Study Guide FOR Mutation Through DNA Technology Exam PDF

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Study Guide for the Exam covering the Genetics chapters of Mutation all the way through DNA technology....

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STUD STUDY Y GUIDE FOR MUT MUTA ATION THROUGH DN DNA A TECHNOL TECHNOLOGY OGY This is a “general guide” for the e exam, xam, howe howev ver anything co covered vered in lecture or assigned in homework is fair game for the e exam. xam.

MUT MUTA ATION AND REP REPAIR AIR Vocabulary:  

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Mutation – the process that produces an alteration in DNA or chromosome structure; in genes, the source of new alleles missense mutation – a mutation that changes a codon to that of another amino acid and thus results in an amino acid substitution in the translated protein. Such changes can make the protein nonfunctional. nonsense mutation – a mutation that changes a codon specifying an amino acid into a termination codon, leading to premature termination during translation of mRNA. neutral missense mutation – a mutation with no perceived immediate adaptive significance or phenotypic effect. silent mutation (sense mutation) – a mutation that alters the sequence of a codon but does not result in a change in the amino acid at that position in the protein. Degenerate – more than one codon can specify the same amino acid or a stop frameshift mutation – an insertion or deletion in the coding region amorph (null/loss of funtion allele) – mutant phenotype results from complete loss of gene function o loss of function hypermorph (gain of function allele) – mutation confers overactive function of the gene o -increase in gene expression, protein works more efficiently, etc… o -gain of function Hypomorph – reduces the function of the gene (level of expression, protein activity, etc..) antimorph (dominant negative) – mutation causes the gene to function in an antagonistic manner to the WT allele (dominant negative: competes with WT allele) conditional mutant (temperature sensitive mutant) – mutations that affect genes that are indispensable for cellular function often result in lethality o conditional mutation  A mutation expressed only under a certain condition; that is, a wild-type phenotype is expressed under certain (permissive) conditions and a mutant phenotype under other (restrictive) conditions. spontaneous vs induced mutations o spontaneous mutations (cellular errors, biochemical variation) - changes in the nucleotide sequence of genes that appear to occur naturally  replication slippage  tautomeric shift  depurination/deamination  oxidative damage  transposons

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induced mutations (extracellular influences, toxins, chemicals, radiation) mutations that result from the influence of exogenous factors  base analogs  alkylating agents and intercalating  UV  Ionizing radiation transition vs transversion mutation o transition – a change from one purine/pyrimidine pair to the other purine/pyrimidine pair  A/T ↔ G/C  T/A ↔ C/G o Transversion – a change between a purine/pyrimidine pair and a pyrimidine/purine pair  A/T ↔ T/A  G/C ↔ C/G  A/T ↔ C/G  G/C ↔ T/A Photolyase – an enzyme that cleaves the cross-linking bonds in thymine dimers photoreactivation repair – a DNA repair system where UV light activates an enzyme photolyase or photoreactivation enzyme (PRE) which splits the dimers. Post-replication repair – a DNA repair system that responds after damaged DNA has escaped repair and has failed to be completely replicated Glycosylase – an enzyme in the BER pathway that recognizes inappropriately paired bases DNA adducts – bulky chemical modifications to bases that cause the double helix to bulge which causes polymerase stalling during transcription. transcription coupled repair – DNA adducts are often targeted for repair during transcription when RNA polymerase II stalls. o

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For the following, know a few different ways each can arise: spontaneous vs induced mutations o spontaneous = cellular errors and biochemical variation o induced = extracellular influences, toxins, chemicals, and radiation Why do repetitive sequences make it more likely for slippage during DNA replication? o Repetitive sequences can confuse DNA polymerase III and it can “lose its place.” Thus spontaneous deletions/insertions may arise in repetitive DNA during replication. How do the following cause mutations? o tautomeric shifts – tautomeric shift of a base can lead to incorporation of the wrong complementary base during DNA replication o base analogs – the Br increases the rate of tautomeric shift and thus adding BrdU to cells increases the chance of mismatch mutations during replication o depurination – the entire base (usually a purine) is lost by breaking the bond to the 1’ C on the deoxyribose sugar o deamination – deamination of C to U and deamination of A to H  Deamination of C to U  Uracil pairs with adenine  One daughter chromosome has a A/T pair  Deamination of A to H  Hypox. Pairs with Cytosine  One daughter chromosome has a G/C pair o chemical mutagens such as EMS and ENU  Ethyl Methanesulfonate (EMS) alkylates guanines GC  AT

