U.S. patent application number 12/243711 was filed with the patent office on 2009-02-19 for using cytosine deaminases to diminish retroelement transfer from pigs to humans.
This patent application is currently assigned to REGENTS OF THE UNIVERSITY OF MINNESOTA. Invention is credited to SCOTT C. FAHRENKRUG, REUBEN S. HARRIS, STEFAN R. JONSSON, REBECCA ST. CLAIRE LARUE.
Application Number | 20090049563 12/243711 |
Document ID | / |
Family ID | 37595856 |
Filed Date | 2009-02-19 |
United States Patent
Application |
20090049563 |
Kind Code |
A1 |
HARRIS; REUBEN S. ; et
al. |
February 19, 2009 |
USING CYTOSINE DEAMINASES TO DIMINISH RETROELEMENT TRANSFER FROM
PIGS TO HUMANS
Abstract
Transgenic pigs that express one or more non-porcine cytosine
deaminases are described as well as methods of making and using
such pigs.
Inventors: |
HARRIS; REUBEN S.; (ST.
PAUL, MN) ; JONSSON; STEFAN R.; (KOPAVOGUR, IS)
; FAHRENKRUG; SCOTT C.; (MINNEAPOLIS, MN) ; LARUE;
REBECCA ST. CLAIRE; (LAUDERDALE, MN) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
PO BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
REGENTS OF THE UNIVERSITY OF
MINNESOTA
SAINT PAUL
MN
|
Family ID: |
37595856 |
Appl. No.: |
12/243711 |
Filed: |
October 1, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11472743 |
Jun 22, 2006 |
|
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12243711 |
|
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60694054 |
Jun 24, 2005 |
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Current U.S.
Class: |
800/17 |
Current CPC
Class: |
A01K 2217/05 20130101;
C12N 9/78 20130101; C12N 2517/02 20130101; A01K 67/0273 20130101;
A01K 2217/075 20130101; A01K 2227/108 20130101; A01K 2207/15
20130101; A01K 2267/025 20130101; A01K 2217/00 20130101; C12N
2740/13011 20130101; A01K 2267/0337 20130101; C12N 15/8509
20130101; A01K 2267/02 20130101; A01K 67/0275 20130101 |
Class at
Publication: |
800/17 |
International
Class: |
A01K 67/027 20060101
A01K067/027 |
Claims
1. A transgenic pig, the nucleated cells of which comprise a
nucleic acid construct, said nucleic acid construct comprising a
transcriptional unit comprising a regulatory region operably linked
to a nucleic acid sequence encoding a non-porcine cytosine
deaminase polypeptide, wherein expression of said non-porcine
cytosine deaminase polypeptide in at least some of the cells of the
pig results, upon co-culture with human cells, in decreased
capability of said cells to transmit porcine endogenous
retroviruses to the human cells.
2. The transgenic pig of claim 1, wherein said regulatory region is
a constitutive promoter.
3. The transgenic pig of claim 1, wherein said porcine regulatory
region is a tissue-specific or an organ-specific promoter.
4. The transgenic pig of claim 1, wherein an insulator element and
an inverted repeat of a transposon flank each side of said
transcriptional unit.
5. The transgenic pig of claim 1, wherein said non-porcine cytosine
deaminase is selected from the group consisting of AID, APOBEC1,
APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3E,
APOBEC3F, APOBEC3G, and APOBEC3H.
6. The transgenic pig of claim 1, wherein said non-porcine cytosine
deaminase is human APOBEC3F or human ABOBEC3G.
7. Cells derived from the transgenic pig of claim 1.
8. Tissue isolated from the transgenic pig of claim 1.
9. Progeny of said transgenic pig of claim 1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation (and claims the benefit
of priority under 35 USC 120) of U.S. application Ser. No.
11/472,743, filed Jun. 22, 2006, which claims the benefit of U.S.
Provisional Application No. 60/694,054, filed Jun. 24, 2005, all of
which are incorporated by reference in their entirety.
TECHNICAL FIELD
[0002] This invention relates to transgenic pigs and porcine cells
that have decreased capability of transmitting retroelements such
as porcine endogenous retroviruses to non-porcine cells and
tissues, and more particularly to transgenic pigs and porcine cells
that contain non-porcine cytosine deaminases.
BACKGROUND
[0003] Treating diabetes remains a substantial burden for patients
and their families, with up to 50% of patients experiencing
devastating secondary complications due to a lifetime of exposure
to elevated glucose levels. Currently the only way to restore and
sustain insulin without the associated risk of hyper- or
hypoglycemia is to replace the patient's insulin-producing cells,
the islets of Langerhans: either by the transplant of a
vascularized pancreas or by the infusion of isolated islets.
However, suitable human pancreas donors are very rare. Pigs provide
a potentially unlimited source of islets for xenotransplantation to
diabetic patients, and can be developed to the point of clinical
applicability, potentially well before other developing
technologies, such as stem cells.
[0004] The potential for transmission of viruses from the donor to
host tissue remains an impediment to the use of xenotransplantation
for the treatment of diabetes. Although rigorous biosecurity and
testing can eliminate most agents from potential donor pigs, one
agent in particular is recalcitrant to this approach. Most, perhaps
all, vertebrate genomes, pigs and humans included, harbor mobile
nucleic acid sequences by prior retroviral infections. Although
most of these are functionally inactive, pig cells contain several
types of ACTIVE retroelements called porcine endogenous
retroviruses (PERVs). These agents are generally innocuous to the
pig, but are of major concern for xenotransplantation. Under
laboratory conditions in which human and pig cells are co-cultured,
transmission of PERVs from pig to human tissue has been
demonstrated (Patience et al. (1997) Nat Med 3, 282-286). It is
unclear what, if any ramifications this transmission would have for
a patient, but the theorized possibility that PERVs alone or in
combination with human agents could cause disease has emerged as a
major hurdle to the widespread application of
xenotransplantation.
SUMMARY
[0005] The invention is based on the expression of non-porcine
cytosine deaminase polypeptides in pig cells and tissues. As
described herein, expression of non-porcine cytosine deaminases
(e.g., human cytosine deaminases) in porcine cells and tissues can
facilitate control of the transmission of retroelements such as
PERVs to human cells. As a result, pigs cells and tissues that
contain non-porcine cytosine deaminases have reduced capability of
transmitting PERVs to human cells and as such, can reduce the risks
associated with xenotransplantation from cross species gene
transfer.
[0006] In one aspect, the invention features a nucleic acid
construct that includes a transcriptional unit, the transcriptional
unit including a porcine regulatory region operably linked to a
nucleic acid sequence encoding a non-porcine cytosine deaminase
polypeptide. An inverted repeat of a transposon can flank each side
of the transcriptional unit. An insulator element also can flank
each side of the transcriptional unit. The nucleic acid construct
further can include a nucleic acid sequence encoding a
transposase.
[0007] The cytosine deaminase polypeptide can be selected from the
group consisting of AID, APOBEC1, APOBEC2, APOBEC3A, APOBEC3B,
APOBEC3C, APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3G, and APOBEC3H. For
example, the cytosine deaminase polypeptide can be an APOBEC3F
polypeptide such as a human APOBEC3F polypeptide or can be an
APOBEC3G polypeptide such as a human APOBEC3G polypeptide. In some
embodiments, the nucleic acid sequence encodes at least two
cytosine deaminase polypeptides (e.g., an APOBEC3F polypeptide and
an APOBEC3G polypeptide). The porcine regulatory region can be a
constitutive promoter or a tissue-specific promoter.
[0008] In another aspect, the invention features an isolated
porcine cell that includes a nucleic acid construct, the nucleic
acid construct including a regulatory region operably linked to a
nucleic acid sequence encoding a non-porcine cytosine deaminase
polypeptide. The cell can be an embryonic cell, a fetal porcine
cell (e.g., a fibroblast), an adult porcine cell (e.g., a dermal
fibroblast), a germ cell (e.g., an oocyte or an egg), a stem cell
(e.g., an adult stem cell or an embryonic stem cell), or a
progenitor cell.
[0009] The invention also features an isolated porcine cell that
includes a non-porcine cytosine deaminase. The cell further can
include a nucleic acid encoding the non-porcine cytosine deaminase.
The cytosine deaminase polypeptide can be selected from the group
consisting of AID, APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C,
APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3G, and APOBEC3H. For example,
the cytosine deaminase polypeptide can be an APOBEC3F polypeptide
such as a human APOBEC3F polypeptide or can be an APOBEC3G
polypeptide such as a human APOBEC3G polypeptide. In some
embodiments, the nucleic acid sequence encodes at least two
cytosine deaminase polypeptides (e.g., an APOBEC3F polypeptide and
an APOBEC3G polypeptide). The regulatory region can be a
constitutive promoter or a tissue-specific promoter.
[0010] In another aspect, the invention features a transgenic pig,
cells derived from the transgenic pig, tissue isolated from the
transgenic pig, and progeny of the transgenic pig. The nucleated
cells of the pig include a nucleic acid construct, which includes a
transcriptional unit that includes a regulatory region operably
linked to a nucleic acid sequence encoding a non-porcine cytosine
deaminase polypeptide. Expression of the non-porcine cytosine
deaminase polypeptide in at least some of the cells of the pig
results, upon co-culture with human cells, in decreased capability
of the cells to transmit porcine endogenous retroviruses to the
human cells. The regulatory region can be a constitutive promoter
or a tissue-specific promoter. An insulator element and an inverted
repeat of a transposon can flank each side of the transcriptional
unit. The cytosine deaminase polypeptide can be selected from the
group consisting of AID, APOBEC1, APOBEC2, APOBEC3A, APOBEC3B,
APOBEC3C, APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3G, and APOBEC3H. For
example, the cytosine deaminase polypeptide can be an APOBEC3F
polypeptide such as a human APOBEC3F polypeptide or can be an
APOBEC3G polypeptide such as a human APOBEC3G polypeptide.
[0011] In another aspect, the invention features a method for
making a transgenic pig. The method includes introducing a
transgenic pig cell into an enucleated pig oocyte to establish a
combined cell, the transgenic pig cell includes a nucleic acid
construct, which includes a transcriptional unit that includes a
regulatory region operably linked to a nucleic acid sequence
encoding a non-porcine cytosine deaminase polypeptide; producing a
porcine embryo from the combined cell; transferring the porcine
embryo to a recipient female; and allowing the porcine embryo to
develop in the recipient female to produce the transgenic pig. An
insulator element and an inverted repeat of a transposon can flank
each side of the transcriptional unit.
[0012] The invention also features a method of making a transgenic
pig. The method includes introducing a nucleic acid construct into
a fertilized egg to produce an injected fertilized egg, where the
nucleic acid construct includes a transcriptional unit that
includes a regulatory region operably linked to a nucleic acid
sequence encoding a non-porcine cytosine deaminase polypeptide;
transferring the injected fertilized egg to a recipient female; and
allowing the injected fertilized egg to develop in the recipient
porcine female to produce the transgenic pig.
[0013] In yet another aspect, the invention features a method for
making a transgenic pig cell. The method includes introducing a
nucleic acid construct into a pig cell, the nucleic acid construct
including a regulatory region operably linked to a nucleic acid
sequence encoding a non-porcine cytosine deaminase polypeptide.
[0014] The invention also features a method for making a transgenic
pig cell. The method includes introducing into a pig cell: a) a
nucleic acid construct that includes a transcriptional unit, the
transcriptional unit including a regulatory region operably linked
to a nucleic acid sequence encoding a non-porcine cytosine
deaminase polypeptide, wherein an insulator element and an inverted
repeat of a transposon flank each side of the transcriptional unit;
and b) a source of a transposase. The source of the transposase can
include a nucleic acid encoding the transposase. The transposon and
the source of transposase can be present on separate nucleic acid
constructs or on the same nucleic acid construct.
[0015] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used to practice the invention, suitable methods and
materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0016] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
DESCRIPTION OF DRAWINGS
[0017] FIGS. 1A and 1B are histograms that indicate APOBEC3G
stimulates mutation in S. cerevisiae by the uracil excision
pathway. (A) APOBEC3G expression causes an increase in the median
frequency of mutation to Can.sup.R. Each X represents the frequency
derived from an independent culture and the median is indicated.
The Y-axis reports the observed number of Can.sup.R colonies per
million viable yeast cells. Yeast expressing the control vector
showed a frequency of spontaneous mutation to Can.sup.R similar to
that reported previously (Huang et al. (2003) Proc Natl Acad Sci
USA 100, 11529-34; Rattray et al. (2002) Genetics 162, 1063-77).
The data are representative of seven independent experiments. FIG.
2 reports the mean of the median values for these experiments). (B)
APOBEC3G and Ugi co-expression triggers a synergistic increase in
the frequency of mutation to CanI. APOBEC3G is tagged with the DNA
binding domain of LexA unless noted. The parameters are identical
to FIG. 1A.
[0018] FIG. 2 is a histogram summarizing the APOBEC3G-dependent
CAN1 mutator phenotype from seven independent experiments, each
performed with 6-8 independent yeast cultures expressing APOBEC3G
or a control vector. The average of the median mutation frequencies
and the corresponding SEMs are shown. The average median mutation
frequency of yeast expressing the vector control was
1.3.times.10.sup.-6; this number was assigned a value of one to
normalize and highlight the magnitude of the APOBEC3G mutator
phenotype (33-fold). Data from FIGS. 1A and 1B, and FIG. 3 are
included with data from three additional experiments. The variation
in values among individual cultures for each experiment and among
the median values of the seven experiments is expected from the
stochastic appearance of Can.sup.R mutants in the growing
cultures.
