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Gene A2b, Which Is Associated with Increased Susceptibility of Navajos to Haemophilus influenzae Type b Disease1
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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gene A2 is
used to encode the majority of anti-Hib Abs, and these are the
highest affinity anti-Hib Abs. Navajos carry a different allele of
the A2 gene segment (A2b) that is defective in its ability to undergo
V-J recombination. The A2b allele has only three nucleotide changes
from the commonly occurring A2a allele, two of which could potentially
affect its ability to recombine. In this study we used two independent
in vitro assays to test whether the nucleotide change found in the A2b
promoter and/or in the A2b recombination signal sequence (RSS) might be
responsible for the decrease in recombination frequency observed in
vivo. Using a luciferase reporter gene assay, we found no significant
difference between A2a and A2b promoter activities. However, the
competition recombination substrate assay showed a 4.5-fold reduction
in the relative frequency of recombination of the A2b RSS compared with
A2a. We show that this decreased frequency is due to a synergistic
effect of the unique nucleotide change present in the heptamer of the
A2b RSS and the shared nucleotide change present in the nonamer of both
A2b and A2a. This in vitro relative frequency of rearrangement is not
significantly different from that observed in vivo; therefore, the A2b
RSS is probably the factor associated with the increased susceptibility
to Hib disease among individuals carrying the A2b
allele. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 
loci being observed
(2). Nonetheless, among Abs mounted in response to both Hib infection
and polysaccharide vaccination, the A2 light chain has been shown to be
by far the most frequently used, and it presents no consistent somatic
mutations. In addition, the A2-containing Abs show the highest avidity
for Hib (3). Together, these data suggest that the presence of a
functional A2 gene in the human locus is a key element for an
efficient anti-Hib response. Navajos and genetically related populations descending from the Na-dene group have a 10-fold increased incidence of Hib disease compared with control populations (4, 5, 6). Previously, we hypothesized that a polymorphism in the A2 gene that impaired its expression might lead to the observed increase disease susceptibility; therefore, we analyzed DNA from healthy Navajo and control individuals. Over half of the Navajos studied, but only one control individual, had a new allele of A2, which we termed A2b (the allele corresponding to the published sequence is termed A2a) (7). The analysis of the relative frequency of recombination of nonproductive A2 rearrangements in A2a/b heterozygous Navajos showed a dramatic decrease in A2b rearrangements compared with A2a, suggesting that the A2b allele is defective in its ability to undergo rearrangement (7). The sequence of the A2b allele shows three nucleotide changes from the published A2a sequence: a one-nucleotide change in the recombination signal sequence (RSS), a one-nucleotide substitution in the promoter 16 bp 5' of the octamer, and a one-nucleotide change in the FR2 (7).
RSSs are necessary cis elements of the V(D)J recombination
process and flank each gene segment to be joined (8, 9). Each RSS
consists of two blocks of sequences, a highly conserved heptamer
(consensus: CACAGTG) and a less well conserved nonamer (consensus:
ACAAAAACC), separated by a spacer of a conserved length of 12 or 23 bp,
but nonconserved sequence. Changes in the first three nucleotides of
the heptamer have been shown to be extremely deleterious, whereas
changes in the other four bases have more various effects (8, 9, 10). In
the nonamer, the presence of three consecutive A residues is necessary
for efficient recombination (10). The A2b RSS has a 1-bp change in the
sixth nucleotide of the heptamer (CACAGAG), and both
the A2a and A2b alleles have a substitution in their nonamer
(ACAGAAACC) (7, 11). Therefore, although these two
positions are not commonly thought to be of critical importance for
recombination, we proposed that the effect of the change in the
heptamer or the combined effects of the two changes in the heptamer and
nonamer were likely to account for the severe decrease in the frequency
of recombination observed in vivo for the A2b gene segment (7). On the
other hand, accessibility of the various V, D, and J loci is a
prerequisite for the initiation of recombination (12, 13, 14).
Transcription of unrearranged gene segments (germline transcripts)
occurs just before rearrangement, and this germline transcription has
been hypothesized to play a role in locus accessibility, although it is
still not clear whether transcription of unrearranged segments is
responsible for, or is a consequence of, the open chromatin structure
at those loci. Therefore, the C
A change in the A2b promoter, which
is located 16 bp upstream of the octamer and which creates a CCAAT box,
is also a potential candidate to influence the ability of the A2b
allele to undergo rearrangement.
