resolution imposed by size of wavelengths of visible light.
How to use model organisms, powerful microscopes, and genetic and biochemical techniques to meaningfully answer questions in cell biology?
-many individual conditions can be varied, such as: length of treatment or temperature, but it is best to vary, or perturb, only one condition, called the independent variable, and hold all others constant. The outcome of the change that is measured (which depends on the independent variable) is called the dependent variable. Why genetic mutants make good experiments: the classical genetic approach is to isolate a naturally occurring mutant form of an organism. It is now possible to artificially alter the DNA of an organism as well.
Caenorhabditis elegans advantages (roundworm)
-study cell differentiation and development in multicellular organisms. -ease of manipulation, relatively short life cycle, and small genome, the first of any multicellular organism to be sequenced. -one of the simplest animals to possess a nervous system. -development from a fertilized egg is remarkably predictable, and the origin and fate of each of its approximately 1000 cells have been mapped out, as have the hundreds of connections among the roughly 200 nerve cells. - worms are transparent, making it is easy to see individual cells under the microscope and to view fluorescently labeled molecules in the living organism
"-omics"
-transcriptomics: recent advanced methods of RNA sequencing allow us to determine the complete set of genes transcribed in a cell. -metabolomics: the analysis of all metabolic reactions happening at a given time in a cell, -lipidomics: the study of all the lipids in a cell, -ionomics: the global study of all the ions in a cell.
Cell Theory: Schwann, 1839:
1. All organisms consist of one or more cells 2. The cell is the basic unit of structure for all organisms. 20 years later, 3rd part. Virchow, 1855: omnis cellula e cellula 3. All cells arise only from preexisting cells.
New discoveries: process
1. Conduct a search of peer-reviewed scientific literature to determine what is known in the specific area of interest. 2. Formulate a hypothesis 3. Design a controlled experiment to test the hypothesis by varying specific conditions while keeping other variables constant. 4. Collect data, interpret the results, and accept or reject the hypothesis; must be consistent not only with the results of the particular experiment but also with prior knowledge.
Modern Cell Biology: Three strands of biological inquiry-
1. Cytology 2. Biochemistry 3. Genetics
Properties of Carbon: Stability
1. Four valence electrons- can form four bonds with other atoms. Associate with each other or other electron deficient atoms by sharing electrons to reach octet. Because four electrons are required to fill the outer orbital or carbon, stable organic compounds have four covalent bonds for every carbon atom. Most likely to share electrons with C, H, O, N, S. 2. Bond energy of approximately 83 cal/mol for C-C 3. Bond energy vs solar radiation. Inverse relationship wavelength of electromagnetic radiation and C-C bond energy. Visible portion of sunlight (400-700nm) is lower in energy than C-C bonds. Otherwise, visible light would break C-C bonds. UV radiation has 300nm waveength, 95kcal/einstein (mole of photons) and can break C-C bonds.
Properties of Carbon: Diversity
1. Great diversity of molecules from very few atoms. Also due to tetravalent nature of carbon. Can makes chains and rings, introduce branching and double bonds to chains.
Two Main factors restricting Leeuwanhoek's understanding of the nature of cells.:
1. Resolving Power of microscopes 2.Descriptive nature of 17th century biology: age of observation with little critical thinking about intriguing architectural details of discoveries
Solvent
A fluid in which another substance can be dissolved
Centrifugation
A means of separating and isolating subcellular structures and macromolecules based on their size, shape, and/or density. Called subcellular fractionation. Study specific parts of cell. Ultracentrifuge: useful for small organelles and macromolecules. Capable of very high speeds—more than 100,000 revolutions per minute—and can thereby subject samples to forces exceeding 500,000 times the force of gravity.
Cell cultures
A model system in which cells can be grown in the laboratory outside their tissue of origin—such as skin cells, muscle cells, and cancer cells. Also can use cells from other organisms Studying isolated cells in culture may not always reflect what happens in the intact organism
Antibody
A protein molecule produced by the immune system that binds one particular target molecule, known as its antigen.
