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James Thomson

EXAM REVIEW : LECTURE 2 :  Two main type of cells : prokaryotes and eukaryotes  Prokaryotes:  Two groups of prokaryotes: eubacteria and archaea - DNA, RNA and protein are composed of 6 elements: H,C,N,O,S,P - No nuclei but a nucleoid, single celled organism, cell wall present, plasma membrane encloses DNA,RNA, proteins. Mitochondria creates ATP (the cell’s energy)  Eukaryotes  Plant, fungi, animals, humans. 1000 times bigger than prokaryotic cells - Usually single-celled but can also be multicellular - Nucleus separates the DNA from the cytoplasm with a membrane. Cytoskeleton. Use phagocytosis to engulf things.  Origins of mitochondria: - Almost all cells contain mitochondria (small bodies in cytoplasm that take up oxygen and harness energy to form ATP that powers the cell’s activities). - Originated from aerobic bacteria that were engulfed by an ancestral anaerobic eukaryotic cell. Both lived in symbiosis; the mitochondria bacterium helping with power generation, and the ancestral eukaryote giving shelter and nourishment to the bacterium.  General attributes of Model organisms: - Rapid development with short life cycles - Small adult (reproductive) size - Readily available (collections or widespread) - Tractability – ease of manipulation or modification - Understandable Genetics  Types of model organisms: - Prokaryote: E.Coli –heterotrophic eubacterium found in the human gut- Synechocystis – free living phototrophic eubacterium- - Eukaryote: o Yeast (Saccharomyces cerevisiae) -minimal eukaryotic model, as closely related to animals as it is to plants. o Arabidopsis- model of flowering plant specie o C.Elegans- nematode worm o Drosophila (fruit fly), mouse and human.  The central dogma; pathway from DNA to protein - DNA, RNA and proteins are all linear chains of information - DNA mRNA(translation)Protein - DNAtRNATransport AA’s for protein synthesis - Information in a nucleic acid sequence (a DNA or RNA sequence) is translated into an AA sequence via a genetic code which is essentially universal among all species. - All cells replicate their hereditary information by templated polymerization (one strand is copied to make another)  The genetic code and nucleic acids - Each nucleotide consists of two parts: a base (either A, T, C or G) and a sugar- phosphate backbone. Each sugar is linked to the next via the phosphate group –phosphodiester bonds- (composed of either 1,2, or 3P’s), creating a long polymer chain. Individual sugar-phosphate groups are asymmetric, giving the strand a polarity or directionality.  What are the differences between DNA and RNA? - DNA: Deoxyribose, ATCG bases, DNA helix is tighter than RNA’s (harder to destroy) - RNA: Ribose, AUCG bases, U used in RNA because it reacts to other molecules more easily, and can therefore be made and destroyed easily.  Nucleic Acid Nomenclature: - Nucleoside monophosphate: Sugar+base+1 phosphate - Nucleoside diphosphate: Sugar+base+2 phosphate - Nucleoside triphosphate LECTURE 3: Molecular Interactions:  4 types of non-covalent attractions: very weak forces but add up to generate strong binding between molecules. - Ionic bonds (electrostatic attractions): result from the attractive forces between oppositely charged atoms. - Hydrogen bonds: polar interaction in which a hydrogen atom is shared by two other atoms- binds DNA on straight line so bases are on the same plane. - Van der Waals attraction: The electron cloud around any non-polar atom will fluctuate and therefore produce a very weak attraction between molecules. - Hydrophobic force –pull the whole DNA helix to the centre-  Base Pairing: - A-T bonds with 2H-bonds - C-G bonds with 3 H-bonds - A and G are purine bases, C, T and U are pyrimidine bases  DNA structure: - DNA forms a double stranded helix; the two strands are complimentary and going into opposite direction. They are antiparallel; one strand is 3’ (ends with the hydroxyl group) to 5’ (ends with the phosphate group), while its partner is 3’ to 5’. - Every 10 nucleotides there is a “turn” in the helix; these turns are called grooves, the major groove and minor groove. The major groove is big enough so that DNA binding proteins can access the DNA bases.  DNA unzipping - important for replication and RNA synthesis - Heating denatures DNA by disrupting H-bonds between bases - The temperature at which DNA denatures is called T(m) (melting temperature) - Denaturation of DNA is a reversible process  Application of DNA denaturation/renaturation capacity - PCR: –Polymerase chain reaction- gene cloning  During the first cycle; double-stranded DNA is heated to separate strands and add primers (short piece of complementary DNA that is needed to start the DNA polymerase –what copies DNA-), after that DNA synthesis happens.  During the second cycle, fragments of DNA from the first cycle are used to make new DNA (and same for all the other cycles.)  Introduction to protein structure: - Primary : AA sequence or protein sequence - Secondary: local folding such as alpha helix or beta sheet - Tertiary: long-range folding such a 3D protein structure - Quaternary: multimeric organization such as multiple polypeptide chains - Supramolecular: large-scale assembly such as viruses making a spherical chain of protein  Proteins: - Proteins are long unbranched polymer chains, formed by stringing amino acid monomers together. There are 20 types of amino acids, and each of them have a side group (R- group) that gives them their distinctive character. - There are 4 categories of amino-acids:  Basic (+charge) - Histidine  Acidic (- charge or neutral)- Aspartic Acid  Polar (uncharged)- Threonine  Non-polar- Phenylalanine  The AA cysteine and disulphide bonds: - Cysteine has the ability to bond to other cysteine AA’s, and forms disulphide bonds used to form bridges, shape and connect to other polypeptide chains and/or proteins. - Disulphide breaks under reduced conditions, therefore proteins can be broken down when environment not stable.  