In this study we used two in vitro assays to test separately the potential effect of each of the promoter and RSS substitutions of the A2b allele. Levels of transcription of the A2a and A2b promoter were analyzed in a luciferase assay, and the individual and combined effects of the nucleotide changes in the A2b RSS on the recombination frequency were assessed in a modified recombination substrate assay. While the sequence change in the A2b promoter showed no significant effect on transcription levels compared with the A2a promoter, there was a 4.5-fold reduction in the relative frequency of recombination of the A2b gene segment compared with that of A2a as determined by the recombination substrate experiments. This indicates that the A2b RSS is at least in a large part responsible for the defective recombination of the A2b gene segment, and consequently, the RSS change may play a significant role in the increased susceptibility to Hib infection among A2b homozygous Navajos.
Thus, a single base change in a position commonly thought to have a modest effect on RSS function can significantly affect the in vivo repertoire and can have severe disease-associated biologic consequences.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The principle and construction of our recombination substrates
have been previously described (15). Competition substrates were
derived from the recombination substrate Vß8.2-Jß1 by multistep
cloning (Fig. 1
A) (16). Two
V segments were inserted on the 5' side of the termination signal
and are therefore competing for rearrangement with the unique J1
segment located on the 3' side of the termination signal. Coding ends
flanked by their RSSs were obtained by PCR from human genomic DNA, with
oligonucleotide primers containing the appropriate restriction sites to
enable insertion into the recombination substrate. Subsequent
modifications of the RSSs were introduced by PCR from the appropriate
clones using mismatched primers. PCR primers were located over 100 bp
upstream the RSS and 10–100 bp downstream the RSS. The two RSS
flanking the V segments on the 5' side of the plasmids were
therefore about 150 bp apart (Comp12, Comp19, Comp16, Comp11, Comp33,
and Comp35) or about 240 bp apart (Comp4 and Comp8). All PCR primer
sequences are available by request.
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18.8 Abelson-Moloney leukemia virus transformed pre-B cells
(20 x 106) were transiently transfected with 20 µg
of purified plasmid (Qiagen, Chatsworth, CA) by electroporation (960
µF, 0.3 kV) and resuspended in 10 ml of RPMI 1640 supplemented with
5 x 10-5 M 2-ME, 10% FCS, 2 mM glutamine, and 1 mM
caffeine. After 48 h, plasmids were recovered from transfected
cells by alkaline lysis, followed by DpnI/SpeI
digestion. Digested plasmids were then electroporated into JM109
Escherichia coli and plated on plates containing ampicillin
(100 µg/ml) and chloramphenicol (5 µg/ml). PCR screening assays
were performed from the colonies by resuspending each
chloramphenicol-resistant bacterial colony directly in the PCR reagent
mixture and amplifying with AF74 and P4 primers (Fig. 1
B)
for 30 cycles (1 min at 94°C, 1 min at 55°C, and 2 min at 72°C).
PCR products from each colony were analyzed by agarose gel
electrophoresis for the size of the product (see Fig. 1). Some of the
clones were simultaneously miniprepped, and their rearrangement status
was confirmed by sequencing. No more than 40 colonies were analyzed
from each transfection, and several transfections were performed with
each construct.
Luciferase constructs
Three sets of constructs were made containing the A2a or A2b
promoters. The enhancerless constructs were made in the pGL3-Basic
luciferase reporter gene vector (Promega, Madison, WI). The other sets
contained either the human intronic enhancer (iE) or the murine
IgH intronic enhancer (Eµ) inserted into the pGL2-Basic luciferase
vector. All enhancer and promoter fragments were generated by PCR. The
entire promoter fragments and the core portions of the enhancer
constructs were sequenced to ensure the absence of Taq-introduced
errors. The A2a and A2b promoters included the region from 18–312 bp
of the A2 sequence (GenBank accession no. M311952). This segment
includes 200 bp 5' of the octamer and extends to 12 bp 5' of the
start of the leader exon. The promoters were inserted into the
XhoI and HindIII sites in the pGL vectors via
sites engineered into the PCR primers. The enhancers were cloned
downstream of the luciferase gene in the BamHI site. The
human enhancer included the region between nucleotides 3598–4089
from GenBank sequence X67858. The heavy chain intronic enhancer
includes bp 21–734 of GenBank sequence M12827.
Luciferase assay
Constructs were transfected into the human cell lines
representing mature B cells (Daudi) and pre-B cells (BLIN-1) (17).
Transfections were performed as described by Fulton et al. (18).
