Exam #2 Flashcards

Question

signal transduction

Answer 1

The regulation of gene expression is influenced by external and internal molecular cues and/or signals. Sensing and responding to environmental fluctuations Single-celled organisms with short doubling times must respond extremely rapidly to their environment. Bacteria are exposed to changing conditions and must be able to adapt to stresses such as nutrient limitation, temperature shifts, varying osmolarity, and transition from exponential growth to stationary phase Adaptation involves changes in gene expression signal transduction transmits information from outside cell to inside allows cells to monitor and react Regulation of protein expression in bacteria mostly occurs at the level of transcription of genes, carried out by RNA polymerase (RNAP) by binding to specific regions on the chromosome (promoters).

Answer 2

microorganisms constantly face changing environment must adapt quickly to survive quorum sensing some organisms can “sense” density of their population allows cells to activate genes useful with critical mass for example, biofilm formation, pathogens′ infective process quorum sensing is a method of cell-to-cell communication affects gene expression and physiological behavior of microbial communities. Production of signal molecules continuous for each cell, BUT…responses only initiated when signal reaches threshold concentration. quorum of bacteria required to produce signal concentrations above threshold; as population increases, concentration of signal increases because more signal producers present some can detect, interfere with signaling molecules of other species can “eavesdrop” and obstruct “conversations” examples: bioluminesence biofilm formation pathogens′ infective process

Answer 3

Gram-negative capable of surviving in wide range of environments. opportunistic pathogen commonly associated with nosocomial infections burn wound infections leading cause of death in severe respiratory infections (cystic fibrosis) quorum sensing has key role in pathogenesis of P. aeruginosa regulates production of extracellular virulence factors regulates expression of antibiotic efflux pumps promotes biofilm maturation Infections with P. aeruginosa difficult to eradicate, due to antibiotic resistance and growth in biofilms.

Answer 4

switch from single-cell, planktonic lifestyle to multicellular, sessile biofilm involves changes in gene expression to produce: adhesins extracellular polysaccharide-containing matrix flagella formation

Answer 5

Quorum sensing in Gram positive cells – small peptides (autoinducing peptides) Peptide signals are not diffusible across the membrane

Answer 6

membrane-spanning sensor modifies internal region in response to specific environmental variations phosphorylates amino acid histidine kinase sensor protein response regulator phosphate group transferred from sensor regulator turns genes on or off in response and a transcriptional regulator known as response regulator

Answer 7

natural selection can play role in gene expression expression of some genes changes randomly in cells enhances survival of at least part of population

Answer 8

antigenic variation - alteration of characteristics of surface proteins - allows pathogens to stay ahead of host defenses Neisseria gonorrhoeae - many genes for pilin (protein subunit of pili) only expresses gene in expression locus randomly moves genes in and out of expression locus immune system responds to dominant pilin type bacteria that have “switched” type survive

Answer 9

phase variation involves switching genes on and off allows E. coli to attach via pili, detach by turning off

Answer 10

-except for “house-keeping” genes, most genes expressed or repressed depending on specific conditions under which cells grow. -competition for scarce resources (nutrients) makes bacterial cells efficient Regulating gene expression is one way to save energy. -gene activity controlled by promoter and regulatory elements that determine whether RNAP will transcribe gene

Answer 11

Genes can be routinely expressed or regulated a set of regulated genes transcribed as single mRNA along with its control sequences is termed operon -lac operon for lactose metabolism separate operons controlled by single regulatory mechanism constitute regulon global control is simultaneous regulation of numerous genes

Answer 12

enzymes can be grouped by type of regulation constitutive enzymes synthesized constantly typically indispensable roles in central metabolism (enzymes of glycolysis) inducible enzymes not routinely produced Synthesize only when needed (β-galactosidase turned on only when lactose present) avoid waste of resources repressible enzymes produced routinely turned off when not required (anabolic pathways such as amino acid synthesis)

Answer 13

Inducible - An inducible system is off unless there is the presence of some molecule (called an inducer) that allows for gene expression. The molecule induces gene expression. Repressible - A repressible system is on except in the presence of some molecule (called a co-repressor) that suppresses gene expression. The molecule is said to repress expression.

