U.S. patent application number 13/606696 was filed with the patent office on 2013-03-07 for activation induced deaminase (aid).
This patent application is currently assigned to MEDICAL RESEARCH COUNCIL. The applicant listed for this patent is Rupert Christopher Landsdowne Beale, Reuben Harris, Michael S. Neuberger, Svend K. Petersen-Mahrt. Invention is credited to Rupert Christopher Landsdowne Beale, Reuben Harris, Michael S. Neuberger, Svend K. Petersen-Mahrt.
Application Number | 20130059931 13/606696 |
Document ID | / |
Family ID | 29424146 |
Filed Date | 2013-03-07 |
United States Patent
Application |
20130059931 |
Kind Code |
A1 |
Petersen-Mahrt; Svend K. ;
et al. |
March 7, 2013 |
ACTIVATION INDUCED DEAMINASE (AID)
Abstract
The invention is directed to a cell comprising a nucleic acid
encoding an Activation Induced Deaminase (AID) polypeptide, a
fusion protein comprising an AID polypeptide, and methods of using
a nucleic acid encoding an AID polypeptide.
Inventors: |
Petersen-Mahrt; Svend K.;
(Cambridge, GB) ; Harris; Reuben; (Cambridge,
GB) ; Neuberger; Michael S.; (Cambridge, GB) ;
Beale; Rupert Christopher Landsdowne; (Cambridge,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Petersen-Mahrt; Svend K.
Harris; Reuben
Neuberger; Michael S.
Beale; Rupert Christopher Landsdowne |
Cambridge
Cambridge
Cambridge
Cambridge |
|
GB
GB
GB
GB |
|
|
Assignee: |
MEDICAL RESEARCH COUNCIL
LONDON
GB
|
Family ID: |
29424146 |
Appl. No.: |
13/606696 |
Filed: |
September 7, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12911292 |
Oct 25, 2010 |
8288160 |
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13606696 |
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10985321 |
Nov 10, 2004 |
7820442 |
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12911292 |
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PCT/GB03/02002 |
May 9, 2003 |
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10985321 |
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Current U.S.
Class: |
514/789 ; 435/18;
435/227; 435/252.33; 435/254.2; 435/320.1; 435/354; 435/366;
435/441 |
Current CPC
Class: |
C12N 2510/00 20130101;
A61P 35/00 20180101; A61P 37/00 20180101; C07K 2319/00 20130101;
C12N 9/78 20130101 |
Class at
Publication: |
514/789 ;
435/227; 435/320.1; 435/18; 435/441; 435/252.33; 435/354; 435/366;
435/254.2 |
International
Class: |
C12N 9/78 20060101
C12N009/78; C12Q 1/34 20060101 C12Q001/34; C12N 15/01 20060101
C12N015/01; C12N 1/19 20060101 C12N001/19; A01N 61/00 20060101
A01N061/00; C12N 1/21 20060101 C12N001/21; C12N 5/10 20060101
C12N005/10; C12N 15/63 20060101 C12N015/63; A61K 45/00 20060101
A61K045/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 10, 2002 |
GB |
0210755.5 |
Jun 14, 2002 |
GB |
0213751.1 |
Jul 29, 2002 |
GB |
0217519.8 |
Claims
1. A cell comprising a nucleic acid encoding an Activation Induced
Deaminase (AID) polypeptide, or an AID variant, derivative or
homologue, and having a mutator phenotype.
2. The cell of claim 1, wherein said cell is a prokaryotic
cell.
3. The cell of claim 1, wherein said cell is an eukaryotic
cell.
4. The cell of claim 1, wherein the AID homologue is Apo1bec-1,
Apobec-1, Apobec3C or Apobec3G.
5. A fusion protein comprising an AID polypeptide, or AID variant,
derivative or homologue thereof, having a mutator phenotype
operably linked to one half of a specific binding pair.
6. The fusion protein of claim 5, wherein said one half of a
specific binding pair is a DNA binding domain.
7. A vector comprising a nucleic acid encoding the fusion protein
of claim 5.
8. A cell comprising a nucleic acid encoding the fusion protein of
claim 5.
9. A method for preparing a gene product having a desired activity,
comprising the steps of: a) expressing a nucleic acid encoding the
gene product in a population of cells according to claim 1; b)
identifying a cell or cells within the population of cells which
expresses a mutant gene product having the desired activity; and c)
establishing one or more clonal populations of cells from the cell
or cells identified in step (b), and selecting from said clonal
populations a cell or cells which expresses a gene product having
an improved desired activity.
10. A method as claimed in claim 9, wherein the nucleic acid
encoding the gene product is operably linked to the second half of
a specific binding pair.
11. A method of directing mutation to a specific gene product of
interest comprising: i) generating a nucleic acid construct
comprising a nucleic acid sequence encoding a specific gene product
operably linked to a DNA binding protein recognition sequence; ii)
transfecting said nucleic acid construct into a population of host
cells expressing the fusion protein of claim 6; ii) incubating said
transfected host cells under conditions suitable for allowing the
specific binding pairing of DNA binding protein to DNA binding
protein recognition sequence to occur; iv) identifying a cell or
cells within the population of cells which expresses a mutant gene
product having the desired activity; and v) establishing one or
more clonal populations of cells from the cell or cells identified
in (iv), and selecting from said clonal populations a cell or cells
which expresses a gene product having an improved desired
activity.
12. A method of identifying components of AID-dependent mutation
activity comprising expressing AID in a cell deficient in
expression or activity of a known gene and assessing mutator
activity compared to activity in a cell expressing said gene.
13. A method of screening for a modulator of AID activity
comprising: (a) expressing AID in a prokaryotic cell; (b)
maintaining the AID-expressing prokaryotic cell in the presence of
a selectable medium; (c) detecting the presence of colonies in the
absence or presence of a test compound, wherein a modified number
of colonies when compared to a sample in the absence of a test
compound is indicative of the ability of the test compound to
modify AID mutator activity.
14. A method of inducing a mutation in a cell comprising
administering an AID polypeptide or functional homologue
thereof.
15. A method for treating a disorder characterized by an increased
mutation rate, comprising administering an agent that modifies AID
functional activity or gene expression.
16. A method of decreasing hypermutation/resistance to a compound
such as an antibiotic in a population of bacteria comprising
modulating activity of a bacterial AID homologue.
17. A construct for use in the method of claim 11, said construct
comprising a coding sequence for the gene product of interest,
wherein said coding sequence is placed under the control of a first
promoter upstream of the coding sequence and further comprising a
second promoter downstream of the coding sequence, wherein said
first and second promoters are arranged in opposing orientation so
as to allow convergent transcription of the coding sequence.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a divisional of copending U.S.
patent application Ser. No. 12/911,292, filed Oct. 25, 2010, which
is a divisional of U.S. patent application Ser. No. 10/985,321,
filed Nov. 10, 2004, issued as U.S. Pat. No. 7,820,442, which is a
continuation of International Application No. PCT/GB03/02002, filed
May 9, 2003, the entirety of which is incorporated herein by
reference.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: One 7,161 Byte
ASCII (Text) file named "711017SequenceListing.TXT," created on
Sep. 5, 2012.
[0003] The present invention identifies that the expression of
Activation Induced Deaminase (AID) and its homologues, such as
Apobec, in cells confers a mutator phenotype and thus provides a
method for generating diversity in a gene or gene product as well
as cell lines capable of generating diversity in defined gene
products. The invention also provides methods of modulating a
mutator phenotype by modulating the expression or activity of AID
or its homologues.
BACKGROUND
[0004] In normal cells, a low mutation rate ensures genetic
stability and this depends on effective DNA repair mechanisms for
repairing the many accidental changes that occur continually in
DNA.
[0005] However, during the generation of antibodies, point
mutations occur within the V-region coding sequence of the antigen
receptor loci and the rate of mutation observed, called somatic
hypermutation, is about a million times greater than the
spontaneous mutation rate in other genes. The antigen receptor loci
are the only loci in human cells that undergo programmed genetic
alterations. However, the mechanisms that allow the nucleotide
changes to be controlled and targeted to the DNA of a precisely
specified part of the genome in this way is not known.
[0006] Functional antigen receptors are assembled by RAG-mediated
gene rearrangement and the isotype switch from IgM to IgG, IgA and
IgE is effected by class switch recombination. Aberrant forms of
RAG-mediated gene rearrangement and class switch recombination have
been shown to underpin many of the chromosomal translocations
associated with lymphoid malignancies. In the case of somatic
hypermutation, it was proposed several years ago by Rabbitts et al
(1984 Nature 309, 592-597) that the chromosomal translocations
which bring the c-myc proto-oncogene into the vicinity of the IgH
locus could make it a substrate for the antibody hypermutation
mechanism. Recent evidence using hypermutating cell lines has
provided evidence in support of this (Bemark, M and Neuberger, M.
S. 2000 Oncogene 19, 3404-3410). A wider role for aberrant
hypermutation came with the finding that several genes apart from
the immunoglobulin V genes can (without being translocated into the
Ig loci) apparently act as substrates for the antibody
hypermutation mechanism in that they exhibit an increased frequency
of point mutation in hypermutating B cells. Recent evidence also
points to a high frequency of mutations in many B cell tumours and
it has been proposed that this is a result of a transient
hypermutation phase caused by the antibody hypermutation mechanism.
In all these cases, the aberrant mutations are largely at dC/dG
residues.
[0007] An uncontrolled and enhanced rate of mutation in
non-antibody producing cells can also be deleterious. For example,
mutations are the hallmark of cancer and the enhanced rate of
mutation in cancer cells may explain their capability to
continually grow and evade the normal human defences. The "mutator
phenotype" hypothesis attributes this phenomenon to an increasing
rate of errors in DNA replication as a tumour grows. According to
this theory, genes encoding proteins normally interacting with
nucleotides such as DNA polymerases and DNA repair enzymes may be
faulty in cancer cells and therefore cause subsequent
mutations.
[0008] In vitro, understanding and harnessing the means for
controlling an enhanced rate of mutation can be usefully employed,
for example, in generating diversity of gene products such as
generating antibody diversity.
[0009] Many in vitro approaches to the generation of diversity in
gene products rely on the generation of a very large number of
mutants which are then selected using powerful selection
technologies. For example, phage display technology has been highly
successful as providing a vehicle that allows for the selection of
a displayed protein (Smith, G. P. 1985 Science, 228, 1315-7; Bass
et al. Proteins. 8, 309-314, 1990; McCafferty et al., 1990 Nature,
348, 552-4; for review see Clackson and Wells, 1994 Trends
Biotechnol, 12, 173-84). Similarly, specific peptide ligands have
been selected for binding to receptors by affinity selection using
large libraries of peptides linked to the C terminus of the lac
repressor Lacl (Cull et al., 1992 Proc Natl Acad Sci USA, 89,
1865-9). When expressed in E. coli the repressor protein physically
links the ligand to the encoding plasmid by binding to a lac
operator sequence on the plasmid. Moreover, an entirely in vitro
polysome display system has also been reported (Mattheakis et al.,
1994 Proc Natl Acad Sci USA, 91, 9022-6) in which nascent peptides
are physically attached via the ribosome to the RNA which encodes
them.
[0010] Artificial selection systems to date rely heavily on initial
mutation and selection, similar in concept to the initial phase DNA
rearrangement involving the joining of immunoglobulin V, D and J
gene segments which occurs in natural antibody production, in that
it results in the generation of a "fixed" repertoire of gene
product mutants from which gene products having the desired
activity may be selected.
[0011] Unlike in the natural immune system, however, artificial
selection systems are poorly suited to any facile form of "affinity
maturation", or cyclical steps of repertoire generation and
development. One of the reasons for this is that it is difficult to
generate enough mutations and to target these to regions of the
molecule where they are required, so subsequent cycles of mutation
and selection do not lead to the isolation of molecules with
improved activity with sufficient efficiency.
[0012] In vivo, after the primary repertoire of antibody
specificities is created by V-D-J rearrangement, and following
antigen encounter in mouse and man, the rearranged V genes in those
B cells that have been triggered by the antigen are subjected to
two further types of genetic modification. Class switch
recombination, a region-specific but largely non-homologous
recombination process, leads to an isotype change in the constant
region of the expressed antibody. Somatic hypermutation introduces
multiple single nucleotide substitutions in and around the
rearranged V gene segments. This hypermutation generates the
secondary repertoire from which good binding specificities can be
selected thereby allowing affinity maturation of the humoral immune
response. In chicken and rabbits (but not man or mouse) an
additional mechanism, gene conversion, is a major contributor to V
gene diversification.
[0013] Much of what is known about the somatic hypermutation
process which occurs during affinity maturation in natural antibody
production has been derived from an analysis of the mutations that
have occurred during hypermutation in vivo (for reviews see
Neuberger and Milstein, 1995 Curr. Opin. Immunol. 7, 248-254; Weill
and Reynaud, 1996 Immunol Today 17, 92-97; Parham, 1998
Immunological Reviews, Vol. 162 (Copenhagen, Denmark: Munksgaard)).
Most of these mutations are single nucleotide substitutions which
are introduced in a stepwise manner. They are scattered over the
rearranged V domain, though with characteristic hotspots, and the
substitutions exhibit a bias for base transitions. The mutations
largely accumulate during B cell expansion in germinal centres
(rather than during other stages of B cell differentiation and
proliferation) with the rate of incorporation of nucleotide
substitutions into the V gene during the hypermutation phase
estimated at between 10.sup.-4 and 10.sup.-3 bp.sup.-1
generation.sup.-1 (McKean et al., 1984; Berek & Milstein,
1988). However, a greater understanding of the steps involved in
these later stages of hypermutation would enable a more diverse
range of gene products to be obtained.
[0014] All three of the above processes, somatic hypermutation,
gene conversion and class-switch recombination, have been shown to
depend upon activity of the protein Activation Induced Deaminase
(AID) (Muramatsu et al. (1999); Muramatsu M. et al. (2000); Revy,
P. et al. (2000); Arakawa, H. et al. (2002); Harris, R. S. et al.
(2002); Martin, A. et al. (2002) and Okazaki, I. et al. (2002))
which has been suggested (by virtue of its homology with Apobec-1
(Muramatsu et al. (1999)) to act by RNA editing. However, evidence
that the three processes could be initiated by a common type of DNA
lesion (Maizels et al. (1995); Weill et al. (1996); Sale et al.
(2001); Ehrenstein et al. (1999)) taken with the fact that first
phase of hypermutation targets dG/dC (Martin et al. (2002); Rada et
al. (1998); Wiesendanger et al. (2000)) has suggested that AID may
act directly on dG/dC pairs in the immunoglobulin locus. However,
to date, the actual function of AID has not been described.