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 N-ethyl-N-nitrosourea (ENU) TA  CG intercalating agents – ethidium bromide (agarose gels), Acridine, and DAPI  Intercalating agents are chemicals that have dimensions and shapes that allow them to wedge between the base pairs of DNA. Wedged intercalating agents cause base pairs to distort and DNA strands to unwind. These changes in DNA structure affect many functions including transcription, replication, and repair. Deletions and insertions occur during DNA replication and repair, leading to frame-shift mutations. o double strand break repair vs nonhomologous end joining  Double strand break repair (homologous recombination repair)  The sister chromatid is used as a template for extending the broken chromosome beyond the break point. Then the strands can separate, and DNA is filled-in and ligated by DNA polymerase I and ligase.  Non-homologous End Joining – a genomic DNA repair mechanism that is induced by double-strand DNA breaks. This type of repair is error prone because broken ends of DNA molecules are randomly ligated together, which may lead to insertions, deletions, translocations, or inversions. o transposable elements – a DNA sequence that can change its position within the genome  ITR – inverted terminal repeats  DR – direct repeats flanking transposon in chromosomal DNA  Transposase – an enzyme that cleaves ITR’s in transposon and in similar sequences within the genome (become DRs), and facilitates transposon integration into genome What are some different ways that mutations affect genes? How do radiation induced mutations differ from the mutations described above? o UV irradiation causes Thymine-Thymine dimers (T^T) o T^T causes a “bulge” in the DNA helix o Causes a deletion in the opposite strand during DNA replication o May stall DNA replication and cause cell death How are thymine-thymine dimers repaired in eukaryotes vs prokaryotes? What are the different types of DNA repair? o Proof-reading and mismatch repair o Post-replication repair o Photo-reactivation repair o Base and nucleotide excision repair o Double-stranded break repair What repair pathway is faulty in xeroderma pigmentosum? What are symptoms of the disorder? o The NER repair pathway is faulty in XP patients. Symptoms of XP include extreme sensitivity to sunlight, high incidence of skin cancer, severe sunburn when exposed to tiny amounts of UV light, freckles, crusting/scaling of skin, blisters, patches of discolored skin, and spider-like blood vessels underneath the skin. What roles do exonucleases/endonucleases play in Base excision repair (BER) and Nucleotide excision repair (NER)? o An exonuclease removes the mismatched base and DNA polymerase replaces it with the correct base. o An endonuclease enzyme creates a nick in the backbone of the unmethylated DNA strand, either 5' or 3' to the mismatch. An exonuclease unwinds and degrades the nicked DNA strand, until the region of the mismatch is reached. Finally, DNA polymerase o

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fills in the gap created by the exonuclease, using the correct DNA strand as a template. DNA ligase then seals the gap. What are some differences between BER and NER? o Base Excision Repair (BER) – the enzyme DNA glycosylase recognizes chemically altered bases and cleaves the base-sugar bond. The enzyme AP endonuclease recognizes damaged bases and removes them. The gap is filled and sealed by DNA polymerase I and ligase. o Nucleotide Excision Repair (NER) – a group of enzymes detects damaged DNA (“bulky”) (T^T and DNA adducts). Damaged nucleotides and adjacent nucleotides (~13) are removed and DNA polymerase and ligase fills and seals. Why is postreplication repair more like a “bandaid” than repair? o Recombination allows for 3 of 4 strands to maintain WT sequence What is the purpose of the Ames test? o The Ames test screens compounds for potential mutagenicity. o The Ames test uses a number of different strains of the bacterium Salmonella typhimurium that have been selected for their ability to reveal the presence of specific types of mutations. o For example, some strains are used to detect base-pair substitutions, and other strains detect various frameshift mutations. Each strain contains a mutation in one of the genes of the histidine operon. The mutant strains are unable to synthesize histidine (his− strains) and therefore require histidine for growth. The assay measures the frequency of reverse mutations that occur within the mutant gene, yielding wild-type bacteria (his+ revertants) (Figure 15.17). The his− strains also have an increased sensitivity to mutagens due to the presence of mutations in genes involved in both DNA damage repair and the synthesis of the lipopolysaccharide barrier that coats these bacteria and protects them from external substances. How is the Ames test carried out? What is the purpose of generating mutants in genetics research? What are some ways mutants can be generated?

