Module One Flashcards
What did Beatle and Tatum show?
That genes work by making enzymes
what were the organisms that Beatle and Tatum worked on?
Fungus- Neurospora
- bread mold
- eukaryotic
what was the system that Beatle and Tatum worked on?
they isolated auxotrophs that could only grow in the presence of arginine
what is an auxotroph?
a micro-organism that can only grow in the presence of a specific growth nutrient such as an amino acid
what did Beatle and Tatum do?
carried out genetic analysis to find the number of loci (genes) in which mutation causes arginine auxotrophy
what did beatle and tatum find?
that there where 3 different genes at 3 different loci
what is a phenotype?
the visible properties of a mutant
what is a genotype?
the specific allele composition of an organism or cell
what is a mutation in classical genetics?
a change in a gene that results in a change in phenotype
what is a mutation in modern genetics?
a heritable change in the DNA sequence of an organism
what is a wild type organism?
the initial “non-mutant” organism
what is a mutant?
an organims carrying a mutation
what is a foward mutation?
a loss of function mutation that generates a mutant from a wild-type organism
what is a reversion?
a mutational event that causes the mutation to revert back to the wild-type
what is an auxotroph?
an organism that can only grow in the presence of a specific nutrient (growth supplement
what is a prototroph?
An organism that can grow without specific supplements
what is a point mutation?
a mutation at a single point (often a single base pair)
what is a substitution mutation?
a mutation where one base pair in the DNA sequence is changed to another
what is a frameshift mutation?
a base pair is lost or incorporated into the DNA sequence.
what is a deletion mutation?
where one or more base pairs are lost from the DNA sequence
what is an insertion mutation?
where one or more base pairs are added to the DNA sequence
what is a spontaneous mutation?
a mutation that occurs in the absence of a mutagen or any other external cause
what is a lesion?
a chemical change in the DNA that is not a mutation, but can lead to a mutation if is remains.
what does the genotype his+ mean?
does not require histidine for growth (denoted His+)
what does the genotype his or hisA mean?
requires histidine for growth (denoted His-)
what does the genotype lac + mean?
Can use lactose as a source of energy (denoted Lac+)
what does the genotype lac or lac Z mean?
Cannot use lactose as a source of energy (denoted Lac-)
what was the basis of the experiment that beadle and Tatum conducted?
carried out genetic analysis to find the number of loci (genes) where mutation caused argine auxotrophy
what where the 3 loci that Beadle and Tatum identified?
3 genes on 3 different chromosomes
arg-1
arg-2
arg-3
what did Beadle and Tatum conclude around the 3 genes identified?
that they each encoded an enzyme that formed a product along the arginine metabolism pathway
what was the function of arg-1?
to convert the precursor into ornithine
what was the function of arg-2?
to convert ornithine to citrulline
what was the function of arg-3?
to convert citrulline to arginine
what did Beadle and Tatum conclude about the arg genes?
that each arg gene encoded a different enzyme that catalyzes a different chemical reaction
what was Beadle and Tatum’s famous hypothesis?
one-gene-one-enzyme
what is the significance of the one-gene-one-enzyme hypothesis?
still fundamentally correct
applies to all proteins, not just enzymes
-does not show how enzymes and genes are related
what does the genotype arg+ mean?
does not require arginine for growth (denoted arg+)
what does the genotype arg, arg-1, or argA mean?
requires arginine for growth (denoted arg-)
why are mutations so important to study?
- mutations cause genetic diversity
- they give insights into the way that genes and their products work
- mutations can cause disease
- mutations can lead to antibiotic resistance in microbes
- they can improve the commercial properties of organisms if enhanced
what are the mutation rates in one generation of humans?
each person has about 40 heritable sequence changes (mutations) compare to their parents
why are microorganisms sued to study genetics?
they allow genetic screens that are not possible for higher eukaryotes and they have the same mechanism and nature of mutation
what is a mutation?
a heritable change to the DNA sequence
what was the experimental system used by Luria and Delbruck?
they were using bacteriophage T1 that infects and kills E.coli bacteria
what is the lytic lifecycle of a bacteriophage?
the cells in infected and then the phage replicates inside and the cell lyses
what did Luria and Delburck fo?
they infected lots of e.coli cells with bacteriophage T1
what was the outcome of the Luria and Delbruck experiments?
most of the E.coli died, but a few survived (must be T1 resistance) and gave colonies that where also resistant to T1
how did the mutant bacteria in the Luria and Delbruck experiment arise?