[0015] The AID homologue Apobec-1 has been identified as playing a
role in modifying RNA. Apobec-1 is a catalytic component of the
apolipoprotein B (apoB) RNA editing complex that performs the
deamination of C.sub.6666 to U in intestinal apoB RNA thereby
generating a premature stop codon. Indeed, the oncogenic activity
of Apobec-1, identified by its overexpression in transgenic mice,
has previously been attributed to its RNA editing activity acting
on inappropriate substrates.
[0016] Deamination of cytosine to uracil can occur in vivo at the
level of nucleotide and in DNA as well as RNA. In the context of
DNA, the low level deamination of cytosine to uracil which takes
place spontaneously (and which might be of relatively minor
significance when it occurs with free nucleotides or in mRNA) can
have major effects, contributing to genome mutation, cancer and
evolution (Lindahl, T. (1993) Nature 362, 709-715). However, to
date, there is no biochemical evidence that APOBEC family members
can trigger such deamination in vitro.
SUMMARY OF THE INVENTION
[0017] The present inventors have demonstrated that expression of
AID in Escherichia coli gives a mutator phenotype yielding DNA
nucleotide transitions at dG/dC. The mutation frequency is enhanced
by deficiency in uracil-DNA glycosylase indicating that AID acts by
deaminating dC residues in DNA.
[0018] In addition, the expression of AID homologues, Apobec-1,
Apobec3C and Apobec3G, including their expression as part of a
fusion protein in E. coli, also yields a mutator phenotype and
these homologues show an increased potency of mutator activity on
DNA sequences when compared to AID.
[0019] Furthermore, deamination of cytosine to uracil in DNA can be
achieved in vitro using partially purified APOBEC1 from extracts of
transformed Escherichia coli. Its activity on DNA is specific for
single-stranded DNA and exhibits dependence on local sequence
context.
[0020] Accordingly, in a first aspect of the invention there is
provided a cell modified to express AID, or an AID variant,
derivative or homologue, and having a mutator phenotype.
[0021] Suitably, the cell is modified to stably express AID, or an
AID variant, derivative or homologue, and having a mutator
phenotype.
[0022] By "stable expression" of a gene is meant that the gene and
its expression is substantially maintained in successive
generations of cells derived from transfected cells. In particular,
the term "stable expression" is not intended to encompass the
transient expression of a protein in a bacterial cell for the
purpose of protein purification.
[0023] In another embodiment, the cell is transiently transfected
to express AID, or an AID variant, derivative or homologue, and
having a mutator phenotype.
[0024] As used herein, "mutator phenotype" means an increased
mutation frequency in the transfected cells modified to express AID
or its homologues when compared to non-modified, non-transfected
cells. Methods for measuring mutation frequency are described
herein. Suitably the mutations are nucleotide transitions at dG/dC
as a result of deamination of dC residues in DNA. The term "mutator
activity" refers to the activity that confers the mutator
phenotype.
[0025] In one embodiment, said cell is a prokaryotic cell, such as
bacteria. Suitable bacteria include E. coli.
[0026] In another embodiment, the cell is a modified eukaryotic
cell in which altered AID expression has been induced by
introduction of AID gene with the proviso that said eukaryotic cell
is not a cell of the human B lymphocyte lineage and, in particular,
is not a human Ramos, BL-2 or CL-01 cell nor a cell derived from
the chicken cell line, DT40. Suitably said cell is derived from
mouse or man and is capable of generating immunoglobulin diversity
through somatic hypermutation or class switching.
[0027] In another embodiment, the AID homologue is Apobec and is,
in particular, selected from Apobec family members such as
Apobec-1, Apobec3C or Apobec3G (described, for example, by Jarmuz
et al (2002)).
[0028] In yet another embodiment, the AID variant is a fusion
protein. Suitably, said fusion protein is AID, Apobec-1, Apobec3C
or Apobec3G in which a heterologous protein or peptide domain has
been fused at either its N- or C-terminus. Preferably, the
heterologous peptide is fused at the amino terminus. Suitably, said
heterologous peptide domain is a binding domain which is one half
of a specific binding pair which can interact with the second half
of said pair to form a complex. Suitable binding pairs include two
complementary components which can bind in a specific binding
reaction. Examples of specific binding pairs include
His-tag--Nickel, DNA binding domain--DNA binding domain recognition
sequence, antibody--antigen, Biotin--Streptavidin etc.
[0029] The data presented herein are consistent with AID or its
homologues activating deamination of dC as an enhancement of the
effect is observed in cells lacking uracil-DNA glycosylase
(UDG).
[0030] Accordingly, in another embodiment, said cell further
comprises a genetic background which confers an enhanced mutator
phenotype effect. In a particularly preferred embodiment, the
genetic background of a prokaryotic cell confers a UDG deficiency
on the cell. Said UDG deficiency is preferably induced by
interfering with UDG expression such as, for example, creating a
ung-background. In some E. coli ung-1 mutants, some back up UDG
activity is provided by the product of the mug gene. Thus, in a
further embodiment, the cell comprises a combined background of
ung- and mug-.
[0031] The introduction of modified expression of AID or an AID
homologue into a cell can increase the mutation rate above the
background mutation rate that would normally be observed in that
cell. Suitably, the modified cell is capable of generating
mutations in a defined gene product. This can be particularly
useful in the generation of gene diversity for example in the
generation of antibody diversity where the defined gene product is
an immunoglobulin V region gene.
[0032] Such cells according to any embodiment of the first aspect
and displaying an enhanced rate of mutation can be useful in a
method for preparing a gene product having a desired activity.
[0033] Preferably the gene product which is desired to mutate is
provided to AID or its homologues as single-stranded DNA. Single
stranded DNA may be provided by introducing single stranded DNA
directly or by introducing double stranded DNA which is later
converted to single stranded, for example, through enzymatic action
such as helicase or transcriptase activity.
[0034] In another aspect of the invention, there is provided a
fusion protein comprising an AID, or AID variant, derivative or
homologue, polypeptide having a mutator phenotype operably linked
to one half of a specific binding pair.
[0035] The term "operably linked" refers to a juxtaposition wherein
the components described are in a relationship permitting them to
function in their intended manner. For example, an AID polypeptide
"operably linked" to one half of a specific binding pair is linked
through ligation of the nucleic acid coding sequences or otherwise
such that a fusion protein is produced in which the mutator
activity of AID is unimpaired whilst allowing the specific binding
pair to form through interaction of the said one half with its
complement.
[0036] In a preferred embodiment, the one half of the specific
binding pair in said fusion protein is a DNA binding domain.
[0037] Preferably, the AID homologue is one of the Apobec family of
proteins and, suitably, is selected from the group consisting of
Apobec-1, Apobec-3G and Apobec-3C.
[0038] In another aspect of the invention, there is provided a
vector for expressing a fusion protein in accordance with the
previous aspect.
[0039] In yet another aspect of the invention, there is provided a
cell modified to express a fusion protein in accordance with that
aspect of the invention.
[0040] The mutator activity of AID can be harnessed to drive
mutation of specific gene products of interest. Accordingly, in a
further aspect of the invention there is provided a method for
preparing a gene product having a desired activity, comprising the
steps of: [0041] a) expressing a nucleic acid encoding the gene
product in a population of cells according to the invention; [0042]
b) identifying a cell or cells within the population of cells which
expresses a mutant gene product having the desired activity; and
[0043] c) establishing one or more clonal populations of cells from
the cell or cells identified in step (b), and selecting from said
clonal populations a cell or cells which expresses a gene product
having an improved desired activity.
[0044] In one embodiment, the nucleic acid encoding the gene
product is available to AID or an AID homologue as single-stranded
DNA.
[0045] Suitably, the nucleic acid encoding the gene product is
operably linked to one component of a specific binding pair. In
this embodiment, a nucleic acid operably linked to the one
component, or second half, of a specific binding pair is ligated in
such a way that the binding of the other component, or first half,
of a specific binding pair can take place. Thus, where the first
half of specific binding pair is linked in a fusion protein to the
AID polypeptide having mutator activity, binding of the first and
second halves of the specific binding pairs brings the mutator
protein into range with the nucleic acid sequence such that
directed mutation of that particular nucleic acid sequence can take
place.
[0046] In a particularly preferred embodiment, the specific binding
pair is a DNA binding protein-DNA binding protein recognition
sequence. In this embodiment, the population of cells comprises
cells expressing a fusion protein being a fusion of AID polypeptide
to a DNA binding protein (or DNA binding domain) and the nucleic
acid sequence encoding the gene product is operably linked to the
DNA binding protein recognition sequence. This would allow the
mutator activity of AID or its homologues to be specifically
directed to the nucleic acid encoding the gene product of
interest.
[0047] Accordingly, in another aspect of the invention, there is
provided a method for directing mutation to a specific gene product
of interest. Suitably said method comprises the steps of: [0048] i)
generating a nucleic acid construct comprising a nucleic acid
sequence encoding a gene product operably linked to a DNA binding
protein recognition sequence; [0049] ii) transfecting said nucleic
acid construct into a population of host cells expressing a fusion
protein in accordance with the invention; [0050] iii) incubating
said transfected host cells under conditions suitable for allowing
the specific binding pairing of DNA binding protein to DNA binding
protein recognition sequence to occur; and [0051] iv) identifying a
cell or cells within the population of cells which expresses a
mutant gene product having the desired activity; and [0052] v)
establishing one or more clonal populations of cells from the cell
or cells identified in step (iv), and selecting from said clonal
populations a cell or cells which expresses a gene product having
an improved desired activity.
[0053] Suitably said host cells may be prokaryotic, bacterial cells
such as E. coli or they may be eukaryotic cells such as yeast or
mammalian cells.
[0054] In one embodiment, the population of cells in accordance
with the invention is derived from a clonal or polyclonal
population of cells which comprises cells capable of constitutive
hypermutation of V region genes.
[0055] The gene product may be an endogenous gene product such as
the endogenous immunoglobulin polypeptide, a gene product expressed
by a manipulated endogenous gene or a gene product expressed by a
heterologous transcription unit operatively linked to control
sequences which direct somatic hypermutation, as described further
below. In this embodiment, the gene product is operably linked to a
nucleic acid which directs hypermutation.
[0056] Alternatively, the gene product may be a heterologous gene
product.
[0057] The nucleic acid which is expressed in the cells of the
invention and subjected to hypermutation may be an endogenous
region, such as the endogenous V region, or a heterologous region
inserted into the cell line of the invention. This may take the
form, for example, of a replacement of the endogenous V region with
heterologous transcription unit(s), such as a heterologous V
region, retaining the endogenous control sequences which direct
hypermutation; or of the insertion into the cell of a heterologous
transcription unit under the control of its own control sequences
to direct hypermutation, wherein the transcription unit may encode
V region genes or any other desired gene product. The nucleic acid
according to the invention is described in more detail below.
[0058] In another embodiment the gene product may be an endogenous
gene product which is not normally subject to hypermutation.
Suitable gene products include genes implicated in disease,
oncogenes and other target genes. Thus, the gene product may be any
gene product in which mutation is desirable.
[0059] In one embodiment, the endogenous or heterologous gene may
be integrated into a chromosome.
[0060] In step b) or step (iv) above, the cells are screened for
the desired gene product activity. This may be, for example in the
case of immunoglobulins, a binding activity. Other activities may
also be assessed, such as enzymatic activities or the like, using
appropriate assay procedures. Where the gene product is displayed
on the surface of the cell, cells which produce the desired
activity may be isolated by detection of the activity on the cell
surface, for example by fluorescence, or by immobilising the cell
to a substrate via the surface gene product. Where the activity is
secreted into the growth medium, or otherwise assessable only for
the entire cell culture as opposed to in each individual cell, it
is advantageous to establish a plurality of clonal populations from
step a) in order to increase the probability of identifying a cell
which secretes a gene product having the desired activity.
Advantageously, the selection system employed does not affect the
cell's ability to proliferate and mutate.
[0061] Preferably, at this stage (and in step c) or step v)) cells
which express gene products having a better, improved or more
desirable activity are selected. Such an activity is, for example,
a higher affinity binding for a given ligand, or a more effective
enzymatic activity. Thus, the method allows for selection of cells
on the basis of a qualitative and/or quantitative assessment of the
desired activity. Successive rounds of selection may allow for
directed evolution in a gene product. Selection of mutants may also
be achieved by growth or selection on selective media as described
herein.
[0062] In a preferred embodiment, the "population of cells" in the
method is a population of prokaryotic cells. In another embodiment,
the "population of cells" is a population of yeast cells.
[0063] The targeted mutation of a specific gene product of interest
can be enhanced by providing the nucleic acid encoding the gene
product in a modified construct. Suitably the construct is arranged
such to favour generation of a single-stranded substrate
oligonucleotide (i.e. the nucleic acid encoding the gene product of
interest). An increased availability of single stranded DNA can be
achieved by providing the substrate oligonucleotide between two
convergent promoters. In one embodiment, this construct favours the
generation of single stranded DNA through DNA bending caused by
promoter activity. In another embodiment, this construct favours
single stranded DNA through bi-directional transcription
activation.
[0064] Accordingly, in another aspect of the invention there is
provided a construct for use in a method in accordance with the
invention said construct comprising a nucleic acid encoding the
gene product of interest wherein said nucleic acid is placed under
the control of a first promoter upstream of the coding sequence and
further comprising a second promoter downstream of the coding
sequence in the opposite orientation. Such a construct may be
referred to as a construct for convergent transcription.
[0065] A number of suitable promoter sequences are known to those
skilled in the art. For example, suitable Prokaryotic promoters
include Activators such as AraBAD, PhoA, Repressors such as Tet,
Lac, Trp, Hybrid Lac/Trp such as Tac, pL and Regulatable hybrids of
pL such as pL-tet or Viral Polymerase, such as T7. Suitable
Eukaryotic promoters include, for example, RNA Polymerase I (e.g.
45S rDNA), RNA Polymerase II (e.g. Gal4, .beta.-Actin, Viral
promoters, such as CMV-IE and Artificial promoters including
Tet-on, Tet-off) or RNA Polymerase III promoters including H1 RNA
and U6 snRNA. In particular, promoters include the PhoB promoter
and inducible promoters such as IPTG inducible Trc promoter.
Suitably said construct is as described in the examples section
herein.
[0066] In another aspect of the invention there is provided a
method of identifying components of AID-dependant mutation activity
comprising expressing AID in a cell deficient in a particular gene
and assessing mutator activity compared to activity in a cell
expressing said gene.
[0067] By "components of AID dependant mutation activity" is meant
aspects or cellular components which contribute to the molecular
role of AID (or its homologues) and includes proteins or nucleic
acid components which interact with AID in its mutator
function.