Regulation of gene expression in prokaryotes Vocabulary:   

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selective pressure auxotrophs - organisms that have lost the ability to synthesize a required molecule through mutation prototrophs – organisms that can synthesize all of their needed organic molecules (proteins, nucleic acids, sugars, fats, vitamins) from a simple mixture of sugar, and inorganic ions, or minimal media. Conjugation – the unidirectional transfer of genetic material through direct cell contact between donor and recipient bacterial cells. F factor – is a plasmid that contains genes that specify formation of F-pili or sex-pili Plasmid – a type of double-stranded circular DNA molecule Transduction - gene exchange between bacteria cells through a bacteriophage virus vector Transformation - the process of extracellular DNA entering a bacterial cell and becoming expressed and replicated by the host cell, the transformant. competent cells – cells that are chemically or electrically treated to increase membrane permeability to DNA housekeeping genes/constitutive

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Constitutive Genes – genes that are always active because their function is always needed (“housekeeping genes”) o Constitutive = always “on” repression vs induction o repressor – are transcribed and translated continuously. The repressor binds to the operator sequence of the lac operon and blocks transcription of lac operon genes. o inducer operator – a site where a repressor binds to inhibit gene expression operon – clustered genes that are coordinately regulated; transcription produces polycistronic mRNA; a common promoter is used for all genes in the operon o Operon Textbook Definition: a genetic unit consisting of one or more structural genes encoding polypeptides, and an adjacent operator gene that regulates the transcriptional activity of the structural gene or genes. polycistronic mRNA – a single transcript is translated into more than one polypeptide allosteric inhibition – the repressor undergoes a conformational change and can no longer bind to the operator ???? catabolite activating protein (CAP) – a catabolite-activating protein; a protein that binds cAMP and regulates the activation of inducible operons allolactose – lactose and it is referred to as the inducer cAMP – cyclic adenosine monophosphate (cAMP) – an important regulatory molecule in both bacteria and eukaryotes adenylyl cyclase – an enzyme that catalyzes the conversion of ATP to cyclic AMP (cAMP) and its activity is inhibited by high concentrations of glucose attenuation – a regulatory process in some bacterial operons that terminates transcription prematurely, thus reducing the production of the mRNA encoding the structural genes in the operon. leader sequences – that portion of an mRNA molecule from the 5’ end to the initiating codon, often containing regulatory or ribosome-binding sites. Riboswitches – secondary structure in RNA binds to a ligand and causes conformational change in another part of RNA o Often result in termination o Ligands can be metal ions or amino acids (and others). sRNAs – small noncoding RNAs – are transcribed from opposite strands of locus, are complementary to coding RNA, and can block or unmask Ribosome Binding Sites (RBS). o

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How did Tatum use auxotrophs to understand conjugation? o Used E. Coli cells. Evidence that genetic recombination (conjugation) occurs between cells of E. Coli. How does recombination in prokaryotes differ from recombination in eukaryotes? How does the F factor/plasmid facilitate conjugation? o Conjugation only happens between opposite F types (F- recipient/F+ donor) How does transformation differ from conjugation? o Conjugation – the unidirectional transfer of genetic material through direct cell contact between donor and recipient bacterial cells. o Transformation – the process of extracellular DNA entering a bacterial cell and becoming expressed and replicated by the host cell, the transformant. How is transformation used for research and commercial purposes?

