The German Mouse Clinic: a platform for systemic phenotype analysis of mouse models.

The German Mouse Clinic (GMC) is a large scale phenotyping center where mouse mutant lines are analyzed in a standardized and comprehensive way. The result is an almost complete picture of the phenotype of a mouse mutant line--a systemic view. At the GMC, expert scientists from various fields of mouse research work in close cooperation with clinicians side by side at one location. The phenotype screens comprise the following areas: allergy, behavior, clinical chemistry, cardiovascular analyses, dysmorphology, bone and cartilage, energy metabolism, eye and vision, host-pathogen interactions, immunology, lung function, molecular phenotyping, neurology, nociception, steroid metabolism, and pathology. The German Mouse Clinic is an open access platform that offers a collaboration-based phenotyping to the scientific community (www.mouseclinic.de). More than 80 mutant lines have been analyzed in a primary screen for 320 parameters, and for 95% of the mutant lines we have found new or additional phenotypes that were not associated with the mouse line before. Our data contributed to the association of mutant mouse lines to the corresponding human disease. In addition, the systemic phenotype analysis accounts for pleiotropic gene functions and refines previous phenotypic characterizations. This is an important basis for the analysis of underlying disease mechanisms. We are currently setting up a platform that will include environmental challenge tests to decipher genome-environmental interactions in the areas nutrition, exercise, air, stress and infection with different standardized experiments. This will help us to identify genetic predispositions as susceptibility factors for environmental influences.

MouseNet database: digital management of a large-scale mutagenesis project.

The Munich ENU Mouse Mutagenesis Screen is a large-scale mutant production, phenotyping, and mapping project. It encompasses two animal breeding facilities and a number of screening groups located in the general area of Munich. A central database is required to manage and process the immense amount of data generated by the mutagenesis project. This database, which we named MouseNet(c), runs on a Sybase platform and will finally store and process all data from the entire project. In addition, the system comprises a portfolio of functions needed to support the workflow management of the core facility and the screening groups. MouseNet(c) will make all of the data available to the participating screening groups, and later to the international scientific community. MouseNet(c) will consist of three major software components:* Animal Management System (AMS)* Sample Tracking System (STS)* Result Documentation System (RDS)MouseNet(c) provides the following major advantages:* being accessible from different client platforms via the Internet* being a full-featured multi-user system (including access restriction and data locking mechanisms)* relying on a professional RDBMS (relational database management system) which runs on a UNIX server platform* supplying workflow functions and a variety of plausibility checks.

The 5' part of the mouse immunoglobulin kappa locus.

The 5' region of the mouse kappa locus comprises 63 Vkappa genes in six contigs of together 1.5 Mb, including one which links the region to the central part of the locus. The structures of the contigs were established by detailed restriction mapping of cosmid clones prepared from libraries of mouse C57BL/6 DNA and of yeast and bacterial artificial chromosomes (YACs, BACs with mouse DNA inserts). Pulsed-field gel electrophoresis of yeast artificial chromosome digests indicated that the gaps between the contigs were 10 to 60 kb, comprising together about 160 kb. The region of the kappa locus described here contains Vkappa1, Vkappa2, Vkappa9/10, Vkappa11, Vkappa12/13, Vkappa20, Vkappa24, Vkappa32, Vkappa33/34 and Vkappa38C genes as well as the VkappaRF gene and, towards the center of the locus, a number of Vkappa4/5 genes. Near the 5' end of the locus interspersed alpha-tubulin gene-like sequences were found. At its 3' side the region borders on the Vkappa4/5 contigs of the central region of the locus which is described in the accompanying report (Eur. J. Immunol. 1999. 29: 2057-2064). Structural details are to be found in the Internet at http://www.med.uni-muenchen.de/biochemie/zach au/kappa.htm. In a concluding section the main features of the structure of the mouse kappa locus are summarized.

The variable genes and gene families of the mouse immunoglobulin kappa locus.

