|
|
|
|
|
|
|
||
Articles |
Locus Is Similarly Well Expressed in Mice and Humans
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
light (L) chain locus in germline configuration were created. The introduced translocus on a yeast artificial chromosome (YAC) accommodates the most proximal Ig variable region (V) gene cluster, including 15 V genes that contribute to >60% of L chains in humans, all J-C segments, and the 3' enhancer. HuIgYAC mice were bred with animals in which mouse Ig
production was silenced by gene targeting. In the –/– background, human Ig was expressed by 
84% of splenic B cells. A striking result was that human Ig was also produced at high levels in mice with normal locus. Analysis of bone marrow cells showed that human Ig and mouse Ig were expressed at similar levels throughout B cell development, suggesting that the Ig translocus and the endogenous locus rearrange independently and with equal efficiency at the same developmental stage. This is further supported by the finding that in hybridomas expressing human Ig the endogenous L chain loci were in germline configuration. The presence of somatic hypermutation in the human V genes indicated that the Ig-expressing cells function normally. The finding that human genes can be utilized with similar efficiency in mice and humans implies that L chain expression is critically dependent on the configuration of the locus.
Key Words: human Ig translocus • light chain expression levels • pre-B cell activation • V gene usage • hypermutation
Abbreviations used: ES, embryonic stem; Hu, human; N, nonencoded; P, palindromic; PFGE, pulsed-field gel electrophoresis; PNA, peanut agglutinin; PP, Peyer's patch; RACE, rapid amplification of cDNA ends; RSS, recombination signal sequence(s); RT, reverse transcriptase; TdT, terminal deoxyribonucleotide transferase; YAC, yeast artificial chromosome.
The light chain component of the Ig protein is encoded by two separate loci, Ig
L chain V-J rearrangement occurs at the transition from pre-B-II to immature B cells, where the surrogate L chain associated with membrane Igµ is replaced by
Here we have introduced a 410-kb yeast artificial chromosome (YAC), which contains most of the V
Southern Blot Analysis.
Hybridoma Production and ELISA Assay.
Flow Cytometry Analysis. and Ig. The proportion of antibodies containing or L chains varies considerably between different species (1–3); in mice the / ratio is 95:5, compared with 60:40 in humans. Two models exist to account for the dominance of Ig expression in the mouse. From the observations that murine Ig-producing myelomas have rearranged L chain genes, whereas Ig-producing cells have the L chain locus in germline configuration, it was proposed initially that rearrangement must occur before rearrangement can begin (4, 5). Although the same observation applies for human B cells, the proportions of - and -producing cells are similar (4), suggesting that other factors are involved. The second proposal is that and loci are equally available for rearrangement at the same time, but the mouse locus is more efficient at engaging the rearrangement process (for review see reference 6). The occasional finding of cells with rearranged and the locus in germline configuration may support this (5, 7, 8). Any influence of antigen selection on the biased / ratio is discounted by the finding that the ratio is similar in fetal liver and in cells that have not encountered antigen (9–13). or (14). Although the timing of L chain rearrangement is essentially defined, the processes that activate L chain locus rearrangement are not fully understood. From locus-silencing experiments, it is apparent that rearrangement is not a prerequisite for recombination (15), but instead that and rearrangements are independent events (16), the activation of which may be affected by differences in the strength of the respective enhancers. Targeted deletion of the 3' enhancer in transgenic mice showed that this region is not essential for locus rearrangement or expression but is required for establishing the / ratio (17). A region that may regulate the accessibility of the human locus has been identified 10 kb downstream of C7 (18, 19). Functional analysis using reporter gene assays identified a core enhancer region flanked by elements that can drastically reduce enhancer activity in pre-B cells (18). Although transfection studies showed that the and 3' enhancer regions appear to be functionally equivalent, the core enhancer motifs are flanked by functional sequences that are remarkably dissimilar. The human Ig locus on chromosome 22q11.2 is 1.1 Mb in size and typically contains 70 V genes and 7 J-C gene segments (20, 21). Approximately half of the V genes and J-C1, 2, 3, and 7 are regarded as functional. The V genes are organized in three clusters, with the members of a particular V gene family contained within the same cluster. There are 10 V gene families, with the largest, VIII, having 23 members. In human peripheral blood lymphocytes, V gene segments of families I, II, and III from the J-C proximal cluster A are preferentially rearranged, with the contribution of the 2a2 V segment (2–14 using a position-based nomenclature; reference 22) being unusually high (23). All gene segments have the same polarity that allows deletional rearrangement (24). The diversity of the Ig repertoire is provided mainly by V-J combination. Additional CDR3 diversity due to N (nonencoded)1 or P (palindromic) nucleotide additions at the V to J junction is present in human sequences, although not as extensively as in IgH rearrangement, but is absent in sequences from mice (25–28), where the TdT (terminal deoxyribonucleotide transferase) activity is downregulated at the time of L chain rearrangement. genes of cluster A and all the J-C segments in germline configuration, into mice that have one or both endogenous Ig alleles disrupted. The translocus shows high expression in both backgrounds, and is able to compete equally with the endogenous mouse locus.