[0019] FIG. 3 is a histogram indicating that APOBEC3G stimulates
mutation in S. cerevisiae by the uracil excision pathway II. Uracil
DNA glycosylase inhibition by Ugi or its ablation by a deletion
caused virtually identical phenotypes. The experimental parameters
are identical to those described for FIG. 1A.
[0020] FIG. 4A-4D demonstrate that APOBEC3G triggers C/G->T/A
transition mutations in S. cerevisiae. (A) Histograms summarizing
the types of mutations found in the CAN1 gene of S. cerevisiae
expressing a control vector or APOBEC3G Data from Lex-APOBEC3G and
untagged APOBEC3G expressing cells were nearly identical and were
pooled for these analyses. Mutations were categorized as
transitions (Trs), transversions (Trv), deletions (Del) or
insertions (Ins). (B) Summary of the base substitution mutations
found in the CAN1 gene of S. cerevisiae expressing a control vector
or APOBEC3G (C) Histograms summarizing the types of mutations found
in the CAN1 gene of S. cerevisiae expressing Ugi and a control
vector or APOBEC3G Labeled as in FIG. 4A. (D) Summary of the base
substitution mutations found in the CAN1 gene of S. cerevisiae
expressing Ugi and a control vector or APOBEC3G.
[0021] FIG. 5 is a graph illustrating the percentage that each base
was found at the indicated position relative to the C/G->T/A
transition mutation site in APOBEC3G expressing cells (n=37). The
base found most frequently is indicated below. APOBEC3G consensus
sites observed in model retroviral substrates, HIV-GFP or MLV-GFP,
are shown (Liddament et al. (2004) Curr. Biol. 14, 1385-91).
Multiple bases had the same percentage and are indicated by the one
letter code where D=A/G/T, R=A/G, S=G/C and Y=C/T. The G/C content
of the CAN1 gene is indicated by the dashed line.
[0022] FIG. 6A-6D indicate that APOBEC3F and APOBEC3G inhibit Ty1
retrotransposition. (A) A schematic depicting retrotransposition by
Ty1 or TyHRT yielding histidine prototrophy or luciferase activity.
GA, PR, IN, RT and LTR represent gag, protease, integrase, reverse
transcriptase and long-terminal repeat, respectively. (B, D)
APOBEC3F or -3G expression diminishes Ty1 or TyHRT
retrotransposition as monitored by the number of His.sup.+
colonies. For each condition, at least eight independent cultures
were analyzed and the error bars depict one standard error of the
mean. (C, E) APOBEC3F or -3G expression diminishes Ty1 or TyHRT
retrotransposition as monitored by luciferase activity. The
conditions were identical to those described above.
[0023] FIG. 7 is a graph of a representative experiment showing the
effect of APOBEC3F or -3G expression on the retrotransposition of a
chromosomal Ty1-his3AI element. APOBEC3F and -3G expression
decreased the frequency of His.sup.+ retrotransposition by 94- and
98-fold, respectively. These effects were likely underestimates
because several of the APOBEC3F and -3G expressing cultures failed
to yield any His.sup.+ colonies (although viable cell counts
indicated that they had grown to saturation). Note that the vector
control level of His.sup.+ retrotransposition in this experiment is
much lower than that shown in FIG. 6, because here the Ty1-his3AI
construct is single-copy and its endogenous promoter is under
cellular control the constructs used in FIG. 6 are expressed from
multi-copy plasmids by a highly efficient GAL promoter]. The error
bars indicate one SEM and they are barely visible for APOBEC3F and
-3G.
[0024] FIG. 8A-8B indicate that APOBEC3F and APOBEC3G induced Ty1
cDNA hypermutations. (A) A schematic depicting the LTR-dependent
His.sup.+ retrotransposition of Ty1 harboring a GFP passenger gene.
His.sup.+ retrotransposition events were pooled and the
GFP-negative variants were recovered for DNA sequencing as
described in the Materials and Methods. GA, PR, IN, RT and LTR
represent gag, protease, integrase, reverse transcriptase and
long-terminal repeat, respectively. (B) A schematic showing all of
the genomic (plus) strand base substitution mutations that were
found in the GFP passenger gene (and surrounding regions) (SEQ ID
NO:1) of His.sup.+/GFP.sup.- retrotransposons. Mutations
attributable to APOBEC3G or -3F expression are indicated above and
below the 1488 bp consensus sequence, respectively. The GFP start
codon is underlined. All of the mutations were recovered from Ty1
experiments, except three G->A substitutions, which were from
TyHRT experiments (one due to APOBEC3F is shown at consensus
position 681; a second due to APOBEC3F and one due to APOBEC3G are
not illustrated because they occurred in HIV RT sequence). All of
these raw sequencing data are summarized in FIG. 9. Four
GFP-negative controls were observed. Two were not recovered by PCR
and two produced smaller PCR products and were not sequenced
(likely deletions).
[0025] FIG. 9 are tables indicating the mutational preferences of
APOBEC3G and APOBEC3F in Ty1 cDNA. (A, C) Summaries of the GFP gene
(and surrounding region) base substitution mutations observed in
pools of His.sup.+ retrotranspositions, which had occurred in the
presence of APOBEC3G or -3F, respectively. (B, D) Base preferences
surrounding the Ty1 cDNA C->T transition sites attributable to
expression of APOBEC3G or -3F, respectively. APOBEC3G shows a clear
preference for 5'-YCC, whereas APOBEC3F prefers 5'-TTC(Y=C or T;
the mutated cytosine is underlined).
[0026] FIG. 10 is a graph indicating human APOBEC3G expression in
pig kidney (PK-15) cells causes a 3-fold decline in PERV transfer
to human 293T cells in a long-term co-culture experiment. The top
two panels are immunoblots showing expression of human APOBEC3G
(HsA3G) in PK-15 cells (center lane), but not in vector only or pig
APOBEC3F (SsA3F) expressing cells (left and right lane,
respectively). Non-specific bands are shown as loading
controls.
[0027] FIG. 11A is a picture of PCR products from a representative
semi-quantitative PCR assay showing that human APOBEC3G potently
diminishes PERV transfer from pig to human cells. FIG. 11B is the
pig specific control.
[0028] FIG. 12 is a graph from a representative quantitative,
real-time PCR assay showing that human APOBEC3G potently diminishes
PERV transfer from pig to human cells.
[0029] FIG. 13 is a sequence alignment of the cow (BtA3F, SEQ ID
NO:8), sheep (OaA3F, SEQ ID NO:9), and pig (SsA3F, SEQ ID NO:10)
APOBEC3F proteins
[0030] FIG. 14 is a sequence alignment of the active site of the
human (HsA3F, SEQ ID NO:11), cow (BtA3F, SEQ ID NO:12), sheep
(OaA3F, SEQ ID NO:13), and pig (SsA3F, SEQ ID NO:14) APOBEC3F
proteins. The conserved motifs are boxed.
[0031] FIG. 15A is a graph of the relative infectivity of HIV-GFP
produced in the presence of a vector control or the indicated A3
protein. To facilitate comparisons, all data were normalized to the
infectivity of HIV-GFP produced in the presence of a vector
control, which was arbitrarily assigned a value of one. The mean
and the SEM of three independent experiments are shown. HIV Vif is
not present in these experiments.
[0032] FIG. 15B is a graph of the relative infectivity of MLV-GFP
produced in the presence of the indicated constructs. Parameters
are identical to those in FIG. 15A.
DETAILED DESCRIPTION
[0033] As described herein, human cytosine deaminases such as
APOBEC3G and 3F are capable of deaminating the genomic DNA of a
eukaryotic cell. Expression of APOBEC3G or its homolog APOBEC3F can
inhibit the mobility of the retrotransposon Ty1 in Saccharomyces
cerevisiae by a mechanism involving the deamination of cDNA
cytosines. This expands the range of cytosine deaminase targets to
include nuclear DNA and endogenous retroelements, which have
pathological and physiological implications, respectively. These
data indicate that the APOBEC3-dependent mechanism of retroelement
restriction is highly conserved and that the range of APOBEC3
substrates may be far broader than originally anticipated. Because
the APOBEC3 proteins do not exist outside of mammals, the results
described herein showing that APOBEC3F or -3G can inhibit yeast Ty1
retrotransposition were unexpected. Therefore, the Ty1 data
described herein not only demonstrate the remarkable conservation
of this mechanism but, importantly, they also show that mammalian
factors (in addition to APOBEC3F or -3G) are not required for
retroelement restriction.
[0034] Furthermore, as demonstrated herein, expression of human
cytosine deaminases in porcine cells reduces the capability of the
porcine cells to transmit endogenous retroelements (e.g.,
retroviruses and retrotransposons) to the human cells. Thus, the
invention provides transgenic pigs and pig cells that express a
non-porcine cytosine deaminase polypeptide. Organs and tissues from
such transgenic pigs are useful for xenotransplantation due to the
decreased risk of transmitting the endogenous porcine retroviruses
to human cells relative to organs and tissues from pigs expressing
endogenous cytosine deaminases.
Non-Porcine Cytosine Deaminases
[0035] As used herein, "cytosine deaminase polypeptide" refers to
any chain of amino acids, regardless of post-translational
modification, that has the ability to deaminate cytosines to
uracils within nucleic acid and that contains the following
zinc-binding cytosine deaminase domain (amino acids provided in
standard one-letter terminology): H/CXE (or another catalytic
residue, e.g., D) X.sub.20-30PCX.sub.2-4C. See, Harris and
Liddament (2004) Nat. Rev. Immunol. 4:868-877. Amino acid
substitutions, deletions, and insertions can be introduced into a
known zinc-binding cytosine deaminase domain and the resulting
polypeptide is a "cytosine deaminase" provided that the polypeptide
retains the ability to deaminate cytosines to uracils.
[0036] Suitable non-porcine, mammalian cytosine deaminase
polypeptides include single domain DNA cytosine deaminases and
double domain DNA cytosine deaminases. For example, single domain
DNA cytosine deaminases include, for example, activation induced
deaminase (AID), APOBEC1, APOBEC2, APOBEC3A, APOBEC3C, APOBEC3D,
APOBEC3E, and APOBEC3H polypeptides. Double domain DNA cytosine
deaminases include, for example, APOBEC3B, APOBEC3F, and APOBEC3G
polypeptides. APOBEC3D and APOBEC3E also can be produced as double
domain cytosine deaminases. See, e.g., Harris and Liddament (2004),
supra; and Jarmuz et al Genomics (2002) 79(3):285-96. APOBEC3G
and/or APOBEC3F are particularly useful. Human APOBEC3G
(apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like
3G, also known as CEM15) uses cytosine to uracil deamination to
inhibit the replication of a variety of retroviruses, including
HIV-1. APOBEC3G localizes predominantly to the cytoplasm of
mammalian cells. In a retrovirus-infected cell, this localization
may facilitate the incorporation of APOBEC3G into viral particles,
which are released from the plasma membrane. APOBEC3G also is
specifically incorporated into virions through an association with
the viral Gag protein and/or viral genomic RNA. Once a retrovirus
enters a cell, its genomic RNA is reverse transcribed, and during
this process, APOBEC3G is capable of deaminating cDNA cytosines to
uracils (C->U). These lesions occur at such a high frequency
that they ultimately inactivate the virus (causing G->A
hypermutation, as read-out on the genomic strand of the virus).
APOBEC3F is a homolog of APOBEC3G and restricts HIV-1 infection by
a similar mechanism. APOBEC3F and -3G deaminate cytosines within
different local contexts, preferring 5'-TC and 5'-CC,
respectively.
[0037] The nucleic acid sequence encoding the cytosine deaminase
can be a cDNA or can include introns or adjacent 5'- or
3'-untranslated regions (e.g., a genomic nucleic acid). For
example, the nucleic acid sequence can encode a human APOBEC3F or
APOBEC3G polypeptide. GenBank Accession Nos. NM.sub.--145298 and
NM.sub.--021822 provide the sequences of the human APOBEC3F and
APOBEC3G cDNAs, respectively. The nucleic acid sequence also can
encode a sheep or cow APOBEC3F polypeptide as described herein. The
sheep and cow APOBEC3F proteins have an active amino terminal DNA
cytosine deaminase domain, which elicits a broader dinucleotide
deamination preference, and are fully resistant to HIV-1 Vif.
[0038] Nucleic acid sequences having silent mutations that do not
change the encoded amino acids or sequence variants that do change
one or more encoded amino acids, but do not abolish enzymatic
function, also can be used. For example, the nucleic acid sequence
of the APOBEC3G used herein differs from the coding sequence of
NM.sub.--021822 by a C to T transition at nucleotide position 588.
This transition is silent and does not change the encoded amino
acid at position 119 (F). In some embodiments, two or more cytosine
deaminase polypeptides (e.g., the human APOBEC3F and APOBEC3G
polypeptides) are encoded on the nucleic acid construct.
Nucleic Acid Constructs
[0039] Nucleic acid constructs of the invention include a nucleic
acid sequence encoding a non-porcine cytosine deaminase. As used
herein, the term "nucleic acid" includes DNA, RNA, and nucleic acid
analogs, and nucleic acids that are double-stranded or
single-stranded (i.e., a sense or an antisense single strand).
