Exam 2 (Ch. 7, 8, & 9) Flashcards
What are some of the MAJOR DIFFERENCES between Prokaryotic and Eukaryotic Transcription and Translation?
EUKARYOTES:
1. A cap is added to the 5’ tail end of mRNA, and a poly A tail is added to the 3’ end
2. mRNA contains introns, which are removed by splicing
3. The mRNA transcript is transported out of the nucleus so that it can be translated in the cytoplasm
4. mRNA is monocistronic; translation begins at the first AUG
PROKARYOTES:
1. mRNA is NOT processed
2. mRNA DOES NOT contain introns
3. Translation of mRNA begins as it is being transcribed
4. mRNA is often polycistronic; translation usually begins at the first AUG that FOLLOWS a ribosome binding site
Are bacteria haploid or diploid?
Haploid
T/F: Bacterial toxins can target eukaroytic enzymes
True
does glucose or lactose gets exhausted first in the Lac Operon graph?
glucose
What is an operon?
a group of structural genes plus sequences that control transcription
includes structural genes, a promoter, and an operator
There are two mechanisms of regulation by repressors.
What occurs in Repressible Control? (e.g. trp)
TRANSCRIPTION ON:
-The repressor alone CANNOT bind to the operator, so transcription continues
TRANSCRIPTION OFF (BLOCKED):
–Corepressor binds to the repressor and ALTERS its shape, enabling it to bind to the operator and blocking transcription
There are two mechanisms of regulation by repressors.
What occurs in Indicible Control?
TRANSCRIPTION OFF (BLOCKED):
-Repressor binds to the operator and blocks transcription
TRANSCRIPTION ON:
-Inducer binds to the repressor and ALTERS its shape, preventing it from binding to the operator
During Lac operon, what happens in transcription when glucose is present, but not lactose?
Transcription not activated AND blocked. Low cAMP-, CAP cannot bind. repressor is bound to operator, blocking polymerase.
What is Positive Inducible Control when it comes to activating transcription?
TRANSCRIPTION OFF (NOT ACTIVATED:
-Activator CANNOT bind to the activator-binding site, thus RNA polymerase cannot bind to the promoter and initiate transcription
TRANSCRIPTION ON:
-Inducer binds to the activator and CHANGES its shape, enabling it to bind to the site.
-RNA polymerase can then BIND to the promoter and initiate transcription
During Lac operon, what happens in transcription when glucose is present and lactose is present?
Transcription not activated. Low cAMP-, CAP cannot bind. Inducer(allolactose) prevents repressor from binding to the operator.
During Lac Operon, what happens in transcription when there is no glucose and no lactose?
Transcription activated, but blocked. High cAMP-, CAP/cAMP complex binds to activator-binding site. Repressor is bound to operator, however, blocking polymerase
What does the Lac Operon control?
controls the expression of genes involved in lactose catabolism
e.g:
-lac Z gene: B-galactosidase
-lac Y gene: Permease
-lac A gene: Thiogalactoside Transacetylase
During Lac Operon, what happens when there is no glucose, but lactose is present
Transciption activated. High cAMP-; CAP/cAMP complex binds to activator-binding site. In addition, inducer (allolactose) prevents repressor from binding to the operator
What is the action of Permease? B-galactosidase?
Permease: actively transports lactose into the cell
B:galactosidase: breaks down the lactose into galactose and glucose
During Lac Operon, what does glucose do?
When glucose is present, glycolysis is not operating, so there isn’t cAMP. cAMP needs to be present to bind to CAP to start transcription. cAMP and CAP need to be binded together to attach RNA polymerase to the promoter.
_________ growth curve occurs in the presence of glucose and lactose.
*BE ABLE TO DRAW THIS AND EXPLAIN IT!
Diauxic
-two growth periods, one on glucose and one on lactose.
-LAG, LOG GROWTH (on glucose), STATIONARY, LOG GROWTH (on lactose), STATIONARY
During Lac Operon, what does lactose do?
Without lactose, the repressor is binded to the operator site, blocking transcription. Lactose needs to be present to have the inducer release the repressor.
Lac Operon is also controlled by Catabolite Repression.
What is Catabolite Repression?