Materials and Methods
Top
Abstract
Materials and Methods
Results
Discussion
References
The HuIgYAC, Introduction into Embryonic Stem Cells, and Derivation of Transgenic Mice.
The 410-kb HuIgYAC, accommodating a 380-kb region (V-JC) of the human L chain locus with V, J, and C genes in germline configuration, was constructed as previously described (29). To allow selection, two copies of the neomycin resistance gene (NEOr) were site-specifically integrated into the ampicillin gene on the left (centromeric) YAC arm. YAC-containing yeast cells were fused with HM-1 embryonic stem (ES) cells (a gift from D. Melton, Department of Pathology, University Medical School, Edinburgh, UK), as previously described (30), and G418-resistant colonies were picked and analyzed 2–3 wk after protoplast fusion. ES cells containing a complete HuIgYAC copy, confirmed by Southern hybridization, were used for blastocyst injection to produce chimeric animals (31). Breeding of chimeric animals with BALB/c mice resulted in germline transmission. Further breeding with –/– mice (32) established the lines for analysis.
Conventional DNA was obtained (33) or high molecular weight DNA was prepared in agarose blocks (34). For the preparation of testis DNA, tissues were homogenized and passed through 70 µM nylon mesh. Pulsed-field gel electrophoresis (PFGE) conditions to separate in the 50–900 kb range were 1% agarose, 180 V, 70 s switch time, and 30 h running time at 3.5°C. Hybridization probes were C2+3 and the left YAC arm probe (LA) comprising LYS2 (29).
Hybridomas were obtained from 3-mo-old HuIgYAC/+/– animals by fusion of splenocytes with NS0 myeloma cells (35). After fusion, cells were plated on 96-well plates such as to obtain single clones. Human and mouse antibody production was determined in sandwich ELISA assays (36) on MaxiSorp plates (Nalge Nunc, Denmark). For the detection of human or mouse Ig, coating reagents were a 1:500 dilution of anti–human L chain mAb HP-6054 (L 6522; Sigma Chemical Co.) or a 1:500 dilution of the 2.3 mg/ml rat anti–mouse mAB (L 2280; Sigma Chemical Co.), respectively. Respective binding was detected with biotinylated antibodies: polyclonal anti–human (B 0900; Sigma Chemical Co.), a 1:1,000 dilution of polyclonal anti–mouse (RPN 1178; Amersham International) or rat anti–mouse Ig (No. 021172D; PharMingen) followed by streptavidin-conjugated horseradish peroxidase (Amersham International). Mouse IgG2a myeloma protein from HOPC1 (M 6034; Sigma Chemical Co.) and human serum IgG (I 4014; Sigma Chemical Co.) were used to standardize the assays. To determine mouse L chain levels, plates were coated with a 1:1,000 dilution of rat anti–mouse , clone EM34.1 (K 2132; Sigma Chemical Co.), and bound Ig was detected using biotinylated rat mAb anti–mouse Ig (Cat. No. 04-6640; Zymed). Mouse myeloma proteins IgG2a and IgG1 (UPC10, M 9144, and MOPC21, M 9269; Sigma Chemical Co.) were used as standards. For detection of mouse IgM, plates were coated with polyclonal anti–mouse µ (The Binding Site, UK) and bound Ig was detected with biotinylated goat anti–mouse µ (RPN1176; Amersham International) followed by streptavidin-conjugated horseradish peroxidase. Mouse plasmacytoma TEPC183, IgM (M 3795; Sigma Chemical Co.) was used as a standard.
Cell suspensions were obtained from bone marrow, spleen, and Peyer's patches (PPs). Multicolor staining was then carried out with the following reagents in combinations (illustrated in Fig. 4): FITC-conjugated anti–human (F 5266; Sigma Chemical Co.), PE-conjugated anti–mouse c-kit (CD117) receptor (clone ACK45, cat. No. 09995B; PharMingen), PE-conjugated anti–mouse CD25 (IL-2 receptor) (clone 3C7, P 3317; Sigma Chemical Co.), biotin-conjugated anti– human (clone G20-193, cat. No. 08172D, PharMingen), biotin-conjugated anti-mouse CD19 (clone 1D3, cat. No. 09654D; PharMingen), followed by Streptavidin-Quantum red (S 2899; Sigma Chemical Co.) or Streptavidin-PerCP (cat. No. 340130; Becton Dickinson) and rat monoclonal anti–mouse L chain (clone MRC-OX-20, cat. MCA152; Serotec, UK) coupled according to the manufacturer's recommendations with allophycocyanin (APC) (PJ25C; ProZyme). Data were collected from 106 stained cells on a FACScalibur® flow cytometer (Becton Dickinson) as previously described (32). Cells were first gated on forward and side scatter to exclude dead cells. To obtain accurate percentage distribution for comparison, cells from normal mice were stained in parallel. In addition, human peripheral blood lymphocytes were purified on Ficoll gradients (1.077 g/ml) and stained with PE-conjugated anti–human CD19 antibody (P 7437, clone SJ25-C1; Sigma Chemical Co.), biotinylated anti–human followed by Streptavidin-Quantum red, and FITC-conjugated anti–human antibodies as above.