Answer 14

must be readily reversible, allow cells to control relative number of transcripts produced two most common are alternative sigma factors and DNA-binding proteins

Answer 15

alternative sigma factors standard sigma factor (σ70 - sigma factor with molecular weight of 70 - "housekeeping" sigma factor or primary sigma factor, transcribes most genes in growing cells. Every cell has a "housekeeping" sigma factor that keeps essential genes and pathways operating) is loose component of RNA polymerase that recognizes specific promoters for genes expressed during routine growth conditions Alternate sigma factors recognize promoters of different architectures – different regulons of different genes; with alternative sigma factors RNAP redirected to new sets of genes Sigma 70 - housekeeping sigma factor - association with RNAP favored because high intracellular level and higher affinity to core RNAP Most housekeeping genes expressed during exponential growth transcribed by holoenzyme containing σ70 and RNA polymerase Alternative σ factors provide a line of response to fluctuating changes in their environment such as heat shock, variation in pH, and osmolarity, nutrient deprivation by effectively reprograming the transcription of sets of specific genes -sporulation in Bacillus subtilis controlled by multiple different alternative sigma factors

Answer 16

DNA-binding proteins can act as repressors or activators repressor blocks transcription (negative regulation) binds to operator, stops RNA polymerase repressors are allosteric: have binding site that alters ability to bind to DNA two general mechanisms induction: repressor binds, blocks transcription inducer binds to repressor, repressor unable to bind Inducible - An inducible system is off unless there is the presence of some molecule (called an inducer) that allows for gene expression. The molecule induces gene expression. repression: repressor unable to bind to DNA corepressor attaches to repressor, complex now binds to DNA and blocks transcription Repressible - A repressible system is on except in the presence of some molecule (called a co-repressor) that suppresses gene expression. The molecule is said to repress expression.

Answer 17

activator facilitates transcription (positive regulation) ineffective promoter preceded by activator-binding site binding of activator enhances ability of RNA to initiate transcription at promoter inducer binding to activator allows binding to DNA///

Answer 18

DNA-binding proteins can act as repressors or activators repressor blocks transcription (negative regulation) binds to operator, stops RNA polymerase repressors are allosteric: have binding site that alters ability to bind to DNA two general mechanisms induction: repressor binds, blocks transcription inducer binds to repressor, repressor unable to bind repression: repressor unable to bind to DNA corepressor attaches to repressor, complex now binds to DNA and blocks transcription

Answer 19

lactose and the lac operon no lactose: repressor prevents transcription lactose present: some converted to inducer allolactose binds to repressor repressor releases operator RNA polymerase transcribes operon only occurs when glucose unavailable

Answer 20

No lactose: lac operon proteins not made, because not needed. If lactose, inducer binds to lac repressor, allows transcription of lac operon genes. When lactose depleted, lac repressor loses inducer, and blocks production of proteins, since they are no longer needed.

Answer 21

glucose and the lac operon carbon catabolite repression (CCR) prevents expression of lac operon in presence of glucose prioritize carbon/energy sources; yields diauxic growth glucose transport system senses glucose catabolite activator protein (CAP) required for transcription functional only when bound by inducer cAMP cAMP made when glucose low Inducer exclusion: lactose transporter blocked during glucose transport

Answer 22

transcription factor CAP (catabolite gene activator protein) forms complex with cAMP (inducer) and is activated to bind to DNA cAMP level varies, depending on growth medium cAMP low when glucose is carbon source: glucose causes inhibition of adenylate cyclase, the enzyme that produces cAMP

Answer 23

low glucose concentration cAMP accumulates binds allosteric site on CAP CAP assumes active shape binds upstream of lac promoter makes it easier for RNA polymerase to bind promoter and start transcription of lac operon increases rate of lac operon transcription high glucose concentration cAMP concentration decreases CAP disengages from lac operon high glucose concentration cAMP concentration decreases CAP disengaged from lac operon low glucose concentration cAMP accumulates binds allosteric site on CAP CAP assumes active shape binds site upstream of lac promoter makes it easier for RNA polymerase to bind adjacent promoter and start transcription of the lac operon increases rate of lac operon transcription.