Nucleic acid analogs can be modified at the base moiety, sugar
moiety, or phosphate backbone to improve, for example, stability,
hybridization, or solubility of the nucleic acid. Modifications at
the base moiety include deoxyuridine for deoxythymidine, and
5-methyl-2'-deoxycytidine and 5-bromo-2'-doxycytidine for
deoxycytidine. Modifications of the sugar moiety include
modification of the 2' hydroxyl of the ribose sugar to form
2'-O-methyl or 2'-O-allyl sugars. The deoxyribose phosphate
backbone can be modified to produce morpholino nucleic acids, in
which each base moiety is linked to a six membered, morpholino
ring, or peptide nucleic acids, in which the deoxyphosphate
backbone is replaced by a pseudopeptide backbone and the four bases
are retained. See, Summerton and Weller (1997) Antisense Nucleic
Acid Drug Dev. 7(3):187-195; and Hyrup et al. (1996) Bioorgan. Med.
Chem. 4(1):5-23. In addition, the deoxyphosphate backbone can be
replaced with, for example, a phosphorothioate or
phosphorodithioate backbone, a phosphoroamidite, or an alkyl
phosphotriester backbone.
[0040] The nucleic acid sequence encoding the cytosine deaminase
can be operably linked to a regulatory region such as a promoter.
Regulatory regions can be porcine regulatory regions or can be from
other species. As used herein, "operably linked" refers to
positioning of a regulatory region relative to a nucleic acid
sequence encoding a polypeptide in such a way as to permit or
facilitate expression of the encoded polypeptide.
[0041] Any type of promoter can be operably linked to a nucleic
acid sequence encoding a cytosine deaminase. Examples of promoters
include, without limitation, tissue-specific promoters,
constitutive promoters, and promoters responsive or unresponsive to
a particular stimulus. Suitable tissue specific promoters can
result in preferential expression of a nucleic acid transcript in
islet cells and include, for example, the human insulin promoter.
Other tissue specific promoters can result in preferential
expression in, for example, hepatocytes or heart tissue and can
include the albumin or alpha-myosin heavy chain promoters,
respectively.
[0042] In other embodiments, a promoter that facilitates the
expression of a nucleic acid molecule without significant tissue-
or temporal-specificity can be used (i.e., a constituitive
promoter). For example, a .beta.-actin promoter such as the chicken
.beta.-actin gene promoter, ubiquitin promoter,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, or
3-phosphoglycerate kinase (PGK) promoter can be used, as well as
viral promoters such as the herpes virus thymidine kinase (TK)
promoter, the SV40 promoter, or a cytomegalovirus (CMV) promoter.
In some embodiments, a fusion of the chicken .beta. actin gene
promoter and the CMV enhancer is used as a promoter. See, for
example, Xu et al. (2001) Hum. Gene Ther. 12(5):563-73; and Kiwaki
et al. (1996) Hum. Gene Ther. 7(7):821-30.
[0043] An example of an inducible promoter is the tetracycline
(tet)-on promoter system, which can be used to regulate
transcription of the nucleic acid. In this system, a mutated Tet
repressor (TetR) is fused to the activation domain of herpes
simplex VP 16 (transactivator protein) to create a
tetracycline-controlled transcriptional activator (tTA), which is
regulated by tet or doxycycline (dox). In the absence of
antibiotic, transcription is minimal, while in the presence of tet
or dox, transcription is induced. Alternative inducible systems
include the ecdysone or rapamycin systems. Ecdysone is an insect
molting hormone whose production is controlled by a heterodimer of
the ecdysone receptor and the product of the ultraspiracle gene
(USP). Expression is induced by treatment with ecdysone or an
analog of ecdysone such as muristerone A.
[0044] Additional regulatory regions that may be useful in nucleic
acid constructs, include, but are not limited to, polyadenylation
sequences, translation control sequences (e.g., an internal
ribosome entry segment, IRES), enhancers, inducible elements, or
introns. Such regulatory regions may not be necessary, although
they may increase expression by affecting transcription, stability
of the mRNA, translational efficiency, or the like. Such regulatory
regions can be included in a nucleic acid construct as desired to
obtain optimal expression of the nucleic acids in the cell(s).
Sufficient expression, however, can sometimes be obtained without
such additional elements.
[0045] Other elements that can be included on a nucleic acid
construct encode signal peptides or selectable markers. Signal
peptides can be used such that the encoded polypeptide is directed
to a particular cellular location (e.g., the cell surface).
Non-limiting examples of selectable markers include puromycin,
adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo,
G418, APH), dihydrofolate reductase (DHFR),
hygromycin-B-phosphotransferase, thymidine kinase (TK), and
xanthin-guanine phosphoribosyltransferase (XGPRT). Such markers are
useful for selecting stable transformants in culture.
[0046] In some embodiments, a nucleic acid sequence encoding a
cytosine deaminase can include a tag sequence that encodes a "tag"
designed to facilitate subsequent manipulation of the encoded
polypeptide (e.g., to facilitate localization or detection). Tag
sequences can be inserted in the nucleic acid sequence encoding the
cytosine deaminase polypeptide such that the encoded tag is located
at either the carboxyl or amino terminus of the cytosine deaminase
polypeptide. Non-limiting examples of encoded tags include green
fluorescent protein (GFP), glutathione S-transferase (GST), and
Flag.TM. tag (Kodak, New Haven, Conn.).
[0047] Nucleic acid constructs can be introduced into embryonic,
fetal, or adult porcine cells of any type, including, for example,
germ cells such as an oocyte or an egg, a progenitor cell, an adult
or embryonic stem cell, a kidney cell such as a PK-15 cell, an
islet cell, a .beta. cell, a liver cell, or a fibroblast such as a
dermal fibroblast, using a variety of techniques. Non-limiting
examples of techniques include the use of transposon systems,
recombinant viruses that can infect cells, or liposomes or other
non-viral methods such as electroporation, microinjection, or
calcium phosphate precipitation, that are capable of delivering
nucleic acids to cells.
[0048] In transposon systems, the transcriptional unit of a nucleic
acid construct, i.e., the regulatory region operably linked to a
nucleic acid sequence encoding a cytosine deaminase polypeptide, is
flanked by an inverted repeat of a transposon. Several transposon
systems, including, for example, Sleeping Beauty (see, U.S. Pat.
No. 6,613,752 and U.S. Patent Publication No. 20050003542), Frog
Prince (Miskey et al. (2003) Nucleic Acids Res. 31(23):6873-81),
and Skipper have been developed to introduce nucleic acids into
cells, including mice, human, and pig cells. The Sleeping Beauty
transposon is particularly useful. A transposase can be encoded on
the same nucleic acid construct or can be introduced on a separate
nucleic acid construct.
[0049] Insulator elements also can be included in a nucleic acid
construct to maintain expression of the cytosine deaminase
polypeptide and to inhibit the unwanted transcription of host
genes. See, for example, U.S. Patent Publication No. 20040203158.
Typically, an insulator element flanks each side of the
transcriptional unit and is internal to the inverted repeat of the
transposon. Non-limiting examples of insulator elements include the
matrix attachment region (MAR) type insulator elements and
border-type insulator elements. See, for example, U.S. Pat. Nos.
6,395,549, 5,731,178, 6,100,448, and 5,610,053, and U.S. Patent
Publication No. 20040203158.
[0050] Viral vectors that can be used include adenovirus,
adeno-associated virus (AAV), retroviruses, lentiviruses, vaccinia
virus, measles viruses, herpes viruses, and bovine papilloma virus
vectors. See, Kay et al. (1997) Proc. Natl. Acad. Sci. USA 94,
12744-12746 for a review of viral and non-viral vectors. Viral
vectors are modified so the native tropism and pathogenicity of the
virus has been altered or removed. The genome of a virus also can
be modified to increase its infectivity and to accommodate
packaging of the nucleic acid encoding the polypeptide of
interest.
[0051] Adenoviral vectors can be easily manipulated in the
laboratory, can efficiently transduce dividing and nondividing
cells, and rarely integrate into the host genome. Smith et al.
(1993) Nat. Genet. 5, 397-402; and Spector and Samaniego (1995)
Meth. Mol. Genet., 7, 31-44. The adenovirus can be modified such
that the E1 region is removed from the double stranded DNA genome
to provide space for the nucleic acid encoding the polypeptide and
to remove the transactivating E1a protein such that the virus
cannot replicate. Adenoviruses have been used to transduce a
variety of cell types, including, inter alia, keratinocytes,
hepatocytes, and epithelial cells.
[0052] Adeno-associated viral (AAV) vectors demonstrate a broad
range of tropism and infectivity, although they exhibit no human
pathogenicity and do not elicit an inflammatory response. AAV
vectors exhibit site-specific integration and can infect
non-dividing cells. AAV vectors have been used to deliver nucleic
acid to brain, skeletal muscle, and liver over a long period of
time (e.g., >9 months in mice) in animals. See, for example,
U.S. Pat. No. 5,139,941 for a description of AAV vectors.
[0053] Retroviruses are the most-characterized viral delivery
system and have been used in clinical trials. Retroviral vectors
mediate high nucleic acid transfer efficiency and expression.
Retroviruses enter a cell by direct fusion to the plasma membrane
and integrate into the host chromosome during cell division.
[0054] Lentiviruses also can be used to deliver nucleic acids to
cells, and in particular, to non-dividing cells. Replication
deficient HIV type I based vectors have been used to transduce a
variety of cell types, including stem cells. See, Uchidda et al.
(1998) Proc. Natl. Acad. Sci. USA 95, 11939-11944.
[0055] Non-viral vectors can be delivered to cells via liposomes,
which are artificial membrane vesicles. The composition of the
liposome is usually a combination of phospholipids, particularly
high-phase-transition-temperature phospholipids, usually in
combination with steroids, especially cholesterol. Other
phospholipids or other lipids may also be used. The physical
characteristics of liposomes depend on pH, ionic strength, and the
presence of divalent cations. Transduction efficiency of liposomes
can be increased by using dioleoylphosphatidylethanolamine during
transduction. See, Felgner et al. (1994) J. Biol. Chem. 269,
2550-2561. High efficiency liposomes are commercially available.
See, for example, SuperFect.RTM. from Qiagen (Valencia,
Calif.).
Transgenic Pigs
[0056] The nucleated cells of the transgenic pigs provided herein
contain a nucleic acid construct described above. As used herein,
"transgenic pig" includes founder transgenic pigs as well as
progeny of the founders, progeny of the progeny, and so forth,
provided that the progeny retain the nucleic acid construct. For
example, a transgenic founder animal can be used to breed
additional animals that contain the nucleic acid construct.
[0057] Tissues obtained from the transgenic pigs and cells derived
from the transgenic pigs also are provided herein. As used herein,
"derived from" indicates that the cells can be isolated directly
from the pig or can be progeny of such cells. For example, brain,
lung, liver, pancreas, heart and heart valves, muscle, kidney,
thyroid, corneal, skin, blood vessels or other connective tissue
can be obtained from a pig. Blood and hematopoietic cells, Islets
of Langerhans, .beta. cells, brain cells, hepatocytes, kidney
cells, and cells from other organs and body fluids, for example,
also can be derived from transgenic pigs. Organs and cells from
transgenic pigs can be transplanted into a human patient. For
example, islets from transgenic pigs can be transplanted to human
diabetic patients.
[0058] Various techniques known in the art can be used to introduce
nucleic acid constructs into non-human animals to produce founder
lines, in which the nucleic acid construct is integrated into the
genome. Such techniques include, without limitation, pronuclear
microinjection (U.S. Pat. No. 4,873,191), retrovirus mediated gene
transfer into germ lines (Van der Putten et al. (1985) Proc. Natl.
Acad. Sci. USA 82, 6148-1652), gene targeting into embryonic stem
cells (Thompson et al. (1989) Cell 56, 313-321), electroporation of
embryos (Lo (1983) Mol. Cell. Biol. 3, 1803-1814), and in vitro
transformation of somatic cells, such as cumulus or mammary cells,
or adult, fetal, or embryonic stem cells, followed by nuclear
transplantation (Wilmut et al. (1997) Nature 385, 810-813; and
Wakayama et al. (1998) Nature 394, 369-374). Pronuclear
microinjection and somatic cell nuclear transfer are particularly
useful techniques.
[0059] Typically, in pronuclear microinjection, a nucleic acid
construct described above is introduced into a fertilized egg; 1 or
2 cell fertilized eggs are used as the pronuclei containing the
genetic material from the sperm head and the egg are visible within
the protoplasm. Linearized nucleic acid constructs can be injected
into one of the pronuclei then the injected eggs can be transferred
to a recipient female (e.g., into the oviducts of a recipient
female) and allowed to develop in the recipient female to produce
the transgenic pigs.
[0060] In somatic cell nuclear transfer, a transgenic pig cell such
as a fetal fibroblast that includes a nucleic acid construct
described above, can be introduced into an enucleated oocyte to
establish a combined cell. Oocytes can be enucleated by partial
zona dissection near the polar body and then pressing out cytoplasm
at the dissection area. Typically, an injection pipette with a
sharp beveled tip is used to inject the transgenic cell into an
enucleated oocyte arrested at meiosis 2. In some conventions,
oocytes arrested at meiosis 2 are termed "eggs." After producing a
porcine embryo (e.g., by fusing and activating the oocyte), the
porcine embryo is transferred to the oviducts of a recipient
female, about 20 to 24 hours after activation. See, for example,
Cibelli et al. (1998) Science 280, 1256-1258 and U.S. Pat. No.
6,548,741. Recipient females can be checked for pregnancy
approximately 20-21 days after transfer of the embryos.
[0061] Standard breeding techniques can be used to create animals
that are homozygous for the cytosine deaminase polypeptide from the
initial heterozygous founder animals. Homozygosity may not be
required, however, to observe a decreased capability of
transmitting PERV to human cells.
