ig genes in b lymphocytes undergo three types of genetic alterations during their development , i.e. , v(d)j recombination , somatic hypermutation ( shm ) , and class switch recombination ( csr ) .
v(d)j recombination takes place in developing b lymphocyte precursors and its biochemical mechanism is well characterized ( 1 ) .
both events occur in activated mature b lymphocytes such as germinal center cells , but outcomes of the events are apparently very different . in shm , mostly point mutations are introduced in ig variable ( v ) region genes , giving rise to ig with high affinity ( 2 ) .
dna cleavages are shown to be introduced in the v region during shm ( 36 ) .
on the other hand , in csr , two switch ( s ) regions located 5 to heavy - chain constant ( ch ) region genes are cleaved and a large dna fragment between the cleavages is excised out from the chromosome to bring in a downstream ig ch region gene to the proximity of a rearranged v gene ( 7 ) .
in addition , neither shm nor csr is prerequisite of the other ( 8 , 9 ) . therefore , it is striking that a defect of aid , a putative rna editing enzyme , virtually abolishes both shm and csr without affecting germinal center formation ( 10 , 11 ) . to explain this unexpected finding , we have proposed a model that aid edits a precursor mrna to synthesize an endonuclease essential for generating dna cleavages in both shm and csr reactions ( 12 ) .
however , it remains to be tested whether aid edits separate pre - mrnas for csr and shm , and thus is involved in different steps in the two genetic events . in the present study we provide the evidence that hypermutation takes place in the unrearranged ig s region under the condition that induces csr but not shm in the v region gene .
the results imply that csr and hypermutation may be mediated , at least in part , by the same molecular machinery .
wild - type ( wt ) and aid mice on ( cba  c57bl/6 )  c57bl/6 back ground were maintained in our animal facility and used 28 mo of age .
spleen b cells were purified by depleting cd43 cells with the magnetic cell sorting system ( macs ; miltenyi biotec ) .
after 58 d cultivation , live cells were harvested and high molecular weight nuclear dna was extracted with sds / proteinase k lysis , followed by phenol / chloroform extraction . in some experiments , switched igg cells were enriched ( 6890% ) or depleted ( 1.56% ) by macs with combination of biotinylated anti - igg1 and anti  igg3 antibodies ( bd pharmingen ) and streptavidin microbeads ( miltenyi biotec ) .
mutation frequencies in each population were determined by sequencing 10,391 and 10,328 bp , respectively , as described in table i. induction of hypermutation in the s region upon csr stimulation dna was extracted from purified spleen b cells cultured with or without lps and il-4 .
the s region ( 1.1 kb ) composed of the 5 and 3 subregions ( 0.5 and 0.6 kb , respectively ) and vj558-jh4 downstream region ( 1.2 kb but 0.5 kb of the jh4 3 flanking sequence was examined ) were amplified , subcloned , and then sequenced .
the switch efficiency to igg1 and igg3 in the lps / il-4 cultured wt cells was 2956% .
statistic tests were done with sas version 2000 software ( sas institute inc . ) .
pcr were performed with the primers shown below using pyrobest dna polymerase ( takara ) that has the 3 exonuclease activity and high fidelity .
after purification , the pcr fragments were digested with ecori or spei and ligated into pbluescript vector .
the ligation mixture was used for transformation and the library was plated without preculturing to avoid amplification of sister clones .
no more than 21 clones were sequenced from a single pcr reaction for the s. clonality of the v region clones were checked by their cdr3 sequences .
nucleotide sequences were determined with abi prism 3100 genetic analyser ( perkinelmer ) . the s region germline sequence of cba and c57bl/6 were determined and compared .
6 and 5 bp polymorphic differences were found in the s and jh4 downstream regions of our interest , respectively , and excluded from mutations .
primers used for s pcr are : 5-ggaattcattccacacaaagactctggacc-3 ; 5-ggaattccagtccagtgtaggcagtaga-3 ( a and d in fig .