Briefly, 1 million cells were transiently transfected with 1 µg of
purified luciferase reporter plasmids using the DEAE-dextran method.
The cells were then cultured in 1.2 ml of RPMI 1640 supplemented with
5 x 10-5 M 2-ME, 10% FCS, and 2 mM glutamine for
24 h, harvested, washed, and lysed in cell culture lysis reagent
(Promega). Twenty microliters of the extract was mixed with 100 µl of
the luciferase assay reagent (Promega), and luciferase activity was
measured for 30 s in a Monolight 2010 Luminometer (Analytical
Luminescence Laboratory, San Diego, CA). For each experiment, three to
five replicate wells were transfected for each construct. In each
experiment, the corresponding vector without any promoter fragment was
also transfected as a negative control for background levels of
transcription. Transfection without any plasmid was used to detect
background luciferase activity, which was minimal. Promoterless pGL2
vectors with either enhancer gave luciferase activity that was
essentially the same as the background luminescence. On the other hand,
the pGL3-Basic vectors had a significantly higher background.
Therefore, in Table IV, the average luciferase units obtained after
transfection with the appropriate promoterless construct was subtracted
from the average luciferase units obtained with the promoter-containing
construct. The ratio of the net increase in luciferase activity with
the A2a promoter to that obtained with the A2b promoter was then
determined. Each construct was transfected in 6–12 separate
experiments, and the SE for each construct is shown.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To analyze the role of the RSSs flanking various gene segments in
the frequency of recombination, we designed a series of recombination
substrates in which two V segments flanked by their RSSs are in
competition for rearrangement with a J1 segment (Fig. 1A). Since both segments are on the same plasmid, this
design controls for variation in transfection efficiency and thus
provides very accurate relative frequencies of recombination. The
status of the recombination was assessed by PCR, using primers located
upstream and downstream of the most external gene segments (AF74 and
P4, Fig. 1B). In this assay, recombination of the external
V gene segment with J1 generates a 370-bp PCR product, while
recombination of the internal V gene generates a 520- to 630-bp PCR
product depending on the gene segments (see Materials and
Methods). Rare background unrearranged clones can be observed as
1360- to 1580-bp PCR products. The reliability of this assay was
confirmed by simultaneously sequencing random clones.
A prerequisite of this study using competition substrates was to
determine whether the internal or external location of the V gene
segments would have an influence on the frequency of recombination. To
do so, we analyzed the relative frequency of recombination of a
substrate containing the same gene segment at both the external and
internal locations. Comp12 contains the same A2b segment at the
external and internal positions, and the relative frequency of
recombination of the external vs internal segments is 49:51% (Table I). This indicates that the location of
the various segments has no effect on their relative frequency of
recombination in our assay.
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To assess the role of the variant heptamer A2b RSS in the
decreased frequency of recombination of the A2b gene segment compared
with A2a, we constructed a series of competition substrates containing
various combinations of those two segments. Comp4 contains an external
A2a segment and an internal A2b segment, while Comp8 is the reverse,
with an external A2b segment and an internal A2a segment (Table I). In
both plasmids similar results were found, with 79–84% of the
recombination happening at the A2a locus regardless of its position in
the plasmid. Fisher’s exact test shows that the distribution of
relative frequency of recombination of A2a vs A2b is not significantly
different in Comp4 and Comp8 (p = 0.3), but is
significantly different from that in the control substrate, Comp12
(p < 0.0001). Since these results confirm the
previous observation that the locations of the gene segments in the
competition plasmids do not influence the outcome of the recombination,
we combined the results obtained for Comp4 and Comp8 to obtain a more
precise estimate of the relative frequencies of recombination of A2a vs
A2b. Table II shows that of 416 clones,
82 ± 3.7% had recombined the A2a segment, and only 18 ±
3.7% had recombined the A2b segment. Thus, these results show that the
single base change in the sixth position of the heptamer of the A2b
segment results in a 4.5-fold difference in its relative frequency of
rearrangement compared with that of A2a.
