HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Klotz, E. L. Articles by Storb, U. Search for Related Content PubMed PubMed Citation Articles by Klotz, E. L. Articles by Storb, U.

Somatic Hypermutation of an Artificial Test Substrate Within an Ig Transgene¹

Emily L. Klotz^2,*, John Hackett, Jr.^3, and Ursula Storb^4,*,

^* Committee on Immunology and Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have characterized a novel substrate for somatic hypermutation, confirming that non-Ig sequences can be targeted for mutation and demonstrating that this substrate allows for the rapid assay for mutations. An artificial sequence containing alternating EcoRV and PvuII sites (EPS) was inserted into the V167 transgene, which is known to be a target for mutation. To assay for somatic hypermutation, the EPS is amplified using flanking transgene primers, and the PCR product is subsequently digested with either EcoRV or PvuII. A mutation is seen as the appearance of a larger fragment, indicating a base change in a restriction enzyme site. The original transgene, V167/EPS, showed evidence of a low level of mutation in both splenic hybridomas and Peyer’s patch-derived or immunized splenic B220⁺ cells with high peanut agglutinin levels. Two derivative lines of V167/EPS were made, V167/POX and V167/PEPS. While none of the V167/POX transgenic lines demonstrated mutation, the V167/PEPS transgene was highly mutated in B220⁺ splenic B cells with high peanut agglutinin levels at a frequency similar to that of endogenous Ig genes. An analysis of splenic RNA from the unimmunized transgenic mice indicated that the levels of stable message in splenic B cells could not be correlated with the mutation seen in GC B cells. The mutable V167/PEPS transgenic line is a unique tool to study somatic hypermutation in vivo.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
While the phenomenon of somatic hypermutation was discovered by examining the Abs generated in response to Ags eliciting a specific heavy and/or light chain response, it has been the construction of Ig transgenes (1, 2) that has allowed for the dissection of the cis requirements for somatic hypermutation. These transgenes can be mutated either as part of the elicited immune response (1) or as passenger genes (3), eliminating the need for production of transgene-encoded Ig molecules. Typically, the assessment of mutation of Ig transgenes is determined by extensive sequencing. The ability of a passenger Ig transgene to undergo mutation suggests that a nonsequencing mutation assay could use a non-Ig gene as the substrate for somatic hypermutation.

The molecular requirements for somatic hypermutation include the Ig enhancers (4). The Ig enhancer requirement is not Ig locus specific, as an Ig enhancer from one Ig gene (i.e., the heavy chain intron enhancer) can target the mutation when used to drive a different Ig gene (i.e., light chain) (5). While a functional promoter is required, it need not be an Ig promoter, as the human ß-globin promoter (4) or the B29 promoter (6) will target the mutation to a transgene. Finally, the promoter seems to have the ability to direct the mutations, as the duplication of an Ig promoter upstream of the C region in a transgene results in mutation of the normally unmutated C region (7).

While it is clear that a functional promoter and Ig enhancer are required for mutation, a comprehensive understanding of the molecular requirements for optimal somatic hypermutation has not been achieved. For example, a chloramphenicol acetyltransferase (CAT)⁵ gene driven by the heavy chain promoter and intronic enhancer was mutated at a low level in hybridomas (8), suggesting that a promoter and a single Ig enhancer alone are sufficient to elicit the process of somatic hypermutation, but may not fulfill the requirements for a high level of mutation. Additionally, a TCRß transgene, driven by the TCRß promoter coupled to the heavy chain intronic enhancer, was mutated at a very low level within B cell hybridomas (9), presumably due to low activity of the TCRß promoter in B cells (10). Non-Ig sequence mutations have been further shown with transgenes in which the human ß-globin, bacterial neo, and bacterial gpt genes were within the context of the VJ region of a transgene and, thus, under the control of the enhancers (11). Combined, these studies suggest that non-Ig genes can be targets for somatic hypermutation, especially when the non-Ig sequences are placed within the context of Ig genes.

While recent advances in the development of in vitro culture systems for somatic hypermutation (12, 13) may allow for further dissection of the cis requirements of mutation, studies regarding the GC formation and antigenic requirements for somatic hypermutation require a system for the easy analysis of somatic hypermutation in vivo. Therefore, we sought to create a mouse carrying an Ig transgene that was capable of undergoing mutation and that contained a non-Ig sequence that could be rapidly assayed for somatic hypermutation.

The V167 transgene had been shown previously to be a target for somatic hypermutation (1, 7, 14, 15). We designed the transgenic construct V167/EPS, which contains an artificial substrate (EPS) within the variable region of a rearranged V167 transgene. The EPS artificial substrate is a 108-bp sequence containing alternating EcoRV and PvuII sites. The advantage of this substrate is that 76% of the 108 bp are contained within the enzyme sites and, thus, can be assayed with great ease without the need for DNA sequencing. We have used this transgene in a number of different transgenic lines to compare the levels of somatic mutation with the levels of transcripts from the different transgenes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
V167/EPS transgenic mice

The V167 transgene derived from the myeloma MOPC167 has been previously described (16). The plasmid containing the rearranged gene (pJRD/VC167) was digested with AgeI (New England Biolabs, Beverly, MA), and the overhang was filled in using Klenow (Boehringer Mannheim, Indianapolis, IN). The EcoRV and PvuII restriction fragment (EPS) was created by ligation of two 114-base oligonucleotides and cloned into the HindIII site in pKSA⁺ (Stratagene, La Jolla, CA) to create pRSa. The EPS fragment (104 bp) was removed from pRSa by digestion with HpaII (Boehringer Mannheim), isolated, and cloned into the filled-in AgeI site present in the V region of pJRD/VC167 to create pJRD/VC167/EPS (Fig. 1A). The EPS was designed with no 5' CTP-GTP 3' (CpGs) to prevent methylation of the construct. One CpG was added to the transgene when the AgeI overhang was filled in, thus duplicating a CpG already contained within the V region. Since the bases are an addition to the transgene, they are shown in Figure 1A as part of the EPS, but are underlined. Insertion of the EPS results in a stop codon; thus, no functional protein will be generated. The transgene (Fig. 1A) was isolated for microinjection from pJRD/VC167/EPS by a double digest with EcoRI (New England Biolabs) and Asp⁷¹⁸ (Boehringer Mannheim). Founder V167/EPS animals were determined by PCR amplification of DNA derived from ear punch samples using primers V8B (5'-GTTTCAGCTCCAGCTTG) and V9B (5'-CTCCTCAGCTCCTGATC) or V3B (5'-GTCAGTGGGGATATTGTGATAACC) and V4 (5'-ACGTCTAGAAGACCACGCTACCTG; Fig. 1B). Transgene status was confirmed by Southern blot analysis of tail DNA digested with BamHI and probed with a murine C probe. The C probe was generated by a random prime reaction using PstI (New England Biolabs)-cut pGEMC (16). CD-1 founder mice were bred to the C57BL/6 background. Two lines of mice were analyzed, 9532 containing two copies of the transgene, and 13 containing 10 to 20 copies of the transgene (high copy line).