[0068] In a further aspect of the invention there is provided a
method of screening for a modulator of AID activity comprising:
[0069] expressing AID in a prokaryotic cell; [0070] maintaining the
AID-expressing prokaryotic cell in the presence of a selectable
medium; [0071] detecting the presence of colonies in the absence or
presence of a test compound wherein a modified number of colonies
when compared to a sample in the absence of a test compound is
indicative of the ability of the test compound to modify AID
mutator activity.
[0072] By "AID activity" is meant activity of AID or any of its
homologues.
[0073] Preferably, the modified number of colonies in the presence
of the test compound is an increased number and is therefore
indicative of enhanced AID-mediated mutation.
[0074] In another aspect of the invention there is provided a
method of conferring a mutator phenotype on a cell comprising
expressing AID or its homologues in a cell.
[0075] Modifying a cell to confer an increased frequency of
mutations by introducing AID expression is equivalent to a method
of introducing mutations into a cell comprising expressing AID, the
mutator protein.
[0076] In another aspect of the invention, there is provided a use
of AID or a functional homologue thereof in triggering mutation in
a cell. In particular, there is provided a use of AID to introduce
nucleotide transitions at dG/dC as a result of deamination of dC
residues in DNA.
[0077] There are several members of the AID/apobec/phorbolin family
in humans (Jarmuz et al. (2002)). Indeed, overexpression of
Apobec-1 is oncogenic in mice (Yamanaka S. et al. (1995)) and
Apobec-1 family members are expressed in many tumour cell lines.
The mutator activity demonstrate herein provides a molecular
explanation for the mechanism for this oncogenesis. Tumour cells
generally show an enhanced rate of mutation compared with
non-tumour cells with mutations at dC/dG being the most common
nucleoside substitutions. Thus, the ability to modulate gene
products that trigger mutation provides a method of treating
disorders characterised by an increased mutation rate, such as
cancer.
[0078] Accordingly, in another aspect of the invention there is
provided a method for treating a disorder characterised by
increased mutations comprising treating an individual having such a
disorder with an agent that modifies AID or AID homologue
functional activity or gene expression. Suitably the disorder is
selected from cancer, autoimmune disease or other disorders in
which increased mutations are correlated with the disease
phenotype.
[0079] In one embodiment said treatment may be prophylactic i.e. a
preventative treatment. This is particularly applicable to
treatment of an individual that may be predisposed to the
development of a specific disorder. For example, an individual may
be predisposed to develop a cancer through, for example,
overexpression of AID or its homologues. In such an individual
prophylactic treatment with an agent that modifies AID or AID
homologue functional activity or gene expression may act to prevent
the condition developing.
[0080] In a preferred embodiment of this aspect, the AID homologue
is Apobec-1, Apobec-3G or Apobec-3C.
[0081] The development of resistance to antibiotics by a population
of bacteria is a problem in treatment of everyday infections. The
ability to decrease the rate at which mutations conferring the
development of antibiotic resistance would be desirable.
Understanding the role of AID in generating mutations along with
the observation that bacterial cells express proteins having a
similar activity to AID (see, for example, Shen et al. (1992);
Navaratnam et al. (1998)) enables modification of an AID-like
mutator activity in bacteria to modify the rate at which antibiotic
resistance arises. Accordingly in another aspect of the invention
there is provided a method of decreasing hypermutation/resistance
to a compound such as an antibiotic in a population of bacteria by
modulating bacterial AID-like activity.
DEFINITIONS
[0082] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art (e.g., in cell culture, molecular
genetics, nucleic acid chemistry, hybridisation techniques and
biochemistry). Standard techniques are used for molecular, genetic
and biochemical methods. See, generally, Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al.,
Short Protocols in Molecular Biology (1999) 4.sup.th Ed, John Wiley
& Sons, Inc.; as well as Guthrie et al., Guide to Yeast
Genetics and Molecular Biology, Methods in Enzymology, Vol. 194,
Academic Press, Inc., (1991), PCR Protocols: A Guide to Methods and
Applications (Innis, et al. 1990. Academic Press, San Diego,
Calif.), McPherson et al, PCR Volume 1, Oxford University Press,
(1991), Culture of Animal Cells: A Manual of Basic Technique, 2nd
Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), and Gene
Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray,
The Humana Press Inc., Clifton, N.J.). These documents are
incorporated herein by reference.
[0083] The abbreviations used herein include: APOBEC1,
apolipoprotein B editing complex catalytic subunit 1; AID,
activation-induced deaminase; TLC, thin-layer chromatography; PEI,
polyethylene imine; UDG, uracil-DNA glycosylase.
[0084] The terms "variant" or "derivative" in relation to AID
polypeptide includes any substitution of, variation of,
modification of, replacement of, deletion of or addition of one (or
more) amino acids from or to the polypeptide sequence of AID.
Preferably, nucleic acids encoding AID are understood to comprise
variants or derivatives thereof.
[0085] Such "modifications" of AID polypeptides include fusion
proteins in which AID polypeptide or a portion or fragment thereof
is linked to or fused to another polypeptide or molecule.
[0086] The term "homologue" as used herein with respect to the
nucleotide sequence and the amino acid sequence of AID may be
synonymous with allelic variations in the AID sequences and
includes the known homologues, for example, Apobec-1 and other
Apobec homologues including Apobec3C, Apobec3G, phorbolin and
functional homologues thereof.
[0087] The "functional activity" of a protein in the context of the
present invention describes the function the protein performs in
its native environment. Altering or modulating the functional
activity of a protein includes within its scope increasing,
decreasing or otherwise altering the native activity of the protein
itself. In addition, it also includes within its scope increasing
or decreasing the level of expression and/or altering the
intracellular distribution of the nucleic acid encoding the
protein, and/or altering the intracellular distribution of the
protein itself. By "AID mutation activity" or "mutator activity" is
meant the functional activity of AID or its homologues to increase
mutation above background.
[0088] The term "expression" refers to the transcription of a genes
DNA template to produce the corresponding mRNA and translation of
this mRNA to produce the corresponding gene product (i.e., a
peptide, polypeptide, or protein). The term "activates gene
expression" refers to inducing or increasing the transcription of a
gene in response to a treatment where such induction or increase is
compared to the amount of gene expression in the absence of said
treatment. Similarly, the terms "decreases gene expression" or
"down-regulates gene expression" refers to inhibiting or blocking
the transcription of a gene in response to a treatment and where
such decrease or down-regulation is compared to the amount of gene
expression in the absence of said treatment.
[0089] The "mutation rate" is the rate at which a particular
mutation occurs, usually given as the number of events per gene per
generation whereas "mutation frequency" is the frequency at which a
particular mutant is found in the population.
[0090] "Hypermutation" or "increased mutation rate" or "increased
mutation frequency" refers to the mutation of a nucleic acid in a
cell at a rate above background. Preferably, hypermutation refers
to a rate of mutation of between 10.sup.-5 and 10.sup.-3 bp.sup.-1
generation.sup.-1. This is greatly in excess of background mutation
rates, which are of the order of 10.sup.-9 to 10.sup.-10 mutations
bp.sup.-1 generation.sup.-1 (Drake et al., 1998 Genetics
148:1667-1686) and of spontaneous mutations observed in PCR. 30
cycles of amplification with Pfu polymerase would produce
<0.05.times.10.sup.-3 mutations bp.sup.-1 in the product, which
in the present case would account for less than 1 in 100 of the
observed mutations (Lundberg et al., 1991 Gene 108:1-6).
[0091] In vivo, hypermutation is a part of the natural generation
of immunoglobulin diversity through generating variable chain (V)
genes. According to one aspect of the present invention therefore,
the cell line is preferably an immunoglobulin-producing cell line
which is capable of producing at least one immunoglobulin V gene. A
V gene may be a variable light chain (V.sub.L) or variable heavy
chain (V.sub.H) gene, and may be produced as part of an entire
immunoglobulin molecule; it may be a V gene from an antibody, a
T-cell receptor or another member of the immunoglobulin
superfamily. Members of the immunoglobulin superfamily are involved
in many aspects of cellular and non-cellular interactions in vivo,
including widespread roles in the immune system (for example,
antibodies, T-cell receptor molecules and the like), involvement in
cell adhesion (for example the ICAM molecules) and intracellular
signalling (for example, receptor molecules, such as the PDGF
receptor). Thus, preferred cell lines according to the invention
are derived from B-cells. According to the present invention, it
has been determined that cell lines derived from antibody-producing
B cells may be isolated which retain the ability to hypermutate V
region genes, yet do not hypermutate other genes.
[0092] "Class switching" or "switch recombination" is the
recombination process in V gene rearrangement that leads to a
change in the constant region of the expressed antibody. "Gene
conversion" is an additional mechanism in the recombination process
which is found to occur in chicken and rabbits (but not in human or
mouse) and contributes to V gene diversification.
[0093] The term "constitutive hypermutation" refers to the ability
of certain cell lines to cause alteration of the nucleic acid
sequence of one or more specific sections of endogenous or
transgene DNA in a constitutive manner, that is without the
requirement for external stimulation. Generally, such hypermutation
is directed. In cells capable of directed constitutive
hypermutation, sequences outside of the specific sections of
endogenous or transgene DNA-are not subjected to mutation rates
above background mutation rates. The sequences which undergo
constitutive hypermutation are under the influence of
hypermutation-recruiting elements, as described further below,
which direct the hypermutation to the locus in question. Thus in
the context of the present invention, target nucleic acid
sequences, into which it is desirable to introduce mutations, may
be constructed, for example by replacing V gene transcription units
in loci which contain hypermutation-recruiting elements with
another desired transcription unit, or by constructing artificial
genes comprising hypermutation-recruiting elements.
[0094] The cell population which is subjected to selection by the
method of the invention may be a polyclonal population, comprising
a variety of cell types and/or a variety of target sequences, or a
(mono-) clonal population of cells.
[0095] A clonal cell population is a population of cells derived
from a single clone, such that the cells would be identical save
for mutations occurring therein. Use of a clonal cell population
preferably excludes co-culturing with other cell types, such as
activated T-cells, with the aim of inducing V gene
hypermutation.
BRIEF DESCRIPTION OF THE TABLES AND FIGURES
[0096] Table 1 shows the results of experiments in which AID was
expressed in E. coli.
[0097] Table 2 shows the results of experiments in which AID and
its homologues, Apobec-1, Apobec3C and Apobec3G were expressed in
E. coli.
[0098] Table 3 shows the results of a second set of experiments in
which AID and its homologues, Apobec-1, Apobec 2, Apobec3C and
Apobec3G were expressed in E. coli.
[0099] Table 4 shows the oligonucleotides used in Example 3.
FIGURE LEGENDS
[0100] FIG. 1 DNA deamination model of Ig gene diversification. For
details, see text.
[0101] FIG. 2 Expression of AID in E. coli yields a mutator
phenotype that is enhanced by UDG-deficiency. (a) Frequencies of
Rif.sup.R mutants generated following overnight culture (+IPTG) of
E. coli KL16 carrying either the AID expression plasmid or the
vector control. Each point represents the mutation frequency of an
independent overnight culture. The fold enhancement by AID
expression is indicated. (b) Mutation frequency of AID- and
vector-transformed, UDG-deficient KL16 ung-1 cells. Performed and
labeled as in (a), but note the differing y-axis scale. (c)
Photograph of representative plates. The mutation frequency
relative to the vector-transformed wildtype control is indicated in
the centre of each plate. See Table 1 for additional data.
[0102] FIG. 3 Nature of the AID-induced Rif.sup.R mutants. (a)
Comparison of the distribution of independent rpoB mutations
identified in Rif.sup.R colonies obtained from AID- and
vector-transformed cells. The data are combined from results
obtained using both KL16 and AB1157 hosts, but the two hosts show
no difference in their mutation spectrum. The underlined sequence
(SEQ ID NO: 23 (nucleic acid sequence) and SEQ ID NO: 24 (amino
acid sequence)) is the region of rpoB which is known (Jin &
Zhou (1996)) to harbour the majority of mutations conferring RifR.
Less than 5% of the Rif.sup.R sequenced clones did not show any
mutations in this region. (b) Comparison of the types of rpoB
nucleotide substitutions identified.
[0103] FIG. 4 Comparison of the independent gyrA mutations
identified in Nal.sup.R colonies of AID- and vector-transformed E.
coli KL16. Less than 5% of the Nal.sup.R clones analysed failed to
show mutations in the sequenced region (SEQ ID NO: 25 (nucleic acid
sequence) and SEQ ID NO: 26 (amino acid sequence)).
[0104] FIG. 5
[0105] (a) Frequencies of Rif.sup.R mutants generated following
overnight culture of cells carrying an APOBEC1 or AID expression
construct or the vector control. Each point represents the mutation
frequency of an independent overnight culture. The median mutation
frequency and the fold enhancement by expression of the mutator are
indicated in which AID and its homologues, Apobec-1, Apobec3C and
Apobec3G were expressed in E. coli.
[0106] (b) Effect of IPTG on APOBEC1-induced mutation to Rif.sup.R.
The mutation observed in the absence of IPTG may well be due to
pTrc99A promoter leakiness. Labeled as in (a).
[0107] (c) Single amino acid changes in APOBEC1 abrogate its
ability to stimulate mutation to Rif.sup.R. Labeled as in (a).
[0108] (d) Comparison of average growth rates of vector- and
APOBEC1 transformed cells propagated in the presence of the inducer
IPTG. Five independent cultures were used for each measurement, but
the standard deviations proved smaller than the symbols.
[0109] FIG. 6 Spectrum of Rif.sup.R mutations found in cells
expressing APOBEC 1.
[0110] (a) Comparison of the distribution of independent Rif.sup.R
mutations within the region of rpoB (SEQ ID NO: 27) found in cells
transformed with vector alone or an APOBEC1 expression construct.
The preferred sites in AID-expressing cells are highlighted by dark
boxes.
[0111] (b) Summary of the types of nucleotide substitutions in rpoB
identified in Rif.sup.R vector- and APOBEC1-transformed cells given
as a percentage of the total database (120 from controls and 136
from APOBEC1-transformed cells).
[0112] FIG. 7 APOBEC1, APOBEC3C and APOBEC3G all stimulate mutation
at dC/dG but with distinct target specificities.
[0113] (a) Schematic of the APOBEC1 family of mutator proteins
depicting the putative zinc-binding deaminase motif and the
conserved leucine-rich region. Other APOBEC1 family members also
contain either single (APOBEC2 and APOBEC3A) or double (APOBEC3B
and APOBEC3F) putative zinc-binding motifs (Madsen et al.).
APOBEC3D and APOBEC3E may be a single protein with two zinc-binding
regions as evidenced by IMAGE clone 3915193 or two separate, single
zinc-binding motif proteins (Jarmuz et al.). For each protein, the
enhancement of mutation to Rif.sup.R yielded by that protein (data
from Table 3), the percentage of the mutations observed that were
nucleotide transitions at dC or dG and the identity of the major
rpoB mutational hotspots observed (the percentage of the total
number of rpoB mutations observed at that hotspot given in
parentheses) are all given. The total number of mutated rpoB
sequences analysed (n) for each APOBEC1 family member is given.
