Homologous recombination between transferred and chromosomal immunoglobulin kappa genes.

Homologous recombination between transferred and chromosomal DNAs provides a means of introducing well-defined, predetermined changes in the chromosomal genes. Here we report that this approach can be used to specifically modify the immunoglobulin genes in mouse hybridoma cells. The test system is based on the Sp6 hybridoma, which synthesizes immunoglobulin M (kappa) specific for the hapten 2,4,6-trinitrophenyl (TNP). As recipient cells, we used the Sp6-derived mutant hybridoma igk14, which has a deletion of the kappa TNP gene and consequently does not synthesize TNP-specific immunoglobulin M. igk14 retains the mu TNP gene and two additional rearranged kappa genes, denoted kappa M21B1 and kappa M21G. As a transfer vector, we used pSV2neo bearing the functionally rearranged TNP-specific V kappa segment. Following DNA transfer by electroporation, we isolated rare transformants which produced normal amounts of the functional kappa TNP chain. Analysis of the DNA of these transformants indicated that in all cases, a functional kappa TNP gene had been formed as the result of a homologous integrative recombination event with the igk14 kappa M21B1 gene. These results suggest that homologous recombination might be used for mapping and introducing immunoglobulin gene mutations and for more conveniently engineering specifically altered immunoglobulins.     

P-element-mediated enhancer detection allows rapid identification of developmentally regulated genes and cell specific markers in Drosophila

We have employed a new technique in Drosophila that allows in vivo detection of genomic regulatory elements using a beta-galactosidase reporter gene. A translational fusion of the reporter gene to the P-transposase gene, which is encoded by the P-transposon of Drosophila, places the expression of beta-galactosidase under the control of the weak P-transposase promoter. Flies carrying single insertions of this P-element construct at different locations in the Drosophila genome frequently stain for beta-galactosidase activity in a temporally and spatially restricted fashion in embryos, larvae and adult ovaries, reflecting the influence of nearby genomic regulatory elements on the P-transposase promoter. This technique is a powerful tool as it can be used to produce very many different cell markers and to isolate developmentally regulated genes in Drosophila. We discuss the implications of our results and the applications of the technique to further the study of Drosophila development.     

Determinant differences between the rabbit and mouse immunoglobulin kappa enhancers impair the activity of the rabbit enhancer in mouse myeloma cells.

Enhancer activity of the rabbit immunoglobulin kappa light chain gene intron conserved region (KICR) was examined in mouse myeloma cells using transient expression experiments. Compared to the homologous region of the mouse kappa light chain gene, the rabbit KICR shows nearly no stimulatory effect on expression of the indicator gene, cat. Experiments with mouse-rabbit chimeric KICRs indicated that differences in the region around the NF-kappa B binding site are responsible for the impaired activity of the rabbit KICR whereas mouse sequences covering the kappa E2 and kappa E3 motifs can be replaced by the equivalent rabbit fragment without affecting enhancer function. Creation of a perfect mouse NF-kappa B target sequence in the rabbit gene only partially restores enhancer activity. Furthermore, mouse and rabbit DNA fragments encompassing the NF-kappa B target sequence behave in an identical manner in an electrophoretic mobility shift assay. The results indicate species-related functional differences in the immunoglobulin kappa light chain gene enhancer and suggest that although the NF-kappa B binding site plays a crucial role in enhancer activity surrounding gene elements are also necessary for full enhancer effect.     

Seed-specific,developmentally regulated genes of peanut

Four cDNAs of seed-specific and developmentally regulated peanut (Arachis hypogaea L.) genes were identified by differential screening of a peanut-seed cDNA library using cDNA probes constructed from mRNAs isolated from immature and mature stages of the seed. Northern analysis, probed with the four cloned cDNAs, indicated that the genes represented by two cDNAs were expressed abundantly early in seed development, while another two were abundantly expressed later at the cell-expansion stages of seed development. These four genes did not show expression in roots, pegs or leaves. However, one of the early expressed genes was seed coat-specific. One of the clones, Psc11, had significant sequence similarity to subtilisin-like genes in Arabidopsis and soybean. Clones Psc32 and Psc33 had significant similarity to the peanut allergen genes Ara h II and Ara h 6, respectively. The sequence of clone Psc12 was unique and did not show significant similarity to any sequence in the databases. One of the four seed-specific clones showed restriction fragment length polymorphism (RFLP) among peanut lines representing the four peanut botanical varieties. These findings indicate that polymorphism exists in peanut seed-storage genes. This contrasts with other genes previously used for genetic mapping of cultivated peanut. Received: 1 September 2000 / Accepted: 4 May 2001     

Structure of two developmentally regulated Dictyostelium discoideum ubiquitin genes.

A previously isolated cDNA clone, pLK229, that is specific for mRNA developmentally expressed during Dictyostelium discoideum spore germination and multicellular development, was used to screen two genomic libraries. Two genomic sequences homologous to pLK229 were isolated and sequenced. Genomic clone p229 is identical to the cDNA clone pLK229 and codes for a polypeptide of 381 amino acids. This polypeptide is composed of five tandem repeats of the same 76-amino-acid sequence. Clone lambda 229 codes for a protein of 229 amino acids, containing three tandem repeats of the identical 76-amino-acid sequence. A computer search for homology to known proteins revealed that the 76-amino-acid repeat was identical to human and bovine ubiquitin except for two amino acid differences.     

The reported germline repertoire of human immunoglobulin kappa chain genes is relatively complete and accurate

We describe a bioinformatic analysis of germline and rearranged immunoglobulin kappa chain (IGK) gene sequences, performed in order to assess the completeness and reliability of the reported IGK repertoire. In contrast to the reported heavy-chain gene repertoire, which includes many dubious sequences, only five IGK variable gene (IGKV) alleles appear to have been reported in error. There was, however, insufficient evidence to justify removing these IGKV genes from the germline repertoire. Bioinformatic analysis of apparent mismatches between reported germline genes and 1,863 expressed IGK sequences suggested the existence of two unreported IGKV polymorphisms. Genomic screening of 12 individuals led to the confirmation of both of these polymorphisms, IGKV1-16*02 and IGKV2-30*02. We also show that in contrast to the heavy chain, the IGK repertoire is dominated by sequences that use just a handful of kappa variable (IGKV) and junction (IGKJ) gene pairs. There is also little modification of IGKV and IGKJ genes by the processes of exonuclease removal and N nucleotide addition. The expressed IGK repertoire therefore lacks diversity and the junction region is particularly constrained. Remarkably, the analysis of a dataset of 435 relatively unmutated rearranged kappa genes showed that ten amino acid sequences account for almost 10% of the rearrangements, with identical sequences being derived from as many as seven independent sources. Such dominant sequences are likely to have important roles in the operation of the humoral immune response.     

Isolation of developmentally regulated genes in Drosophila melanogaster

We used a molecular approach to search for maternally expressed genes in Drosophila melanogaster. The relative merits of differential and competition screens were analyzed in a series of reconstruction experiments using either purified phage plaques or derivative DNA sequences. In the course of this study, we isolated 5 clones whose RNA level varies during early embryogenesis. Three gastrula differential clones, b4, b8 and d3, are present in numerous copies in the genome; clone b4 hybridizes with the copia-like B104 repetitive sequence described by Scherer et al. We also isolated 2 maternally-expressed genes, not previously identified in either classical genetic or similarly molecular-based screens. These clones, b11 and d6, map at cytogenetic positions 98F and 4F respectively, on the polytene chromosome map.