exam 2

mutation

• modification in the gene sequence of DNA in a gene often resulting in an alteration in the protein encoded by the gene

  • spontaneous

  • induced

spontaneous mutation

• stable inheritable changes in the base sequence of DNA

• can occur as a result of:

  • base substitutions

  • removal/addition of nucleotides

  • transposable elements

vertical gene transfer

• mutation passed onto progeny

• inherited

reversion

• change in a cells genotype and phenotype to its original state through a change in the mutated gene

base substitutions

• most common mistake

• results from mistakes during DNA synthesis

• incorrect base is incorporated into DNA

• point mutations:

  • missense mutation

  • nonsense mutation

  • null/knockout mutation

point mutations

occur when one base pair is changed

missense mutation

mutation resulting from amino acid substitution

nonsense mutation

mutation that changes an amino acid codon to a stop codon

null/knockout mutation

mutation that inactivates a gene

removal/addition of nucleotides

• shifts the translational reading frame

  • shifts the codon

• frameshift mutation

frameshift mutation

• affects all amino acids downstream from addition or deletion

• mutations frequently result in premature stop codons

induced mutations

• essential for understanding genetics

• mutations can be intentionally produced to demonstrate fxn of particular gene/set of genes

how can mutations be induced?

via:

• chemical mutagens

• transposition

• radiation

chemical mutagens

• type of induced mutation

• chemical modification of purines and pyrimidines

• base analogs

• intercalating agents

chemical modification of purines and pyrimidines

• increases frequency of mutations as DNA replicates (base substitution)

• nitrous acid

• alkylating agents

base analogs

• chemicals that are structurally similar to nitrogenous bases but have slightly altered base pairing properties (base subst)

• may result in pairing w/ wrong base as complementary strand is being synthesized

intercalating agents

• molecules that insert themselves between adjacent bases

• increase freq. of frameshift mutations, create spaces btwn bases

what is a common intercalating agent

ethidium bromide

ethidium bromide

is a common intercalating agent

• potential carcinogen

• used to stain DNA in gel electrophoresis

transposition

common procedure used to induce mutation in laboratory via transposons

transposons

• genes that move from one replicon to another site in the same replicon, or to another replicon within the same cell

• move spontaneously from gene to gene

• disrupts proper fxn of gene, product is generally nonfunctional

insertion mutation

type of induced mutation

• gene that receives the transposon will undergo null/knockout mutation

types of radiation

• UV light

• X-ray

UV light

• causes covalent bonding btwn adjacent thymine bases to ‘buckle out’ (DNA distortion) by forming thymine dimers

• repair system will remove bond and insert correct/unbonded thymines or wrong bases

• causes skin cancer due to DNA damage

• used as sterilant

X-rays

• causes single and double stranded breaks in DNA

• breaks are often lethal

direct selection

• involves inoculating population of bacteria on medium in which only mutants will grow

• used to select antimicrobial resistant and/or auxotrophic mutants reverted to phototrophic organisms

• ex: streptomycin exists in all bacteria, killing off everything not interested in so only organisms w/ mutation survive + grow

indirect selection

• Required to isolate organisms that require growth factor that parent strain does not have (auxotrophic mutants)

• Replica plating w/ velvet

• looking for mutation that causes loss of something

• identified by growing in presence of histidine (example) and check for organisms that grow when histidine is removed

mutation selection

testing for carcinogens

carcinogens

• cancer causing chemicals

• many mutagens are also carcinogens

• microbes used to test potential carcinogenic activity, tests are based on effect chemical has on microbial DNA

AMES test

• assumes that freq of reversions is increased by mutagens and that most mutagens are carcinogens

• tests rate of reversion of salmonella autotroph and potential lethality

• Reversion freq: testing for ability of chemical to alter DNA, looking for change in mutation rate.

horizontal gene transfer

genes transferred from one cell to another

what are the 3 natural mechanisms used to transfer genes btwn bacteria?

• DNA mediated transformation

• transduction

• conjugation

DNA mediated transformation

• bacteria reaching out and grabbing DNA and pulling it back into the cell, usually with pillus/pilli.

• transfer of naked DNA from environment to recipient cell

• cells rupture during stationary + death phase

• recipient cell picks up portion of naked DNA and integrates it into chromosome

• occurs when cells are ‘competent’

transduction

bacterial DNA is transferred by a bacterial virus (bacteriophage)

• bacteriophages live off bacteria and replicate within bacterial cell. Sometimes DNA from old bacteria will be taken in and incorporated

conjugation

• intentional DNA transfer when 2 bacterium are in contact w/ each other via sex pilus

• mediated by plasmid (hold antibiotic resistant genes)

• bacteria become antibiotic resistant due to conjugation

stages of natural transformation

1. entry of DNA; only single strands enter, double strands are degraded

2. integration of donor DNA; via H-bonding, enzymes cleave donor DNA and set in place

3. mismatch repair; removes DNA that doesn’t match (donor or recipient), replaced w/ correct nucleotides