[0062] Once transgenic pigs have been generated, expression of
cytosine deaminase polypeptides can be assessed using standard
techniques. Initial screening can be accomplished by Southern blot
analysis to determine whether or not integration of the construct
has taken place. For a description of Southern analysis, see
sections 9.37-9.52 of Sambrook et al., 1989, Molecular Cloning, A
Laboratory Manual, second edition, Cold Spring Harbor Press,
Plainview; NY. Polymerase chain reaction (PCR) techniques also can
be used in the initial screening. PCR refers to a procedure or
technique in which target nucleic acids are amplified. Generally,
sequence information from the ends of the region of interest or
beyond is employed to design oligonucleotide primers that are
identical or similar in sequence to opposite strands of the
template to be amplified. PCR can be used to amplify specific
sequences from DNA as well as RNA, including sequences from total
genomic DNA or total cellular RNA. Primers typically are 14 to 40
nucleotides in length, but can range from 10 nucleotides to
hundreds of nucleotides in length. PCR is described in, for example
PCR Primer: A Laboratory Manual, ed. Dieffenbach and Dveksler, Cold
Spring Harbor Laboratory Press, 1995. Nucleic acids also can be
amplified by ligase chain reaction, strand displacement
amplification, self-sustained sequence replication, or nucleic acid
sequence-based amplified. See, for example, Lewis (1992) Genetic
Engineering News 12, 1; Guatelli et al. (1990) Proc. Natl. Acad.
Sci. USA 87, 1874-1878; and Weiss (1991) Science 254,
1292-1293.
[0063] Expression of a nucleic acid sequence encoding a cytosine
deaminase polypeptide (e.g., an APOBEC3F and/or APOBEC3G
polypeptide) in the tissues of transgenic pigs can be assessed
using techniques that include, without limitation, Northern blot
analysis of tissue samples obtained from the animal, in situ
hybridization analysis, Western analysis, immunoassays such as
enzyme-linked immunosorbent assays, and reverse-transcriptase PCR
(RT-PCR). Expression of a non-porcine cytosine deaminase
polypeptide in at least some of the cells of the pig can result,
upon co-culture with human cells, in a decreased capability of the
cells to transmit PERV to the human cells.
[0064] Decreased capability to transmit PERV can be assessed, for
example, by a co-culture assay. Transgenic pig cells and human
cells (e.g., 293T cells) can be physically separated by a thin
membrane with 1 micron-sized pores and co-cultured for
approximately 50 generations or 25 days. Such a membrane permits
free diffusion of small molecules including viral particles but
does not permit diffusion of cells. At the end of the culturing
period, the human cells can be harvested and tested for PERV
reverse transcriptase activity (as a measure of infectivity) using
an ELISA assay (e.g., from Cavidi Tech, Uppsala, Sweden). It is
understood that a particular phenotype in a transgenic animal
typically is assessed by comparing the phenotype in the transgenic
animal to the corresponding phenotype exhibited by a control
non-human animal that lacks the transgene.
[0065] Transgenic pigs of the invention can be bred with other
animals of interest (e.g., animals with transplantation-compatible
backgrounds such as pigs with an inactivated .alpha.-1,3 galactosyl
transferase gene). The resulting progeny animals may be
particularly useful for xenotransplantation due to the decreased
risk of transmitting endogenous retroviruses to human cells and the
decreased risk of hyperacute rejection. Such animals can be
produced by, for example, crossing (a) a transgenic pig expressing
a non-porcine cytosine deaminase polypeptide with (b) a transgenic
pig with an inactivated .alpha.-1,3 galactosyl transferase gene.
Alternatively, a single line of transgenic pigs can be produced by
initially preparing the pigs using the appropriate transgenes.
[0066] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Methods and Materials for Examples 2-7
[0067] Yeast Strains. Yeast mutation assays were done in L40 (MATa
his3.DELTA.200 trp1-901 leu2-3112 ade2 LYS2::(lexAop).sub.4-HIS3
URA3::(lexAop).sub.8-lacZ GAL4) (1). Retrotransposition assays were
done in DG1251 (MATa ura3-167 trp1-hisG spt3-101 h is 3 .DELTA.200)
or GRY 1990, a derivative of DG1251 in which E. coli
.beta.-galactosidase is constitutively expressed from the yeast
PGK1 promoter (Nissley et al., (1996) Nature 380, 30; Nissley et
al. (1998) Proc. Natl. Acad. Sci. USA 95, 13905-10). Endogenous
retrotransposition assays were carried in DG1141 [MAT .alpha.
trp1-hisG ura3-167 his3 .DELTA.200 Ty1-2y2his3AI; (Curcio and
Garfinkel (1991) Proc. Natl. Acad. Sci. USA 88, 936-40). L40
ung1::kanMX4 was constructed by amplifying the ung1::kanMX4
cassette from yeast deletion strain 36067 (R. Wright, University of
Minnesota), transforming L40 with the resulting PCR product and
selecting G418-resistant colonies (Wach et al. (1994) Yeast 10,
1793-1808)). ung1 deletion was confirmed by PCR and screening for a
modest CAN1 mutator phenotype.
[0068] Plasmids. Constructs were based on pHybLex-Zeo or pJG4-5
(Invitrogen, Carlsbad, Calif.). The LexAAPOBEC3G fusion protein was
constructed by subcloning APOBEC3G from pAPOBEC3G-IRES-bleo (Harris
et al. (2003) Cell 113, 803-809) using NotI and PstI. Untagged
APOBEC3G in pHybLex-Zeo contains a 5 bp insertion between the LexA
and APOBEC3G open reading frames.
[0069] Ugi was subcloned from pEF-Ugi (Di Noia and Neuberger (2002)
Nature 419, 43-48) as an EcoRI and NotI fragment into pcDNA3.1
(Invitrogen). It was subcloned into pYES3-CT using Hind III and
NotI (Invitrogen). Ugi expression was confirmed using the CAN1
mutation assay. The wild-type HIV-1 Vif sequences were amplified by
PCR from HIV-1 YU-2 and IIIB proviral plasmids (M. Malim, Kings
College London), digested with NcoI and BamHI and were first cloned
into similarly cut pTrc99A (AP Biotech). Vif was subsequently
subcloned into pHybLex-Zeo using a NcoI and PstI digest and finally
into pJG4-5 using EcoRI and SphI. APOBEC3F was subcloned from
pTrc99A-APOBEC3F (Liddament et al. (2004) Curr. Biol. 14,
1385-1391) into both pHybLex-Zeo and pJG4-5 using EcoRI and
SalI.
[0070] Galactose (GAL)-inducible his3AI marked versions of Ty1
(pGALTy1) and TyHRT (pHART21) were described previously (Nissley et
al., 1996, supra; Nissley et al., 1998, supra).
[0071] Yeast Mutation Assays. pHybLex-Zeo, pJG4-5, pYES3-CT and
their derivatives were transformed into L40 and selected using a
synthetic complete medium containing zeocin (300 .mu.g/mL) and
lacking tryptophan (SC+ZEO-TRP) (Ausubel et al. (2002) (John Wiley
and Sons, Inc.). Several thousand viable cells from independent
colonies were used to inoculate 2.5 ml SC+GAL+RAF+ZEO-TRP (2%
galactose, 1% raffinose, 300 .mu.g/mL zeocin). Cultures were grown
at 30.degree. C. for 3-4 days, concentrated 5-fold and a fraction
was plated to SC+CAN-ARG (30 .mu.g/mL CAN) to obtain canavanine
resistant (Can.sup.R) mutants. Viable cell counts were obtained by
plating a dilution to rich medium. Viable cells were counted after
2 days and Can.sup.R colonies were counted after 3-4 days of
incubation at 30.degree. C. The CAN1 gene of Can.sup.R colonies was
amplified by PCR and sequenced as previously reported (Marsischky
et al. (1996) Gene Dev. 10, 407-420). Accurate values for the
mutation frequencies were obtained by using multiple independent
cultures (6-8) for each strain in each experiment and by repeating
each experiment at least twice and as many as seven times.
Sequencher (Genes Codes Corp) was used for mutational analyses.
[0072] Immunoblotting. Cell pellets from a 10 mL log phase culture
were washed with 1 mL 20% trichloroacetic acid (TCA), resuspended
in 50 .mu.L 20% TCA, and then lysed by vortexing with an equal
volume of glass beads at 4.degree. C. The supernatant was
centrifuged to pellet the proteins. Pelleted proteins were
resuspended in 100 .mu.L SDS-gel loading buffer, separated by
SDS-PAGE, transferred to a PVDF membrane and probed with antibodies
to APOBEC3G (Newman et al. (2005) Curr. Biol. 15, 166-170), LexA
(Invitrogen), or Vif (Fouchier et al (1996) J. Virol. 70,
8263-8269; Simon et al. (1997) J. Virol. 69, 4166-4172; Simon et
al. (1997) J. Virol. 71, 5259-5267).
[0073] Ty1 Retrotransposition Assays. Ty-his3AI, TyHRT-his3AI,
Ty-lucAI or TyHRT-lucAI plasmids were co-transformed with pJG4-5,
pJG4-5-APOBEC3G or pJG4-5-APOBEC3F into DG1251 or GRY 1990 (Gietz
et al. (1995) Yeast 11, 355-360) and selected using
SC-URA-TRP+GLC.
[0074] his 3AI transformants were grown in SC-URA-TRP+GLC to
saturation. Approximately 10.sup.6 cells were subcultured in 1 ml
of SC-URA-TRP+GAL for 12 hrs and an aliquot was plated to SC-HIS.
Cell viability was determined by plating a dilution to rich medium.
Retrotransposition was quantified by determining the frequency of
His+ colonies.
[0075] lucAI transformants were grown 1 day in SC-URA-TRP+GLC.
Cells were transferred to SC-URA-TRP+GAL and grown for an
additional 2 days at 30.degree. C. to induce retroelement
expression and reverse transcription. Retrotransposition was
quantified by measuring the relative active levels of luciferase to
.beta.-galactosidase. All incubations for plasmid-based Ty1 assays
were at 30.degree. C.
[0076] For endogenous retrotransposition assays, DG1141 was
transformed with pJG4-5, pJG4-5-APOBEC3G, or pJG4-5-APOBEC3F.
Single colonies were resuspended in water and 10-50,000 cells were
transferred to 2 mL SC-TRP+GAL and grown at 20.degree. C. for 7-10
days until the cultures reached saturation. Dilutions of the
starting and ending cultures were plated to rich media to determine
the number of viable cells and the equivalent of 1 mL of the
saturated culture was plated to SC-HIS to score retrotransposition
events.
[0077] Ty1 DNA Sequencing. Retrotransposed Ty1 and TyHRT cDNAs were
isolated by growing His.sup.+ colonies overnight in 10 ml SC-HIS at
30.degree. C. and preparing DNA with a standard glass bead/phenol
extraction method. The resulting DNA was used to amplify a 1,026
(Ty) or 971 (TyHRT) bp region spanning the RT gene and HIS3 using
5'-TTC ATG TGG GAC ACT AGA GAT (TyRT, SEQ ID NO:15) or 5'-CCT GAG
TGG GAG TTG TTA (TyHRT, SEQ ID NO:2) and 5'-TAT GAT ACA TGC TCT GGC
CAA (HIS3, SEQ ID NO:3). PCR products were purified (Qiagen) and
sequenced with 5'-GT CTG CGA GGC AAG AAT GAT (SEQ ID NO:4).
GFP-negative retrotransposition events were obtained from pools of
His.sup.+ colony genomic DNA by transformation into E. coli.
GFP-negative colonies were identified using fluorescent light and
the resident plasmid DNA was amplified (as above except the product
was 2.1 kb for Ty RT) and sequenced using 5'-C GTT ATC CGG ATC ATA
TGA (SEQ ID NO:5) and 5'-G TAG TTC CCG TCA TCT TGA (SEQ ID
NO:6).
Example 2
APOBEC3G Stimulates Mutation in Saccharomyces cerevisiae Via the
Uracil Excision Pathway
[0078] To test whether human APOBEC3G could elicit its hallmark
mutator activity in yeast, a LexA-APOBEC3G fusion protein was
expressed in the haploid strain L40 and the accumulation of
mutations that conferred resistance to the toxic amino acid
canavanine was monitored. Liquid cultures were grown from
individual colonies expressing APOBEC3G or a control vector and
then plated onto a solid medium containing canavanine. The numbers
of canavanine-resistant (Can.sup.R) colonies were determined after
3-4 days growth. In contrast to cells expressing a control vector,
those expressing LexAAPOBEC3G showed a 20-fold increase in the
median frequency of Can.sup.R mutation, suggesting that APOBEC3G
was capable of deaminating cytosines within yeast genomic DNA (FIG.
1A; FIG. 2).
[0079] To begin to determine whether the LexA-APOBEC3G-induced
mutator phenotype occurred by a C->U deamination mechanism, it
was asked whether a uracil DNA glycosylase deficiency would
exacerbate this phenotype. Since most DNA-based organisms use
uracil DNA glycosylase to rid their genomes of uracil (Barnes and
Lindahl (2004) Annu. Rev. Genet. 38, 445-476), it was likely that
if this were the mechanism then many of the APOBEC3G-induced uracil
lesions would have been repaired and that the observed mutation
frequency would be an underestimate of APOBEC3G activity. Indeed,
yeast expressing both APOBEC3G and a uracil DNA glycosylase
inhibitor (Ugi) protein showed a 320-fold increase in the median
frequency of mutation to CanR (FIG. 1B). This stimulation was
approximately 6-fold and 26-fold higher than that observed in
LexA-APOBEC3G-expressing and in Ugi-expressing yeast cells,
respectively, indicating that many of the APOBEC3G-dependent
uracils were repaired by a uracil excision mechanism.