[0114] (b) Distribution of rpoB mutations in Rif.sup.R mutants
obtained using bacteria transformed with different APOBEC family
members. There are 26 sites within the sequenced region of rpoB
where a single nucleotide substitution can yield Rif.sup.R; at 11
of these sites, Rif.sup.R can be achieved by a transition at dC or
dG. The percentage of the total number of Rif.sup.R mutations
obtained with each APOBEC family member that occurred at each of
these 11 sites is indicated. Mutations at other sites are not
indicated (an omission which is mainly of significance to the
depiction of the vector control).
[0115] FIG. 8a shows pRB700 construct comprising the Bacillus
subtilis gene SacB under the control of the E. coli promoter for
PhoB.
[0116] FIG. 8b shows the pRB740 construct comprising a variant SacB
cassette under the control of the PhoB promoter and also under the
control of the strong IPTG inducible Trc promoter downstream and in
the opposite orientation.
[0117] FIG. 9 shows the results of mutation analysis in mutants in
the SacB cassette.
[0118] FIG. 10a shows mutation frequency in constructs when
transcription is induced in either or both directions.
[0119] FIG. 10b shows the results of mutation frequency analysis.
pRB700 and pRB740 are described in FIG. 9. Vector control and
APOBEC-1 expression plasmids pTrc99a and pRH200 are as described
(Harris et al 2002 Mol Cell. 10(5):1247-53). Growth media all
include 100 .mu.g/ml carbenicillin and 1 mM IPTG to maintain and
induce expression plasmids. LB=Luria Bertani medium. Min
MOPS=Minimal MOPS medium (Neidhardt et al 1974. Culture medium for
enterobacteria. J Bacteriol. 119:736-47) using 0.1% glycerol as
carbon source supplemented with 2 .mu.M Zn.sup.2+ and 0.1%
casamino-acids (C, D, G, H) or bacto-peptone (I).
[0120] FIG. 11 shows a table of results for mutation analysis.
[0121] FIG. 12 shows the results of assaying for DNA deaminase
activity in crude extracts using the TLC-based assay.
[0122] A, Schematic representation of the TLC-based deaminase
assay. .alpha.-[.sup.32P]dCMP-labelled single-stranded DNA was
incubated with the indicated extracts, purified, digested with P1
nuclease and analysed by TLC in one of two buffer systems.
[0123] B, Analysis by TLC in either the LiCl [panel (i)] or
CH.sub.3COOH+LiCl [panels (ii) and (iii)] buffer systems of the
assay products of .alpha.-[.sup.32P]dCMP-labelled single-stranded
DNA incubated with sonic extracts of E. coli transformants that
carry plasmids directing the overexpression of APOBEC1, APOBEC2, a
mutant APOBEC1 (harbouring an E63->A substitution) or dCTP
deaminase (DCD). Controls are provided by extracts from E. coli
transformed with vector only (-) as well as by substrate DNA that
has been subjected to chemical deamination using bisulfite. The
plasmid/host strain combination used for recombinant protein
expression was pTrc99/E. coli KL16 except where (as indicated) the
pET vector was used (in which case the host strain was BL21DE3) or
where activity was monitored using the E. coli SO177 host (which is
deficient in both dcd and cdd deaminases). The migration of dUMP,
dCMP and [.sup.32P] inorganic phosphate (Pi) markers is indicated.
The abundance of wild-type and E63->A mutant APOBEC1
polypeptides in extracts was monitored by Western [lower part of
panel (iii)].
[0124] FIG. 13 shows APOBEC1 fractionation.
[0125] A. Ion-exchange chromatography on Sepharose Mono-Q.
Clarified lysates of APOBEC1 (and APOBEC1[E63->A])-expressing E.
coli were loaded onto Mono-Q. The presence of APOBEC1 polypeptide
was detected by Western blot [panel (ii)]. Deaminase activity was
monitored by both TLC- and UDG-based assays [panels (i) and (iii)]
in the total lysate (T), the flow through (FT) and in the 800 and
1000 mM-salt washes.
[0126] B, Gel filtration of the concentrated high (>1 M) salt
eluate from the Mono-Q column on Sephacryl S200. Fractions were
analysed by: (i) SDS/PAGE; bands were excised and analysed by
MALDI-TOF following in-gel trypsin digestion. The bands yielding
peptide sequences derived from APOBEC1 and ribosomal proteins L1,
2, 6 and 9 and S4 are indicated. M, molecular weight markers. (ii)
Western blotting for APOBEC1; (iii) TLC-based and (iv) UDG-based
deaminase assays, which were performed on samples of the total
clarified bacterial lysate (T) as well as on the eluate from the
Mono-Q. The UDG-based deaminase assay was performed using
3'-.alpha.-[.sup.32P]-labelled SPM274; note that some of the
3'-label is removed during the incubation. The percentage of label
associated with the 26-base product of the deamination/cleavage (as
opposed to 40-base input oligonucleotide) is indicated.
[0127] FIG. 14 shows specificity of APOBEC1-mediated DNA
deamination using the UDG-based assay.
[0128] A, Schematic representation of the UDG-based deaminase
assay. 5'-biotinylated (circle) oligonucleotides that were
3'labelled (asterisk) with fluorescein or
.alpha.-[.sup.32P]dideoxyadenylate were incubated with
APOBEC1-containing (or control) samples prior to streptavidin
purification, UDG-treatment and PAGE-urea analysis.
[0129] B, Partially purified APOBEC1 as well as the E63->A
mutant were tested for their ability to deaminate 3'-fluorescein
conjugated oligonucleotide SPM168 using the UDG-based assay. The
fluorescence scan of the gel, including controls performed without
UDG treatment or without APOBEC1, is shown with the positions of
the expected products and size markers indicated.
[0130] C, Time-course of SPM168 deamination by partially purified
APOBEC1.
[0131] D, Inclusion of RNAase (1 .mu.g) or of tetrahydrouridine
(THU; 20 nmoles, 2 nmoles, or 200 pmoles) does not inhibit the
activity of APOBEC1.
[0132] E, Deaminating activity is specific for a single-stranded
substrate. The assay was performed using 3'-fluorscein-labelled
oligonucleotide SPM168 in the presence of the indicated ratio of
either oligonucleotide SPM171 (which is complementary to SPM168) or
SPM201 (which is not).
[0133] F, Comparison of 3'-fluorescein labelled oligonucleotides
SPM168 (left three lanes) and SPM163 (right three lanes) as targets
for deamination by 0.5, 1 and 2 .mu.l of APOBEC1.
[0134] FIG. 15 Autoradiographs showing hybridisation of APOBEC1,
APOBEC3G, and ubiquitin (control) probes to matched pairs of tumour
(T) and corresponding normal (N) cDNA samples derived from a
variety tissues using a cancer profiling array (Clontech).
DETAILED DESCRIPTION OF THE INVENTION
[0135] The fact that AID, a homologue of Apobec-1 (which deaminates
C in RNA), is required for all three programmes of diversification
of rearranged immunoglobulin genes (Muramatsu M. et al. (2000);
Revy, P. et al. (2000); Arakawa, H. et al. (2002); Harris, R. S. et
al. (2002) and Martin, A. et al. (2002)) and that the initiation of
all three programmes could be explained by DNA modification at
dG/dC (Martin et al. (2002), Maizels et al. (1995), Weill et al.
(1996); Sale et al. (2001), Ehrenstein et al. (1999) Rada et al.
(1998) and Wiesendanger et al. (2000)) led the present inventors to
the model presented in FIG. 1. The hypothesis set out herein is
that AID mediates the deamination of a small number of C residues
within the Ig loci. Conventionally, this would trigger base
excision repair (Lindahl T. (2000)) with uracil being removed by
uracil-DNA glycosylase (UDG) and, following cleavage at the abasic
site by an apyrimidic endonuclease (APE), a dC residue would be
reinserted by a DNA polymerase/deoxyribophosphodiesterase. If,
instead of being repaired, the DNA strand harbouring the dU residue
were used to template DNA synthesis, then the consequence would be
a dC.fwdarw.dT (and dG.fwdarw.dA) transition. Alternatively, if DNA
synthesis occurred over the abasic site, both transitions and
transversions would be generated although a transition bias might
still be observed if the polymerase used for the lesion bypass
preferentially inserted dA residues. Thus, the stage at which
polymerase bypass of the original lesion occurred as well as the
preferences of the polymerase used would affect the transition bias
of the hypermutation. This could account for the otherwise puzzling
observation that whereas mutation in mouse and man as well as in
the hypermutating Ramos B cell line exhibits a marked transition
preference (Sale et al. (1998)), no such preference is evident in
the mutations exhibited by the XRCC2-deficient chicken DT40 B cell
line (Sale et al. (2001)).
[0136] Templated repair of the deamination-induced lesion by a V
pseudogene would lead to gene conversion; such repair would be
dependent on the RAD51 paralogues XRCC2, XRCC3 and RAD51B (Sale et
al. (2001)). The second phase of mutation (yielding mutations at
dA/dT) which is observed in vivo in man and mouse would be
triggered by MSH2/MSH6 recognition of the dU/dG mismatch itself or
of some intermediate in its correction (Rada et al. (1998);
Wiesendanger et al. (2000)), and would presumably occur by some
form of patch repair. Repair partnered on another switch region
could lead to switch recombination. For switching, where there is
an indication for a role of non-homologous end-joining (Manis et
al. (2002); Peterson et al. (2001)), one might imagine that
deamination of proximal dCs on opposite strands could generate the
staggered DNA breaks proposed by Chen et al (2001).
[0137] A central prediction of this model is that AID has the
ability to trigger dC.fwdarw.dU deamination in DNA. Such an
activity would presumably be largely restricted to its
physiological target (the Ig loci) since a rampant DNA deaminase
activity would likely be harmful to the cell.
[0138] The results presented herein suggest that, whereas
functional Ig genes are generated by RAG-mediated rearrangement,
subsequent diversification is triggered by AID-mediated deamination
of dC residues within the immunoglobulin locus with the outcome
(gene conversion, switch recombination or mutation phases 1/2)
dependent upon the way in which the initiating dU/dG lesion is
resolved.
[0139] As well as AID, the APOBEC/AID family contains several
members that are capable of mutating DNA, triggering nucleotide
substitutions at dC/dG by a process which, given its sensitivity to
uracil-DNA glycosylase, is likely to be dC deamination.
[0140] The physiological functions of the other APOBEC family
members are unknown. Whereas APOBEC1 shows relatively restricted
tissue distribution, APOBEC3G is much more widely expressed.
Hybridisation experiments suggest that some APOBEC family members
are well expressed in a variety of cancers (FIG. 8) and cancer cell
lines.
[0141] Quite apart, however, from the normal physiological
functions of the APOBEC family members, the fact that several of
the members can display a DNA mutator activity (taken together with
the observation that transgenic expression of APOBEC1 is oncogenic
in mice) raises the possibility that they might contribute to the
`spontaneous` dC deamination that occurs in normal cells as well as
the elevated mutation rates proposed to be associated with many
human cancers. Indeed, in the large database of p53 mutations in
human cancers (where nearly 13,000 single base changes have been
identified scattered over a large number of positions in the gene)
over 50% of the substitution mutations (and over 60% of the silent
mutations) are nucleotide transitions at dC/dG with roughly half of
these dC/dGs being at dCpdG dinucleotides.
Measuring an Enhanced Mutation Rate in Cells as an Indication of a
Mutator Phenotype
[0142] Hypermutating cells or cells having a mutator phenotype may
be identified by a variety of techniques, including sequencing of
target sequences, selection for expression loss mutants, assay
using bacterial MutS protein and selection for change in gene
product activity. Methods for measuring mutation rates include
fluctuation analysis (described, for example, by Luria and Delbreck
(1943) and Capizzi and Jameson (1973)). In this, the generation of
clones showing resistance to a selection media. Suitable selection
media for prokaryotic cells include rifampicin, nalidixic acid,
valine and fucose. Cells selected according to this procedure are
cells in which mutation has occurred in a gene or genes which
enable the effect of the selection media to be overcome. Other ways
of determining mutation rates include direct sequencing of specific
portions of DNA or indirect methods such as the MutS assay (Jolly
et al., 1997 Nucleic Acids Research 25, 1913-1919) or monitoring
the generation of immunoglobulin loss variants.
[0143] In a preferred embodiment of the invention, the method
involves generating mutations in a target nucleic acid which
encodes an immunoglobulin. Immunoglobulin loss may be detected both
for cells which secrete immunoglobulins into the culture medium,
and for cells in which the immunoglobulin is displayed on the cell
surface. Where the immunoglobulin is present on the cell surface,
its absence may be identified for individual cells, for example by
FACS analysis, immunofluorescence microscopy or ligand
immobilisation to a support. In a preferred embodiment, cells may
be mixed with antigen-coated magnetic beads which, when sedimented,
will remove from the cell suspension all cells having an
immunoglobulin of the desired specificity displayed on the
surface.
[0144] The technique may be extended to any immunoglobulin
molecule, including antibodies, T-cell receptors and the like. The
selection of immunoglobulin molecules will depend on the nature of
the clonal population of cells which it is desired to assay
according to the invention.
[0145] Alternatively, mutations in cells according to the invention
may be identified by sequencing of target nucleic acids, such as V
genes, and detection of mutations by sequence comparison. This
process may be automated in order to increase throughput.
[0146] In a further embodiment, cells which hypermutate V genes may
be detected by assessing change in antigen binding activity in the
immunoglobulins produced in a clonal cell population. For example,
the quantity of antigen bound by a specific unit amount of cell
medium or extract may be assessed in order to determine the
proportion of immunoglobulin produced by the cell which retains a
specified binding activity. As the V genes are mutated, so binding
activity will be varied and the proportion of produced
immunoglobulin which binds a specified antigen will be reduced.
[0147] Alternatively, cells may be assessed in a similar manner for
the ability to develop a novel binding affinity, such as by
exposing them to an antigen or mixture of antigens which are
initially not bound and observing whether a binding affinity
develops as the result of hypermutation.
[0148] In a further embodiment, the bacterial MutS assay may be
used to detect sequence variation in target nucleic acids. The MutS
protein binds to mismatches in nucleic acid hybrids. By creating
heteroduplexes between parental nucleic acids and those of
potentially mutated progeny, the extent of mismatch formation, and
thus the extent of nucleic acid mutation, can be assessed.
[0149] Where the target nucleic acid encodes an gene product other
than an immunoglobulin, selection may be performed by screening for
loss or alteration of a function other than binding. For example,
the loss or alteration of an enzymatic activity may be screened
for.