When glucose is LOW, cAMP is HIGH, binds to Catabolite Activator Protein (CAP) to induce transcription
In presence of lactose, allolactose is produced, BINDS to repressor, FALLS OFF operator so that transcription can proceed
ESSAY QUESTION
What happens in the following situation of a Lac Operon:
- No glucose, no lactose
Transcription ACTIVATED, but BLOCKED
-High cAMP
-CAP/cAMP complex binds to the activator-binding site, but a repressor is bound to the operator, blocking RNA polymerase
ESSAY QUESTION
What happens in the following situation of a Lac Operon:
- Glucose present, no lactose
Transcription NOT ACTIVATED and BLOCKED
-Low cAMP
-CAP CANNOT bind, and the repressor is bound to the operator, blocking RNA polymerase
ESSAY QUESTION
What happens in the following situation of a Lac Operon:
- Glucose present, lactose present
Transcription NOT ACTIVATED
-Low cAMP
-CAP CANNOT bind. At the same time, the inducer (allolactose) prevents the repressor from binding to the operator
ESSAY QUESTION
What happens in the following situation of a Lac Operon:
- No glucose, lactose present
Transcription ACTIVATED
-High cAMP
-CAP/cAMP complex binds to the activator-binding site and the inducer (allolactose) prevents the repressor from binding to the operator
what is the purpose of the trp operon
the purpose of the trp operon is to make enzymes needed in the biosynthetic pathway for making tryptophan
what happens if there is plenty of trp present
if there is plenty of trp is present, the trp will serve as a co-repressor, activating the repressor to stop transcription
what does UTR mean?
untranslated region
trp is a repressible operon.
What happens when tryptophan is LOW?
- The trp repressor is normally inactive
- It cannot bind to the operator
- So, transcription takes place
trp is a repressible operon.
What happens when tryptophan is HIGH?
- Tryptophan binds to the repressor and makes it active
- The trp repressor binds to the operator and shuts transcription off
What are the TWO roles of the hairpin loops in the trp operon?
WHEN TRP IS HIGH:
-Hairpin prevents transcription; produces the attenuator that terminates transcription
WHEN TRP IS LOW:
-Hairpin allows transcription; attenuator never forms, thus allowing transcription to continue
A regulator gene produces a repressor in an inducible operon. A geneticist isolates several constitutive mutations affecting this operon (meaning constitutive expression of operon).
- Where might these constitutive mutations occur?
- How would the mutations cause the operon to be constitutive?
- the repressor is active and binds to the operator, inhibiting transcription. For transcription to occur, an inducer must bind to the repressor making it unable to bind the operator.
- Constitutive mutations might occur in the regulator gene, altering the repressor so it can’t bind to the operator. Alternatively, constitutive mutations might occur in the operator so that the repressor cannot bind.
T/F: One mutation has a greater effect in a haploid organism than in a diploid organism
TRUE
What is the difference between a Wild-Type and a Mutant?
Wild-Type: organism normally found in nature
Mutant: an organism that has an acquired changed from mutation
What is an Auxotroph?
organisms that NEED added nutrients
What is a Spontaneous Mutation?
mutation that occurs randomly, but at a predictable rate
can be spontaneous from errors in replication, repair, and recombination (movement of large sections of DNA)
Mutants will rarely emerge as a significant part of the population.
What is a time they are?
when they are selected from (e.g., in the presence of an antibiotic)
What are the three types of methods for selecting Mutants?
- Direct Selection
- Indirect Selection
- Penicillin Enrichment
What is Direct Selection?
for selection of antibiotic-resistant mutants
Placed on an agar plate containing an antibiotic and one without (e.g., Transformation lab)
What is Indirect Selection?
for auxotrophic mutants
uses cotton velvet as a stamp; an enriched complex medium and synthetic medium is used
Enriched complex medium = ALL colonies grow
Synthetic medium = auxotrophs DO NOT grow
What is Penicillin Enrichment?
selects for auxotrophic mutants (another way)
Penicillin is added to synthetic medium, which kills actively multiplying cells
Most prototrophs are KILLED; auxotrophs SURVIVE because they cannot multiply in the medium
What is Transformation in bacteria?
transfer of “naked” DNA
What is Transduction in bacteria?
transfer of DNA via bacteriophage
What is Conjugation in bacteria?
transfer of DNA via direct contact between two bacterial cells
List the steps that occur in DNA-mediated Transformation.
