The tissue polarity gene nemo carries out multiple roles in patterning during Drosophila development.

Drosophila nemo was first identified as a gene required for tissue polarity during ommatidial development. We have extended the analysis of nemo and found that it participates in multiple developmental processes. It is required during wing development for wing shape and vein patterning. We observe genetic interactions between nemo and mutations in the Notch, Wingless, Frizzled and Decapentaplegic pathways. Our data support the findings from other organisms that Nemo proteins act as negative regulators of Wingless signaling. nemo mutations cause polarity defects in the adult wing and overexpression of nemo leads to abdominal polarity defects. The expression of nemo during embryogenesis is dynamic and dsRNA inhibition and ectopic expression studies indicate that nemo is essential during embryogenesis.

Analysis of T cell receptor beta chain expression by isoelectric focusing following gene amplification and in vitro translation.

We describe a new approach to analysis of T cell receptor diversity based on isoelectric focusing of in vitro translation products of amplified V region genes. The method is illustrated by analysis of V beta 2 profiles in peripheral blood lymphocytes from normal donors. The primers used for V beta 2 analysis spanned the V-(D-)J junction and included the segment from amino acid residue position 53 in the variable region to residue 132 of the constant region. The isoelectric focusing patterns display approximately 13-14 bands of varying intensity. Differences in expression of V beta 2-derived peptides were detected in comparisons of the isoelectric focusing profiles from different individuals, suggesting that the method may be useful for detecting genetically determined, immune response related or disease associated differences in Tcr V region expression. The major isoelectric focusing bands have been interpreted as representing groups of V beta 2 sequences sharing J beta region and NDN region charge similarity. Quantitative differences were detected in V beta 2 profiles of CD4 and CD8 T cell subpopulations indicating there may be selection for different charge characteristics in NDNJ sequences in the two T cell subsets. The method provides a new dimension for the detection of perturbations in the T cell repertoire.

Monoclonal hybridoma screening by analysis of immunoglobulin light chains.

A technique is described for the characterization of immunoglobulin light chains of hybridomas in culture. Immunoglobulins biosynthetically labelled with 14C are obtained from culture supernatants. Following complete reduction and alkylation light chains are separated from heavy chains and most other labelled contaminants by urea-formate gel electrophoresis. They are subsequently analyzed by isoelectric focusing using a simple transfer procedure. The method can be used to analyze up to 30 samples at a time and has a potential for the distinction of 750 light chains. The technique is especially useful (1) to determine the monoclonality of antibodies at an early stage in production, (2) to identify and classify antibodies having different structures but similar specificities, (3) to identify any alterations which may occur in quantity or quality of antibodies in long term culture, (4) to identify different hybridomas which produce antibodies of identical light chain subgroup.

Polymorphism of kappa variable region (V kappa-1) genes in inbred mice: relationship to the Ig kappa-Ef2 serum light chain marker.

We describe here the cloning and complete sequence analysis of the rearranged kappa variable region gene from the V kappa-1A-producing myeloma (C.AL20-TEPC-105). A 5'-flanking region probe from the V kappa-1A gene has been used to study the V kappa-1 germ-line gene family in strains of mice differing at the Ig kappa-Ef2 locus. All Ef2a strains examined possess an identical pair of BamHI restriction fragments strongly hybridizing to the 5' probe. Surprisingly, only two of the six strains of mice previously designated Ef2b (NZB and C58) possessed clearly altered restriction fragment sizes for V kappa-1 genes. The remaining four Ef2b strains, namely BDP/J, CE/J, I/LnJ, and P/J, appear to carry V kappa-1 genes similar to those of Ef2a strains. It is suggested that these strains may carry a third form of the V kappa-1A gene, differing in the protein coding region but indistinguishable at the DNA level with the use of BamHI or EcoRI. Alternatively, these strains may fail to express V kappa-1A light chains due to a regulatory defect involving this subgroup of kappa genes.

Recombination between kappa chain genetic markers in the mouse.

In this study we report the first instance of recombination between kappa chain genetic markers in the mouse. The recombination frequency, 0.45% (95% limits, 0.12-1.61), is similar to that previously found for recombination between the kappa chain locus and the Lyt-2,3 locus (0.3%, 95% limits, 0.05-1.6), but is relatively low in comparison with that found at the heavy chain locus (0.41-5.4%). Lyt-2,3-typing of the recombinants permits a partial ordering of the kappa chain and Lyt-2,3 loci as (Lyt-2,3, Igk-Ef1) - Igk-Ef2. Light chains controlled by the two kappa markers include the Vk-(ser) subgroup (controlled by Igk-Ef1) and Vk-1 (controlled by Igk-Ef2). One of the recombinants has been recovered in a homozygous state ("NAK") and should be suitable for Vk gene mapping studies.

Recombination between kappa chain genetic markers and the Lyt-3 locus.

Recombination has been detected for the first time between chromosome 6 loci controlling kappa chain expression in normal mouse serum immunoglobulin and the Lyt-3 locus. The recombination event occurred at the 26th or 27th backcross generation during the derivation of the Lyt-2a, Lyt-3a-congenic line B6.PL(85NS). The line is now homozygous for the Lyt-2a, Lyt-3a allele(s) at N30F13 and homozygous animals express the Igk-Ef1b allele derived from C57BL/6. The frequency of recombination has been estimated to be 0.30% based on the present results and previous studies in which no recombination was detected. The results rule out the hypothesis that the Lyt-3 locus itself controls the light chain phenotype observed in normal serum immunoglobulin.

Ef2: a new LY-3-linked light-chain marker expressed in normal mouse serum immunoglobulin.

A new light-chain marker has been detected in normal mouse serum immunoglobulin light chains by gel isoelectric focusing. The marker (Ef2) involves the presence of two major and several minor bands in the normal light-chain IF profiles. Strains expressing the marker IF bands are designated Igk-Ef2a, whereas those lacking the bands are Igk-Ef2b. The majority of inbred strains are Igk-Ef2a. Strains found to be Igk-Ef2b are NZB/BlNJ, BDP/J, C58/J, I/LnJ, CE/J, and P/J. The strain distribution of the alleles differs from the distribution of alleles at the Ly-2 and Ly-3 loci, suggesting the new marker may represent a separate locus. Genetic studies have shown that Igk-Ef2 locus is closely linked to Igk-Ef1 and Hd loci on Chromosome 6, indicating that it is also closely linked to Ly-3. The relative importance of the bands controlled by the Igk-Ef2 locus suggests that the entire normal light-chain pool could be controlled by as few as 100 such loci.