|
genes, PP cells were stained with FITC-conjugated peanut agglutinin (PNA) (L 7381; Sigma Chemical Co.) and PE-conjugated anti–mouse B220 antibodies (P 3567; Sigma Chemical Co.). Double-positive cells were sorted on the FACStarPlus flow cytometer (Becton Dickinson) as previously described (32) and 5 x 103 cells were lysed in denaturing solution (37). 5'RACE was carried out as described below with one modification: 2 µg of carrier RNA was added to the cell lysates before RNA extraction and precipitation.
Cloning and Sequencing of 5'RACE Products.
Spleen RNA was prepared as previously described (37) and for cDNA preparation 2–3 µg of RNA was ethanol precipitated and air dried. For rapid amplification of 5' cDNA ends (5'RACE) (38) first strand cDNA was primed with oligo(dT)22, and 100 U of Super Script II reverse transcriptase (GIBCO BRL) was used at 46°C according to the manufacturer's instructions with 20 U of placental RNAse inhibitor (Promega). The DNA/RNA duplex was passed through 1 ml G-50 equilibrated with TE (10 mM Tris-HCl, pH 7.8, and 1 mM EDTA) in a hypodermic syringe to remove excess oligo(dT). For G-tailing, 20 U of TdT (Cambio, UK) was used according to standard protocols (39). Double-stranded cDNA was obtained from G-tailed single-stranded cDNA by addition of oligonucleotide Pr1 (see below), 100 µM dNTP, and 2.5 U of Klenow fragment (Cambio), followed by incubation for 10 min at 40°C. After heating the reaction for 1 min at 94°C and extraction with phenol-chloroform, the double-stranded cDNA was passed through G-50 to remove primer Pr1. PCR amplifications, 35 cycles, were carried out in the RoboCycler Gradient 96 Thermal Cycler (Stratagene) using oligonucleotides Pr2 and Pr3. For PCR of PP cDNA 50 cycles were used: 40 cycles in the first amplification and 10 cycles in additional amplifications. Pfu Thermostable Polymerase (Stratagene) was used instead of Taq polymerase to reduce PCR error rates. The amplification products were purified using a GENECLEAN II kit (BIO 101) and reamplified for five cycles with primers Pr2 and Pr4 to allow cloning into EcoRI sites. Oligonucleotide for 5'RACE of V genes were: Pr1, 5'-AATTCTAAAACTACAAACTGCCCCCCCCA/T/G-3'; Pr2, 5'-AATTCTAAAACTACAAACTGC-3' (sense); Pr3, 5'-CTCCCGGGTAGAAGTCAC-3' (reverse); and Pr4, 5'-AATTCGTGTGGCCTTGTTGGCT-3' (reverse nested).
V PCR products of 500 bp were cut out from agarose gels and purified on GENECLEAN II. The DNA was incubated in 50 mM Tris-HCl, pH 7.4, and 10 mM MgCl2, with 100 µM dGTP/dCTP and 1 U of Klenow fragment for 10 min at room temperature. Under these conditions the Klenow fragment removed the 3' ends of the PCR products (AATT) leaving ligatable EcoRI overhangs. DNA was ligated with EcoRI-restricted pUC19, transformed into competent E. coli XL1Blue, and colonies were selected on X-Gal/IPTG/amp plates. Plasmid DNA prepared from white colonies was used for sequencing. Sequencing of both strands was done on the ABI 373 automated sequencer (Applied Biosystems, Inc.) in the Babraham Institute Microchemical Facility.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Locus. translocus (Fig. 1) was assembled as a YAC by recombining one YAC containing about half of the human V gene segments with three overlapping cosmids containing V and J-C gene segments and the 3' enhancer (29). This produced a 410-kb YAC accommodating a 380-kb region of the human L chain locus containing 15 V genes regarded as functional, 3 Vs with open reading frames not found to be expressed, and 13 V pseudogenes (40). This HuIgYAC was introduced into ES cells by protoplast fusion (30) and chimeric mice were produced by blastocyst injection (31). The ES cell clone used for blastocyst injection showed a 450-kb NotI fragment corresponding to HuIgYAC, as identified by PFGE and Southern hybridization with probes to the 3' end of the construct, identifying the C2+3 regions, and to the left centromeric YAC arm at the 5' end, identifying the LYS2 gene (data not shown). Germline transmission was obtained, and PFGE analysis of testis DNA from one animal is illustrated in Fig. 2. A NotI fragment larger than 380 kb is necessary to accommodate this region of the HuIgYAC, and the 450-kb band obtained indicates random integration involving the single NotI site 3' of J-C and a NotI site in the mouse chromosome. Digests with EcoRI/HindIII and hybridization with the C2+3 probe further confirmed the integrity of the transferred HuIgYAC (Fig. 2). The results indicated that one complete copy of the HuIgYAC was integrated in the mouse genome.
