BISC300: Microbiology Exam 3
Ribosomes: Bacteria vs. Eukaryotes
- Bacteria: 70S, 30S small subunit with 16S rRNA subunit, 50S large subunit with 5S and 23S rRNA subunits - Eukaryotes: 80S, 40S small subunit with 18S rRNA subunit, 60S large subunit with 5S, 5.8S, and 28S rRNA subunits
Addition of 3' cap: Bacteria vs. Eukaryotes
- Bacteria: No - Eukaryotes: Yes
Addition of 5' cap: Bacteria vs. Eukaryotes
- Bacteria: No - Eukaryotes: Yes
Splicing or pre-mRNA: Bacteria vs. Eukaryotes
- Bacteria: No - Eukaryotes: Yes
Shine-Dalgarno sequence in mRNA: Bacteria vs. Eukaryotes
- Bacteria: Present - Eukaryotes: Absent
Simultaneous transcription and translation: Bacteria vs. Eukaryotes
- Bacteria: Yes - Eukaryotes: No
Strand elongation: Bacteria vs. Eukaryotes
- Bacteria: core + σ = holoenzyme - Eukaryotes: RNA Polymerases I, II, or III
Amino acid carried by initiator tRNA: Bacteria vs. Eukaryotes
- Bacteria: fMet - Eukaryotes: Met
Number of polypeptides encoded per mRNA: Bacteria vs. Eukaryotes
- Bacteria: monocistronic or polycistronic - Eukaryotes: monocistronic
Transformant selection
- Bacterium does not take up plasmid, is not ampicillin resistant - Bacterium takes up nonrecombinant plasmid with intact lacZ gene - bacterium takes up recombinant plasmid, cannot produce beta-galactosidase enzyme - the plasmids are combined with a culture of actively growing bacteria. some cells do not take up plasmids, others take up non recombinant plasmids, and few take up the recombinant plasmids. - bacteria are cultured on a plate with ampicillin and a substance that changes color when exposed to the beta-galactose enzyme. cells that did not take up plasmids are killed by ampicillin. cells w/ non recombinant plasmids grow colonies that change colors. cells w/ recombinant plasmids grow white colonies
Freezing
- Below -2C - stops metabolism, may kill microbes
Do eukaryotes encode operons? - Yes - No
- No
If a single point mutation causes a codon to change from amino acid coding to a start codon what term do we associate with that mutation? - Missense - Nonsense - Silent - Frameshift
- Nonsense - Was coding for amino acids
Sanger DNA Sequencing (Old school)
- PCR + gel electrophoresis are used to sequence DNA - reaction uses old ddNTPs ( - each ddNTP incorporates a fluorophore - produces a series of truncated products - these are separated by electrophoresis - fluorescence used to read terminal bases in order of size
RNA Transcription Termination
- RNA Polymerase must dissociate from DNA template and release newly made RNA - DNA template has repeated nucleotide sequences that act as termination signals, causing RNA Polymerase to stall and release from the DNA template, freeing the RNA transcript.
sigma factor
- RNA polymerase comprises six polypeptide subunits - five of which compose the polymerase core enzyme responsible for adding RNA nucleotides to a growing strand, the sixth subunit is known as sigma (σ) - the σ factor enables RNA polymerase to bind to a specific promoter, thus allowing for the transcription of various genes. There are various σ factors that allow for transcription of various genes.
DNA Replication Elongation
- RNA primase synthesizes a short 4-10 nucleotide long primer with a free 3'-OH group that is complementary to the template DNA - RNA primase does not require a free '3-OH group to synthesize an RNA molecule - once the primer is made, DNA Polymerase III can use the primer to start synthesizing the DNA - this results in short DNA pieces known as Okazaki fragments that are separated by RNA primers - primers have to get replaced by DNA bases - primers are removed by exonuclease activity and DNA polymerase I fills in the gaps - DNA ligase fills in the gaps between Okazaki fragments - catalyzes bonds btwn 3' and 5'
Example of genotype and phenotype
- Serratia marcescens has a gene for red pigment - when grown at 38C, the bacteria expresses that gene, resulting in the production of the red pigment, giving the bacteria the phenotype of being red - at 37C, the bacteria doesn't express the red pigment gene, resulting in the phenotype of beige bacteria - the genotype has not changed, the bacteria still has the gene for red pigment, it's just not expressing that gene - therefore, no protein is made and the phenotype changes
What causes mutations?
- Spontaneous mutations - Induced mutations
Radiation
- Strong ionizing radiation - Ionizing radiation - Nonionizing radiation
Hyperbaric oxygen therapy
- air pressure 3x higher than normal - inhibits metabolism and growth of anaerobic microbes - forces oxygen in as many places as possible, inhibiting growth of anaerobic bacteria
Genome
- all DNA present in a cell or virus - bacteria and archaea have one set (haploid - 1N) - eukaryotes have two sets (diploid - 2N) - for an RNA virus, the genome is made of RNA
What parts of a plasmid are required for antibiotic resistance to be expressed by the bacteria? - origin of replication - multiple cloning site - promoter - antibiotic resistance gene - transgene
- antibiotic resistance gene - needed as base gene - promoter - drive the expression of the antibiotic gene - origin of replication - needed or else the plasmid won't be maintained
Restriction Enzymes
- bacteria naturally encode restriction enzymes - these evolved as a natural defense system against foreign DNA - restriction enzymes recognize and cut at a specific DNA sequence - sticky vs. blunt ends - sticky ends can basepair with another piece of similarly cut DNA, resulting in annealing, DNA can be ligated together forming a new strand - bacteria prevent digestion of their own DNA using restriction-modification systems
Generalized transduction
- bacteriophages can pick up any portion of the host's genome - any piece of chromosomal DNA can be transferred to a new host by accidental packaging into the phage head during phage assembly
RNA Transcription Elongation
- begins when sigma factor is released after 10-20 nucleotides - RNA is synthesized in the 5'-3' direction - DNA is unwound ahead of RNA Polymerase and rewound behind it - ~40 nucleotides per second - In illustrations, RNA polymerase is the oval, RNA temporarily accesses the genome to make a copy then binds back together - polycistronic, meaning that they encode multiple polypeptides
Ionizing radiation
- can cause deamination of cytosine to uracil - this is used to kill microbes to sterilize medical devices and foods, because of its dramatic nonspecific effect in damaging DNA, proteins, and other cellular components
Pasteurization
- can vary, one type is 72C for 15sec - denatures proteins and laters membranes - does not render the food sterile, reduces the number of spoilage causing microbes while maintaining food quality - LTH (low temperature holding - 63C for 30min) - HTST (high temp short time - 72C for 15sec) - UHT (ultra high temp - 138 for >2sec)
tRNA
- carries the correct amino acid to the site of protein synthesis in the ribosome - structure is critical to function, short, stable - contains amino acid binding site and mRNA binding site
Methods that kill microbes
- carry the suffix -cide or -cidal - bactericide kill bacteria, viricides kill viruses, fungicides kill fungi - these methods can lead to complete sterility
Induced mutations
- caused by exposure to mutagens - chemical agents or radiation; mutagen exposure can increase the rate of mutation more than 1000 fold - mutagens are often also carcinogens. however, whereas nearly all carcinogens are mutagenic, not all mutagens are necessarily carcinogenic
Spontaneous mutations
- caused by mistakes in the process of DNA replication - error rate of DNA polymerase is 1 incorrect base per billion base pairs replicated
What did we know before the discovery of genetic material?