Model organisms
A species that is widely studied, well characterized, and easy to manipulate, and has particular advantages, making it useful for experimental studies. Examples: -bacterium Escherichia coli -yeast Saccharomyces cerevisiae -fruit fly Drosophila melanogaster
Solute
A substance that dissolves in a fluid.
Electronegativity
Affinity for electrons (O, N, S higher than C or H).
Differential interference contrast microscopy
Also uses optical modifications to exaggerate differences in refractive index. Make possible to see living cells clearly. Light waves have crests and troughs, and the precise positions of these maxima and minima as light travels are known as the phase of the light. Technique enhances and amplifies slight changes in the phase of transmitted light as it passes through a structure having a different density than the surrounding medium
Calorie
Amount of energy needed to raise the temperature of water by 1 degree celcius. Kilocalorie=1000 calories. C-C: 83kcal/mol. C-N: 70 kcal/mol C-O: 84 kcal/mol C-H: 99 kcal/mol C=C: 146 kcal/mol C_=C: 212 kcal/mol
Heat of vaporization
Amount of energy required to convert 1 gram of a liquid into vapor. High for water because H-bonds must be disrupted. Makes H2O an excellent coolant- explains sweating.
Specific heat
Amount of heat a substance must absorb per gram to raise the temperature by 1 degree Celcius. For H2O: 1.0 calorie/g. In most solvents, heat energy would cause an increase in solvent molecule motion, increasing temerature. But in H2O, the energy is used to break the H-bonds, buffering H2O against temperature change. Large temp change would cause problems for cells.
Covalent bond
Atoms associate with each other or with other electron-deficient atoms, allowing adjacent atoms to share a pair of electrons, one from each atom, so that each atom's outer orbital has a full set of eight electrons including shared electrons. Single bond- sharing one pair of electrons Double bond- sharing two pairs of electrons Triple bond- sharing three pairs of electrons. Still four covalent bonds total (ex two double bonds or on double and two singles, etc)
Ion
Atoms or molecules that are charged because they have gained or lost an electron or proton (H+).
Properties of carbon: Tetrahedral
Can form stereoisomers. When four different atoms/groups of atoms are bonded to four corners of tetrahedral structure, two different spatial configurations possible. Not superimposable- mirror images.
Reduction
Carbon containing compounds gain electrons. Typically biosynthetic and requires energy.
Oxidation
Carbon containing compounds lose electrons to other atoms like molecular oxygen. Typically involves degradation and releases energy.
Asymmetric carbon
Carbon with four different atoms/groups of atoms attached. Compound with n asymmetric carbons will have 2^n possible stereoisomers.
Mass Spectrometry
Commonly used to determine the size and composition of individual proteins; allows to determine the identity and characteristics of individual proteins
H2O and cohesivness
Due to H-bonds Accounts for: -high surface tension -high boiling point -high specific heat -high heat of vaporization
Light Microscope
Earliest tool of cytologists. Identify membrane bound structures called organelles- "little organs" (plant/animal-not in bacteria) such as: -nuclei -mitchondria -chloroplasts
Phase constant microscopy
Enhances contrast in unstained cells by amplifying variations in refractive index within specimen; especially useful for examining living, unpigmented cells. Make possible to see living cells clearly. Light waves have crests and troughs, and the precise positions of these maxima and minima as light travels are known as the phase of the light. Technique enhances and amplifies slight changes in the phase of transmitted light as it passes through a structure having a different density than the surrounding medium
Magnification Power
Enlarges objects to X times their normal size (eg: 30X= 30 times normal size). Limited Hooke's observations. Made it difficult to learn about internal organization of cells.
In vitro
Experiments conducted in the laboratory using purified chemicals and cellular components.
In silico
Experiments using computers to test new hypotheses involving vast amounts of data
Transmission Electron Microscope (TEM)
Forms an image from electrons that are transmitted through the specimen.