Genetic Manipulation of Cells
[0150] Cells modified to express AD or its homologues are cells in
which AID protein expression (or AID homologue protein expression)
has been induced by means, for example, of transfecting host cells
with a vector encoding AID protein. Such transfection may be stable
or transient transfection.
[0151] "Vector" refers to any agent such as a plasmid, cosmid,
virus, autonomously replicating sequence, phage, or linear
single-stranded, circular single-stranded, linear double-stranded,
or circular double-stranded DNA or RNA nucleotide sequence that
carries exogenous DNA into a host cell or organism. The recombinant
vector may be derived from any source. In the context of the
present invention, the vector is for stable expression of AID and
is, therefore, capable of genomic integration or autonomous
replication but maintained throughout division cycles of the host
cell.
[0152] An expression vector includes any vector capable of
expressing a coding sequence encoding a desired gene product that
is operatively linked with regulatory sequences, such as promoter
regions, that are capable of expression of such DNAs. Thus, an
expression vector refers to a recombinant DNA or RNA construct,
such as a plasmid, a phage, recombinant virus or other vector, that
upon introduction into an appropriate host cell, results in
expression of the cloned DNA. Appropriate expression vectors are
well known to those with ordinary skill in the art and include
those that are replicable in eukaryotic and/or prokaryotic cells
and those that remain episomal or those which integrate into the
host cell genome. For example, DNAs encoding a heterologous coding
sequence may be inserted into a vector suitable for expression of
cDNAs in mammalian cells, e.g. a CMV enhancer-based vector such as
pEVRF (Matthias, et al., 1989).
[0153] Construction of vectors according to the invention employs
conventional ligation techniques. Isolated plasmids or DNA
fragments are cleaved, tailored, and religated in the form desired
to generate the plasmids required. If desired, analysis to confirm
correct sequences in the constructed plasmids is performed in a
known fashion. Suitable methods for constructing expression
vectors, preparing in vitro transcripts, introducing DNA into host
cells, and performing analyses for assessing gene product
expression and function are known to those skilled in the art. Gene
presence, amplification and/or expression may be measured in a
sample directly, for example, by conventional Southern blotting,
Northern blotting to quantitate the transcription of mRNA, dot
blotting (DNA or RNA analysis), or in situ hybridisation, using an
appropriately labelled probe which may be based on a sequence
provided herein. Those skilled in the art will readily envisage how
these methods may be modified, if desired.
[0154] Vector-driven protein expression can be constitutive or
inducible. Inducible vectors include either naturally inducible
promoters, such as the trc promoter, which is regulated by the lac
operon, the IPTG promoter which is inducible by IPTG and the pL
promoter, which is regulated by tryptophan, the MMTV-LTR promoter,
which is inducible by dexamethasone, or can contain synthetic
promoters and/or additional elements that confer inducible control
on adjacent promoters. Other promoters include E. coli promoters
such as PhoB.
[0155] Methods for introducing the vectors and nucleic acids into
host cells are well known in the art; the choice of technique will
depend primarily upon the specific vector to be introduced and the
host cell chosen. Plasmid vectors will typically be introduced into
chemically competent or electrocompetent bacterial cells. Vectors
can be introduced into yeast cells by spheroplasting, treatment
with lithium salts, electroporation, or protoplast fusion.
Mammalian and insect cells can be directly infected by packaged
viral vectors, or transfected by chemical or electrical means.
Methods for Generating Fusion Proteins
[0156] AID or any of its homologues or derivatives, including
Apobec-1, may be generated as fusion proteins comprising the AID
protein or a portion that retains its mutator activity coupled to a
DNA binding domain or one half of a specific binding pair.
Preferably the fusion protein will not hinder the mutator activity
of the protein sequence. Methods for generating fusion proteins
will be familiar to those skilled in the art and include generation
of expression vectors comprising the AID nucleic acid sequence
linked or ligated to the nucleic acid sequence encoding a DNA
binding domain.
Methods for Preparing and Selecting Immunoglobulins or Other
Surface Expressed Proteins.
[0157] The process of hypermutation is employed, in nature, to
generate improved or novel binding specificities in immunoglobulin
molecules. Thus, by selecting cells according to the invention
which produce immunoglobulins capable of binding to the desired
antigen and then propagating these cells in order to allow the
generation of further mutants, cells which express immunoglobulins
having improved binding to the desired antigen may be isolated.
[0158] A variety of selection procedures may be applied for the
isolation of mutants having a desired specificity. These include
Fluorescence Activated Cell Sorting (FACS), cell separation using
magnetic particles, antigen chromatography methods and other cell
separation techniques such as use of polystyrene beads.
[0159] Separating cells using magnetic capture may be accomplished
by conjugating the antigen of interest to magnetic particles or
beads. For example, the antigen may be conjugated to
superparamagnetic iron-dextran particles or beads as supplied by
Miltenyi Biotec GmbH. These conjugated particles or beads are then
mixed with a cell population which may express a diversity of
surface immunoglobulins. If a particular cell expresses an
immunoglobulin capable of binding the antigen, it will become
complexed with the magnetic beads by virtue of this interaction. A
magnetic field is then applied to the suspension which immobilises
the magnetic particles, and retains any cells which are associated
with them via the covalently linked antigen. Unbound cells which do
not become linked to the beads are then washed away, leaving a
population of cells which is isolated purely on its ability to bind
the antigen of interest. Reagents and kits are available from
various sources for performing such one-step isolations, and
include Dynal Beads (Dynal AS; http://www.dynal.no), MACS-Magnetic
Cell Sorting (Miltenyi Biotec GmbH; http://www.miltenyibiotec.com),
CliniMACS (AmCell; http://www.amcell.com) as well as Biomag,
Amerlex-M beads and others. Similar techniques can be used for
non-immunoglobulin surface expressed molecules where selection for
their surface expression can be through recognition by a specific
binding partner.
[0160] Fluorescence Activated Cell Sorting (FACS) can be used to
isolate cells on the basis of their differing surface molecules,
for example surface displayed immunoglobulins. Cells in the sample
or population to be sorted are stained with specific fluorescent
reagents which bind to the cell surface molecules. These reagents
would be the antigen(s) of interest linked (either directly or
indirectly) to fluorescent markers such as fluorescein, Texas Red,
malachite green, green fluorescent protein (GFP), or any other
fluorophore known to those skilled in the art. The cell population
is then introduced into the vibrating flow chamber of the FACS
machine. The cell stream passing out of the chamber is encased in a
sheath of buffer fluid such as PBS (Phosphate Buffered Saline). The
stream is illuminated by laser light and each cell is measured for
fluorescence, indicating binding of the fluorescent labelled
antigen. The vibration in the cell stream causes it to break up
into droplets, which carry a small electrical charge. These
droplets can be steered by electric deflection plates under
computer control to collect different cell populations according to
their affinity for the fluorescent labelled antigen. In this
manner, cell populations which exhibit different affinities for the
antigen(s) of interest can be easily separated from those cells
which do not bind the antigen. FACS machines and reagents for use
in FACS are widely available from sources world-wide such as
Becton-Dickinson, or from service providers such as Arizona
Research Laboratories (http://www.arl.arizona.edu/facs/).
[0161] Another method which can be used to separate populations of
cells according to the affinity of their cell surface protein(s)
for a particular antigen is affinity chromatography. In this
method, a suitable resin (for example CL-600 Sepharose, Pharmacia
Inc.) is covalently linked to the appropriate antigen. This resin
is packed into a column, and the mixed population of cells is
passed over the column. After a suitable period of incubation (for
example 20 minutes), unbound cells are washed away using (for
example) PBS buffer. This leaves only that subset of cells
expressing immunoglobulins which bound the antigen(s) of interest,
and these cells are then eluted from the column using (for example)
an excess of the antigen of interest, or by enzymatically or
chemically cleaving the antigen from the resin. This may be done
using a specific protease such as factor X, thrombin, or other
specific protease known to those skilled in the art to cleave the
antigen from the column via an appropriate cleavage site which has
previously been incorporated into the antigen-resin complex.
Alternatively, a non-specific protease, for example trypsin, may be
employed to remove the antigen from the resin, thereby releasing
that population of cells which exhibited affinity for the antigen
of interest.
Insertion of Heterologous Transcription Units
[0162] In order to maximise the chances of quickly selecting an
antibody variant capable of binding to any given antigen, or to
exploit the AID-dependant hypermutation system for
non-immunoglobulin genes, a number of techniques may be employed to
engineer cells according to the invention such that their
hypermutating abilities may be exploited.
[0163] In a first embodiment, transgenes are transfected into a
cell according to the invention such that the transgenes become
targets for the directed hypermutation events.
[0164] As used herein, a "transgene" is a nucleic acid molecule
which is inserted into a cell, such as by transfection or
transduction. For example, a "transgene" may comprise a
heterologous transcription unit as referred to above, which may be
inserted into the genome of a cell at a desired location. The
"transgene" may be the nucleic acid encoding the gene product of
interest.
[0165] The plasmids used for delivering the transgene to the cells
are of conventional construction and comprise a coding sequence,
encoding the desired gene product, under the control of a promoter.
Gene transcription from vectors in cells according to the invention
may be controlled by promoters derived from the genomes of viruses
such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma
virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus and
Simian Virus 40 (SV40), from heterologous mammalian promoters such
as the actin promoter or a very strong promoter, e.g. a ribosomal
protein promoter, and from the promoter normally associated with
the heterologous coding sequence, provided such promoters are
compatible with the host system of the invention.
[0166] Transcription of a heterologous coding sequence by cells
according to the invention may be increased by inserting an
enhancer sequence into the vector. Enhancers are relatively
orientation and position independent. Many enhancer sequences are
known from mammalian genes (e.g. elastase and globin). However,
typically one will employ an enhancer from a eukaryotic cell virus.
Examples include the SV40 enhancer on the late side of the
replication origin (bp 100-270) and the CMV early promoter
enhancer. The enhancer may be spliced into the vector at a position
5' or 3' to the coding sequence, but is preferably located at a
site 5' from the promoter.
[0167] Advantageously, a eukaryotic expression vector may comprise
a locus control region (LCR). LCRs are capable of directing
high-level integration site independent expression of transgenes
integrated into host cell chromatin, which is of importance
especially where the heterologous coding sequence is to be
expressed in the context of a permanently-transfected eukaryotic
cell line in which chromosomal integration of the vector has
occurred, in vectors designed for gene therapy applications or in
transgenic animals.
[0168] Eukaryotic expression vectors will also contain sequences
necessary for the termination of transcription and for stabilising
the mRNA. Such sequences are commonly available from the 5' and 3'
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA.
[0169] Transgenes according to the invention may also comprise
sequences which direct hypermutation. Such sequences have been
characterised, and include those sequences set forth in Klix et
al., (1998; Eur. J. Immunol. 28:317-326), and Sharpe et al., (1991;
EMBO J. 10:2139-2145), incorporated herein by reference. Thus, an
entire locus capable of expressing a gene product and directing
hypermutation to the transcription unit encoding the gene product
is transferred into the cells. The transcription unit and the
sequences which direct hypermutation are thus exogenous to the
cell. However, although exogenous the sequences which direct
hypermutation themselves may be similar or identical to the
sequences which direct hypermutation naturally found in the
cell
[0170] The endogenous V gene(s) or segments thereof may be replaced
with heterologous V gene(s) by homologous recombination, or by gene
targeting using, for example, a Lox/Cre system or an analogous
technology or by insertion into hypermutating cell lines which have
spontaneously deleted endogenous V genes. Alternatively, V region
gene(s) may be replaced by exploiting the observation that
hypermutation is accompanied by double stranded breaks in the
vicinity of rearranged V genes.
[0171] Furthermore, enhanced targeting of mutation can be achieved
by inducing convergent promoters upstream and downstream of the
desired gene and therefore inducing transcription in both
directions. Deamination of dC in vitro by APOBEC-1 has be
demonstrated to be dependent on the single-strandedness of the
substrate oligonucleotide as described herein. The increase in
availability of single-stranded DNA can be induced by convergent
transcription or by a combination of transcription and DNA bending
caused by promoter activation. Suitable types of promoter include
the PhoB promoter. Other Prokaryotic promoters include Activators
(e.g. AraBAD, PhoA), Repressors (e.g. Tet, Lac, Trp, Hybrid Lac/Trp
such as Tac, pL, Regulatable hybrids of pL such as pL-tet) and
Viral Polymerase (e.g. T7). Suitable Eukaryotic promoters include
promoters recognised by RNA Polymerase I (e.g. 45S rDNA) RNA
Polymerase II (e.g. Gal4, .beta.-Actin or Viral, such as CMV-IE and
Artificial, especially Tet-on, Tet-off) RNA Polymerase III)(e.g. H1
RNA, U6 snRNA).
DNA Binding Domain and Specific DNA Recognition Sequences
[0172] Transcription factors bind DNA by recognising specific
target sequences generally located in enhancers, promoters, or
other regulatory elements that affect a particular target gene. The
target sequences for a number of transcription factors are well
known to those skilled in the art. Transcription factors having
specific DNA target or recognition sequences include the yeast
transcription factors such as GAL4, bacterial proteins such as the
repressor protein Lex A and mammalian transcription factors such as
estrogen receptor.
[0173] The DNA binding domain within such proteins serves to bind
the protein to the target sequence or "DNA binding protein
recognition sequence" and therefore bring the protein to a set
location within a DNA sequence.
[0174] One particular type of transcription factor binding site is
named a "response element" which is a particular DNA sequence which
causes a gene to respond to a regulatory transcription factor.
Examples include the heat shock response element (HRE) and the
glucocorticoid response element (GRE). A number of hormone response
elements are also known to those skilled in the art. Response
elements contain short consensus sequences which are the target or
recognition for the DNA binding domains found within the
corresponding inducible transcription factors such that, for
example, transcription factors induced by a heat shock response
bind HREs, glucocorticoid-induced factors bind GREs etc. Other
examples include the binding of estrogen receptor via a DNA binding
domain to the specific DNA binding protein recognition sequence
called the ERD or estrogen response domain. The interaction of
transcription factors and response elements are described, for
example in Genes VI, Lewin, Oxford University Press, 1997.
Comparisons between the sequences of many transcription factors
suggest that common types of motif can be found that are
responsible for binding to DNA. Such motifs include the zinc finger
motif, the helix-turn-helix or the helix-loop-helix. Other such
motifs are known to the person skilled in the art.
[0175] The interaction between a DNA binding domain and a DNA
binding protein recognition sequence can be used to direct mutation
to a specific nucleic acid sequence. One way of directing mutation
in this way is described as follows: an expression construct for
expressing a fusion protein comprising Apobec with the estrogen
receptor DNA binding domain (ERD) (Schwabe et al. Cell. 1993 Nov.
