Gene and cluster-specific expression of the Iroquois family members during mouse development.

Mammalian homologues of the Drosophila Iroquois homeobox gene complex, involved in patterning and regionalization of differentiation, have recently been identified (Mech. Dev., 69 (1997) 169; Dev. Biol., 217 (2000) 266; Dev. Dyn., 218 (2000) 160; Mech. Dev., 91 (2000) 317; Dev. Biol., 224 (2000) 263; Genome Res., 10 (2000) 1453; Mech. Dev., 103 (2001) 193). The six members of the murine family were found to be organized in two cognate clusters of three genes each, Irx1, -2, -4 and Irx3, -5, -6, respectively (Peters et al., 2000). As a basis for further study of their regulation and function we performed a comparative analysis of the genomic organization and of the expression patterns of all six Irx genes. The genes are expressed in highly specific and regionalized patterns of ectoderm, mesoderm and endoderm derived tissues. In most tissues the pattern of expression of the clustered genes, especially of Irx1 and -2 and of Irx3 and -5, respectively, closely resembled each other while those of Irx4 and -6 were very divergent. Interestingly, the expression of cognate genes was found to be mutually exclusive in adjacent and interacting tissues of limb, heart and the laryncho-pharyncheal region. The results indicate that the Irx genes are coordinately regulated at the level of the cluster.

Bambi is coexpressed with Bmp-4 during mouse embryogenesis.

Signaling of TGF-beta superfamily members is tightly controlled by an elaborate network of regulators (for recent review see Trends Genet. 15 (1999) 3; Genes Dev. 14 (2000) 627). Recently, the transmembrane protein BAMBI (BMP and activin membrane-bound inhibitor) has been shown to interfere with Bmp and activin-like signaling by inhibiting Tgf-beta type I receptor activation (Nature 401 (1999) 480). In striking contrast to other Bmp antagonists like noggin (Cell 86 (1996) 599) or chordin (Cell 86 (1996) 589), BAMBI is strictly coexpressed with Bmp-4 during early Xenopus embryogenesis. The grouping of genes according to their shared complex spatial expression pattern and their involvement in the same biological signaling pathway has been referred to as synexpression group. This concept facilitates prognoses about the roles of a group member with unknown function. Apparently, only a minority of genes is organized in synexpression groups and up to now they have mainly been described in yeast and Xenopus (for review see Nature 402 (1999) 483). In the frog, BAMBI is a member of the Bmp-4 synexpression group (Nature 401 (1999) 480). We identified two murine homologues of BAMBI one of which, named Bambi-psi, is a pseudogene. We show that the spatiotemporal expression pattern of Bambi closely matches that of Bmp-4 during mouse embryonic development. Moreover, we show that Bambi expression is induced in mouse embryonic fibroblasts by Bmp-4. Hence, we provide first evidence for the existence of an evolutionarily conserved Bmp-4 synexpression group in mammals.

Organization of mouse Iroquois homeobox genes in two clusters suggests a conserved regulation and function in vertebrate development.

Iroquois proteins comprise a conserved family of homeodomain-containing transcription factors involved in patterning and regionalization of embryonic tissues in both vertebrates and invertebrates. Earlier studies identified four murine Iroquois (Irx) genes. Here we report the isolation of two additional members of the murine gene family, Irx5 and Irx6. Phylogenetic analysis of the Irx gene family revealed distinct clades for fly and vertebrate genes, and vertebrate members themselves were classified into three pairs of cognate genes. Mapping of the murine Irx genes identified two gene clusters located on mouse chromosomes 8 and 13, respectively. Each gene cluster is represented by three Irx genes whose relative positions within both clusters are strictly conserved. Combined results from phylogenetic, linkage, and physical mapping studies provide evidence for the evolution of two Irx gene clusters by duplication of a larger chromosomal region and dispersion to two chromosomal locations. The maintenance of two cognate Irx gene clusters during vertebrate evolution suggests that their genomic organization is important for the regulation, expression, and function of Irx genes during embryonic development.