Discoveries in Biochemistry
Friedrich Wöhler, 1828: demonstrated that urea, an organic compound of biological origin, could be synthesized in the laboratory from an inorganic starting material, ammonium cyanate. Before: living organisms not governed by chemistry/physics Louis Pasteur, 30 years later: living yeast cells were responsible for the fermentation of sugar into alcohol. Eduard and Hans Buchner, 1897: fermentation could take place with isolated extracts from yeast cells—that is, the intact cells themselves were not required. Enzymes responsible. Gustav Embden, Otto Meyerhof, Otto Warburg, and Hans Krebs, 1920's-30's: described the enzymatic steps in the Embden-Meyerhof pathway for glycolysis for glucose breakdown and the Krebs cycle for energy production. Fritz Lipmann, 1920's-30's: showed that the high-energy compound adenosine triphosphate (ATP) is the principal energy storage compound in most cells. Melvin Calvin, 1940's-50's: traced the fate of 14C-labeled carbon dioxide (14CO2) in illuminated algal cells that were actively photosynthesizing. Led to Calvin Cycle. First pathway discovered with radioisotopes.
Chromatography
General term describing a variety of techniques by which a mixture of molecules in solution is separated into individual components; separate molecules based on their size, charge, or affinity for specific molecules or functional groups.
Genetics
Goes back 150 years, but most present understanding in the last 75 years. Important Discovery: -demonstration that, in all organisms, DNA (deoxyribonucleic acid) is the bearer of genetic information; encodes variety of proteins/RNA molecules responsible for most functional/structural features of cells. genome- all DNA
Discoveries in Genetics
Gregor Mendel, 1866: Studied pea plants; laid out the principles of segregation and independent assortment of the "hereditary factors" known today as genes. Walther Flemming, 1880: identified chromosomes, threadlike bodies seen in dividing cells. Called the division process mitosis. Chromosome number soon came to be recognized as a distinctive characteristic of a species and was shown to remain constant from generation to generation. Wilhelm Roux, 1883: chromosomes themselves might be the actual bearers of genetic information Carl Correns, Ernst von Tschermak, and Hugo de Vries, 1900: Rediscovery of Mendel. Chromosome theory of heredity. Thomas Hunt Morgan and his students, Calvin Bridges and Alfred Sturte-vant, 1900-1920: Used Drosophila melanogaster, the common fruit fly, as their experimental model organism, they identified a variety of morphological mutants of Drosophila and were able to link specific traits to specific chromosomes. Johann Friedrich Miescher, 1869: discovery of DNA. Salmon sperm and human pus from surgical bandages, isolated and described what he called "nuclein." 75 years ahead of time. Robert Feulgen, 1914: Staining technique, DNA implicated as important component of chromosomes. 1930: DNA composed of 4 nucleotides. Proteins more diverse and thouht to carry genetic info. Oswald Avery, Colin MacLeod, Maclyn McCarty 1944: DNA could "transform" a nonpathogenic strain of bacteria into a patho-genic strain, causing a heritable genetic change. Alfred Hershey and Martha Chase, 1952: showed that DNA, and not protein, enters a bacterial cell when it is infected and genetically altered by a bacterial virus. George Beadle and Edward Tatum, 1940's: bread mold Neurospora crassa, formulated the "one gene-one enzyme" concept, asserting that each gene controls the production of a single, specific protein James Watson, Francis Crick, Rosalind Franklin 1953: Double helix model for DNA structure which immediately suggested how replication during cell division could occur by precise base pairing between complementary strands. 1960s: discovery of the polymerase enzymes that synthesize DNA and RNA and the "cracking" of the genetic code, which specifies the relationship between the order of nucleotides in a DNA or RNA molecule and the order of amino acids in a protein. Jacques Monod and François Jacob, 1960's: deduced the mechanism responsible for regulating bacterial gene expression.
In vivo
Hypotheses tested in living cells and organisms. Uses a variety of model organisms.
Microtome
Instrument developed for rapid and efficient preparation of very thin (several um) tissue slices of biological samples
Translation
It involves a language change—from the nucleotide sequence of an RNA molecule to the amino acid sequence of a polypeptide chain.