5; 75(3):567-78) is constructed as described below. The expression
construct is expressed in yeast/E. Coli using standard transfection
procedures. The yeast/E. Coli host cell is also engineered such
that the desired target gene is also linked to a short ERD
recognition sequence (Schwabe et al., 1993).
Screening for Modulators of AID Activity
[0176] Compounds having inhibitory, activating, or modulating
activity can be identified using in vitro assays for activity
and/or expression of AID or its homologues including APOBEC1,
APOBEC 2, APOBEC 3c and APOBEC 3G, e.g., ligands, agonists,
antagonists, and their homologs and mimetics.
[0177] Modulator screening may be performed by adding a putative
modulator test compound to a cell expressing AID (or its
homologues) in accordance with the invention, and monitoring the
effect of the test compound on the function and/or expression of
AID. A parallel sample which does not receive the test compound is
also monitored as a control. The treated and untreated cells are
then compared by any suitable phenotypic criteria, and in
particular by comparing the mutator phenotype of the treated and
untreated cells using methods as described herein.
[0178] The invention is further described below, for the purposes
of illustration only, in the following examples.
EXAMPLES
Example 1
[0179] A plasmid containing a human AID cDNA expressed under
control of the lac promoter was transformed into E. coli strain
KL16 and its effect on the frequency of mutation to
rifampicin-resistance (Rif.sup.R) measured by fluctuation analysis
(FIG. 2 and Table 1).
[0180] The AID expression plasmid was generated by cloning the
human AID cDNA (Harris et al. (2002)) on an NcoI-HindIII fragment
into pTrc99A (Pharmacia; gift of R. Savva). E. coli strains KLI6
(Hfr (PO-45) relAl spoT1 thi-1) and its ung-1 derivative (BW310) as
well as AB1157 and its nfi-1::cat derivative (BW1161) were from B.
Weiss; GM1003 (dcm-6 thr-1 hisG4 leuB6 rpsL ara-14 supE44 lacY1
tonA31 tsx-78 galK2 galE2 xyl-5 thi-1 mtl-1 mug::mini-Tn10)
derivatives carrying ung-1 and/or mug::mini-Tn10 mutations were
from A. Bhagwat.
[0181] APOBEC1 and APOBEC2 expression constructs were generated by
subcloning the rat APOBEC1 cDNA (Bam-HI-SalI fragment of
pSB202.sup.27 gift from N. Navaratman and J. Scott) or the human
APOBEC2 cDNA (NcoI-BsaA1 fragment from IMAGE clones 341062).
APOBEC3C was amplified from the Ramos human Burkitt lymphoma cell
line cDNA using oligonucleotides 5' NNNGAATTCAAGGCTGAACATGAATCCACAG
(SEQ ID NO: 1) and 5' NNNNNGTCGACGGAGACCCCTCACTGGAGA (SEQ ID NO:
2). APOBEC3G was amplified from IMAGE clone 1284557 using
oligonucleotides 5'-NNNGAATTCAAGGATGAAGCCTCACTTCAGA (SEQ ID NO: 3)
and 5' NNGACTGCAGOCCATCCTTCAGTTTTCCTG (SEQ ID NO: 4).
[0182] The E63A, W90S and C93A substitutions in APOBEC1 were
introduced by site-directed mutagenesis using the following
oligonucleotide pairs:
5'ACCAACAAACACGTTGcAGTCAATTTCATAGAAA (SEQ ID NO: 5)
/TTTCTATGAAATTGACTgCAACGTGTTT GTTGGT (SEQ ID NO: 6),
5'ACCTGGTTCCTGTCCTcGAGTCCCTGTGGGGAG (SEQ ID NO: 7)
/CTCCCCACAGGGACTCgAGGACAGGAACC AGGT (SEQ ID NO: 8),
and CTGTCCTGGAGTCCCgcTGGGGAGTGCTCCAGG (SEQ ID NO: 9)
[0183] /CCTGGAGCACTCCCCAgcGGGACTCCAGGAC AG (SEQ ID NO: 10)
(substitutions in lower case).
[0184] All constructs were verified by DNA sequencing and were
identical to published sequences (Madsen et al. (1999), J. Invest.
Dermatol. 113, 162-169; Jarmuz et al, Anant et al. (2001), Am. J.
Physiol. Cell Physiol., 281, C1904-1916) or to existing GenBank
entries (APOBEC1: NM.sub.--012907.1; APOBEC2:
NM.sub.--006789.1).
[0185] The plasmids were transformed into E. coli strain KL16 and
their effect on the frequency of mutation to rifampicin-resistance
(Rif.sup.R) measured by fluctuation analysis (Tables 2 and 3).
Mutation Assays
[0186] Mutation frequencies were measured by determining the median
number of colony-forming cells surviving selection per 10.sup.9
viable cells plated. Each median was determined from 8-16
independent cultures grown overnight to saturation in rich medium
supplemented with 100 .mu.g/mL carbenicillin and 1 mM IPTG (unless
indicated otherwise). Rif.sup.R and Nal.sup.R colonies were
selected on rich medium containing 100 .mu.g/ml rifampicin and 40
.mu.g/ml nalidixic acid respectively. Valine- and fucose-resistant
mutants were selected on minimal M9 medium containing 0.2%
glucose/40 .mu.g/ml L-Valine and 0.1% L-arabinose/0.2% D-fucose
respectively.
[0187] In multiple experiments performed in the presence of the
transcriptional inducer IPTG, the AID-transformed cells generated
Rif.sup.R colonies at a frequency some 4-8 fold higher than
vector-transformed controls. This stimulation was evident in
different genetic backgrounds (KL16, GM1003 and AB1157), was
dependent upon AID (monitored.+-.IPTG) and was not peculiar to the
selection applied, being also clear when mutation to nalidixic acid
(Nal)-, valine- or fucose-resistance was monitored (FIG. 2 and
Table 1). The variation in the mutation enhancement observed in the
different selections could reflect differences in the types and
abundances of mutations that confer resistance.
[0188] In similar multiple experiments, cells transformed with
Apobec-1 generated Rif.sup.R colonies at a much higher frequency
(several hundred fold) than vector-transformed controls. Cells
transformed with other Apobec homologues, Apobec3C and Apobec3G,
also showed an increased frequency (10-20 fold) of mutation to
Rif.sup.R compared to the vector-transformed controls.
[0189] For the experiments shown in FIG. 5 and Table 3, all
measurements were performed using KL16 or its ung-1 derivative
BW310 transformed with vector alone or an expression construct as
indicated. Mutation frequencies were measured by determining the
median number of colony-forming cells surviving selection per
10.sup.9 viable cells plated. Each median presented in FIG. 5 and
Table 3 was determined from 12-16 independent cultures grown
overnight to saturation in rich medium supplemented with 100
.mu.g/mL carbenicillin and 1 mM IPTG (with the exception of control
experiments in which the inducer IPTG was omitted, FIG. 5b).
IPTG-induced expression of APOBEC1 or its homologues conferred no
obvious defect in cell growth or viability (e.g. APOBEC1, FIG. 5c).
Rif.sup.R mutants were selected on rich medium containing 100
.mu.g/ml rifampicin and sequenced. Only about 1% of the Rif.sup.R
colonies failed to contain mutations in the region of rpoB
sequenced [nucleotides 1525-1722, numbering from the initiating
ATG; GenBank AE000472].
[0190] The nature of the Rif.sup.R and Nal.sup.R mutants was
determined by directly amplifying and sequencing the relevant
section of the rpoB [627 bp PCR product amplified using
5'-TTGGCGAAATGGCGGAAAACC (SEQ ID NO: 11) and
5'-CACCGACGGATACCACCTGCTG (SEQ ID NO: 12)] or gyrA [521 bp PCR
product amplified using oligonucleotides 5'-GCGCGGCTGTGTTATAATTT
(SEQ ID NO: 13) and 5' TTCCGTGCCGTCATAGTTATC (SEQ ID NO: 14)].
[0191] If the AID-mediated enhancement in mutation frequency is due
to a stimulation of dC deamination, the pattern of mutation to
Rif.sup.R should show a shift toward dC.fwdarw.dT and dG.fwdarw.dA
transitions. Sequence of the rpoB gene in multiple independent
Rif.sup.R colonies, revealed that this is indeed the case. Such
transitions account for 79% of the mutations scored in the
AID-transformed cells but only for 31% of the mutations in the
vector transformed controls (FIG. 3a, b). Given the extent of
mutation stimulation by AID, the data are consistent with the
entire AID-mediated enhancement being due to transitions at dG/dC.
A similar conclusion was obtained by examining the spectrum of gyrA
mutations in the Nal.sup.R colonies--despite the fact that the
selected mutations appeared restricted to essentially three
nucleotide positions. Thus, whereas 34% of the gyrA mutations
amongst the control transformants are nucleotide transitions at
dG/dC, the percentage increases to 71% in the AID transformants
(FIG. 4)
[0192] It is notable that there is a striking difference in
mutation distribution between the AID transformants and controls.
Analysis of the rpoB mutations amongst the vector-transformed
control cells reveals that dC.fwdarw.dT transitions at positions
Ser512, Ser522, His526, Ser531, Pro564 and Ser574 can all confer
Rif.sup.R. However, transitions at only some of these positions
(His526, Ser531 and Ser574) are enhanced amongst the AID
transformants whereas other positions (Ser522 and Pro564) show
little sign of increased mutation. Even more striking is the fact
that a common dG.fwdarw.dA transition in the AID transformants
(Arg529) is not seen at all in the controls (FIG. 3). Similar
evidence of specific targeting comes from gyrA. Whereas
dC.fwdarw.dT transitions at Ser83 and dG.fwdarw.dA transitions at
Asp87 can both confer Nal.sup.R, it is the C.fwdarw.T transitions
at Ser83 that are selectively enhanced by AID (FIG. 4). Despite
this strong evidence that AID-dependent mutation is non-random,
presumably depending upon local sequence environment, we cannot
discriminate on these datasets whether this sequence preference
reflects a hotspot preference similar to that of the dG/dC-biased
phase antibody hypermutation (Rada et al. (1998)) since there are
only a limited number of base substitutions that can yield the
selected phenotypes.
[0193] If AID-induced mutations in the E. coli transformants are
indeed occurring through deamination of dC, an enhancement of the
effect would be expected in cells lacking uracil-DNA glycosylase
(UDG). This is indeed the case. Although both UDG deficiency and
ectopic expression of AID are sufficient in themselves to yield a
mutator phenotype, AID expression in an ung background yielded a
mutation frequency that was much greater than the sum of their
independent mutation frequencies (FIG. 2 and Table 1). A similar
effect was seen in E. coli expressing Apobec-1 (see Table 2).
[0194] In E. coli ung-1 mutants, some back-up uracil
DNA-glycosylase activity may be provided by the product of the mug
gene (Sung et al. (2001) and Mokkapati et al. (2001)). It is found
that whilst the AID mutator effect is not significantly higher in a
mug.sup.- than mug.sup.+ background, the mug mutation allows at
most a slightly augmented AID-mutator effect when combined with
ung-1 (Table 1). If AID were to act by deaminating dG rather than
dC, an increased mutation frequency in a background deficient in
endonuclease V (encoded by nfi) might be anticipated since this
enzyme is implicated in the repair of deoxyxanthosine.sup.21,22.
This does not occur; the mutation frequency displayed by
AID-transformed nfi-1 cells approximates the sum of the frequencies
that are independently attributable to AID and nfi-1 (Table 1).
[0195] The data strongly suggest that AID mediates the deamination
of dC residues in the DNA. The homology of AID to Apobec-1 and
cytidine deaminases (Muramatsu et al (1999)) obviously argues in
favour of a close involvement of AID in the DNA deamination process
itself. The preferential targeting of mutation to the
immunoglobulin loci in lymphocytes presumably depends on proteins
with which AID associates. Given that the cis-regulation of both
switch recombination and hypermutation is linked to the
transcription regulatory elements (Manis et g. (2002); Betz et al
(1994)), it would appear likely that AID is recruited either
directly or indirectly by transcription- or chromatin-associated
factors.
[0196] APOBEC1-transformed bacteria grown in the presence of the
transcriptional inducer IPTG displayed massively elevated
frequencies of Rif.sup.R mutation (FIG. 5a). This enhancement was
confirmed by fluctuation analyses of Rif.sup.R mutants observed in
three independent experiments (Table 3 top). In comparison to
vector-transformed cells, the median enhancement by APOBEC1 ranged
from 440-, to 700-fold (mean of 530), whereas that attributable to
AID ranged from 3.8 to 13-fold (mean of 7.8 in agreement with data
above). The observed increases were due to APOBEC1 since
experiments performed in the absence of the inducer IPTG resulted
in a significantly diminished effect (FIG. 5b). Furthermore, single
amino acid changes E63A, W90S and C93A (which are located at or
close to the proposed Zn.sup.2+-coordination domain at the active
site of APOBEC1 (Navaratnam et al) abolished the enhancement (FIG.
5c). The stimulation was not specific to the selection or the locus
(Rif.sup.R mutations map largely to the rpoB gene) since it was
also clear when resistance to nalidixic acid was selected (due
mostly to mutations in the gyrA gene) (Table 3 (top)). Mutation
frequencies at gyrA were significantly lower than at rpoB; this
likely reflects restrictions in numbers of base substitutions that
each locus permits (both genes are essential and fewer sites appear
mutable in gyrA). It is notable that whilst APOBEC1 yields a
mutator phenotype in these assays as strong as that achieved with
some of the most potent E. coli mutators (e.g. mismatch
repair-defective strains (Schapper (1993), J. Bio. Chem. 268,
23762-23765)), the increased mutation load due to APOBEC1
expression caused no obvious defects in cell growth or viability
(FIG. 5d). This might reflect the nature of the lesions introduced
by APOBEC1 expression.
[0197] If, like AID, the observed stimulation of mutation is due to
increased dC deamination, then this should be apparent in the
spectrum of Rif.sup.R mutations--a bias toward dC/dG.fwdarw.dT/dA
transition mutations would result. This was confirmed by sequencing
rpoB gene PCR products from purified Rif.sup.R colonies selected
from APOBEC1-transformed cultures (as well as from AID-transformed
and vector-transformed controls). By comparison with
vector-transformed controls, APOBEC1-transformed cells showed a
dramatic shift in mutation spectrum, from 27% (32/120 mutations) to
100% (136/136 mutations) transition mutations at dC/dG (FIG. 2).
Consistent with the results presented above, AID-transformed E.
coli gave a somewhat less dramatic shift to 82% (102/124)
transitions at dC/dG (FIG. 7) reflecting the fact that AID, at
least in this system, is a less potent mutator than APOBEC1.