1 a , respectively ) with 30 cycles of 94c for 30 s , 62c for 30 s , 72c for 1 min .
primers used for sequencing in addition to common primers for pbluescript are : 5-ggaattcgtaaggagggacccaggctaag-3 ; 5-ggaattcttccagaatcccaggattgcc-3 ( b and c in fig .
the 3 subregions of nonswitched alleles were amplified by nested pcr as follows : the first step , 20 cycles of 98c for 10 s , 68c for 7 min , with the primer a and 3-agcccatgctagctcagcctcacataa-5 ( 3 of the s core ) ; the second step , 15 cycles of 98c for 10 s , 68c for 80 s , with the b and d primers . for vj558-jh4 downstream region pcr
, primers described ( 13 ) were used with 35 cycles of 98c for 10 s , 68c for 80 s. distribution of mutations in the s region .
( a ) a total of 58 point mutations and 6 deletions shown in table i ( closed and hatched symbols ) and table ii ( open symbols ) are mapped by triangles and rectangles , respectively , on the s region ( bar ) .
position from the transcription start site ( arrow ) is indicated by a scale below .
lengths ( bp ) of deletions ( a  f ) are : a , one ; b , 17 ; c , 30 ; d , 52 ; e , 7 ; f , 19 .
the s nucleotide sequence of the c57bl/6 mouse tail dna is shown by upper case letters .
sequences that are predicted to form s / l structures are underlined ( references 12 and 34 ) .
conditions used for the s / l prediction were : the ionic conditions ( mm ) , [ na ] = 150 , [ mg ] = 0.5 ; the folding temperature , 37c ; the maximum distance between paired bases , 25 .
recombinant retrovirus constructs ( pmx - aid - ires - gfp ) to express aid or aid , inactive mutant of aid , and preparation and infection of retroviruses were described before ( 14 ) .
wild - type ( wt ) and aid mice on ( cba  c57bl/6 )  c57bl/6 back ground were maintained in our animal facility and used 28 mo of age .
spleen b cells were purified by depleting cd43 cells with the magnetic cell sorting system ( macs ; miltenyi biotec ) .
after 58 d cultivation , live cells were harvested and high molecular weight nuclear dna was extracted with sds / proteinase k lysis , followed by phenol / chloroform extraction . in some experiments , switched igg cells were enriched ( 6890% ) or depleted ( 1.56% ) by macs with combination of biotinylated anti - igg1 and anti  igg3 antibodies ( bd pharmingen ) and streptavidin microbeads ( miltenyi biotec ) .
mutation frequencies in each population were determined by sequencing 10,391 and 10,328 bp , respectively , as described in table i. induction of hypermutation in the s region upon csr stimulation dna was extracted from purified spleen b cells cultured with or without lps and il-4 .
the s region ( 1.1 kb ) composed of the 5 and 3 subregions ( 0.5 and 0.6 kb , respectively ) and vj558-jh4 downstream region ( 1.2 kb but 0.5 kb of the jh4 3 flanking sequence was examined ) were amplified , subcloned , and then sequenced .
the switch efficiency to igg1 and igg3 in the lps / il-4 cultured wt cells was 2956% .
statistic tests were done with sas version 2000 software ( sas institute inc . ) .
pcr were performed with the primers shown below using pyrobest dna polymerase ( takara ) that has the 3 exonuclease activity and high fidelity .
after purification , the pcr fragments were digested with ecori or spei and ligated into pbluescript vector .
the ligation mixture was used for transformation and the library was plated without preculturing to avoid amplification of sister clones .
no more than 21 clones were sequenced from a single pcr reaction for the s. clonality of the v region clones were checked by their cdr3 sequences .
nucleotide sequences were determined with abi prism 3100 genetic analyser ( perkinelmer ) . the s region germline sequence of cba and c57bl/6 were determined and compared .