- Transforming DNA (double-stranded) attaches to the recipient cell surface
- Single strand of donor DNA enters
- Pairing of donor DNA with homologous region of recipient chromosome
- Integration of single-stranded donor DNA by breakage and reunion
- Transformed cells multiply; non-transformed cells die when placed on antibiotic medium
What does it mean when a cell is “competent?”
refers to its ability to take up foreign DNA from the environment
Can you induce competence?
Yes - depending on the bacteria it must be in a particular part of the growth curve or a special physiological condition
Means such as electroporation is a way in which to allow DNA into a cell, thus making it competent
What is Electroporation?
an electric field generates pores in bacterial membranes allowing the entry of the exogenous DNA molecule
Explain Transduction
Transfer of DNA from one cell to another through a replicating virus (known as phages)
- Phage enzymes degrade the bacterial DNA
- Phage genome now controls the cell’s replication
-Make new viral particles
-Pack bacterial DNA into a phage protein coat - Cell lyses
- Release of bacterial phage.
- Recipient incorporates donated DNA by recombination.
What are the steps in which Conjugation is able to occur between two bacteria cells?
- The donor must have the gene for pili or sex pili, which is located on the F plasmid (i.e., they are F+)
- Donor cell (F+) connects to the recipient (F-) with its pilus
- Pilus draws cells together
- One strand of the F pilus transfers to the Recipient
- The Recipient now synthesizes the complementary strand of the F plasmid to become an F+ cell with a pilus
- The Donor synthesizes a complementary strand restoring its plasmid.
What is the difference between the following:
- F’
- F-
- HFR
- H+
- F’ = plasmid has come back out of HFR
- F- = no plasmid (recipient cells)
- HFR = plasmid has incorporated into chromosome
- H+ = has plasmid
What are 2 virulence factors found on plasmids?
-Adhesions
-Enterotoxin
If 75% of one E. coli strain’s genes exist in all strains (the “core genome”), where is the remainder located?
made up of mobile genetic elements such as plasmids, transposons, bacteriophages, and genomic islands
What are Transposable Elements?
Segments of DNA that are 700 - 40,000 bp in length that move (transpose) from one location in a DNA molecule to another.
Often causes mutation if the gene is interrupted
What do Transposons contain?
Transposons all contain plaindromes
e.g., GAATTC is the palindrome of CTTAAG
they vary in their sequence in genetics, this is called an inverted repeat
What are Insertion Sequences in relation to Transposons?
short repeat sequences flanking the transposase gene
-moves to DIFFERENT locations in DNA in the SAME cell
T/F: Transfers may only occur between organisms of the same species.
FALSE - most transfer occurs between organisms of the same species, but occasionally interspecies transfer may occur
What is a plasmid?
circular, double-stranded DNA replicon; smaller than chromosomes;
Generally codes only for NON-essential genetic information
What is a Genomic Island?
A large fragment of DNA in a chromosome or plasmid
Codes for genes that allow cell to occupy SPECIFIC environmental locations
What are some applications for genetically engineered bacteria?
-vaccines
-pharmaceutical proteins for treatment
-DNA production for study and vaccines
T/F: the gene for Bt toxin protects plants from insect larvae.
TRUE
How many cells do we have in our body? How many bacteria cells?
Cells in our body = 10 - 40 TRILLION
Bacteria cells = 100 - 400 TRILLION
Explain the steps in cloning a gene (genomic library)
- The total DNA is isolated from the organism (e.g., Bacterium X)
- Restriction enzymes break the DNA into genomic fragments
- Genomic fragments join vector molecules - forming “Recombinant Molecules”
- These recombinant molecules are introduced into bacterial cells (e.g., E. coli)
- A population of bacterial cells (e.g., E. coli) together contain ALL of the fragments of the genome from Bacterium X
What is the function of Restriction Enzymes?
cut DNA at specific sequences
these leave jagged and “sticky ends” on the DNA
How does a cut DNA sequence join a vector molecule?
a restriction enzyme cuts the cloning vector, leaving “sticky end”
The “sticky ends” of the DNA sequence insert into the “sticky ends” of the cloning vector - forming the Recombinant DNA
List 2 Restriction Enzymes, how they cut, and the sequence they cut
- EcoER1 - cuts cohesively (“sticky ends”)
5’ - G|AATTC - 3’
3’ - CTTAA|G - 5’ - Hae3 - cuts bluntly
5’ - GG|CC - 3’
3’ - CC|GG - 5’
What is the Restriction Site?