Single exchanges of amino acids in the basic region change the specificity of N-Myc.

We exchanged specific amino acids in the basic region of the murine N-Myc protein and tested the mutant proteins for their DNA binding specificity. The amino acids we exchanged were chosen in analogy to residues of the homologous basic regions of bHLH and bZIP proteins. Mutant N-Myc peptides were expressed in Escherichia coli and specific DNA binding was monitored by gel shift experiments. For this we used palindromic target sequences with systematic base pair exchanges. Several mutants with altered DNA binding specificity were identified. Amino acid exchanges of residues -14 or -10 of the basic region lead to specificity changes (we define leucine 402 of N-Myc as +1; comparable to GCN4 see (1)). The palindromic N-Myc recognition sequence 5'CACGTG is no longer recognized by the mutant proteins, but DNA fragments with symmetrical exchanges of the target sequence are. Exchanges at position -15 broaden the binding specificity. These data were used to build a computer based model of the putative interactions of the N-Myc basic DNA binding region with its target sequence.

Surface IgM mediated regulation of RAG gene expression in E mu-N-myc B cell lines.

Transgenic mice carrying either the c-myc or N-myc oncogene deregulated by the immunoglobulin heavy chain enhancer element (E mu) develop both pre-B and B cell lymphomas (E mu-c-myc and E mu-N-myc lymphomas). We report here that B cell lines derived from these tumors, as well as a line derived from v-myc retroviral transformation, simultaneously express surface immunoglobulin (a hallmark of mature B cells) as well as a common subset of genes normally restricted to the pre-B stage of development-including the recombinase activating genes RAG-1 and RAG-2. Continued RAG-1 and RAG-2 expression in these lines is associated with VDJ recombinase activity detected with a VDJ recombination substrate. Cross-linking of the surface immunoglobulin on these lines with an anti-mu antibody leads to rapid, specific and reversible down-regulation of RAG-1 and RAG-2 gene expression. We also find that a small but significant percentage of normal surface immunoglobulin bearing bone marrow B cells express the RAG-1 gene. These findings are discussed in the context of their possible implications for the control of specific gene expression during the pre-B to B cell transition.

Determination of the DNA sequence recognized by the bHLH-zip domain of the N-Myc protein.

The DNA-binding domain of the murine N-Myc protein, comprising the basic helix-loop-helix-zipper (bHLH-zip) region was expressed as a fusion protein in E. coli. The affinity purified glutathione-S-transferase-N-Myc fusion protein (GST-N-MYC) was used to select the N-Myc specific DNA-recognition motif from a pool of random-sequence oligonucleotides. After seven rounds of binding-site selection, specifically enriched oligonucleotides were cloned and sequenced. Of 31 individual oligonucleotides whose sequences were determined, 30 contained a common DNA-motif, defining the hexameric consensus sequence CACGTG. We confirm by mutational analysis that binding of the N-Myc derived bHLH-zip domain to this motif is sequence-specific.

Mechanism of endogenous myc gene down-regulation in E mu-N-myc tumors.

Transgenic mouse lines carrying the N-myc oncogene deregulated by the immunoglobulin heavy-chain enhancer spontaneously develop B-lymphoid tumors (R. Dildrop, A. Ma, K. Zimmerman, E. Hsu, A. Tesfaye, R. DePinho, and F. W. Alt, EMBO J. 8:1121-1128, 1989; H. Rosenbaum, E. Webb, J. M. Adams, S. Cory, and A. W. Harris, EMBO J. 8:749-755). Permanent cell lines derived from these tumors (E mu-N-myc cell lines) express extremely high levels of the N-myc transgene but little or no detectable endogenous N-myc or c-myc. We have employed nuclear run-on assays to show that down-regulation of endogenous N- and c-myc expression occurs at the transcriptional level. To determine whether the lack of endogenous myc gene transcription is a direct effect of high-level N-myc transgene expression, we have generated Abelson murine leukemia virus (A-MuLV)-transformed cell lines from prelymphomatous E mu-N-myc mice (A-MuLV/E mu-N-myc cell lines). Although these A-MuLV/E mu-N-myc lines express very high levels of the N-myc transgene, they continue to transcribe the endogenous c-myc gene. These findings demonstrate that high-level N-myc gene expression alone does not necessarily lead to down-regulation of endogenous myc gene expression and suggest that events associated with transformation by N-myc may be critical to this process.

