Lewin Benjamin (ed.) Genes IX
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event
involves the
genes
of or'ly
7ne of the
homologous
chromosomes, and as a resulr
the
alleles on
the other chromzsome are
not
expressed
in
the same
cell.This
phenomenon
is called allelic
exclusion.
The occurrence of allelic
exclusion compli-
cates
the analysis
of somatic recombination.
A
probe
reacting with a
region that has rearranged
on one
homolog will also
detect the allelic
sequences
on
the other homolog. We
are there-
fore compelled
to analyze
the different
fates of
the
two chromosomes
together.
The usual
pattern
displayed
by a rearranged
active
gene
can be
interpreted in terms of
a dele-
tion
of the
material between the
recombining
V and
C loci.
Two
types of
gene
organization
are seen
in
active cells:
.
Probes to the
active
gene
may
reveal
one
rearranged copy and
one
germline
copy.
We assume, then,
that
joining
has
occurred
on one chromosome,
whereas
the
other chromosome
has
remained
unaltered.
.
TWo different
rearranged
patterns may
be
found,
indicating that the
chro-
mosomes
have suffered
independent
rearrangements.
In
some
of
these
instances,
material between
the recom-
bining
V and C
gene
segments
is entirely
absent
from the cell line.
This is most
easily
explained by
the occurrence
of
independent
deletions
(resulting
from
recombination)
on each chromosome.
When two
chromosomes
both
lack the
germline
pattern,
usually
only one of
them
has
passed
through a
productive
rearrangement
to
generate a functional
gene. The other
has
suffered
a
nonproductive
rearrangement;
this
may take several
forms,
but in each
case
the
gene
sequence cannot
be expressed
as
an
immunoglobulin
chain.
(It
may be incomplete,
for example
because
D-J
joining
has
occurred
but
V-D
joining
has not
followed; or
it may be
aberrant,
with
the
process
completed
but fail-
ing to
generate a
gene
that
codes for
a func-
tional
protein.)
The
coexistence
of
productive
and
nonpro-
ductive
rearrangements
suggests
the existence
of
a feedback
loop to control the
recombination
process. A model
is outlined
in
,
,l
:r::
'
r
Suppose
that
each cell
starts with
two loci
in
the
unrearranged
germline
configuration
Ig0.
Either
of these
loci
may be rearranged
to
gen-
erate a
productive
gene
Ig+ or a nonproductive
gene Ig-.
If the
rearrangement
is
productive, the
syn-
thesis of
an active
chain
provides a trigger
to
prevent
rearrangement
of the
other
allele.
The
active cell
has the
configuration
Ig0/lg+.
If the
rearrangement
is
nonproductive,
it
creates
a cell
with the
configuration
Ig0ttg-.
There is
no impediment
to
rearrangement
of
the
remaining
germline allele.
If this
rearrange-
ment
is
productive,
the expressing
cell
has
the
configurationlg+llg-.
Again,
the
presence
of
an
active
chain
suppresses
the
possibility of fur-
ther rearrangements.
TWo successive
nonproductive
rearrange-
ments
produce
the
cell
Ig-lIg-.In
some
cases
an
Ig-lIg-
cell can
try
yet again.
Sometimes
the
observed
patterns of
DNA
can
only
have
been
generated
by
successive
rearrangements.
The crux
of the
model
is that
the cell
keeps
trying to
recombine
V
gene segments
and
C
gene
segments
until
a
productive
rearrange-
ment is achieved.
Allelic
exclusion
is caused
by the
suppression
of
further
rearrangement
as soon as
an active
chain
is
produced.
The use
of this
mechanism
in
vivo
is demonstrated
by
the
creation
of transgenic
mice
whose
germline
has a
rearranged
immunoglobulin
gene.
i';.:,.,,'r
,, li A successful
rearrangementto
produce
an
active
light
or
heavy
chain
suppresses
further
rearrangements
of the
same
type,
and
results
jn
aLletic
exctusion.
23.9
Attetic
Exclusion
Is
Triggered
by
Productive
Rearrangement
583
Expression
of
the transgene
in
B cells
sup-
presses
the
rearrangement
of endogenous
genes.
Allelic
exclusion
is independent
for
the
heavy-
and
light-chain
loci.
Heavy-chain genes
usually
rearrange
first.
Allelic
exclusion
for
light
chains
must
apply
equally
to
both families
(cells
may have
either
aclive
r
or
i,light
chains).
Ir is
Iikely
that
the
cell rearranges
its
r
genes
first,
and
tries
to rearrange
l,
only if
both K
attempts
are
unsuccessful.
There
is an interesting paradox
in
this series
of
events.
The
same
consensus
sequences
and
the same
V(D)J
recombinase
are involved
in
the
recombination
reactions
at H, r,
and l, loci,
and
yet
the
three loci
rearrange
in a
set order.