Site on double-stranded DNA that contains the recognition sequence of a restriction enzyme…to be cut
there are over 200 of these!
Why are Restriction Enzymes important? How are they used?
-provides the means to modify DNA and construct new forms
-use of high copy plasmid will produce more protein
Why is it so difficult to produce a new antibiotic?
-Expensive
-Long trials (mice - primates, Phase I, Phase II….)
-Must pass all trials, which doesn’t alway happen (can fail at any time!)
What is a cDNA library?
a collection of synthetic DNA that has been transcribed from a specific mRNA through a reaction using the enzyme reverse transcriptase
What is PCR?
Polymerase Chain Reaction - Amplifies specific sequence of interest
- Heating to denature DNA
- Cooling to 50C to allow added primers to anneal to the single-stranded templates
- DNA synthesis occurs at 72C
- Mid-length strands synthesized and used as templates
What is Microarray Analysis?
can be used to detect CERTAIN DNA SEQUENCES (uses digested genomic DNA)
can be used to look at GENE EXPRESSION (uses cDNA)
-labeled with different fluorescent dyes
-washing unhybridized target and scanning
What is a Colony Blot?
can be used to detect a SPECIFIC DNA sequence within a colony
screen for colonies that have taken up gene of interest
What is FISH
used for detection of chromosomal abnormalities or to ID bacteria in a biofilm
Describe DNA Sequencing for bacteria
- PCR with fluorescent chain-terminating ddNTPs
- Size separation by capillary gel electrophoresis
- Laser excitation and detection by sequencing machine
what are resistance plasmids?
They are genes for resistance to one or more antimicrobial drugs or heavy metals. Certain cells can transfer resistance plasmids to other cells, making others resistant
eg: R plasmids involves stains of E. Coli that have acquired resistance to the antimicrobials amplicillin, tetracycline, and kanamycin from a strain of bacteria pseudomonas
What are bacteriocin plasmids?
plasmids that carry genes for protein-aceous toxins called bacteriocins, which kill bacteria cells of the same or similar species that lack the plasmid. This way, a bacterium containing this plasmid can kill its competitors.
What are virulence plasmids?
virulence plasmids- carry instructions for structures, enzymes, or toxins that enable a bacterium to become pathogenic.
eg: E coli, normal resident of the human gastrointestinal tract, can cause diarrhea when it carries plasmids that code for certain toxins
what are constitutive genes?
genes that are expressed at all times; contantly transcribed and translated and play a persistent role in phenotypes.
What are two types of regulation of transcription in bacteria?
induction and repression
what are operons?
special arrangements of prokaryotes genes that play roles in gene regulation.
What are the parts of an operon?
genes, promoter, operator.
Their genes encode for enzymes and structures such as channels and carrier proteins.
what are inducible operons, and what do they do?
Inducible operons- are usually inactive and must be induced by inducers. Such operons often code for proteins that transport and catabolize nutrients that are only sometimes available to a cell
What are repressible operons and what do they do?
Repressible operons operate in reverse fashion-they are typically continually active until repressors deactivate them. They often produce proteins that are involved in anabolic pathways.
What is tryptophan operon?
a repressible polycistronic operon consisting of a promoter, an operator, and five genes that code for enzymes involved in synthesis of tryptophan.
Define Antisepsis and give examples.
Antisepsis reduce the number of microorganisms and viruses on LIVING TISSUE
iodine, alcohol
frequent disinfectants whose strength has been reduced to be safe for living tissues.
What does the operon do when tryptophan is available?