View larger version (20K): [in this window] [in a new window]   FIGURE 1. Diagram of the V167/EPS transgene and primers. A, Diagram of the V167/EPS transgene and sequence of the EPS (not to scale). The EPS fragment was cloned into the AgeI site in the variable region of the V167 transgene. The transgene was prepared for microinjection by digesting the plasmid with Asp718 and EcoRI. The sequence of the EPS fragment is given below the transgene diagram. Underlined bases indicate those bases added to the transgene by filling in the AgeI site. Seventy-six bases are included in the EcoRV and PvuII sites. B, Diagram of the V167/EPS transgene from the leader to the 5' end of the J-C intron. Primers used for analysis of transgenic mice, cloning, and sequencing are diagrammed below the transgene. The italicized primers (Box1 and VOx6) are VOx-specific primers, but are included to indicate their locations within the gene. The Vmu+1 and Box1 primers have a 1-bp mismatch from the leader sequence and create a BglII site upon amplification. C, Line diagrams of the EcoRV and PvuII sites within the EPS. The line diagrams represent the EPS that contains alternating EcoRV and PvuII sites. The restriction enzyme sites are lettered from A to G for EcoRV and from A to F for PvuII. Complete digestion results in a ladder containing 10-, 12-, 14-, 16-, 18-, and 20-bp fragments for EcoRV and a ladder containing 11-, 13-, 15-, 17-, and 19-bp fragments for PvuII.

V167/PEPS transgenic mice

To create pJRD/VC167/PEPS, pJRD/VC167/EPS was digested with HpaI (New England Biolabs), Asp⁷¹⁸, and SpeI (New England Biolabs). The 800-bp HpaI/SpeI fragment was gel isolated and cloned into HincII/SpeI-cut pKSA⁺. The resulting plasmid was digested with Asp⁷¹⁸ and SpeI and ligated to pJRD/VC167/EPS digested with Asp⁷¹⁸ and SpeI, resulting in pJRD/VC167/PEPS. Thus, the region upstream of the promoter has been reduced from 4 to 0.6 kb in the V167/PEPS transgene (Fig. 2). The V167/PEPS transgene was isolated for microinjection from pJRD/VC167/PEPS by double digestion with Asp⁷¹⁸ and EcoRI. About 19 bases of pKSA⁺ polylinker remained (from the HincII site to the Asp⁷¹⁸ site).

View larger version (9K): [in this window] [in a new window]   FIGURE 2. Diagram of the V167/POX and V167/PEPS transgenes (not to scale). In the V167/POX transgene, the upstream, leader, and a portion of the L-V intron of V167/EPS have been replaced with those of the VOx transgene. The V167/PEPS transgene is identical the V167/EPS transgene, except that the region upstream of the promoter has been reduced from approximately 4 to 0.6 kb.

V167/POX transgenic mice

The V167/POX transgene (Fig. 2) is identical with V167/PEPS, except that the promoter has been switched with that of the VOx gene. Mel22, a plasmid containing the VOx transgene (gift of Dr. M. Neuberger), was digested with EcoRI and BamHI. The 5-kb EcoRI/BamHI fragment containing the upstream, V, and C regions of VOx was ligated to EcoRI/BamHI-digested pUC18. The resulting plasmid (Mel22 clone 6) was used as a template for PCR amplification using the Pfu polymerase and the primers 1233 (New England Biolabs) and VOx6. 1233 is a vector primer; VOx6 is an antisense primer in the leader V intron of VOx and creates a SpeI site for cloning (5'-GCAACTAGTGGACACAAATTCCCCA, the SpeI site is underlined). The PCR product was digested with EcoRI and SpeI, gel-isolated, and cloned into pKSA⁺ digested with SpeI and EcoRI. The sequence integrity of the PCR-generated insert was confirmed by automated sequencing (AmpliTaq FS, Perkin-Elmer, Norwalk, CT) with T3 and T7 primers. The plasmid was subsequently digested with Asp⁷¹⁸ and SpeI. The approximately 800-bp Asp⁷¹⁸-SpeI insert was ligated into pJRD/VC167/EPS digested with Asp⁷¹⁸ and SpeI, resulting in pJRD/VC167/POX. The V167/POX transgene was isolated by digestion with EcoRI. About 44 bases of polylinker remained (from the EcoRI site to the Asp⁷¹⁸ site of pKSA⁺).

Southern blot analysis and subsequent breeding of the V167/POX founder animals were performed as described above for the V167/PEPS mice. Three founder animals were generated, POX10, POX39, and POX55. From the Southern blot analysis of kidney DNA from the F₁ mice, the POX10 contains approximately nine copies and POX39 contains approximately four copies. The POX55 founder had two different transgene integrations, resulting in the POX55A and POX55B sublines with approximately four and seven copies, respectively. Sublines were determined by both transgene copy number quantitation and restriction enzyme analysis of the EPS. POX55B contains at least one copy of the V167/POX transgene with a mutation in the EPS (EcoRV, site D).

Immunization

Mice were immunized i.p. with 2 x 10⁸ SRBC (Cappel, West Chester, PA) in saline on days 0 and 21 and sacrificed on day 24 according to the method of Rogerson (17).

B220⁺ PNA^high B cell isolation

The isolation of B220⁺ PNA^high cells is a modification of the PP isolation protocol described by González-Fernández et al. (18). PP were isolated by dissection from the small intestine of an unimmunized mouse, or the spleen was removed from an SRBC-immunized mouse. The respective organs were disaggregated using a nylon cell strainer (Falcon Labware, Lincoln Park, NJ). For the splenic cell suspension, the RBCs were lysed, and the debris was removed from the remaining cells by a nylon strainer. For the PP cell suspension, cells were washed twice in DMEM with 10% FBS (HyClone, Logan, UT). Phycoerythrin-coupled anti-CD45R (clone RA3-6B2; Life Technologies, Gaithersburg, MD) and FITC-coupled PNA (Sigma, St. Louis, MO) were diluted and added to cells for a 20-min incubation on ice. After washing, B220⁺ PNA^high cells were isolated using a FACStar Plus (Becton Dickinson).

Generation of hybridomas from V167/EPS mice

V167/EPS transgenic mice were immunized i.p. with 2 x 10⁸ SRBC (Organon Teknika, West Chester, PA) in saline on days 0 and 7 and were sacrificed on day 14. For the phosphorylcholine (PC)-KLH immunizations, the mice were immunized i.p. with PC-KLH (100 µg; gift from Dr. J. Kenny, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD) on day 0 in CFA (Life Technologies). One month later mice were given a secondary immunization of PC-KLH (100 µg) in IFA (Life Technologies). Tertiary immunization was performed 1 mo later and consisted of PC-KLH (100 µg) in saline. Mice were sacrificed 6 days after the last immunization.

