Hypervariable loci in the human gut virome
Samuel Minota, Stephanie Grunberga, Gary D. Wub, James D. Lewisc, and Frederic D. Bushmana,1
aDepartment of Microbiology,bDivision of Gastroenterology, andcDepartment of Biostatistics and Epidemiology, Perelman School of Medicine at the
University of Pennsylvania, Philadelphia, PA 19104
Edited by Jeffrey H. Miller, University of California, Los Angeles, CA, and accepted by the Editorial Board January 24, 2012 (received for review November
Genetic variation is critical in microbial immune evasion and drug
resistance, but variation has rarely been studied in complex
heterogeneous communities such as the human microbiome. To
begin to study natural variation, we analyzed DNA viruses present
in the lower gastrointestinal tract of 12 human volunteers by
determining 48 billion bases of viral DNA sequence. Viral genomes
mostly showed low variation, but 51 loci of ∼100 bp showed ex-
tremely high variation, so that up to 96% of the viral genomes
encoded unique amino acid sequences. Some hotspots of hyper-
variation were in genes homologous to the bacteriophage BPP-1
viral tail-fiber gene, which is known to be hypermutagenized by a
unique reverse-transcriptase (RT)-based mechanism. Unexpectedly,
other hypervariable loci in our data were in previously undescribed
gene types, including genes encoding predicted Ig-superfamily pro-
teins. Most of the hypervariable loci were linked to genes encoding
RTs of a single clade, which we find is the most abundant clade
among gut viruses but only a minor component of bacterial RT
populations. Hypervariation was targeted to 5′-AAY-3′ asparagine
codons, which allows maximal chemical diversification of the en-
coded amino acids while avoiding formation of stop codons. These
findings document widespread targeted hypervariation in the hu-
man gut virome, identify previously undescribed types of genes tar-
motivate studies of hypervariation in the full human microbiome.
deep sequencing|diversity-generating retroelement|mutagenesis|
major tropism determinant
targeted changes in DNA. The vertebrate adaptive immune
system is based on covalent DNA rearrangements that diversify
genes encoding Ig-domain antigen-binding proteins. In response,
viral and cellular pathogens encode genetic systems that vary
antigens bound by host antigen receptors (1, 2).
In this study we begin to characterize patterns of sequence
variation in heterogeneous natural communities, using the human
microbiome as a model. We chose to study viral samples because
they represent a medically important microbiome component, but
containa smalleraggregate genomesizethanthefullmicrobiome,
allowing sequencing to a depth that permits empirical assessment
A newly discovered mechanism of targeted hypermutation,
particularly pertinent here, involves the Bordetella bacteriophage
BPP-1, which has been shown to vary the sequence of the gene
encoding its phage tail fiber to bind divergent cell-surface recep-
tors (3–6). The phage-encoded major tropism determinant (MTD)
hypermutation by a reverse transcriptase (RT)-dependent mech-
anism (7, 8). The 3′ part of the tail-fiber gene is duplicated in the
phage genome, and the duplicated template repeat (TR) is tran-
scribed and reverse-transcribed in an error-prone fashion. The
mutated copy is then incorporated into the MTD gene variable
repeat (VR), leading to very high mutation rates. Diversity-gen-
erating systems involving related RTs and genes encoding C-type
lectin folds have been inferred from prokaryotic genome se-
quences (3, 4), but only the BPP-1 system has been characterized
ey aspects of host–parasite interactions are mediated by
Here we have used the Solexa/Illumina HiSeq method to in-
terrogate 48 billion bases of DNA sequence from populations of
gut DNA viruses, which allowed us to identify regions of targeted
hypervariation in the primary sequence data. We found that RT-
associated hypervariation systems were present in 11 of 12 sub-
jects examined, and act on a much wider range of gene types
than was known previously. Analysis of the sequence information
further specifies the chemical logic of the mutational targeting
and suggests that the most common role of RTs in the gut virome
is targeted hypervariation.
Sequence and Assembly of 48 Gb of Gut Viral DNA. To study diversity
in natural populations of the human virome, we collected stool
samples from 12 healthy individuals (three per subject) over a
2-mo period, then purified viral particles by sequential filtration,
banding in CsCl density gradients, and treatment with nuclease,
amplified, and then sequenced using the Solexa/Illumina HiSeq
paired-end sequencing platform. A total of 495,053,311 reads
were generated, averaging 97.2bpin length.Asan empirical error
control, 153 million reads were determined for DNA from phage
ΦX174, showing an accuracy of 99.94%. A total of 48 Gb of data
were collected, the largest survey of viral sequences yet reported.