Properties of H2O: solvent due to polarity
Many molecules in cells are also polar and can form H-bonds with water. Hydrophilic: substances that have a high affinity for H2O and dissolve readily (polar molecules and ions) Hydrophobic: Molecules that are not very soluble in H2O. (nonpolar)
Cellular Size: Units.
Micrometer (um): One millionth of a meter (10^-6). 1 inch=25000um (approx.) -Most bacterial cells are a few um in length. -Plant/animal cells: 10-20 times larger than bacterial cells. -Mitochondria/chloroplasts: a few um (comparable to bacteria) -If seen with light microscope: use um Nanometers (nm): One billionth of a meter (10^-9) -used for molecules and subcelluar structures too small to be seen with light microscope. -Ribosome: 25-30nm diameter -used for cell membranes, microtubules, microfilaments, DNA Angstrom (A) 0.1nm. -Used in cell biology for measuring dimensions in proteins/DNA. Size of H atom
Steroisomers
Non superimposable mirror images of two structures with same formula
Compound Microscope
One lens (eyepiece) magnifies the image created by second lens (the objective). Allowed higher magnification and better resolution. 1830's. Structures size 1um could be seen clearly.
Properties of H2O: Polar
Polarity accounts for water's cohesiveness, temperature-stabilizing capacity, and solvent properties. H2O is bent, not linear, with two H's bonded to O at 104.5 degree angles. Causes asymmetry in the molecule. Oxygen atom is highly electronegative and "hogs" the electrons giving it a partial negative charge and the H's a partial positive charge. O also has two lone pairs. Makes molecule highly polar. Forms H bonds- one O can "bond" two H's. Caused extensive 3D network of H bonds which, while weak individually, are strong en masse. In liquid water, the hydrogen bonds between adjacent molecules are constantly being broken and re-formed, with a typical bond having a half-life of a few mi-croseconds.Each molecule of H2O Hbonded to at least 3 others at a given time in liquid. In ice, the hydrogen bonding is still more extensive: rigid, hexagonal crystal-line lattice with every oxygen hydrogen-bonded to hydrogens of two adjacent molecules and every water molecule therefore hydrogen-bonded to four neighboring molecules
Messenger RNA (mRNA)
RNA that is translated into protein; carries a genetic message from DNA to macromolecular complexes known as ribosomes, where protein synthesis actually takes place.
Null hypothesis
Rather than try to prove a hypothesis, scientists typically try to rephrase the hypothesis as its opposite and try to prove it. Failure to confirm this null hypothesis in a sufficiently large number of attempts is indirect evidence that the hypothesis is correct. The certainty of its correctness increases with the number of experimental samples and the number of times the results are replicated.
Transcription
Refers to RNA synthesis using DNA as a template to emphasize that this phase of gene expression is simply a transfer of information from one nucleic acid to another so the basic "language" remains the same.
Limit of resolution:
Refers to how far apart adjacent objects must be to appear as separate entities. The smaller the limit of resolution, the greater the resolving power-ability to see finer details of structure. Example: If limit is 400nm, objects must be 400nm apart. 200nm would be a better microscope. Theoretical limit of resolution: half the size of the wavelength of light used (max magnification 1000X-1400X). For visible light (400nm-700nm), limit of resolution is about 200-350nm.
Electrophoresis
Refers to several related techniques that use an electrical field to separate macromolecules based on their mobility through a semisolid gel. Travel at different rates depending on size and charge.
Scanning Electron Microscope (SEM)
Scans the surface of the specimen and forms an image by detecting electrons that are deflected from its outer surface.
Recombinant DNA Techology
Scientists can create recombinant DNA molecules containing DNA sequences from two different sources using restriction enzymes.
Transfer RNA (tRNA)
Serve as intermediaries that recognize the coded base sequence of an mRNA and bring the appropriate amino acids to the ribosome for protein synthesis.
