Class 4: Microbiology
bacterial - reproduction
stationary phase = carrying capacity = maximum population an environment can sustain binary fission = big increase in population size - no change in genetic phase (vs Conjugation = big increase in genetic diversity but NO CHANGE IN POPULATION SIZE)
auxotrophs "-"
- means they cannot do something ex: arg (-): cannot synthesize (make chemically or produce) on its own, needs it to survive; results from a mutation in a gene coding for an enzyme in a synthetic pathway (agr+ = they can synthesize it themselves) aa- means that they need that amino acid to survive
retroviruses (+ssRNA) aka +RNA lysogenic virus
- must encode RT (RNA dep DNA POL) but doesn't have to carry it due to the 3 genes produced - will undergo lysogeny - integrate into host genome as provirus - must also encode RdRp because host does not have enzymes to replicate RNA genome (this will make -ssRNA and RdRp can be used to then make +ssRNA) 3 main retroviral genes: 1. gag (codes for viral capsid proteins( 2. pol (polymerase, codes for RT) 3. env (envelope codes for viral envelope proteins) Issues with this virus 1. they never have to excise themselves from the host genome 2. RNA POL have no editing capability so mutations are likely
dsDNA virus
- often encode enzymes required for dNTP synthesis and DNA replication - these viruses often have large genomes that include genes for enzymes in deoxyribonucleotide synthesis (make DNA) and DNA replication - can use hosts enzymes
Artificial chromosomes
- plasmids can only carry inserts up to a certain size - if large inserts are required, artificial chromosomes can be used - bacterial artificial chromosomes (BACs) carry inserts of 100 - 350 kilobase pairs - yeast artificial chromosomes (YACs) carry inserts between 100- 3500 kbs - BACs can easily carry up to 350,000 base pairs of DNA and YACs can contain up to 3 million base pair inserts
Eukaryotic plasmids
- require many of the same components as bacterial plasmids - eukaryotes use different selection agents - usually puromycin or neomycin - they also require different promoters in expression plasmids, as well as a pol-adenylation signal downstream of the inserted gene, to terminate transcription - eukaryotic plasmids can be introduced into mammalian host cells via transfection (similar to transformation) - cells can be chemically transfected using calcium phosphate precipitates, or plasmid packaging in liposomes - these lipid vesicles mask the plasmid, but deliver it to the interior of the cell by fusing with the plasma membrane - non-chemical transfections = electroporation, optical transfection with lasers, or shooting the DNA coupled to a gold nanoparticle into a cell nucleus using a gene gun - viruses can also deliver DNA into eukaryotic cells via viral transduction. Transduced cells can express genes carried by the viral vector
Gel electrophoresis
- separating by size or by charge - wells are (-) end and opp end is (+) - Decreasing the percent of the polyacrylamide (and the density) will increase your resolution capacity, and proteins with similar molecular weights will be separated by a greater distance - The proteins are negatively charged because during preparation they are denatured (and thus travel through the gel in a denatured state) - fragments of DNA (or RNA) in an electrophoresed gel can be transferred to a more solid and stable membrane in blotting techniques
viroids
- short piece of circular +ssRNA (200-400 bases) with extensive self-complimentarity (can base pair with itself to create double stranded regions) - DO NOT CODE FOR PROTEINS - lack capsids - replicate independently - cause disease - some are catalytic ribozymes (=an RNA molecule capable of acting as an enzyme.) - some, when replicated produce siRNAs that can silence normal gene expression (=small-interfering RNAs, mess with mRNA) - use coinfected virus'es RDRP to make (-)ssRNA - must be coinfected (must be inside capsid of another virus to infect cell of another virus) - act as miRNAs or SiRNAs to block translation RNA POL creates -ssRNA strand from +ssRNA circular strand
facultative anaerobes
Can use oxygen when it is present, but are able to continue growth by using fermentation or anaerobic respiration when oxygen is not available - can use ETC
obligate anaerobes
carry out fermentation or anaerobic respiration and cannot survive in the presence of O2 ex: A researcher isolates a microbe from the human gut. She determines that it has a thick cell wall, grows only in the absence of oxygen, and requires supplemental arginine. bacterial flora of the gut = Mesophilic obligate anaerobes - soil bacteria
Ultimate energy source and where carbon chains come from
photo = sun chemo = ATP auto = make own carbon from CO2 hetero = eat other critters photoautotrophs = plants, photosynthetic bacteria chemoheterotrophs = animals photohetrotrophs = carnivorus plants chemoautotrophs = deep sea vent bacteria, cave bacteria
Cells that undergo the productive cycle dont have a cell wall but rather a