- cells contain nucleic acids, lipids, polysaccharides, and proteins - most would guess that proteins store genetic information we knew that there was inheritance but not what it was based upon
Prokaryotes Genome Organization
- chromosomes are typically circular - haploid, contain one copy of each gene/chromosome - DNA supercoiled by topoisomerase (enzyme that maintain the structure of supercoiled chromosomes) - no histones
Eukaryotes Genome Organization
- chromosomes are typically linear - contain multiple distinct chromosomes - diploid, many contain two copies of each chromosome - DNA supercoiled by topoisomerase (enzyme that maintain the structure of supercoiled chromosomes) and packed with histones (DNA binding proteins) to form chromatin (DNA with these attached proteins)
Noncoding DNA
- chromosomes have a significant amount of noncoding DNA which do not encode proteins or stable RNA products - often found in areas prior to coding sequences as well as intergenic regions (i.e., DNA sequences located between genes) - can represent ~98% of genome in eukaryotes - can contribute to the regulation of transcription or translation through the production of small noncoding RNA molecules - roles in DNA packaging, and chromosomal stability
Phenotype
- collection of observable characteristics - product of the array of proteins being produced by the cell at a given time - the phenotype may change in response to environmental signals that affect which genes are expressed - cells carefully regulate expression of their genes, only using genes to make specific proteins when those proteins are needed
Nonsense mutation
- converts a codon encoding an amino acid (a sense codon) into a stop codon (a nonsense codon) - results in shortened proteins, typically not functional
Mismatch Repair
- correct just after the replication machinery has moved - enzymes recognize the incorrect nucleotide, excise it and replace it with the correct
Proofreading
- corrected by DNA polymerase via proofreading - the DNA polymerase ensures the complementary base to the template strand is correct - if an incorrect base was added, DNA polymerase releases the wrong nucleotide and adds the correct base
Ribosomes
- critical cellular machinery responsible for translation - ribosomes are composed of rRNAs (ribozymes) and structural rRNAs as well as polypeptides - recall that rRNAs can be structural and catalytic - dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation
Which control method functions by inhibiting metabolism? - gamma rays - dehydration - filtration - UV rays - high sugar addition
- dehydration - high sugar addition
Boiling
- denatures proteins and alters membranes - 100C at sea level
Dry-heat oven
- denatures proteins and alters membranes, dehydration, dessication - 170C for 2hrs
In-Use Test of evaluating effectiveness
- determine whether an actively used solution of disinfectant in a clinical setting is microbial contaminated - put on plate and look
Simple dessication
- drying - inhibits metabolism - dried fruits, jerkey
Silent mutations
- due to degeneracy of the genetic code, a point mutation (one base pair being changed) will commonly result in the same amino acid being incorporated into the resulting polypeptide despite the sequence change - this would have no effect on the protein's structure
Genetic Diversity in Sexual Reproduction
- each parent contributes their genetic information which is combined to produce new combinations of parental genotypes in diploid offspring - this occurs by assortment of chromosomes during meiosis and cross over events - this genetic diversity allows offspring to survive in changing or inconsistent environments
Why is E. Coli a model organism?
- easy to grow in the lab - short generation time - single, relatively small chromosome (4.6 million basepairs) - 42 minutes replication, 1,000 nucleotides incorporated per second
rRNA
- ensures the proper alignment of mRNA, tRNA, and ribosome during protein synthesis - catalyzes peptide bond formation between amino acids - structure is critical to function, longer stable - 60% of the ribosomes mass is rRNA (ribosomal RNA) - provides the location where the mRNA binds - rRNA ensures the proper alignment of the mRNA, tRNA, and the ribosomes; the rRNA of the ribosome also has an enzymatic activity (peptidyl transferase) and catalyzes the formation of the peptide bonds between two aligned amino acids during protein synthesis
oriC is an A-T rich region of DNA where DNA replication starts. Mutation of the nucleotides to G-C nucleotides will help the helicase to separate DNA strands more easily - True - False
False
While humans are not microbes, their individual cells such as red blood cells, cancer cell lines, white blood cells, etc. are considered microbes - True - False
False cells being isolated doesn't make them microbes
Which nucleotide pairings are found in RNA? - A:T - G:C - A:U - G:U - A:C
G:C and A:U
How does penicillin work?
Penicillin inhibits the transpeptidation reaction during cell wall synthesis.
During DNA Replication what protein creates primers? - DNA Polymerase - Single-stranded binding protein - Primase - Helicase
Primase
Lac operon activation by a catabolite activator protein
- glucose is the preferred carbon source of all cells - E. Coli always uses its preferred carbon source first - as ATP cells decrease due to lower glucose levels, EllA enzyme is phosphorylated. EllA activates adenylyl cyclase which converts ATP to cyclic AMP - cAMP is important signaling molecule in cells and as levels begin to rise, the cell can switch to turning on the lac operon at a higher rate - as cAMP increases it binds to the catabolite activator protein, binds to the promoter region of the lac operon, activating it. this increases the binding ability of RNA Polymerase to the region, resulting in more transcription - repressor also has to be released, so glucose must be depleted and lactose must be present
Induction
- in the absence of lactose, the lac repressor binds the operator and transcription is blocked - in the presence of lactose, the lac repressor is released from the operator and transcription proceeds at a slow rate - lac operon bound to the operator, physically preventing RNA Polymerase from transcribing when lactose is not present - when lactose is present in the cell, it is converted to allolactose - allolactose is the inducer, it binds to the repressor and causes a conformational change which releases it from the DNA, allowing transcription to begin
Repression
- in the absence of tryptophan, the trp repressor dissociates from the operator and RNA synthesis proceeds - when tryptophan is present, the trp repressor binds the operator and RNA synthesis is blocked - many repressible operons encode for biosynthetic pathways - when levels of tryptophan are low, the operon is turned on and more tryptophan can be made. once tryptophan reaches high enough levels, tryptophan binds to the repressor, allowing it to bind to the operator and block transcription
Fomites
- inanimate objects that may carry microbial contamination - may be treated w/ more aggressive control methods or for longer time, to achieve lower levels of contamination - living tissue is more fragile and either requires gentler methods or we have to tolerate greater contamination levels
RNA Transcription
- information encoded in the DNA sequence of a gene is transcribed into a strand of RNA (RNA transcript) - mRNA acts as a mobile molecular copy of the DNA sequence - transcription bubble - site of RNA synthesis, requires DNA double helix to unwind - DNA acts as the template (using the anti-sense strand) - because RNA product is complementary to template DNA, it is identical to the non-template DNA strand (sense strand) - RNA is synthesized in 5'-3' direction but doesn't require 3'-OH for initiation (doesn't need a primer)
Translation of mRNA to Protein
- information encoded on mRNA is translated into a polypeptide - sequence by the ribosome - mRNA is read from the 5'-3' direction - polypeptides are synthesized in the N terminal -> C terminal direction - transcription and translation are often coupled/simultaneous in Bacterial Archaea - as RNA Polymerase is making the RNA, ribosomes can attach and start making the protein polypeptide - multiple ribosomes can translate a single mRNA - polyribosome (or polysome) , which is why bacteria can rapidly increase levels of proteins in the cell
Simple steps of DNA Replication
- initiation - elongation - termination
Prokaryotes vs Eukaryotes
- main difference is that prokaryotes can simultaneously transcribe and transition in the cytoplasm - eukaryotes must separate the process with transcription happening in the nucleus and translation happening in the cytoplasm
Extrachromosomal DNA
- many cells have additional molecules of DNA outside the chromosomes - this includes chromosomes from any organelles such as mitochondria (all cells) and chloroplasts (plants and algae) - circular chromosomes in these organelles is a trace of their prokaryotic origins, supports endosymbiotic theory - genomes of some DNA viruses are maintained independently in host cells. in these cases, these viruses are another form of extrachromosomal DNA.
