Behavioural analysis of congenic mouse strains confirms stress-responsive Loci on chromosomes 1 and 12.

The way in which animals respond to stressful environments correlates with anxiety-related behaviour. To begin identifying the genetic factors that influence anxiety, we have studied the stress-responsiveness of inbred mouse strains using a modified form of the open field activity test (OFA), termed the elevated (e) OFA. In particular, two strains show high (DBA/2J) or low (C57BL/6J) stress-responsiveness in the eOFA. Genetic studies of an F(2) intercross between these two strains previously identified two regions, on chromosomes (Chr) 1 and 12, linked to anxiety-related behaviour. To confirm that these regions contain loci for stress-responsiveness, we established separate congenic mouse strains for the linked Chr1 and Chr12 regions. Each congenic strain harbours a DBA/2J-derived interval encompassing the linked region on the C57BL/6J genetic background: the congenic intervals are between, but not including approximately 48.6 Mb and approximately 194.8 Mb on Chr1, and approximately 36.2 Mb and the distal end of Chr12. Cohorts of DBA/2J, C57BL/6J and congenic mice were analysed for a series of stress-responsive phenotypes using the eOFA test. Both congenic strains had significantly different stress-responsive phenotypes compared to the low-stress C57BL/6J parental strain, but the DBA/2J-derived Chr12 interval had a greater genetic effect than the DBA/2J-derived Chr1 interval for changing the behavioral phenotype of the parental C57BL/6J mouse strain. These results confirmed the presence of stress-responsive loci on Chr1 and Chr12. New stress-related phenotypes were also identified, which aided in comparing and differentiating DBA/2J, C57BL/6J and congenic mice.

Molecular cloning of a C-type lectin superfamily protein differentially expressed by CD8alpha(-) splenic dendritic cells.

Dendritic cells (DC) are potent antigen presenting cells that activate naive T cells. It is becoming increasingly clear that DC are not a homogeneous cell population, but comprise different subpopulations that differ in ontogeny and function. To further the molecular characterisation of DC, we screened for genes that were differentially expressed amongst DC subsets and could therefore give insight into their varying biological functions. Using Representational Difference Analysis (RDA) we identified a gene (CIRE) that is expressed at higher levels in the myeloid-related CD8alpha(-) DC than in the lymphoid-related CD8alpha(+) DC. CIRE is a 238 amino acid type II membrane protein, of approximately 33 kDa in size, whose extracellular region contains a C-type lectin domain. Northern blot analysis revealed that CIRE is almost exclusively expressed in DC and was not detected in organs such as heart, brain, kidney, liver, and thymus. T cells failed to express message for CIRE, whilst B cells expressed very low levels. These data here further substantiated by Northern blot analysis of 18 cell lines of various origins (myeloid, macrophage, B and T cell) where only one cell line, which was of myeloid origin and could give rise to DC, expressed mRNA for CIRE. Semi-quantitative RT-PCR suggested that CIRE is down-regulated upon activation. CIRE shares 57% identity with human DC-SIGN, a molecule that has been shown to be the ligand of ICAM-3 and that is also a receptor that binds HIV and facilitates trans-infection of T cells.

Cloning and characterization of the genes encoding the ankyrin repeat and SOCS box-containing proteins Asb-1, Asb-2, Asb-3 and Asb-4.

Members of the suppressor of cytokine signalling (SOCS) family of proteins have been shown to inhibit cytokine signalling via direct interactions with JAK kinases or activated cytokine receptors. In addition to their novel amino-terminal regions and SH2 domains that mediate these interactions, the SOCS proteins also contain carboxy-terminal regions of homology called the SOCS box. The SOCS box serves to couple SOCS proteins and their binding partners with the elongin B and C complex, possibly targeting them for degradation. Several other families of proteins also contain SOCS boxes but differ from the SOCS proteins in the type of domain or motif they contain upstream of the SOCS box. We report here the cloning, characterization, mapping and expression analysis of four members of the ankyrin repeat and SOCS box-containing (Asb) protein family.

Cloning, expression analysis, and chromosomal localization of murine and human homologues of a Xenopus mix gene.

We report the cloning and chromosomal localization of murine and human Mix genes, members of a subclass of paired-like homeobox genes of which the Xenopus laevis Mix.1 gene is the founding member. The murine Mix gene was mapped to the distal region of chromosome 1 and the human region to the syntenic region 1q41-42. Northern analysis and RT-PCR of murine adult and embryonic tissues demonstrated that Mix expression was restricted to the early embryo. Whole-mount in situ hybridization revealed patchy but symmetrical Mix expression in visceral endoderm of embryonic day (E)5.5 embryos. In slightly older embryos, the expression was skewed to one side of the embryo and by E6.5, at the onset of gastrulation, expression was seen in the epiblast, visceral endoderm, nascent mesoderm, and the primitive streak. This expression pattern was maintained in mid- and late-streak embryos. In early bud-stage embryos, expression was strongest in the proximal two thirds of the streak, extending to the base of the allantois. By the headfold-stage, expression was confined to the remnant of the primitive streak in the caudal region of the embryo and, after E8.0, in the caudal notochord and tail bud mesoderm. Mix transcripts were no longer detectable after embryonic day 9.5.

