Microbial Genome
Organisation
Lecture 6 - DNA re-arrangements and gene
expression
Prof. Duncan Shaw
The material for this lecture is from these sources:

"Genes and Genomes" by M. Singer and P. Berg, University Science Books
1991, Chapter 10

Weiser JN et al (1989), "The molecular mechanism of phase variation of H.
Influenzae lipopolysaccharide"; Cell 59, 657-665

Hammerschmidt S et al (1996), "Capsule phase variation in Neisseria
meningitidis"; Molec.Microbiol. 20, 1211-1220

Borst P & Greaves DR (1987), "Programmed gene rearrangements altering
gene expression"; Science 235, 658-667
Some further reading: a review of the adaptive mutation hypothesis:

Rosenberg SM (1994), "In pursuit of a molecular mechanism for adaptive
mutation"; Genome 37, 893-899
Click on one of these links to move to a specific topic, or browse through the
whole lecture:
Introduction
DNA re-arrangements
Yeast mating-types
The cassette model
Other examples
Slipped strand mispairing
My home page
Introduction
You have already had lectures on genome mapping of various kinds, and next term
those of you who take the module "Chromosome Organisation and development"
will hear more about the organisation of genomes. Mostly, these lectures will have
used the paradigm of a genome as an entity whose structure is stable. But there are
several important ways in which the organisation of a genome can change (as well
as mutation, etc). Some classes of DNA re-arrangements result in changes of gene
expression and hence of the phenotype of the organism. It's this type of change that
will be covered in this lecture.
DNA re-arrangement - programmed and unprogrammed
There is a distinction to be made between programmed and unprogrammed DNA
re-arrangements.

Programmed re-arrangements appear to have a "purpose" - they are involved
in the regulation of gene expression, maybe in response to an external
stimulus. Examples include immunoglobulin and T-cell receptor genes,
phase variation in bacteria such as Salmonella, Neisseria and Haemophilus,
and yeast mating-type switching.

Unprogrammed re-arrangements could be viewed as a class of mutation.
They include duplication and transposition of repeated sequences,
transposons, and viral genomes, and translocations between chromosomes in
eukaryotes. If this affects gene expression, it is random and non-specific
(and will usually result in the loss of the gene's function).
Just to confuse things, I should say that this distinction is arbitrary and arises from
scientists' need to classify and assign a purpose to events. The same molecular
mechanism may underlie both programmed and unprogrammed DNA rearrangements, but the outcomes can be quite different.
Yeast mating-type switching
Many yeasts, including Saccharomyces cerevisiae (the example that is used here)
can exist as haploid or diploid forms. Diploids are heterozygous for the matingtype locus, MAT, and haploid cells can be either MATa or MATalpha.
This picture shows the life cycle
of the yeast. The red and blue
cells are a and alpha types. The
difference between homo- and
hetero-thallic is that the former
have an active HO gene and can
switch spontaneously between
mating type, and therefore a
single spore can give rise to a
self-fertile population, whereas
the latter do not have HO and
maintain the same mating type
during the haploid growth cycle.
Genetic analysis (i.e. the use of
mutants) shows that the
following genes are required for
mating-type switching to occur:

MAT

HO, which codes for an
endonuclease

HMLalpha (for MATa ->
MATalpha switch)

HMRa (for MATalpha -> MATa switch)
All except HO are on yeast chromosome 3. The "cassette" model was proposed to
explain mating-type switching, which occurs at too high a frequency to be due to
normal mutation events. The model proposes that the HMLalpha and HMRa loci
contain "silent copies" of alpha and a mating type genes. Replicas of either can be
copied into MAT, the active locus, where they are expressed.
This model was supported by the following experiments:
1. Southern blot analysis.
The MATalpha allele was cloned by complementation of a MATalpha-, HO- yeast
mutant. This was used as a probe on Southern blots of yeast DNA to investigate
the structures of the MAT, HMLalpha and HMRa loci:
The identity of all these genes was confirmed by
cloning and sequencing. The experiment shows
that the MAT locus, which is expressing the
genetic information in it, changes in length
between a and alpha forms, but the silent
HMLalpha and HMRa loci stay the same.
2. Electron microscopy of DNA hybrids
To see which parts of each gene were homologous and which were different, all
pairs of genes were mixed, denatured, reannealed and visualised by EM:
The cassette model
These and other data were
put together into the
cassette model:
The gene products shown
are regulatory proteins,
that control the ability of
the yeast to mate (and
other aspects of its
phenotype).

alpha1 is a positive
regulator, that switches on genes required for the alpha phenotype, including
alpha factor, a secreted pheromone

