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Molecular Bio & Genetics

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Laura Parker

Molecular Bio & Genetics (Nathans) Mutation and DNA Repair Lecture -mutation: any inherited alteration that does not involve uptake of foreign genetic material -allele: one of the different forms of a gene that can exist at a single locus -wild type: what you would find in the wild -phenotype: detectable outward manifestations of a specific genotype -genotype: specific allelic composition of a cell -prototroph: wild type or revertants that do not require the added compound -auxotroph: mutant that is deficient in the synthesis of an essential compound -conditional mutation: mutant phenotype is observed only under certain circumstances -permissive condition: environ conditions under which a conditional mutant shows the wt phenotype -nonpermissive condition: doesn’t allow the protein to be normal, brings out the mutant phenotype -selection: a protocol that allows you to eliminate by killing all of the non-mutant organisms and allows only the mutants to survive -enrichment: selecting for auxotrophs by performing a screen in which you selectively kill of the normal cells/bacteria and eventually isolate the cells/bacs with the deleterious mutation -screen: a detection system, a set of circumstances in which a mutant allele will make its presence know at the phenotypic level -replica plating: transferring bacterial colonies growing on an agar plate with a sterile velvet disc to several secondary plates in such a way that the spatial pattern of bacterial colonies is preserved -leaky mutant: mutations in less crucial areas of a protein will most likely have less deleterious effect, often resulting in “leaky” or partly inactivated mutants -insertion: -deletion: -substitution: -frameshift: insertion or deletion of nucleotide pair or pairs, causing a disruption of the translational reading -nonsense: a bp substitution results in the creation of one of three stop codons, terminating the polypeptide chain -missense mutation: one amino acid is substituted for another -reversion: the production of the wt gene from a mutant gene -suppression: 2 site reversion in which an alteration somewhere else restores the activity, it suppresses the effect of the 1 mutation -intragenic: suppressor of mutant on the same gene -extragenic: suppressor of mutant on another gene -complementation: the production of a wt phenotype when two diff mutations are combined in a diploid or heterokaryon -mutagen: agent that is capable of increasing the mutation rate -base analogue: a chemical whose molecular structure mimics that of a DNA base; because of the mimicry, the analog may act as a mutagen -deamination: process of removing the amine group -akylating agent: a chemical agent (i.e. mutagen) that can add alkyl groups to another molecule -intercalating agent: a chemical that can insert itself b/w the stacked bases at the center of the DNA double helix, possibly causing a frame-shift mutations -Ames test: test to detect possible chemical carcinogens; based on mutagenicity in the bacterium Salmonella -SOS repair: error-prone process whereby gross structural DNA damage is circumvented by allowing replication to proceed past the damage through imprecise polymerization -direct repair: repair of a DNA lesion once it occurs by reversing it directly to the normal base (e.g. DNA photolyase repair of thymine dimmers, alkyltransferase protein, base excision repair, nucleotide excision repair) -base excision repair: a chemically modified base can be cut off of the attached ribose at the N-glycosidic bond leaving an ‘AP’ (AP= apurinic or apyrimidinic) site. DNA Pol and DNA ligase eventually repair the gap. -nucleotide excision repair: distortion in DNA caused by damage can be repaired by the excision of a patch of 12 bases by the avrABC nuclease system -mismatch repair: a repair mechanism that is able to recognize errors even after DNA replication has already occurred; this mech can detect DNA mismatches that occur in DNA replication -xeroderma pigmentosa: results from a defect in any of the genes effecting nucleotide excision repair Molecular Genetic Methods Lecture (part 1) Restriction enzyme: cut across both strands of a DNA molecule at a specific and relatively small (4-8bp) recognition sequence Clone: a group of genetically identical cells or individs derived by asexual division from a common ancestor; a section of DNA that has been inserted into a vector molecule, such as a plasmid or a phage chromosome, and then replicated to form many copies Plasmid vector: autonomously replicating extrachromosomal DNA containing an origin of DNA replication, an antibiotic resistance gene, and a region that is not essential for propagation into which a piece of foreign DNA can be inserted Viral vector: phage chromosome used to carry a cloned DNA segment cDNA: ‘complementary DNA’ produced by synthesis of a complementary DNA strand by reverse transcriptase using mRNA as a template strand cDNA libraries: allow one to analyze mRNA structure and abundance by working with DNA copies of the sequence which are more stable and more easily manipulated than RNA copies Denaturation: separation of the two strands of the DNA double helix without breaking the major bonds of its chains Renaturation: reassociation of complementary single strands of nucleic acids into double- stranded helical forms. Southern blot: transfer of