plasma membrane animal cells budding helps acquire a coating of lipid bilayer = envelope = animal viruses *animal cells*
Flagella
monotrichous amphitrichous peritrichous
virus
obligate intracellular parasite protein (capsid) + nucleic acid bacteria virus = bacteriophage *can't have both RNA and DNA
blotting
transfer of DNA or proteins from an electrophoresis gel to a nitrocellulose of PVDF membrane
phagocytosis and pinocytosis
two types of endocytosis - both involve non-specific uptake - phago = large-scale uptake - pino = small-scale uptake
+ssRNA lysogenic virus
= +ssRNA must first get DNA genome = creates ssDNA via RT (=RdDp) - now host enzymes will be used to make this double stranded =dsDNA *dsDNA will get inserted into host cell genome dsDNA fates: - transcribe and translate using host ribosomes to make viral proteins - transcribe only with host RNA polymerase to make (+)ssRNA; this will ne packaged in new viral progeny *theoretically, +ssRNA lysogenic virus does not have to carry RT with it, because it could be immediately translated by host as is - viral protein transcribed by host may create RdDp, but carrying RT is better for virus Problems with this virus 1. permanently in genome 2. very rapid mutation via - RdDp (+ssRNA to ssDNA) - transcribing dsDNA into +ssRNA via host RNA POL
enveloped viruses
= a lipid bilayer, acquired by budding through the host cell membrane can only grow in animal cells (if the viral host is eukaryotic, and since the virus has an RNA genome, it is likely that the genome has a poly-A tail to mimic eukaryotic RNA and facilitate translation.
bacteria shape: circular
= blank - coccus; Staph
bacteria shape: rod shaped
= blank bacillus; E. Coli
subviral particles
= infections agents smaller and simpler than viruses ex: prions and virioids
opportunistic bacterial infection
= infections occurring due to bacteria, fungi, viruses, or parasites that normally do not cause a disease, but become pathogenic when the body's defense system is impaired ex: a Staphlycoccal infection in a wound. Staphylococcus is a normal part of the skin's flora and is helpful in combating other pathogenic organisms unless it ends up on damaged or exposed tissue, such as an open wound, where it can then cause an opportunistic infection. Streptococcus is not part of the throat's flora nor H. pylori in the gut. Food poisoning is just that - the exotoxins produce the symptoms rather than an actual infection.
Conjugation (gram + or gram - do this?)
= involves transfer of DNA (F- plasmid) via sex pilus and requires cell to cell contact - conjugation is a feature of Gram-negative bacteria. = means of increasing genetic diversity in a bacterial population by exchanging DNA (plasmid, F+ or genomic Hfr) via conjugation bridge
High Frequency Conjugation
= plasmid incorporated into genome; some of the genome will be sent over conjugation bridge to other bacteria - even though F-factor is an extrachromosomal element it can become integrated into bacterial chromosomes via recombination = Hfr
transduction (think lysogenic cycle)
= removal of piece of host genome during excision - this can get transferred to next host - means of acquiring genetic diversity for bacteria
bacteria shape: spiral
= spirella
exotoxins
= toxic substances secreted by both gram (+) and gram (-) - help bacteria compete with other bacteria (i.e mammalian gut) ex: botulism
Transduction
= transfer of DNA from one bacterium into another via bacteriophage (lysogenic) - bacteriophage accidentally take a piece of genome to the next bacteriophage - viruses can deliver DNA into eukaryotic cells this way (transduced cells express the genes carried by the viral vector) **requires the presence of a lysogenic phage** A bacteriophage is a type of virus that infects bacteria. In fact, the word "bacteriophage" literally means "bacteria eater," because bacteriophages destroy their host cells. All bacteriophages are composed of a nucleic acid molecule that is surrounded by a protein structure *relate to lysogenic cycle*
Prions can
Mutant bad prions ca: 1. arise spontaneously due to mutation 2. be heritable 3. be transmitted through consumption of infected tissues
A benefit of the lysogenic viral cycle
- During the lysogenic viral cycle, the viral genome integrates into the host genome but remains mostly silent - With every round of host cell division, the viral genome is replicated . ** Bacterial lysis is a characteristic of the lytic cycle, not the lysogenic cycle
lac + vs. lac -
+ = bacteria capable of surviving on minimal medium that has disaccharide lactose as the only carbon source (no glucose) - = incapable of growing with lactose as its only carbon source (mutation in lactase enzyme)
-ssRNA
- (-)ssRNA = a template for mRNA - must carry and encode RDRP, will be used right away to make (+)ssRNA - host does not have this enzyme
bacterial conjugation
