Epilepsy, hyperalgesia, impaired memory, and loss of pre- and postsynaptic GABA(B) responses in mice lacking GABA(B(1)).

GABA(B) (gamma-aminobutyric acid type B) receptors are important for keeping neuronal excitability under control. Cloned GABA(B) receptors do not show the expected pharmacological diversity of native receptors and it is unknown whether they contribute to pre- as well as postsynaptic functions. Here, we demonstrate that Balb/c mice lacking the GABA(B(1)) subunit are viable, exhibit spontaneous seizures, hyperalgesia, hyperlocomotor activity, and memory impairment. Upon GABA(B) agonist application, null mutant mice show neither the typical muscle relaxation, hypothermia, or delta EEG waves. These behavioral findings are paralleled by a loss of all biochemical and electrophysiological GABA(B) responses in null mutant mice. This demonstrates that GABA(B(1)) is an essential component of pre- and postsynaptic GABA(B) receptors and casts doubt on the existence of proposed receptor subtypes.

Monitoring and interpreting the intrinsic features of somatic hypermutation.

We have used both normal and transgenic mice to analyse the recruitment and targeting of somatic hypermutation to the immunoglobulin loci. We compare methods for analysing hypermutation and discuss how large databases of mutations can be assembled by PCR amplification of the rearranged V-gene flanks from the germinal centre B cells of normal mice as well as by transgene-specific amplification from transgenic B cells. Such studies confirm that hypermutation is preferentially targeted to the immunoglobulin V gene with the bcl6 gene, for example, escaping this intense mutational targeting in germinal centre B cells. We review our data concerning the nature of the hypermutation domain and the targeting of hotspots within that domain. We consider how enhancer-mediated recruitment of hypermutation to the immunoglobulin loci operates in a clonally maintained fashion and illustrate how both the degree of expression and demethylation of the transgene broadly correlate with its mutability.

Multiple sequences from downstream of the J kappa cluster can combine to recruit somatic hypermutation to a heterologous, upstream mutation domain.

Recruitment of somatic hypermutation to the Ig kappa locus has previously been shown to depend on the enhancer elements, Ei/MAR and E3'. Here we show that these elements are not sufficient to confer mutability. However, hypermutation is effectively targeted to a chimeric beta-globin/Ig kappa transgene whose 5' end is composed of the human beta-globin gene (promoter and first two exons) and whose 3' end consists of selected sequences derived from downstream of the J kappa cluster (Ei/MAR, C kappa + flank and E3'). Thus, multiple downstream Ig kappa sequences (all derived from 3' of the J kappa cluster) can combine to recruit mutation to a heterologous mutation domain. The location of this hypermutation domain is defined by the position of the transcription start site and this applies even if the Ig kappa Ei/MAR is positioned upstream of the promoter. Hotspots within the mutation domain are, however, defined by local DNA sequence as evidenced by a new hotspot being created within the beta-globin domain by a mutation within the transgene. We propose that multiple, moveable Ig kappa sequences (that are normally located downstream of the transcription start site) cooperate to bring a hypermutation priming factor to the transcription initiation complex; a mutation domain is thereby created downstream of the promoter but the local sequence defines the detailed pattern of mutation within that domain.

Cells strongly expressing Ig(kappa) transgenes show clonal recruitment of hypermutation: a role for both MAR and the enhancers.

The V regions of immunoglobulin kappa transgenes are targets for hypermutation in germinal centre B cells. We show by use of modified transgenes that the recruitment of hypermutation is substantially impaired by deletion of the nuclear matrix attachment region (MAR) which flanks the intron-enhancer (Ei). Decreased mutation is also obtained if Ei, the core region of the kappa3'-enhancer (E3') or the E3'-flank are removed individually. A broad correlation between expression and mutation is indicated not only by the fact that the deletions affecting mutation also give reduced transgene expression, but especially by the finding that, within a single mouse, transgene mutation was considerably reduced in germinal centre B cells that poorly expressed the transgene as compared with strongly expressing cells. We also observed that the diminished mutation in transgenes carrying regulatory element deletions was manifested by an increased proportion of B cells in which the transgene had not been targeted at all for mutation rather than in the extent of mutation accumulation once targeted. Since mutations appear to be incorporated stepwise, the results point to a connection between transcription initiation and the clonal recruitment of hypermutation, with hypermutation being more fastidious than transcription in requiring the presence of a full complement of regulatory elements.

Rapid methods for the analysis of immunoglobulin gene hypermutation: application to transgenic and gene targeted mice.