What
ensures
that
heavy
rearrangement
pre-
cedes
light
rearrangement,
and that r
precedes
l,?
The loci
may
become
accessible
to the
enz).rne
at different
times.
possibly
as
the result
of
tran-
scription.
Transcription
occurs
even
before
rearrangement,
although
of course
the
prod-
ucts
have
no
coding function.
The
transcrip-
tional
event
may
change
the
structure
of
chromatin,
making
the
consensus
sequences
for recombination
available
to
the enzyme.
@
The
RAG Proteins
Catalyze
Breakage
and
Reunion
r
The
RAG
proteins
are necessary
and
sufficient for
the
cleavage
reaction.
.
RAGl
recognizes
the nonamer
consensus
sequences
for
recombination.
RAG2
binds to
RAGl
and
cteaves
at the heptamer.
.
The reaction
resembles
the
topoisomerase-tike
resotution
reaction
that
occurs
in
transposition.
r
It
proceeds
through
a hairpin
intermediate
at the
coding
end; opening
of the hairpin
is
responsibte
for
insertion
of extra
bases
(P
nucleotides)
in
the
recombined gene.
o
Deoxynucteoside
transferase
inserts
additional.
N
nucteotides
at
the coding
end.
o
The
codon
at the
site of
the V-(D)J
joining
reaction
has
an extremely
variabte
sequence
and
codes
for
amino
acid
96 in
the antigen-binding
site.
.
The
doubte-strand
breaks
at
the coding
joints
are
repaired
by
the same
system
invotved
in
nonhornologous
end-joining
of damaged
DNA.
o
An
enhancer
in
the
C
gene
activates
the
promoter
of the
V
gene
after recombination
has
qenerated
the
intact
immunogtobutin
gene.
The proteins
RAGI
and
RAG2
are
necessary
and
sufficient
to
cleave
DNA
for V(D)J
recom-
CHAPTER
23
Immune
Diversitv
bination.
They
are coded
by two
genes.
sepa-
rated
by
<10
kb
on the
chromosome,
whose
transfection
into fibroblasts
causes
a suitable
substrate
DNA to
undergo
the
V(D)J
joining
reaction.
Mice that
lack
either RAGL
or RAG2
are unable
to recombine
their immunoglobu-
lins
or T cell
receptors,
and
as a result
have
immature
B lymphocytes
and T
lymphocytes.
The
RAG
proteins
together
undertake
the
cat-
alytic
reactions
of cleaving
and
rejoining
DNA,
and
also
provide
a
structural framework
within
which the
reactions
occur.
RAGI
recognizes
the
heptamer/nonamer
signals with
the appropriate
l2l23
spacing
and
recruits
RAG2
to the
complex.
The
nonamer
provides
the site for
initial recognition,
and
the
heptamer
directs
the site
of cleavage.
The
reactions
involved
in
recombination
are
shown in
Fl#{Jftil
f^1.1.+.
The
complex
nicks
one strand
at
each
junction.
The
nick
has 3'-OH
and
5'-P
ends. The
free 3'-OH
end then
attacks
the
phosphate
bond
at the corresponding
posi-
Iion in
the other
strand
of the
duplex.
This
creates
a hairpin
at the coding
end,
in
which
the 3'end
of one
strand is
covalently
linked
to the 5'
end
of
the other
strand;
it leaves
a blunt
double-
strand
break
at the
signal
end.
This
second
cleavage is
a transesterification
reaction
in
which
bond
energies
are
conserved.
It resembles
the
topoisomerase-like
reactions
calalyzedby
the resolvase
proteins
of
bacterial
transposons (see
Section
2I.9,
TnATfansposi-
tion
Requires
Tlansposase
and Resolvase).
The
parallel
with these
reactions
is
supported
further
by a homology
between
RAGI
and bacterial
invertase proteins
(which
inyert
specific
seg-
ments
of DNA
by
similar
recombination
reac-
tions). In
fact,
the RAG proteins
can
insert
a
donor
DNA
whose
free
ends
consist
of the
appropriate
signal
sequences (heptam
er
-
|
2 | 23
-
spacer
nonamer)
into
an
unrelated
target
DNA
in
an in vitro
transposition
reaction.
This
sug-
gests
that somatic
recombination
of immune
genes
evolved
from
an ancestral
transposon.
It
also
suggests
that
the RAG
proteins
are
respon-
sible
for
chromosomal
translocations
in
which
Ig
or TCR loci
are
connected
to other
loci.
The hairpins
ar the
coding
ends
provide
the
substrate
for
the next
stage
of reaction.
If
a sin-
gle-strand
break
is introduced
into
one
strand
close
to the
hairpin,
an
unpairing
reaction
at
the end
generates
a single-stranded
protrusion.
Synthesis
of a complement
to the
exposed
sin-
gle
strand
then
converts
the
coding
end to
an
extended
duplex.
This
reaction
explains
the
introduction
of P
nucleotides
at
coding
ends;
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