Transcript Complementation

Genetics and Cell Biology
(D211P1)
Dr Lee Garratt
Plant Sciences, Sutton Bonington Campus
[email protected]
Lecture content:
 Complementation
 Collinearity of Gene & Protein
 Genetic Code
 tRNA & Translation
 Bacterial RNA Transcription
 The lac Operon
What is Genetics?
 Genetics tries to answer four questions:
A) What are genes – what is their structure?
B) How do genes work?
C) How are genes passed on to the next generation?*
D) How do genes change with evolution?
*Dr
Ian Taylor will cover (C).
Complementation
Haploid and diploid organisms
DIPLOID organisms
have two copies of
each chromosome.
HAPLOID organisms
have one copy of each
chromosome.
 and of each gene.
 and of each gene.
Haploid and diploid organisms
 Most higher organisms are diploid,
though most go through a haploid
stage in the life cycle. This may be
extensive in some (e.g. mosses).
 Bacteria and viruses are haploid.
Haploid and diploid organisms
 If a gene is mutated in a haploid organism, the
effect will be seen immediately as a mutant
phenotype.
 In a diploid organism, this may not happen
because the unmutated (wild type) copy of the
gene will be dominant over the mutated one.
 Dominance is related to complementation.
Microorganisms
Microorganisms are good for genetic
studies because:
1. They are small and simple so they have
few genes.
2. Some grow on simple defined media –
inorganic salts & sugar.
3. The generation time is very short (20
minutes for some bacteria).
Microorganisms
 Yeast is a microorganism that is normally
haploid but can be induced to fuse and grow
as a diploid.
 Yeast has no visible features so we can’t get
mutations that change eye colour etc. but we
can get nutritional mutants.
 One such mutant cannot make the amino acid
proline and will only grow if you add proline
to the growth medium.
Dominance
Take a haploid yeast cell with a
normal proline biosynthesis gene
and put it on a minimal medium
with no added proline.
Will it grow?
Yes
Dominance
Take a haploid yeast cell with a
mutated proline biosynthesis
gene and put it on a minimal
medium with no added proline
Will it grow?
No
Dominance
Take a diploid yeast cell with two
normal proline biosynthesis
genes and put it on a minimal
medium with no added proline
Will it grow?
Yes
Dominance
Take a diploid yeast cell with two
identical mutated proline biosynthesis
genes and put it on a minimal medium
with no added proline
Will it grow?
No
Dominance
These are both homozygous (Greek homos –
same). They have two identical chromosomes.
What about a heterozygous (Greek heteros –
other) cell in which each of the same pair of
chromosomes is different?
Dominance
Take a diploid yeast cell with one
mutated proline biosynthesis gene
and one normal one and put it on a One good copy
minimal medium with no added
of the gene is
proline
enough
Will it grow?
Yes
Dominance
• Each gene instructs the cell how to make a
protein – in this case an enzyme needed to
make proline. The cell only needs one set of
instructions.
• The normal gene is able to specify an enzyme
that works and is dominant. The mutated
gene has lost this ability and is recessive.
Mutants
1
2
Each mutation is likely to be different, even if it
occurs in the same gene. However, if we take a
different proline requiring mutant and repeat the
dominance test, we will get the same result.
The yeast will grow without proline.
Mutants
2
1
Take a diploid yeast cell with two
different mutated proline biosynthesis
genes and put it on a minimal medium
with no added proline
Will it grow?
No
Complementation
3
1
Take a diploid yeast cell with another
two mutated proline biosynthesis genes
and put it on a minimal medium with
no added proline.
Will it grow?
Yes
Complementation
3
2
Take a diploid yeast cell with another
combination of different mutated
proline biosynthesis genes and put it
on a minimal medium with no added
proline.
Will it grow?
Yes
Complementation
• This is one case where two wrongs can
sometimes make a right. Genes that behave in
this way are said to complement each other.
• This obeys rules. A given pair of genes will
either always complement to give a wild-type
phenotype, or always not complement.
Complementation
• Two mutant genes that always do complement
each other are said to be in different
complementation groups.
• Two that always don’t complement are said to
be in the same complementation group.
• Different groups are given different letters proA,
proB, proC etc
Complementation
• Complementation groups arise because many
biochemical changes require several chemical
reactions.
• Each reaction is carried out by a different
enzyme and each enzyme is specified by a
different gene.
• There isn’t just one proline gene, there are
several: proA, proB, proC etc.
Complementation
• Complementation is the standard genetic test to
find out whether two genes have the same
function.
• “Genes” defined by a complementation test
should not strictly be referred to as genes but as
cistrons. In many cases, a cistron is the same as a
gene but not always – One example is the lac
operon, to be discussed in a later lecture.
Cross-Feeding
• Cross-feeding is related to complementation
because it arises when mutations affect different
steps in a biochemical pathway.
• This can be seen in mutants of the bacterium
Escherichia coli (often shortened to E. coli). E.
coli is always haploid but it can be made diploid
for part of its only chromosome (merodiploid) by
a genetic trick – which we won’t go into now.
Cross-Feeding
• Tryptophan requiring mutants of E. coli fall into
several complementation groups: trpA, trpB,
trpC, trpD and trpE.
• When put onto a minimal agar plate with no
tryptophan, these will not grow but when several
different types are spread in a particular pattern
there is sometimes a little growth.
Cross-Feeding
Cross-Feeding
• The mutants grow very slightly where
they are near to another mutant. They
must be getting some diffusible
substance through the agar and it can’t
be tryptophan.
Cross-Feeding
• A test shows that :
• The trpB mutant needs tryptophan (nothing else
will do).
• The trpA mutant needs either tryptophan
or indole.
Cross-Feeding
IGP
A
A
B
TrpA Indole
enzyme
Indole
Trp TrpB
enzyme
B
Complementation
Complementation is
similar but everything is
going on in the same cell.
Complementation
Compound 1
A
A
Enzyme A
Compound 2
B
B
Enzyme B
Proline