From the spleen, a single cell suspension was made, RBCs were removed by lysis in 0.17 M NH₄Cl, and remaining cells were washed in DMEM (Sigma). Spleen cells and Sp2/0 (gift from Dr. J. Miller, University of Chicago, Chicago) were fused at a 2:1 or a 1:1 ratio. The resulting fusions were selected and maintained in DMEM supplemented with 10% horse serum GG free (Life Technologies), 1x MEM nonessential amino acids (Life Technologies), 50 µg/ml streptomycin, 50 U/ml penicillin, 10 mM HEPES (Life Technologies), 1x NCTC-109, and 1x hypoxanthine-aminopterin-thymidine (Sigma). Resulting hybridomas were analyzed for IgG secretion by ELISA and for maintenance of the transgene by PCR.

ELISA for hybridoma IgG and IgM production

Hybridoma supernatants were diluted and added to microtiter plates coated with rabbit anti-mouse IgG (Fc specific) Ab (Jackson ImmunoResearch Laboratories, West Grove, PA), and bound Ab was detected by a peroxidase-labeled rabbit anti-mouse IgG/IgM (H+L) Ab (Jackson ImmunoResearch Laboratories).

Analysis of the EPS in hybridomas

Hybridoma DNA was amplified using primers V8B and V9B (25 cycles of 94°C for 20 s, 60°C for 30 s, and 72°C for 30 s) with Taq DNA polymerase. After 20 cycles of amplification, 2.5 µCi of [-³³P]dATP was added to the PCR reaction, and the remaining five cycles were completed. The PCR product was digested with either EcoRV (New England Biolabs) or PvuII (New England Biolabs). The digests were analyzed by running the samples on an 18% acrylamide/5% glycerol gel. The gel was then dried and exposed to x-ray film. Amplifications were repeated to confirm that the mutations detected were not due to Taq DNA polymerase errors.

Cloning and analysis of the EPS substrate in the V167/EPS, V167/PEPS, and V167/POX transgenes

The transgenes were cloned from DNA from hybridomas or B220⁺ PNA^high cells by amplification with Pfu DNA polymerase (Stratagene) using JRH2 (5'-GACCACGCTACCTGCAG) as the 3' primer. The 5' primer for the V167/EPS and V167/PEPS transgenics is Vmu+1 (5'-CCTGGGGGTGCTTATGTTCTAGATCTCTGG). The 5' primer for the V167/POX transgenics is Box1 (5'-GTGCAGATCTTCAGCTTCCTGC). The PCR products were digested with BglII (New England Biolabs). The 5' BglII sites (underlined) were introduced by a single base change from the transgenic sequence. The BglII fragment was cloned into the BamHI site of pUC18. Clones were screened for the presence of mutations within the EPS fragment by amplification with Taq DNA polymerase using EKVK1 (5'-ATTTGATGTCCACCCGTG) and EKVK2 (5'-TCAACTGATAATGAGCCCTC) or V8B and V9B for 30 cycles of 94°C for 15 s, 60°C for 20 s, and 72°C for 30 s. The PCR product was digested with either EcoRV or PvuII. The samples were run on an 18% acrylamide/5% glycerol gel, which was subsequently stained with ethidium bromide. Amplifications were repeated to confirm that the mutations detected were not due to Taq DNA polymerase errors.

Sequencing of the EPS and flanking transgenic DNA

Bacterial clones containing the EPS and flanking sequence were grown, and plasmid DNA was isolated using either the Wizard Plus Minipreps Kit (Promega, Madison, WI) or the QIAprep Spin Miniprep Kit (Qiagen, Chatsworth, CA). The cloned transgene was sequenced using Sequenase version 1.0 and the dGTP nucleotide kit, and combinations of the following primers (Fig. 1B) were used: 1233, 1224, 1212 (New England Biolabs), EKVK1, EKVK2, EPSAS1 (5'-ATACACACCCACATCCTCAGCC), V7 (5'-GAGTGAAGGCTGAGGATGTG), JRH1 (5'-GATGTAGATTCAGGTGC), seq4 (5'-CAGGAGCTGAGGAGATTGTC), and V10A (5'-CTGCAGGAGATGGAAAC).

For the analysis of the PEPS4 clones, a combination of sequencing and restriction enzyme digestion was used. The PEPS4 transgenics contain four copies of the V167/PEPS transgene. The initial sequencing was performed on the seven clones containing EPS mutations and 10 randomly chosen unmutated clones. Flanking mutations were present in three of the seven mutated clones. To insure that the mutations were not germline-encoded mutations, 24 clones needed to be analyzed to be 96% confident that each copy of the transgene had been analyzed at least twice (see Estimation of number of transgene copies to be analyzed). The four mutations found in the E7M clone were determined not to be germline encoded by sequence analysis of at least 25 clones. For the A2 and B4 2/13 mutations, the clones containing the transgene copies were amplified by V3B and V8B (30 cycles of 94°C for 15 s, 60°C for 20 s, and 72°C for 30 s) and digested. The A2 mutation results in a loss of a PstI site, and the B4 2/9 mutation results in the gain of a HaeIII site. Both sites were analyzed in 28 clones. As the mutations did not appear twice, they are the result of somatic hypermutation.

Estimation of the number of transgene copies to be analyzed

To be certain that the mutations observed were not simply an artifact of integration, we needed to be confident that we had sequenced at least two copies of each of the four V167/PEPS transgenes. Let s₁, ... , s₄ represent the number of sampled copies of transgenes one through four (_i⁴ = 1 S_i = n). Assuming that the pool of PCR products is very large, and that each of the transgenes is equally represented, then the probability of obtaining any particular combination s₁, ... , s₄ from a sample of n clones may be written as n!( )ⁿ/s₁! x ... x s₄!). Summing these probabilities over the set {s₁, ... , s₄ : s₁ 2, ... , s₄ 2} yields the probability of obtaining at least two copies of each transgene. Thus, we computed that a sample of 24 clones provided a 96% chance of sampling at least two copies of each of the four transgenes.

Calculation of mutation frequency for the EPS

Within the EPS, 76 bp are within restriction enzyme sites. The mutation frequency (mutations/base) for the hybridomas from the 9532 V167/EPS line was calculated as (number of hybridomas with a mutation in a restriction site)/(number of hybridomas x 2 copies of the transgene x 76). Mutation frequencies for the clones derived from the B220⁺ PNA^high sorted cells were calculated as (number of mutant restriction enzyme sites)/(number of clones x 76).

Splenic RNA preparation

Spleens were removed from unimmunized mice. The V167/POX and V167/PEPS mice were F₁ animals. The P5'C mouse carries a modified V167 transgene and has been described previously (7). Both the V167/EPS and P5'C mice have been crossed to C57BL/6 for multiple generations. Two C57BL/6 mice were included as controls. The spleens were Dounce homogenized in 5 ml of STAT-60 containing phenol and guanidinium thiocyanate (Tel-Test, Friendswood, TX). The RNA was extracted, precipitated, resuspended in diethylpyrocarbonate H₂O, and stored at -70°C. DNase treatment was performed using the MessageClean kit (GenHunter, Nashville, TN).