|
|
Expression Is Dominant in Mouse –/– Animals. L chain repertoire for the production of authentic human antibodies, the HuIgYAC mice were bred with mice in which endogenous Ig production was silenced by gene targeting (32). In these –/– mice, the mouse Ig titers are elevated compared with +/+ strains (32, 41). Serum titrations (Fig. 3) showed that human Ig antibody titers in HuIgYAC/–/– mice are between 1 and 2 mg/ml, which is up to 10-fold higher than average mouse Ig levels. Interestingly, in the HuIgYAC/–/– mice, the mouse Ig production returned to levels similar to that found in normal mice. High numbers of human Ig+ cells were also identified in flow cytometric analysis of splenic B cells from HuIgYAC/–/– mice (Fig. 4 A), with human expressed on the surface of 84% of the B cells and mouse Ig+ expressed on <5% (data not shown).
|
Expression Equals Mouse Ig Production. production in heterozygous HuIg YAC+/+/– mice allowed a detailed comparison of expression and activation of endogenous versus transgenic L chain loci present at equal functional numbers. Serum analysis (Fig. 3) of mice capable of expressing both human and mouse showed similar titers for human and mouse L chains. Human Ig levels in HuIgYAC/+/+ transgenic mice were similar to those in HuIgYAC/+/– mice. Total Ig levels in HuIgYAC+/+/– mice were 1–2 mg/ml, with an average contribution of 51% mouse Ig, 43% human Ig, and 6% mouse Ig. As is also seen in human serum, the analysis of individual HuIgYAC/+/– animals showed there were variations in the / ratios. Three of the HuIgYAC/+/– mice produced somewhat higher levels, whereas in two mice the human levels were higher than the Ig titers. In HuIgYAC/+/– mice, high translocus expression was also found in B220+ B cells from different tissues, with 38% of spleen cells expressing human and 45% expressing mouse (Fig. 4 A). As illustrated, these values closely resemble the levels in human volunteers with 34% Ig+ versus 51% Ig+ in CD19+ peripheral blood lymphocytes. In HuIgYAC/ +/+ mice, which carry a wild-type locus, the levels of Ig are 25% and endogenous levels are 60% (Fig. 4 A). It is likely that these differences in expression levels are dependent on the number of active gene loci.
To assess the developmental stage at which the high contribution of the human translocus becomes established, we examined surface L chain expression by bone marrow cells of HuIgYAC/+/– mice. For this, B cell lineage marker CD19 and specific antibodies to human and mouse were used in four-color staining with the early B cell markers c-kit (CD117) and CD25. Fig. 4 B shows that surface L chain expression (human or mouse ) was detectable on a similar small proportion of B cells at each of these stages of development, which suggests that human and mouse L chain rearrangements are simultaneous. The specificity of the staining detecting mouse and human on small numbers of early B cells, which has been reported independently (42), was verified by the absence of similar positive cells in the analysis of bone marrow from control mice (data not shown).
DNA Rearrangement and Diversification of a Highly Active Human Translocus.
To further clarify the potential of the L chain translocus to contribute to the antibody repertoire, we analyzed human and mouse L chain production using individual hybridoma clones from HuIgYAC/ +/– mice. Results from two fusions suggest that human and mouse L chain–producing cells were present in the spleen of HuIgYAC/–/+ mice at similar frequencies. Furthermore, in the hybridomas the amounts of human Ig (2–20 µg/ml) or mouse Ig (4–25 µg/ml) were very similar. To determine whether Ig rearrangement precedes Ig, as found in mice and humans (4, 5), the configuration of the endogenous Ig and the human translocus were analyzed in these hybridomas. Southern blot hybridization of randomly picked hybridoma clones showed that in 11 human Ig expressers, 7 had the mouse locus in germline configuration, 1 clone had mouse Ig rearranged, and 3 clones had the mouse locus deleted, whereas in 19 mouse Ig expressers, all but 2 had the human Ig locus in germline configuration. This result suggests that there is no locus activation bias and further emphasizes that the human translocus performs with similar efficiency as the endogenous locus.