Answer 24

No lactose, no glucose No glucose, lactose present Glucose present, no lactose Lactose present, glucose present (Know the diagram)

Answer 25

Beta galactosidase hydrolyzes lactose to produce glucose and galactose (lac Z) Lactose is both inducer for beta-galactosidase (lac Z) expression, and, when expressed, is its substrate

Answer 26

experimental inducer - not hydrolyzed by beta-galactosidase IPTG able to induce the operon, but it cannot be hydrolyzed by beta-galactosidase. (IPTG can bind repressor to change its conformation so it will not bind at operator.) induction of gene and performance of gene's function can be separated.

Answer 27

analyzing a prokaryotic DNA sequence (+) strand used to represent sequence of RNA transcript ATG in (+) DNA indicates possible start codon computers search for open reading frames (ORFs) stretches of nucleotides generally longer than 300 bp begin with start codon, end with stop codon ORF potentially encodes protein other characteristics (upstream sequence serving as ribosome-binding site) also suggest ORF encodes protein can be compared with published sequences presumed function can be assigned

Answer 28

analysis of total microbial genomes in environment can study all microorganisms and viruses in community not limited to just those that grow in culture can track changes in composition of microbiota of individual over time (healthy, diseased) compare microbiota at different body sites also between different individuals study microbial life in oceans, soils new understanding of extent of biodiversity may lead to discoveries of useful compounds (for example, antibiotics) Tremendous amount of data presents challenges

Answer 29

spontaneous mutations: base substitutions, deletion or addition of nucleotides, transposons induced mutations: chemical mutagens, transposition, radiation repair of damaged DNA proofreading (DNA polymerase), mismatch repair, modified bases, thymine dimers, SOS repair mutant selection: direct and indirect selection, mutant screening

Answer 30

transformation competence, Griffith’s experiment transduction conjugation mobile gene pool transposons, plasmids, genomic islands, phage DNA

Answer 31

by mutation or by horizontal gene transfer

Answer 32

A genetic change alters an organism’s genotype. gene mutations - spontaneous and induced This can have a profound impact on bacteria because they are haploid. Because of this, a change in genotype can easily alter the observable characteristics of an organism, its phenotype

Answer 33

deletion of gene for tryptophan biosynthesis yields mutant that only grows if tryptophan supplied growth factor required; mutant termed auxotroph geneticists compare mutants to wild type typical phenotype of strains isolated from nature wild-type E. coli strain is prototroph strains designated by three-letter abbreviations for example, Trp– cannot make tryptophan streptomycin resistance designated StrR

Answer 34

An auxotroph requires an organic growth factor in order to grow. A prototroph has no such requirement

Answer 35

randomly determined; having a random probability distribution or pattern that may be analyzed statistically but may not be predicted precisely.

Answer 36

mutations passed to progeny occasionally change back to original state: reversion large populations contain mutants (example: cells in colony) environment selects cells that grow under its conditions -antibiotics select for resistant bacteria if present -environment does not cause mutations* -single mutation rare; two even rarer -physicians may use two antibiotics to reduce resistance chance is product of mutation rate for each

Answer 37

Replica plating: reproduce colonies from an original plate to new plates by “stamping” the original plate with velvet then stamping empty plates with the same velvet. Bacteria from each colony are picked up then deposited on new plates by the velvet. Did the colonies on the new plate evolve antibiotic resistance? Result: Penicillin-resistant bacteria were in population before they encountered penicillin, they did not evolve resistance in response to exposure to the antibiotic. …the environment does not cause mutations…spontaneous mutations caused by normal processes occur randomly at infrequent characteristic rates….

Answer 38

base substitution addition or deletion transposable elements

Answer 39

base substitution most common incorrect nucleotide incorporated during DNA synthesis point mutation is change of a single base pair base substitution leads to three possible outcomes silent mutation: wild-type amino acid missense mutation: different amino acid resulting protein may only partially function termed leaky nonsense mutation: specifies stop codon yields shorter protein base substitutions may affect translation genetic code: three nucleotides = codon redundancy: code is degenerate

Answer 40

base substitutions a mutation that inactivates gene is termed a null or knockout mutation “silent mutation” sometimes used to indicate mutation that does not alter protein function base substitutions more common in aerobic environments reactive oxygen species (ROS) produced from O2 can oxidize nucleobase guanine DNA polymerase often mispairs with adenine

Answer 41

impact depends on number of nucleotides three pairs changes one codon one amino acid more or less impact depends on location in protein one or two pairs yields frameshift mutation different set of codons translated -premature stop codon shortened, nonfunctional protein knockout mutation A frameshift mutation affects all the amino acids inserted after the frameshift occurs

Answer 42

Deleting one nucleotide results in frame shift , resulting in a change of all amino acids translated beyond the deletion. Deleting three nucleotides results in only the deletion of one amino acid.