The raw sequences were assembled into contigs using the
deBruijn graph-based assembler SOAPdenovo (10). The depth of
sequencing for the gut viral contigs averaged 49× and ranged up
to 3,000× (Fig. 1A). There were 78 contigs longer than 1 kb that
assembled as complete circles, indicating probable completion of
the viral genome sequence. Circular assemblies could arise either
concatemers, which are intermediates in the replication of many
DNA viruses. The mean number of contigs per subject longer
than 1 kb was 1,390, ranging from 573 to 3,390.
Protein functions were inferred by comparing the conceptual
translation of predicted ORFs to a curated database of protein
families. A broad range of viral functions were identified in the
encoded proteins (Fig. 1B), as observed previously (9, 11). On
average, 72% of the ORFs did not resemble any recognizable
protein family, emphasizing the immense diversity of novel genes
in gut viral populations.
To assess the relationship to known viral genome sequences,
Information (NCBI) RefSeq collection of viral genomes. The five
database sequences with the most extensive similarity are shown
Author contributions: S.M., S.G., G.D.W., J.D.L., and F.D.B. designed research; S.M. and
S.G. performed research; S.M. and F.D.B. analyzed data; and S.M. and F.D.B. wrote
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. J.H.M. is a guest editor invited by the Editorial
Data deposition: Contigs containing variable regions listed in Table S1 have been depos-
ited in the GenBank database.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| March 6, 2012
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in Fig. 1C. Most of the recognizable viral sequences showed
matches to prokaryotic viruses (DNA bacteriophages). Regions
of similarity were typically short patches (median alignment
length 202 bp at e-value ≤ 10−5), supporting the idea that bac-
teriophage functions are commonly organized in genetic cassettes
(12, 13). Only one well-characterized virus known to replicate on
human cells was detected: human papillomavirus type 6b (Fig.
1C), which was only found within a single subject and sequenced
to a depth of 23-fold. The next best hit to a eukaryotic virus was
unconvincing, indicating that this viral fraction in healthy subjects
is overwhelmingly composed of bacterial viruses.
Hypervariable Loci in Gut DNA Viruses. To investigate sequence
variation within each contig, we aligned the raw reads back to
that multiple small regions showed extremely high variation against
a background of low variation. Comparison of filtering criteria led
us to focus on regions of at least 90 bp that were sequenced to
a depth of at least 5×, contained a proportion of unique sequences
at least 5%, yielding 36 regions of the highest variability.
Analysis of these regions revealed that 12 resembled the di-
versity-generating retroelement of phage BPP-1 described above
(6, 8). This system is comprised of ∼100-bp repeat regions, the
donor TR, the targeted VR, and an RT, which is required to
(6).Because of the central roleof RT in thisprocess, we identified
all of the genes encoding RT-like sequences within the full
collection of contigs, revealing 185 genes. Duplicated sequences
can potentially break contig assemblies, so we manually inspected
all of the RT-containing contigs to identify broken assemblies
near the RT sequences suggestive of TR/VR pairs. We repaired
33 contigs through a combination of directed resequencing and
manual realignment of shotgun Solexa/Illumina reads. This pro-
cess increased the number of variable regions to 51, those variable
regions falling within a TR/VR pair to 36, and those with both an
RT and a TR/VR pair to 29 (Fig. S1 and Table S1). In every case
where a variable region was near an RT, it also contained a TR/
VR pair. Such elements were found in 11 of 12 subjects studied.
Based on the resemblance to BPP-1, we refer to these systems as
“diversity-generating retroelements” below. Of these retroele-
ments, 18 of 29 were found in contigs that could be tentatively
assigned to a specific bacteriophage family (Table S1).