[0080] In yeast, the major uracil DNA glycosylase is Ung1p (uracil
DNA N-glycosylase 1 protein). Ung1p and most other Ung proteins
from bacteria to humans are strongly inhibited by Ugi (Mol et al.,
(1995) Cell 82, 701-708). However, Ugi-resistant uracil excision
activities occur in mammalian cells, such as those elicited by the
SMUG1 and TDG1 proteins (Barnes and Lindahl (2004) supra). To
eliminate the possibility that some of the APOBEC3G-induced uracils
might be repaired by auxiliary systems in yeast, homologous
recombination was used to construct an Ung1p deletion strain, L40
ung1::kanMX4. This strain showed levels of Can.sup.R mutation
virtually indistinguishable from Ugi-expressing cells in the
presence or absence of APOBEC3G (FIG. 3). Thus, the majority of
APOBEC3G-induced lesions in yeast were repaired by an
Ung1p-dependent mechanism. Together with the exquisite specificity
that Ung1p has for uracil, these data indicated that the
APOBEC3G-dependent mutator phenotype was attributable to a DNA
cytosine deamination mechanism.
[0081] APOBEC3G is localized predominantly to the cytoplasm of
mammalian cells. Therefore, it was surprising that its expression
in yeast caused high mutation frequencies. To ensure that the high
mutation frequencies were not attributable to the DNA binding
properties of the LexA tag, the CAN1 mutation frequency was
monitored of cells expressing either LexA-APOBEC3G or untagged
APOBEC3G. Little difference in the overall median frequencies of
Can.sup.R mutation was observed demonstrating that the DNA binding
domain of LexA was not responsible for the APOBEC3G-dependent
mutator phenotype (FIG. 1B).
Example 3
APOBEC3G Triggers Predominantly C/G->T/A Transition Mutations in
Yeast
[0082] CAN1 encodes a membrane-spanning arginine transporter that
must be inactivated for growth to occur in the presence of the
toxic arginine analog canavanine. A wide variety of base
substitution, insertion, deletion and more complex mutations can
confer CanR [e.g., (Huang et al., (2003) Proc. Natl. Acad. Sci. USA
100, 11529-11534; Rattray et al (2002) Genetics 162, 1063-1077]. To
further investigate the mechanism of the APOBEC3G-induced mutator
phenotype, the CAN1 gene of a large number of CanR colonies was
sequenced. In agreement with previous studies, cells containing a
control vector displayed a wide range of CAN1 mutations including
transitions (26%), transversions (43%), insertions (3%) and
deletions (28%) (FIG. 4A-4B). In contrast, the vast majority (90%)
of the CAN1 mutations in APOBEC3G expressing cells were C/G->T/A
transitions. APOBEC3Ginduced transitions occurred at the expense of
other types of mutations, accounting for the elevated Can.sup.R
mutation frequency (FIG. 4A-4B).
[0083] Yeast lacking Ung1p due to Ugi expression also displayed an
increased level of C/G->T/A transition mutations (64%), as would
be expected of cells lacking uracil excision repair (FIG. 4C-4D).
However, 5/7 of these transitions occurred at positions that were
not mutated in APOBEC3G expressing cells. Co-expression of Ugi and
APOBEC3G resulted in an even stronger C/G->T/A transition bias
(95%), and 19/21 of these mutations occurred at sites that were
also mutated in APOBEC3G expressing (Ugi negative) yeast cells
(FIG. 4C-4D). These data further demonstrated that APOBEC3G is
capable of triggering genomic hypermutation in yeast by a C->U
deamination mechanism.
Example 4
The Local APOBEC3G Mutation Preference in Yeast is Nearly Identical
to that Observed in Model Retroviral Substrates
[0084] A closer examination of the C/G->T/A transitions
triggered by APOBEC3G expression revealed that 37/37 occurred
within the dinucleotide 5'-CC, which could be found on either
strand of the DNA duplex (FIGS. 4 and 5). APOBEC3G expression alone
triggered C/G->T/A transition mutations at 14 distinct sites
within the CAN1 gene. Co-expression of APOBEC3G and Ugi caused
C/G->T/A transition mutations at 6 identical and 2 additional
sites. The three most frequently APOBEC3G mutated 5'-CC
dinucleotide sites, C.sub.356, C.sub.656, and C.sub.1195, accounted
for 48% of the total combined APOBEC3G- and
APOBEC3G-plus-Ugi-dependent base substitution mutations. The
extended sequence preference of APOBEC3G in the yeast system was
compared to that defined previously in model HIV and MLV retroviral
systems as 5'-YCCA [Y=C or T]. Interestingly, APOBEC3G exhibited a
strikingly similar 5'-CCCA preference in yeast (FIG. 5), indicating
that its preference as observed in other systems was intact.
[0085] It is further notable that in addition to a large number of
C/G->T/A transition mutations, four deletions and a single
insertion were detected in the CAN1 gene of APOBEC3G-expressing
yeast cells (FIG. 4; combined data including the Ugi experiments).
Three of five of these alterations occurred either in or
immediately adjacent to a preferred or potential APOBEC3G hotspot,
5'-CCC. In contrast, only 1/12 of the deletions and insertions
found in control vector containing cells occurred at similar sites.
The remainder (11/12) were distributed throughout the CAN1 gene and
were presumably caused by a variety of mechanisms. The presence of
deletions and insertions associated with APOBEC3G hotspots
suggested that C->U deamination events are able to precipitate
gross genomic instability. This is further supported by our
observation that a small (approximately 5%) proportion of Can.sup.R
mutants failed to yield a CAN1 gene-specific PCR product,
potentially representing larger-scale lesions.
Example 5
Affect of HIV-1 Vif on APOBE3G-Induced Yeast Hypermutation
[0086] In primates such as humans and chimpanzees, Vif counteracts
the anti-retroviral activity of APOBEC3G by targeting it for
proteasomal degradation. Vif accomplishes this by binding to
APOBEC3G. Some data suggest that this association alone may
directly impair APOBEC3G function (Stopak et al. (2003) Mol. Cell.
12, 591-601). Therefore, it was assessed whether the interaction
between Vif and APOBEC3G could be detected using this yeast assay
system.
[0087] HIV-1 Vif, derived from the YU2 or the IIIB provirus, was
expressed alongside APOBEC3G using yeast two-hybrid bait or prey
vectors. All possible pairwise combinations were tested for the
ability to drive the yeast two-hybrid reporter genes lacZ or HIS3.
No significant .beta.-galactosidase activity or histidine
prototrophy was observed despite repeated attempts (data not
shown). This result was not attributable to an expression failure
as both proteins could be detected in cell lysates by
immunoblotting.
[0088] However, because some weak or transient interactions may
escape detection by the yeast two-hybrid assay, it was reasoned
that the sensitive CAN1 mutation assay might provide a more robust
method for monitoring this interaction. To examine whether HIV-1
Vif could affect APOBEC3G-mediated hypermutation in yeast, the
Can.sup.R mutation frequencies of cells co-expressing Vif and
APOBEC3G were compared with those of cells expressing either
protein alone. The robust hypermutability of APOBEC3G was not
significantly affected by HIV-1 Vif co-expression. Therefore, a
Vif-APOBEC3G interaction in yeast was not detected.
Example 6
APOBEC3F and APOBEC3G Inhibit Ty1 Retrotransposition
[0089] To explore the possibility that APOBEC3 proteins function to
impede the mobility of endogenous retroelements that replicate
using LTR sequences, the ability of the yeast retrotransposon Ty1
to replicate was assayed in the presence of APOBEC3G or its homolog
APOBEC3F. Ty1 activity was monitored using an intron-disrupted
retrotransposition indicator gene (FIG. 6A). Ty1 RNA expression,
splicing, reverse transcription and integration yield functional
reporter gene cDNA copies, encoding either histidine prototrophy or
luciferase activity.
[0090] The ability of Ty1-his3AI to retrotranspose was monitored in
the presence of human APOBEC3F or -3G (FIG. 6B). In comparison to
cells containing a control vector, an average of 51% or 70% fewer
His.sup.+ colonies were detected in the presence of APOBEC3F or
-3G, respectively. Slightly larger APOBEC3-dependent declines in
Ty1-lucAI retrotransposition were observed, as monitored by the
relative levels of luciferase present in liquid cultures (FIG. 6C).
However, an almost total inhibition (94-98%) was observed when
retrotransposition of a genomic Ty1-his3AI element was assayed in
the presence of APOBEC3F or -3G, suggesting that the ratio of
APOBEC3 protein to retrotransposition intermediate (and/or Ty host
factors) is a key determinant of this inhibitory mechanism (FIG.
7). Together, these data clearly demonstrated that APOBEC3F or -3G
can inhibit Ty1 retrotransposition.
[0091] To assess whether the APOBEC3-dependent inhibition of
retrotransposition in yeast could be influenced by the reverse
transcriptase or the integration pathway, similar assays were
performed with Ty1 constructs in which the normal reverse
transcriptase was replaced with that from HIV-1 [TyHRT]. TyHRT
integration occurs predominantly by homologous recombination,
whereas Ty1 integration mostly uses its own integrase.
Retrotransposition of both TyHRT-hisAI and TyHRT-lucAI (i.e., the
accumulation of HIV-1 reverse transcriptase products) was also
inhibited by APOBEC3F or APOBEC3G expression (FIGS. 6D, E). Levels
of inhibition were roughly similar to those observed with Ty1
reverse transcriptase, indicating that neither the reverse
transcriptase nor the integration pathway were key effectors of the
APOBEC3-imposed retrotransposition block. These data further
highlight the utility of the yeast Ty1 system for studying aspects
of both APOBEC3 and HIV-1 biology.
Example 7
Ty1 Restriction by APOBEC3F and APOBEC3G Involves a cDNA Cytosine
Deamination Mechanism
[0092] As cDNA C->U deamination is a hallmark anti-retroviral
activity of APOBEC3F and -3G, it was asked whether this could
account for the observed Ty1 retrotransposition block. If so, it
was expected that an inordinate number of retrotransposon minus
strand C->T transition mutations would be found amongst the
His.sup.+ integrants (equivalent to plus strand G->A
transitions). Over 26 and 47 kbp was sequenced of TyRT-HIS3
template generated in the presence of APOBEC3F and -3G,
respectively, and only two C->T transitions were found among the
APOBEC3G-exposed templates. One occurred within a dinucleotide
consensus 5'-GC that is rarely preferred by this protein, and it
therefore likely represents a reverse transcription or PCR error.
The second occurred within the trinucleotide 5'-CCC, which is the
most common APOBEC3 Gpreferred site. However, this meager number of
base substitutions may have been in part due the fact that
functional His.sup.+ (and not His.sup.-) integrants were analyzed.
It is further possible that uracil residues within the
retrotransposon cDNA triggered its degradation, as hypothesized
originally for retroviruses (Harris et al. (2003) Cell 113,
803-809).
[0093] Therefore, to address the former possibility and to enrich
for mutations, a modified version of the Ty-his3AI system was used
in which a GFP cassette was placed upstream of his3AI (FIG. 8A).
This enabled the selection of His.sup.+ integrants and a subsequent
screen for unselected GFP-negative variants. Twenty independent GFP
mutants were recovered from retrotransposition experiments in which
APOBEC3G was expressed. Each sequence contained at least one
mutation and as many as 15 mutations. In total, 57 base
substitution mutations were identified and 47 of these were minus
strand C->T transitions (FIG. 8B, FIG. 9A). Almost all of the
APOBEC3G-dependent transitions occurred within the consensus
5'-YCC, identical to the preferred cytosine deamination consensus
site in the CAN1 gene and in a variety of other systems (e.g.,
compare FIG. 9B and FIG. 5). Moreover, many of the C->T
transitions occurred at positions that were identical to those
observed previously in GFP-encoding HIV or MLV. A similar
strand-specific transition bias and sequences with multiple
transitions were found in GFP-negative templates produced in the
presence of APOBEC3F (FIG. 9C; FIG. 8B). However, in contrast to
APOBEC3G, the APOBEC3F-dependent mutations occurred within a
distinct 5'-TTC consensus [FIG. 9D; observed previously with an HIV
substrate]. Thus, Ty1 retrotransposition can be inhibited by
APOBEC3F and -3G and much (and possibly all) of this effect can be
attributed to a cDNA cytosine deamination mechanism.
Example 8
Expression of Human APOBEC3G in Pig Cells Reduces Transfer of
Porcine Endogenous Retrovirus (PERV) to Human 293T Cells
[0094] A construct for expression of human APOBEC3G was produced
using the cytomegalovirus (CMV) promoter to drive expression and
the neomycin gene as a selectable marker. To assess whether the
APOBEC3G protein can inhibit PERV transmission to human cells, the
construct was stably introduced into pig kidney PK-15 cells (ATCC #
CCL-33) using Fugene.RTM. 6 reagent (Roche Applied Science,
Indianapolis, Ind.) and cells were selected for neomycin
resistance. PK-15 cells were chosen for these experiments because
the PERVs residing in these cells were able to infect human 293T
cells in simple supernatant mixing experiments (Patience et al.,
(1997) Nat Med 3, 282-286). (Note that PERVs can transmit as
solution-soluble cell-free particles and/or through cell-cell
contact). APOBEC3G expression in PK-15 cells was confirmed using
specific antibodies (Newman et al. (2004) Curr Biol. 15(2):
166-70).