During Griffith's experiment, the Staph bacteria he used naturally liked to take up DNA from the environment. If the Staph he used wasn't naturally competent (able to take up environmental DNA), what would have been his experimental results? - mice would've died in all treatments - mice would've survived all treatments - mice would've only survived exposure to living non-virulent Staph - mice would've only died by exposure to living virulent Staph
- mice would've only died by exposure to living virulent Staph
Physical delivery
- microinjection is another technique for introducing DNA into eukaryotic cells - a miroinjection needle containing recombinant DNA is able to penetrate both the cell membrane and nuclear envelope - gene gun is good for crushing through cell walls with macromolecule
Modifying agents
- modify normal DNA bases, resulting in different base-pairing rules - ex: nitrous acid deaminates cytosine, converting it to uracil. uracil then pairs with adenine in a subsequent round of replication, resulting in the conversion of a GC base pair to an AT base pair. - nitrous acid also deaminates adenine to hypoxanthine which base pairs with cytosine instead of thymine, resulting in the conversion of a TA base pair to a CG base pair
DNA Replication Termination
- much less is known about termination of replication - resulting complete circular genomes of prokaryotes are concatenated at the origin of replication, meaning that the circular DNA chromosomes are interlocked and must be separated from each other - these are separated by topoisomerase IV, introducing double stranded breaks into the DNA, allowing them to separate - it then reseals the circular chromosomes - this is unique to prokaryotes bc of their circular chromosomes
Transposition
- process where DNA independently excises from one location in a DNA molecule and integrates elsewhere - "cut and paste" moves from 1 area of DNA to another (nonreplicative) - "copy and paste" leave a copy and moves to a new DNA molecule (replicative) - doesn't only occur in prokaryotes - transposons can move within a DNA molecule, from one DNA molecule to another, or even from one cell to another, they have the ability to introduce genetic diversity
RNA Transcription Initiation
- promotor region - DNA sequence onto which the transcription machinery binds and initiates transcription - initiation site - nucleotide pair in the DNA double helix that corresponds to the site from which the first 5' RNA nucleotide is transcribed - at the -10 and -35 positions within the DNA prior to the initiation site (designated +1), there are two promoter consensus sequences, or regions that are similar across all promoters and across various bacterial species. - -10 has the TATA box with the sequence TATAAT - -35 is where the sigma factor binds, allowing RNA Polymerase to bind - Sigma factor binds RNAP, binds -35 of promotor - RNA Polymerase unwinds DNA at -10 of promotor, strands are easy to separate because of the A's and T's - RNA is synthesized
How to fix mutations?
- proofreading - mismatch repair
Western blots to detect translation
- separation gel -> blotting tank where proteins are transferred to a nitrocellulose sheet (blockt) -> immune-staining of the blot with labeled antibodies -> autoradiography -> develop and fix autoradiograph -> antigen bands visualized - can detect: relative amounts of specific proteins and what types of cells have specific proteins
mRNA
- serves as intermediary between DNA and protein - used by ribosome to direct synthesis of protein it encodes - largely unstructured, short, unstable, single stranded - information from DNA is transmitted to the ribosome for protein synthesis using mRNA - if DNA serves as the complete library of cellular information, mRNA serves as a photocopy of specific information needed at a particular point in time that serves as the instructions to make a protein - mRNA carries the message from the DNA, which controls all of the cellular activities in a cell. If a cell requires a certain protein to be synthesized, the gene for this product is "turned on" and the mRNA is synthesized through the process of transcription - mRNA then interacts with ribosomes and other cellular machinery to direct the synthesis of the protein it encodes during the process of translation
Alternate sigma factors
- sigma factors recognize promoter regions, allowing RNA Polymerase to bind - bacteria can change which sigma factor is expressed and thus which genes are transcribed
Dideoxynucleotide
- similar in structure to deoxynucleotide but missing 3' hydroxyl group. when this is incorporated into a DNA strand, DNA synthesis stops - we use this to determine the sequence of a piece of DNA by stopping replication and aligning the fragments by size and reading the sequencing
RNA Structure
- single stranded RNA can fold upon itself, stabilized by short areas of complementary base pairing; in some cases it can form complex 3D structures
Intercalating agents
- slide between the stacked nitrogenous bases of the DNA double helix, distorting the molecule and creating atypical spacing between nucleotide base pairs - this results in DNA polymerase either skipping several nucleotides (creating a deletion) or inserting extra nucleotides (creating an insertion) -> frameshift mutation
Plasmids
- some prokaryotes commonly maintain circular segments of DNA that may contain one or a few genes not essential for normal growth - replicate when the cell divides - can contain important genes which can be exchanged through horizontal gene transfer
Conjugation via Hfr cell
- sometimes the F plasmid integrates into the bacterial chromosome via recombination - this results in Hfr cell, referring to the high frequency of recombination seen when recipient F- cells receive genetic information from Hfr cells - the integrated F plasmid is imprecisely excised from the chromosome producing a new plasmid called F' that carries the plasmid plus chromosomal DNA adjacent to the integration site. because the F' plasmid contains chromosomal DNA, it's more likely to integrate. - sometimes Hfr cells try to transfer the entire chromosome like it's a huge F plasmid -- this takes a long time but since contact between bacterial cells is transient, it's unusual for this to happen - historically bacterial genomes are measured in minutes because Hfr cells were often used to map genomes based on conjugation
DNA Structure
- specific base pairs form due to hydrogen bonding patterns: Adenine-Thymine, Guanine-Cytosine - this allows nucleic acid strands to hybridize - the strands run in antiparallel directions, the 3' end on one strand faces the 5' end on the other strand, forming the double helix - the phosphodiester backbone is made up of the sugars and phosphates on the outside with the nitrogenous bases on the inside - minor groove is when the backbone is close together - major groove is when the backbone is far apart
Genotype
- specific set of genes an organism possesses
Griffith transformation mouse test (1928)
- strain R is non-virulent because it lacks a capsule, strain S is virulent and has a capsule which allows it to evade the host immune response - live, Strain R doesn't kill mice - live, Strain S kills mice - killed, Strain S doesn't kill mice - live Strain R mixed with killed, Strain S does kill mice! - how does this occur? bacteria can pick up "traits" from the environment - a chemical factor from the virulent strain transforms the nonvirulent strain, but which chemical is it?