RPA template

To create a template for in vitro transcription for the antisense probe for the RPA, a portion of the V167/POX transgene present in all three transgenes was cloned into pKSA⁺. A 550-bp fragment was amplified from pJRD/VC167/POX with RPA1 (5'-CAACTGCAGTCTCAGACCGGAACA) and JRH2 using Pfu polymerase. The fragment was subsequently digested with PstI and BglII. The PstI site is created at the 5' end of the fragment by the RPA1 primer (the PstI site is underlined). The resulting product was gel isolated and cloned into pKS digested with BamHI and PstI. Positive clones were confirmed by both PCR and restriction digest. The resulting plasmid (V167/EPS RPA6) contains a 521-bp fragment of the transgene that, when transcribed with the T7 RNA polymerase, creates an antisense probe for the RPA. The 521-bp fragment contains (from 5' to 3'): V region (13 bp), the EPS (108 bp), V region (105 bp), J region (37 bp), and J-C intron (258 bp; see Fig. 5A). For in vitro transcription, the V167/EPS RPA6 plasmid was linearized with EcoRI.

View larger version (49K): [in this window] [in a new window]   FIGURE 5. RPA analysis of the V167/EPS, V167/POX, and V167/PEPS transgenics. A, Line diagrams of the V167/EPS RPA6 probe and the protected fragments. The full-length V167/EPS RPA6 probe is diagrammed with the regions of the transgene labeled. The 108-base EPS is flanked by V region segments, a 5' segment of 13 bases and a 3' segment of 105 bases. The J region is 37 bases, and the J-C intron segment is 258 bases. In the RPA analysis, the predicted protected fragment for the V167/EPS, V167/POX, and V167/PEPS transgenes is a 263-base fragment representing the EPS, the flanking V region segments, and the J region (VEVJ). The control V167 mouse (P5'C) does not contain the EPS, resulting in a protected fragment of 142 bases. B, A typical RPA analysis of the V167/EPS, V167/POX, and V167/PEPS transgenics. Splenic RNA from unimmunized mice was analyzed by RPA to quantitate transgenic message. Two probes were used: V167/EPS RPA6 (A) and pTRI RNA 18S. Using the V167/EPS RPA6 probe, the mature transgenic message results in a protected fragment (VEVJ) of 263 bases. This band is present in the V167/PEPS (PEPS4), V167/POX (POX10, POX39, POX55A), and V167/EPS (9532) mice (mouse lines are in parentheses). P5'C is a mouse from a different V167 line, one that does not contain the EPS. As a result, the protected transgene fragment is 142 bases. The B6 mice are control C57BL/6 mice. The undigested pTRI RNA 18S probe is 128 bases, and the protected fragment runs as a doublet at 80 bases. The marker in the last lane is an end-labeled 123-bp marker.

RPAs were performed with the following probes: V167/EPS RPA6 (described above) and pTRI RNA 18S (Ambion, Austin, TX), an 18S +RNA probe. Both probes were generated using the MAXIscript kit (Ambion) with T7 RNA polymerase and labeled [-³²P]CTP. The probes were incubated with approximately 2 µg of splenic RNA for 8 to 9 h at 42°C. The low sp. act. 18S probe was present in at least a fivefold molar excess. For the subsequent RNase reaction, the RNase (RNase A and RNase T1) was diluted 1/100. The RNase reaction was conducted for 30 min at 37°C. The protected fragments were analyzed on either 5 or 6% acrylamide denaturing gels (1.00 mm thick). The marker was a 123-bp marker (Life Technologies) end labeled with [-³²P]dCTP using T4 DNA polymerase (New England Biolabs). Gels were dried and exposed to the phosphorimager screen for 9 to 12 h. Using the ImageQuant version 1.11 software, both the protected V167/EPS RPA6 and 18S bands were quantitated. The 18S probe results in a protected doublet at 80 bases. For the 18S quantitation, values for both of the doublet bands were combined. To determine the level of transgene transcription relative to 18S for each mouse, the counts of the transgenic band were divided by those of the 18S bands.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
V167/EPS transgene and transgenic mice

Figure 1A contains the nucleotide sequence for the 108-base EPS and a diagram of its placement within the Ig transgene. Amplification with flanking primers and complete digestion with either EcoRV or PvuII yields a ladder of small DNA fragments as diagrammed in Figure 1C. Figure 3 is a typical digest of the PCR product. The EcoRV digestion results in fragments of 10, 12, 14, 16, 18, and 20 bp in length. The PvuII digestion yields fragments of 11, 13, 15, 17, and 19 bp in length. The smaller fragments (underlined) are not visible on the gel, probably due to melting of the dsDNA fragments. In addition to the ladder, digestion with either enzyme results in larger bands representing the flanking DNA. Loss of one of the restriction sites due to a point mutation will result in the loss of two smaller bands and the appearance of a larger band as seen in the lanes for clones 27 (PvuII mutation) and 43 (EcoRV mutation). Any of the 76 bases within the EPS are contained within restriction enzyme sites, and thus, substitutions of these bases are detectable by digestion. All the sites have been labeled by letter (Fig. 1C); thus, the EcoRV site mutated in clone 43 is site E, and the PvuII site mutated in clone 27 is site E.

View larger version (92K): [in this window] [in a new window]   FIGURE 3. An example of a digestion of PCR-amplified EPS. Plasmid clones of the V167/EPS transgene derived from PP B220+ PNAhigh cells were amplified by PCR using primers flanking the EPS and digested with either EcoRV or PvuII. Digestion with EcoRV results in a ladder of 20, 18, 16, 14, 12, and 10 bases, and digestion with PvuII results in a ladder of 19, 17, 15, 13, and 11 bases (underlined bases are not visible on gel). In addition to the ladder, digestion with either enzyme results in two larger DNA fragments representing the 5' and 3' flanking DNA. The 3' fragments run at the top of the gel. The 5' fragments are 45 bases (EcoRV) and 50 bases (PvuII) and run in the middle of the gel. Clone 43 has a mutation in an EcoRV site as evidenced by the larger band (34 bases) and the absence of the two lower bands. Clone 27 has a mutation in a PvuII site, resulting in a 36-base band and the absence of two lower bands). E, EcoRV; P, PvuII.