Fluorescence Microscopy
Shows the locations of specific molecules in the cell. Fluorescent substances absorb ultraviolet radiation and emit visible light. The fluorescing molecules may occur naturally in the specimen, but more often are made by tagging the molecules of interest with fluorescent dyes or antibodies. Can detect specific proteins, DNA sequences, or other molecules that are made fluorescent by coupling them to a fluorescent dye or binding them to a fluorescently labeled antibody. Use two or more such dyes or antibodies, each emitting light of a different color: can follow the distributions of different kinds of molecules in the same cell. Limitation: viewer can focus on only a single plane of the specimen at a time, yet fluorescent light is emitted throughout the specimen, blurring the image.
Functional groups
Specific arrangements of atoms that confer characteristic chemical properties on the molecules to which they are attached. At neutral ph, many are ions/proton (H+)
Model systems
Systems developed to study cellular processes directly in living cells and organisms.
Resolving Power
The ability to see fine details of structure.
Bond energy
The amount of energy required to break one ole of a bond. Expressed in calories
Dependent variable
The outcome of the change that is measured
DNA transformation
The process of introducing DNA into cells.
Hydrocarbons
The resulting compounds when only hydrogen atoms are bonded to carbon atoms in linear or branched chains or in rings Limited use in biochemistry because insoluble in H2O. Used in cell membranes- tails of phospholipids.
Cytology
The study of cellular structure. Cyto=hollow vessel. Three centuries ago: -Light microscope:initial development -electron microscopy/other advance optical techniques- increased understanding of cell structure/function
Bright Field Microscopy
Type of microscopy described. White light is passed directly through a specimen that is either stained or unstained and the background (field) is illuminated. Staining: enhances contrast Limitations: Specimen must often be chemically fixed (preserved), dehydrated, embedded in paraffin or plastic for slicing into thin sections, and stained to highlight otherwise transparent features. No longer alive, so features observed could be distortions caused be slide preparation.
Polar bond
Unequal sharing of electrons. The more electronegative atom will have electron ore than half the time, giving that atom a partial negative charge and the other atom (C or H) a partial positive charge. More soluble in H2O, more chemical reactivity.
Polarity
Uneven distribution of charge within a molecule.
Superresolution Light Microscopy
Use imaging and computational methods so advanced that they can see beyond the theoretical limit of resolution. These methods allow us to visualize structures 50-100 nm in size, which, until the past few years, were believed impossible to see with any light microscope.
Electron Microscope
Uses a beam of electrons that is deflected and focused by an electromagnetic field instead of visible light and optical lenses. Max Knoll/Ernst Ruska 1931. Because the wavelength of electrons is so much shorter than the wavelengths of visible light, the practical limit of resolution for the electron microscope is much better—generally about 100 times better than a light microscope, or 2 nm. Magnification 100000X. Two Designs: -Transmission Electron Microscope (TEM) -Scanning Electron Microscope (SEM)
Confocal Miscroscopy
Uses lasers and special optics to focus illuminating beam on a single plane within the specimen. Only those regions within a narrow depth of focus are imaged. Regions above and below the selected plane of view appear black rather than blurry. Overcomes limitation of fluorecence.
Digital Video Microscopy
Uses video cameras to collect digital images for computer storage. By attaching a highly light-sensitive digital video camera to a light microscope, re-searchers can observe cells for extended periods of time using very low levels of light. This image intensification is particularly useful to visualize fluorescent molecules present at low levels in living cells and even to see and identify individual macromolecules (DNA, proteins)
Hydrogen Bond
Water molecules are attracted to each other so that the electronegative oxygen atom of one molecule is associated with the electropositive hydrogen atoms of adjacent molecules. Noncovalent interaction, approximately 1/10 as strong as a covalent bond.
Hypothesis
a tentative explanation that can be tested experimentally or via further observation. Often, a hypothesis takes the form of a model that appears to provide a reasonable explanation of the phenomenon in question.
Ribosomal RNA (rRNA)
integral components of the ribosome itself.
Genomics
the study of all the genes of an organism