tRNAs
- structural RNA molecules w/ specific 3D structure formed by base pairing within the ssRNA molecule - have an anticodon (bonds w/ mRNA codon) and the CAA amino acid binding end (amino acid binding site) - bacteria have 60-90 tRNAs in cytoplasm - each binds to a specific codon and adds the corresponding amino acid - translate RNA language into protein - highly specific and interact w/ 3 factors: aminoacyl tRNA syntheses, ribosomes, and mRNA
Translation Elongation
- tRNAs bring amino acids one by one to add to polypeptide chain - 50S subunit has 3 sites: A, P, E - A - binds incoming charged aminoacyl tRNAs - P - binds charged tRNAs with amino acid that have formed a peptide bond but not yet dissociated from the tRNA - carries the growing polypeptide chain - E - releases dissociated tRNAs so they can be recharged w/ new aa - Peptide transferase - RNA based ribozyme integrated into the 50S subunit, catalyzes peptide bond formation btwn amino acids - a new tRNA arrives at A site, 23S rRNA catalyzes peptide transferase - polypeptide chain in P site is transferred to A site, new amino acids is bound to C term of polypeptide - tRNA with polypeptide moves from A to P site - empty tRNA moves from P to E and dissociates - ribosome moves down 1 codon to keep growing polypeptide chain
Sterilization
- used on fomites - completely eliminates all vegetative cells, endospores, and viruses from an inanimate item (autoclave, radiation)
Disinfection
- used on fomites - destroys or reduces microbial load of an inanimate item through application of heat or antimicrobial chemicals (like bleach)
Sanitization
- used on fomites - reduces microbial load of an inanimate item to safe public health levels through application of heat or antimicrobial chemicals (detergents, industrial strength cleaners)
Degerming
- used on living tissue - reduces microbial load on skin or tissue through gentle to firm scrubbing and the use of mild chemicals (soap)
Antisepsis
- used on living tissue - reduces microbial load on skin ot tissue through application of an antimicrobial chemical (betadine, hydrogen peroxide_
A macrobroth dilution assay to determine the minimal inhibitory concentration (MIC) of amoxicillin on E. coli is shown below. What is the MIC?
0.05 µg/mL
Monocistronic Expression
1 protein per transcript - eukaryotes
Rank these growth control techniques from LOWEST microbial contamination on top to HIGHEST microbial contamination on the bottom: - degerming - antisepsis - sanitization - disinfection - sterilization
1.) sterilization (lowest) 2.) disinfection 3.) sanitization 4.) antisepsis 5.) degerming (highest)
How many standard amino acids are there? - 4 - 20 - 22 - 64
20
How many nucleotides make up a codon? - 1 - 2 - 3 - 4 - 5 - 6
3
Phosphodiester bonds are formed between which carbons on the nucleotide? - 1 - 2 - 3 - 4 - 5
3 and 5
Which of the statements are true about the differences between DNA and RNA: - DNA has an extra oxygen on the 2' carbon - RNA is typically double-stranded, while DNA is typically single-stranded - 3 nitrogenous bases are shared between DNA and RNA - Cellular genomes are based on RNA
3 nitrogenous bases are shared between DNA and RNA
T7 phage's simple replisome
4 proteins - gp5 - DNA polymerase 1 - gp4 - helicase/primase - gp2.5 - single strand binding protein - trx - thioredoxin 3 proteins from the phage and 1 from the E. Coli host
Prokaryotic Ribosomes
70S ribosome composed of: - small 30S subunit, contains 15S rRNA; binds mRNA template - is like the surface that everything sits on - large 50S subunit, contains 5S and 23S rRNA; binds tRNAs, squishes the mRNA
Eukaryotic Ribosomes
80S ribosome found in the cytoplasm and rough ER, composed of: - small 40S subunit - contains 18s rRNA; binds mRNA template - large 60S subunit - contains 5S, 5.8S, 28S rRNA; binds tRNAs 70S ribosomes in the mitochondria and chloroplasts.. why?
Flow of information in the cell
DNA -> RNA -> Protein - heritable genetic information is stored in DNA - instructions for protein synthesis are transcribed into messenger RNA - mRNA is translated into protein by the ribosome which incorporates ribosomal RNA - translation involves transfer RNA
Which technique fits this general description: DNA is neither created nor destroyed, it is simply moved around? - Polymerase Chain Reaction (PCR) - DNA Gel electrophoresis - Protein polyacrylamide gel electrophoresis - Western Blot
DNA gel electrophoresis
Which technique that we talked about uses an electrical current to move molecules? - DNA gel electrophoresis - SDS-Polyacrylamide gel electrophoresis - Restriction enzyme digestion - PCR test -Western Blotting
DNA gel electrophoresis
Which atoms are unique to which macromolecule in the Hershey Chase Experiment? - Sulfur to protein: Carbon to DNA - Nitrogen to protein: Carbon to DNA - Phosphorus to protein: Sulfur to DNA - Sulfur to protein: Phosphorus to DNA - Nitrogen to protein: Phosphorus to DNA
Sulfur to protein: Phosphorus to DNA
If the Avery-MacLeod-McCarty experiment was performed and the researchers had found that virulence was no longer transferred after treatment with RNAse, which molecule would they have included was the molecule of inheritance? - DNA - RNA - Protein - Lipid
RNA
Overview of Molecular Cloning
Vector - Gene for antibiotic resistance in plasmid, Foreign DNA, part of it is taken out, put into the plasmid which is opened up. come together, becomes recombinant DNA. can be cloned as a plasmid.