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Transformation is used for mapping traits, studying molecular mechanisms, and making transgenic bacteria. What are some different strategies employed to prepare cells to take up DNA? What is the difference between constitutive genes and regulated genes? o Constitutive Genes – genes that are always active because their function is always needed (“housekeeping genes”)  Example: genes that encode for glucose metabolism o Regulated Genes – gene activity is induced or repressed in response to the needs of the cell or organism.  Example: genes that encode for lactose metabolism  Lactose induces expression of lactose metabolism genes so that it may be used as a source of Carbon. How do regulated genes help give rise to different cell types and functions? What are some genes that would be constitutive in human cells? Why might genes be repressed or activated? o Gene regulation is all about the process of genes being repressed or activated. The process of turning genes on and off (repressing or activating) is known as gene regulation. Gene regulation is an important part of normal development. Genes are turned on and off in different patterns during development to make a brain cell look and act different from a liver cell or a muscle cell, for example. Gene regulation also allows cells to react quickly to changes in their environments. What are the 3 genes found in the lac operon? o The lac operon consists of three structural genes, and a promoter, a terminator, regulator, and an operator. The three structural genes are: lacZ, lacY, and lacA. (Wikipedia) What are their functions? o IacZ = β-Galactosidase Gene – encodes for β-Galactosidase, an enzyme whose primary role is to convert the disaccharide lactose to the monosaccharides glucose and galactose. This conversion is essential if lactose is to serve as the primary energy source in glycolysis. o IacY = Permease Gene – specifies the primary structure of permease, an enzyme that facilitates the entry of lactose into the bacterial cell. o IacA = Transacetylase Gene - codes for the enzyme transacetylase. While its physiological role is still not completely clear, it may be involved in the removal of toxic by-products of lactose digestion from the cell. How is the lac operon regulated? o The lac operon uses a two-part control mechanism to ensure that the cell expends energy producing the enzymes encoded by the lac operon only when necessary. (Wikipedia) How does lactose result in induction of the lac operon? o In the absence of lactose, the lac repressor, lacI, halts production of the enzymes encoded by the lac operon.[3] The lac repressor is always expressed unless a coinducer binds to it. In other words, it is transcribed only in the presence of small molecule co-inducer. (Wikipedia) How does the cell regulate the lac operon if glucose is present? o In the presence of glucose, the catabolite activator protein (CAP), required for production of the enzymes, remains inactive, and EIIAGlc shuts down lactose permease to prevent transport of lactose into the cell. (Wikipedia) What would be the effect on the regulation of the lac operon in both the presence of lactose and in the absence of lactose given the following mutations: o

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a mutated operator sequence  the nucleotide sequence of the operator DNA is altered and will not bind with a normal repressor molecule. The result is the same: The structural genes are always transcribed. a loss of function mutation in the repressor  Expression of lac operon genes is constitutive (always on)  the repressor protein is altered or absent and cannot bind to the operator region, so the structural genes are always turned on

 a loss of function mutation in permease  unable to use lactose as an energy source a loss of function mutation in β-galactosidase  unable to use lactose as an energy source

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In the absence of lactose, the enzymes encoded by the genes are not needed and the expression of genes encoding these enzymes is repressed. When lactose is present, it indirectly induces the activation of the genes by binding with the

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repressor.* If all lactose is metabolized, none is available to bind to the repressor, which is again free to bind to operator DNA and to repress transcription. (textbook) What is the difference between alternative splicing and polycistronic mRNA? o Multiple genes vs. single gene o Multiple translation start sites vs. single translation start sites Why does cellular compartmentalization in eukaryotes and lack of organelles in prokaryotes affect how gene expression is regulated in both systems? How does regulation of the trp operon differ from regulation of the lac operon? Why is the lac operon considered inducible while the trp operon is considered repressible? o Trp is a corepressor that binds to a repressor and activates it (allosteric activation) How does attenuation help regulate expression of the trp operon? 16.6 Would trp operon attenuation be possible if prokaryotes had organelles and a nucleus? 16.6 Why are leader sequences found in many operons encoding enzymes of amino acid biosynthesis pathways? 16.6

Regulation of gene expression in eukaryotes Vocabulary:      

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reverse genetics - a tool for molecular genetic investigation RNA interference - RNAi – a cellular response to the presence of double-stranded RNA that results in the sequence-specific degradation of mRNA dsRNA siRNA - small interfering RNAs – lab synthesized small dsRNA molecules Dicer - an enzyme that cuts dsRNA into fragments 20-25 bp long with 2-3 bp overhangs RISC - RNA-induced silencing complex (RISC) – a multi-protein mach...