In this report 118 mouse Vkappa genes are described which, together with the 22 Vkappa genes reported previously (T. Kirschbaum et al., Eur. J. Immunol. 1998. 28: 1458-1466) amount to 140 genes that had been cloned and sequenced in our laboratory. For 73 of them cDNAs are known, i. e. they have to be considered functional genes, although 10 genes of this group have 1-bp deviations from the canonical promoter, splice site or heptanucleotide recombination signal sequences. Twenty Vkappa genes have been defined as only potentially functional since they do not contain any defect, but no cDNAs have been found (yet) for them. Of the 140 Vkappa genes 47 are pseudogenes. There are indications that two to five Vkappa genes or pseudogenes exist in the kappa locus which we have not yet been able to clone. The 140 Vkappa genes and pseudogenes were assigned to 18 gene families, 4 of them being one-member families. This differs from previous enumerations of the families only by the combination of the Vkappa9 and Vkappa10 families and by the addition of the Vkappa dv gene as a new separate family. Sequence identity usually was 80% or above within the gene families and 55-80% between genes of different families. Many of the mouse Vkappa gene families show significant homologies to the human ones, indicating that in evolution Vkappa gene diversification predated the divergence of the primate and rodent clades.

Clustered and interspersed gene families in the mouse immunoglobulin kappa locus.

Although numerous solitary germ-line V kappa genes and two small V kappa contiguously cloned gene regions (contigs) are known, no attempts to systematically elucidate the structure of the kappa locus of the mouse have been reported so far. As a first step to this aim we screened a cosmid library of C57BL/6J mouse DNA with 18 probes that are more or less specific for the different V kappa gene families. Ninety-one V kappa gene-containing cosmid clones were characterized by detailed restriction mapping and hybridizations. Several contigs were constructed from overlapping clones. The contigs and the still unlinked cosmid clones cover 1.6 Mb. Many of the cosmid clones were localized on chromosome 6 where the kappa locus is known to reside; no evidence for the existence of dispersed V kappa genes (orphons) was obtained. Eighty-five strong hybridization signals were assigned to distinct V kappa gene families, while for 11 weak signals the assignment was less definite. As to the distribution of gene families within the locus the following situation emerged: there are both, groups of genes which belong to one V kappa gene family ("clusters") and groups in which genes of different families are interspersed. The interspersion of gene families seems to be more pronounced than has been assumed so far. Additional V kappa genes which are known to exist will have to be isolated from other gene libraries of the same mouse Ig kappa haplotype.

The human immunoglobulin kappa locus: pseudogenes, unique and repetitive sequences.

The human kappa locus contains 25 pseudogenes. After seven of them were described earlier the structures of the remaining 18 are reported now, thus completing the description of all human V kappa genes and pseudogenes. Most of the pseudogenes carry several defects each. Alignments of the pseudogene sequences and comparison with the consensus sequences of the potentially functional V kappa genes indicate that, on PCR amplification of genomic DNA aimed at certain genes of the latter class, also some of the pseudogenes would be coamplified. Unique sequences, which qualify as sequence tagged sites (STS), were defined across the locus. The occurrence of 15 repetitive elements of the LINE1 type in the locus is described. The 15 sequenced Alu elements were assigned to the known Alu subfamilies of different evolutionary age. One of the Alu elements was found only in one of the copies of the kappa locus. It must, therefore, have been inserted after the duplication step which may have taken place about one million years ago. This element belongs to an Alu subfamily known to have been mobile until recently. Some aspects of the evolution of the V kappa pseudogenes and orphons (i.e. V kappa genes located outside the kappa locus) are also discussed.

The human immunoglobulin kappa locus. Characterization of the partially duplicated L regions.