Analysis of the V167/EPS transgene in splenic hybridomas

To determine whether the non-Ig EPS sequence could be a target for somatic hypermutation, two different lines of mice carrying the V167/EPS transgene were immunized, and hybridomas were generated. IgG⁺ hybridomas were selected for analysis as they represented Ag-stimulated B cells. DNA from IgG⁺ hybridomas was amplified by PCR using primers flanking the EPS, and the PCR product was digested. Since the hybridomas contain multiple copies of the transgene, the presence of mutations was ascertained by the appearance of a larger band and not the loss of smaller bands. The results are summarized in Table I. Hybridomas from a mouse from the V167/EPS 13 (high copy) line were obtained after immunization with SRBC. Of the 21 IgG⁺ hybridomas, two contained mutated V167/EPS transgenes. One hybridoma (no. 77) contained a mutation in an EcoRV site (site C). The second hybridoma (no. 99) contained two mutations, each within an EcoRV site (sites C and F). By amplification and cloning of the transgene from the hybridomas, it was determined that the two mutations in hybridoma 99 are on independent copies of the transgene. While hybridomas 77 and 99 share the same mutated restriction enzyme site (EcoRV site C), the base changes that resulted in the loss of the EcoRV site are different. The substitution in hybridoma 77 is a G to a C; in hybridoma 99 the change is from an A to a T (GATATC; underlined bases are mutated).

View this table: [in this window] [in a new window]   Table I. EPS mutations in V167/EPS splenic hybridomas

Analysis of the V167/EPS transgene in PP-derived B220⁺ PNA^high cells

Previous work with transgenic mice has shown that B220⁺ PNA^high cells isolated from the PP contain a highly mutated population of B cells (18). To assess whether mutations in the V167/EPS transgene can be assayed easily in a pool of B cells without the need for hybridoma production, B220⁺ PNA^high cells were isolated from the PP of a two-copy V167/EPS transgenic (line 9532) mouse. The transgene was amplified and cloned from the B220⁺ PNA^high and B220⁺ PNA^low cell populations and analyzed by restriction enzyme digestion. Of 64 clones analyzed, two carried mutations within the EPS, resulting in a mutation frequency of 0.411 x 10^-3 mutations/base. Figure 3 is a gel of the digests of the two mutated clones and five unmutated clones. The site E EcoRV mutation in clone 43 results in the appearance of a 34-bp fragment, and the site E PvuII mutation in clone 27 results in a 36-bp fragment. These two mutations represent somatic hypermutation and not germline mutations as determined by statistical analysis (see Materials and Methods). As a control, 37 clones from B220⁺ PNA^low cells, representing nonmutated B cells were analyzed, and no mutations were found. As an additional control for PCR-induced error, kidney DNA was amplified and cloned. Of the 59 clones obtained, no mutations were detected.

To determine mutations in transgene sequences outside the EPS, the leader V intron was sequenced. Of the 64 B220⁺ PNA^high-derived clones described above, 35 were sequenced. The two clones with EPS mutations were not mutated in the leader V intron. Four clones without EPS mutations had mutations in the intron. Three had one mutation, and one had two. From the sequence data, the resulting mutation frequency is approximately 0.76 x 10^-3 mutations/base and is similar to the mutation frequency of 0.411 x 10^-3 mutations/base calculated by the EPS analysis of the clones.

V167/POX and V167/PEPS transgenic mice

While the V167/EPS transgene seemed to be capable of undergoing somatic hypermutation, the mutation frequency was not statistically significantly higher than the published error frequency of Pfu (19), the high fidelity polymerase used to amplify the EPS and flanking transgene. As a result, we designed two new transgenic constructs in an effort to make the V167/EPS a better target for somatic hypermutation.

Since there is evidence that the promoter targets mutation within an Ig gene (7), the V167/EPS transgene may mutate at a low frequency due to poor transcription in the mutating GC B cells, the centroblasts. The low transcription could be the result of a "weak" promoter, since the V167 contains a nonconsensus TATA box (ACAAAA). The VOx promoter has been able to target mutation to a number of transgenes (4, 11, 18, 20) and contains a TATA box (TTTAAA) that is closer to the consensus sequence. Thus, for the V167/POX transgene (Fig. 2), the promoter of V167/EPS was replaced with the promoter of the VOx transgene. The V167/POX transgene contains the upstream, promoter, and leader regions of VOx and part of the VOx leader V intron. As a result, the V167/POX transgene has a shorter upstream region than that of the original V167/EPS transgene, since the region upstream of the TATA box was shortened from 4 kb to approximately 600 bp. Previous studies have shown that this upstream sequence is not critical for optimal somatic hypermutation (4). However, to control for any influence this shortening may have on the V167/POX transgene, we created a second derivative construct, V167/PEPS, in which the upstream region of V167/EPS has been shortened to match V167/POX. The lines of V167/POX and V167/PEPS mice are listed in Table II.

View this table: [in this window] [in a new window]   Table II. Summary of EPS mutations and frequencies in splenic B220+ PNAhigh cells from SRBC-immunized V167/PEPS and V167/POX

Analysis of the V167/POX and V167/PEPS transgenes in SRBC-immunized mice

Representative F₁ mice from the V167/POX and V167/PEPS lines were immunized i.p. with SRBCs, and transgenic DNA was amplified from B220⁺ PNA^high splenic cells. As a control, the V167/EPS line described above (line 9532) was also immunized and analyzed for mutation. Table II summarizes the analysis of the transgenic mice. The V167/EPS line demonstrated the same mutation frequency as that in the PP-derived cells, 0.411 x 10^-3 mutations/base. In contrast, none of the four V167/POX lines demonstrated mutation in the analysis of a similar number of clones.

Analysis of the V167/PEPS transgene in the PEPS4 line revealed mutations within the EPS. Of 119 clones, seven were mutated that contained a total of 12 mutant restriction enzyme sites. Based on this analysis, the mutation frequency is 1.33 x 10^-3 mutations/base. As discussed below, this might be an underestimation of the mutation frequency, as a single enzyme site may carry multiple mutations. Two mutant clones contained three mutated restriction sites each, one contained two sites, and four contained one site each. Figure 4 is a gel of the PCR and subsequent digestion of the seven mutated and two unmutated clones. The mutations in the EcoRV and PvuII sites are listed in Table III. The actual base changes were determined by sequencing. Sequencing revealed that the mutant EcoRV site G in E7M contains two mutations, either of which would result in the loss of cutting at that EcoRV site (Table III). Thus, the number of mutations in the EPS based on the loss of cutting is an underestimate, as there are 13, not 12, mutations within the restriction enzyme sites.

View larger version (75K): [in this window] [in a new window]   FIGURE 4. Digestion of PCR-amplified EPS from PEPS4 clones. Plasmid clones of the V167/PEPS transgene from splenic B220+ PNAhigh cells were amplified by PCR using primers flanking the EPS (V9B and V8B) and digested with either EcoRV or PvuII (see Fig. 3 for description of ladder). With the V8B and V9B primers, the 5' fragments are 63 bases (EcoRV) and 68 bases (PvuII), and the 3' fragments are 150 bases (EcoRV) and 160 bases (PvuII). Clones A1 and H7 are unmutated. Clones B4 2/9, B4 2/13, C4, E7M, F5M, and G4 are all mutated (see Table III for a list of the mutations). Clones E7M and F5M each contain two inserts. One insert is mutated; the other is not. As a result, all the lower ladder bands are present, but some bands are less intense. For example, one of the two inserts in clone E7M contains a mutation in a PvuII site (site E). Thus, a 36-bp band is present due to the mutated insert. However, there is not a disappearance of the 17- and 19-bp ladder bands but only a reduction in their intensity. E, EcoRV; P, PvuII.