6 and 5 bp polymorphic differences were found in the s and jh4 downstream regions of our interest , respectively , and excluded from mutations .
primers used for s pcr are : 5-ggaattcattccacacaaagactctggacc-3 ; 5-ggaattccagtccagtgtaggcagtaga-3 ( a and d in fig .
1 a , respectively ) with 30 cycles of 94c for 30 s , 62c for 30 s , 72c for 1 min .
primers used for sequencing in addition to common primers for pbluescript are : 5-ggaattcgtaaggagggacccaggctaag-3 ; 5-ggaattcttccagaatcccaggattgcc-3 ( b and c in fig .
the 3 subregions of nonswitched alleles were amplified by nested pcr as follows : the first step , 20 cycles of 98c for 10 s , 68c for 7 min , with the primer a and 3-agcccatgctagctcagcctcacataa-5 ( 3 of the s core ) ; the second step , 15 cycles of 98c for 10 s , 68c for 80 s , with the b and d primers . for vj558-jh4 downstream region pcr
, primers described ( 13 ) were used with 35 cycles of 98c for 10 s , 68c for 80 s. distribution of mutations in the s region .
( a ) a total of 58 point mutations and 6 deletions shown in table i ( closed and hatched symbols ) and table ii ( open symbols ) are mapped by triangles and rectangles , respectively , on the s region ( bar ) .
position from the transcription start site ( arrow ) is indicated by a scale below .
lengths ( bp ) of deletions ( a  f ) are : a , one ; b , 17 ; c , 30 ; d , 52 ; e , 7 ; f , 19 .
the s nucleotide sequence of the c57bl/6 mouse tail dna is shown by upper case letters .
sequences that are predicted to form s / l structures are underlined ( references 12 and 34 ) .
conditions used for the s / l prediction were : the ionic conditions ( mm ) , [ na ] = 150 , [ mg ] = 0.5 ; the folding temperature , 37c ; the maximum distance between paired bases , 25 .
recombinant retrovirus constructs ( pmx - aid - ires - gfp ) to express aid or aid , inactive mutant of aid , and preparation and infection of retroviruses were described before ( 14 ) .
we reasoned that csr target s regions might receive extensive sequence alterations like shm even without actual csr upon stimulation of b cells if shm and csr share a common mechanism for dna cleavage . to assess this possibility , we examined dna sequences of the s region in splenic b lymphocytes stimulated with lps and il-4 . to avoid pcr artifacts due to highly g rich repetitive sequences in the s core region ( 15 )
, we chose to analyze the upstream flanking region to the s core sequence ( hereafter called the s region ) , in which practically frequent csr can take place ( 16 , 17 ) .
we also analyzed such s regions that are located on nonswitched alleles , using nested pcr .
we found a significant number of mutations accumulating in the s region of spleen b cells stimulated with lps and il-4 but not of unstimulated b cells ( table i ) .
the mutations are located mainly in the 3 subregion of the s region ( fig .
the mutation frequency observed was 4.5  10/bp and the fraction of mutated clones reached 21.6% of sequenced clones .
these mutations in the s region are independent of csr because ( a ) the mutation frequency in s regions on nonswitched alleles is as high as that of total s ( table i ) , ( b ) the mutation frequency was not significantly changed between switched and unswitched b cells ( 4.8  10/bp and 2.9  10/bp , respectively ; p = 0.58 , fisher 's exact test ) , and ( c ) no csr junctions were included in the s region sequenced although frequent mutations are found in the proximity ( most often within 10 bp ) of csr junctions ( 18 , 19 ) . furthermore , no mutations were found in a non - ig gene , c - myc ( total 9,430 bp sequences of 26 clones ) , excluding the possibility of nonspecific genomewide hypermutation due to dna damage and repair .
most importantly , these mutations are not a part of shm in the v region because lps and il-4 stimulation could not induce hypermutation in vj558-jh4 downstream regions ( table i ) . the vj558 family is shown to constitute the major vh population in c57bl mice and vj558-jh4 downstream regions are known to accumulate mutations in in vivo activated b cells ( 13 ) .
the absence of shm induction by lps and il-4 stimulation in vitro is consistent with the previous reports ( 2022 ) .
a few heavily mutated clones exist in both before and after the stimulation in wt samples , which are likely due to memory cells .
direct comparison of the mutation rate in the s region to that in the v region is not straightforward because unlike shm this mutation frequency represents unselected clones in in vitro primary cultures .
nonetheless , the v region of a human b lymphoma cell line , ramos spontaneously accumulates 2.3  10/bp mutations during 2 wk ( 3 ) , the frequency of which is slightly higher than but comparable to the present data .