Answer 43

transposons (jumping genes) can move from one location to another process is transposition gene insertionally inactivated function destroyed most transposons have transcriptional terminators blocks expression of downstream genes in operon

Answer 44

induced mutations result from outside influence agent that induces change is mutagen geneticists may use mutagens to increase mutation rate two general types: chemical, radiation chemical mutagens may cause base substitutions or frameshift mutations some chemicals modify nucleobases change base-pairing properties increase chance of incorrect nucleotide incorporation nitrous acid (HNO2) converts cytosine to uracil base-pairs with adenine instead of guanine

Answer 45

Alkylating agents add alkyl groups onto nucleobases nitrosoguanidine adds methyl group to guanine base-pairs with thymine

Answer 46

base analogs resemble nucleobases have different hydrogen-bonding properties can be mistakenly incorporated by DNA polymerase 5-bromouracil resembles thymine, often base-pairs with guanine 2-amino purine resembles adenine, often pairs with cytosine

Answer 47

flat molecules that intercalate (insert) between adjacent base pairs in DNA strand pushes nucleotides apart, produces space causes errors during replication if in template strand, base pair added to synthesized strand if in strand being synthesized, a base pair deleted -often results in premature stop codon ethidium bromide is common intercalating agent carcinogen

Answer 48

transposons can be used to generate mutations transposon inserts into cell’s genome generally inactivates gene into which it inserts

Answer 49

1. ultraviolet irradiation forms thymine dimers covalent bonds between adjacent thymine cannot fit into double helix; distorts molecule replication and transcription stall at distortion cell will die if damage not repaired mutations result from cell’s SOS repair mechanism X rays cause single and double-strand breaks in DNA double-strand breaks often produce lethal deletions X rays can alter nucleobases

Answer 50

Enormous amount of spontaneous and mutagen-induced damage to DNA if not repaired, can lead to cell death; cancer in animals in humans, two genes associated with breast cancer code for DNA repair enzymes; mutations in either result in 80% probability of breast cancer mutations are rare; alterations in DNA generally repaired before being passed to progeny several different DNA repair mechanisms

Answer 51

proofreading mismatch repair light repair (photoreactivation) and dark repair (excision repair) glycosylases SOS repair BRCA 1 and 2 are human DNA repair factors – pathway repairs double stranded breaks in DNA

Answer 52

During replication, DNA polymerase sometimes incorporates wrong nucleotide mispairing slightly distorts DNA helix recognized by enzymes mutation prevented by repairing before DNA replication two mechanisms: proofreading, mismatch repair proofreading by DNA polymerase verifies accuracy can back up, excise nucleotide incorporate correct nucleotide very efficient but not flawless

Answer 53

The removal of an incorrect base and the incorporation of the correct base in its place. In bacteria, DNA polymerases have the ability to proofread, using 3' → 5' exonuclease activity. When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA and excises the mismatched base.

Answer 54

fixes errors missed by DNA polymerase enzyme cuts sugar-phosphate backbone another enzyme degrades short region of DNA strand methylation of DNA indicates template strand methylation takes time, so newly synthesized strand is unmethylated DNA polymerase, DNA ligase make repairs mismatch repair DNA mismatch repair (MMR) corrects mismatched base pairs mainly caused by DNA replication errors. The mechanisms and proteins involved in the reactions of MMR are highly conserved from bacteria to humans.

Answer 55

glycosylase removes oxidized nucleobase another enzyme cuts DNA at this site DNA polymerase removes short section; synthesizes replacement DNA ligase seals gap

Answer 56

Several methods to repair damage from UV light photoreactivation: light repair enzyme uses energy from light breaks covalent bonds of thymine dimer only found in bacteria excision repair: dark repair enzyme removes damage DNA polymerase, DNA ligase repair

Answer 57

SOS repair: last-ditch repair mechanism SOS response - inducible DNA repair system -allows bacteria to survive sudden increases in DNA damage induced following extensive DNA damage photo-reactivation, excision repair unable to correct DNA and RNA polymerases stall at unrepaired sites several dozen genes in SOS system activated includes a DNA polymerase that synthesizes even in extensively damaged regions has no proofreading ability, so errors made result is SOS mutagenesis LexA repressor regulates transcription of all SOS genes.