4. cell multiplication; cells multiply under select conditions where non-transformed cells will not grow

types of transduction

• generalized: any gene of donor can be transferred

• specialized: only specific genes can be transferred

mis-packaging of DNA during viral replication (transduction)

• mispackaged phase infects new bacterial cell and inserts donor DNA

• donor DNA is integrated into cell by homologous recombination

plasmid

a self replicating extrachromosomal piece of DNA and can code for traits (antibiotic resistance) that give bacteria advantages

R plasmids

group of plasmids that confer resistance to many antimicrobial agents

self transmissible R plasmids

carry all of the genetic info they need to transfer. contain different genes that can replicate outside the chromosome

mobilizable R plasmid

encode for some, but not all, of the info needed to transfer

4 fundamental tools of biotech

1. restriction enzymes

2. gel electrophoresis

3. DNA probes

4. primers

restriction enzymes

naturally occurring enzymes that cut DNA into fragments (restriction fragments), cut in predictable and controllable manner

resriction fragments

• generated by restriction enzymes

• can be joined w/ new fragments

• enzymes produce jagged cuts (sticky ends) or blunt cuts (blunt ends), phosphate backbones (ends) fuse together by DNA ligase to form new strand

gel electrophoresis

• Gel matrix contains ⊕ and ⊖ poles, DNA is ⊖ charged and is pulled toward ⊕ pole

• DNA is dropped into wells in the gel

• separates DNA fragments based on size

  • large fragments remain high up

  • small fragments appear to be lower (more motile)

• gel must be stained to view DNA (often w/ ethidium bromide)

DNA probes

• only bind to complementary segments. Pieces of dna that has detection (enzyme)

• used to locate nucleotide sequences in DNA/RNA

• ‘probe’= single stranded DNA tagged w/ marker

• probe hybridizes to complementary fragment of interest

4 applications of probe tech

• colony blotting

• southern blotting

• fluorescence in situ hybridization (FISH)

• DNA/RNA microarrays

colony blotting

• detects specific DNA sequences in colonies grown on agar plates

• colonies are transferred on nylon membrane

• used to determine which cells contain gene of interest

southern blotting

• uses probes to detect DNA sequences in restriction fragments separated from gel electrophoresis

southern vs northern vs western blotting

fluorescence in situ hybridization (FISH)

• probe is fluorescently labeled to detect specific nucleotide sequences

• detects seq. inside intact cell

• specimen viewed under fluorescence microscope

• used to identify specific properties of bacteria

DNA microarray tech

• studies gene expression under certain conditions

• DNA arrays are solid supports w/ fixed patterns of different single stranded DNA fragments attached

• entire DNA specimen is labeled

• allows researchers to screen sample multiple seq. simultaneously

what diseases can alterations in DNA sequences cause?

1. sickle cell anemia: single base pair change in gene, blood cells are misshapen and break down bc the hemoglobin cannot carry oxygen

2. cystic fibrosis: 3 base pair deletion, cells produce excess fluid that builds up in lungs (mucus)

dideoxychain termination

elements for termination rxn:

• single stranded DNA template

• primer (anneals to template)

• DNA polymerase

• one of the nucleotide bases is labeled w/ marker to detect

  polyacrylamide gel electrophoresis used to separate DNA fragments by size

dideoxynucleotides

lack 3’ OH, incorporation causes chain termination

automated DNA sequencing

• most automated systems use fluorescent dyes to detect newly synthesized DNA

• gel electrophoresis used to separate fragments into colored bands

• laser is used to detect color variations- order of color reflects nucleotide seq.

primers

• single stranded DNA binds to seq of DNA

• used in in vitro DNA synthesis

• primers serve as fragments for addition of DNA nucleotides (PCR)

polymerase chain reaction (PCR)

• amplifies large amount of DNA with very specific sequences from a small sample

• millions of copies within hours

• technique exploits specificity of primers

  • allows selective replication of chosen region, ‘ target DNA’

• staring w/ double stranded DNA mol.,process involves # of amp. cycles

• DNA is exponentially amplified

3 step amplification cycle in PCR

1. double stranded DNA denatured by heat

2. primers anneal to complementary seq. of target DNA and DNA synthesis occurs w/ hear stable DNA polymerase

3. duplication of target DNA

DNA cloning

process of producing copies of DNA

• cloned DNA generally combined w/ carrier mol. called vector

• insures replication of target DNA

researching gene fxn + regulation

• studied by gene fusion

• joining gene is being studied to reporter gene

• reporter gene encodes observable trait, which makes it possible to determine conditions that may affect gene activity

taxonomy

science that studies organisms in order to order + arrange them

3 areas of taxonomy

1. identification

2. classification

3. nomenclature

identification is the process….