1 a ) , which is in agreement with previous reports that internal deletions of s ( s and s1 ) regions can occur upon csr induction ( 23 , 24 ) . these data indicate that csr stimulation of b cells induces wide spread cleavage in s dna , which can cause point mutations as well as deletions . to determine if the hypermutation in the s region also depends on aid , we analyzed spleen b cells from aid mice ( 10 ) in parallel with those of wt mice .
altogether the 109.5 kb sequences of the s region were mutation free in aid b cells ( table i ) .
we conclude that the recombination - uncoupled hypermutation in the s region is mediated by the function of aid . to exclude the possibility that an apparent induction of the hypermutation in the s region represents the outgrowth of the population already with mutations , most likely memory b cells , we transfected aid primary b cells with aid - expressing retroviruses . as aid b cells
have no background mutations , population changes in the cell culture would not affect the result .
splenic b cells that had been stimulated with lps and il-4 1 d before virus infection were harvested for analysis 5 d after infection .
29% of infected aid cells , which were distinguished by green fluorescent protein ( gfp ) coexpressed
bicistronically with aid , switched to igg whereas only a background level of igg cells was found in uninfected cells ( fig .
the switch efficiency of rescued aid cells is comparable to that ( 29.5% ) of wt cells without aid virus infection .
by contrast , a control virus carrying a deletion mutant aid ( aid ) did not rescue csr ability of aid cells .
purified spleen b cells from aid ( reference 10 ) and wt mice were stimulated with lps ( 25 g / ml ) and il-4 ( 75 /ml ) . on day 1 ,
the culture were split into three , and two of them were infected with either aid or aid expressing virus . on day 6 , cells were harvested and stained with anti - b220 , anti - igg1 , anti - igg3 ( bd pharmingen ) , and propidium iodide ( pi ) .
b220 and pi - negative cells were electronically gated and their igg and gfp expressions are shown .
we then analyzed the sequence of the s region in aid b cells infected with aid virus .
virus - uninfected cells were removed by enrichment of igg cells or gfp cells by cell sorting .
clearly , only aid - virus transfected cells mutated their s region while aid - virus infected cells did not ( table ii ) .
the mutation frequencies of igg and gfp cells were 1.2  10 and 1.3  10 per bp , respectively , which are almost the same as the frequency in wt cells infected with aid virus ( 1.2  10/bp ) .
almost all mutations are found in the 3 subregion in consistence with virus non - infected wt cells ( table i , fig .
, practically no mutations were observed in vj558-jh4 downstream regions in aid or wt b cells infected with aid virus ( table ii ) .
these results indicate that the mutations of the s region are introduced de novo upon csr stimulation without shm in the v region , and absolutely dependent on aid , implicating that both recombination and hypermutation in s regions may be catalyzed by a certain common reaction regulated by aid .
retrovirus - mediated aid transfection rescues the hypermutation phenotype in aid b cells cells were infected and selected as indicated .
the percentage of virus - infected cells within the selected populations was measured by expression of gfp coexpressed with aid .
data were obtained and presented as in table i. p = 0.031 ( fisher 's exact test ) .
if so , the s hypermutation may be biased to stem and loop ( s / l ) structures as shown for csr cleavage sites ( 12 , 18 , 25 ) . therefore , we mapped the hypermutation sites to the predicted s / l structures on both strands of the s region , and found significant bias of the mutation to the s / l structures ( table iii , fig . 1 b ) .