Answer 58

Without SOS repair, the cell would not be able to multiply.

Answer 59

Mutants rare, difficult to isolate two main approaches direct selection: cells inoculated onto medium that supports growth of mutant but not parent -antibiotic-resistant mutants exposed to antibiotic Mutant selection – antibiotic resistance – direct selection

Answer 60

Mutants rare, difficult to isolate two main approaches direct selection: cells inoculated onto medium that supports growth of mutant but not parent -antibiotic-resistant mutants exposed to antibiotic indirect selection: isolates auxotroph from prototrophic parent strain more difficult since parents will grow on any media on which auxotroph can grow replica plating

Answer 61

take 1 x 109 cells/ml E. coli treat with chemical mutagen (such as EMS – ethylmethane sulfonate - alkylating agent) plate on glucose plate replica plate onto medium with lactose - only carbohydrate source mutant unable to grow (would be lac- mutant)

Answer 62

penicillin enrichment of mutants sometimes used increases proportion of auxotrophs in broth culture penicillin kills growing cells prototrophs auxotrophs survive penicillinase then added cells plated on rich medium

Answer 63

screening for possible carcinogens carcinogens cause many cancers; most are mutagens animal tests expensive, time-consuming mutagens increase low frequency of spontaneous reversions Ames test measures effect of chemical on reversion rate of histidine-requiring Salmonella auxotroph uses direct selection if mutagenic, reversion rate increases relative to control rat liver extract added since non-carcinogenic chemicals often converted to carcinogens by animal enzymes additional tests on mutagenic chemicals to determine if carcinogenic

Answer 64

can demonstrate recombinants with auxotrophs combine two strains for example, His–, Trp– with Leu–, Thr– spontaneous mutants unlikely colonies that can grow on glucose-salts medium most likely acquired genes from other strain

Answer 65

transformation: naked DNA uptake by bacteria transduction: bacterial DNA transfer by viruses conjugation: DNA transfer during cell-to-cell contact

Answer 66

DNA replicated only if replicon has origin of replication plasmids, chromosomes DNA fragments added to chromosome via homologous recombination only if sequence similar to region of recipient’s genome

Answer 67

naked DNA not within cell or virus cells release DNA when lysed ->addition of DNase prevents transformation demonstration of transformation in S. pneumoniae only encapsulated cells pathogenic CAPSULES: Rough strain doesn’t form capsule = rough colonies on plate Smooth strain forms a capsule shiny round colonies on plate demonstration of transformation in Streptococcus pneumoniae only encapsulated cells pathogenic!

Answer 68

transformation recipient cell must be competent most take up regardless of origin some accept only from closely related bacteria (DNA sequence) process tightly regulated Streptococcus pneumoniae has two-component regulatory system high concentration bacteria (quorum sensing) only fraction of population becomes competent

Answer 69

1. Double stranded DNA binds to plasma membrane 2. Enzymes cut the DNA 3. Broken down to one strand 4. Protein binds the DNA and replaces part of the bacterium DNA dsDNA bound to the cell surface fragmentation of dsDNA occurs upon binding ssDNA fragments transported across the membrane via transformation pseudopilus, evolutionarily related to type IV pili transport possibly driven by proton motive force. retraction (disassembly) of pseudopilus allows exogenous DNA to cross peptidoglycan

Answer 70

RecA gene family plays central role in homologous recombination during bacterial transformation The RecA protein is essential for transformation in Bacillus subtilis and Streptococcus pneumoniae . Expression of the RecA gene is induced during development of competence for transformation in these organisms. RecA protein interacts with entering single-stranded DNA (ssDNA) to form RecA/ssDNA nucleofilaments that scan the chromosome for regions of homology and bring the entering ssDNA to the corresponding region, where strand exchange and homologous recombination occur.