Amino Acid review facts - Groups of AA’s with similar properties tend to be clustered in the codon table - Codons of AA’s with similar properties tend to have fewer mutational steps between them - One random mutation in a codon is less likely to result in a dramatic change in amino acid properties than two random mutations  Synthesis of Proteins - Possess a carboxylic acid group (COOH) that is also called the “C-terminus” and an amino group called the “N-terminus”, and both are linked by a single carbon atom called the α- carbon. AA’s are always presented from the N- terminus to the C-terminus, so read from left to right. - A condensation reaction between the C-terminus and the N-terminus of two AA’s creates a peptide bond - Their chemical variety comes from the side chain that is attached to the α- carbon. AA’s make proteins, which are polymers of amino acids joined head to tail in a long chain that is then folded into a 3D structure unique to each type of protein.  The Alpha helix- secondary protein structure - An α-helix is generated when a single polypeptide chain twists around on itself to form a rigid cylinder. - A hydrogen bond forms between every fourth peptide bond, linking the C=O of one peptide bond to the N-H of another - α-helices can wrap around each other to form a particularly stable structure called coiled coil. This structure can form when two or three α-helices have most of their non-polar (hydrophobic) side chains on one side, so they can twist around each other with hydrophobic side chains facing inwards.  The Beta sheet- secondary protein structure - Core of many proteins; form either from neighbouring parallel polypeptide chains or from a long polypeptide chain that fold back and forth upon itself (antiparallel chains) - Very rigid structure held together by hydrogen bonds that connect the peptide bonds  Tertiary structures: 3D polypeptide chain - Held together by  Hydrophobic interactions  Non-covalent bonds  Covalent disulphide bonds (from AA cysteine)  Each of the protein molecules folds into a precise 3-D form with reactive sites, or domains on its surface. These reactive sites bind specifically to other molecules, and show as a groove in the surface of the polypeptide to act as enzymes to catalyse reactions that make or break covalent bonds. - Example: The protein Src has three domains specialized for different functions:  SH2 and SH3 domains both perform regulatory functions  Kinase domain is a functional domain that can be activated/deactivated to form the kinase enzyme  Quaternary Structure: - Complex of more than one polypeptide chain  Hemoglobin; formed of different subunits (2 alpha, 2 beta)  Each subunit is a separate polypeptide.  Sickle cell anemia is caused by a mutation in the Beta subunit  Hemoglobin function: - Transports O 2rom lungs to tissues - Heterozygotes for the sickle cell anemia mutation in the Beta globin gene are partially protected against malaria - Frequency of the sickle cell allele has reached highest levels in Africa and India (evolution led because of the high levels of malaria) - Related molecule, myoglobin has only one subunit and is found in muscle tissue  Large scale protein assemblies: - Protein molecules often serve as subunits for the assembly of large structures such as enzyme complexes, ribosomes, viruses and membranes - In many viruses, identical protein subunits pack together to create a spherical shell (capsid) that protects the DNA and RNA LECTURE 4:  Human Genome sequence: - 3 billion base pairs per genome - One maternal + one paternal genome - ~25 000 genes spread across 23 chromosomes - XX female, XY male - Only 1.5% of the human genome encode for proteins and 50% of your genome is repetitive DNA - Many different parts in a genome: o Exons, introns, transposons, retroviral-like elements (remnants from viruses from the past)  Packaging of DNA in the cell - In prokaryotes: o In a non-packaged state, even a small prokaryotic genome would occupy too much space of the cell volume o In prokaryotes, the DNA is condensed through folding and twisting about 1000 fold. Bound together, DNA + protein form the nucleoid o Important components of this packaging are: Positively charged polyamines Nucleoid-associated proteins Supercoiling of DNA by enzyme topoisomerase  In eukaryotes o The nucleus encloses the genome to protect it o The genome is packed into chromosomes in order to make more space in the cells (23 pairs) o Each chromosome contains a single, long linear DNA molecule and associated proteins; the association of the two is called chromatin o Chromatin is tightly packaged, but the DNA must remain accessible for transcription, replication and repair. o Coding sequences are called exons, non-coding sequences are called introns  Hybridization: - Fluoresence In Situ Hybridization can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence complementarity.  The cell cycle: - Interphase: gene expression and chromosome replication. - M-phase: Happens when DNA is completely replicated, mitosis occurs, - chromosomes condense, are pulled apart and nucleus is divided into 2 daughter nuclei. A nuclear envelope reforms and in the final step of M-phase, two daughter cells are produced.  Nucleosomes: - Nucleosome is the basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound in sequence around 4 histone protein cores. - There are 4 core histones proteins (H2A, H2B, H3 & H4) and there are a pair of each in each octamer core, and one linker histone (H1). - The nucleosome core particle can be released from isolated chromatin by digestion of the linker histone with a nuclease (enzyme that breaks down DNA linker).  