- Female (or F-) bacteria can undergo conjugation with both male (F+) and Hfr strains - Hfr strains can only mate with female bacteria, not other Hfr strains - Conjugation increases the number of F+ cells. However, when bacteria divide by binary fission, plasmid distribution in the two daughter cells is relatively unregulated. This can result in a male parental cell giving both male and female daughter cells - The conjugation bridge (or sex pilus) is formed by the F+ (or Hfr) cell. During conjugation, nucleic acids (either a plasmid or a chromosome) are transferred to the female cell across this bridge
productive cycle
- ONLY ANIMAL CELLS can go through the productive cycle because viruses cannot bud out of a cell with a cell wall (bacteria have cell walls) - the host genome typically stays intact, viral particles bud out of the host cell - Viral genome integration into the host genome DOES NOT OCCUR ONLY HAPPENS IN ANIMAL CELLS - no lysis of host cell - instead uses budding (= virus cell stimulates a vesicle to form from the host cell membrane) - the vesicle the virus buds from will have host envelope an viral proteins embedded into it (camoflauged from host immune system) - envelope will be able to fuse with next hosts cell membrane
Reverse transcriptase (RT)
- RNA-dependant DNA polymerase. - Unlike DNA-dependant DNA polymerase, which is used in eukaryotic cellular DNA replication, RT does not have any proofreading mechanism. Thus, many errors occur during the creation of DNA from the viral RNA - gives rise to drug resistance in HIV patients
Exocytosis
- the release of material from the cell using a vesicle
provirus state of HIV
- There is no viral genome transcription or translation; only passive replication of the genome occurs - When the host cell replicates its genome during S phase, the viral genome is passively replicated along with it. - Each of the resulting daughter cells thus receives a copy of viral DNA; now instead of just one infected cell, there are two ( - The virus does not replicate independently of the host, and there is no viral transcription or translation during this stage - If this were to occur, there would be viral proteins present in the host cell's cytoplasm; this could jeopardize the viability of the host cell or alert the immune system to viral infection.
western blot
- allows you to detect presence of certain proteins within a sample and also serves as a diagnostic tool ex: whether cancer cells express certain tumor-promoting growth receptors on their surface - Antibodies are used to identify proteins - Proteins are separated via gel electrophoresis, transferred to a membrane, and then probed with a primary antibody
prions
- do not follow central dogma because they are self-replicating proteins - a misfolded version of a protein that already exists - they are naturally occurring in mammals; axons of neurons - resistant to chemicals or heat - can be genetically linked (through mutations in genes that code for prions) - long incubation periods (years to decades) Characteristics: - no DNA or RNA - no membranes - no organelles - very small - extremely stable (forms beta-sheets) 2 Types: 1. normal prions (good) 2. bad/mutant prions (bad prions can induce normal prions to misfold) **normal and mutant prions have the exact same primary structure, what differs is their folding in the secondary and tertiary structures
naked viruses
- do not have envelopes because they escape host via lytic mechanism - all phages and plant viruses
+ssRNA
- must encode RDRP but do not have to carry it - must encode RT (RdDp) to create viral DNA genome
obligate aerobes
- require oxygen to live - have ETC
Immunohistochemistry
- similar to ISH but is specific for proteins instead of nucleic acids - protein expression in a tissue
Transfection
- similar to transformation, but for eukaryotic cells (not bacteria) - eukaryotic plasmids are introduced into a mammalian host via transfection
Horizontal gene transfer
- transformation - transduction - conjugation - high frequency conjugation
Restriction endonucleases
- used in Recombinant DNA technology - are bacterial enzymes that recognize specific sequences of DNA and cut the double-stranded molecule in two pieces - a nuclease is an enzyme that cuts nucleic acids - an endo-nuclease cuts in the middle of a DNA chain
Receptor-mediated endocytosis
- uses clathrin-coated pits on the cell surface as the basis for endosomes in which to package material that binds to the receptor and bring it into the cell - specific uptake
How HIV generates new viral genome copies from HIV provirus
DDRP HIV provirus genome = DNA HIV viral = +ssRNA - The HIV provirus refers to the HIV genome that has been reverse transcribed into DNA and inserted into the host genome (also DNA). - Thus, the enzyme that generates new copies of the viral genome must be DNA-dependent (i.e., read DNA). - Note that this DNA-dependant RNA polymerase is not a virally encoded enzyme, but rather a normal host enzyme used to transcribe its own genome. - HIV manipulates this host enzyme into transcribing the integrated provirus. This creates new HIV RNA genomes that will eventually be encapsulated by the viral protein capsid.