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). Although our in vivo
data showed a greater disparity between the relative recombination
frequencies, Fisher’s exact test showed that this in vivo distribution
of the relative frequency of A2a/A2b recombination was not
significantly different from the results found in vitro
(p = 0.44). Since our in vitro sample size was
much larger than that in vivo, the ratio of 82:18 may reflect more
accurately the relative frequency of A2a/A2b recombination. These
results indicate that the TA substitution in the sixth position of
the heptamer of the A2b RSS is sufficient to account for the decreased
frequency of recombination observed in vivo for the A2b allele compared
with A2a. Individual and synergistic effects of the substitutions in the heptamer and nonamer on the frequency of recombination
In addition to the TA change in the A2b heptamer, both the A2a
and A2b alleles had an AG substitution in their nonamer compared
with consensus RSS motifs (Table I). Hence, the decreased frequency of
recombination observed in A2b could be either the result of the change
in the heptamer alone or a synergistic effect of the combination of
changes in the heptamer and nonamer. To test whether the combined
effect of the nucleotide substitution in the heptamer and nonamer is
similar to the combined effect of each substitution individually, we
designed new constructs containing various combinations of each
substitution and compared them to an A2 segment flanked by a consensus
RSS. Comp19 shows the relative frequency of recombination of the
A2a/A18 RSS vs a consensus RSS, and therefore the impact of the nonamer
substitution alone, while Comp33 shows the impact of the heptamer
substitution alone (Table III). (A2a and
A18 are duplicated genes, and have identical RSS sequences and very
similar coding end sequences, the only differences between the terminal
portion of the coding ends being 9, 17, and 24 bp 5' of the RSS. Both
are equivalently amplified by our PCR primers, and these minor coding
region differences are unlikely to be significant.) The change in the
nonamer alone results in a relative frequency of recombination of
28 ± 7.2% compared with a consensus nonamer (Comp19), equivalent
to a ratio of 2.6, and the change in the heptamer alone results in a
relative frequency of recombination of 27 ± 8% compared with a
consensus heptamer (Comp33), also with a ratio of 2.7. If the effects
of the nucleotide changes in the heptamer and nonamer are cumulative,
we would expect that an RSS containing both changes (A2b) would
rearrange sevenfold less well (2.6 x 2.7) than a consensus RSS.
Comp35 shows that the relative frequency of recombination of the A2b
RSS vs a consensus RSS is 7 ± 4.2%, or a ratio of 13.3 (Table III). The relative frequency of recombination is therefore decreased
twofold from the expected cumulative effects of the two nucleotide
changes. This indicates that the effects of the nucleotide changes in
the heptamer and nonamer decrease the frequency of recombination in a
synergistic manner.
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To assess the potential role of the CA nucleotide substitution
present in the A2b promoter, the transcription activity of the A2a and
A2b promoter regions was tested in a luciferase reporter gene assay.
Since the relevant transcription that might affect recombination is
that of the unrearranged V genes that occurs in pre-B cells before L
chain rearrangement, we transfected BLIN-1, an acute leukemic pre-B
cell line (17). Because Ig transcription activity is lower in pre-B
cells than in B cells, we also transfected Daudi cells, a mature B cell
lymphoma line. Three sets of vectors were made (pGL3/enhancerless,
pGL2/Eµ, and pGL2/iE). pGL2/Eµ and pGL2/iE contain the murine
IgH intronic enhancer and the human intronic enhancer,
respectively. Since the enhancers are very distant from the V gene
promoters before rearrangement, germline transcription is thought to be
enhancer independent or at least independent of any enhancer yet
described. Therefore, in the first set of vectors we placed the V
promoters in the enhancerless pGL3-Basic luciferase reporter vector.
pGL3-Basic was chosen over the closely related pGL2-Basic, since no
promoter activity was observed when strong V gene promoters were placed
in pGL2-Basic without enhancers (data not shown). pGL3-Basic is a more
sensitive vector, although it has a higher background, and with this
vector we were able to detect luciferase activity driven by these
promoters in the absence of any enhancer.
The results are displayed in two ways. In Fig. 2, the black bars represent the average
of the actual mean luciferase units for each construct from every
experiment. These data also display the magnitude of the relative
activities of the different constructs and their promoterless controls.
Within each experiment, the replicate wells were extremely similar, but
the magnitude of the maximal luciferase activity among experiments did
vary significantly. Hence, the calculations in Table IV are the most valid for comparing the
relative promoter values of A2a vs A2b. In these calculations, the net
increase in luciferase activity over the background activity given by
the appropriate promoterless construct was determined for each
promoter-containing construct in each experiment. For each experiment,
the ratio of this net luciferase activity of A2a to that for A2b was
determined. The ratios from all of the experiments were averaged, and
the SE was calculated.