IgH enhancer-mediated deregulation of N-myc gene expression in transgenic mice: generation of lymphoid neoplasias that lack c-myc expression.

We have generated transgenic mouse lines that carry one of three different constructs in which the murine N-myc gene is expressed under the control of the immunoglobulin heavy chain transcriptional enhancer element (E mu-N-myc genes). High-level expression of the E mu-N-myc transgenes occurred in lymphoid tissues; correspondingly, many of these E mu-N-myc lines reproducibly developed pre-B- and B-lymphoid malignancies. The E mu-N-myc transgene also appeared to participate in the generation of a T cell malignancy that developed in one E mu-N-myc mouse. These tumors and cell lines adapted from them expressed exceptionally high levels of the E mu-N-myc transgene; the levels were comparable to those observed in human neuroblastomas with highly amplified N-myc genes. In contrast, all of the E mu-N-myc cell lines had exceptionally low or undetectable levels of the c-myc RNA sequences, consistent with the possibility that high-level N-myc expression can participate in the negative 'cross-regulation' of c-myc gene expression. Our findings demonstrate that deregulated expression of the N-myc gene has potent oncogenic potential within the B-lymphoid lineage despite the fact that the N-myc gene has never been implicated in naturally occurring B-lymphoid malignancies. Our results also are discussed in the context of differential myc gene activity in normal and transformed cells.

A new V gene expressed in lambda-2 light chains of the mouse.

We have partially sequenced the light chain variable regions expressed in three IgM-producing hybridomas generated from newborn mice or from manipulated animals suppressed for IgM production. In these lines a new V gene (V-lambda-X), exhibiting less than 60% homology to any known lambda or kappa V gene, is rearranged to J-lambda-2. The light chains produced by these cells contain the lambda-2 constant domain, but are not recognized by goat antisera raised against conventional mouse lambda light chains.

Timing, genetic requirements and functional consequences of somatic hypermutation during B-cell development.

While somatic antibody mutants are rare in the preimmune repertoire and in primary immune responses, they dominate secondary and hyperimmune responses. We present evidence that somatic hypermutation is restricted to a particular pathway of B-cell differentiation in which distinct sets of B-cell clones are driven into the memory compartment. In accord with earlier results of McKean et al. (1984) and Rudikoff et al. (1984), somatic mutation occurs stepwise in the course of clonal expansion, before and after isotype switch, presumably at a rate close to 1 X 10(-3) per base pair per generation. At this rate, both selectable and unselectable mutations accumulate in the rearranged V region genes. The distribution of replacement mutations in the V regions shows that a fraction of the mutations in CDRs is positively selected whereas replacement mutations are counterselected in the FRs. By constructing an antibody mutant through site-specific mutagenesis we show that a point mutation in CDR1 of the heavy chain, found in most secondary anti-NP antibodies, is sufficient to increase NP binding affinity to the level typical for the secondary response. Somatic mutation may contribute to the immune repertoire in a more general sense than merely the diversification of a specific response. We have evidence that clones producing antibodies which no longer bind the immunizing antigen can be kept in the system and remain available for stimulation by a different antigen. Somatic mutations are 10 times less frequent in DJH loci than in either expressed or non-expressed rearranged VDJH or VJ loci. We therefore conclude that a V gene has to be brought into the proximity of the DJH segment in order to fully activate the hypermutational mechanism in these loci.

Analysis of somatic mutation and class switching in naive and memory B cells generating adoptive primary and secondary responses.