Either the presence of the T1 bacteriophage induced some of the bacteria to become resistance or the mutation arose spontaneously in the bacterial population and was selected for once the pacteriophage was added
what is the fluctuation test?
expose different cultures (batches) of bacteria to T1 bacteriophage
spread the cultures to allow the growth of surviving bacteria
count the number of surviving colonies
conduct an experimental comparison
what is a jackpot culture?
when there are so many revertant/mutant bacteria
what is the conclusion for Luria and Delbruck from the fluctuation test?
that the mutation arises spontaneously and randomly in bacteria
they can be selected for by environmental conditions
what were the limitations of the Luria and Delburcks experiment?
- bacteria are rapidly killed and so they have no time to adapt
- it can take some time for the mutation to arise in the population (several generations)
why are all E.coli not resistant to T1 phage?
T1 resistance E.coli are less able to acquire iron, and so when the T1 phage is not present, the wild-type e.coli out compete with them
what is a mutagen?
an agent that is capable of causing an increased rate of mutation
how do we test for mutation?
direct- use of lab animals (good lab model for humans
who developed the Ames Test?
Bruce Ames and colleagues
what is the principle of the Ames test?
uses strains of bacterium Salmonella typhimurium that are AUXOTROPHS for histidine
bacteria are spread onto a plate that does not contain histidine and any bacteria thst grow are revertants
when does the Ames test show a likely mutagen?
when the test compound increases the number of revertants by a lot
how does the Ames test detect different types of mutation?
there are different strains of Salmonella with different kinds of mutations that can select from different types of bacteria
what are some limitations of the Ames experiment?
many chemicals are not mutatgenic in bacteria but they are in humans because they get metabolised into the active from in the mammalian liver
how can we reduce the limitation of the Ames Test?
prepare liver extracts and incorporate them into the medium used in the Ames Test
what was the experimental system that Benzer used?
bacteriophage T4
Why did Benzer use bacteriophage?
they are viruses that infect bacteria and they are easily and rapidly grown
they have simple genetics
they must have similar genetics to the host cell
analysis of billions is easy
what are bacteriophage plaques?
“holes” in a lawn of bacteria where the bacteriophage has infected and killed the bacteria
what e.coli strains can wild-type T4 infect?
Both E.coli K and B
what e.coli strains can rll mutant bacteriophage infect?
only E.coli B
no E.coli K
what is complementation?
production of wild-type phenotype when two mutant haploid genomes are present in the same cell
what was the complementation system that Benzer used?
both rll mutant 1 and rll mutant 2 infected the same E.coli cell
when does complementation occur?
when the mutations are in different genes
when does no complementation occur?
when the mutations are in the same gene
what is recombination?
a process that generated new gene combinations
how did Benzer screen to see if recombination was occurring?
E.coli strain B (both phages can infect) were infected with pairs of mutant phage
the progeny of these phages was infected into E.coli K(only wild-type an grow)
the bacteria that caused plaques was then known to have undergone recombination
what does the number of bacterial plaques on the bacteria lawn indicate in Benzer’s experiment?
the frequency of recombination events
what did Benzer conclude from rll phage mutation analysis?
that genetic maps are linear
most mutations are changes at only one mutable site
mutations can cause the deletion of one or more mutable sites
some sites are prone to mutation
what is a hotspot on a gene?
a region of the gene that is prone to mutation
why were phages good for Benzer’s experiment?
he had a powerful screening system that allowed him to select for recombinants
what is complementation?
lets you tell if mutations are in the same of different genes; progeny are genetically identical to parents
what is recombination?
results in new combinations of mutations
how did Benzer use complementation?
to determine the number of rll genes
how did Benzer use recombination?
to map the relative locations of mutations
what is required for a mutation to occur spontaneously?
DNA synthesis
how do spontaneous mutations occur?
can involve error by DNA polymerase
can arise through the tautomerisation of bases
what is tautomerisation?
a base transiently flips into a different configuration (isomer) that has different pairing properties
How do tautomers cause mutation?
they can change the way that a base pairs and this can mean that the wrong base pairs when the new strand is being synthesised and so the new strand contains a mutation
how is the rate of spontaneous mutation reduced in the cell?
DNA polymerase “checks” each new base-pair
It can remove the wrong base if it is idenfifed
How effective is DNA polymerase at checking the base pairing?
over 99% effective
why is proofreading the DNA so important?
it reduces the frequency of spontaneous mutation
what is the mismatch repair system?
can detect non-matched base pairs of bases in the DNA
Determines which base is wrong
can remove the incorrect base
what detects the mismatched proteins?