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show that the transcription levels of
the luciferase gene are somewhat reduced in enhancerless A2b/pGL3/-
vector compared with those in the A2a/pGL3/- vector in the BLIN-1
pre-B cells, although they are essentially the same in Daudi cells. The
A2a and A2b promoters were also placed into a pGL2 vector into which we
had inserted 600 bp of the human intronic enhancer
(A2a/pGL2/iE). The luciferase activity from this set of plasmids was
low, however, and only minimal activity over background was obtained
after transfection of BLIN-1 pre-B cells (data not shown). In the Daudi
cells, the A2a promoter was somewhat better than A2b, although
transcription levels were low for both (Fig. 2). The reason why the
enhancer activity from this fragment was so low is not clear. Although
we anticipated somewhat higher activity, this result was not totally
unpredictable, as the activity of the murine intronic enhancer in
murine pre-B cells and B cells was previously shown to be low (18). In
mature B cells, both iE and 3'E are needed for optimal activity.
However, since we are primarily interested in the promoter activity in
pre-B cells where the 3'E is very weak and in which there is no
synergy between the two enhancers, we did not create a double enhancer
construct (18).
Since the activities of the promoters were so weak in the presence of
iE, we also made a third set of vectors in which the A2a and A2b
promoters were placed into pGL2 vectors into which we had previously
inserted the murine intronic enhancer (pGL2/Eµ). These vectors gave
high promoter activity, and the A2a and A2b promoters showed equivalent
activity in both pre-B cells and B cells (Table IV).
These results suggest that the nucleotide substitution in the A2b
promoter region is minimally deleterious to the transcription activity
in the absence of any enhancer or with the enhancer and displays no
difference in activity in the presence of the strong Eµ enhancer.
These data strongly suggest that differential transcriptional activity
is unlikely to be a major factor in the reduced rearrangement of A2b.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test the role of the variant A2b RSS, we constructed a competition
recombination substrate assay consisting of in vitro recombination of a
plasmid containing the A2b and A2a gene segments in competition for
rearrangement with the J1 gene segment, and compared their relative
frequencies of recombination. The results show that the single T to A
substitution present at the sixth position of the A2b heptamer leads to
a 4.5-fold decrease in the relative frequency of rearrangement of the
A2b gene segment compared with that of A2a. These relative frequencies
of recombination found in vitro (82:18) are similar to and not
statistically significantly different from our previous in vivo
observation (89:11) (7). Since our in vitro sample size is much larger
than that in vivo, this may more accurately reflect the relative
frequency of A2a vs A2b recombination and suggests that the
substitution in the A2b RSS alone is sufficient to explain the
decreased recombination rate observed for the A2b allele. Thus, this
one-nucleotide change in the heptamer might be the main genetic factor
responsible for higher susceptibility to Hib infection in populations
carrying the A2b allele.
Changes in the heptamer and nonamer result in a broad range of deleterious effects on the recombination efficiency, depending on their nature and location (8, 9, 10). Some changes almost abolish recombination, while others have been described as more minor effects. Importantly, we show in this study that a four- to fivefold drop in the relative frequency of recombination, commonly considered a modest decrease, might, in fact, result in a dramatic under-representation of the gene segment in the primary B cell repertoire and eventually lead to a disease-associated inability to mount a specific Ab response. This suggests, therefore, that it may be important to consider the biologic effects of minor changes in the RSS in the light of these results.
In the study by Akamatsu et al. using a different recombination
substrate system, an RSS with the same change in the sixth position of
the heptamer as that present in A2b rearranged at 39.6 ± 5% the
frequency of a consensus RSS (10). This corresponds to a 2.5-fold
decrease or a (72:28%) relative frequency of recombination for the
consensus RSS vs the RSS with the altered heptamer, respectively. The
estimation of the effect of the T to A change in the heptamer in that
report is therefore about 2-fold lower than that observed in this study
between A2a and A2b (Table I, Comp4 and Comp8) and over 3 times lower
than our in vivo observation (Table II). Since the construct in
Akamatsu et al. study contained the heptamer change in the context of a
consensus nonamer, and since both A2a and A2b have an additional
nucleotide change in the nonamer, one explanation for the apparent
difference in results was that the effect of the nucleotide changes in
the heptamer and nonamer in A2b would be synergistic, e.g., would be
worse than a simple cumulative effect of each of the changes. We
directly tested this hypothesis by comparing the individual and
combined effects of the two substitutions in additional competition
substrates, containing a consensus RSS as one of the two segments. In
agreement with Akamatsu et al., we found that a T to A substitution at
the sixth position of the heptamer leads to a 2.7-fold reduction of
rearrangement (73:27%) compared with that of a consensus RSS. In
addition, we found a similar 2.6-fold relative reduction for the A to G
substitution at position 4 of the nonamer (72:28%), while Akamatsu et
al. found a slightly more deleterious effect (79:21%, ratio 3.7).
