Naming genes, mutations and complementation Flashcards
how are bacterias named
genus with capital
species lower case
all in italics
how are genotype and phenotype linked
genotype directly affects the phenotype, if have a mutation may lose or gain a characteristic
what is the genotype
genetic material of an individual
what is the phenotype
the observable characteristics
what affects phenotype
environment
what are the 3 essential features in bacterial gene
a promoter
transcribed region
terminator region
what is the promoter for in bacterial gene structure
All the sequences in the DNA required for expression and regulation of a gene
These sequences are NOT all included in the mRNA
what is the transcribed region in bacterial gene structure for
makes mRNA
Can include more than one ORF (operon)
These sequences ARE included in the mRNA
what terminator sequence in bacterial gene structure for
determines where mRNA finishes
Defines end of gene or operon
Often a hair-pin loop
how is mRNA transcribed from prokaryotic gene structure
mRNA formed 5’ to 3’
what is included in mRNA structure
5’ UTR
translated region
terminator sequence
where is the 5’ UTR
before the AUG start codon
where is the translated region
Each ORF (open reading frame, must have a RBS) is preceded by a ribosome binding site (RBS)
what is the terminator sequence for
Normally a hair-pin loop
Gives mRNA stability
how are genes identified
First studies defined genes in terms of observable properties
i.e. by PHENOTYPE
how are phenotypes written
as non-italics and often
with a superscript letter to indicate further details
how is something that is resistant to ampicillin antibiotic written
Amp^r
how is something that is sensitive to ampicillin antibiotic written
Amp^s
what does His ^- mean
strain can’t make histidine (would need histidine in media for growth)
what does Lac- mean
can’t break down lactose (not necessarily mutant, may have never done it in the first place, so would not be a mutant varies in different strains
how were genes responsible for phenotypes identified
Mostly by finding a loss-of-function mutant strain
what is a mutant
strain containing a mutation in its genome
how are genotypes written
3 letter codes, in lower case and italics. An additional capital letter indicates a specific gene
what indicates gene defined by a mutation, first one found
xyz-1
what indicates gene linked to a phenotype, first gene = A
xyzA
what indicates gene defined as first mutation in gene A
xyzA1
what is a lacZ gene
gene that encodes the enzyme ß-galactosidase
what is a lacY gene
gene that encodes the enzyme lactose permease
what does the number mean lacZ19
No other lac mutation will have the number 19 (not just lacZ mutations)
what is needed for tryptophan biosynthesis
several genes
how may a Trp^- phenotype be formed
trp mutant may have a mutation in one of a number of genes to give the Trp- phenotype
how would trp mutants be named as they are found
named trp-1, trp-2 etc
what happens to the trp name if analysis shows which gene is mutated
a letter can replace the number: trpA or trpB etc.
if particular mutations identified what happens to trp name
numbers are added: trpA1 or trpB2
what is Δ
deletion
what is ::
insertion
what is :
gene fusion
what is xyz’
xyz is the gene
3’ end of gene missing
what is ‘xyz
xyz is the gene
5’ end of gene missing
what is xyz^-
undefined mutation with gene function lost
what is (pXY123)
plasmid XY123 inside cell
what can cause mutations
chemical
physical
biological
what can cause chemical mutations
Ethyl Methyl Sulphonate (EMS)
what can cause physical mutations
UV
what can cause biological mutations
transposons
what conditions are ideal in selection
Ideally we use Selective conditions
i.e. when either the mutant or ‘wild-type’ will not grow
what does the selection method depend on
nature of the mutation and its effect on the bacterium
what is an auxotrophic mutant
require something for growth that the wild-type (prototrophic) bacteria don’t
examples of auxotrophic mutants
- wild-type will grow on minimal medium of inorganic salts and glucose
- an E. coli trp mutant won’t grow on this medium unless tryptophan is also added
how are trp mutants found
we need to use replica plating colonies on to media +/- tryptophan
what happens in selection by replica plating
Put all these colonies on and agar plate, hope some have the trp mutant
Want to know which ones are – need to characterize these further
Press agar plate onto velveteen ,get an imprint
Press onto two different plates: one with minimal medium, other with complete that contains trp, (cant make trp if it is mutant for it)
Workout which grow on complete but not minimal
what are the replica plating problems
In practice using velvet replica plating has a number of drawbacks
It’s easy to smear colonies together
often not all colonies are lifted
transferred colonies can be confused with contaminating bacteria
what is a better method than replica plating
often cleaner/clearer to use sterilised wooden cocktail sticks to duplicate colonies in a grid on plates
which are the easiest mutants to select
those which gain a new phenotype as the mutation introduces a marker gene
e.g. resistance to antibiotics (or other biocides)
The only things that grow on a plate containing the biocide
or gaining a new trait
Cells that grow have a visible phenotype
what is used for selection by new traits
green fluorescent protein
bioluminescence genes (lux)
lacZ - blue/white colony screening
what does GFP do
Good marker on plasmids –if they’re green, taken up plasmid and expressing GFP marker
what causes bacteria to glow (selection by new traits)
Plasmid contains many lux genes put into bacteria e.g. e.coli will glow
Use as selection markers
what does lacZ show
Determine if bacteria has taken up a particular plasmid
what is complementation
ability of a cloned DNA fragment to ‘complement’ a mutation
i.e. the conversion of the mutant phenotype back to wild type
what happens in plasmid complementation - lacZ or lac^- produced
beta-galactosidase mutant
produces lacZ or Lac^-
is white colonies
what happens in plasmid complementation lacZ^- (placZ^+) or Lac^+
beta-galactosidase insert lacZ gene on a plasmid
forms complemented mutant
lacZ^- (placZ^+) or Lac^+
blue colonies