When tryptophan is available, it binds to the repressor, thus activating it. The activated repressor then binds to the operator, halting the movement of RNA polymerase and transcription. Tryptophan stops its own synthesis.
Define Aseptic and give examples.
Aseptic refers to an ENVIRONMENT OR PROCEDURE free of pathogenic contaminants.
surgical fields, handwashing, flame sterilization
What happens in attenuation if tryptophan is low?
When the cells is starved for tryptophan, the amount of Trp-tRNA drops. region 2 will pair with region 3 to signal antitermination. The RNA polymerase is allowed to proceed and create more tryptophan
define suffixes -cide + -cidal and give examples.
suffixes indicating DESTRUCTION of a type of microbe.
bactericide, fungicide, germicide
germicides include ethylene oxide, propylene oxide, and aldehydes
After RNA polymerase has transcribed region 2, what happens?
After RNA polymerase has transcribed region 2, regions 1 and 2 can pair to form a loop structure. This causes RNA polymerase to pause and allow the ribosome to catch up to it.
Define degerming and give examples.
removal of microbes by MECHANICAL MEANS
**handwashing, alcohol swabbing, at site of injection.
-chemicals play a secondary role in mechanical removal of microbes.
What happens when there is plenty of tryptophan?
When there is enough trytophan, the ribosome continues to the stop codon at the end of region 1, preventing pairing of 2 and 3. Instead, 3 and 4 pair which signals for termination.
Define disinfection and give examples?
disinfection means destruction of most microorganisms and viruses on NONLIVING TISSUE
phenolics, alcohols, aldehydes, soaps
the term is used primarily in relation to pathogens.
Define pasteurization and give examples.
refers to using heat to destroy pathogens and reduce the number of spoilage microorganisms in foods and beverages.
pasteurized milk and fruit juices
Define sanitization and give examples.
Removal of pathogens from objects to meet public health standards.
washing tableware
define the suffixes -stasis + -static, and give examples.
suffixes indicating inhibition, but not complete destruction, of a type of microbe
bacteriostatic; fungistatic; virustatic
-germistatic agents including some chemicals, refrigeration, and freezing
define Sterilization and give examples.
destruction of ALL microorganisms and viruses in or on an object.
Preparation of microbiological culture media and canned food
typically steamed under pressure, incineration, or ethylene oxide gas.
CHP 9 ANTIMICROBIAL ACTIVITY OF GERMICIDES
ESSAY QUESTION
what level of germicide does endospore requires?
Endospore–
yes–Sterilant is needed
some–only some endospores are killed with high level
CHP 9 ANTIMICROBIAL ACTIVITY OF GERMICIDES
ESSAY QUESTION
what level of germicide does Mycobacteria requires?
Sterilant-yes
High level-yes
intermediate level-varies
CHP 9 ANTIMICROBIAL ACTIVITY OF GERMICIDES
ESSAY QUESTION
what level of germicide does other bacteria requires?
Sterilant- yes
High level- yes
intermediate level- yes
low level- varies
CHP 9 ANTIMICROBIAL ACTIVITY OF GERMICIDES
ESSAY QUESTION
what level of germicide does all fungi requires?
Sterilant- yes
High level- yes
Intermediate level- yes
low level- varies
CHP 9 ANTIMICROBIAL ACTIVITY OF GERMICIDES
ESSAY QUESTION
what level of germicide does nonlipid viruses requires?
sterilant- yes
high level- yes
intermediate level- varies
low level- no
CHP 9 ANTIMICROBIAL ACTIVITY OF GERMICIDES
ESSAY QUESTION
what level of germicide does lipid viruses requires?
sterilant- yes
high level- yes
intermediate level- yes
low level- varies
Death rate in microorganisms is normally __________ over time
CONSTANT - constant percentage of the extant population is killed each minute (usually 90%)
A particular sterilization process takes 25 minutes to kill all organisms in a sample containing 100,000 (10^5) microbes.
How long does it take to eliminate 90% of the organisms using this process?
5 minutes
25 minutes = 0 microbes
25 minutes / 5 log = 5 minutes
T/F: While bacterial death rate is normally constant over time, it varies drastically between bacterial species
TRUE
What factors would you consider when choosing an antimicrobial treatment?
RESISTANCE!