[0095] PK-15 cells expressing a vector control, human APOBEC3G
(hA3G), or pig APOBEC3F (SsA3F) were co-cultured with human 293T
cells for 25 days (approximately 50 cell generations); the two cell
types were physically separated by a thin membrane with 1
micron-sized pores, which permitted free diffusion of small
molecules including viral particles but it did not permit diffusion
of cells. After 25 days, whole cell protein extracts were prepared
from the 293T cells using standard procedures. Cell lysates (10
.mu.g) were tested for PERV reverse transcriptase (RT) activity (as
a measure of PERV transfer to the human cells) using the C-type
retrovirus RT.TM. Activity ELISA assay; performed as recommended by
the manufacturer, Cavidi Tech, Uppsala, Sweden. Little activity was
detected in 293T cells grown in the presence of human
APOBEC3G-expressing PK-15 cells, in contrast to controls where
significant levels of infection were detected (FIG. 10). Results in
FIG. 10 are shown as relative fold inhibition of RT activity
normalized to RT activity in 293T cells cultured with PK-15 cells
expressing an empty vector. This experiment indicates that
expression of human A3G (but not expression of additional pig
APOBEC3F) in PK-15 cells inhibits PERV transfer from PK-15 cells to
293T cells.
[0096] Semi-quantitative and quantitative, real-time PCR assays
were performed to monitor for the presence of integrated PERV DNA
in human 293T cells. Semi-quantitative PCR was performed using 75
ng of template genomic DNA from human 293T cells and primers
(forward 5'-AA CCC TTT ACC CTT TAT GTG GAT-3', SEQ ID NO:16;
reverse 5'-AA AGT CAA TTT GTC AGC GTC CTT-3', SEQ ID NO:17) made to
the PERV pol gene (product size: 196 bp). As indicated in FIG. 11A,
very little PERV DNA was detected in 293T cells grown in the
presence of human APOBEC3G-expressing PK-15 cells, in contrast to
other samples where significant levels were detected. To ensure
that the human cell co-culturing compartment was not contaminated
by pig cells (i.e., micro-chimerism), PCR also was performed using
primers specific to pig DNA (forward 5'-GG AAC CTG CAA CCT ATG
GAA-3', SEQ ID NO:18; reverse 5'-GG TGT GGC CCT AAA AAG ACA-3', SEQ
ID NO:19) (351 bp product). The left panel of FIG. 11B shows that
no pig PCR products were detected in 293T samples from the
co-culture experiment. The right panel contains positive and
negative controls. Micro-chimerism was not detected.
[0097] Quantitative, real-time PCR assays were performed in 25
.mu.L reactions containing 10 ng of 293T genomic DNA, 100 nM
primers, and 2.times. iQ SYBR Green super mix (BioRad, Hercules,
Calif.) and run on an iCycler iQ Multicolor Real-Time PCR detection
System (BioRad, Hercules, Calif.). Thermocycler conditions were
95.degree. C. for 5 min followed by 50 cycles of 95.degree. C. for
15 seconds and 60.degree. C. for 30 seconds. A melting curve
analysis directly followed the cycling to verify amplification of
the PERV pol gene PCR product (amplified as discussed above). The
human beta-actin gene (housekeeping gene) was amplified as an
internal control using the following primers: Forward, 5'-AT CAT
GTT TGA GAC CTT CAA-3' (SEQ ID NO:20) and reverse, 5'-A GAT GGG CAC
AGT GTG GGT-3' (SEQ ID NO:7) (product size: ca. 100 bp). All data
were normalized and PERV gene copies are presented per 100,000
beta-actin copies (FIG. 12). PERV transfer was apparent after 20
days of continuous co-culture in the vector control cells, whereas
little transfer occurred in the presence of human APOBEC3G. Thus,
expression of human APOBEC3G in pig PK-15 cells inhibited PERV
transfer from pig PK-15 cells to human 293T cells.
Example 9
Artiodactyl Double Deaminase Domain APOBEC3F Proteins
[0098] NCBI BLAST searches were performed using the human and mouse
A3 deaminase domains as query polypeptides. Several artiodactyl
(cloven hoof ungulates) ESTs were identified, which suggested the
presence of at least one A3 protein in cattle (Bos taurus (Bt),
GenBank Accession No. BE684372, Smith et al, Gen. Res. 11(4):
626-630, 2001) and pigs (Sus scrofa (Ss), GenBank Accession No.
BI346898, Fahrenkrug et al., 2002, Mamm Genome, 13, 475-478).
Corresponding cDNA clones were obtained, sequenced and shown to
encode A3 proteins with two putative zinc-binding, cytosine
deaminase domains. The orthologous sheep (Ovis aries, Oa) double
domain A3 cDNA sequence was obtained using a combination of
degenerate PCR and nested 3' prime RACE. All three of these A3
proteins were similar in size to the 373 amino acid HsA3F protein,
except the pig A3 protein, which was slightly longer due to a
unique C-terminal, serine-rich extension. The cow, sheep, and pig
A3 proteins are referred to herein as BtA3F (SEQ ID NO:8), OaA3F
(SEQ ID NO:9) and SsA3F (SEQ ID NO:10), respectively. An alignment
of the amino acid sequences of BtA3F, OaA3F, and SsA3F is shown in
FIG. 13.
[0099] Amino acid alignments of the active deaminase domains (plus
five residues on each side) were made using Clustal W software
(Higgins et al. 1994, Methods Mol. Biol. 25:307-18). The cow and
sheep A3 active sites were 78% identical. Both the cow and the
sheep proteins shared a lower level of identity with the pig
protein (56%). The active sites of these artiodactyl A3 proteins
were 56-62% identical to HsA3F (FIG. 14).
[0100] To test whether the artiodactyl A3F proteins have the
capacity to deaminate cytosines within single-strand DNA, the
intrinsic mutator activity of these proteins was monitored using an
E. coli based mutation assay. Rifampicin resistance (RifR) is
attributable to base substitution mutations in the E. coli RNA
polymerase B (rpoB) gene, and it occurs in approximately one of
every five million bacterial cells. This assay therefore provides a
robust measure of intrinsic DNA cytosine deaminase activity. See,
for example, Hache et al. (2005) J Biol Chem, 280, 10920-10924;
Harris et al. (2002) Molecular Cell, 10, 1247-1253. Expression of
each of the artiodactyl A3 proteins increased the RifR mutation
frequency in E. coli from 3- to 7-fold, levels that were higher
than those attributable to HsA3F but slightly lower than that those
caused by HsA3G. BtA3F and SsA3F expression triggered a HsAID-like
increase in RifR mutation frequency.
[0101] Artiodactyl A3F DNA cytosine deamination preferences were
examined by sequencing the rpoB gene of at least 100 independent
RifR mutants. In contrast to HsA3F and HsA3G, which preferentially
deaminate cytosines at rpoB nucleotide positions 1721 and 1691,
5'-TC and 5'-CC, respectively, the artiodactyl A3F proteins showed
less biased rpoB mutation spectra. OaA3F preferentially deaminated
cytosine 1576, which is part of a 5'-GC dinucleotide. SsA3F also
preferred cytosine 1576. However, SsA3F also clearly deaminated
cytosine 1586, which is part of a 5'-AC dinucleotide. The main
conclusion from the RifR mutation assays was that all three of the
artiodactyls A3F proteins were capable of deaminating DNA cytosines
and triggering a corresponding shift in the pattern of C/G->T/A
transition mutations within the rpoB mutation substrate. Since the
intrinsic DNA cytosine deamination preferences of HsA3F and HsA3G
are apparent in retroviruses like HIV-1, these data suggest that
the physiological dinucleotide substrates of OaA3F and SsA3F will
be 5'-GC, and 5'-RC, respectively (R=A or G).
[0102] As an initial step toward understanding the potential
retroelement targets of the artiodactyl A3F proteins, the
sub-cellular distribution of these proteins was determined by live
cell fluorescence microscopy. Approximately 7,500 HeLa cells were
seeded on LabTek chambered coverglasses (Nunc). After 24 hrs of
incubation, the cells were transfected with 200 ng of the
pEGFP-A3-based DNA constructs. After an additional 24 hrs of
incubation, images of the live cells were collected using a Zeiss
Axiovert 200 microscope at 400.times. total magnification. In
contrast to HsA3B and an eGFP control, which localized to the
nucleus and the entire cell, respectively, the artiodactyl A3F
proteins and MmA3 (mouse) were predominantly cytoplasmic, with
punctate bodies apparent in some cells. This pattern of
localization is identical to that seen for HsA3F and HsA3G,
indicating that the artiodactyl A3F proteins might similarly
function to inhibit the replication of LTR-dependent retroviruses
such as HIV or MLV.
Example 10
Retrovirus Restriction by Artiodactyl A3F Proteins
[0103] It was tested whether the artiodactyl A3F proteins could
inhibit the infectivity of HIV- and MLV-based retroviruses. In
these systems, a GFP gene embedded in proviral DNA provides a
measure of both transfection efficiency (which correlates directly
with virus production levels) and of viral infectivity. 293T cells
were grown in Dulbecco's modified Eagle's medium (Invitrogen)
containing 10% fetal bovine serum (Gemini Bioproducts), penicillin,
and streptomycin (Invitrogen). HIV-GFP [also called CS-CG] was
produced by FuGENE.RTM. 6 (Roche Applied Sciences)-mediated
transfection of 50-70% confluent 293T cells with a plasmid mixture
containing 0.22 .mu.g of CS-CG, 0.14 .mu.g of pRK5/Pack1 (Gag-Pol),
0.07 .mu.g of pRK5/Rev, 0.07 .mu.g of pMDG (VSV-G Env), and 0.5
.mu.g of an APOBEC expression or empty vector control plasmid as
described previously (Liddament et al. (2004) Curr Biol
14:1385-1391). After an incubation period of 48 hr,
virus-containing supernatants were clarified by low speed
centrifugation, filtered (0.45 .mu.m), and quantified using a
reverse transcriptase activity based ELISA (Cavidi Tech). Reverse
transcriptase-normalized supernatants were applied to fresh 293T
cells, and infection was allowed to proceed for 96 hr. Infectivity
(GFP fluorescence) was then measured by flow cytometry
(FACSCalibur, BD Biosciences). For experiments requiring the
recovery of retroviral DNA for hypermutation analyses, the viral
supernatants were treated with 50 units/ml DNase (Sigma) prior to
293T cell infection.
[0104] Expression of HsA3F and HsA3G caused 4- and 24-fold
reductions in the infectivity of HIV-GFP. MmA3 also was capable of
strongly inhibiting HIV-GFP. In comparison, expression of BtA3F,
OaA3F or SsA3F caused 30-, 8- and 29-fold decreases in the
infectivity of HIV-GFP, respectively (FIG. 15A). These potent
anti-HIV activities demonstrated that the artiodactyl A3F proteins
have at least one retrovirus restriction activity. These results
further imply that the artiodactyl A3F proteins are able to
specifically associate with the HIV Gag/genomic RNA complex and
thereby gain access to assembling virus particles.
[0105] HIV-GFP infectivity also was monitored in the presence or
absence of HIV-1 Vif and human, artiodactyl or mouse A3 proteins.
Expression of HIV-1 Vif neutralized HsA3G and HsA3F (although the
latter to a lesser extent) and caused a proportional recovery of
HIV-GFP infectivity. Expression of HIV-1 Vif did little to enhance
the infectivity of HIV-GFP produced in the presence of MmA3 or any
of the artiodactyl A3F proteins. Thus, the artiodactyl A3F proteins
were fully resistant to HIV-1 Vif.
[0106] Expression of MmA3 has little effect on the infectivity of
MLV, presumably because MLV excludes (or simply avoids) this A3
protein (FIG. 15B). In contrast, HsA3F and HsA3G inhibit the
infectivity of MLV-based retroviruses, but to a lesser extent than
HIV-based viruses (FIG. 15B). Therefore, to begin to ask whether
the artiodactyl A3F proteins possess broad, HsA3F- or HsA3G-like,
or narrow, MmA3-like retrovirus restriction potentials, the
infectivity of MLV-GFP produced in the presence of these A3
proteins was monitored. Interestingly, much like the HsA3F and
HsA3G proteins, expression of the artiodactyl A3F proteins reduced
the infectivity of MLV-GFP by 2- to 4-fold (FIG. 15B). Thus, the
HIV-GFP and MLV-GFP infectivity data combined to suggest that the
artiodactyl A3F proteins have a relatively broad retrovirus
restriction potential.
Example 11
The N-Terminal Zinc-Binding, Deaminase Domain of the Artiodactyl
A3F Proteins Catalyzes C->U Deamination
[0107] To work-out the mechanism of retrovirus restriction by
artiodactyl A3F proteins and to test whether the N- or the
C-terminal (or both) zinc-binding domain of these proteins
catalyzes DNA cytosine deamination, the conserved glutamate (E) of
each active site was changed to glutamine (Q) using site-directed
mutagenesis and the resulting mutants were tested for HIV-GFP
restriction activity.
[0108] As reported previously, the glutamate of both the N- and the
C-terminal zinc-binding domain of HsA3G contributes to inhibiting
HIV infectivity, but the C-terminal catalytic glutamate appears
more important. Both the N- and the C-terminal BtA3F zinc-binding
domain E.fwdarw.Q mutants appeared to retain full levels of
anti-HIV activity. In contrast, the N-terminal OaA3F and SsA3F
zinc-binding domain E.fwdarw.Q mutants were less able than the
corresponding C-terminal domain mutants to inhibit the infectivity
of HIV-GFP. This result was particularly clear for SsA3F. These
data were essentially the inverse of the HsA3F and HsA3G E.fwdarw.Q
mutant studies, and they therefore suggested that the N-terminal,
zinc-binding domain of these proteins catalyzes retroviral cDNA
C.fwdarw.U deamination. MmA3 was clearly distinct, as both the N-
and the C-terminal zinc-binding domain glutamates were required for
HIV-GFP restriction.