The L regions are parts of the C kappa proximal (p) and distal (d) copies of the human immunoglobulin kappa locus and are therefore called the Lp and Ld regions. The two regions with their 25 V kappa genes and pseudogenes have now been cloned, thus completing the cloning of the kappa locus. Lp has been linked to the neighboring Ap and B regions, while Ld was linked to Ad. There is good evidence that at the other side of Ld, i.e. towards the centromere, the end of the locus has been reached. Most of the cloning and linking was achieved by chromosomal walking, employing cosmid and phage lambda clones. No such clones could be found for three small gaps. Two of them were closed by a polymerase chain reaction strategy; the third one was characterized by genomic blot hybridization experiments and eventually bridged by a yeast artificial chromosome clone. Early in evolution, a stretch of about 25 kb which comprised three V kappa genes near the 5' end of the L region precursor must have been duplicated, such that the later duplication of large parts of the kappa locus resulted in the appearance of two very similar three-gene regions in each, Lp and Ld. Two deletions in the central parts of the L regions, on the other hand, must have occurred after the duplication of the locus, since they are found in Lp and Ld in different positions.

The V kappa genes of the L regions and the repertoire of V kappa gene sequences in the human germ line.

Only 14 of the 25 V kappa genes and pseudogenes had been found before as parts of the L regions. The cloning and linking described in the accompanying report allowed us now to assign to Lp or Ld some V kappa genes which had been found before on scattered clones. In addition the sequences of several still unknown genes are reported here, thus completing the publication of the V kappa genes of the kappa locus as far as they are potentially functional or have only one or two 1-bp defects. Of the V kappa genes of the kappa locus, 32 are potentially functional, 16 have minor defects, 3 have both potentially functional and slightly defective alleles and 25 are pseudogenes which amounts to a repertoire of 76 V kappa-related gene sequences. The V kappa genes of the L regions are, within the subgroups, particularly similar to each other, which is in part due to common evolutionary origins and in part caused by gene conversion-like events. One donor-acceptor pair could be clearly identified, since converted and not-converted alleles of the acceptor gene were found. In other cases the duplicates of the converted genes served as non-converted controls.

Of orphons and UHOs. Delimitation of the germline repertoire of human immunoglobulin kappa genes.

Two problems in defining the germline repertoire of immunoglobulin kappa genes were investigated. One concerns putative transposed V kappa genes (orphons), the other one weak hybridization signals which may or may not turn out to be V kappa genes (UHOs). It was shown by sequencing that the three V kappa genes Z2, Z3 and Z4 are very closely related to the Z1 and V118 genes and to two other genes which had been localized on chromosomes 1 and 22, i.e. outside the kappa locus on chromosome 2. It is therefore likely that also the Z2-Z4 genes are orphons and not part of the kappa locus. Two UHOs turned out not to contain V kappa-like structures. This together with previous results makes it likely that we have detected all germline V kappa genes with the available hybridization probes.

The human immunoglobulin kappa locus. Characterization of the duplicated A regions.

The central regions of the kappa locus, the so-called A regions, have been fully characterized on cosmid and phage lambda clones. The regions, which are parts of the C kappa-proximal and -distal copies of the locus and are, therefore, called Ap and Ad regions, comprise about 140 kb each and contain together 30 V kappa genes and pseudogenes. The A regions have been linked on their 5' sides to the O regions and on their 3' sides to the L regions. Chromosomal walking has eliminated a previous gap in the Ap region. Detailed restriction maps of the Ap and Ad regions and the sequences of 9 V kappa genes are reported. Four events, which have occurred in evolution probably after the duplication of the A region, were identified: the insertion of an Alu element in Ad; the insertion of part of a LINE element in Ap; the deletion of a 17.5-kb fragment including one V kappa gene from Ap; the sequence divergence of duplicated V kappa gene regions which ranges among the five pairs studied here from 0 to 14 bp per kb and converted two genes to pseudogenes while their duplicates stayed functional. An analysis of the A regions of the lymphoid cell lines RPMI 6140 and GM607 confirmed the previous finding that the V kappa-J kappa rearrangement in these cell lines had occurred by deletion and inversion mechanisms, respectively. Thus, the structural data contribute to the understanding of the evolution and the functioning of the A regions of the kappa locus.