electrophoretically separated DNA fragments from the gel to an absorbent sheet. This sheet is then immersed in a solution containing a labeled probe that will bind to a fragment of interest Northern blot: transfer of electrophoretically separated mRNA fragments from the gel to an absorbent sheet, which is then immersed in a labeled probe that will bind to the RNA of interest Hybridization probe: A hybridization probe is a short piece of DNA (on the order of 100- 500 bases) that is denatured (by heating) into single strands and then radioactively labeled, usually with phosphorus (32P or 33P). The radioactive phosphorus is incorporated into one of the bases of DNA (adenine, guanine, cytosine, or thymidine) and these labeled bases are incorporated into the backbone of the DNA strands by incubation with a polymerase enzyme Restriction map: DNA RFLPs are used as reference loci for mapping in relation to known genes other RLFP loci Synthetic DNA: the solid phase chemical synthesis of DNA fragments by growing a nucleotide chain anchored to a resin PCR: method of amplifying specific DNA segments that exploits certain features of DNA replication DNA chip: a highly miniaturized array of target DNA sequences immobilized on a solid surface and the DNA probe is tagged with a fluorescent compound Molecular Genetics Methods Lecture (part 2) Western Blot: membrane carrying an imprint of proteins separated by electrophoresis; can be probed with a labeled antibody to detect a specific protein Protein tags: to produce a protein by introducing the cloned DNA that codes for it into bacterial cells, we can change the coding DNA so that we will produce a derivative of the original protein that has additional sequences that help visualize the protein Affinity purification: makes use of specific binding interactions between molecules. A particular ligand is chemically immobilized or “coupled” to a solid support so that when a complex mixture is passed over the column, only those molecules having specific binding affinity to the ligand are purified Epitope: A unique shape or marker carried on an antigen's surface that triggers a corresponding antibody response Yeast two-hybrid system: a purely genetic approach to identifying which other proteins bind to a particular protein of interest; The yeast two hybrid screen is designed to identify relevant protein-protein interactions using a bait protein to fish out target proteins. A postive signal is identified by in vivo reconstitution of an active transcription complex. The one hybrid screen is designed to fish out DNA binding proteins that recognize a DNA sequence cloned upstream of yeast reporter genes. Tissue culture: maintenance or growth of tissues, in vitro, in a way that may allow differentiation and preservation of their architecture and/or function Transfection: introduction of DNA into cultured cells; ‘stable transfection’ refers to that added DNA ends up integrating into genome and its stably maintained Transgenic animals: An animal into whose genome foreign DNA has been introduced to study the function of that DNA Embryonic stem cells: Cells of the early embryo that can give rise to all differentiated cells, including germ line cells; introduce DNA segment into ES cells and select for homologous integration, inject ES cell line with the correct targeting event into blastocysts, and implant blastocyst into a foster mother, the pups will be chimeras, if the ES cells contributed to the germ-line then the ES genotype can be passed to the next generation Knockout mouse: A gene knockout is a genetically engineered organism that carries one or more genes in its chromosomes that has been made inoperative or simply saying one or more genes have been knocked out of that particular organism. Knockout is accomplished through a combination of techniques, beginning in the test tube with a plasmid, a bacterial artificial chromosome or other DNA construct, and proceeding to cell culture. Individual cells are genetically transformed with the construct and--for knockouts in multi-cellular organisms--ultimately fused with a stem cell from a nascent embryo. The construct is engineered to recombine with the target gene, which is accomplished by incorporating sequences from the gene itself into the construct. Recombination then occurs in the region of that sequence within the gene, resulting in the insertion of a foreign sequence to disrupt the gene. With its sequence interrupted, the altered gene in most cases will be translated into a nonfunctional protein, if it is translated at all. In diploid organisms, which contain two alleles for most genes, and may as well contain several related genes that collaborate in the same role, additional rounds of transformation and selection are performed until every targeted gene is knocked out. Gene therapy: add the normal version of a gene to the somatic cells of an individual who had inherited a defective version Genetically engineered vaccines: genes required for virulence can be deleted so that reversion to the pathogenic state is impossible, and additional immunogenic proteins can be added to the vaccine strain to produce a vaccine that protects against multiple strains of a pathogen Stem cell therapy: the process of injecting stem cells into an organism in the hopes that the stem cells will differentiate into the same type of cell as the damaged cells, and replace them Bacterial cells Prokaryote: Organisms, namely bacteria and cyanobacteria (formerly known as blue- green algae), characterized by the possession of a simple naked DNA chromosome, occasionally two such chromosomes, usually of circular structure, without a nuclear membrane and possessing a very small range of organelles, generally only a plasma membrane and ribosomes Eubacteria: The commonly occurring prokaryotes that live in water or soil, or within larger organisms; Archaebacteria and Eubacteria differ from each other in their metabolic abilities, the composition of their membranes, and the structure of their ribosomes Archaea: A group of prokaryotic single-celled microorganisms that constitute the recently recognized Archaea phylogenetic domain. Archaea can be distinguished from bacteria in that their cell walls do not have murein, a peptidoglycan-containing muramic acid. Another unique feature of archaea is the presence of isopranyl ether lipids in their cell membranes Gram stain :a staining technique used to classify bacteria; bacteria are stained with gentian violet and then treated with Gram's solution; after being decolorized with alcohol and treated with safranine and washed in water, those that retain the gentian violet are Gram-positive and those that do not retain it are Gram-negative Cocci: spherical bacteria Bacilli: rod-shaped bacteria Coupled transcription/translation: the ribosome follows closely apace behind the RNA polymerase Periplasmic space: periplasmic proteins (usually hydrolytic enzymes) reside in this ‘space’ between the inner and outer membrane of gram-negative bacteria Flagellum: A long, thread-like organelle used by many microscopic organisms for locomotion and feeding Pilus: thin proteinaceous tubes projecting out from the bacterial surface that function in attachment to cell surfaces and for conjugation and DNA transfer Fimbria: an appendage in many gram-negative bacteria that is thinner than a flagellum Capsule (glycocalyx): slimy layer of polysacchrides (or possibly protein/glycoprotein) polymerized at the outer surface of a bacterial cell varying in thickness according to species Peptidoglycan: Large polymer that provides much of the strength and rigidity of bacterial cell walls, in gram-positive cells that makes up around 50% of total cell weight, while in gram negative cells it makes up as little as 1-2% Teichoic acids: are characteristic features of gram positive cells that exist in two forms, membrane and cell wall; can account for up to 50% of cell wall mass, are immunogenic, and serve as attachments for capsular polysacchrides Penicillin: a β-lactam antibiotic used in the treatment of bacterial infections caused by susceptible, usually Gram-positive, organisms; kills cells by lysis as bactericidal drug Outer membrane: a part of gram-negative cell envelope; a phospholipid bilayer that is more permeable to charged ions than cytoplasmic membrane Lipopolysaccharide: an essential constituent of the outer layer of gram-negative membrane made up an O-specific polysaccharide joined to a core region, which is turn attached to lipid A which is anchored in the outer membrane outer leaflet O-antigen: a species-specific repeating polysaccharide that extends from the core of the gram-negative cell wall into medium Lipid A: a disaccharide phosphate unit to which are joined several fatty acids of various types; the fatty acids are anchored hydrophobically in the outer leaflet of the outer membrane of a gram-negative cell Braun’s lipoprotein: as the final component of the gram-negative cell wall, this specialized lipoprotein attaches the peptioglycan layer to the outer membrane Bacterial Gene Transfer Bacteriophage (bacterial virus): consist of an inner core of DNA or RNA and an external protein coat that protects the phage genome and mediates binding to a specific attachment site on the bacterial surface Lysogenic infection: the phage genome enters into a dormant state within the bacterium, replicating once per cell division either as an extrachromosomal element or by virtue of its integration into the host chromosome Lytic infection: replication of the phage DNA and expression of genes encoding the phage proteins, assembly of hundreds of progeny phage, and lysis of the host cell Lambda repressor: the lysogenic pattern of gene expression consists of production of the lambda repressor, a protein that binds to P Lnd P aRd blocks their transcription. At concentrations capable of inactivating P and P , lambda repressor activates transcription L R of its own gene, but at higher concentrations it represses its own synthesis. The repressor/repressor gene feedback loop maintains the lysogenic state indefinitely Induction: 1. The relief of repression for a gene or set of genes under negative control by a repressor. 