Process of characterizing in order to group them

classifications arrange organisms into…

similar and/or related groups

nomenclature is the system of…

assigning names

what are the methods used to identify prokaryotes

1. microscope morphology

2. metabolic capabilities

3. serology

why is understanding organisms’ phylogeny important

1. it assists in classification and allows for organized classification of newly recognized organisms

2. molecular techniques make genetic relatedness possible

taxonomic hierarchies

• Species – group of related isolates or strains

• Genus – group of related species

• Family – collection of similar genera

• Order – collection of similar families

• Class – collection of similar orders

• Phylum – collection of similar classes

• Kingdom – collection of similar phyla

• Domain – collection of similar kingdoms

microscopic morphology

can be used to determine size, shape and staining characteristics

• gram stain: gram ⊕ and gram ⊖, narrows down possibilities

• special stains (acid fast): present unique characteristics of organism

metabolic capabilities

culture characteristics, colony morphology can give clues to identity

• Green pigment of Pseudomonas aeruginosa

• β-hemolytic colonies of Streptococcus pyogenes

  (beta=“complete” hemolysis)

biochemical tests

more conclusive identification;

most tests rely on pH indicators or chemical rxn that results in color change when compound is degraded

• pH can be:

  • acidic (fermentation of sugars)

  • alkaline (prod. of CO2, raises pH)

  • no change (bacteria not growing or utilizing specific nutrient source)

3 biochemical tests

1. catalase test

2. sugar fermentation

3. urease test

catalase test

bacteria that prod. catalase break down hydrogen peroxide to release oxygen, bubbles are formed

sugar fermentation

fermentation of sugars result in acid prod. causing pH indicators to change in color

urease test

breakdown or urea by urease enzyme releases ammonia and CO2 creating an alkaline environment within tube, indicated by pink color

macconkey agar

• identifies lactose fermentation, gram ⊖ enteric pathogens (differential) and inhibits gram ⊕ (selective)

• fermentation of lactose turns medium red/pink due to acidic environment

biochemical typing

used to identify species and strains by tracing specific biochemical characteristics (biovar/biotype)

serological typing

identification made based off differences in serological molecules that react w/ antibodies, characteristics called serovar/serotype

serology

• technique observing interaction between antibodies and antigens

• available for rapid detection of organisms like E.coli (O157:H7)

sequencing ribosomal RNA genes

• little genetic variance in rRNA

• 16s rRNA is ‘gold standard’ for identifying unknown bacteria + determining evolutionary relationships

advantages w/ sequencing RNA

• how the ‘tree of life’ was determined

• identifies organisms that can’t be grown in culture

genomic typing

restriction fragment length polymorphisms (RFLPs)

• restriction enzymes digest DNA from each organism

• resolved w/ pulse field gel electrophoresis

• polymorphisms: variations in fragments btwn organisms

• national molecular sub-typing network for food-borne disease surveillance catalogs RFLPs of certain food-borne pathogens

antibiograms

• identify organisms based on antibiotic susceptibility

• disc infused w/ antimicrobials placed on inoculated plate

  • clear = microbial susceptibility

phototroph

harvest energy from sunlight

photoautotroph

obtains carbon from CO2 (self producing)

photoheterotroph

obtains carbon from organic compounds

anoxygenic phototroph

phototroph that does not produce O2

• likely first photosynthesizing organisms

• oxidize hydrogen sulfide or organic molecules when making NADPH

• include purple and green bacteria

• habitats (aquatic): bogs, lakes, superficial layer of mud

purple bacteria

• anoxygenic phototroph

• gram ⊖, appear red/orange or purple due to pigments used in photosynthesis

purple sulfur bacteria

• anoxygenic phototroph

• sulfur rich springs

• prefer hydrogen sulfide to generate reducing power

• most are strict anaerobes but some can grow aerobically and w/o light

purple non-sulfur bacteria

• anoxygenic phototroph

• found in moist soil, bogs and paddy fields

• prefer organic molecules to generate reducing power

• most grow aerobically and in absence of light

green bacteria

• anoxygenic phototroph

• gram ⊖, typically green/brown

green sulfur bacteria

• anoxygenic phototroph

• sulfur rich habitats

• use hydrogen sulfide to generate reducing power

• gas vesicles

• strict anaerobes

green non-sulfur bacteria

• anoxygenic phototroph

• filamentous growth

• use organic molecules to generate reducing power

• can grow aerobically and in absence of light

oxygenic phototroph

phototroph that does produce O2

• uses H2O as source of e^-

• oxidation of water liberates oxygen

• cyanobacteria thought to be earliest organism of this kind and converted CO2 → O2

chemotroph

harvests energy by oxidizing chemicals

chemolithotroph

• anerobic chemotroph

• oxidizes reduced inorganic chemicals to produce energy

• use alternate terminal e^- acceptor other than oxygen (CO2/sulfur)

• usually archaea

chemoorganotroph

oxidizes organic chemicals

ex: fungi, bacteria, protists, animals, archaea

methanogens

• anaerobic chemotroph

• archaea

• produce energy by oxidizing H2 and using CO2 as terminal e^- acceptor

  • creates methane and H2O

• found in sewage, swamps, marine sediments and mammalian digestive tracts

• highly sensitive to oxygen- anaerobic chambers used to cultivate