Other Methods of Studying the Genome
Exome and Targeting Sequencing - sequencing only the exons of the genome Karyotyping - order all chromosomes 1 - 22 plus sex chromosomes for a genome wide view of genetic information - stain chromosomes to highlight structural features - major genetic changes (millions of bps), aneuploidy (abnormal number of chromosomes), and some insertions, deletions, or translocations can be revealed Fluorescence in situ Hybridiztion (FISH) - uses fluorescently labeled probes to locate the positions of specific DNA sequences on chromosomes - detects presence/absence of specific DNA sequences on chromosomes (translocations)
During a Gram staining procedure, a bacterial culture does not appear as either Gram-positive or Gram-negative. What structural attribute could make bacteria appear Gram-indeterminate? A. Presence of a thick cell wall B. Presence of an outer lipid bilayer C. Presence of an inner lipid bilayer D. Presence of a cell wall with a waxy composition
The presence of a cell wall with a waxy composition could make bacteria appear Gram-indeterminate . Gram-negative bacteria have both inner and outer lipid bilayers; it is in fact the outer lipid bilayer that stains the classic pink-to-red color that indicates Gram-negativity. Gram-positive bacteria have a thick well wall which stains purple as part of the process. A change in cell wall composition, particularly to something more waxy, would interfere with the staining process, making the results indeterminate. Mycobacterium are an example of this and alternative acid fast staining techniques are used with them.
A pregnancy test is an ELISA set up to detect human chorionic gonadotropin (hCG), a hormone specific to pregnancy. How would this ELISA be configured? A. A primary antibody specific for hCG, blood or urine sample, a secondary antibody specific for hCG B. Blood or urine sample, a primary antibody specific for hCG, a secondary antibody specific for the primary antibody's constant region C. Blood or urine sample, a primary antibody specific for hCG, a secondary antibody specific for the primary antibody's antigen binding site D. A primary antibody specific for hCG, blood or urine sample, a secondary antibody specific for the primary antibody
a primary antibody specific for hCG, blood or urine sample, a secondary antibody specific for hCG. ELISA tests utilize the specificity of antibodies to identify antigen or antibody in a patient's blood or urine sample. hCG in this case is acting as the antigen; anti-hCG antibodies are fixed to a microtiter well plate, and the blood or urine sample is applied. If hCG is present, it will bind to the primary antibody. A wash removes any unbound substances. The secondary antibody is specific to a different region on hCG and is covalently bound to an enzyme that produces a color change; the secondary antibody will bind to hCG and application of the substrate will produce the color change.
Tolerant anaerobes
can grow in the presence or absence of oxygen but do not use it in their metabolism - no ETC aka aertolerant anaerobes
Which of the following viruses should be able to reproduce successfully if they carry RNA-dependent RNA polymerase into their host cell? I.(+) RNA virus II.(-) RNA virus III.ds DNA virus
- Item I is true: (+) RNA viruses must at least code for RNA-dependent RNA polymerase in their genome. However, if they are carrying the enzyme, they should be able to use it to make their template strand of RNA (the (-) strand) for later genome replication. - Item II is true: (-) RNA viruses must always carry at least one copy of RNA-dependent RNA polymerase in order to make strands of (+) mRNA that can be used for translation into protein. - Item III is true: while a ds DNA virus would not require RNA-dependent RNA polymerase to make mRNA or replicate its genome, it could still reproduce if the enzyme was present. It would simply ignore the enzyme.
A strain of bacteria is isolated which consumes oxygen during metabolism and utilizes carbon dioxide as its source of carbon. Which of the following descriptions could characterize this strain of bacteria? I. Obligate aerobe II. Autotroph III. Facultative anaerobe
- Item I is true: this strain of bacteria uses oxygen during metabolism so it could be an obligate aerobe - Item II is true: since the bacteria use carbon dioxide as their source of carbon, the strain is an autotroph ( - Item III is true: the bacteria could be a facultative anaerobe, since these organisms can (and will) use oxygen during metabolism when it is available Note that without knowing more about what this strain of bacteria does in the absence of oxygen, we cannot distinguish between obligate aerobe and facultative anaerobe, however the question only asks about what the bacteria could be.