[0109] Although both the N- and the C-terminal domain E.fwdarw.Q
mutants of the human and the artiodactyl A3 proteins still showed
significant levels of anti-retroviral activity, it was surmised
that bonafide catalytic site mutants should be unable to catalyze
retroviral cDNA C.fwdarw.U deamination [although they may still
inhibit retroviral infectivity]. Minus strand uracils template the
incorporation of plus strand adenines, ultimately manifesting as
retroviral plus strand G.fwdarw.A hypermutations. Therefore, to
directly test which zinc-binding domain(s) catalyzes DNA cytosine
deamination and to gain additional insight into the artiodactyl A3F
retrovirus restriction mechanism, the GFP gene from the
aforementioned HIV-GFP infectivity experiments was amplified by
high-fidelity PCR, cloned and subjected to DNA sequence analyses.
HIV-GFP produced in the presence of a control vector showed a low
base substitution mutation frequency, 0.00014 mutations per base,
which is attributable to errors in reverse transcription and PCR.
In contrast, viruses produced in the presence of HsA3F, HsA3G, all
three of the artiodactyl A3F proteins or MmA3 showed between 30-
and 80-fold more base substitution mutations, which were almost
exclusively retroviral G.fwdarw.A transition mutations. HsA3G with
a C-terminal domain E.fwdarw.Q mutation failed to cause retroviral
hypermutation, although this variant still significantly inhibited
HIV-GFP infectivity. The HsA3F C-terminal zinc-binding domain
mutant was still able to modestly inhibit HIV-GFP infectivity,
without obvious signs of retroviral hypermutation.
[0110] E.fwdarw.Q substitutions in the N-terminal (but not the
C-terminal) domain of all three of the artiodactyl A3F proteins
abolished the accumulation of retroviral hypermutations. Thus,
these data combined to demonstrate that the N-terminal,
zinc-binding deaminase domain of the artiodactyl A3F proteins is
catalytic and that both deaminase-dependent and -independent
activities are required for full levels of retrovirus
restriction.
Example 12
Retroviral Hypermutation Properties of Artiodactyl A3F Proteins
[0111] As described above, the rpoB mutation spectra of BtA3F,
OaA3F and SsA3F suggested that these proteins would trigger
retroviral hypermutation patterns biased toward 5'-YC, 5'-GC, and
5'-R C, respectively (R=A or G). To test this prediction, the types
of base substitution mutations and the local retroviral cDNA
deamination preferences attributable to expression of the
artiodactyl A3F proteins was examined. In terms of the dinucleotide
mutation preferences, the base immediately 5' of the targeted
cytosine is a crucial target site determinant. HsA3F and HsA3G
overwhelmingly preferred 5'-CC (84%) and 5'-TC (84%), respectively,
whereas MmA3 preferred 5'-TC (61%) and 5'-CC (29%). Like mouse A3,
the cow and the sheep A3F proteins appeared to prefer a pyrimidine
(Y) 5' of the deaminated cytosine (93% and 79%, respectively).
However, roughly paralleling the E. coli rpoB mutation data, the
pig A3F protein preferred 5'-GC (47%). This is notable because this
constitutes the only example of an A3 protein preferring
5'-purine-C (the immunoglobulin gene deaminase AID also has this
preference). In addition, all of the A3 proteins characterized in
these analyses preferred a pyrimidine at the -2 position (which was
invariably a T, except for HsA3G which preferred C>T).
Example 13
Engineered Pigs Expressing APOBEC3F and/or APOBEC3G
[0112] Skin fibroblasts from a 9-year old prize boar were
transfected with expression constructs encoding human APOBEC3F,
human APOBEC3G, or both, and placed under G418 selection. Resistant
colonies were picked and expanded. Colonies expressing the
APOBEC3F, APOBEC3G, or both were identified by RT-PCR.
[0113] Enucleation and Donor Cell Transfer. In vivo matured ova
were surgically recovered from donor animals between 46 and 50 hrs
after HCG administration. Immediately prior to enucleation,
expanded cumulus and corona cells were removed from both types of
ova by blunt dissection and repeated pipetting of the ova in HEPES
buffered North Carolina State University 23 (NCSU-23, Petters and
Wells (1993) J Reprod Fertil Suppl. 48:61-73) medium supplemented
with 0.1% hyaluronidase. Groups of ova were transferred into 5
.mu.l droplets of HEPES buffered NCSU-23 containing 10% fetal calf
serum, 2.5 .mu.g/ml cytochalasin B (CB) and 5 .mu.g/ml Hoechst
33343, which were arranged in a column on the lid of a 9
mm.times.50 mm Petri dish. Enucleation was achieved by physically
removing the polar body and adjacent cytoplasm, containing the
metaphase II plate, using an ES cell transfer pipette. Whole cell
transfer was accomplished using an ES cell transfer pipette
(Eppendorf, Westbury, N.Y.) with a sharp, beveled tip (inner
diameter 10-25 .mu.m depending on cell type).
[0114] Donor cells (i.e., transfected skin fibroblasts) were
synchronized in presumptive G0/G1 by serum starvation (0.5%) for 24
h. Microdrops containing oocytes were spiked with a small volume of
donor cells that had been trypsinized not more than 3 h prior to
enucleation.
[0115] Fusion and Activation of oocytes. Donor cells were injected
into the perivitelline space and pressed against the oocyte's
membrane. Cell-cytoplast couplets were fused within 2 h after
enucleation. Groups of 5-10 couplets were manually aligned between
the electrodes of a 1 mm gap fusion chamber (BTX, San Diego,
Calif., USA) overlaid with mannitol fusion medium (0.28 M mannitol,
0.2 mM MgSO.sub.4.times.7H.sub.2O, 0.01% PVA). Couplets were fused
by exposure to a single pulse of 150 V/mm for 60 us. Following
fusion, couplets were cultured in HEPES buffered NCSU+10% fetal
calf serum from 0.5 to 1.5 h before activation. Couplets were
activated by placing them in 1 mm gap fusion chamber overlaid with
mannitol medium supplemented with 0.1 mM CaCl.sub.2.times.2H.sub.2O
and exposing them to two 60 microsecond pulses of 150V/mm.
[0116] In vitro culture of cloned embryos. After activation
treatments, the reconstructed cloned embryos were thoroughly washed
and cultured in 50 .mu.l drops of NCSU23 supplemented with 1% MEM
non essential amino acid, 2% BME amino acids and 0.4 mg/ml BSA for
5 days at 38.5.degree. C. in 5% CO.sub.2 in air without a medium
change. Following 120 h in culture, fetal calf serum (10%) was
added to all microdrops containing reconstructed embryos. The rates
of development were examined daily for in vitro cleavage after
activation and cleaved embryos at 2-4 cell stages were selected for
transfer.
[0117] Superovulation and embryo transfer. Pubertal crossbred gilts
aged 8 to 10 months were synchronized with Regumate (containing
0.4% altrenogest; 10 mg/day; Intervet, Boxmeer, Netherlands) mixed
in commercial feed and given each morning for 17-19 days. All donor
gilts were injected with 2,000 IU PMSG (Folligon & Chorulon)
and 80 h later with 1,000 IU hCG (Folligon & Chorulon).
Recipient gilts were injected with half the dosage of PMSG and hCG
administered to the donors. Oocytes were surgically collected 46-50
h after hCG injection by flushing from the oviduct with HEPES
buffered NCSU-23.
[0118] To produce cloned pigs, reconstructed embryos were
surgically transferred into the oviduct of each synchronized foster
mother by 20-24 h after activation. Nuclear transfer embryos
(N=385) were transferred to three recipients, 2302, 5570 and 2175
on day 1. One week later, an additional group of reconstructed
embryos (N=360) was transferred to three additional recipients,
2306, 5638 and 2211. An ultrasound scanner (Aloka SSD-500, JAPAN)
with an attached 3.5 MHz transabdominal probe was used to check
pregnancies at 25 and 35 days after embryo transfer; five of 6
recipients possessed at least 1 fetus at this time (83%). Five
pregnant pigs were obtained. Pregnant recipients were reexamined by
ultrasound again at approximately 30 days prior to the expected
date of parturition. Recipient 2211 was not pregnant and exhibited
estrus approximately 1 month after transplantation, giving an
overall 67% pregnancy rate.
[0119] One week prior to the date of projected farrowing, all gilts
were moved to farrowing crates. Gilts 2302, 5570 and 2175 were
given 2 mL of PGF.sub.2.alpha. on day 113, 112 and 111 of gestation
and 2 mL of oxytocin 24 hrs later. On day 118 after embryo
transfer, one 700 g male clone was manually removed from recipient
5570. On the same day, recipient 2175 passed one degenerated mummy.
The remaining recipient, 2302, who had exhibited substantial milk
let-down following her shot of oxytocin, was removed from the crate
and given an injection of 15 mL of Lidocaine in a vertebral disk
between one set of lower lumbar vertebrae. After waiting 20 minutes
for the anesthesia to take effect, a high flank incision was made
and both horns of the uterus were exposed. Neither horn contained
any fetuses or mummies. However, the endometrium in both horns
exhibited extensive cystic hyperplasia, which gave the uterus the
appearance of being gravid. Recipient 2302 then was given a general
anesthesia (acepromazine+ketamine) and euthanized. Recipient 2175
was also anesthetized and euthanized and her uterus examined for
the presence additional mummies or fetuses; none were found.
[0120] On day 112 of gestation, recipient 5638 received 2 mL of
PGF.sub.2.alpha.. Oxytocin (2 mL) was administered at 6 AM the
following day. At 7:20 AM, the first of five male clones was
manually removed from the recipient. By 11:00 AM, a total of 5 male
clones had been delivered. Thus, 6 live-born transgenic clones were
generated from a total of 6 recipients.
[0121] All clones exhibited from moderate to severe arthrogryposis
in the rear legs, which greatly reduced their mobility. Clone 1
(700 g) was manually removed from recipient 5570 but was unable to
nurse so was photographed and euthanized the next day. Clones 2
(1000 g) and 3 (700 g) died within a few hours of birth. Clone 4
(700 g), who had a fairly severe case of arthrogryposis, died later
on the same day. Clones 5 (1200 g) and 6 (1200 g) were manually fed
hourly, via syringe and mouse feeding needle, with Esbilac (milk
replacement formula). Clone 6's health visibly improved during this
nursing period, while clone 5's health declined. Clone 5 was
extremely weak and no longer swallowing Esbilac and was euthanized
the next day. Clone 6 was returned to his dam and was viable for 2
weeks, but had major abscesses on the tops of his rear feet and his
swollen front pasterns. Daily injections of Tylan 200 and
penicillin did not resolve this condition so the piglet was
euthanized and cells were harvested.
[0122] Epigenetic reprogramming may be deficient in cloned embryos.
Nuclei can be more effectively reprogrammed by passing them through
multiple rounds of cloning and fetal fibroblast isolation before
carrying piglets to term. Transgenic cells already produced can be
used to generate reconstructed embryos, which would then be
implanted and left to develop for about 40 days before the
termination of pregnancy. Fibroblasts would then be isolated from
these fetuses and cultured briefly before use in another round of
somatic cell nuclear transfer to generate new piglets. Another
alternative would rely on the use of fetal fibroblasts to start
with, i.e., using fetal fibroblasts to generate new transgenic
cells expressing the APOBEC proteins, and then generating pigs by
somatic cell nuclear transfer as described above. Most successful
pig cloning experiments have utilized cells derived from fetal
fibroblasts, as opposed to the aged boar used in this example.
Other sources of cells, including embryonic or adult stem cells
also can be used.
OTHER EMBODIMENTS
[0123] While the invention has been described in conjunction with
the foregoing detailed description and examples, the foregoing
description and examples are intended to illustrate and not to
limit the scope of the invention, which is defined by the scope of
the appended claims. Other aspects, advantages, and modifications
are within the scope of the claims.