Binary fission
a form of asexual reproduction in which one cell divides to form two identical cells
Transcription
a gene is composed of DNA that is "read" or transcribed to produce an RNA molecule
Regulon
a group of operons all controlled simultaneously, usually in response to an environmental signal
Dispersive Model for DNA
all strands have a mix of parental and new DNA
Bioinformatics
analysis of large comic data sets
Which organisms contain polycistronic operons? - bacteria - archaea - eukaryotes
bacteria and archaea
Metals
bind to proteins and inhibit enzyme activity
Single stranded binding proteins
bind to single stranded DNA to prevent hydrogen bonding between DNA strands, reforming double stranded DNA
Nucleotide
building block of nucleic acid
Conjugation
use of a hollow tube (conjugation plus) to transfer genes between cells - F pilus of an F+ cell comes in contact with an F- cell - pilus retracts, bringing the two cell envelopes into contact and the cytoplasmic bridge forms at the site of the pilus - rolling circle replication of the F plasmid occurs in the F+ cell - ssDNA is transferred through the cytoplasmic bridge to the F- cell, which synthesizes the complementary strand, resulting in the complete plasmid - the F- cell is now an F+ cell and can mate with other F- cells - there are genes on the F plasmid to encode proteins preventing conjugation between F+ cells
Asepsis
using aseptic technique to maintain sterility and prevent contamination with microbes failure of aseptic technique may put a clinical patient at risk for sepsis, a systemic inflammatory response to a systemic infection
Strong ionizing radiation
x-rays and gamma rays can cause single and double stranded breaks in the DNA backbone through the formation of hydroxyl radicals on radiation exposure
Chromosome
contains the vast majority of an organism's genome
Molecular Cloning Pieces: Ligation
covalent linkage of two pieces of DNA
Chemical food preservatives
decrease pH and inhibit enzymatic function
Alcohols
denature proteins and disrupt membranes
Phenolics
denature proteins and disrupt membranes
Bisbiguanides
disruption of cell membranes
Old School Biotechnology
domestication and breeding of plants for farming domestication and breeding of animals fermentative processes for food, fuel, and drink
Semiconservative Model for DNA
double helix separates and each strand serves as a template for new synthesis
Codon
each amino acid is defined within the mRNA by a triplet of nucleotides
Transcriptomics
entire mRNA produced by cells or organisms
Proteomics
entire protein population of a cell or organisms
DNA Polymerase I
exonuclease activity removes RNA primer and replaces it with newly synthesized DNA
Binary fission results in: - one cell created from two diverse cells - genetically diverse progeny cells - genetically identical progeny cells (clonal) - none of the above
genetically identical progeny cells (clonal)
Sliding clamp
helps hold DNA Polymerase III in place when nucleotides are being added
Mutation
heritable change in the DNA sequence of an organism
Alkylating Agents
inactivation of enzymes and nucleic acid
Natural Food Preservatives
inhibition of cell wall synthesis
Topoisomerase IV
introduces single-stranded break into concatenated chromosomes to release them from each other, then reseals the DNA
Wild type
most commonly observed phenotype
Translation
one major type of RNA molecule, called messenger RNA (mRNA), provides the information for the ribosome to catalyze protein synthesis
Helicase
opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases
Peroxygens
oxidation and destabilization of cellular macromolecuels
Halogens
oxidation and destabilization of cellular macromolecules
Supercritical gases
penetrates cells, forms carbonic aid, lowers intracellular pH
Which was the first natural antimicrobial agent discovered?
penicillin
Molecular Cloning Pieces: Vector
plasmid that exogenous DNA (insert) is cloned into for expression in bacterial, plant, and mammalian cells
Meta-omics
population based genomics, transcriptomics, or proteomics
Posttranslational modifications
posttranslational modifications before newly made peptides become biologically active: 1. removal of translated signal sequences 2. proper folding into 3D structure facilitated by chaperone proteins 3. proteolytic processing of an inactive polypeptide to activate it 4. chemical modifications of individual amino acids (e.g. phosphorylation, methylation, or glycosylation)
Molecular Cloning Pieces: Transformaiton
process of introducing exogenous DNA into a bacterial cell
Mutant
recognizable phenotypic change compared to wild type
Topoisomerase II (DNA gyros)
relaxes supercoiled chromosome to make DNA more accessible for the initiation of replication; helps relieve the stress on DNA when unwinding, by causing breaks and then resealing the DNA
Ligase
seals the gaps between the Okazaki fragments on the lagging strand to create one continuous DNA strand
Polycistronic Expression
several proteins per trasncript - prokaryotes, organelles, lac operon
Thymine Dimer Repair - Nucleotide excision repair (dark repair)
the pyrimidine dimer is removed by helicase and DNA Polymerase and DNA ligase replace it with the correct nucleotide
Biotechnology
the science of using living systems to benefit humankind
Vertical Gene Transfer
the transmission of genetic information from generation to generation
Bacterial messenger RNAs are processed in which ways? - 5' cap - intron splicing - poly-A tail - translated by 70S ribosomes
translated by 70S ribosomes
Strand elongation using: Bacteria vs. Eukaryotes
- Bacteria use DNA Polymerase III - Eukaryotes use pol δ, pol ε
Will carbapenems affect eukaryotic cells?
No because eukaryotic cells do not have a cell wall
Rate of DNA replication: Bacteria vs. Eukaryotes
- Bacteria replicate at 1000 nucleotides per second - Eukaryotes replicate at 100 nucleotides per second
RNA Primer removal using: Bacteria vs. Eukaryotes
- Bacteria use DNA Polymerase I - Eukaryotes use RNase H
Genome structure: Bacteria vs. Eukaryotes
- Bacteria have single circular chromosomes - Eukaryotes have multiple linear chromosomes
Refrigeration
- 0C-7C - inhibits metabolism, slows or arrests cell division
High pressure processing
- 100-800MPa - denatures proteins and can cause cell lysis - used in preservation of food
Genetic Code chart
- 3 nucleotides/codon = 64 possible combinations - degeneracy is redundancy in genetic code, mainly due to the wobble position - wobble position = third codon, sometimes it doesn't matter what letter is there, there may be other tRNAs in there but it will still give you the same aa - stop codon/nonsense codon - terminate protein synthesis - start codon - initiates protein synthesis
Telomerase: Bacteria vs. Eukaryotes
- Bacteria don't have - Eukaryotes have
Number of origins per chromosome: Bacteria vs. Eukaryotes
- Bacteria have a single origin per chromosome - Eukaryotes have multiple origins per chromosome
DNA Sequencing (New School)
- DNA is attached to a microscope slide - fluorescent nucleotides are detected as they interact w/ polymerase - a billion bases read in a single run
Cellular Genome Function
- DNA is responsible for inheritance, passed from parent to offspring - must be replicated with accuracy to preserve integrity - a genome contains the full complement of DNA within a cell and is organized into smaller, discrete units called genes that are arranged on chromosomes and plasmids. - DNA codes for the expression of proteins, which are necessary for organism survival - DNA regulates protein expression based on growth and reproduction needs based on the environment
Starting Idea for DNA Replication
- DNA structure was first proposed by Watson and Crick and an X-ray diffraction of DNA shows a double stranded nature, proposed double helix - double stranded DNA gave a hint as to how replication could occur - if 2 strands were separated you'd have 2 templates for synthesis of new strands
Molecular Cloning Pieces: Insert
- DNA that is cloned into vector (usually a plasmid) - typically contains a gene of interest
Bacterial translation and transcription cannot happen simultaneously because the ribosomes are located in the cytoplasm separated by the nuclear envelope. - True - False
- False - Prokaryotes don't have nuclei
Due to DNA's negative charge, it easily diffuses through the cell membrane. - True - False
- False - it's heavily non-polar and difficult to diffuse
Reduce water activity
- addition of salt or water or sugars. at very high concentrations, the amount of available water n microbial cells is reduced dramatically due to osmosis - inhibits metabolism and can cause lysis
Nonionizing radiation in Sterilization
- UV light - less energetic, less penetrating - causes thymine dimers to form between adjacent thymines in DNA
RNA
- Uracil replaces Thymine - sugar is ribose rather than deoxyribose - most RNA molecules are single stranded, some are double stranded
Heat killing
- a function of temperature and time - serious microorganisms respond differently to high temps - boiling doesn't kill all microbes! some endospores survive up to 20 hours of boiling!