northern blot
- RNA is separated via gel electrophoresis - Once separated, transferred to a nitrocellulose membrane and detected via radiolabeled nucleic acid probe - RNA is probed with either DNA or RNA - is used to probe RNA sequences - enables you to determine whether specific gene products (normal or pathologic) are being expressed (if their mRNA is present in a cell, they are probably being translated to protein) - Retroviruses are a type of (+)sense single-stranded RNA viruses, viroids are sequences of RNA, and transcripts are composed of RNA (bacterial genomes are composed of DNA; Southern blotting would be a better choice)
Analyzing gene expression
- RT-PCR and qPCR give information on which genes are being transcribed in a given cell population (use RT to turn RNA into cDNA; cDNA will then undergo PCR to determine gene expression) - western blot = directly test protein expression, and is limited to only the amount of starting lysate and the availability of antibodies specific for the protein being studied
DNA fingerprinting
- Short tandem repeat analysis for DNA fingerprinting - .is a method by which scientists can distinguish different DNA samples, but does NOT generate a large amount of DNA (police departments use) - since the DNA of any 2 people if 99% identical, DNA fingerprinting exploits stretches of repetitive and highly variable DNA = polymorphisms 2 methods: - restriction fragment length polymorphism (RFLP) - short tandem repeat (STR) analysis
If a Hfr cell mates with an F- bacterium, the female bacterium could end up female (F-), male (F+), or Hfr
- The Hfr strain has the F factor in the genome, male (F+) bacteria contain the F factor as a plasmid, while the female bacteria (F-) has no F factor. - Both male (F+) and Hfr strains can mate with female strains - The Hfr cell keeps a copy of its genome and passes a copy to the female cell, through the conjugation bridge (or sex pilus). The female cell can receive a portion of the Hfr chromosome, or a copy of the whole thing (if the cells stay connected for a longer time). - The F factor is the final gene transferred during conjugation; if the F factor doesn't make it over to the female cell, she will stay female (but may acquire new genetic traits). If the F factor is transferred, it can end up in a plasmid (the cell would then be male, F+) or the genome (the cell would then be Hfr).
DNA Sequencing and Genomics
- a method to determine gene sequences - precursor for developing primers in PCR reactions Sanger Method: - the ringed ribose of a dNTP has a nitrogenous base at 1'C, a hydrogen at 2'C (OH occupies this site in RNA), a OH at the 3'C, and a string of 3 phosphates at the 5'C - the OH at the 3'C serves as the binding site for another dNTP (without this free 3'OH, dNTPs couldn't be linked together and DNA synthesis would not be possible) - method utilizes a modified dNTP that lacks the 3'C OH - these dideoxynucleotide triphosphates (ddNTPs) maintain their 5'C triphosphate moiety and can be incorporated into a normally growing DNA molecule - but since they are lacking the 3' OH, no further bases can be added to them - the ddNTPs terminate strand elongation at the point of their insertion - allows a DNA sequence to be read from the bottom up of a gel
Recombinant DNA
- a recombinant protein is one which has been obtained by transcribing and translating a novel combination of DNA (recombinant DNA) from different organisms ex: the gene for human insulin can be placed in a bacterial plasmid - bacteria with the plasmid will then produce insulin that can be used to treat diabetes
restriction fragment length polymorphism (RFLP)
- a type of DNA fingerprinting - uses restriction endonucleases to cut 10-100 bp stretches of polymorphic DNA into small fragments (these fragments vary in size and are unique to the individual) - the RFLPs are separated via gel electrophoresis - southern blotting techniques analyze the sample - membrane is probed with radiolabeled DNA oligonucleotides that base-pair with specific RFLP sequences - membrane is visualized - polymorphic DNA, even though recovered from the same chromosomal region, will yield unique band distribution for each person - when RFLPs are recovered from DNA sequences within genes, mutations can be detected ex: sickle cell disease is caused by a single base substitution in the beta chain of hemoglobin - the substituted valine at the 6th position (normally glutamic acid) will introduce a novel restriction site within the gene - when cut with restriction endonucleases, the point mutation generates a different sized RFLP (when compared to the normal gene cut with the same enzymes)
Short Tandem Repeats (STR)
- a type of DNA fingerprinting Step 1: - PCR amplifies 5-10 bp stretches of highly polymorphic and repetitive DNA located within non-coding regions (introns) of the genome - the STRs vary in sequence and number of repeats found at each locus - to profile an individual a sample of DNA is obtained and the polymorphic DNA is amplified with PCR Step 2: - thee amplified STRs are separated via gel electrophoresis and analyzed with southern blotting
Quantitative PCR (qPCR)
- aka real-time PCR - the PCR product is both detected and quantified as either an absolute number of copies of as a relative amount normalized to a control - amplified DNA is detected as the reaction progresses = real time - detection process can use a dye that is fluorescent and binds DNA or a fluorescent oligonucleotide probe that hybridizes to the sequence of interest - can be performed using DNA or cDNA templates - so it can therefore give information on the presence and abundance of a particular DNA sequence in samples (if DNA is template) or on gene expression (if cDNA is template)
ELISA (enzyme-linked immunosorbent assay) = color change