Answer 71

bacterial DNA transfer by viruses (bacteriophages) transfer of bacterial DNA enclosed in a phage head from one bacterium to another generalized transduction: any genes of donor cell specialized transduction: specific genes transfer of old bacterial host DNA to new bacterial host via bacteriophage

Answer 72

conjugation: DNA transfer between bacterial cells via specialized conjugal pilus requires contact between donor, recipient cells pilus - structure used by bacteria during conjugation transfer of genetic material between a donor and a recipient cell conjugative plasmids direct their own transfer replicons F plasmid (fertility) of E. coli most studied other plasmids encode resistance to some antibiotics spread resistance easily via conjugation

Answer 73

F plasmid of E. coli F+ cells have, F– do not encodes proteins, including F pilus sex pilus brings cells into contact enzyme cuts plasmid single strand transferred complementary strands synthesized both cells are now F+ F plasmid also contains an origin of transfer.

Answer 74

involves Hfr cells (high frequency of recombination) F plasmid is integrated into chromosome via homologous recombination process is reversible F′ plasmid results when small piece of chromosome is removed with F plasmid DNA F′ is replicon Hfr cell produces F pilus transfer begins with genes on one side of origin of transfer of plasmid (in chromosome) part of chromosome transferred to recipient cell chromosome usually breaks before complete transfer (full transfer would take ~100 minutes) recipient cell remains F– since incomplete F plasmid transferred

Answer 75

Quorum sensing mediates coordinated shifts in group behavior – conjugation and competence

Answer 76

Genomics reveals surprising variation in gene pool of even a single species perhaps 75% of E. coli genes found in all strains termed core genome of species remaining make up mobile gene pool plasmids, transposons, genomic islands, phage DNA plasmids found in many bacteria and archaea some eukarya usually dsDNA with origin of replication - generally nonessential; cells survive loss

Answer 77

resistance to antimicrobial medications, heavy metals (mercury, arsenic) often two parts R genes RTF (resistance transfer factor) codes for conjugation often broad host range normal microbiota can transfer to pathogens

Answer 78

transposons provide mechanism for moving DNA can move into other replicons in same cell simplest is insertion sequence (IS) encodes only transposase enzyme, inverted repeats composite transposons include one or more genes integrate via non-homologous recombination Transposons yielded vancomycin-resistant Staphylococcus aureus strain patient infected with S. aureus susceptible to vancomycin also had vancomycin-resistant strain of Enterococcus faecalis -transferred transposon-containing plasmid to S. aureus

Answer 79

natural genetic engineering Agrobacterium tumefaciens causes crown gall different properties, produces opine, plant hormones piece of tumor-inducing (Ti) plasmid called T-DNA (transferred DNA) is transferred to plant, incorporated into plant chromosome

Answer 80

genomic islands: large DNA segments in genome originated in other species nucleobase composition very different from genome G-C base pair ratio characteristic for each species may provide different characteristics: utilization of energy sources acid tolerance development of symbiosis ability to cause disease pathogenicity islands

Answer 81

resisting phage infection by degrading foreign DNA restriction enzymes and a modification enzyme restriction enzyme: recognizes short nucleotide sequence then cuts at that sequence modification enzyme protects cells own DNA by adding methyl groups to self DNA restriction enzymes can’t degrade methylated (self) DNA

Answer 82

Some bacterial genomes include very small pieces of phage DNA. The bacteria have survived phage infections and retained small segments of that invader’s DNA, incorporated into the bacterial genome. Segments are used to recognize and destroy that specific invading DNA in the future, providing the cell with a form of adaptive immunity.

Answer 83

three mechanisms confer bacterial resistance to phage infection: 1. preventing phage attachment 2. restriction/modifiction systems 3. CRISPR Bacteria and their viral predators (bacteriophages) locked in constant battle. To proliferate in phage-rich environments, bacteria have impressive arsenal of defense mechanisms. In response, phages have evolved counter-strategies to evade these antiviral systems.

Answer 84

Recognition of host receptor molecules A limiting factor for phage invasion of a bacterial cell is the ability of the phage to bind to a receptor on the host bacterial cell surface. If a bacterium alters or covers a receptor, that cell becomes resistant to any phage that requires the receptor for attachment. Bacteria can lose or alter the target receptor of phages Can produce an extracellular matrix of polysaccharides that blocks phage attachment Can produce competitive inhibitors that bind to the phage attachment site.