Mut proteins
what is the process of removing the wrong bases in the mismatch repair system?
DNA is broken by Mut H
Excising the DNA including the wrongly incorporated base
A new section of DNA is synthesised and then ligated
how does the mismatch repair system know when the pairing is wrong?
the system can distinguish between the original and new strand
how are the original and new DNA strands distinguishable?
DNA inside E.coli is chemically modified by methylation one it is a complete strand (original). New DNA is not yet methylated
Adinine bases in the DNA are also chemically modified (does not effect pairing)
what is the model for indel mutations?
Streisinger mutation
how do spontaneous mutations occur?
can involve error by DNA polymerase
can arise through the tautomerisation of bases
what is tautomerization?
a base transiently flips into a different configuration (isomer) that has different pairing properties
How do tautomers cause mutation?
they can change the way that base pairs and this can mean that the wrong base pairs when the new strand is being synthesized and so the new strand contains a mutation
how is the rate of spontaneous mutation reduced in the cell?
DNA polymerase “checks” each new base-pair
It can remove the wrong base if it is idenfifed
How effective is DNA polymerase at checking the base pairing?
over 99% effective
why is proofreading the DNA so important?
it reduces the frequency of spontaneous mutation
what is the mismatch repair system?
can detect non-matched base pairs of bases in the DNA
Determines which base is wrong
can remove the incorrect base
what detects the mismatched proteins?
Mut proteins
how does 2-amino purine act as a mutagen?
-if it occurs during replication it leads to an A-T basepair being changed to an A-C and subsequently G-C
how does the mismatch repair system know when the pairing is wrong?
the system can distinguish between the original and new strand
how are the original and new DNA strands distinguishable?
DNA inside E.coli is chemically modified by methylation once it is a complete strand (original). New DNA is not yet methylated
Adenine bases in the DNA are also chemically modified, 6 methyl adenine (does not affect pairing)
what is the model for indel mutations?
Streisinger mutation
what is required for a mutation to occur spontaneously?
DNA synthesis
how do spontaneous mutations occur?
can involve error by DNA polymerase
can arise through the tautomerisation of bases
what is tautomerization?
a base transiently flips into a different configuration (isomer) that has different pairing properties
how does an apurinic site cause a mutation?
during DNA replication the lack of purine means that there is a “blank” where the purine should be
a base (often adenine) may be inserted opposite the blank
the can change the sequence of base-pairs
how is the rate of spontaneous mutation reduced in the cell?
DNA polymerase “checks” each new base-pair
It can remove the wrong base if it is idenfifed
How effective is DNA polymerase at checking the base pairing?
over 99% effective
why is proofreading the DNA so important?
it reduces the frequency of spontaneous mutation
what is the mismatch repair system?
can detect non-matched base pairs of bases in the DNA
Determines which base is wrong
can remove the incorrect base
what detects the mismatched proteins?
Mut proteins
what is the process of removing the wrong bases in the mismatch repair system?
DNA is broken by Mut H
Excising the DNA including the wrongly incorporated base
A new section of DNA is synthesized and then ligated
how does the mismatch repair system know when the pairing is wrong?
the system can distinguish between the original and new strand
how is EMS damage repaired?
Repair proteins- alkyltransferase
The (alkyl) ethyl group is transferred from the base in the DNA to the protein
what is the model for indel mutations?
Streisinger mutation
what is the frequency of spontaneous mutations after prevention has occurred?
1 per 10^9
what is a mutator bacterial strain?
bacteria that have a higher mutation rate
how are photodimers repaired?
Nucleotide excision repair
-similar to the repair of apurinic sites
Photolyase enzyme
-uses energy from white light to convert photodimers back to pyrimidines (photo repair)
do different mutagens cause different types of mutations?
yes
how do chemicals act as mutagens?
work by modifying base-pairing properties
DNA polymerase then incorporates wrong bases during DNA replication
what is EMS (ethyl methane sulphonate)?
- an alkylating agent
- causes GC-AT mutations
how does EMS work as a chemical mutagen?
adds an ethyl group to the G which creates a pre mutagenic legion
on the second replication if it remains it stays it becomes a mutation
what is Holliday’s model for recombination?