Hypermutation of immunoglobulin genes is a key process in antibody diversification. Little is known about the mechanism, but the availability of rapid facile assays for monitoring immunoglobulin hypermutation would greatly aid the development of culture systems for hypermutating B cells as well as the screening for individuals deficient in the process. Here we describe two such assays. The first exploits the non-randomness of hypermutation. The existence of a mutational hotspot in the Ser31 codon of a transgenic immunoglobulin V gene allowed us to use PCR to detect transgene hypermutation and identify cell populations in which this mutation had occurred. For animals that do not carry immunoglobulin transgenes, we exploited the fact that hypermutation extends into the region flanking the 3'-side of the rearranged J segments. We show that PCR amplification of the 3'-flank of VDJH rearrangements that involve members of the abundantly-used VHJ558 family provides a large database of mutations where the germline counterpart is unequivocally known. This assay was particularly useful for analysing endogenous immunoglobulin gene hypermutation in several mouse strains. As a rapid assay for monitoring mutation in the JH flanking region, we show that one can exploit the fact that, following denaturation/renaturation, the PCR amplified JH flanking region DNA from germinal centre B cells yields mismatched heteroduplexes which can be quantified in a filter binding assay using the bacterial mismatch repair protein MutS -Wagner et al. (1995) Nucleic Acids Res. 23, 3944-3948-. Such assays enabled us, by example, to show that antibody hypermutation proceeds in the absence of the p53 tumour suppressor gene product.

Deregulation of PAX-5 by translocation of the Emu enhancer of the IgH locus adjacent to two alternative PAX-5 promoters in a diffuse large-cell lymphoma.

Analyses of the human PAX-5 locus and of the 5' region of the mouse Pax-5 gene revealed that transcription from two distinct promoters results in splicing of two alternative 5' exons to the common coding sequences of exons 2-10. Transcription from the upstream promoter initiates downstream of a TATA box and occurs predominantly in B-lymphocytes, whereas the TATA-less downstream promoter is active in all Pax-5-expressing tissues. The human PAX-5 gene is located on chromosome 9 in region p13, which is involved in t(9;14)(pl3;q32) translocations recurring in small lymphocytic lymphomas of the plasmacytoid subtype and in derived large-cell lymphomas. A previous molecular analysis of a t(9;14) breakpoint from a diffuse large-cell lymphoma (KIS-1) demonstrated that the immunoglobulin heavy-chain (IgH) locus on 14q32 was juxtaposed to chromosome 9p13 sequences of unknown function [Ohno, H., Furukawa, T., Fukuhara, S., Zong, S. Q., Kamesaki, H., Shows, T. B., Le Beau, M. M., McKeithan, T. W., Kawakami, T. & Honjo, T. (1990) Proc. Natl. Acad. Sci. USA 87,628-632]. Here we show that the KIS-1 translocation breakpoint is located 1807 base pairs upstream of exon 1A of PAX-5, thus bringing the potent Emu enhancer of the IgH gene into close proximity of the PAX-5 promoters. These data suggest that deregulation of PAX-5 gene transcription by the t(9;14)(pl3;q32) translocation contributes to the pathogenesis of small lymphocytic lymphomas with plasmacytoid differentiation.

The targeting of somatic hypermutation.

Somatic hypermutation does not occur randomly within immunoglobulin V genes but, rather, is preferentially targeted to certain nucleotide positions (hot spots) and away from others (cold spots). Cold spots often coincide with residues essential for V gene folding. Hotspots, which appear to be strategically located to favour affinity maturation, are most frequently located in the CDRs (particularly CDR1) though conserved hotspots are also found at the base of FR3. Hotspots are in part created by local DNA sequence and the strong biases of codon usage in V genes indicate that the genes have evolved such that somatic hypermutation is targeted to those parts of the V where it is likely to prove most useful. These features of mutational hotspots and biased codon usage are also evident in V genes of lower animals suggesting that diversification by strategic targeting of non-templated mutation may have evolved early in antigen receptor evolution.

Targeting of non-Ig sequences in place of the V segment by somatic hypermutation.

Affinity maturation of antibodies is characterized by localized hypermutation of the DNA around the V segment. Here we show, using mice containing single or multiple transgene constructs, that an immunoglobulin V kappa segment can be replaced by human beta-globin or prokaryotic neo or gpt genes without affecting the rate of hypermutation; the V gene itself is not necessary for recruiting hypermutation. The ability to target hypermutation to heterologous genes in vivo could find more general applications in biology.