- antigen-antibody interactions to determine the presence of either - ELISA tests utilize the specificity of antibodies to identify antigen or antibody in a patient's blood or urine sample. ANTIGEN SAMPLE - when testing for the presence of a specific antigen in a sample, the wells are coated with an antibody specific for that antigen. Then the sample is added, and if the antigen is present it will bind to the antibodies. The wells are washed and a secondary antibody (specific for the antigen) is added; the secondary antibody is linked to a detection enzyme ANTIBODY SAMPLE - When testing for the presence of antibody in a patient's blood, the antigen for which that antibody is specific must be bound to the microtiter well plate. Neither secondary antibody (the one conjugated to an enzyme) nor the detection enzyme itself can ever be bound to the well plate or false-positive results would occur. ex: good technique for viral infections: - serum from a patient suspected to be infected with HIV is loaded into wells that are coated with HIV coat proteins - if the serum contains anti-HIV antibodies (indicating infection), the antibodies will adhere to the proteins (antigens present in the wells) on the wells, bind enzyme-linked antibodies, and effect a color change
exonucleases
- cut nucleotides from the ends of DNA chains (like DNA POL)
southern blot
- detect presence of specific sequences within a heterogeneous sample of DNA - also allows you to isolate and purify target sequences of DNA for further study Step 1: Separate DNA fragments on a gel Step 2: Transfer fragments to a nitrocellulose filter Step 3: Probe the filter for the target DNA sequence with hybridized probes
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
- detect relative expression of gene products - does not measure the actual expression or abundance of proteins, rather provides a gauge of gene transcription by measuring the relative amount of target mRNAs - first isolate all mRNAs within a cell population - convert to more stable cDNA via RT - library of cDNAs is subject to PCR, using primers (nucleotides) specific for a certain gene of interest - if the gene was actively transcribed at the time of harvest, its mRNA will have yielded a cDNA, which will be amplified by the PCR reaction and visualized on a gel
PCR
- detecting and amplifying specific DNA sequences, screening hereditary and infectious diseases, cloning genes, and fingerprinting DNA - The requirements for the polymerase chain reaction are similar to what is required in a cell for DNA replication: - DNA polymerase - template DNA - DNA primers (note that in euk. cellular replication RNA primer is needed) - dNTPs (free nucleotides) (note RNA polymerase is used during transcription, not replication, so it is not needed for this technique) Cycles through 3 temperatures: high temperature for template denaturing, low temperature for primer annealing and medium temperature for polymerization - The DNA polymerases used in PCR are derived from thermophilic bacteria so that they will survive the heat necessary to denature the sequences in the first place and then be active only in certain temperature ranges to control their activity. - the number of DNA molecules at the end of the PCR experiment is equal to 2^n, where n = the number of rounds of PCR
In situ hybridization (ISH)
- determine expression of a gene of interest in a tissue or in an embryo - permeabilized to open cell membrane - labeled probe binds transcript of interest - enzyme linked antibody binds probe - substrate for enzyme is added and complex is detected - it can be determined where transcripts are expressed on a multicellular level - DETERMINES LOCATION OF RNA TRANSCRIPTS
Embryonic Stem Cells
- found in the inner cell mass of the blastocyst and are the only stem cells in humans which are pluripotent - pluripotent cells are able to differentiate into any of the 3 germ layers and can generate all of the over 220 cell types in the human body - can replicate indefinitely
Adult Stem Cells
- found in various tissues and function in tissue repair and regeneration = multipotent = they can produce many cell ypes - usually tissue specific with more differentiation come from: - bone marrow (to regenerate blood cells) - adipose tissue (to regenerate fat tissue) - blood (hematopoietic stem cells that create all other blood cells)
Complementary DNA (cDNA)
- gives information on gene expression (because made from an RNA precursor) - is produced from fully spliced eukaryotic mRNA via reverse transcriptase obtained from retroviruses - cDNAs carry the complete coding sequences for genes but lack introns (so they are smaller than the genomic sequence of the gene) - once a cDNA is ligated into a bacterial plasmid and bacteria are transformed with this plasmid, they can produce the protein encoded by the cDNA
Endospore formation
- gram + cells form endospores under unfavorable conditions - these are tough and thick shells made of PG - inside the endospore is; genome, ribosomes, and RNA - all of which are required for the spore to become metabolically active when conditions become favorable (T > 100C) - germination = metabolic reactivation of an endospore - 1 spore per bacterial cell; so bacteria cannot increase their population through endospore formation (produced during stationary phase, spores are like hibernation)
Flow cytommetry
- looks at individual cells, as they are passed in a liquid stream through a laser (gives information which fluorescent molecules are bound to individual cells) - gives information on cell size and how many cells in the sample express each of the markers that were labeled
Radioimmunoassays = radioactivity change
- similar to ELISA, but use radiolabled antigen and antibodies instead of enzyme-linked antibodies - the presence of target proteins or antibodies is assayed by measuring the amount of radioactivity instead of a color change - are a type of competition assay where the radiolabeled version is offset by the unlabeled version and the decrease in radioactivity indicates the level of the unknown being measured. This process is done with known concentrations of each to establish the standard curve to which results from unknowns can then be compared. ex: Radiolabeled antigen is mixed with antibody followed by antigen without the radiolabel and the decrease in radioactivity is measured as the amount of unlabeled antigen increases ex: a known amount of radiolabled antigen is mixed with a known amount of antibody and the total amount of radioactivity is measured - then unlabeled antigen is added in increasing amounts; the unlabeled antigen displaces the radiolabeled antigen so that less radioactivity is measured - this data formulates a standard curve, and the steps are repeated using the patient's serum instead of the unlabeled antigen - the radioactivity is measured and compared to the standard curve to determine the amount of antigen