Structure and packaging of a nucleosome: - Each of the core histones has an N-terminal amino-acid “tail” which extends out of the DNA-histone core. These histone tails affect the further stacking of the nucleosome and can also be modified to affect its structure. - Chromatin remodelling complexes u-sing the energy of ATP hydrolysis- push the DNA out of its bound on the octameric histone core and loosens it in order to make DNA available to other proteins in the cell  Chromatin packing: - A short region of DNA double helix turns into the “beads-on-a-string” form of chromatin after being wrapped around core histone octamers. Nucleosomes are then tightly packed together to form the mitotic chromosome shape we all know - The net result is that each DNA molecule has been packaged into a chromosome that is 10 000 shorter than its extended length - Chromatin is also remodelled into loops to alter access to DNA.  There are two different kinds of chromatin: Heterochromatin Euchromatin Highly condensed chromatin Relatively non-condensed chromatin Mitotic & meiotic chromosomes Centromeres and telomeric regions “Heterochromatic” regions of “Euchromatic” regions of interphase interphase chromosomes = regions chromosomes = regions where genes where gene expression is suppressed tend to be expressed - Covalent modification of histones, the presence of chromatin remodelling complexes, and RNA polymerase complexes modulate the reversible switching from euchromatic to heterochromatic regions. o Methylation leads to heterochromatin o Acetylation leads to decondensation of chromatin  Genes move to different regions of the nucleus when their expression changes: - The interior of the nucleus is very heterogeneous, and different nuclear neighbourhoods have distinct effects on gene expression. Therefore, a gene will move to a neighbourhood that will favour its expression o Eg: Nuclear neighbourhood for gene silencing. LECTURE 5:  DNA is semi-conservative; the two new daughter DNA molecules are half “old” and half “new”  There are two types of cells; germ cells (transmit genetic information) and somatic cells (form the body of the organism)  The direction in which DNA is replicated is unidirectional: o Always from the 5’ to the 3’ end with the help of the enzyme DNA polymerase. . This characteristic of DNA polymerase means that the daughter strands synthesize through different methods. o The first strand, which replicates nucleotides one by one, is called the leading strand; the other strand, which replicates in chunks, is called the lagging strand.  The DNA replication machinery begins replication at the origin of replication. Origins of replication include an initiator protein called Orc (Origin recognition complex), a stretch of DNA that is AT rich (easy to unwind) and one binding site for proteins.  Experiment to identify origins of replication in yeast Ars = autonomously replicating sequence - This experiment was looking at the effect of deleting various replication origins in yeast. It proved that removing a few origins have little effect, because replication forks that begin at neighbouring origins of replication can continue into regions that lack their origin. But if many are removed it results in the loss of the chromosomes as the cells divide, because it replicated too slowly. - This is the reason why eukaryotes carry an excess of origins; to ensure that the complete genome can be replicated in a timely manner if some origins fail to function.  How does DNA replication proceed in bacteria? - DNA replication starts at a single origin of replication, and the two replication forks assembled proceed in opposite directions until they meet halfway around the chromosome  What happens at the DNA replication forks? 1. An initiator protein is at the replication origin, and DNA helicase binds to the initiator protein. 2. Both DNA binding proteins (SSB) and DNA Helicase work together to untwist the helix at locations called replication origins (helicase works from 5’ to 3’ on the lagging strand). SSB stabilize the single strands of DNA to help the DNA helicase, and prevent the formation of hairpin helices. 3. In the leading strand; the nucleotides are added in the 5' to 3' direction. Triggered by RNA primase -which adds the first nucleotide to the nascent chain- the DNA polymerase simply sits near the replication fork, adding nucleotides one after the other, preserving the proper anti-parallel orientation. A sliding clamp keeps the polymerase firmly on the DNA when it is moving, but releases it as soon as it encounters a double-stranded region of DNA. 4. In the lagging strand; replication happens in small segments, called Okazaki fragments. Primase moves with the replication fork and synthesizes numerous RNA primers, each of which triggers the growth of an Okazaki fragment. These fragments are stretches of 100 to 200 nucleotides in humans that are synthesized in the 5' to 3' direction away from the replication fork. Each subsequent Okazaki fragment starts at the replication fork and continues until it meets the previous fragment. These fragments are then stitched together by DNA ligase, creating a continuous strand. Lecture 6 – Replication (II) DNA Replication mechanism – Replication fork  DNA replication is performed by a large multienzyme complex: this “machine” is composed of proteins and powered by nucleoside triphosphate hydrolyses. STEPS REPLICATION  At the front of replication fork: DNA helicase opens the DNA helix.  2 DNA polymerases: one is situated on the leading strand, whilst the other works on the lagging strand. - DNA polymerase on leading strand works in a continuous
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