note that the majority of mutations coupled with csr are located in the proximity ( within 10 bp ) of the csr junctions but do not necessarily coincide with cleavage sites ( 18 ) .
this is because mutations are probably introduced during error prone dna synthesis to repair cleavages .
accordingly , the present level of the coincidence between s mutations and s / l structures well supports the assumption that both csr and s mutations may be mediated by an enzyme recognizing s / l structures .
mutation bias to rgyw / wrcy and s / l structure mutations in the 3 subregion were analyzed for association with sequence and structural motifs .
mutations which associate or do not associate with the rgyw / wrcy motif were counted as ( + ) and (  ) , respectively .
s / l structure was predicted by a computer program that is developed by zuker as described and shown in fig . 1 b. the two strands were analyzed separately for association with the s / l structure and the results are combined .
s region hypermutation shares important features with v region shm : aid dependency , high frequency point mutations with occasional deletions , and mutations biased to transition ( 64% ) and to the rgyw / wrcy motif ( r , purine ; y , pyrimidine ; w , a / t ; table iii ) ( 2 ) .
it is therefore reasonable to assume that either a similar mechanism activated by aid or aid itself is responsible for hypermutations in the s as well as v region .
it has been shown that the defect in the non - homologous end - joining ( nhej ) system affects csr but not shm ( 2629 ) , and that aid mice show no obvious abnormality in the nhej system ( 10 ) , indicating that csr and shm differ in the repair mechanism .
these results taken together suggest that a common step of csr and shm , most likely at dna cleavage rather than dna repair , may be regulated by aid .
it is well established that csr and shm are independent events in b cells ( 8 , 9 ) .
in fact , under the present conditions both csr and hypermutation in s regions occur concurrently without shm in the v region .
there must be another level of regulation to distinguish the targets of the aid - activated system .
as aid deficiency does not affect germline transcription and nhej repair , aid is likely to be involved in cleavage of the s region during csr ( 10 ) .
this conclusion is confirmed by the recent finding that accumulation of h2ax and nbs1 at double strand breakages in the igh locus during csr is dependent on aid ( 30 ) .
the present finding suggests that csr and shm are likely to be mediated by the same enzyme .
it is therefore less likely that aid edits separate pre - mrnas for csr and shm .
these results taken together argue for , but do not prove , the hypothesis that aid edits a precursor mrna to synthesize an endonuclease essential for generating dna cleavage in both shm and csr reactions ( 12 ) .
alternatively , aid itself may have dna attaching activity although we consider it less likely because ( a ) aid does not bind to double - stranded or single - stranded s sequences ( data not shown ) and ( b ) aid is not associated with the s core region by chromatin immunoprecipitation assay of csr - induced b cell chromatin ( data not shown ) .
we have further proposed that the endonuclease introduces nicks by recognizing secondary structures such as stem and s / l in s and v regions , which are formed transiently by transcription - promoted strand separation ( 12 ) .
nicking in the v region will be repaired by exonuclease and error - prone dna synthesis ( 3133 ) , followed by mismatch repair and ligation . by contrast ,
in s regions frequent nicking generates staggered cleavages ( 18 ) which are also repaired by exonuclease and error - prone dna synthesis , followed by the nhej repair system ( 2628 ) .
 somatic hypermutation ( shm ) and class switch recombination ( csr ) cause distinct genetic alterations at different regions of immunoglobulin genes in b lymphocytes : point mutations in variable regions and large deletions in s regions , respectively . yet 
 both depend on activation - induced deaminase ( aid ) , the function of which in the two reactions has been an enigma . here 
 we report that b cell stimulation which induces csr but not shm , leads to aid - dependent accumulation of shm - like point mutations in the switch  region , uncoupled with csr . 
 these findings strongly suggest that aid itself or a single molecule generated by rna editing function of aid may mediate a common step of shm and csr , which is likely to be involved in dna cleavage . 