Answer 85

1. Epithelial cells secrete mucus. 2. Phage adhere to mucus through Iq-like domains. 3. Adherent phage form anti-microbial layer. 4. Mucus-adherent phage have increased chance of replicative success 5. Phage and bacteria are shed with mucus.

Answer 86

resisting phage infection by degrading foreign DNA restriction enzymes and a modification enzyme restriction enzyme: recognizes short nucleotide sequence then cuts at that sequence modification enzyme protects cells own DNA by adding methyl groups to self DNA restriction enzymes can’t degrade methylated (self) DNA

Answer 87

Some bacterial genomes include very small pieces of phage DNA. The bacteria have survived phage infections and retained small segments of that invader’s DNA, incorporated into the bacterial genome. Segments are used to recognize and destroy that specific invading DNA in the future, providing the cell with a form of adaptive immunity. CRISPR - 3 steps: invader DNA incorporation CRISPR array transcribed and processed by Cas proteins CRISPR interference: the invader’s DNA is recognized by complementarity to the crRNA and is neutralized

Answer 88

Q: Describe three mechanisms that confer bacterial resistance to phage infection. A: 1. Preventing phage attachment 2. Restriction/Modification systems 3. CRISPR Q: How do modification enzymes protect host cell DNA from restriction enzymes? A: By methylating certain nucleobases which then are not recognized by the restriction enzyme. Q: How does a bacterial cell acquire a historical record of phage infections? A: By incorporating a piece of the entering phage genome into its own genome.

Answer 89

Many uses for genetically engineered bacteria: protein production DNA production research tools relies on DNA cloning

Answer 90

…a set of techniques used to insert recombinant DNA from a prokaryotic (or eukaryotic) source into a replicating vehicle such as a plasmid or a viral vector. Cloning refers to making numerous copies of a DNA fragment of interest, such as a gene.

Answer 91

Use of microbiological and biochemical techniques to solve practical problems recombinant DNA techniques possible to genetically alter organisms can isolate genes from one, transfer to another genetic engineering numerous uses: agriculture, medicine, law enforcement

Answer 92

-recognize 4 to 6 base-pair nucleotide sequence and cut each strand of DNA -typically palindrome -generates restriction fragments -cohesive ends (sticky ends) are complementary, can anneal -allows creation of recombinant DNA molecules Mix the two ends in the presence of ATP and an enzyme, T4 DNA ligase. *T4 bacteriophage During ligation reaction, hydrogen bonds form between overhangs on the fragments, then ligase repairs phosphate backbone (phosphodiester bond)

Answer 93

plasmids - circular pieces of DNA found naturally in bacteria plasmids can carry antibiotic resistance genes, genes for receptors, toxins or other proteins plasmids can replicate separately from the genome of the organism -must have origin of replication plasmids can be engineered to be useful cloning vectors vector is usually modified plasmid or bacteriophage -has origin of replication -carries cloned DNA -must have restriction site(s) multiple-cloning site useful -selectable marker ampicillin common second marker helpful distinguish recombinant plasmids from intact vector

Answer 94

isolate DNA cut with restriction enzymes join insert (DNA fragment) with vector (plasmid) to generate independently replicating recombinant molecule introduce into host Can place gene (insert) into high-copy-number vector host bacteria will make large amounts of the protein each gene copy can be transcribed and translated

Answer 95

Second marker: lac Z gene will be interrupted if plasmid takes up insert. if insert, there will be no beta-galactosidase expression so beta-galactosidase (lac Z) will not cleave X-gal. when X-gal is cleaved, colonies are blue multiple-cloning site in gene insert interrupts lacZ′ intact vector —> blue colonies recombinant molecule —> white colonies

Answer 96

Engineered plants and animals: transgenic Protein production safer, more economical -gene for human insulin cloned into bacteria previously, insulin extracted from animals sometimes caused allergic reactions -vaccine production: clone gene for specific proteins: -vaccines for hepatitis B and cervical cancer -foot-and-mouth disease of domestic animals genetically engineered eukaryotes yeasts can be engineered like bacteria; provide model Ti plasmid from Agrobacterium tumefaciens used to generate corn, cotton, potatoes that produce Bt toxin from Bacillus thuringiensis toxic only to insects and their larvae soybean, cotton, corn engineered to resist biodegradable plants with improved nutritional value rice containing β-carotene; iron

Answer 97

example: insect resistant crops Bacillus thuringeiensis is a bacterium that naturally produces a protein (Bt toxin) with insecticidal properties. used as an insect-control strategy for many years in agriculture and gardening. recently, plants developed (using Ti plasmid from Agrobacterium sp.) that express a recombinant form of bacterial protein, which may effectively control some insect predators. Environmental issues associated with the use of transgenic crops have not been resolved.