alpha2 is a negative regulator that turns off a-specific genes

in diploid cells, a1 and alpha2 combine to inhibit alpha1 (and hence all the
genes it regulates) and repress HO, and turn on the meiosis pathway if the
diploid cells are starved
Why aren't HML and HMR expressed all the time? They have the same DNA
sequence as MAT which is expressed. The reason is that the HML and HMR loci
are "silenced" by the products of the SIR genes (Silent Information Regulators).
These proteins interact with regions of DNA ~1000bp upstream of the loci that are
transposed (HML and HMR). These DNA sequences are called "silencers". They
can be turned around or moved up to 2.5kb away and they are still active. So in the
cassette model, transposition moves the genes from a transcriptionally silent site to
a transcriptionally active one. Silencing is believed to act through a localised
change in the structure of chromatin.
The mechanism by which the genetic information in MAT is replaced by HML or
HMR is known as "gene conversion". It is a process that is found in many aspects
of genetics and it works as shown below. The initial event is cutting of the DNA at
specific sites by the HO
endonuclease:
As you can see, the outcome
of this process is that the
original DNA sequence (blue)
is replaced by the homologous
but different sequence (red),
but the red sequence itself is
left unchanged.
Other examples of gene regulation by DNA re-arrangement
Trypanosomes
Trypanosomes are protozoan organisms that can live as parasites in either the
tsetse fly or mammals (including humans, cattle). They cause sleeping sickness in
humans.
This is the life cycle of the trypanosome. VSG
stands for variable surface glycoprotein, which
is a major component of the coat of the
organism, and the main antigenic determinant.
During the course of the life cycle, the VSG can
switch between a number of different types.
During the course of infection, about 100
different VSG types can be produced. The
trypanosome cycles through a series of types
and this keeps it one step ahead of the host's
immune defences. Altogether, there are about
1000 different VSG genes in the trypanosome's
genome, but at a given time, only one gene is
being actively transcribed. The rate of switching
between types is about 1/1,000,000 per cell
division.
The repertoire of VSG types produced by a
single population of trypanosomes is called a serodeme.
The diagram shows how different VSG genes
become the active one. A VSG gene is
transcribed when it is in an "active site", close
to a telomere. All the other VSG genes are
silent.
To be activated, a VSG gene must be
transposed from a silent site to an active site,
close to a telomere. When this happens, the
gene that was previously at the active site is
lost.
This is believed to happen by gene
conversion, as described above for yeast
mating types. There are regions of homology
upstream and downstream from each VSG
gene, that initiate the gene conversion
process. The upstream region of homology
includes a few copies of a 70bp repeat. Gene
conversion is the most likely mechanism
because (1) the gene copy at the active site is lost and (2) the amount of upstream
and downstream DNA that is transposed can vary between different occurrences of
the same gene replacement.
Although there are several sites close to telomeres where VSG genes can be
transposed, not all of these sites are active. Why should this be, since there are no
obvious differences between the actual DNA sequence at these sites? The answer
is not known for sure but may involve silencing due to modification of C bases in
the non-active sites, or changes in chromatin structure that inhibit access to
transcription factors. Compare this with yeast mating-type switching.
Phase variation in Haemophilus influenzae

H influenzae is a gram negative bacterium, that infects the respiratory tract
where it can be involved in septicemia and meningitis

It has a cell-surface lipopolysaccharide (LPS) that can have different
structural forms

The LPS switches between forms at a rate of 1/100 per cell division - this is
called phase variation

Genetic experiments showed that a locus, licABCD, is required for phase
variation
This shows the first few bases of the licA
gene. It contains a number of tandem repeats
of the tetranucleotide CAAT (N = between 27
and 32 copies). When the gene from H
influenzae strains with different LPS was
sequenced, it was found that they also had
different CAAT numbers
Different CAAT numbers cause the reading
frame of the protein in the upstream region to
be shifted. This diagram shows some
examples. In some cases (e.g N=29) there are
no in-phase Met codons upstream and so no
protein is produced. N=30 or 31 produce
protein forms that differ in their N-terminal
regions.
Phase variation in Neisseria meningitidis

N meningitidis is another human pathogen, the invasive form of which is
associated with meningitis.

It has 12 different antigenic forms, that differ due to variation in the
polysaccharide capsule

The gene involved is siaD, which codes for an enzyme of polysaccharide
biosynthesis

When the siaD gene was sequenced from N. meningitidis strains of different
pathogenicity, a stretch of repeated Cs was found near the 5' end.
In the wild-type strain there are 7 Cs and a
functional protein is made. In mutants with
different polysaccharide capsules, there are
different numbers of Cs so the reading
frame of the protein is disrupted, and a
non-functional protein is made.
It is possible to get revertants to wild-type
from a mutant strain, and in these the
number of Cs has gone back to 7.
A common molecular mechanism - slipped-strand
mispairing
Both of the last 2 examples (H. influenzae and N. meningitidis) have something in
common - the mechanisms of phase variation are controlled by a DNA sequence
that is a simple sequence repeat (SSR), in one case a tetranucleotide repeat, and in
the other a mononucleotide.
SSRs are known to be prone to a high rate of mutation via a mechanism called
slipped strand mispairing (or replication slippage).
This picture illustrates slipped-strand
mispairing. The second frame shows the 2
DNA strands dissociating, e.g. during DNA
replication. Because it is a repeated DNA
sequence, it can re-anneal in either the
correct way or shifted as in the 3rd frame.
The mispaired DNA sequence is recognised
as a replication error by the DNA repair
system. One way in which it could be
repaired is by nicking both strands and
inserting an extra base opposite each
mispaired base (4th frame).
There are some other examples of mutations
in SSRs that cause a change in phenotype in human genetic disease, for example. This
will be covered in the Honours module on
Chromosome Organisation and
Development.
It is possible, but not proven, that under conditions where a mutation in a SSR
would be favourable, e.g. to change the antigenic properties of the organism, the
cell might up-regulate its DNA repair system to cause the process shown in the
diagram to be accelerated. There is an interesting (though controversial) example
of this in the phenomenon of adaptive mutation, where starved, non-dividing
bacteria can acquire mutations that might allow them to start growing again.
The end of the lecture!