Localization of Idd11 using NOD congenic mouse strains: elimination of Slc9a1 as a candidate gene.

Type 1 diabetes is a multigenic autoimmune disease, the genetic basis for which is perhaps best characterized in the nonobese diabetic (NOD) mouse model. We previously located a NOD diabetes susceptibility locus, designated Idd11, on mouse Chromosome (Chr) 4 by analyzing diabetic backcross mice produced after crossing NOD/Lt with the nondiabetic resistant strain C57BL/6 (B6) strain. In order to confirm Idd11 and further refine its location, three NOD congenic mouse strains with different B6 derived intervals within Chr 4 were generated. Two of the congenic strains had a significant decrease in the cumulative incidence of diabetes compared with NOD/Lt control mice. The third NOD congenic strain, containing a B6 interval surrounding the Slc9a1 locus, was not protected against diabetes. These results define a new distal boundary for Idd11 and eliminate the Slc9a1 gene as a candidate. The Idd11 locus has now been definitively mapped to a 13cM interval on mouse Chr 4.

Construction and characterization of two anti-sweetener single chain antibodies using radioligand binding, fluorescence and circular dichroism spectroscopy.

Two single-chain antibodies (scFv) that bind the superpotent sweetener ligand, NC-174, were generated from mouse monoclonal antibodies (mAb) NC6.8 (IgG, kappa) and NC10.14 (IgG, lambda). These scFv were constructed by cloning the variable region sequences of the mAb, connecting them in tandem with a 25-amino-acid polypeptide linker, and expressing them in E. coli using the pET-11a system. The recombinant proteins were purified using Ni(2+)-NTA-agarose by virtue of a hexahistidine sequence introduced to the C-terminus of the heavy chain variable region during the cloning process. The secondary structure and ligand binding properties of the two scFv, the parent mAbs and proteolytically derived Fab fragments were examined using radioligand binding, circular dichroism (CD) and fluorescence spectroscopy. The far-UV CD spectra of both scFv possessed predominantly beta character, as did those of the Fab, and the near-UV CD spectral data for scFvNC10.14, NC6.8 and NC10.14 Fab indicated that chromophore perturbation occurred upon ligand binding. The affinity constants determined for the two scFv, Fab and mAb were nearly equivalent.

Alanine scanning mutagenesis of an alphabeta T cell receptor: mapping the energy of antigen recognition.

The T cell receptor (TCR) from the alloreactive T lymphocyte 2C recognizes a nonamer peptide QL9 complexed with the MHC class I molecule H2-Ld. Forty-two single-site alanine substitutions of the 2C TCR were analyzed for binding to QL9/Ld and anti-TCR antibodies. The results provided a detailed energy map of T cell antigen recognition and indicated that the pMHC and clonotypic antibody epitopes on the TCR were similar. Although residues in each Valpha and Vbeta CDR are important in binding pMHC, the most significant energy for the TCR/QL9/Ld interaction was contributed by CDRs 1 and 2 of both alpha and beta chains. The extent to which the individual energy contributions are directed at class I helices or peptide was also assessed.

Reactivity and epitope mapping of single-chain T cell receptors with monoclonal antibodies.

To examine further the structure of the T cell receptor (TCR) and the specificity of mAbs generated against the native protein, the TCR was expressed in Escherichia coli as a single chain in which the variable regions of the alpha and beta chains are joined by a 25 amino acid linker. Five single-chain TCR that have different alpha and/or beta variable (V) regions were examined with the anti-V beta 8 region mAbs KJ16 and F23.1 and the anti-V alpha 8 mAbs KT50, KT65 and B21.14. Each of the mAbs reacted with one or more of the single-chain receptors. Western blot analysis demonstrated that the intrachain disulfide bonds were required for proper epitope conformation and recognition of the TCR by the antibodies. KT50, KT65 and B21.14 antibodies distinguished between two related V alpha regions that differed at only six residues. A model of the V regions of the TCR based on immunoglobulin (Ig) structure suggests that three of these six variant residues are in the putative CDR1 of the receptor and possibly accessible to antibody. To test this possibility, site-directed mutagenesis of the unreactive V alpha region demonstrated that the combination of all three residues restored binding by the anti-V alpha 8 antibodies. In addition, these three complimentarity determining regions (CDR) residues are likely to be in close proximity to the putative CDR3 which also influenced binding of the antibodies. The epitopes recognized by the V alpha-specific antibodies are thus predicted to reside closer to the putative binding site than the epitopes previously determined to be recognized by the anti-V beta 8 antibodies, KJ16 and F23.1. Finally, the specificities of KT50 and KT65 as determined with the E. coli expression system suggests an explanation for previous observations about the differences in the T cell populations that are recognized by these antibodies.