Short hairpins near the VR are essential for activity in the BPP-
1 system (14). Similar hairpin sequences were found in only 13 of
the above 29 elements (Table S1). Evidently some of the novel
diversity-generating retroelements may use different initiation
mechanisms that do not involve hairpin structures. Of those 13
hairpins, 6 were found within ORFs, raising the question of how
All of the 29 variable regions adjoining both an RT and a TR/
VR pair were within an intact ORF longer than 500 bp, allowing
the targeted genes to be analyzed. We used BLASTp alignments
and the homology-based structural prediction pipeline Phyre2 to
analyze each ORF (Table S1) (15). Fourteen ORFs had the
hypervariable region near the 3′ end of the coding region and
Lactococcus phage jj50
0 - 24X Coverage (27kb)
Lactococcus phage bIL67
0 - 49X Coverage (22kb)
Bacteroides phage B40-8
0 - 1510X Coverage (45kb)
Streptococcus phage SM1
0 - 29X Coverage (35kb)
0 kb5 kb10 kb
Human papilloma virus type 6b
0 - 39X Coverage (7.8kb)
Phage DNA packaging
Phage other structural
as a point, the length is shown on the x axis and the depth of reads mapped to each contig on the y axis. Circular contigs are shown in red. (B) Assignment of
gene functions from viral contigs using the Pfam database of protein families; assignment of Pfam domains to viral functions is described in ref. 9. The
proportion of sequences that were assigned for each function is indicated on the y axis. On average, 21% of each sample was assigned to a Pfam protein
family. (C) The five viral RefSeq genomes with the most similarity to sequences generated in this study. Vertical lines indicate the depth of sequencing at that
position, and colored lines indicate mismatches with the reference sequence. The range of coverage is noted to the left of each plot. Blue boxes below each
genome indicate annotated genes.
Assembly, functional assessment, and identification of viral sequences. (A) Summary of contigs assembled from viral sequences. Each contig is shown
Minot et al.PNAS
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100%), resembling the well-studied MTD of BPP-1 (percentage of
identity 15–33%) (Fig. 2A; the arrow indicates the direction of
information transfer inferred from the BPP-1 model).
Unique Gene Types at Hypervariable Loci. Surprisingly, six hyper-
variable ORFs encoded proteins that aligned to cadherins,
invasins, and fibronectins, which contain Ig-superfamily β-sand-
wich domains (Table S1). Proteins aligned with modest percent
identity (11–15%), but structure predictions suggested multiple
β-sandwich domains with high confidence (Phyre2 confidence
scores of 97–99%). Several of these proteins were also homol-
ogous to each other (80–90% identity), despite being isolated
from different individuals. Ig-superfamily proteins are common
in bacteriophage (16–18), but genes encoding Ig-superfamily
proteins were not previously known to be subject to hyper-
variation by an RT-associated mechanism. The VRs for Ig-
superfamily proteins were in the middle of the target ORF, so
that the TR and VR were separated by an average of 1,938 bp. In
comparison, the MTD-like TR and VR are separated by an av-
erage of only 356 bp. The greater distance between repeats in Ig-
superfamily genes may have hindered their detection in previous
studies based on DNA sequence comparisons (3, 6).
Three hypervariable ORFs also encoded predicted leucine-
rich repeat proteins N terminal to the hypervariable region
(Phyre2 confidence score of 95–97%). These proteins were all
embedded in large ORFs, ranging from 1,716 to 2,181 predicted
amino acids. One of these also had a C-type lectin fold in the
hypervariable region. The remaining eight ORFs containing
hypervariable regions did not have convincing similarity to
known proteins, and may represent still further types of proteins
subject to targeted hypermutagenesis.
Hypervariation at Adenine Residues. Adenine residues were tar-
geted in all of the hypervariable regions identified here (Fig. 3A).
The 5′-AAY-3′ sequences were particularly strongly affected
2200_scaffold2278 - 36 kb
10 kb20 kb30 kb
Phage tape measure
33 kb34 kb35 kb
10 kb20 kb30 kb40 kb
2011_scaffold3 - 43 kb
38 kb39 kb40 kb
C-type lectin fold
lectin fold. (B) Hypervariation in a gene predicted to encode an Ig-superfamily fold. In A and B, the Upper section shows the contig of origin, with gray vertical
lines showing sequencing depth and boxes showing annotated proteins. The indicated area is expanded below to show the TR, the corresponding VR, RT, and
the ORF that contains the targeted VR. The inferred direction of information transfer between the TR and VR is shown with an arrow. The Lower section of
each plot shows an alignment of the sequences spanning the TR and VR for each element (white space indicates gaps between reads). Above the VR sequence
is a barplot indicating the proportion of bases in the VR that differ from the consensus base in the TR. DNA bases are indicated by colors as indicated on the
sides of the panels.
RT-associated hypervariable regions from the human gut virome. (A) Hypervariation in a gene predicted to encode a protein with an MTD-like C-type
| www.pnas.org/cgi/doi/10.1073/pnas.1119061109Minot et al.