View this table: [in this window] [in a new window]   Table III. EPS mutations in V167/PEPS4 clones

Sequencing was conducted on the seven mutant clones and the 10 unmutated clones. The region sequenced encompasses an approximately 700-bp region from within the L-V intron to the first 30 bases of the J-C intron. As detected by sequencing, none of the clones without EPS mutations contained flanking mutations. Of the seven EPS-mutated clones, three carried additional mutations. Clone A2 had one mutation, a G to A change in FWR1 (Cys to Tyr in codon 23). Clone B4 2/13 had one mutation, a A to C in FWR3 (Asp to Ala in codon 60). E7 M contained four additional mutations. One mutation was a T to an A in codon 1, resulting in a stop codon. The second mutation is a silent mutation in codon 47 in FWR2 (A to G). The last two mutations were C to T mutations in the J-C intron.

Thus, the EPS sequence in the PEPS4 mouse clearly is a very good substrate for mutation in germinal center B cells. As a control, we have analyzed mutations in B cells from an unimmunized PEPS4 mouse. In 180 DNA clones, not a single one had a mutation in either an EcoRV or a PvuII site. The mutations found in PNA^high cells from immunized mice must therefore be due to the somatic hypermutation process.

Transgene expression

Given the role of the promoter in targeting mutation (7), we wanted to assess the levels of transgenic transcription in the original V167/EPS mice (line 9532) and the lines containing the two derivative transgenes to determine whether the frequency of mutation could be linked to the level of transcription. We analyzed the transgene expression in total RNA from the spleens of unimmunized animals by RPA. The V167/EPS RPA6 probe is diagrammed in Figure 5A. The control probe was an 18S ribosomal RNA probe that in its undigested form runs at 128 bases and in its protected form runs at 80 bases as a doublet. Figure 5B shows a typical RPA. The protected fragment for the V167/EPS, V167/POX, and V167/PEPS mice runs at 263 bases. The P5'C line contains a modified V167 that has been shown previously to be targeted for mutation (7) and was included as a point of comparison. P5'C does not contain the EPS, but does include the downstream V and J transgene segments. As a result, the protected fragment runs at 142 bases.

The two large endogenous bands present (Fig. 5B) in all the lanes and flanking the 263-base transgenic band are somewhat puzzling. The upper of these two bands could be a protected fragment representing the endogenous J region and the intron segment (295b), as the probe was designed to contain intron sequence. This suggests that the RNA may be contaminated with DNA. However, both doublet bands were present after DNase treatment of the RNA samples. Therefore, these bands may represent prespliced message or remaining DNA contamination after DNase treatment. We do not know what the two bands at about 220 and 110 bases in the RPAs with the POX mice represent.

While it remains possible that there is some DNA contamination, its presence does not affect the quantitation of transgenic message, as the quantitated bands do not include intron sequence. The transgenic message bands at 263 bases (or 142 bases for P5'C) were compared with the 18S doublet at 80 bases. Table IV summarizes the relative transcription values for the experiment shown in Figure 5B and for an average of three experiments. All the mice express the transgenes to some level. In this analysis, there seems to be no obvious link between the level of transgene expression and the frequency of somatic hypermutation. The V167/PEPS4 and V167/POX10 lines have similar expression levels (Table IV), but the EPS in the V167/PEPS4 line is mutated, whereas the EPS in the V167/POX10 line is not (Table III). Additionally, the transgene in the V167/EPS 9532 line is mutated (0.41 x 10^-3 mutations/base) at a similar frequency as the transgene in the P5'C line (0.39 x 10^-3 mutations/base) (7), but the V167 transgene in the P5'C line is expressed at levels six- to eightfold higher than the V167/EPS transgene (Table IV).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The EPS sequence has been designed to prevent DNA methylation. It contains no 5'CpG3' dinucleotides. In previous V167 transgenes, into which we had inserted bacterial sequences, transcription and mutation was abolished (J. Hackett, Jr., E. L. Klotz, and U. Storb, unpublished observations). The inserted ß-galactosidase or supF transfer RNA sequences from Escherichia coli as well as the flanking mouse Ig sequences were highly methylated in the B lymphocytes of many different transgenic lines. The fact that the same transgene with the extraneous EPS insert is transcribed and can be mutated supports the idea that transcription is required for somatic mutation and suggests that hypermethylation interferes with somatic mutation.

The V167/PEPS transgene in the PEPS4 line was mutated in B220⁺ PNA^high cells isolated from immunized spleen. As determined by the EPS mutations, the mutation frequency is 1.33 x 10^-3 mutations/base. The mutation frequency is similar to that seen for both endogenous genes (17, 24) and other transgenes (4, 18). Additionally, the types of mutations seen are characteristic of somatic hypermutation, as most are single base changes, and transitions are favored over transversions. Sequencing revealed that, in addition to the mutations within the EPS, mutations in the flanking transgene were present.

Previously, a major hot spot for somatic hypermutation has been described as the AGY serine codon (20, 25). This sequence seems to remain a hot spot even when it is out of frame in non-Ig sequences (11). The AGY triplet (Y = C or T) is contained within the PvuII sites (CAGCTG) in the EPS. Since the EPS contains a number of potential hot spots, the prediction was that the EPS would be highly mutated, and the majority of the mutations would be in the PvuII sites. However, all the mutations found within the hybridomas were within EcoRV sites. This suggests that somatic hypermutation can occur in a sequence without necessarily targeting every hot spot. The B220⁺ PNA^high sorted cell clones from the V167/EPS and V167/PEPS mice contained equal numbers of both EcoRV and PvuII mutations. Interestingly, the EPS seems to be more highly mutated than the flanking transgene DNA. Further analysis of the PEPS4 mice has shown that this preference is typical of a large number of transgene sequences and may offer further insights into the mechanism of somatic hypermutation.⁶

Our initial analysis of the V167/EPS transgenics suggested that some somatic hypermutation of the transgene was occurring, but the frequency was too low to make these mice useful as a tool for the study of mutation. We were concerned that the low frequency of mutation was linked to the quality of the V167 promoter, as we previously suggested a model of somatic hypermutation based on a transcription-mediated mechanism (7). The V167 TATA box differs from the consensus (TATAAA) in the first three bases (ACAAAA). In an in vitro transcription study with both human and yeast TFIID, Wobbe and Struhl made single base substitutions in the TATA box and examined the resulting transcription. As single base substitutions, all of the first three bases decreased transcription to between 1 and 30% of the transcription of a TATAAA template (26). Thus, the low mutation frequency observed in the V167/EPS transgenic mice could be due to poor transcription of the transgene. Ideally, the analysis of transgene transcription should be performed using mutating centroblasts, as the transcription of Ig genes in these cells may be different from that in other B cell populations. Unfortunately, currently there are no good markers for the isolation of murine centroblasts. As an alternative, we examined stable transgenic message in unimmunized spleens. All the transgenes are expressed, suggesting that the nonconsensus V167 TATA box is sufficient for transcription in unactivated B cells.