Trait
- a genetically determined characteristic - in this class, trait broadly to mean whatever message or goal you have - phenotype, protein production, metabolite, disruption of a certain area of DNA - in order to manipulate traits, we are often manipulating DNA
Conditional mutation
- a missense mutation apparent only under certain environmental conditions - could be advantageous but only under the right environmental conditions - ex: temperature sensitive mutation
During the third round of amplification, the DNA Polymerase is copying what? - an original strand - a new strand - half new and half original - none of the above
- a new strand - as the bottom one replicates, it becomes internal and becomes the new template for the next round of amplification - will start to be half new and half original because the dsDNA can be replicated too
Eukaryotic RNA Transcription
- eukaryotes use three different polymerases, RNA polymerases I, II, and III, all structurally distinct from the bacterial RNA polymerase. each transcribes a different subset of genes. - monocistronic, meaning that they each encode only a single polypeptide - protein-encoding primary transcripts (pre-mRNA), the RNA molecules directly synthesized by RNA polymerase, must undergo several processing steps to protect these RNA molecules from degradation during the time they are transferred from the nucleus to the cytoplasm and translated into a protein - pre-mRNA is first coated with RNA-stabilizing proteins to protect it from degradation while it is processed and exported out of the nucleus - a special 7-methylguanosine nucleotide, the 5' cap is added to the 5' end of the growing transcript. once elongation is complete, another processing enzyme then adds a string of approximately 200 adenine nucleotides to the 3' end, called the poly-A tail
Denaturing of DNA
- exposing DNA to high heat or chemicals can break the hydrogen bonds between complementary bases, separating them into two single strands (ssDNA) - can be put back together if under the right, favorable conditions
Ionizing radiation in Sterilization
- exposure to X-rays or gamma rays and high energy electron beams - penetrates cells, directly damages biological molecules. causes DNA mutations, leading to cell death - x-rays and gamma rays penetrate paper and plastic to sterilize packaged materials
Incineration
- exposure to flame - destroy by burning
Alarmones
- expressed in response to stress - small intracellular nucleotide derivatives - change which genes are expressed
Transposase
- facilitates recombination between inverted repeats
Ribozymes
- folded RNA that can do catalysis
Disk-Diffusion method of evaluating effectiveness
- for chemical agents - filter disks containing chemical placed on an agar plate inoculated with bacterium - compound diffuses and causes a zone of inhibition of microbial growth - size of zone correlates w/ potency of compound
Transduction
- genes are transferred between cells in a virus - bacteriophage are viruses that infect bacteria, they mediate transduction - transduction can result in new pathogenic properties for the receiving bacteria
Transposons
- genetic elements of DNA that include inverted repeat sequences on their ends and a gene that encodes an enzyme called a transposase - is cut from its original location and inserted into a new location - can include additional genes (genetic diversity) - can move from one DNA molecule to another OR to new cells - found in prokaryotes and eukaryotes
New School Biotechnology
- genetic manipulation or genetic engineering - the direct alteration of an organisms genetics to achieve desirable traits - utilizes recombinant DNA technology to manipulate DNA in vitro - transgenic organism - an organism that contains recombinant DNA form another organism - gene edited organism - an organism that had its genetics altered with synthetic and targeted methods, but no addition of foreign DNA
Testing the models of DNA replication
- grew E. coli for several generations in a medium containing a "heavy" isotope of nitrogen (15N), was incorporated into the DNA. this is labeled the parental DNA. the culture was then shifted into a medium containing 14N, allowed to grow for one generation, DNA was isolated - ultracentrifugation allowed the DNA to form bands according to its density - if conservative model is correct, there should be 2 distinct bands. if semiconservative/dispersive was correct, DNA should settle at an intermediate level bc it would contain 15N and 14N. - after the first round, the bands were in the intermediate position, ruling out conservative. - the cells were allowed to grow for one more generation in the 14N medium then spun again - if semiconservative was correct, two distinct bands were expected. if dispersive was correct, the bands should be at an intermediate level again. - after being spun, one DNA band was at the intermediate position and the other corresponded to the band of 14N DNA.
Generalized transduction is only performed by the lytic phage and specialized transduction is specific to lysogenic phage. - true - half true, half false - half false, half true - false
- half false, half true - first half is false, second half is true - generalized transduction is when chunks of genome is packaged into phage and goes on to infect the next cell. both lysogenic and lytic phages can do this. - lysogenic phages are the only ones who can integrate their DNA then get it back out so they're the only one who can do specialized
Which of the following can be considered biotechnology? - harvesting polysaccharides from red algae to make agarose - using an organic pesticide like bacillus thuringiensis bacteria to kill insects on your squash - using radiation to kill bacteria - putting uranium metabolizing bacteria into mines to reduce soluble uranium - using manure to fertilize your garden
- harvesting polysaccharides from red algae to make agarose - using an organic pesticide like bacillus thuringiensis bacteria to kill insects on your squash - using manure to fertilize your garden
Gel Electrophoresis
- how are fragments of DNA separated and analyzed in the lab? - samples w/ DNA fragments are loaded into wells on an agarose gel slab - a voltage is applied across the gel, causing DNA fragments to migrate - smaller fragments migrate faster, so DNA fragments separate by size - DNA fragments all have the same negative charge per bp - DNA migrates towards positive electrode in an electric field - gel matrix slows down larger fragments - shorter pieces run faster
Which of these control methods would be viable for use on human skin without excessive damage? - incineration - hyperbaric chamber - freezing at -70C - autoclave - boiling
- hyperbaric chamber
Methods that inhibit microbial growth
- identified by the suffix -stat or -static - bacteriostatic for bacteria, fungistatic for fungi - in the context of infection, these may allow the immune system an opportunity to clear an infection
Consequences of Conjugation
- important for genetic diversity and sharing of key traits - R (resistance) plasmids, encode genes for antimicrobial resistance and genes that control conjugation, can transfer between cells of the same or different species, may encode multiple resistances - plasmid may also encode toxins, and special metabolic enzymes - special plasmids may also undergo transfer to plant or animal cells
DNA Replication Initiation
- initiation occurs at origin of replication (oriC in E. Coli), very A-T rich - DnaA protein that starts and initiates the replication at the origin of replication, has some specific bases/patterns that it needs to see to bind - since DNA is supercoiled, it's relaxed by topoisomerase II (or DNA gyrase) - DNA helicase separates the DNA strands (breaks hydrogen bonds between base pairs, A-T is easier to pull apart) - DNA opens, Y-shaped structures called replication forks are formed. two replication forks are formed at the origin of replication, allowing for bidirectional replication - single stranded binding proteins prevent ssDNA from rewinding into dsDNA - DNA pol III is responsible for replication, it can only add nucleotides in the 5' to 3' direction, works great on the leading strand because DNA polymerase can continually add nucleotides because it's synthesizing in the 5' to 3' direction - sliding clamp helps keep DNA Pol III in place
Electroporation
- introduce the gene to the cell - apply the electric pulse, pores form in the cell membrane, pulse generates a short-lived positive charge on one side of the cell's interior and a negative charge on the opposite side; the charge difference draws negatively charged DNA molecules into the cell - after the electric pulse, the pores reseal and the gene remains in the cell