(e.g., Fig. 2, Lower). This substitution pattern in 5′-AAY-3′,
which encodes asparagine, allows access to many different
chemistries in the encoded amino acid side chains while sup-
pressing creation of stop codons, as was originally pointed out for
the MTD system (3). The size of the dataset reported here
allowed us to carry out statistical analysis of the placement of the
5′-AAY-3′ relative to the three possible reading frames, which
showed that the 5′-AAY-3′ sequences were overwhelmingly in
the asparagine-encoding frame (Fig. 3B) (P < 10−163). Thus,
variable region sequences have evolved to take advantage of
asparagine-codon diversification while suppressing other types
RT Gene Populations in Gut DNA Viruses and Bacteria. We next took
advantage of the above data to annotate functions of gut virome
RT genes. All of the RT sequences previously associated with
hypervariable regions (3, 6, 19) cluster in a monophyletic clade
containing the BPP-1 RT (Fig. 4A, cluster marked “DGR” for
diversity-generating retroelements). In our dataset, we found
that most of the new RTs clustered in this group (n = 99), in-
cluding all of the RTs found to be associated with hypervariable
regions (Fig. 4A, green symbols). There was no obvious corre-
lation between RT phylogeny and targeted gene type. Far fewer
gut virome RTs clustered with group II intron RTs (n = 8), and
retron RTs (n = 6). We observed two previously undescribed
groups of RT sequences. Five sequences fall into “novel 1” (Fig.
4A), which is most similar to the Unk2 family (19). Seven
sequences fall into “novel 2” (Fig. 4A), which is a sister clade to
the retron RTs. The average pair-wise distance of the pooled
RTs associated with diversity generating systems described here
is 1.14, which greatly increases the diversity of this group (pre-
viously 0.90) and rivals the diversity of the large retroviral/LTR
retrotransposon RT clade (1.20).
We compared the distribution of RT clades in gut DNA
viruses described here to that of their bacterial hosts. The bac-
terial genomes were dominated by the RT clades associated with
group II introns and retrons, and thus differed from the DNA
viruses, where RTs associated with diversity-generating retro-
elements dominated (Fig. 4B).
We report that DNA viruses of the human gut are rich in hyper-
variable regions, and that these are associated with template re-
substitutions was so high that up to 96% of alleles in hypervariable
regions encoded unique protein sequences. Most of the RT genes
in the virome dataset were in the clade linked to diversity-gener-
ating retroelements. Thus, targeted hypervariation appears to be
the major role of RTs in DNA viruses of the human gut.
Surprisingly, several of the genes subject to hypervariation were
predicted to encode Ig-superfamily proteins. Thus, both gut
Proportion of sites
* ACDEFGH I K L MNPQRST VWY
VR amino acid
TR amino acid
Heatmap showing the relationship of positions in the TR (y axis) to the
resulting nucleotides in the VR (x axis). Of 15,447 mutated bases, 14,930
(97%) are located at adenine-positions relative to the TR. (B) Amino acid
substitution heatmap showing the relationship of codons in the TR (y axis) to
the resulting codons in the VR (x axis). Of 11,462 mutated codons, 9,212
(80%) are located at asparagine (N) codons in the TR.
Characteristics of RT-associated hypervariation in the gut virome. (A)
Group II Intron
Group II Introns
human gut. (A) Phylogentic tree of RT sequences.
Each sequence was aligned to a position-specific
scoring matrix to construct a multiple sequence
alignment. The tree was constructed using the
maximum-likelihood method. Green circles in-
dicate RT sequences on viral contigs from this
dataset that contain hypervariable regions and
TR/VR pairs. Purple circles indicate other RT
sequences from this dataset; the remaining
leaves indicate reference sequences from the
NCBI. RT clades were adapted from refs. 6 and 19,
and are indicated by gray lines. The bootstrap
support ofinternalnodesis indicatedbythecolor
of internal branches as described in the key.
Clades are marked according to refs. 6 and 19:
Abi, abortive-phage-infection; DGR, diversity
generating retroelements; G2L, group II intron-
like families; Hpdn, hepadnaviruses; LTR, LTR
retrotransposons; PLE, Penelope-like elements;
Rpls, retroplasmid; Telo, telomerase; Unk, unknown families (19). The scale bar indicates the log-corrected distance metric used by FastTree, adapted from
BLOSUM45.Distancesrangefrom 0, indicatinga perfectmatch,to 3, indicatingno overlap.(Scale bar,1.0).(B) Relativeproportions of RTsinviruses studied here,
the RefSeq phage genome database, and the RefSeq bacterial genome database.
RT sequences found inDNAviruses ofthe
Minot et al.PNAS
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viruses and vertebrate antigen receptors have evolved to use Ig
domainsasscaffolds fordisplaying highlydiversified polypeptides.