[0198] The mutation spectra revealed striking local differences
between vector-, APOBEC1- and AID-transformed cells with respect to
the specific dC/dG pairs targeted. Whereas, in keeping with AID,
the majority of dC/dG to dT/dA transitions in rpoB in
AID-transformed cells clustered at C1576 (45/124 mutations) and
G1586 (23/124 mutations), those in APOBEC1-transformed cells showed
a quite distinct distribution with major hotspots at C1535 (39/136
mutations) and C1592 (74/136 mutations) (FIG. 6a and FIG. 7). Thus,
the entire enhancement of Rif.sup.R mutation frequency observed in
APOBEC1-transformed cells occurs via transitions at dC/dG base
pairs but with the local targeting specificity being remarkably
different from that of AID.
[0199] The different local targeting specificities of APOBEC1 and
AID strongly argues that both proteins are involved in a dC
deamination process, generating dU/dG lesions in DNA. Given this
likely mode of action, one would expect that the stimulation of
mutation by APOBEC1 (like that by AID) would be enhanced in cells
lacking uracil-DNA glycosylase (UDG), an enzyme that specifically
recognises dU in DNA and initiates base excision repair of dU/dG
lesions (Lindahl). UDG-deficiency (ung-1) and APOBEC1 expression by
themselves enhance mutation about 10- and 500-fold, respectively.
APOBEC1 expression in UDG-deficient cells further increases levels
of mutation to about 2600-fold above vector-transformed ung.sup.+
cells, a much more than additive effect demonstrating that APOBEC1
is capable of triggering dU/dG lesions (Table 3 top). Despite the
additional mutation load in ung-1 cells, sequence analysis of the
mutations conferring Rif.sup.R revealed that, as for AID, the
mutational targeting by APOBEC1 in an ung-1 background was
essentially the same as in ung.sup.+ cells (data not shown).
[0200] At least six other APOBEC1-like proteins exist in humans
(Madsen et al; Jarmuz et al; Anant et al). APOBEC2 (also called
APOBEC1-related cytidine deaminase-1, ARCD-1) is found on
chromosome 6p21.1 and the others, termed APOBEC3A through APOBEC3G
(also termed phorbolins or ARCDs) are encoded on chromosome
22q12-q13. They all contain a region homologous to the putative
Zn.sup.2+-binding cytidine deaminase motif of APOBEC1. This
suggested that the mutator activities of these proteins might also
be conserved and prompted us to ask whether these homologues might
also work on DNA. Expression of APOBEC3C and APOBEC3G,
representative members of the chromosome 22 cluster (FIG. 7a), but
not APOBEC2, triggered increases in the frequencies of mutation to
Rif.sup.R and Nal.sup.R in E. coli (Table 3 bottom). The
stimulation of mutation by APOBEC3G is significantly greater when
monitored by the frequency of Rif.sup.R rather than Nal.sup.R
clones (Table 3). This may reflect the relatively strong target
preference of APOBEC3G (see below) taken together with the fact
that there are many more dC/dG targets in rpoB than in gyrA that
can confer resistance to the relevant antibiotic. The mutation
frequencies to Rif.sup.R achieved with both APOBEC3C and APOBEC3G
was also further elevated in an ung-1 background indicating that
they, like APOBEC1 and AID, potentiate dU/dG mispairs, substrates
for UDG and subsequent repair (Lindahl). In contrast, cells
transformed with a human APOBEC2 expression construct showed
neither increased mutation frequencies (ung.sup.+ or ung.sup.-
backgrounds; Table 3 bottom) nor a significantly altered rpoB
mutation spectrum (data not shown).
[0201] That APOBEC3C and APOBEC3G also act like APOBEC1 and AID is
supported further by a near complete shift in the spectrum of
mutations that yield Rif.sup.R, from 27% (32/120)
dC/dG.fwdarw.dT/dA transitions in vector-transformed cells to 94%
(102/108) and 88% (81/92) in APOBEC3C- and APOBEC3G-transformed
cells respectively (FIG. 7). Moreover, a direct comparison of the
dC/dG base pairs targeted by APOBEC3C and APOBEC3G with those
targeted by APOBEC1 and AID revealed obvious biases, the most
striking of which was mutation of C1691 bp APOBEC3G (71/92
mutations compared to 4/120 for vector-transformed cells).
APOBEC3C, on the other hand, shared one hotspot with APOBEC1
(44/108 at C1535) and another with AID (23/108 at C1576), and
appeared to be slightly more promiscuous causing dC/dG->dT/dA
transition mutations at eight positions in rpoB (FIG. 7b).
Example 2
APOBEC1 Expressed in E. coli can be Used to Mutate a Heterologous
Gene Integrated into the Chromosome.
[0202] The Bacillus subtilis gene SacB is toxic to E. coli in the
presence of sucrose. SacB is cloned under the control of the E.
coli promoter for PhoB and the cassette integrated into the
chromosome of E. coli strain DH10b at the Lambda phage attachment
site using pRB700 (FIG. 8a), a derivative of the CRIM system
plasmid pSK50A-uidA2 (Haldimann et al 1996 Proc Natl Acad Sci USA.
93(25):14361-6., Haldimann and Wanner 2001 J. Bacteriol.
183(21):6384-93). The PhoB promoter is active under conditions of
low inorganic phosphate availability. Thus mutants in the SacB
cassette can be selected by growing independent colonies
transfected with either an APOBEC-1 expression construct or a
control plasmid to saturation (using one fortieth of the colony as
the inoculum) and plating on minimal MOPS medium containing 5%
sucrose and limiting phosphate.
[0203] PCR and subsequent sequencing of the integrated SacB genes
in these sucrose resistant colonies demonstrates that spontaneous
mutants at this locus arise primarily by transposon insertion (and
therefore generate a significantly larger than expected PCR
product). This accounted for 13/16 spontaneous mutations. In
contrast, point mutations predominate when APOBEC-1 is expressed.
Furthermore, these point mutations are overwhelmingly (32/33)
transitions at C and G, consistent with these mutations arising by
deamination of dC as expected (FIG. 9).
Enhanced Targeting of Mutation can be Achieved by Inducing
Convergent Promoters Upstream and Downstream of the Desired
Gene.
[0204] The dependence of mutation caused by APOBEC-1 at this locus
on transcription is investigated. APOBEC-1 increases the mutation
frequency at SacB approximately 12-fold when colonies are grown in
rich medium, and growth in medium containing limiting phosphate
does not appear to enhance mutation at this locus. To investigate
the possibility that transcription in both directions might be
required to show an increase in mutation frequency, a variant SacB
cassette in pRB740 is created, under the control of the same PhoB
promoter, additionally placing the strong IPTG inducible Trc
promoter downstream of the SacB gene in the opposite orientation
(FIG. 8b).
[0205] The mutation frequency following growth in rich medium with
IPTG of SacB in this case is comparable to that of the original
cassette without the convergently orientated Trc promoter, and so
is the spectrum of point mutations obtained (20/20 are transitions
at C or G), indicating that this variant SacB cassette does not
mutate with an appreciably higher frequency when transcription is
induced only in the antisense direction.
[0206] However, following growth in limiting phosphate together
with IPTG, the mutation frequency is enhanced approximately 1000
fold above that achieved either by APOBEC-1 without the downstream
promoter under the same conditions, or with the downstream promoter
in rich medium (FIG. 10a, b).
[0207] Thus, activation of convergent promoters located on opposite
sides of the gene is able to enhance APOBEC-1 induced mutation at
that locus very appreciably. Furthermore, expression of the less
mutagenic APOBEC family members AID and APOBEC-3G under these
conditions of convergent transcription also gives rise to a
significant increase in mutation frequency above background and a
shift towards the expected PCR product size, indicating that
transposon insertions are responsible for a lower proportion of the
observed mutants. The expected PCR size product is obtained in
10/10 and 7/10 cases for AID and APOBEC3G respectively, compared to
only 2/10 and 5/10 respectively under non-transcribing conditions
(FIG. 11).
[0208] Under conditions of bi-directional transcription,
transitions at C or G account for 8/8 and 5/5 of the point
mutations observed for AID and APOBEC3G respectively (FIG. 11).
Taken together, these results demonstrate that targeted deamination
by members of the APOBEC family can be achieved if the desired gene
to be targeted is placed between convergent promoters (FIG.
11).
Example 3
Deamination of Cytosine to Uracil in DNA can be Achieved In Vitro
Using Partially Purified APOBEC1 from Extracts of Transformed
Escherichia coli.
Plasmids and Bacteria
[0209] The pTrc99- and pET-based expression vectors for rat APOBEC1
and its E63->A mutant, for human APOBEC2 and for E. coli dCTP
deaminase as well as the E. coli host strains have been described
previously (Rada et al. (2002) Curr. Biology 12, 1748-1755,
Randerath K., and Randerath E. (1967) Method Enzymol. 12, 323-347).
The pTrc99- and pET-based vectors differ both in the nature of the
promoter used (pTrc99 uses the trp/lac hybrid promoter whereas pET
uses the T7 promoter) and in the length of heterologous peptide
linked to the amino-terminus of the recombinant protein (9 amino
acid with pTrc99 but 34 amino acid with pET (Rada et al. (2002)
Curr. Biology 12, 1748-1755., Randerath K., and Randerath E. (1967)
Method Enzymol. 12, 323-347)).
Oligodeoxyribonucleotides
[0210] The oligodeoxyribonucleotides used are listed in Table
4.
Preparation of Recombinant APOBEC1
[0211] A 2 ml overnight culture of a fresh E. coli transformant
grown in LB, 0.2% Glucose, 50 .mu.g/ml carbenicillin was diluted
into 300 ml of the same medium and grown at 37.degree. C. to an
A.sub.600 of 0.8. The culture was chilled on ice for 20 min and
then incubated with aeration for 16 h at 16.degree. C. in the
presence of inducer (1 mM IPTG). Cells were harvested by
centrifugation, washed and resuspended in 20 ml H buffer (50 mM
Tris.HCl, pH7.4, 50 mM KC1, 5 mM EDTA, 1 mM DTT and a protease
inhibitor cocktail [Roche]).
[0212] Following sonication and ultracentrifugation (100,000 g for
45 min), the supernatant was passed through a 0.2 .mu.m filter and
applied to a Sepharose Fast-Flow Mono-Q column (Amersham
Biosciences; 10 ml bed volume). After washing with seven column
volumes of buffer H, bound proteins were eluted in buffer H
supplemented with increasing salt concentrations (from 50 to 1500
mM Cl) collecting 15 ml fractions. Fractions and flow-through were
concentrated one-hundred fold using VivaSpin concentrators (M.sub.r
10,000 cut-off) (VivaScience) and assayed. Samples eluting with
1000-1500 mM salt were pooled and loaded in a volume of 0.5 ml onto
a HighPrep Sephacryl S-200 High-Resolution 16/60 gel-filtration
column (Amersham Biosciences) in buffer H. Fractions (1 ml) were
collected and concentrated twenty fold before analysis.
TLC-Based Deaminase Assay
[0213] Samples (2-4 .mu.l) were incubated at 37.degree. C. for 5 h
in 20 .mu.l of buffer R (40 mM Tris pH 8, 40 mM KCl, 50 mM NaCl, 5
mM EDTA, 1 mM DTT, 10% glycerol) containing 75,000 cpm of
.alpha.-[.sup.32P]dC-labelled single-stranded DNA (prepared by a 3
min heating to 95.degree. C. of the products of asymmetric PCR
amplification of the lad region in pTrc99 performed using
.alpha.-[.sup.32P]dCTP (3000 Ci/mmol)). Following phenol extraction
and ethanol precipitation, the DNA was digested with Penicillium
citrinum P1 nuclease (Sigma) overnight at 37.degree. C. (Grunau C.,
Clark S. J., and Rosenthal A. (2001) Nucleic Acids Res. 29, E65)
and the P1 digests then subjected to thin layer chromatography on
PEI-cellulose in either (i) 0.5 M LiCl at 4.degree. C. or (ii) at
room temperature in 1 M CH.sub.3COOH until the buffer front had
migrated 2.5 cm and then in 0.9 M CH.sub.3COOH:0.3M LiCl (Cohen, R.
M., and Wolfenden, R. (1971) J. Biol. Chem. 246, 7561-7565).
Products were detected using a phosphorimager. Chemical deamination
of cytosine in DNA using bisulfite/hydroquinone was performed as
described (Yamanaka et al. (1995) Proc. Nat. Acad. Sci. USA, 92,
8483-8487).
UDG-Based Deaminase Assay
[0214] Samples (1-2 .mu.l) were incubated at 37.degree. C. for 2 h
in 10 .mu.l of buffer R with 5'-biotinylated oligonucleotides that
either were synthesized with fluorescein at their 3'-ends (3 pmol
of oligonucleotide per reaction) or were 3'-labelled by ligation
with .alpha.-[.sup.32P]dideoxyadenylate (100,000 cpm; 0.1 pmol)
using terminal deoxynucleotidyl transferase.
[0215] Reactions were terminated by heating to 90.degree. C. for 3
min and oligonucleotides purified on streptavidin magnetic beads
(Dynal), washing at 72.degree. C. (except in FIG. 2A, where the
streptavidin purification step was omitted). Deamination of
cytosine in the oligonucleotides was monitored by incubating the
bead-immobilised oligonucleotides at 37.degree. C. for 30 min with
excess uracil-DNA glycosylase (0.5 units UDG; enzyme and buffer
from NEB) and then bringing the sample to 0.15M in NaOH and
incubating for a further 30 min. The oligonucleotides were then
subjected to electrophoresis on 15% PAGE-urea gels which were
developed by either fluorescence detection or phosphorimager
analysis.
Western Blotting
[0216] Western blot detection of APOBEC1 following SDS/PAGE of
samples that had been diluted 20-100 fold was performed using a
goat-anti-APOBEC1 serum (Santa Cruz Biotechnology), developing with
horseradish peroxidase-conjugated donkey anti-goat immunoglobulin
antiserum (Binding Site, Birmingham, UK). Low-range molecular
weight markers were from BioRad.
Results
DNA Deamination Assay in Cell Extracts
[0217] Since, of all the APOBEC family members tested, APOBEC1
displayed the most potent mutator activity in the E. coli mutation
assay (Randerath K. and Randerath E. (1967) Method Enzymol 12,
323-347), APOBEC1-transformed E. coli were investigated in order to
see if DNA deamination activity in vitro using cell extracts could
be detected.
[0218] Initially, the UDG-based deaminase assay was tried, working
with an oligodeoxyribonucleotide substrate. However, no evidence of
deamination was obtained using double-stranded oligonucleotide
substrates whereas single-stranded oligonucleotides were rapidly
degraded by both APOBEC1 and control extracts (data not shown). The
possibility that the DNA deaminating activity might be specific for
single-stranded substrates but that this activity might be masked
by non-specific nucleases was investigated. An assay that would be
less sensitive to contaminating nucleases (FIG. 12A) was
devised.