The capacity of the human locus to produce a diverse antibody repertoire is further documented by the V-J rearrangement. Sequences were isolated from spleen and PP cells by 5'RACE PCR amplification to avoid bias from specific V gene primers. The use of individual V genes is indicated by the triangles in Fig. 1, and shows that a substantial proportion of the V genes on the translocus are being used in productive rearrangements, with V3-1 and V3-10 being most frequently expressed. In V-J rearrangements, J2 was used preferentially and J3 and 1 were used less frequently, whereas, as expected, J4, 5, and 6 were not used as they are adjacent to 
Cs. Extensive variability due to N or P sequence additions, which is found in human but not mouse L chain sequences (25, 27, 28), was not observed. Sequences obtained by RT-PCR from FACS®-sorted PP germinal center B cells (B220+/PNA+) revealed that somatic hypermutation is operative in HuIg YAC mice (Fig. 5). We identified 11 unique V-J rearrangements with two or more changes in the V region, excluding the CDR3, which may be affected by V-J recombination. The majority of mutations lead to amino acid replacements, but there was no preferential distribution in CDR1 and CDR2.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
to L chain expression varies considerably between different species (1–3, 43, 44), and in mice the low L chain levels are believed to be a result of inefficient activation of the mouse locus during B cell differentiation (for review see reference 6). The Ig (40%) to Ig (60%) ratio in humans is more balanced and suggests that both and play an equally important role in immune responses. This is supported by the finding that the mouse V genes are most similar to the less frequently used distal human V gene families, whereas no genes comparable to the major contributors to the human V repertoire are present in the mouse locus (40). With the HuIgYAC, these V genes are available, and are able to make a significant contribution to the antibody repertoire. The 410-kb HuIgYAC translocus accommodates V gene region cluster A containing at least 15 functional V genes (see Fig. 1). In humans, cluster A is the main contributor to the antibody repertoire, with V 2-14 (2a2) expressed most frequently at 27% in blood lymphocytes (23). We also find expression of V 2-14 in the translocus mice, but the main contributors to the L chain repertoire were 3-1 (the V gene most proximal to the J-C region) and 3-10, both of which are expressed at 3% in humans. Although the validity of conclusions about the contribution of different genes is dependent on the numbers examined, the overexpression of V3-1 (11 sequences) and V3-10 (8 sequences) in the 31 sequences obtained may imply that rearrangement or selection preferences are different in mice and humans. Analysis of recombination signal sequences (RSS) in mouse L chains showed that and RSS differ significantly, and that those genes with the highest similarity to consensus RSS rearrange most frequently (45). The RSS of V3-1 and V3-10 show a 100% match with the mouse consensus sequence, which may explain their frequent expression in the translocus mice. In addition, most human V RSS match the established consensus sequence significantly better than mouse Vs (21, 45).
We found extensive somatic hypermutation of many rearranged human Ig sequences, indicating that they are able to participate in normal immune responses. The levels of mutation in B220+/PNA+ PP cells from HuIgYAC translocus mice were similar to what has been reported for mouse L chains (46). Rather unexpected was the pattern of somatic hypermutation with similar numbers of silent and replacement point mutations found in the complementarity-determining and framework regions. Somatic hypermutation is usually associated with a higher level of replacement mutations in CDRs and more silent mutations in the framework regions, and the distribution observed here may argue against efficient antigen selection having taken place. Interestingly, however, L chain sequences obtained from human peripheral blood lymphocytes also showed high numbers of mutations in framework 2 (23). Part of framework 2 lies at the interface of the VL and VH domains and it has been suggested that this region may be important for optimal H and L chain interaction and, in particular, interaction of the human L chain and the endogenous mouse H chain (26).
In the mouse, unlike in humans, L chain diversification due to untemplated nucleotide addition is essentially absent, because TdT expression has been downregulated by the stage at which L chain rearrangement takes place (28, 47). This concept is challenged by the observation that mouse L chain rearrangement can occur at the same time as VH to DJH rearrangements (48) or even earlier (42). Our results also show L chain rearrangement at the pre-B cell stage with a similar number of human - or mouse -expressing B cells also expressing c-kit+ or CD25+ (see Fig. 4). Although the cell numbers are small, the results suggest that there is no preferential activation of either the human translocus or the endogenous locus. However, despite this early activation, there is no accumulation of N or P nucleotide diversity in the rearranged human L chains, unlike rearranged L chains from human peripheral B cells (27). The small number (<1%) of human Ig+/mouse Ig+ double positive spleen and bone marrow cells may indicate that haplotype exclusion at the L chain level is less strictly controlled than is IgH exclusion (49).