Evolution may have converged on these β-sheet–rich domains
because they are relatively rigid and so can maintain their folds
despite primary sequence diversification, as has also been sug-
gested for the C-type lectin fold (3). The placement of diversified
regions on Ig domains appears to differ between vertebrates and
phage. Although more complete structural characterization is
needed, modeling suggests that the phage Ig-superfamily domains
may be diversified along one surface and into the adjoining linker
between domains, but the vertebrate antigen receptors are di-
versified in loops between β-sheets within an Ig domain. The
mechanism of diversification in phage clearly differs from that in
the vertebrate immune system—the phage genes are diversified
by error-prone reverse transcription (8), but the Gnathostomata
immune system is diversified by V(D)J recombination, which
involves DNA double strand breaks (20), and targeted deami-
nation by activation-induced cytidine deaminase (21).
The functions of the previously undescribed viral hypervariable
genes found here are not fully clarified. Hypervariable genes may
encode viral structural proteins targeted by human IgA, which is
secreted into the gut in large amounts, so that diversification of
viral structural proteins may allow immune evasion. However,
a role in ligand binding may be more likely—a weakness of the
immune evasion model is that only specific short regions are
targeted for hypermutagenesis, so the remainder of the protein
could still be antigenic.
Some of the hypervariable Ig proteins may be homologs of T4
highly immunogenic outer capsid (hoc) protein, which encodes
an Ig protein related in sequence to those studied here. Hoc
decorates T4 heads by binding to sixfold symmetric vertices in
hexameric capsomeres, thereby providing a polyvalent binding
moiety on the outside of phage heads. Hoc is proposed to me-
diate binding of T4 to surfaces such as the Escherichia coli host
cell (22), and has also been used for phage display to create new
binding specificities for biotechnology applications (23). If the
Ig-superfamily proteins studied here are also accessory head
proteins, they may mediate binding of viral particles to candidate
host cells or environmental materials, allowing selection to
enrich for those binding specificities that optimize reproductive
success. The most useful binding specificities may differ widely
during replication in the human gut or after shedding in feces,
but hypervariation allows optimization in each new environment.
Further research will be needed to clarify the full biological roles
of these viral diversity generating systems.
Gut Viral DNA Sequencing and Assembly. Details can be found in the SI
Methods. Briefly, stool samples were collected from healthy subjects as de-
scribed previously (9, 24). Viral DNA was purified by filtration and density
ultracentrifugation (9) to a purity of ∼99.9% by 16S rDNA quantitative PCR
HiSEq. 2000 using 100-bp paired-end chemistry. Sequences were trimmed to
Q35 (using FASTX v0.0.13) and assembled within each subject using SOAP-
denovo (v1.05) and a k-mer size of 63. Reads were mapped back to those
contigs within each subject using Burrows-Wheeler Aligner (v0.5.9-r16).
Functions encoded by these contigs were predicted using RPSBLAST (v2.2.20)
and the NCBI Conserved Domain Database.
Analysis of Hypervariable Loci. VariableregionswerefoundusingRscriptsthat
analyzed the variability of reads mapped back to these novel contigs. ORFs
containing hypervariable loci were translated using custom scripts and sub-
mitted to Phyre2 (15), using a confidence threshold of 95%. RT sequences
associated with hypervariable loci were aligned to the curated RT position-
specific scoring matrix PF00078 using hmmalign (HMMER v3.0), and the
resulting approximately maximum-likelihood tree was generated by Fast-
Tree. R scripts, BAM alignment files of the 29 contigs found in Table S1, and
RT sequence alignments are available upon request.
ACKNOWLEDGMENTS. We thank members of the G.D.W., J.D.L., and F.D.B.
laboratories for help and suggestions; Scott Sherril-Mix for the gift of a useful
script; and the Penn Genome Frontiers Institute. This work was supported by
Human Microbiome Roadmap Demonstration Project UH2DK083981 (G.D.W.,
F.D.B., and J.D.L. are co-Principal Investigators); National Institutes of Health
(NIH) Grant T32AI060516 (to S.M.); a grant with the Pennsylvania Department
of Health; NIH Grant AI39368 (to G.D.W.); the Molecular Biology Core of The
Center for Molecular Studies in Digestive and Liver Diseases (P30 DK050306);
the Joint Penn-Children’s Hospital of Philadelphia Center for Digestive, Liver,
and Pancreatic Medicine; NIH instrument Grant S10RR024525 and NIH Clinical
Research Resources; and the Crohn’s and Colitis Foundation of America.
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| www.pnas.org/cgi/doi/10.1073/pnas.1119061109Minot et al.