More than 150 novel variants of HLA class I genes detected in German Stem Cell Donor Registry and UCLA International Cell Exchange samples.

High throughput analysis using amplicon-based next-generation sequencing (NGS) of HLA class I genes in samples of registered stem cell donors of the German Stem Cell Donor Registry Düsseldorf revealed 151 novel variants. In addition, four new variants were identified in well-defined samples obtained from the UCLA International Cell Exchange program. New alleles included 37 HLA-A, 57 HLA-B, and 61 HLA-C variant alleles. All variants were confirmed by NGS of HLA-A, HLA-B, and HLA-C genes including the respective 5' and 3' untranslated regions as well as Sanger sequence analysis. Mainly, the variants encompass single nucleotide changes in intronic as well as exonic parts of the genes. We identified intronic variations in 114 new alleles, nonsynonymous nucleotide changes in 25 alleles, synonymous nucleotide changes in nine alleles, and three hybrid alleles. Four alleles carry exonic deletions or insertions resulting in frameshift of peptide translation. Two novel alleles of HLA-C were shown to result in splicing defects of the transcript. Two alleles showed exonic as well as intronic changes. Thirty-four of the new alleles were found in multiple samples.

Organisation of regulatory elements in two closely spaced Drosophila genes with common expression characteristics.

Sperm tail proteins that are components of a specific structure formed late during spermatid elongation have been found to be encoded by the Mst(3)CGP gene family. These genes have been demonstrated to be regulated both at the transcriptional as well as at the translational level. We report here on the dissection of the regulatory regions for two members of the gene family, Mst84Da and Mst84Db. While high level transcription and negative translational control of Mst84Da is mediated by a short gene segment of 205 nt (-152/+53), Mst84Db expression is controlled by a number of distinct regulatory elements with different effects that all reside within the gene itself. We identify a transcriptional control element between +154 and +216, a translational repression element around +216 to +275 and an RNA stability element within the 3'UTR. Irrespective of the final common expression characteristics, correct regulation for any individual member of the gene family seems to be achieved by very different means. This confirms earlier observations that did not detect any other sequence elements in common apart from the TCE (translational control element).

Establishment and characterization of four cell lines derived from human head and neck squamous cell carcinomas for an autologous tumor-fibroblast in vitro model.

To study interactions between tumor cells and stromal elements, we established carcinoma cell lines as well as tumor-derived and skin fibroblast cultures from four patients with squamous cell carcinoma of the head and neck. For the characterization of the tumor cell lines we a) determined population doubling times, b) assessed morphological features by light and electron microscopy, c) investigated the expression of typical markers by immunohistochemistry, including various intermediate filaments and surface antigens, d) compared these findings with expression patterns in the respective original tumor specimens, e) evaluated p53 mutations in tumor specimens and cell lines, f) performed chromosome analysis, g) investigated the tumorigenicity in athymic mice, and h) tested the formation of both tumor and mixed tumor-fibroblast multicellular spheroids. Tumor cell cultures were considered established cell lines when maintained and passaged over a period of two years after primary explantation. The in vitro morphology of the cell lines showed well preserved characteristics of squamous cell carcinoma, and electron microscopy as well as immunohistochemistry revealed their squamous type of differentiation. All cell lines presented the same p53 genotype as the respective original tumors. Furthermore, they were successfully xenotransplanted into nude mice and formed both pure and mixed three dimensional spheroids. This experimental model allows the in vitro and in vivo investigation of various tumor-fibroblast interactions.

Mpv37 mouse strain--a model for the relationship between the kidney and the inner ear.

Using the Mpv17-negative mouse strain, which developed inner ear and kidney dysfunction, we confirm a strong relationship between the kidney and the inner ear. Both organs have specialized epithelia involved in active ion transport, which are separated from the vessels by a basement membrane of similar composition. Our recent results indicate that the glomerular and the stria vascularis basement membrane are simultaneously affected in early stages. Concomitant deposits of IgG during the progressive development of the disease support the idea of a shared antigen. Understanding the pattern of the development of the degeneration will provide a basis towards understanding the essential role of the Mpv17 protein in the structures of both organs and may provide a basis for future therapeutic intervention.