- alignment of DNA sequences
- breakage of DNA strands, exchange and rejoining of the DNA
- branch migration, giving heteroduplex (hybrid) DNA where each strand is derived from a different parent molecule
- resolution leaves two DNA molecules with new combinations of alleles
what is a heteroduplex?
a DNA molecule in which each strand is derived from a different parent molecule
what is Aflatoxin?
a chemical produced by fungi
how does Aflatoxin work as a mutagen?
chemically reacts with guanine (G) bases in DNA
Causes GC to TA mutations
What happens when aflatoxin B reacts with DNA?
generates apurinic sites
this can lead to mutation
what is depurination?
a purine base (adenine or guanine) is lost from the DNA
what bases are purines?
adenine and guanine
what is an apurinic site?
a site without a purine
how does an apurinic site cause a mutation?
during DNA replication the lack or purine means that there is a “blank” where the purine should be
a base (often adenine) may be inserted opposite the blank
the can change the sequence of base-pairs
what is “bypass” DNA polymerase?
DNA polymerase that can synthesise past “damaged” DNA (lesions) such as apurinic sites
How accurate is “bypass” DNA pol?
not as accurate as regular DNA polymerase
What is TLS?
trans-lesion synthesis
how do chemical mutagens work?
alter the bases in the DNA, affecting base pairing
“mimic” normal bases and become incorporated into the DNA, late pair with the “wrong” base
absence of a “pairable” base during DNA synthesis
all mechanisms require DNA replication to become mutations
what is a pre-mutagenic lesion?
a change to the DNA that may lead to a mutation
how does a pre-mutagenic lesion arise?
at least one round of DNA replication must occur to produce a pre-mutagenic lesion
DNA repair takes place at the pre-mutagenic lesion- once the mutation is “established”, it is too late
when do DNA repair systems repair damages?
before it is replicated
how is EMS damage repaired?
Repair proteins- alkytransfersese
The (alkyl) ethyl group is transferred from the base in the DNA to the protein
How are apurinic sites repaired?
an enzyme (AP endonuclease) recognizes an apurinic site and cuts the strand of DNA that contains it
the defective DNA and some adjacent DNA is then removed by a set of enzymes (excision exonucleases)
the gap is filled in by DNA synthesis
what is the enzyme involved in apurinic site recognition?
AP endonuclease
how does Ultrviolet light work as a mutagen?
Adjacent thymidine (T) bases in the DNA can become covalently cross-linked (photodimers)
These fail to base-pair properly during DNA replication
Translesion DNA polymerases can replicate past these but may incorporate the wrong base
how are photodimers repaired?
Nucleotide excision repair
-similar to the repair of apurinic sites
Photolyase enzyme
-uses energy from white light to convert photodimers back to pyrimidines (photorepair)
are the mechanisms for mutation and repair the same in bacteria and eukaryotes?
yes
what is a chiasma (chiasmata)
the physical appearance of crossing over
what are the features of crossing over?
- due to the exchange of genetic material (DNA) between corresponding chromosomes
- must involve the breaking and rejoining of DNA
- molecular mechanisms are best understood in microorganisms
what are the steps in recombination?
double strand break erosion invasion and displacement polymerisation resolution to crossover by nicks
what is Holiday’s model for recombination?
- alignment of DNA sequences
- breakage of DNA strands, exchange and rejoining of the DNA
- branch migration, giving heteroduplex (hybrid) DNA where each strand is derived from a different parent molecule
- resolution leaves two DNA molecules with new combinations of alleles
what is a heteroduplex?
a DNA molecule in which each strand is derived from a different parent molecule
what is the key intermediate in the Holliday model?
- proposed to arise during the recombination process after strand exchange and branch migration
- has been observed experimentally, using electron microscopy
- resolution of this structure gives two possible outcomes
how does recombination occur at the molecular level?
RecBCD protein complex nicks, unwinds and degrades the DNA to generate single-stranded DNA
RecA protein coats the single stranded DNA and then catalyses base-pairing of the DNA with the “target” double-stranded molecule proteins (RuvA and RuvB) cause branch migration
RuvC protein then resolves Holliday junctions by DNA cleavage
what evidence supports the current model of recombination?
- genetic information from eukaryotic systems (fungi)
- visualisation of DNA in Holliday-type structures
- the phenotypes of bacterial mutants lacking relevant proteins
- the biochemical reactions catalysed by relevant proteins
how are double-strand breaks repaired?
- deradation of DNA strands
- Strand invasion, DNA synthesis
- Formation of Holliday junction
- Resolution can lead to crossover