In transgenic mice carrying Ig regions in germline configuration on minigene constructs, efficient DNA rearrangement and high antibody expression levels are rarely achieved. Competition with the endogenous locus can be eliminated using Ig knockout strains, in which transgene expression is usually improved (50). Poor transloci expression levels could be a result of the failure of human sequences to work efficiently in the mouse background or, alternatively, of the absence of locus-specific control regions that are more likely to be included on larger transgenic regions (51–53). Recently we addressed this question in transgenic mice by the introduction of different sized minigene- and YAC-based human L chain loci (53). The result showed that neither the size of the V gene cluster nor the V gene numbers present were relevant to achieving high translocus expression levels. The YAC-based loci contained downstream regions of the human locus, and it is possible that the presence of an undefined region with cis-controlled regulatory sequences may have been crucial in determining expressibility and subsequently L chain choice. The HuIgYAC contains equivalent regions from the human Ig locus, which may promote the use of the translocus in the L chain repertoire. Hybridomas from HuIgYAC+/ +/– mice show no evidence for a bias in L chain locus selection during development, as demonstrated by the absence of rearrangement of the nonexpressed locus. This is in contrast with what is seen in Ig expressing mouse and human B cell clones (4, 5), and supports the model that and rearrangements are indeed independent (15, 54) and that poor Ig expression levels in mice may be the result of inefficient signals acting during recombination (16). A possible signal that initiates L chain recombination has been identified through gene targeting experiments where the 3' enhancer was deleted (17). In these mice, the / ratio was reduced from 20:1 in normal mice down to 1:1, and the locus was largely in germline configuration in -expressing cells, as we also see in the HuIgYAC+/+/– hybridoma clones. The high level of human Ig expression in the HuIgYAC+/+/– mice could be due to the strength of the downstream enhancer of the human locus. An analysis of human L chain enhancer activities identified three synergistic modules at the 3' end of the locus which constitute a powerful pre-B cell specific enhancer that appears to be stronger than the corresponding enhancer (55). Analysis of the mouse 3' enhancer suggests the biased / ratio in mice may be a direct result of the differences in locus specific regulation provided by the respective enhancers (19, 56). The results suggest that strength and ability of the human 3' enhancer to function in the mouse background may be the reason that and loci can compete equally at the pre-B cell stage to initiate L chain rearrangement, resulting in the similar levels of human Ig and mouse Ig seen in the HuIgYAC+/+/– mice.
|
Acknowledgments |
|---|
sequences and helpful discussions. We are grateful to Drs. D. Melton for provision of the HM-1 ES cells, E. Corps for hybridoma production, B. Goyenechea for help with the Southern hybridization, and N. Miller for helping with the flow cytometry. This work was supported by the Biotechnology and Biological Sciences Research Council and the Babraham Institute.
Submitted: 14 December 1998
Revised: 8 March 1999
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
1 Hood L, Gray WR, Sanders BG & Dreyer WY. Light chain evolution: antibodies, Cold Spring Harbor Symp Quant Biol, 1967, 32, 133–146.
2 McIntire KR & Rouse AM. Mouse immunoglobulin light chains: alterations of : ratio, Fed Proc, 1970, 19, 704.
3 Arun SS, Breuer W & Hermanns W. Immunohistochemical examination of light-chain expression (lambda/ kappa ratio) in canine, feline, equine, bovine and porcine plasma cells, Zentralbl Veterinarmed A, 1996, 43, 573–576.[Medline]
4 Hieter PA, Korsmeyer SJ, Waldmann TA & Leder P. Human immunoglobulin light-chain genes are deleted or rearranged in -producing B cells, Nature, 1981, 290, 368–372.[Medline]
5 Coleclough C, Perry RP, Karjalainen K & Weigert M. Aberrant rearrangements contribute significantly to the allelic exclusion of immunoglobulin gene expression, Nature, 1981, 290, 372–378.[Medline]
6 Selsing, E., and L.E. Daitch. 1995. Immunoglobulin genes. In Immunoglobulin Genes. Second Edition. T. Honjo and F.W. Alt, editors. Academic Press, London, UK. 193–203.
7 Berg J, McDowell M, Jäck HM & Wabl M. Immunoglobulin gene rearrangement can precede gene rearrangement, Dev Immunol, 1990, 1, 53–57.[Medline]
8 Abken H & Bützler C. Re-organization of the immunoglobulin kappa gene on both alleles is not an obligatory prerequisite for Ig lambda gene expression in human cells, Immunology, 1991, 74, 709–713.[Medline]
9 Takemori T & Rajewsky K. Lambda chain expression at different stages of ontogeny in C57BL/6, BALB/c and SJL mice, Eur J Immunol, 1981, 11, 618–625.[Medline]
10 McGuire KL & Vitetta ES. / shifts do not occur during maturation of murine B cells, J Immunol, 1981, 127, 1670–1673.[Abstract]
11 Kessler S, Kim KJ & Scher I. Surface membrane and light chain expression on spleen cells of neonatal and maturing normal and immune-defective CBA/NB mice: the ratio is constant, J Immunol, 1981, 127, 1674–1678.[Medline]
12 Lejeune JM, Briles DE, Lawton AR & Kearney JF. Estimate of the light chain repertoire size of fetal and adult BALB/cJ and CBA/J mice, J Immunol, 1982, 129, 673–677.[Abstract]
13 Rolink A, Streb M & Melchers F. The / ratio in surface immunoglobulin molecules on B lymphocytes differentiating from DHJH -rearranged murine pre-B cell clones in vitro. , Eur J Immunol, 1991, 21, 2895–2898.[Medline]
14 Osmond DJ, Rolink A & Melchers F. Murine B lymphopoiesis: towards a unified model, Immunol Today, 1998, 19, 65–68.[Medline]
15 Zou YR, Takeda S & Rajewsky K. Gene targeting in the Ig locus: efficient generation of chain-expressing B cells, independent of gene rearrangements in Ig, EMBO (Eur Mol Biol Organ) J, 1993, 12, 811–820.[Medline]
16 Arakawa H, Shimizu T & Takeda S. Re-evaluation of the probabilities for productive rearrangements on the and loci, Int Immunol, 1996, 8, 91–99.