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. IV. The less frequently expressed VL are heterogeneous.

We previously demonstrated that the human anti-Haemophilus influenzae type b polysaccharide (Hib-PS) VL repertoire is dominated by a product of the V kappa II gene, A2, and that V kappa II-A2 anti-Hib-PS antibodies have little or no somatic mutation in VL. To further study this VL repertoire, we studied non-A2 anti-Hib-PS antibodies that were identified either serologically or by amino-terminal amino acid sequence analysis. Of 15 non-A2 anti-Hib-PS antibodies from 12 vaccinated adults, we found four V lambda, five V kappa I, one non-A2 V kappa II, four V kappa III, and one V kappa IV antibodies. As expected, all but two of these subjects also produced V kappa II-A2 antibodies. Interestingly, one of these subjects lacks the A2 gene in the germ line. However, both subjects who did not produce detectable V kappa II antibody did produce normal amounts of total anti-Hib-PS antibody after vaccination. Candidate V kappa genes for the non-A2 antibodies were identified by comparison of up to 60 VL amino acid residues, including CDR1 and CDR2, with all sequenced V kappa genes. V kappa I antibodies appear to be products of three newly sequenced V kappa I genes, O8, O18, and L11, that are reported here. The O8 and O18 genes encode identical amino acid sequences. The non-A2 V kappa II antibody is a likely product of the A1 or A17 genes, the V kappa III antibodies are likely products of the A27 gene, and the V kappa IV antibody is a product of the single V kappa IV gene, B3. Unlike V kappa II-A2 antibodies, the V kappa I, V kappa III, and V kappa IV antibodies differed by one to five CDR residues from the germ line product of the candidate genes, suggesting the presence of somatic mutations. Thus, anti-Hib-PS antibodies can be divided into two types, the most frequently observed A2 antibodies with little or no somatic mutation and non-A2 antibodies that likely contain somatic mutations.

Polymorphisms and haplotypes in the human immunoglobulin kappa locus.

By comparing the restriction patterns of the DNA from 23 unrelated individuals 16 polymorphisms were defined which allowed us to differentiate between the duplicated copies Op, Ap, Lp and Od, Ad, Ld of the kappa locus (p for the C kappa proximal, d for the distal copy). Some of these duplication-differentiating polymorphisms or DDP revealed also allelic differences between individuals; they are therefore restriction fragment length polymorphism (RFLP) markers at the same time. Three RFLP in the single copy B-J kappa-C kappa region were included into the study. Three basic haplotypes were derived from the combined genotype data, haplotypes N, G and 11. The latter haplotype in which the whole distal copy of the kappa locus is missing was found three times among the 46 haploid genomes studied. The genotypes of the family members of an individual who is homozygous for haplotype 11 are consistent with Mendelian inheritance. Haplotypes N and G are distinguished from each other by eight RFLP markers. Six additional haplotypes, which were found in one or several individuals each, can be derived from the basic haplotypes N and G by hypothetical recombination and/or mutation events.

The J kappa proximal region of the human K locus contains three uncommon V kappa genes which are arranged in opposite transcriptional polarities.

The structure of one of the V kappa gene-containing regions of the locus coding for the human immunoglobulin light chains of the kappa type is described. This so-called B region contains three genes: B1, B2 and B3. According to its sequence B1 is a pseudogene which does not fit well into the present subgroup classification. In lymphoid cell lines the B1 gene region is frequently deleted. B2 and B3 are the previously reported EV15 and V kappa IV genes. The transcriptional polarity of the B1 gene is found to be opposite to one of the B2 and B3 genes. This observation together with the fact that the B region is proximal to the J kappa C kappa gene segment leads to the conclusions to the mechanism of the V kappa-J kappa recombination and allows us to explain the formation of the recombination products in a particular cell line by two consecutive inversions.