Horizontal gene transfer
- introduction of genetic material from one organism to another organism within the same generation - more common among evolutionarily related organisms, but can occur in between two species that are in close proximity - most prevalent in prokaryotes - only small pieces of prokaryotic genome can be transferred like this at any one time
Splicing of pre-mRNA
- it is essential that all of the intron-encoded RNA sequences are completely and precisely removed from a pre-mRNA before protein synthesis so that the exon-encoded RNA sequences are properly joined together to code for a functional polypeptide. - the process of removing intron-encoded RNA sequences and reconnecting those encoded by exons is called RNA splicing - facilitated by the action of a spliceosome containing small nuclear ribonucleo proteins (snRNPs)
Translation initiation
- mRNA template - small 30S ribosome - 3 initiation factors (IF1, IF2, IF3) - a special initiator tRNA carrying N-formyl-methionine (fMet-tRNAfMet) - GTP (energy source) - transitional complex forms, tRNA brings first amino acid in peptide chain to bind to start codon on mRNA - 30S subunit is bound by IF3, preventing the 50S subunit form binding - the 16S rRNA binds the Shinde-Dalgarno sequence (also known as the ribosomal binding site AGGAGG), positioning the 30S subunit and releases IF3 - IF1 interacts and blocks tRNA from binding - IF2 helps to guide the initiator tRNA (fMET) to bind to the mRNA - after this occurs, the 50S subunits come in and binds, forming an intact ribosome
When would be a poor time to use sterilization to control microbial growth? - on metal dental tools - when inoculating a bacterial culture - preparing apple slices for lunch - cleaning your toilet - the surface of your skin before surgery
- preparing apple slices for lunch - when inoculating a bacterial culture - the surface of your skin before surgery
Transformation
- naked DNA is taken up from the environment - shown in Griffiths' experiment - natural competence - they actively bind to environmental DNA, transport it across their cell envelopes into their cytoplasm, and make it single stranded. - DNA is typically derived from lysed cells that release their contents into the environment - ssDNA can recombine into the bacterial genome -> new phenotypes! - plasmid uptake - plasmids can also be taken up by competent bacteria - relatively inefficient in nature because not a lot of free DNA is present in the environment - transformation involves making dsDNA ssDNA. this occurs as the DNA is transported across the bacterial membrane and cell wall, the ssDNA is then bound by a protein called DprA which loads DNA onto RecA, which mediates homologous recombination into the chromosome via DNA repair pathway
Gene regulation
- not all genes in the genome are expressed at the same time - gene expression is a highly regulated process - in bacteria, gene expression is regulated at the level of transcriptoin - bacteria don't want to waste energy making proteins they don't need at the moment, therefore, most bacterial gene regulation is dependent upon the environment and what the bacteria needs to thrive under those specific conditions
Nucleic Acids and Phosphodiester Bonds
- nucleotides polymerize to form strands of nucleic acid, called the phosphodiester backbone - phosphodiester bonds link phosphate to ribose 3' carbon - nucleic acids are directional, 5'C at one end, 3'C at the other end, generally written 5'->3'
Deoxyribonucleotides
- nucleotides that compose DNA - three components of a deoxyribonucleotide are: a five-carbon sugar called deoxyribose, a phosphate group, and a nitrogenous base - 4 nitrogenous bases: Cytosine, Thymine, Adenine, Guanine
Ribonucleotide
- nucleotides that compose RNA - three components of a ribonucleotide are: a five-carbon sugar called ribose, a phosphate group, and a nitrogenous base - 4 nitrogenous bases: Cytosine, Uracil, Adenine, Guanine
Plasmid or Vector
- origin of replicatoin - antibiotic resistance - allows for only elimination of cells not carrying the plasmid - MCS - multiple cloning site - series of restriction sites engineered very close together so that you can place your DNA of interest there - promoters - these can be constitutive, indictable, or even repressive - transgenes - genes, ususally, that you want to express
Filtration
- physical separation of microbes from air or liquid - uses filters with pores of specific sizes - ideal when liquids contain heat sensitive components - high efficiency particulate air (HEPA) filters have pores ~0.3um - filter out bacteria, endospores, and many viruses - efficiency of HEPA filters 99.97% for particles of 0.3um diameter or more Membrane filters - porous membranes with defined pore sizes, microbes removed by physical screening
Ways to control microbial growth
- physical: heat, radiation - chemical: gas, liquid - mechanical removal: filtration - biological: virus, toxin
The Operon
- proteins with related functions are encoded together in the genome under a single promoter - allows simple regulation of entire pathways - repressive, indictable, and constitutive operons - eukaryotes do not have operons - each operon also includes a regulatory region that can influence its expression, this can include transcription factors - repressors are transcription factors that suppress/prevent transcription of genes by binding to the DNA at the operator site thus preventing RNA Polymerase from binding - activators are transcription factors that promote transcription and help RNA Polymerase bind to the promoter - there are also inducers - a different type of regulatory molecule that can activate or repress transcription by binding to the repressor or activator - of course, there are some genes that are always needed such as those involved w/ DNA replication or core metabolism, these are always expressed and referred to as constituitive
Lyophilization
- rapid freezing under vacuum so that water is lost by sublimation. combines both exposure to cold temperatures and desiccation - inhibits metabolism
Repair of Thymine Dimers
- recall that UV light can induce thymine dimers, which can interrupt DNA replication or transcription either stalling it or resulting in a frameshift mutation - this is a very common error because most organisms are exposed to UV light on a regular basis - there are 2 types of repair:
Molecular Cloning Pieces: Restriction Enzyme
- recognizes a specific DNA sequence and cleaves the DNA
Translation Termination
- release factor recognizes stop codon, translational complex dissociates and completed polypeptide is released
Frameshift mutation
- results from insertion or deletion of 1 or 2 base pairs in the coding region of the gene - can change every amino acid after the point of the mutation - the new reading frame may also include a stop codon before the end of the coding sequence - resulting proteins nearly always nonfunctional
Missense mutation
- results in a different amino acid being incorporated into the resulting polypeptide - the effect depends on how chemically different the new amino acid is from the wild-type amino acid and location
Rolling Circle Replication
- results in the synthesis of a single new copy of the circular DNA - begins with the enzymatic nicking of one strand of the double-stranded circular molecule at the double-stranded origin (dso) site. - DNA Polymerase III binds to the 3'-OH group of the nicked strand and begins to unidirectionally replicate the DNA using the un-nicked strand as a template, displacing the nicked strand as it does so - completion of DNA replication at the site of the original nick results in full displacement of the nicked strand, which may then recircularize into a single-stranded DNA molecule - RNA primate then synthesizes a primer to initiate DNA replication at the single-stranded origin (sso) site of the ssDNA molecule, resulting in a dsDNA molecule identical to the other circular DNA molecule
Genes
- segments of DNA molecules - individual genes contain the instructional code necessary for synthesizing various proteins, enzymes, or stable RNA molecules
Avery- MacLeod-McCarty experiment (1944)