Plasmids
- small circular ds-DNA molecules found in bacteria - capable of autonomous replication = replication that is independent of chromosome replication - used alongside recombinant techniques to propagate and express foreign genes in bacteria - contain multiple cloning site, which has restriction sites for dozens of restriction enzymes - this means that the plasmid can be digested and any desired sequence with complementary ends can be ligated into the plasmid - plasmids also have a drug resistance gene, which helps select and isolate bacteria possessing the plasmid from other bacteria Bacterial expression plasmids need: - a prokaryotic promotor and start site; this allows expression of an inserted gene in the bacterial host Bacterial Transformation: - once inside the bacteria, the plasmid will be exposed to the host's replication machinery, which replicates the plasmid
MicroARRAys = (RNAaa)
- study relative RNA amounts between 2 samples or compare RNA levels in one sample to a normal reference - are a way to determine transcript levels that are being expressed by a population of cells. - RNA is harvested from the cells, fluorescently labeled and applied to a chip. - chip contains binding sites for every known gene, which act as probes to determine transcript levels in samples - Microarrays are not used to determine directly which proteins are expressed by a cell. - microarray data is quantitative and actual folding changes can be calculated based on color and intensity
Primers for PCR
- suitable primers have a high GC content and have G or C base pairs at the 5' and 3' ends
lytic cycle
- the host genome is commonly degraded to generate free dNTP building block - Viral genome integration into the host genome DOES NOT OCCUR - The virus particles are released when the host cell bursts in the lytic cycle STEPS: 1. transcribe and translated viral genome early genes: hydrolase (destroys host cell genome), capsid proteins - digests host genome (prok) this stops the host cell from taking resources from virus - nucleotides from host genome can be used for the replication of viral genome 2. replicate viral genome - produce viral progeny - some use host enzymes, some encode their own 3. lysis of host and release of new viral particles late genes: lysozyme (cleaves host cell membrane)
uracil auxotrophs
- they must be supplied with uracil as they cannot synthesize their own
lysogenic cycle
- think dormant stage 1. integrate viral genome with host genome - main differences from lytic cycle is that virus i in latent phase where viral genome is in stasis - the viral genome can integrate itself into the host genome or it can just float their on its own - binding proteins prevent replication of viral genome*** - viral genome is silent due to viral-encoded repressor proteins 2. normal host activity, including reproduction - viral genome is activated upon host cell stress 3. excision and lytic cycle - must excise viral genome out of host genome before it can copy it or else too large - some of the host genome can also be excised and packaged into viral particles. This new bit of DNA can be transferred to the next host on subsequent infection **The viral genome is activated and excised when the host cell is stressed, and will enter the lytic or productive cycle. If the virus waits until the host cell is dead, it will certainly not be able to reproduce ***the phage is silent and genes are not expressed and viral progeny are not produced = dormancy = is due to transcription of phage genes being blocked by a phage encoded repressor protein that binds to specific DNA elements in phage promotors (operators) - think like lac operon how when repressor is bound to operator no lactose genes are transcribed
Short tandem repeat analysis for DNA fingerprinting
- utilizes patterns of repetitive DNA within the intronic part of the genome to identify individuals - Short tandem repeat analysis looks at polymorphic, repetitive sequences within introns to identify a person.
Gram + bacteria
-envelope absorbs crystal violet stain and appears deep purple -have thick layer of peptidoglycan (made of AA and sugar) -contain lipoteichoic acid (activates human's immune system so it provides protection from host's immune system)
Gram - bacteria
-envelope absorbs safranin and appears pink-red - due to the presence of an outer membrane and a thinner peptidoglycan wall. Endotoxins are commonly found in the outer membrane of Gram-negative bacteria, and the cell wall is found between the two membranes -contain smaller amount of peptidoglycan - have outer and inner membrane - periplasmic space - gives rise to antibiotic resistance -contains lipopolysaccharides (triggers immune system response in humans)
Advantages of productive cycle
1. virus is camouflaged from host organism's immune system 2. exit from host gains envelope, and entry into host is fusion a. budding - virus simply activates vesicle formation pathway that already existed in cell b. fusion - productive cycle allows virus to make more virus - could be able to reinfect that same host again - envelope provides some immune protection AND makes for easier infection of next host (animal viruses are taken up with their capsid and uncoated inside the host cell)
A single plasmid is isolated from bacterial cells. When the plasmid is digested with a restriction enzyme and then subjected to gel electrophoresis, two bands are observed on the gel. How many restriction sites for this enzyme are present in this plasmid?