Despite the caveat of the potential differences in Ig gene transcription between centroblasts and unimmunized splenic B cells, the question remains as to why the V167/EPS and V167/PEPS transgenes mutate, whereas the V167/POX transgenes do not. Two explanations could account for the lack of mutation in the V167/POX transgenes. The first explanation is that while transcription drives somatic hypermutation (7), the level of transcription may not correlate with the frequency of somatic hypermutation. In this study we have shown that the level of stable transcripts as a measure of transcription of an Ig transgene in unimmunized splenic cells does not correlate with the frequency of mutation in GC B cells upon immunization. These data suggest that perhaps there is no direct relationship between the rate of transcription and the frequency of somatic hypermutation. However, a more definitive answer to the question of the influence of transcription on mutation awaits the analysis of murine centroblasts.

The second possibility to explain the lack of mutation in the expressed V167/POX transgenes is that the transgenes may be influenced by position-effect variegation (PEV) due to the site of chromosomal integration. PEV has been an explanation of the inhibition of gene expression in yeast and Drosophila (reviewed in 27 . Additionally, PEV has been seen in transgenic mice carrying a ß-galactosidase gene under the control of the regulatory element of -globin. The ß-galactosidase transgene expression in multiple lines of mice was heterocellular, as some RBCs expressed the transgene and others did not (28).

PEV could have two possible effects in the V167 transgenics. The V167 transgene expression could be heterocellular in the B cells, resulting in transgenic mouse lines with different percentages of centroblasts that express the transgene. As judged by FACS analysis, heterocellularity within a single mouse has been seen with other transgenes (5). Secondly, it is possible that position influences not transcription but other molecular requirements for somatic hypermutation. Thus, while the transcription levels of some of the EPS transgenics may be similar, the different transgene integration sites may allow unequal access to the postulated mutator factor (see Footnote 6).

Curiously, the V167 transgene with the shortened upstream region (PEPS) showed many more mutations than the same transgene with a 4-kb upstream region. While this may be a fortuitous coincidence, there were two transgenes with the long upstream region and low mutation (this paper) and two with the short upstream region (PEPS4 and another one; our unpublished observations) that had high and intermediate levels of somatic mutation. A similar observation was made with another set of transgenes in which those with a short upstream region also had higher mutation frequencies (29). These findings may suggest that there is an inhibitory region to transcription (the levels of transcripts were lower in 9532 than in PEPS4; Table IV) or hypermutation in this upstream region.

The EPS transgene has already been a useful tool in the identification of cis-acting sequences involved in somatic mutation (see Footnote 6). Because of its great ease of analysis, one can easily study 50 or more gene copies per day. It will also be helpful in determining trans-acting factors that influence somatic mutation and in studying alterations in somatic mutation in various immunologic states, including the roles of GC formation and antigenic stimulation. These questions require an analysis of somatic hypermutation in vivo, where the V167/PEPS transgene will be a reliable target that undergoes mutation at a frequency similar to that seen for endogenous Ig genes.

	Acknowledgments

 
We thank James Kenny (National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD) for the gift of PC-KLH, to Michael S. Neuberger (Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K.) for Mel22, and to Jim Miller (University of Chicago, Chicago, IL) for the gift of the Sp2/0 cell line. We are indebted to Phil Schumm for the statistical analysis, and Karen Kage for her assistance with DNA sequencing. We thank Grazyna Bozek and Darryl Stern for help maintaining the mouse lines, and Brad Kurtz, Andrew Peters, and Richard Hodes for critical reading of the manuscript. We are grateful to Julie Auger for her excellent technical assistance with flow cytometry.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant GM38649 and National Institutes of Health Training Grants GM07183 and AI07090 (to E.K.). The FACStar Plus is maintained by the University of Chicago Cancer Research Center Flow Cytometry Core Facility supported by National Institutes of Health Grant P30CA14599. J.H. was supported by postdoctoral and special fellowships from the Leukemia Society of America.

² Current address: Experimental Immunology Branch, National Cancer Institute, Building 10, Room 4B10, 10 Center Dr., MSC 1360, Bethesda, MD 20892-1360.

³ Current address: Department 90D, Abbott Laboratories, 1401 N. Sheridan Rd., North Chicago, IL 60064.

⁴ Address correspondence and reprint requests to Dr. Ursula Storb, Department of Molecular Genetics and Cell Biology, Cummings Life Science Center, 920 E. 58th St., Chicago, IL 60637.

⁵ Abbreviations used in this paper: CAT, chloramphenicol acetyltransferase; EPS, EcoRV-PvuII sequence; CpGs, CTP-GTP 3', PNA, peanut agglutinin; PP, Peyer’s patches; PC, phosphorylcholine; KLH, keyhole limpet hemocyanin; RPA, ribonuclease protection analysis; PEV, position-effect variegation.

⁶ U. Storb, E. Klotz, J. Hackett, K. Kage, G. Bozek, T. E. Martin. 1998. A hypermutable insert in an immunoglobulin transgene contains hotspots of somatic mutation and sequences predicting highly stable structures in the RNA transcript. J. Exp. Med., In press.