- tested Streptococcus pneumonia, Strain R is non-virulent, Strain S is virulent - treated heat killed cells with enzymes to break down DNA, RNA, or protein, and then test transformation - starting with heat killed S, no enzyme for control, add R, S cells still present, transformation occurred - starting with heat killed S, protease (protein degrading enzyme), add R, S cells still present, transformation occurred - starting with heat killed S, RNAse (RNA degrading enzyme), add R, S cells still present, transformation occurred - starting with heat killed S, DNAse (DNA degrading enzyme), add R, S cells not present, transformation did not occur - RESULT: S cells were only not present when DNA was degraded, showing that transformation does not occur without DNA
Genetic code
- the "language" of the cell composed of 20 amino acids - the relationship between the mRNA codon and the amino acid is the genetic code - genetic code is basically universal - DNA makes mRNA - mRNA makes protein using the genetic code
Delivery
- the act or manner of giving or sending forth - in our case this will often be how to deliver the DNA - there are a wide variety of tools that can be used to damage a cell just enough to let DNA in but not kill the cells/organism
Specialized transduction
- the bacteriophages pick up only specific portions of the host's DNA - when lysogenic phages are excised from the bacterial chromosome, pieces of the bacterial chromosome on each side of the phage integration site may also be acquired
Thymine Dimer Repair - Direct repair (light repair)
- the enzyme photolyase binds to the thymine dimer and in the presence of visible light, breaks apart the dimer, restoring the base pairing of the thymines with complementary adenines on the opposite DNA strand
Central Dogma
- the flow of information from DNA to RNA to Protein - DNA transcribed to make mRNA, mRNA is the template for protein translation which requires rRNA and tRNA and the ribosome to make the protein
Human insulin made by bacteria
- the insulin gene was isolated from the DNA of human cells - the gene was introduced into a bacterial plasmid - this recombinant plasmid was introduced into bacteria - these transformed bacteria now secrete human insulin
Eukaryotic DNA Replication
- the leading strand is continuously synthesized by the eukaryotic polymerase enzyme pol δ, while the lagging strand is synthesized by pol ε. - RNase H, instead of a DNA polymerase I, removes the RNA primer which is replaced with DNA nucleotides - when the replication fork reaches the end of the linear chromosome, there is no place to make a primer for the DNA fragment to be copied at the end of the chromosome. these ends, also known as telomeres (noncoding repetitive sequences), thus remain unpaired and, over time, they may get progressively shorter as cells continue to divide - telomerase contains a catalytic part and a built-in RNA template. it attaches to the end of the chromosome, and complementary bases to the RNA template are added on the 3' end of the DNA strand. Once the 3' end of the lagging strand template is sufficiently elongated, DNA polymerase can add the nucleotides complementary to the ends of the chromosomes. In this way, the ends of the chromosomes are replicat
Gene Expression
- the processes of transcription and translation collectively - synthesis of a specific protein with a sequence of amino acids that is encoded in the gene
Lagging strand
- the strand being built by DNA Polymerase III that goes away from the fork - as DNA unwinds, it has to start again from the unwind
Leading strand
- the strand being built by DNA Polymerase III that goes towards the inside of the fork - as it unwinds it keeps going
Mechanisms of Horizontal Gene Transfer
- transformation - transduction - conjugation
Transformation in the lab
- transformation is the process of foreign DNA into cells - some bacteria are naturally competent, some are not - bacteria can be made competent in the lab by becoming chemically competent or electrocompetent - conjugation can be used to introduce foreign DNA (F-plasmid mediated) - bacteriophage can be used to introduce foreign DNA (phagemid mediated)
Which mechanism of diversity is NOT considered horizontal gene transfer? - transduction - transposition - conjugation - transformation
- transposition
Efficacy of Microbial Killing
- treatments do not kill all microorganisms instantly, killing is a somewhat random process. microbial populations usually die exponentially, which can be plotted on a microbial death curve, giving a straight line on a semi-log plot - efficacy of a killing agent is measured by the D-value, decimal reduction time
Match the antimicrobial agent to its mode of action.
- trimethoprim - acts as an antimetabolite - penicillin - inhibits cell wall synthesis - tetracycline - inhibits the 30S ribosomal subunit - fluoroquinolone - inhibits nucleic acid synthesis - chloramphenicol - inhibits the 50S ribosomal subunit - artemisinin - produces reactive oxygen species - atovaquone - competes with coenzyme Q for electrons - imidazole - inhibits ergosterol synthesis - pentamidine - affects DNA and RNA, and, therefore, protein production - quinoline - interferes with heme detoxification - bacitracin - inhibits cell wall synthesis - diarylquinoline - inhibits ATP synthase - isonicotinic acid hydrazide - acts as an antimetabolite - polymyxin - disrupts cell membranes - rifampin - inhibits nucleic acid synthesis
Autoclave
- typical settings: 121C for 15min at 15psi - uses heat + pressure - denatures proteins and alters membranes - rely on moist-heat sterilization and raise temps above the boiling point of water, considered the most effective method of sterilization
Nonionizing radiation
- ultraviolet light, can induce dimer formation between two adjacent pyrimidine bases, commonly two neighboring thymines, which become covalently linked. cause stall in DNA replication and transcription - DNA polymerase may proceed and replicate the dimer incorrectly, potentially leading to frameshift or point mutations
Hershey and Chase DNA Phage experiment (1952)
- used E. coli and phage T2; phage only have protein and DNA. - labeled DNA with 32P, labeled protein with 35S - allowed the phage to infect the bacteria, then separated the phage and bacteria - bacteria infected with phage containing 32P-labeled DNA produced 32P-labeled phage. Bacteria infected with 35S-labeled phage produced unlabeled phage. - RESULT: DNA must be the storage molecule
PCR (Polymerase Chain Reaction)
- used to make many copies of DNA sequences in vitro - start w/ a DNA template you want to copy - heat is used in place of helicases to separate DNA strands - customized DNA primers have sequences that allow them to bind to specific sites on the template - a thermostable DNA polymerase starts at the primer and synthesizes a copy of the template strand - this is repeated in cycles - product strands from one cycle serve as templates in the next cycle - in each cycle, the reaction steps through different temperatures that allow individual steps to happen - denaturation - high temperature separates template DNA strands - annealing - lower temperature allows primers to bind template DNA - extension - optimal temperature for polymerase to synthesize new strand - this process is a chain reaction bc the product of each round serves as a template for the next
Genetic Variability: Combo of 2 organisms
- vertical gene transfer, genetic diversity - when we think of genetic transfer, most of us think of vertical gene transfer - the transmission of genetic info
Genome Sizes Vary
- viral genomes are typically smaller than most bacterial genomes, encoding only a few genes, because they rely on their hosts to carry out many of the functions required for their replication - plants and animal genomes are much larger
Both vancomycin and penicillin target the bacterial cell wall and have the same mode of action. - True - False
False
An antibiotic that kills only Gram-negative bacteria can be classified as:
Narrow spectrum
Ampicillin is a semisynthetic antimicrobial agent. - True - False
True
Antimicrobial agents may be the metabolites naturally made by microorganisms or synthetically made. - True - False
True
If antibiotic treatment is needed for immunocompromised individuals who develop infections, the medications should be bactericidal rather than bacteriostatic. -True -False
True
Plasmids can have naturally occurring restriction enzyme cutting sites. - True - False
True
Surfactants
lowers surface tension of water to help w/ washing away of microbes, and disruption of cell membranes
DNA Polymerase III
main enzyme that adds nucleotides in the 5'-3' direction
Other omics
metabolomics, glycomics, lipidomics
Genomics
study and comparison of entire genomes
Primase
synthesizes RNA primers needed to start replication
Conservative Model for DNA Replication
the 2 parental strands of DNA remain together, a completely new molecule of DNA is synthesized