-Site to be treated
-Cost, how fast they act, stable
-How well do they kill
Some organisms are more resistant to antimicrobials than others.
List a couple of the MOST SUSCEPTIBLE microbes and a couple of the MOST RESISTANT microbes
MOST SUSCEPTIBLE:
1. Most Gram+ bacteria
2. Enveloped viruses
MOST RESISTANT:
1. Prions
2. Endospores
T/F: disinfectant work better in warmer environments
True
What germicidal chemicals target DNA?
Ethylene oxide, Aldehydes and radiation target the DNA of bacteria.
what two structures of the bacteria does phenolics target as a germicidal chemical
denatures cytoplasmic membrane and proteins
what structure of the bacteria does halogens target as a germicidal chemical
denatures proteins
what structures of the bacteria does aldehydes target as a germicidal chemical
denatures proteins and DNA
Soaps and detergents contain quaternary ammonium compounds (QUATS)
What does this do?
mimics the polar head group of the cell membrane, entering it and disrupting its integrity
an ANTISEPTIC that kills bacteria and other microorganisms
If Alcohol impacts cell health and aid in sterilization by targeting protein structure - would you select 100%, 70%, or 50% ethanol to clean a surface? Why?
70% ethanol - the membrane is disrupted while not evaporating too quickly
100% = evaporates too quickly
50% = harder to disrupt membrane
What chemical methods have high level of activity?
aldehydes, gaseous agents, oxidizing agents (ozone, peroxides), and enzymes against target substrate.
what chemical methods have low level of activity?
surfactants and heavy metals
what chemical methods have intermediate levels of activity?
alcohols, halogens
what chemical methods have intermediate to low levels of activity
phenol, phenolics, antimicrobials
How are the effects on proteins different for the following:
Heat-
Heat or pH-
heavy metals-
heat - complete denaturation
heat or pH - incorrect folding (ENZYME INACTIVE)
heavy metal - active sites blocked (Substrate CANNOT access the active site)
what is the active phenolic ingredient in Lysol
ortho-phenylphenol
T/F: triclosan is now a banned phenolic ingredient that was found in many anti-bacterial soaps
TRUE
what does non-ionizing do to DNA?
creates abnormal DNA bonds
What is the difference between a disinfectant and an antiseptic?
Disinfectant = destruction of most microorganisms and viruses on NONLIVING TISSUE (e.g., surfaces)
Antiseptic = reduction in the number of microorganisms and viruses on LIVING TISSUE (e.g., iodine, alcohol)
What is the difference between sanitization and sterilization?
Sanitization = removal of pathogens from objects to meet public health standards
Sterilization = destruction of ALL microorganisms and viruses in or on an object
A particular sterilization process takes 8 minutes to kill all organisms in a sample containing 100,000,000 (10^8) microbes.
How long does it take to eliminate 90% of the organisms using this process?
60 seconds
8 mins = 0 microbes
8 mins / 8 log = 1 minute (or 60 seconds)
What is the Thermal Death Time?
the time it takes to sterilize a volume of liquid at a set temperature
What would be more effective:
Dry heat or Moist heat?
MOIST HEAT
-dry heat is less effective because it requires higher temps for longer times because heat is transferred less readily
autoclave needs 121C for 15 mins to sterilize…an oven at the same temp will take 16 hours
Why might filtration (instead of autoclaving) be used as a method of sterilizing certain types of
growth media?
Some of this may be heat-sensitive, so filtration sterilizes without disrupting the media with heat
List some Preservation Methods and examples
- Radiation - surface of fruits, vegetables
- Chemical Preservatives - toothpaste, cosmetics, food items
- Low-Temp Storage - refrigeration, freezing (foods)
- Reducing Available Water - adding salt (meat) or sugar (jam), drying foods (jerky)
Why is autoclaving needed rather than just boiling in order to sterilize media, glassware, etc?
Autoclaving reaches much higher temperatures in less time (moist heat) compared to others, where boiling-resistant bacteria will not survive
How does UV/ionizing radiation work as an anti-microbial treatment? Examples?
This damages the DNA of the microbes
-Fruits/vegetables
-Spices
-Meats
-Medical and Dental Supplies