Received for publication January 13, 1998. Accepted for publication March 16, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. O’Brien, R. L., R. L. Brinster, U. Storb. 1987. Somatic hypermutation of an immunoglobulin transgene in transgenic mice. Nature 32:405.
2. Sharpe, M. J., M. Neuberger, R. Pannell, M. A. Surani, C. Milstein. 1990. Lack of somatic mutation in a light chain transgene. Eur. J. Immunol. 20:1379.[Medline]
3. Sharpe, M. J., C. Milstein, J. M. Jarvis, M. S. Neuberger. 1991. Somatic hypermutation of immunoglobulin may depend on sequences 3' of C and occurs on passenger transgenes. EMBO J. 10:2139.[Medline]
4. Betz, A. G., C. Milstein, A. González-Fernández, R. Pannell, T. Larson, M. S. Neuberger. 1994. Elements regulating somatic hypermutation of an immunoglobulin gene: critical role of the intron enhancer/matrix attachment region. Cell 77:239.[Medline]
5. Klotz, E. L., U. Storb. 1996. Somatic hypermutation of a 2 transgene under the control of the enhancer or the heavy chain intron enhancer. J. Immunol. 157:4458.[Abstract]
6. Tumas-Brundage, K., T. Manser. 1997. The transcriptional promoter regulates hypermutation of the antibody heavy chain locus. J. Exp. Med. 185:239.[Abstract/Free Full Text]
7. Peters, A., U. Storb. 1996. Somatic hypermutation of immunoglobulin genes is linked to transcription initiation. Immunity 4:57.[Medline]
8. Azuma, T., N. Motoyama, L. E. Fields, D. Y. Loh. 1993. Mutations of the chloramphenicol acetyl transferase transgene driven by the immunoglobulin promoter and intron enhancer. Int. Immunol. 5:121.[Abstract/Free Full Text]
9. Jr Hackett, J., C. Stebbins, B. Rogerson, M. M. Davis, U. Storb. 1992. Analysis of a T cell receptor gene as a target of the somatic hypermutation mechanism. J. Exp. Med. 176:225.[Abstract/Free Full Text]
10. Storb, U., A. Peters, E. Klotz, B. Rogerson, Jr J. Hackett. 1996. The mechanism of somatic hypermutation studied with transgenic and transfected target genes. Semin. Immunol. 8:131.[Medline]
11. Yélamos, J., N. Klix, B. Goyenechea, F. Lozano, Y. L. Chui, A. González Fernández, R. Pannell, M. S. Neuberger, C. Milstein. 1995. Targeting of non-Ig sequences in place of the V segment by somatic hypermutation. Nature 376:225.[Medline]
12. Källberg, E., S. Jainandunsing, D. Gray, T. Leanderson. 1996. Somatic hypermutation of immunoglobulin V genes in vitro. Science 271:1285.[Abstract]
13. Denépoux, S., D. Razanajaona, D. Blanchard, G. Meffre, J. D. Capra, J. Banchereau, S. Lebecque. 1997. Induction of somatic hypermutation of a human B cell line in vitro. Immunity 6:35.[Medline]
14. Jr Hackett, J., B. J. Rogerson, R. L. O’Brien, U. Storb. 1990. Analysis of somatic mutations in transgenes. J. Exp. Med. 172:131.[Abstract/Free Full Text]
15. Rogerson, B., Jr J. Hackett, A. Peters, D. Haasch, U. Storb. 1991. Mutation pattern of immunoglobulin transgenes is compatible with a model of somatic hypermutation in which targeting of the mutator is linked to the direction of DNA replication. EMBO J. 10:4331.[Medline]
16. Storb, U., C. Pinkert, B. Arp, P. Engler, K. Gollahon, J. Manz, W. Brady, R. L. Brinster. 1986. Transgenic mice with µ and genes encoding antiphoshorylcholine antibodies. J. Exp. Med. 164:627.[Abstract/Free Full Text]
17. Rogerson, B. J.. 1995. Somatic hypermutation of VHS107 genes is not associated with gene conversion among family members. Int. Immunol. 7:1225.[Abstract/Free Full Text]
18. González-Fernández, A., C. Milstein. 1993. Analysis of somatic hypermutation in mouse Peyer’s patches using immunoglobulin light-chain transgenes. Proc. Natl. Acad. Sci. USA 90:9862.[Abstract/Free Full Text]
19. Lundberg, K. S., D. D. Shoemaker, M. W. W. Adams, J. M. Short, J. A. Sorge, E. J. Mathur. 1991. High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108:1.[Medline]
20. Betz, A. G., C. Rada, R. Pannell, C. Milstein, M. S. Neuberger. 1993. Passenger transgenes reveal intrinsic specificity for the antibody hypermutation mechanism: clustering, polarity, and specific hot spots. Proc. Natl. Acad. Sci. USA 90:2385.[Abstract/Free Full Text]
21. Jacob, J., G. Kelsoe, K. Rajewsky, U. Weiss. 1991. Intraclonal generation of antibody mutants in germinal centres. Nature 354:389.[Medline]
22. Jacob, J., G. Kelsoe. 1992. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. II. A common clonal origin for periarteriolar lymphoid sheath-associated foci and germinal centers. J. Exp. Med. 176:679.[Abstract/Free Full Text]
23. Siekevitz, M., C. Kocks, K. Rajewsky, R. Dildrop. 1987. Analysis of somatic mutation and class switching in naive and memory B cells generating adoptive primary and secondary responses. Cell 48:757.[Medline]
24. Weiss, U., R. Zoebelein, K. Rajewsky. 1992. Accumulation of somatic mutants in the B cell compartment after primary immunization with a T-cell dependent antigen. Eur. J. Immunol. 22:511.[Medline]
25. Betz, A. G., M. S. Neuberger, C. Milstein. 1993. Discriminating intrinsic and antigen-selected mutational hotspots in immunoglobulin V genes. Immunol. Today 14:405.[Medline]
26. Wobbe, C. R., K. Struhl. 1990. Yeast and human TATA-binding proteins have nearly identical DNA sequence requirements for transcription in vitro. Mol. Cell. Biol. 10:3859.[Abstract/Free Full Text]
27. Karpen, G. H.. 1994. Position-effect variegation and the new biology of heterochromatin. Curr. Opin. Gen. Dev. 4:281.[Medline]
28. Robertson, G., D. Garrick, W. Wu, M. Kearns, D. Martin, E. Whitelaw. 1995. Position-dependent variegation of globin transgene expression in mice. Proc. Natl. Acad. Sci. USA 92:5371.[Abstract/Free Full Text]
29. Wu, P., L. Claflin. 1996. 5' cis-elements and V-gene hypermutation. FASEB Abstr. 10:978.

This article has been cited by other articles:

				S. Unniraman and D. G. Schatz Strand-Biased Spreading of Mutations During Somatic Hypermutation Science, August 31, 2007; 317(5842): 1227 - 1230. [Abstract] [Full Text] [PDF]

				N. Kim, G. Bozek, J. C. Lo, and U. Storb Different Mismatch Repair Deficiencies All Have the Same Effects on Somatic Hypermutation: Intact Primary Mechanism Accompanied by Secondary Modifications J. Exp. Med., July 1, 1999; 190(1): 21 - 30. [Abstract] [Full Text] [PDF]

				G. S. Shapiro, K. Aviszus, D. Ikle, and L. J. Wysocki Predicting Regional Mutability in Antibody V Genes Based Solely on Di- and Trinucleotide Sequence Composition J. Immunol., July 1, 1999; 163(1): 259 - 268. [Abstract] [Full Text] [PDF]

				U. STORB, A. PETERS, N. KIM, H.M. SHEN, G. BOZEK, N. MICHAEL, J. HACKETT, E. KLOTZ, J.D. REYNOLDS, L.A. LOEB, et al. Molecular Aspects of Somatic Hypermutation of Immunoglobulin Genes Cold Spring Harb Symp Quant Biol, January 1, 1999; 64(0): 227 - 234. [Abstract] [PDF]

				U. Storb, E. L. Klotz, J. Hackett Jr., K. Kage, G. Bozek, and T. E. Martin A Hypermutable Insert in an Immunoglobulin Transgene Contains Hotspots of Somatic Mutation and Sequences Predicting Highly Stable Structures in the RNA Transcript J. Exp. Med., August 17, 1998; 188(4): 689 - 698. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Klotz, E. L. Articles by Storb, U. Search for Related Content PubMed PubMed Citation Articles by Klotz, E. L. Articles by Storb, U.