17 Gorman JR, van der Stoep N, Monroe R, Cogne M, Davidson L & Alt FW. The Ig 3' enhancer influences the ratio of Ig versus Ig B lymphocytes, Immunity, 1996, 5, 241–252.[Medline]
18 Glozak M & Blomberg BB. The human immunoglobulin enhancer is controlled by both positive elements and developmentally regulated negative elements, Mol Immunol, 1996, 33, 427–438.[Medline]
19 Asenbauer H & Klobeck HG. Tissue-specific deoxyribonuclease I-hypersensitive sites in the vicinity of the immunoglobulin C lambda cluster of man, Eur J Immunol, 1996, 26, 142–150.[Medline]
20 Frippiat JP, Williams SC, Tomlinson IM, Cook GP, Cherif D, Le Paslier D, Collins JE, Dunham I, Winter G & Lefranc MP. Organization of the human immunoglobulin lambda light-chain locus on chromosome 22q11.2, Hum Mol Genet, 1995, 4, 983–991.
21 Kawasaki K, Minoshima S, Nakato E, Shibuya K, Shintani A, Schmeits JL, Wang J & Shimizu N. One-megabase sequence analysis of the human immunoglobulin gene locus, Genome Res, 1997, 7, 260–261.
22 Giudicelli V, Chaume D, Bodmer J, Muller W, Busin C, Marsh S, Bontrop R, Marc L, Malik A & Lefranc M-P. IMGT, the international ImMunoGeneTics database, Nucleic Acids Res, 1997, 25, 206–211.
23 Ignatovich O, Tomlinson IM, Jones PT & Winter G. The creation of diversity in the human immunoglobulin V(lambda) repertoire, J Mol Biol, 1997, 268, 69–77.[Medline]
24 Combriato G & Klobeck H-G. V and J-C gene segments of the human immunoglobulin light chain locus are separated by 14 kb and rearrange by a deletion mechanism, Eur J Immunol, 1991, 21, 1513–1522.[Medline]
25 Foster SJ, Brezinschek H-P, Brezinschek RI & Lipsky PE. Molecular mechanisms and selective influences that shape the gene repertoire of IgM+B cells, J Clin Invest, 1997, 99, 1614–1627.[Medline]
26 Ignatovich, O. 1998. The creation of diversity in the human immunoglobulin V repertoire. Ph.D. thesis. University of Cambridge, Cambridge, UK.
27 Bridges SL, Lee SK, Johnson ML, Lavelle JC, Fowler PG, Koopman WJ & Schroeder HW. Somatic mutation and CDR3 length of immunoglobulin light chains expressed in patients with rheumatoid arthritis and in normal individuals, J Clin Invest, 1995, 96, 831–841.[Medline]
28 Victor KD, Vu K & Feeney AJ. Limited junctional diversity in light chains, J Immunol, 1994, 152, 3467–3475.[Abstract]
29 Popov AV, Bützler C, Frippiat J-P, Lefranc M-P & Brüggemann M. Assembly and extension of yeast artificial chromosomes to build up a large locus, Gene, 1996, 177, 195–201.[Medline]
30 Davies, N.P., A.V. Popov, X. Zou, and M. Brüggemann. 1996. Human antibody repertoires in transgenic mice: manipulation and transfer of YACs. In Antibody Engineering: A Practical Approach. J. McCafferty, H.R. Hoogenboom, and D.J. Chiswell, editors. IRL Press, Oxford. 59–76.
31 Hogan, B., R. Beddington, F. Costantini, and E. Lacy. 1994. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 497 pp.
32 Zou X, Xian J, Popov AV, Rosewell IR, Müller M & Brüggemann M. Subtle differences in antibody responses and hypermutation of light chains in mice with a disrupted constant region, Eur J Immunol, 1995, 25, 2154–2162.[Medline]
33 Wurst, W., and A.L. Joyner. 1993. Production of targeted embryonic stem cell DNA. In Gene Targeting. A.L. Joyner, editor. IRL Press, Oxford. 33–61.
34 Herrmann BG, Barlow DP & Lehrach H. A large inverted duplication allows homologous recombination between chromosomes heterozygous for the proximal t complex inversion, Cell, 1987, 48, 813–825.[Medline]
35 Galfré G & Milstein C. Preparation of monoclonal antibodies: strategies and procedures, Methods Enzymol, 1981, 73, 3–46.[Medline]
36 Tijssen, P. 1985. Practice and theory of enzyme immunoassays. In Laboratory Techniques in Biochemistry and Molecular Biology. Volume 15. R.H. Burdon and P.H. Knippenberg, editors. Elsevier, Amsterdam.
