Chapters 11/12 (cont.)

point mutation/base mutation

when a single nucleotide is changed in a DNA sequence

3 types of point mutation/base substitutions

1.) Missense mutation

2.) Nonsense mutation

3.) Silent mutation

missense mutation

base substitution results in the change of an amino acid

nonsense mutation

base substitution results in a nonsense (stop) codon

silent mutation

has no effect on the protein sequence

frameshift mutation

insertion or deletion of one or more nucleotide pairs, shifts the translational reading frame, causes changes in many amino acids downstream from the site of the original mutation

inversion

occurs when a fragment of DNA is flipped in orientation in relation to the DNA on the other side

what causes mutations?

a “mistake” by DNA polymerase that fails to be repaired

chemical mutagens

chemicals that directly or indirectly cause mutations

physical agents of mutations

cosmic rays, X-rays, UV radiation (causes pyrimidine dimers)

chemical agents of mutations

reactive oxygen molecules (like H₂O₂), superoxide radicals, nitrous acid, acridine orange

2-aminopurine

inserted in place of A but base pairs with C → converts AT to GC base pair

6-bromouracil

inserted in place of T but base pairs with G → converts AT to GC base pair

nitrous oxide

deaminates C to U → converts GC to AT base pair)

4 types of DNA repair

1.) Base excision repair

2.) Methyl mismatch repair

3.) SOS repair

4.) DNA recombination

base excision repair

recognizes a specific damaged base and removes it from the DNA backbone

methyl mismatch repair

requires recognition of the methylation pattern in DNA bases

SOS repair

coordinated cellular response to damage that can introduce mutations in order to save the cell

DNA recombination

the process of “crossing over” and exchange of 2 DNA helices

4 levels of gene expression

1.) Changing the DNA sequence

2.) Control of transcription

3.) Translational control

4.) Post-translational control

changing the DNA sequence

some microbes change the DNA sequence to activate or disable a particular gene (ex. phase variation)

control of transcription

transcription can be regulated by protein repressors, activators, and alternative sigma factors

translational control

control of transcription initiation sequences that recognize specific repressor proteins

post-translational control

control of proteins that are already made (ex. activate, deactivate, or degrade the protein)

operon

a group of genes that are transcribed together as a single mRNA molecule, often encoding proteins involved in a related metabolic pathway

promoter

a DNA sequence that initiates transcription, where RNA polymerase binds

operator

a DNA sequence that acts as a binding site for a repressor protein, which can block transcription

how do genes respond to changes inside/outside the cell? - 3 ways

1.) Sensing the intracellular environment

2.) Global regulators

3.) Sensing the extracellular environment

sensing the intracellular environment

different regulatory proteins bind to specific compounds to determine the compound’s concentration (ex. dtx, the diphtheria toxin gene)

global regulators

proteins that affect the expression of many different genes (ex. cAMP receptor protein (CRP) of E. coli and related species)

sensing the extracellular environment

a common mechanism used by bacteria to sense outside the cell and transmit that info inside that relies on a series of 2-component protein phosphorylation relay systems (ex. sensor kinase PhoQ in Salmonella sense magnesium and pH outside the cell)

genetic recombination - vertical gene transfer

occurs during reproduction between generations of cells

genetic recombination - horizontal gene transfer/lateral gene transder

the transfer of genes between cells of the same generation

plasmids

mostly circular, ds, extrachromosomal DNA; self replicating

transposons

genes that can move from one chromosome to another; cannot replicate outside a larger DNA molecule, include a transposase gene

transformation

importing free DNA from the environment into bacterial cells

transformasome

responsible for the uptake of extracellular DNA and its subsequent transformation/integration into bacterial genome

a bacterial cell is considered competent when it is

capable of taking up foreign DNA from its environment

electroporation

a brief electrical pulse “shoots” DNA across the membrane

transduction

gene transfer is mediated by a bacteriophage (bacterial virus) vector

conjugation

gene transfer requires contact between donor and recipient cells - LARGER QUANTITIES OF DNA ARE TRANSFERRED COMPARED TO TRANSDUCTION OR TRANSFORMATION

DNA plasmids have

certain DNA sequences that can be cut by a specific enzyme called a restriction endonuclease

the restriction sequence of a plasmid is usually a

“palindrome” in which the sequence of base pairs reads the same forwards and backwards

the names of restriction enzymes reflect what?

the genus and species of the source organism

AluI and HaeIII do what?

cut DNA

AluI comes from

Arthrobacter luteus

HaeIII comes from

Haemophilus aegyptius

BamHI, HindIII, and EcoRI produce what?

“sticky” ends (staggered cut)

BamHI comes from

Bacillus amyloliquefaciens

HindIII comes from

Haemophilus influenzae RD

EcoRI comes from

Escherichia coli RY13

4 steps of DNA electrophoresis

1.) DNA sample loading

2.) Application of electrical field (DC)

3.) DNA separation

4.) UV transillumination and documentation

gel electrophoresis

a technique commonly used to separate biological molecules based on size and biochemical characteristics, such as charge and polarity

agarose gel electrophoresis

widely used to separate DNA (or RNA) of varying sizes that may be generated by restriction enzyme digestion or by other means, such as the PCR

4 steps of DNA recombination

1.) Plasmid vector preparation

2.) Foreign DNA preparation

3.) Annealing

4.) Ligation

plasmid vector preparation

• a plasmid (circular DNA) is used as a vector

• EcoRI endonuclease cuts at a specific cleavage site, creating sticky ends (overhanging ssDNA)

foreign DNA preparation

foreign DNA (containing the gene of interested) is also cut with EcoRI at matching sites, producing complementary sticky ends

annealing

the sticky ends of the foreign DNA and plasmid align and pair due to complementary base pairing

ligation

DNA ligase seals the sugar-phosphate backbone, creating a recombinant plasmid (chimera) that contains both plasmid and foreign DNA)

gene fusion

transposition of genes from one location of the chromosome to another (fusion of 2 genes together)

depending on the design of gene fusion, a function may be

inactivated or placed under the control of a different regulatory sequence