2. Regarding temperate phages, the process causing a prophage to become virulent. Generalized transduction: the encapsidation of a fragment of bacterial DNA in a phage particle and subsequent transfer of this DNA into a recipient bac cell in which the transduced fragment can recombine into the genome by double crossing over Specialized transduction: occurs when a prophage (e.g. lambda) excises imperfectly and carries w/ it a segment of flanking bacterial DNA. It’s called ‘specialized’ because only the genes immediately flanking the phage integration site are transduced. Specialized transduction generates a hybrid DNA molecule part of which contains bacterial sequences and part phage sequences. Transformation: the direct uptake of free DNA, following adsorption to the cell surface the DNA is cleaved by a surface endonuclease and then one strand enters the cell (the other is degraded). The single-stranded DNA can displace its homologue from the chromosome and form a heteroduplex with complementary strand. DNA replication and/or mismatch repair can permanently convert the recipient allele to the transforming allele Conjugative plasmid: A plasmid that can effect its own intercellular transfer by means of conjugation (the union of two bacterial cells, during which chromosomal material is transferred from the donor to the recipient cell); this transfer is accomplished by a bacterium being rendered a donor, usually with specialized pili F factor (fertility factor): a bacterial genetic extrachromosomal element whose presence confers donor ability -In bacterial gene transfer, one organism receives genetic information from a donor; the recipient is changed by that information Tra operon: region on the F factor that encodes over twenty genes that are involved in conjugation. The products of these genes include the structural gene for the sex pilus, proteins involved in pilus assembly, and proteins required for DNA processing before and during conjugation oriT (origin of transfer): a specific DNA sequence on the F factor at which conjugtive transfer replication initiates Unidirectional transfer: the mode of conjugative transfer of either plasmid or chromosomal DNA from one cell to another by cell-cell contact. DNA transfer is not reciprocal, therefore the two cells that participate are referred to as "donor" and "recipient". F-plus: In E. coli, a cell having a free fertility factor; a male cell F-minus: In E. coli, a cell having no fertility factor; a female cell F-prime factor: a fertility factor into which a part of the bacterial chromosome has been incorporated Hfr: In addition to existing as a plasmid, the F factor can become integrated into the bacterial chromosome. A cell containing an integrated F factor can donate chromosomal - DNA to an F recipient. Just as for transfer of F DNA, this transfer of chromosomal genes requires cell to cell contact and is unidirectional. Cells containing an integrated F factor are termed Hfr since they donate chromosomal genes to an F recipient giving rise to recombinants at high (H) frequency (fr). Cointegrate: two plasmids joined together; the product of the fusion of two circular elements to form a single, larger circle Bacterial Gene Regulation Activator: Attenuator: a region adjacent to the structural genes of the trp operon; this region acts in the presence of tryptophan to reduce the rate of transcription from the structural genes Biofilm: an immobile, structured community of bacteria (or fungus) encased in a self- produced polymeric polysaccharide matrix and adherent to an inert or living surface CAP (Catabolite Activator Protein): binds front of and adjacent to RNA polymerase, it increases affinity of the polymerase for the promoter and increases the transcription rate cis-dominant mutation: the ability of a gene to affect genes next to it on the same chromosome; implies the physical interaction of an element with the genes directly in contact with it Constitutive mutant: Elongation: Horizontal gene transfer: transduction, transformation, & conjugation Inducer: an environmental agent that triggers transcription from an operon Initiation (transcriptional): Initiation (translational): lac operon: lacI gene: gene that encodes the repressor in the lac operon system mero-diploid: Operator (e.g. lacO): a DNA region at one end of an operon that acts as the binding site for repressor protein Polycistronic mRNA: a mRNA that encodes more than one protein Promoter: Quorum sensing: a means of prokaryotic cell-cell communication that allows bacterial cells to modulate their behavior as a function of cell density via signaling molecules Regulon: A group of transcriptional units or operons of related function scattered around at different sites that are coordinately controlled by a regulator Release factor: RNA secondary structure: Sigma factor: subunit of RNA polymerase that specifies the site of transcription initiation; diff sigma factors recognize diff promoter sequences, allowing sporulation- specific genes to be expressed coordinately Site-specific recombination: Response regulator: Transcription unit: Translational control: trans-acting mutation: TRAP (trp RNA attenuation protein): promotes terminator formation and transcription attenuation in the B. subtilis operon trp operon: Two-component system: a signal transduction system comprised of sensor histidine kinases and their response regulator substrates by which bacteria sense and respond to extracellular signals Drugs, Sex and Transposition Acetyltransferase: Aminoglycoside: A group of antibiotics (such as gentamicin) that inhibit bacterial protein synthesis and are particularly active against Gram-negative bacteria Beta-lactamase: a type of enzyme responsible for bacterial resistance to beta-lactam antibiotics (e.g. penicillin).They have a common a four-atom ring known as a beta- lactam. The lactamase enzyme breaks that ring open, deactivating the molecule's antibiotic properties dam methylase/methylation: Methylation at the N 6 position of adenine in the sequence GATC by dam methylase, an enzyme found in most laboratory strains of E. coli. dam methylation may prevent cleavage by some restriction enzymes. Direct repeat: Donor site: Insertion sequence (IS): smallest units of transposition in bacterial cells; they can hop from one region from one region of the genome to another, from chromosome to plasmid or phage genome. IS sequences don’t themselves carry drug resistance genes, but do carry the genes required for the transposition process; these tnp genes encode transposase proteins Integrase: a protein produced by a virus that enables genetic material that is helpful to the virus, proviral DNA, to be integrated into the DNA of the infected cell. Inverted repeat: An inverted repeat is a sequence of nucleotides that is the reversed complement of another sequence further downstream. For example, 5'--- GACTGC....GCAGTC---3'. Inverted repeats define the boundaries in transposons. LINE (L1): a human retrotransposon that also encodes RT, and transposases via an RNA intermediate. Yet it lacks LTRs, the IN and PR proteins of retrovirus, instead encoding an endonuclease (EN) protein that nicks target sites in the host chromosome. This type of element moves by Target Primed Reverse Transcription (TPRT) LTR retrotransposon: Multiple drug resistance protein: human cancer cells become resistant to multiple chemoteherapeutic drugs simultaneously. These cells overexpress a normal cellular protein, encoded by the mdr gene, that apparently acts as a pump for these drugs, pumping them out of the cell Non-LTR retrotransposon: retrotransposons w/o long terminal repeats Palindrome: both strands of DNA have the same nucleotide sequence but in antiparalllel orientation Phosphotransferase: category of enzymes with the quality of catalyzing phosphorylation Reporter gene: reporter-gene constructs, pieces of cis-regulatory DNA are fused near a transcription unit that can express a reporter protein, a protein whose presence can be easily monitored R (resistance)-factor: large, conjugable plasmids similar to the F factor, are responsible for much clinically drug resistance. It consists of insertion sequences (IS), transposons (Tn), and integrons Retrotransposon: makes a full-length RNA copy of its genome, which is then reverse transcribed to DNA by the enzyme reverse transcriptase (RT). The DNA copy made by the RT is then integrated into the genome. This rxn is carried out by a transposon- encoded integrase protein (IN), which resembles bac transposases. SINE (Alu, non-LTR): a mobile repeat sequence abundant in humans. This element is transcribed by RNA polymerase III. Alu sequences don’t encode proteins but can move and cause disease when they disrupt genes. They’re known to highjack LI encoded proteins in order to retrotranspose. Staggered break: Target site duplication: Transposase: Transposon (Tn): segment of DNA able to move to a new site in host genome without any sequence homology b/w the transposon and target. These elements usu encode the proteins for such movement. Chromosomes Adjacent segregation: in a reciprocal translocation, this segregation produces balanced gametes that have either a normal chromosome complement or the two translocation chromosomes (balanced) Alternate segregation: yields unbalanced gametes in combinations of trisomy and monosomy Aneuploidy: an aneuploid cell results when nondisjunction takes place, and such cells either have an extra chromosome or are missing a chromosome. Centromere: specialized region of DNA on each eukaryotic chromosome; it acts as a site for the binding of the kinetochore proteins Chiasmata: cross-shaped structures commonly observed between nonsister chromatids in meiosis; the site of crossing over Chromatid: one of the two side-by-side replicas produced by chromosome division Crossover: the exchange of corresponding chromosome parts between homologs by breakage and reunion Diploid: two copies of the genome; A cell having two chromosome sets or an individual having two chromosome sets in each of its cells. Down syndrome: most common type of viable human aneuploid; can be caused by a Robersonian translocation or more commonly by nondisjunction of chromosome 21 in a normal parent that leads to trisomy 21 Euploid: the # of chromosomes in a basic set is called the monoploid # (x). Organisms with multiples of the monoploid # of chromosomes are called euploid Fluorescent in situ hybridization (FISH): A technique in which a fluorescently labeled DNA probe is used to hybridize with and therefore detect a particular chromosome or gene with the help of fluorescence microscopy. Fragile site: Chromosomal anomaly that appears as a region of decondensed or partially broken mitotic chromosomes under specific karyotyping conditions Gamete: specialized haploid cell that fuses with a gamete from the opposite sex or mating type to form a diploid zygote Giemsa banding (G-banding): A complex of stains specific for the phosphate groups of DNA. Used in Giemsa banding (or G-banding) to stain chromosomes and often used to create a karyotype.It is used to identify chromosomal aberrations like translocations Haploid: A cell having one chromosome set or an organism composed of such cells. Karyotype: the entire chromosome complement of an individual or cell, as seen in mitotic metaphase Klinefelter syndrome: A human clinical syndrome that results from disomy for the X chromosome in a male, which results in a 47,XXY male. Many of the affected males are mentally deficient, have underdeveloped testes, and are taller than average. Meiosis: is the process during which chromosome number is reduced to i
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