Answer 98

lytic phage infections lytic or virulent phages exit host cell is lysed productive infection: new particles formed T4 phage (dsDNA) as model entire process takes ~30 minutes attachment phage exploits bacterial receptors genome entry T4 lysozyme degrades cell wall tail contracts, injects genome through cell wall and membrane synthesis of proteins and genome early proteins translated within minutes nuclease degrades host DNA protein modifies host’s RNA polymerase to not recognize its own promoters late proteins are structural proteins (capsid, tail) produced toward end of cycle assembly (maturation) some components spontaneously assemble, others require protein scaffolds release lysozyme produced late in infection; digests cell wall cell lyses, releases phage burst size of T4 is ~200

Answer 99

option of lytic infection or incorporation of DNA into host cell genome lysogenic infection infected cell is lysogen lambda (λ) phage as model Temperate phages generally do not kill the bacterial cells they infect. PROPHAGE: latent form of phage DNA that is present in lysogenic bacteria

Answer 100

lambda (λ) phage: linear chromosome complementary single-stranded overhangs at ends join inside host resulting circular molecule either directs lytic infection or integrates into E. coli chromosome phage enzyme integrase inserts DNA at specific site site specific recombination integrated phage DNA termed prophage replicates with host chromosome -can be excised by phage-encoded enzyme -results in lytic infection repressor prevents excision, maintains lysogenic state

Answer 101

-if DNA damaged (UV light exposure), SOS repair system turns on, activates a protease -protease destroys repressor, allows prophage to be excised, enter lytic cycle -called phage induction; allows phage to escape damaged host SOS repair system turns on, activates a protease: RecA !!!!!!

Answer 102

During normal growth, the LexA repressor binds promoter region of SOS genes and prevents their expression. When the cell senses DNA damage, the LexA repressor undergoes self-cleavage and the SOS genes are de-repressed. RecA specifically binds single-stranded DNA (ssDNA), promotes LexA cleavage (inducing the SOS response). cI has structural similarity to lexA and in lysogens it is cleaved -cleavage allows expression of lytic genes In the SOS response, RecA cleaves LexA, removing SOS response repression. cI also forms a repressor. cI is the only factor needed to maintain the lysogenic state once it is established cI forms a repressor. cI is the only factor needed to maintain the lysogenic state once it is established. RecA destroys cI. PR and PL promoters are no longer repressed and switch on, and the cell returns to the lytic sequence of expression events.

Answer 103

lysogen immune to superinfection (infection by same phage) Repressor maintaining integrated prophage also binds to operator on incoming phage DNA, prevents gene expression: immunity to superinfection

Answer 104

generalized transduction -results from packaging error during phage assembly -some phages degrade host chromosome; fragments can be mistakenly packaged into phage head -these phages cannot direct phage replication cycle termed generalized transducing particles following release, can bind to new host, inject DNA DNA may integrate via homologous recombination, replacing host DNA any gene from donor cell can be transferred

Answer 105

-excision mistake during transition from lysogenic to lytic cycle of temperate phage -short piece of flanking bacterial DNA removed; piece of phage DNA remains -excised DNA incorporated into phage heads; defective particles released -can bind to new host, inject DNA -bacterial genes may integrate via homologous recombination -only bacterial genes adjacent to integrated phage DNA transferred

Answer 106

…either exclusively episomally replicating phage or temperate phage that are chromosomally integrated, but can be induced to start episomal replication episome: a genetic element inside some bacterial cells, especially the DNA of some bacteriophages, that can replicate independently of the host and also in association with a chromosome with which it becomes integrated. single-stranded DNA phages used to produce only single- stranded recombinant DNA -look like long fibers cause productive infections -host cells not killed, but grow more slowly -M13 phage as model attaches to protein on F pilus of E. coli single-stranded DNA genome enters cytoplasm

Exam #2 Flashcards

(130 cards)