[0219] The bacterial extracts were incubated with
.alpha.-[.sup.32P]dC-labelled single-stranded DNA which was then
purified, digested with nuclease P1 and subjected to thin-layer
chromatography to test for the presence of .alpha.-[.sup.32P]dUMP.
Clear evidence of dC deamination in this assay was detected using
extracts of E. coli expressing two different APOBEC1 constructs but
not from control extracts or from extracts made from E. coli cells
carrying plasmids expressing mutant APOBEC1, APOBEC2 or dCTP
deaminase (none of which function as DNA mutators in the bacterial
assay (Randerath K. and Randerath E. (1967) Method Enzymol 12,
323-347)) (FIG. 12B). The DNA deaminase activity was evident in
APOBEC1-transformants of a mutant E. coli deficient in both dcd-
and cdd-encoded deaminases (FIG. 12B (iii)). That the product of
APOBEC1 action was indeed dUMP is indicated by the co-migration of
the radioactive product with dUMP in two distinct buffer
systems.
[0220] These results suggested fractionation of the extracts of
APOBEC1-transformed E. coli to see if the DNA deamination activity
could be sufficiently separated from non-specific nucleases so as
to be detectable using the oligonucleotide cleavage assay.
Partial Purification
[0221] Pilot experiments revealed that ion-exchange chromatography
could be used to obtain samples of APOBEC1 that contained
diminished non-specific nuclease activity. Thus, whilst only a
proportion of the APOBEC1 polypeptide bound to the Mono-Q column
(around 10-20% based on ECL quantitation of the Western blot
assay), elution of this bound fraction with >0.8 M CI yielded a
sample that displayed cytosine-DNA deamination activity (as
monitored using the TLC-based assay) but containing diminished
non-specific nuclease activity in the UDG-based assay (FIG. 13A).
These fractions were then concentrated and subjected to gel
filtration (FIG. 13B). The major APOBEC1 peak eluted in fractions
7-9 (corresponding to an M.sub.r of 95-140,000) co-eluting with
peak DNA deaminating activity. Indeed, with these fractions from
the gel filtration column, DNA deamination could now readily be
detected by the UDG-based assay using a single-stranded
oligonucleotide substrate (although the peak fractions also
contained activity that removed the 3'-label from the
oligonucleotide). Mass spectrometric analysis of proteins in
fraction 9 following SDS/PAGE revealed the recombinant APOBEC1
migrating at the position marked by the asterisked in FIG. 13B(i)
although the majority of the bands derived from ribosomal
proteins.
Characteristics of the DNA Deaminating Activity
[0222] The UDG-based deaminase assay was used to monitor the
specificity and characteristics of the partially purified APOBEC1
(FIG. 14A). Samples were incubated with a single-stranded
oligodeoxyribonucleotide (with or without its complement) which
contained internal dC residue(s) and that was 5'-biotinylated as
well as 3'-labelled. After purification on streptavidin, the
oligonucleotide was treated with UDG (plus alkali), resulting in
site-specific cleavage if the oligonucleotide had been subjected to
dC->dU deamination. Thus, deamination is read out by the
appearance of the specific cleavage product following PAGE-urea
analysis.
[0223] The partially-purified wild type protein (but not the
E63->A mutant) showed clear activity on a single-stranded
oligonucleotide with the cleavage being dependent on the subsequent
incubation with UDG (FIG. 14B, C). The deaminating activity was not
inhibited by tetrahydrouridine (which inhibits cytidine deaminases
(Frederico et al. (1990) Biochemistry, 29, 2532-2537)) or by RNAse
(FIG. 14D). Strikingly [and consistent with our inability to detect
deamination on double-stranded oligonucleotide substrates using
crude extracts of bacterial transformants (see above)], the
activity was blocked if a complementary (but not if an irrelevant)
oligonucleotide was titrated into the assay (FIG. 14E). Examination
of the cleavage products generated in the UDG-based assay suggests
that not all dC residues are equally susceptible to
APOBEC1-mediated deamination. It is clear, for example, that in
oligonucleotide SPM168 the third cytosine in the sequence TCCGCG is
much less favoured than the other two (FIG. 14B-E). Similarly,
evidence of specificity comes from comparing various related
oligonucleotides as substrates, where all the data taken together
point to deamination being especially disfavoured when a purine is
located immediately 5' of the cytosine (FIG. 14F).
Discussion
[0224] The results described here provide biochemical evidence that
APOBEC1-mediated deamination of cytosine to uracil can occur on
single-stranded DNA, is dependent on local sequence context and is
abolished by mutation of the APOBEC1 zinc-coordination motif.
Unlike AID (where genetic evidence indicates that the natural
physiological substrate of deamination is DNA (Harris et al. (2002)
Mol. Cell. 10, 1247-1253, Wagner et al. (1989) Proc. Nat. Acad.
Sci. USA, 86, 2647-2651), the major physiological substrate of
APOBEC1 is clearly apolipoprotein B RNA (Teng et a (1993) Science
260, 1816-1819, Blanc, V. and Davidson, N. O. (2003) J. Biol. Chem.
278, 1395-1398). Nevertheless, the observation that misexpression
of APOBEC1 in transgenic mice predisposes to cancer suggests that
APOBEC1-mediated DNA deamination could well be of pathological
relevance.
[0225] Given the abundance of APOBEC1 polypeptide in the peak
fraction from the gel filtration column, it appears that--on
average--each molecule of recombinant APOBEC1 is responsible for in
the order of a single deamination event in a 10 minute incubation
in the UDG-based assay. Crude calculations indicate that if the
.about.500 molecules of APOBEC1 expressed in each E. coli
transformant displayed a DNA deamination activity of this order in
vivo and if this were targeted randomly to all cytosine residues in
the genome, then this could, in principle, be more than sufficient
to account for the several thousand-fold enhanced mutation
frequencies seen at the rpoB and other loci in UDG-deficient E.
coli following 20 generations of growth (Randerath K. and Randerath
E. (1967) Method Enzymol 12, 323-347). Similarly, somatic
hypermutation of immunoglobulin variable genes by targeted
AID-mediated dC deamination may involve a single and most probably
less than ten targeted dC deamination events in each B lymphocyte
cell cycle.
[0226] The results provide information about the preferred target
of APOBEC1-mediated DNA deamination. The in vitro assay reveals a
clear sensitivity to the local sequence context of the dC residue
to be deaminated. The results obtained here suggest there may be
bias against a 5'-flanking purine residue. This would accord well
with the in vivo data where a near-total restriction to mutation at
dC residues with a 5'-flanking pyrimidine is seen at the rpoB locus
(Randerath and Randerath).
[0227] The in vitro assay also reveals that APOBEC1 deamination is
targeted to single-stranded DNA and, indeed, was undetectable on
double-stranded DNA. This specificity for single-stranded DNA is in
accordance with the fact that the natural substrate of APOBEC1 is
most likely single-stranded RNA (Blanc and Davidson) and,
presumably, the same active site in APOBEC1 is used for both types
of polynucleotide. Furthermore, spontaneous deamination of cytosine
is also much more rapid in single- (as opposed to double-) stranded
DNA which may explain the correlation with transcription of the DNA
target gene described herein and where convergent promoters
increase the availability of single-stranded DNA to APOBEC-1.
Example 4
Expression of Apobec-1 Fusion Proteins
[0228] The Apobec-1 expression plasmid was generated as described
above but a nucleic acid encoding rat Apobec1 with an aminoterminal
fusion encoding:
TABLE-US-00001 (SEQ ID NO: 15)
Met-His-His-His-His-His-His-His-His-Tyr-Asp-Ile-Pro-Thr-Ala-Ser-Glu-Asn-Le-
u-Tyr- Phe-Gln-Gly-Ser- joining to the initiator Met of
Apobec-1
[0229] The expression construct was expressed from in E. coli
strain BL21 DE3 (purchased from Novagen) and the effect on the
frequency of mutation to rifampicin-resistance (Rif.sup.R) measured
by fluctuation analysis as described above.
[0230] The results are as follows:
TABLE-US-00002 Rif R colonies vector alone 42 35 28 23 His-Apobec-1
3000 3000 2000 1500
(The numbers are numbers of Rifr colonies in 4 independent
experiments, the experiments being performed as in Tables 1 and
2).
[0231] This demonstrates that the Apobec fusion protein with a
His-tag fused to its N-terminus retains mutator activity in E.
coli.
Example 5
Hybridisation Experiments
[0232] A cancer profiling array was obtained from Clontech (Cat.
No. 7757-1) and hybridised as directed with the following
.sup.32P-dCTP-labeled human cDNA probes: APOBEC1 (IMAGE clone
2107422), APOBEC3G (IMAGE clone 1284557) and ubiquitin (control
provided with array). The array was hybridised first with APOBEC1,
subsequently with APOBEC3G, and finally with ubiquitin. After each
hybridisation the probe was removed by boiling in 0.5% SDS.
Hybridisation images, shown in FIG. 15, were visualised with the
Typhoon Phosphoimaging System (Pharmacia) and ImageQuant software.
Data are grouped by tissue to facilitate comparison, although the
entire blot (representing all tissues shown) was hybridised
simultaneously as a single filter in each experiment (i.e. with
each probe) and the autoradiographic image subsequently separated
by computer manipulation (without adjusting gain or
background).
Results
[0233] APOBEC1 expression appears to be restricted to
gastrointestinal tissues (colon, stomach, rectum, and small
intestine), whereas APOBEC3G was expressed to some extent in all
tissues examined. Perhaps most notable is the fact that for some
tumour samples, APOBEC1 (colon and rectum) and APOBEC3G (breast and
kidney) appear better expressed than in corresponding normal
tissues (only intra-hybridisation pairs should be considered). Note
also that for APOBEC1 hybridisation of stomach samples the opposite
may be the case.
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[0264] All publications mentioned in the above specification, and
references cited in said publications, are herein incorporated by
reference. Various modifications and variations of the described
methods and system of the present invention will be apparent to
those skilled in the art without departing from the scope and
spirit of the present invention. Although the invention has been
described in connection with specific preferred embodiments, it
should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
which are obvious to those skilled in molecular biology or related
fields are intended to be within the scope of the following claims.
Sequence CWU 1
1
27131DNAArtificial SequencePCR primer 1nnngaattca aggctgaaca
tgaatccaca g 31230DNAArtificial SequencePCR primer 2nnnnngtcga
cggagacccc tcactggaga 30331DNAArtificial SequencePCR primer
3nnngaattca aggatgaagc ctcacttcag a 31430DNAArtificial SequencePCR
primer 4nngactgcag cccatccttc agttttcctg 30534DNAArtificial
SequencePCR primer for mutagenesis 5accaacaaac acgttgcagt
caatttcata gaaa 34634DNAArtificial SequencePCR primer for
mutagenesis 6tttctatgaa attgactgca acgtgtttgt tggt
34733DNAArtificial SequencePCR primer for mutagenesis 7acctggttcc
tgtcctcgag tccctgtggg gag 33833DNAArtificial SequencePCR primer for
mutagenesis 8ctccccacag ggactcgagg acaggaacca ggt
33933DNAArtificial SequencePCR primer for mutagenesis 9ctgtcctgga
gtcccgctgg ggagtgctcc agg 331033DNAArtificial SequencePCR primer
for mutagenesis 10cctggagcac tccccagcgg gactccagga cag
331121DNAArtificial SequencePCR primer 11ttggcgaaat ggcggaaaac c
211222DNAArtificial SequencePCR primer 12caccgacgga taccacctgc tg
221320DNAArtificial SequencePCR primer 13gcgcggctgt gttataattt
201421DNAArtificial SequencePCR primer 14ttccgtgccg tcatagttat c
211524PRTArtificial SequenceSynthetic N-terminal fusion sequence
15Met His His His His His His His His Tyr Asp Ile Pro Thr Ala Ser 1
5 10 15 Glu Asn Leu Tyr Phe Gln Gly Ser 20 1640DNAArtificial
Sequenceoligodeoxyribonucleotide 16attattatta ttagctattt atttatttat
ttatttattt 401742DNAArtificial Sequenceoligodeoxyribonucleotide
17attattatta ttccgcggat ttatttattt atttatttat tt
421840DNAArtificial Sequenceoligodeoxyribonucleotide 18attattgtta
ttatcaattt gtttatttgt ttatttattt 401940DNAArtificial
Sequenceoligodeoxyribonucleotide 19attattgtta ttatcgattt gtttatttgt
ttatttattt 402040DNAArtificial Sequenceoligodeoxyribonucleotide
20attattgtta ttaactattt gtttatttgt ttatttattt 402142DNAArtificial
Sequenceoligodeoxyribonucleotide 21aaataaataa ataaataaat aaatccgcgg
aataataata at 422226DNAArtificial Sequenceoligodeoxyribonucleotide
22gttctggcaa atattctgaa atgagc 2623209DNAArtificial SequenceRpoB
vector DNA - see figure 3 23ggttccagcc agctgtctca gtttatggac
cagaacaacc cgctgtctga gattacgcac 60aaacgtcgta tctccgcact cggcccaggc
ggtctgaccc gtgaacgtgc aggcttcgaa 120gttcgagacg tacacccgac
tcactacggt cgcgtatgtc caatcgaaac ccctgaaggt 180ccgaacatcg
gtctgatcaa ctctctctg 2092466PRTArtificial SequenceRpoB protein
sequence - figure 3 24Ser Gln Leu Ser Gln Phe Met Asp Gln Asn Asn
Pro Leu Ser Glu Ile 1 5 10 15 Thr His Lys Arg Arg Ile Ser Ala Leu
Gly Pro Gly Gly Leu Thr Arg 20 25 30 Glu Arg Ala Gly Phe Glu Val
Arg Asp Val His Pro Thr His Tyr Gly 35 40 45 Arg Val Cys Pro Ile
Glu Thr Pro Glu Gly Pro Asn Ile Gly Leu Ile 50 55 60 Asn Ser 65
2524DNAArtificial SequencegyrA vector DNA sequence -figure 4
25ggtgactcgg cggtctatga cacg 24268PRTArtificial SequenceGyrA
Protein sequence- figure 4 26Gly Asp Ser Ala Val Tyr Asp Thr 1 5
27209DNAArtificial SequenceRpoB vector for Apobec - figure 6
27ggttccagcc agctgtctca gtttatggac cagaacaacc cgctgtctga gattacgcac
60aaacgtcgta tctccgcact cggcccaggc ggtctgaccc gtgaacgtgc aggcttcgaa
120gttcgagacg tacacccgac tcactacggt cgcgtatgtc caatcgaaac
ccctgaaggt 180ccgaacatcg gtctgatcaa ctctctctg 209
* * * * *
References