Sequence CWU 1
1
2011488DNAArtificial SequenceSynthetic 1aagaaaccaa ttattaaagg
cttacttact gatagtagat caacgatcag tataattaag 60tctacaaatg aagagaaatt
tagaaacaga ttttttggca caaaggcaat gagacttaga 120gatgaagtat
caggtaataa tttatacgta tactacatcg agaccaagaa gaacattgct
180gatgtgatga caaaacctct tccgataaaa acatttaaac tattaactaa
caaatggatt 240cattagatcg cgcgcggatc cgccgattca ttaatgcagc
tggcacgaca ggtttcccga 300ctggaaagcg ggcagtgagc gcaacgcaat
taatgtgagt tagctcactc attaggcacc 360ccaggcttta cactttatgc
ttccggctcg tatgttgtgt ggaattgtga gcggataaca 420atttcacaca
ggaaacagct atgaccatga ttacgccaag cttgcatgcc tgcaggtcga
480ctctagagga tccccgggta ccggtagaaa aaatgagtaa aggagaagaa
cttttcactg 540gagttgtccc aattcttgtt gaattagatg gtgatgttaa
tgggcacaaa ttttctgtca 600gtggagaggg tgaaggtgat gcaacatacg
gaaaacttac ccttaaattt atttgcacta 660ctggaaaact acctgttcca
tggccaacac ttgtcactac tttctcttat ggtgttcaat 720gcttttcccg
ttatccggat catatgaaac ggcatgactt tttcaagagt gccatgcccg
780aaggttatgt acaggaacgc actatatctt tcaaagatga cgggaactac
aagacgcgtg 840ctgaagtcaa gtttgaaggt gatacccttg ttaatcgtat
cgagttaaaa ggtattgatt 900ttaaagaaga tggaaacatt ctcggacaca
aactcgagta caactataac tcacacaatg 960tatacatcac ggcagacaaa
caaaagaatg gaatcaaagc taacttcaaa attcgccaca 1020acattgaaga
tggatccgtt caactagcag accattatca acaaaatact ccaattggcg
1080atggccctgt ccttttacca gacaaccatt acctgtcgac acaatctgcc
ctttcgaaag 1140atcccaacga aaagcgtgac cacatggtcc ttcttgagtt
tgtaactgct gctgggatta 1200cacatggcat ggatgagctc tacaaataat
gaattccaac tgagcgccgg tcgctaccat 1260taccaacttg tctggtgtca
aaaataatag gcctactagt cggcgcgcgg atccatctgc 1320agctttaaat
aatcggtgtc actacataag aacacctttg gtggagggaa catcgttggt
1380accattgggc gaggtggctt ctcttatggc aaccgcaaga gccttgaacg
cactctcact 1440acggtgatga tcattcttgc ctcgcagaca atcaacgtgg agggtaat
1488218DNAArtificial SequencePrimer 2cctgagtggg agttgtta
18321DNAArtificial SequencePrimer 3tatgatacat gctctggcca a
21420DNAArtificial SequencePrimer 4gtctgcgagg caagaatgat
20519DNAArtificial SequencePrimer 5cgttatccgg atcatatga
19619DNAArtificial SequencePrimer 6gtagttcccg tcatcttga
19719DNAArtificial SequencePrimer 7agatgggcac agtgtgggt
198385PRTBos taurus 8Met Gln Pro Ala Tyr Arg Gly Tyr Ser Gln Met
Pro Trp Thr Arg Asp1 5 10 15Ser Ser Glu His Met Ala Arg Leu Asp Pro
Glu Thr Phe Tyr Phe Gln20 25 30Phe Cys Asn Leu Leu Tyr Ala Asn Arg
Arg Asn Cys Ser Tyr Ile Cys35 40 45Tyr Lys Val Glu Arg Arg Lys Tyr
His Ser Arg Ala Ser Phe Asp Trp50 55 60Gly Val Phe His Asn Gln Val
Tyr Gly Gly Thr Arg Cys His Thr Glu65 70 75 80Leu Arg Phe Leu Ser
Trp Phe His Ala Glu Lys Leu Arg Pro Asn Glu85 90 95Arg Tyr His Ile
Thr Trp Phe Met Ser Trp Ser Pro Cys Met Lys Cys100 105 110Ala Lys
Glu Val Ala Asp Phe Leu Gly Arg His Gln Asn Val Thr Leu115 120
125Ser Ile Phe Thr Ser Arg Leu Tyr Lys Phe Gln Glu Glu Gly Ser
Arg130 135 140Gln Gly Leu Leu Arg Leu Ser Asp Gln Gly Ala His Val
Asp Ile Met145 150 155 160Ser Tyr Gln Glu Phe Lys Tyr Cys Trp Lys
Lys Phe Val Tyr Ser Gln165 170 175Arg Arg Pro Phe Arg Pro Trp Lys
Lys Leu Asp Arg Asn Tyr Gln Arg180 185 190Leu Val Glu Glu Leu Glu
Asp Ile Leu Gly Asn Thr Met Asn Leu Leu195 200 205Arg Glu Val Leu
Phe Lys Gln Gln Phe Gly Asn Gln Pro Arg Val Pro210 215 220Ala Pro
Tyr Tyr Arg Arg Lys Thr Tyr Leu Cys Tyr Gln Leu Lys Gln225 230 235
240Arg Asn Asp Leu Thr Leu Asp Arg Gly Cys Phe Arg Asn Lys Lys
Gln245 250 255Arg His Ala Glu Ile Arg Phe Ile Asp Lys Ile Asn Ser
Leu Asp Leu260 265 270Asn Pro Ser Gln Ser Tyr Lys Ile Ile Cys Tyr
Ile Thr Trp Ser Pro275 280 285Cys Pro Asn Cys Ala Asn Glu Leu Val
Asn Phe Ile Thr Arg Asn Asn290 295 300His Leu Lys Leu Glu Ile Phe
Ala Ser Arg Leu Tyr Phe His Trp Ile305 310 315 320Lys Ser Phe Lys
Met Gly Leu Gln Asp Leu Gln Asn Ala Gly Ile Ser325 330 335Val Ala
Val Met Thr His Thr Glu Phe Glu Asp Cys Trp Glu Gln Phe340 345
350Val Asp Asn Gln Ser Arg Pro Phe Gln Pro Trp Asp Lys Leu Glu
Gln355 360 365Tyr Ser Ala Ser Ile Arg Arg Arg Leu Gln Arg Ile Leu
Thr Ala Pro370 375 380Ile3859372PRTOvis aries 9Met Pro Trp Ile Ser
Asp His Val Ala Arg Leu Asp Pro Glu Thr Phe1 5 10 15Tyr Phe Gln Phe
His Asn Leu Leu Tyr Ala Tyr Gly Arg Asn Cys Ser20 25 30Tyr Ile Cys
Tyr Arg Val Lys Thr Trp Lys His Arg Ser Pro Val Ser35 40 45Phe Asp
Trp Gly Val Phe His Asn Gln Val Tyr Ala Gly Thr His Cys50 55 60His
Ser Glu Arg Arg Phe Leu Ser Trp Phe Cys Ala Lys Lys Leu Arg65 70 75
80Pro Asp Glu Cys Tyr His Ile Thr Trp Phe Met Ser Trp Ser Pro Cys85
90 95Met Lys Cys Ala Glu Leu Val Ala Gly Phe Leu Gly Met Tyr Gln
Asn100 105 110Val Thr Leu Ser Ile Phe Thr Ala Arg Leu Tyr Tyr Phe
Gln Lys Pro115 120 125Gln Tyr Arg Lys Gly Leu Leu Arg Leu Ser Asp
Gln Gly Ala Cys Val130 135 140Asp Ile Met Ser Tyr Gln Glu Phe Lys
Tyr Cys Trp Lys Lys Phe Val145 150 155 160Tyr Ser Gln Arg Arg Pro
Phe Arg Pro Trp Lys Lys Leu Lys Arg Asn165 170 175Tyr Gln Leu Leu
Ala Ala Glu Leu Glu Asp Ile Leu Gly Asn Thr Met180 185 190Asn Leu
Leu Arg Glu Thr Leu Phe Lys Gln Gln Phe Gly Asn Gln Pro195 200
205Arg Val Pro Pro Pro Tyr Tyr Arg Arg Lys Thr Tyr Leu Cys Tyr
Gln210 215 220Leu Lys Glu Leu Asp Asp Leu Met Leu Asp Lys Gly Cys
Phe Arg Asn225 230 235 240Lys Lys Gln Arg His Ala Glu Ile Arg Phe
Ile Asp Lys Ile Asn Ser245 250 255Leu Asn Leu Asn Pro Ser Gln Ser
Tyr Lys Ile Ile Cys Tyr Ile Thr260 265 270Trp Ser Pro Cys Pro Asn
Cys Ala Ser Glu Leu Val Asp Phe Ile Thr275 280 285Arg Asn Asp His
Leu Asn Leu Gln Ile Phe Ala Ser Arg Leu Tyr Phe290 295 300His Trp
Ile Lys Pro Phe Cys Arg Gly Leu His Gln Leu Gln Lys Ala305 310 315
320Gly Ile Ser Val Ala Val Met Thr His Thr Glu Phe Glu Asp Cys
Trp325 330 335Glu Gln Phe Val Asp Asn Gln Leu Arg Pro Phe Gln Pro
Trp Asp Lys340 345 350Leu Glu Gln Tyr Ser Ala Ser Ile Arg Arg Arg
Leu Gln Arg Ile Leu355 360 365Thr Ala Pro Thr37010418PRTSus scrofa
10Met Asp Pro Gln Arg Leu Arg Gln Trp Pro Gly Pro Gly Pro Ala Ser1
5 10 15Arg Gly Gly Tyr Gly Gln Arg Pro Arg Ile Arg Asn Pro Glu Glu
Trp20 25 30Phe His Glu Leu Ser Pro Arg Thr Phe Ser Phe His Phe Arg
Asn Leu35 40 45Arg Phe Ala Ser Gly Arg Asn Arg Ser Tyr Ile Cys Cys
Gln Val Glu50 55 60Gly Lys Asn Cys Phe Phe Gln Gly Ile Phe Gln Asn
Gln Val Pro Pro65 70 75 80Asp Pro Pro Cys His Ala Glu Leu Cys Phe
Leu Ser Trp Phe Gln Ser85 90 95Trp Gly Leu Ser Pro Asp Glu His Tyr
Tyr Val Thr Trp Phe Ile Ser100 105 110Trp Ser Pro Cys Cys Glu Cys
Ala Ala Lys Val Ala Gln Phe Leu Glu115 120 125Glu Asn Arg Asn Val
Ser Leu Ser Leu Ser Ala Ala Arg Leu Tyr Tyr130 135 140Phe Trp Lys
Ser Glu Ser Arg Glu Gly Leu Arg Arg Leu Ser Asp Leu145 150 155
160Gly Ala Gln Val Gly Ile Met Ser Phe Gln Asp Phe Gln His Cys
Trp165 170 175Asn Asn Phe Val His Asn Leu Gly Met Pro Phe Gln Pro
Trp Lys Lys180 185 190Leu His Lys Asn Tyr Gln Arg Leu Val Thr Glu
Leu Lys Gln Ile Leu195 200 205Arg Asn Thr Met Asn Leu Leu Lys Glu
Asn Ile Phe Ile Gln Gln Phe210 215 220Gly Asn Gln Pro Arg Val Leu
Ala Pro Tyr Tyr Leu Arg Lys Thr Tyr225 230 235 240Leu Cys Tyr Gln
Val Lys Gly Pro Asp Asp Ser Ile Leu Asp Lys Gly245 250 255Cys Phe
Gln Asn Lys Lys Lys Arg His Ala Glu Ile Arg Phe Ile Asp260 265
270Lys Ile Asn Ser Leu Asn Leu Asp Gln Asn Gln Cys Tyr Arg Ile
Ile275 280 285Cys Tyr Val Thr Trp Ser Pro Cys His Asn Cys Ala Lys
Glu Leu Val290 295 300Asp Phe Ile Ser Asn Arg His His Leu Ser Leu
Gln Leu Phe Ala Ser305 310 315 320Arg Leu Tyr Phe His Trp Val Arg
Cys Tyr Gln Arg Gly Leu Gln Arg325 330 335Leu Gln Ala Lys Arg Val
Ser Val Ala Val Met Lys Gly Pro Glu Phe340 345 350Lys Asp Cys Trp
Glu Lys Phe Val Asp His Gln Gly Arg Ser Phe Pro355 360 365Ser Trp
Glu Lys Leu Glu Gln Tyr Ser Glu Ser Ile Ser Arg Arg Leu370 375
380Ser Arg Ile Leu Arg Phe Ala Asn Gln Asn Asn Leu Glu Asp Ser
Phe385 390 395 400Arg Asp Leu Arg Leu Gly Ser Pro Ser Pro Ser Ser
Ser Arg Ser Asp405 410 415Ser Arg1145PRTHomo sapien 11Pro Glu Thr
His Cys His Ala Glu Arg Cys Phe Leu Ser Trp Phe Cys1 5 10 15Asp Asp
Ile Leu Ser Pro Asn Thr Asn Tyr Glu Val Thr Trp Tyr Thr20 25 30Ser
Trp Ser Pro Cys Pro Glu Cys Ala Gly Glu Val Ala35 40 451245PRTBos
taurus 12Gly Gly Thr Arg Cys His Thr Glu Leu Arg Phe Leu Ser Trp
Phe His1 5 10 15Ala Glu Lys Leu Arg Pro Asn Glu Arg Tyr His Ile Thr
Trp Phe Met20 25 30Ser Trp Ser Pro Cys Met Lys Cys Ala Lys Glu Val
Ala35 40 451345PRTOvis aries 13Ala Gly Thr His Cys His Ser Glu Arg
Arg Phe Lys Ser Trp Phe Cys1 5 10 15Ala Lys Lys Leu Arg Pro Asp Glu
Cys Tyr His Ile Thr Trp Phe Met20 25 30Ser Trp Ser Pro Cys Met Lys
Cys Ala Glu Leu Val Ala35 40 451445PRTSus scrofa 14Pro Asp Pro Pro
Cys His Ala Glu Leu Cys Phe Leu Ser Trp Phe Gln1 5 10 15Ser Trp Gly
Leu Ser Pro Asp Glu His Tyr Tyr Val Thr Trp Phe Ile20 25 30Ser Trp
Ser Pro Cys Cys Glu Cys Ala Ala Lys Val Ala35 40
451521DNAArtificial SequencePrimer 15ttcatgtggg acactagaga t
211623DNAArtificial SequencePrimer 16aaccctttac cctttatgtg gat
231723DNAArtificial SequencePrimer 17aaagtcaatt tgtcagcgtc ctt
231820DNAArtificial SequencePrimer 18ggaacctgca acctatggaa
201920DNAArtificial SequencePrimer 19ggtgtggccc taaaaagaca
202020DNAArtificial SequencePrimer 20atcatgtttg agaccttcaa 20
* * * * *