However, when both changes are present simultaneously in the RSS (A2b
RSS), our results show a dramatic 13.3-fold decrease in
recombination (93:7%) relative to the A2 RSS with consensus heptamer
and nonamer, which is about 2-fold more than the product of each change
individually. These results indicate a synergistic effect of the
changes in the heptamer and nonamer of the A2b RSS.
Our results suggest that the effect on recombination of changes in one of the RSS motifs is dependent on the context of the other RSS motif. Recent DNA binding studies indicate that nonamer and heptamer motifs fulfill different primary functional roles during the first steps of the recombination (19, 20, 21). In these studies, the nonamer was revealed as the critical element for initial recognition and binding by RAG-1, while the heptamer was mainly involved in directing the cleavage step after engagement of the RAG-1/RAG-2 complex on the DNA, although it was also described to enhance RAG binding. Both nonamer and heptamer could therefore modulate the frequency of rearrangement, by controlling either the binding or the cleavage activity. Here, we show that changes in the heptamer and nonamer in the A2b RSS synergistically decrease the frequency of recombination, suggesting that the effect of the additional change in the A2b heptamer could be related to the destabilization of the RAG-1/2 complex, as a result of the poorer binding to the nonconsensus A2 nonamer.
We also tested whether the change in the promoter of the A2b gene,
which creates a CCAAT element 14 bp 5' of the essential octamer, might
have any effect on transcription. To mimic the transcription that
occurs from unrearranged genes before V(D)J rearrangement and that is
tightly correlated with accessibility for rearrangement (12, 13, 14), we
established an assay in which we could measure luciferase activity in a
construct in the absence of any enhancer. We also tested these
promoters in the presence of two different enhancers. Our results show
no significant difference between the two promoters, although the A2a
promoter was slightly better in the absence of any enhancer or with the
enhancer. Thus, if germline transcription is required for
rearrangement accessibility, then the A2b promoter change is unlikely
to play any role in the reduced rearrangement potential of the A2b
allele. However, the promoter may be involved in promoting chromatin
restructuring to create accessibility of the downstream V gene
independent of germline transcription. Thus, our results do not exclude
the possibility that a CCAAT binding protein may bind to the A2b
promoter and block the chromatin restructuring necessary for
accessibility of the A2b gene.
In summary, these results show a 4.5-fold decrease in the relative frequency of recombination of the A2b segment compared with that of A2a. Moreover, we show a 13.3-fold decrease in the A2b segment compared with segments flanked by consensus RSSs. Together, these data indicate that the nucleotide variations from consensus in the heptamer and nonamer of A2b are synergistic, presumably by destabilizing the interaction of the RAG-1/RAG-2 complex with the RSS. Importantly, our results suggest that a 4- to 5-fold decrease in the relative recombination rate of the A2b gene segment is genetically associated with high susceptibility to Hib disease, suggesting that minor changes in the frequency of recombination of gene segments can have important biologic consequences.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ann J. Feeney, The Scripps Research Institute, Department of Immunology IMM-22, 10550 North Torrey Pines Rd., La Jolla, CA 92037. E-mail address:
3 Abbreviations used in this paper: Hib, Haemophilus influenzae type b; RSS, recombination signal sequence; RAG-1, recombination-activating gene-1.
Received for publication May 18, 1998. Accepted for publication July 21, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene rearrangement correlates with induction of germline gene transcription. Cell 58:1001.[Medline]
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G. S. Williams, A. Martinez, A. Montalbano, A. Tang, A. Mauhar, K. M. Ogwaro, D. Merz, C. Chevillard, R. Riblet, and A. J. Feeney Unequal VH Gene Rearrangement Frequency Within the Large VH7183 Gene Family Is Not Due to Recombination Signal Sequence Variation, and Mapping of the Genes Shows a Bias of Rearrangement Based on Chromosomal Location J. Immunol., July 1, 2001; 167(1): 257 - 263. [Abstract] [Full Text] [PDF]
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 L. Hougs, L. Juul, A. Svejgaard, and T. Barington Structural Requirements of the Major Protective Antibody to Haemophilus influenzae Type b Infect. Immun., May 1, 1999; 67(5): 2503 - 2514. [Abstract] [Full Text] [PDF]
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