Clonal progeny of naive B cells (producing a primary antibody response) and of memory B cells (producing a secondary response) were identified in a cell transfer system. Primary response clones are typically derived from IgM precursors and express unmutated V regions. Multiple isotype switches occur in these clones. Secondary response clones derive from IgG1 precursors and express highly mutated V regions. Additional switches do not occur. With one exception, there was no evidence for somatic mutation during clonal expansion. The generation of mutated memory cells may thus represent a distinct differentiation pathway. Evidence is presented that, in this pathway, mutants that have lost antigen binding specificity but that remain available for stimulation by a different antigen arise upon antigenic stimulation.

VH-gene expression in murine lipopolysaccharide blasts distributes over the nine known VH-gene groups and may be random.

VH-gene expression in hybridomas derived from lipopolysaccharide-activated B cells was analyzed. Isolated cytoplasmic RNA was hybridized to probes representing the 9 known VH-gene groups or subjected to mRNA sequencing. In the collection of hybridomas VH genes of all 9 groups are expressed at frequencies which in most correlate reasonably well with the relative complexities of the groups. In 51 out of 54 RNA samples VH-gene transcripts could be identified and corresponded to one of the known VH-gene groups. It therefore appears that the latter essentially represent the VH-gene cluster of the mouse.

A V region determinant (idiotope) expressed at high frequency in B lymphocytes is encoded by a large set of antibody structural genes.

Idiotope Ac38, a V region determinant of the lambda 1 chain-bearing, germ line encoded antibody B1-8, is expressed at high frequency (approximately 1/40) in lambda 1 chain-bearing B cells. Here, we describe the isolation of lambda-positive hybridomas from C57BL/6 mice which had been immunized with antibody Ac38, the antibody recognizing idiotope Ac38. In Northern blot analysis, mRNA isolated from 10 such hybridomas hybridizes with a cDNA probe from the VH gene expressed in the cell line B1-8. Amino acid sequence analysis of the VH regions of four of the hybridoma proteins reveals that they are all derived from related, though distinct, germ line VH genes. In one case the sequence data suggest that extensive somatic mutation has taken place. Only one of the four sequences derives from the same VH gene that is expressed in the cell line B1-8. Together with earlier evidence, the present data demonstrate that the Ac38 idiotope is a marker for at least five VH and three D region genes in the C57BL/6 germ line. This explains the high frequency at which this idiotope is expressed in the B cell population. In addition, our sequence determinations identify two VH genes in the C57BL/6 strain which are closely related (and possibly allelic) to two known BALB/c VH genes. One of these genes is the gene expressed in the BALB/c myeloma MOPC 104E.

A new classification of mouse VH sequences.

Renate Dildrop has compared heavy chain variable-region sequences of mouse immunoglobulins at the amino acid level and determined the homology within the coding region of the entire VHgene segment. The resulting homology plot defines seven separate groups of VHgene sequences which differ from the five groups of variable-region sequences in the previous classification of Kabat et al.' and accordfully with the recently identified chromosomal clusters of VHgenes.

Immunoglobulin V region variants in hybridoma cells. II. Recombination between V genes.

The mouse hybridoma line B1-8.delta 1 secretes a monoclonal IgD, lambda 1 anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody with defined idiotypic determinants. Two spontaneous V-region variants (B1-8.V1/V2) with altered idiotope pattern were selected and the structural variation was located to the variable region of the heavy chain. The amino acid sequences of the B1-8. delta 1 and variant heavy chain V regions were determined. The variant VH regions are identical. Wild-type and variant VH regions differ in 10 positions. Single amino acid exchanges are found in the first and second framework at positions 20 and 43. The majority of replacements (eight substitutions) is clustered in the second complementary-determining region (CDR 2). There are no differences in CDR 1 and CRD 3 and the JH region. The variant, which at first glance appears to have undergone a series of point mutations, arose by recombination, possibly gene conversion, between the rearranged VDJ gene of the wild-type (B1-8.delta 1) and a neighbouring germ line VH gene encoding all of the substitutions.