37 Chomczynski P & Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal Biochem, 1987, 162, 156–159.[Medline]
38 Frohman MA, Dush MK & Martin GR. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer, Proc Natl Acad Sci USA, 1988, 85, 8998–9002.
39 Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, K. Struhl, and J.A. Smith, editors. 1995. Current Protocols In Molecular Biology. Massachusetts General Hospital, Boston, MA; Harvard Medical School, Boston, MA; University of Alabama, Birmingham, AL; Wiley & Sons, New York.
40 Williams SC, Frippiat J-P, Tomlinson IM, Ignatovich O, Lefranc M-P & Winter G. Sequence and evolution of the human germline V repertoire, J Mol Biol, 1996, 264, 220–232.[Medline]
41 Chen J, Trounstine M, Kurahara C, Young F, Kuo C-C, Xu Y, Loring JF, Alt FW & Huszar D. B cell development in mice that lack one or both immunoglobulin light chain genes, EMBO (Eur Mol Biol Organ) J, 1993, 12, 821–830.[Medline]
42 Novobrantseva TI, Martin VM, Pelanda RM, Muller W, Rajewsky K & Ehlich A. Rearrangement and expression of immunoglobulin light chain genes can precede heavy chain expression during normal B cell development in mice, J Exp Med, 1999, 189, 75–88.
43 Saitta M, Iavarone A, Cappello N, Bergami MR, Fiorucci GC & Aguzzi F. Reference values for immunoglobulin kappa and lambda light chains and the kappa/ lambda ratio in children's serum, Clin Chem, 1992, 38, 2454–2457.
44 Hood L, Gray WR & Dreyer WY. On the mechanism of antibody synthesis: a species comparison of L-chains, Proc Natl Acad Sci USA, 1966, 55, 826–835.
45 Ramsden DA & Wu GE. Mouse light-chain recombination signal sequence mediate recombination more frequently than do those of light chain, Proc Natl Acad Sci USA, 1991, 88, 10721–10725.
46 Gonzalez-Fernandez A, Gupta SK, Pannell R, Neuberger MS & Milstein C. Somatic mutation of immunoglobulin lambda chains: a segment of the major intron hypermutates as much as the complementarity-determining regions, Proc Natl Acad Sci USA, 1994, 91, 12614–12618.
47 Li Y-S, Hayakawa K & Hardy RR. The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver, J Exp Med, 1993, 178, 951–960.
48 Hardy RR, Carmack CE, Shinton SA, Kemp JD & Hayakawa K. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow, J Exp Med, 1991, 173, 1213–1225.
49 Harada K & Yamagishi H. Lack of feedback inhibition of V gene rearrangement by productively rearranged alleles, J Exp Med, 1991, 173, 409–415.
50 Brüggemann M & Neuberger MS. Strategies for expressing human antibody repertoires in transgenic mice, Immunol Today, 1996, 17, 391–397.[Medline]
51 Green LL & Jakobovits A. Regulation of B cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes, J Exp Med, 1998, 188, 483–495.
52 Zou X, Xian J, Davies NP, Popov AV & Brüggemann M. Dominant expression of a 1.3 Mb human Ig locus replacing mouse light chain production, FASEB J, 1996, 10, 1227–1232.[Abstract]
53 Xian J, Zou X, Popov AV, Mundt CA, Miller N, Williams GT, Davies SL, Neuberger MS & Brüggemann M. Comparison of the performance of a plasmid-based human Ig minilocus and YAC-based human Ig transloci for the production of a human antibody repertoire in transgenic mice, Transgenics, 1998, 2, 333–343.
54 Nadel B, Cazenave P-A & Sanchez P. Murine lambda gene rearrangements: the stochastic model prevails over the ordered model, EMBO (Eur Mol Biol Organ) J, 1990, 9, 435–440.[Medline]
55 Asenbauer H, Combriato G & Klobeck H-G. The immunoglobulin lambda light chain enhancer consists of three modules which synergize in activation of transcription, Eur J Immunol, 1999, 29, 713–724.[Medline]
56 Hagman J, Rudin CM, Haasch C, Chaplin D & Storb U. A novel enhancer in the immunoglobulin lambda locus is duplicated and functionally independent of NF kappa B, Genes Dev, 1990, 4, 978–992.
57 Eagle H. Propagation in a fluid medium of a human epidermoid carcinoma strain KB, Proc Soc Exp Biol Med, 1955, 89, 362–364.
58 Taub RA, Hollis GF, Hieter PA, Korsmeyer S, Waldmann TA & Leder P. Variable amplification of immunoglobulin light chain genes in human populations, Nature, 1983, 304, 172–174.[Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| TABLE OF CONTENTS |
|
) indicate V gene use in functionally Ig