2 - Plasmids are circular DNA, so if the plasmid is cut in two places it will produce two fragments. - The general rule when cutting circular DNA with restriction enzymes is that the number of fragments you end up with is the same as the number of restriction sites.
Transformation
= uptake of short fragments of (naked, freely floating) DNA by naturally transformable bacteria (could be bacteria under stress) - can still do via plasmid ** is the process by which bacterial cells acquire genetic information from the external environment ** ex: A scientist takes 10 μL of culture containing E. coli cells and puts them in a tube with 4 fmol of a plasmid. The tube is placed on ice for half an hour, then resuspended while in a 42°C water bath for 10 seconds. The bacteria are plated on selective media and grown overnight at 37°C
endotoxins
= when bacteria (gram - ) die & their disintegrated outer membranes are released into circulation; cells of immune system release so many chemicals that patient goes into septic shock - can cause out immune systems to react and die
Which of the following could be found in a bacterial cytoplasm, but NOT in an animal cell cytoplasm? A. RNA polymerase B. Glycolytic enzymes C. mRNA D. Ribosomes
A - Both bacteria and animal cells will have glycolytic enzymes, mRNA, and ribosomes in their cytoplasms as this is where glycolysis and protein translation take place in both types of cells - However, RNA polymerase will only be found in bacterial cytoplasms because in eukaryotes, transcription (RNA synthesis) occurs in the nucleus (choice A will be found in bacterial cytoplasm, but not in animal cell cytoplasms and is the correct answer choice).
Which of the following is NOT true about bacterial conjugation? A. It can be used to map the bacterial genome. B. It changes an F- "female" bacterium into an F+ "male" bacterium. C. It involves the transfer of either plasmid DNA, genomic DNA, or both, between two bacteria. D. It increases the genetic diversity and the size of a bacterial population.
D. It increases the genetic diversity and the size of a bacterial population. - should increase genetic diversity but not size Hfr bacteria can be used in genomic mapping experiments, in which conjugation is allowed to proceed for varying amounts of time, and the order in which genes are transferred to the recipient determines the order of those genes in the genome. At the end of conjugation, the F- "female", having received the F plasmid, is now an F+ "male"
All of the following processes occur in bacteria EXCEPT: A. electron transport. B. regulation of transcription. C. simultaneous transcription and translation. D. mRNA processing
D. mRNA processing Prokaryotic (bacterial) mRNA can be immediately translated; no processing is required (mRNA processing does not occur in bacteria). This is one reason why transcription and translation can proceed simultaneously (simultaneous transcription and translation does occur in bacteria and can be eliminated). Although not compartmentalized as in eukaryotes, bacteria do participate in electron transport on their cell membranes (electron transport does occur and can be eliminated). Lastly, regulation of transcription does occur; in fact some of the best-understood transcriptional regulation mechanisms are found in bacteria (e.g., the lac operon, regulation of transcription occurs).
In the creation of an expression vector, what step is necessary to prepare both a gene of interest and the plasmid that will carry it for ligation? A. Ensure both genetic pieces are single stranded. B. Treat both with proteases to avoid contaminating interactions from proteins. C. Allow exonucleases to proofread both sequences. D. Digest both genetic sequences with the same restriction endonuclease.
digest both genetic sequences with the same restriction endonuclease To create ends that can be connected, the same restriction endonuclease must be used to both open the plasmid and digest the ends of the gene of interest so it can be ligated into the plasmid. Both pieces need to be double-stranded, not single. Proofreading of any type would have already been done in synthesizing the sequences. At this stage, the genetic constructs are already isolated from proteins so proteases are not needed.
Shared characteristics between mitochondria and prokaryotes include which of the following? A. Internal organelles B. Possession of an electron transport system C. Mitosis D. Motility
possession of an electron transport system. Mitochondria have the electron transport system that eukaryotic cells rely on for their energy production. Aerobic prokaryotes (bacteria) also have an electron transport system. Mitochondria do not have internal organelles (they are an internal organelle!) and prokaryotes do not possess any organelles. Mitochondria do not move, prokaryotes can. Mitosis is the process by which eukaryotic cells divide and reproduce, mitochondria do not under go mitosis. Prokaryotes reproduce via binary fission.
What is the best experimental method to analyze the effect of tdh2 gene deletion on the rate of histone acetylation? Comparing histone acetylation in wild-type and Δtdh2 cells by: A.Western blot B.Southern blot C.Northern blot D.RT-PCR
posttranslational modification of proteins such as histone acetylation is analyzed by Western blotting
