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Mrjp1 gene belongs to the honeybee mrjp gene family encoding the major royal jelly proteins (MRJPs), secreted by nurse bees into the royal jelly. In this study, we have isolated the genomic clone containing the entire mrjp1 gene and determined its sequence. The mrjp1 gene sequence spans over 3038 bp and contains six exons separated by five introns. Seven mismatches between the mrjp1 gene sequence and two previously independently published cDNA sequences were found, but these differences do not lead to any change in the deduced amino acid sequence of MRJP1. With the aid of inverse polymerase chain reaction we obtained sequences flanking the 5' ends of other mrjp genes (mrjp2, mrjp3, mrjp4 and mrjp5). Putative promoters were predicted upstream of all mrjp genes (including mrjp1). The predicted promoters contain the TATA motif (TATATATT), highly conserved both in sequence and position. Ultraspiracle (USP) transcription factor (TF) binding sites in putative promoter regions and clusters of dead ringer TF binding sites upstream of these promoters were predicted computationally. We propose that USP, as a juvenile hormone (JH) binding TF, might possibly act as a mediator of mrjp expression in response to JH. Mrjp1's genomic locus is predicted to encode an antisense transcript, partially overlapping with five mrjp1 exons and entirely overlapping with the putative promoter and predicted transcriptional start point of mrjp1. This finding may shed light on the mechanisms of regulation of mrjps expression. Southern blot analysis of genomic DNA revealed that all so far known members of mrjp gene family (mrjp1, mrjp2, mrjp3, mrjp4 and mrjp5) are present as single-copy genes per haploid honeybee genome. Although MRJPs and the yellow protein of Drosophila melanogaster share a certain degree of similarity in aa sequence and although it has been shown that they share a common evolutionary origin, neither structural similarities in the gene organization, nor significant similarities between intron sequences of mrjp1 gene and fourteen yellow-like genes of D. melanogaster were found.

13 páginas, 2 figuras, 3 tablas.-- et al., Human chromosome Xp11.3–Xp11.23 encompasses the map location for a growing number of diseases with a genetic basis or genetic component. These include several eye disorders, syndromic and nonsyndromic forms of X-linked mental retardation (XLMR), X-linked neuromuscular diseases and susceptibility loci for schizophrenia, type 1 diabetes, and Graves' disease. We have constructed an ∼2.7-Mb high-resolution physical map extending from DXS8026 to ELK1, corresponding to a genetic distance of ∼5.5 cM. A combination of chromosome walking and sequence-tagged site (STS)-content mapping resulted in an integrated framework and transcript map, precisely positioning 10 polymorphic microsatellites (one of which is novel), 16 ESTs, and 12 known genes (RP2, PCTK1, UHX1, UBE1, RBM10, ZNF157, SYN1, ARAF1, TIMP1, PFC, ELK1, UXT). The composite map is currently anchored with 89 STSs to give an average resolution of ∼1 STS every 30 kb. By a combination of EST database searches and in silico detection of UniGene clusters within genomic sequence generated from this template map, we have mapped several novel genes within this interval: a Na+/H+ exchanger (SLC9A7), at least two zincfinger transcription factors (KIAA0215 and Hs.68318), carbohydrate sulfotransferase-7 (CHST7), regucalcin (RGN), inactivation-escape-1 (INE1), the human ortholog of mouse neuronal protein 15.6, and four putative novel genes. Further genomic analysis enabled annotation of the sequence interval with 20 predicted pseudogenes and 21 UniGene clusters of unknown function. The combined PAC/BAC transcript map and YAC scaffold presented here clarifies previously conflicting data for markers and genes within the Xp11.3–Xp11.23 interval and provides a powerful integrated resource for functional characterization of this clonally unstable, yet gene-rich and clinically significant region of proximal Xp., We thank the Guide Dogs for the Blind Association (D. L. T.) and The Wellcome Trust (A. J. H.) for funding this research. A. M. was supported by a grant from the German Ministry for Education and Research.

X-linked cone-rod dystrophy (COD1) is a retinal disease that primarily affects the cone photoreceptors; the disease was originally mapped to a limited region of Xp11.4. We evaluated the three families from our original study with new markers and clinically reassessed all key recombinants; we determined that the critical intervals in families 2 and 3 overlapped the RP3 locus and that a status change (from affected to probably unaffected) of a key recombinant individual in family 1 also reassigned the disease locus to include RP3 as well. Mutation analysis of the entire RPGR coding region identified two different 2-nucleotide (nt) deletions in ORF15, in family 2 (delAG) and in families 1 and 3 (delGG), both of which result in a frameshift leading to altered amino acid structure and early termination. In addition, an independent individual with X-linked cone-rod dystrophy demonstrated a 1-nt insertion (insA) in ORF15. The presence of three distinct mutations associated with the same disease phenotype provides strong evidence that mutations in RPGR exon ORF15 are responsible for COD1. Genetic heterogeneity was observed in three other families, including the identification of an in-frame 12-nt deletion polymorphism in ORF15 that did not segregate with the disease in one of these families.

We searched for novel genes as candidates of X-linked dystonia parkinsonism (XDP) in the critical interval of Xq13.1 that harbors the disease locus (DYT3). A gene, ACRC (acidic repeat containing), was discovered by a combination of in silico and "wet" experiments. ACRC is composed of at least 12 exons and 11 introns. It is expressed in all tissues tested, including skeletal muscle, liver, kidney, pancreas, heart, lung, and brain. Highest levels of expression are found in skeletal muscle. The ACRC protein is characterized by a previously undescribed acidic repeat tract of 21 units of 8–10 amino acids. The N-terminal portion of the protein is highly acidic (pI=3.2), and the C-terminal region is basic (pI=10.2). There are nuclear localization signals in its C-terminal portion. Extensive mutation analysis of the transcribed region of the gene, including intron-exon boundaries and the 5' and 3' untranslated intervals, did not reveal a mutation in XDP patients. Exclusion of a mutation in the transcribed portion of this and all other known genes within the DYT3 critical interval suggests that XDP is most likely caused by a mutation in a regulatory region of a gene within the critical interval, or by a structural rearrangement.

A number of distinct, partly non-overlapping genetic loci have been reported for the complete type of X-linked congenital stationary night blindness (CSNB1), suggesting genetic heterogeneity. In order to refine the localization of the CSNB1 gene and to demonstrate genetic homogeneity, linkage analysis was performed in two large CSNB1 families. Clinical features consistent with the diagnosis of CSNB1 were documented in five patients from a German seven-generation kindred by full ophthalmological examination including psychophysical and electroretinographical testing. Haplotype analysis in 30 members of the large German family was performed with 38 polymorphic markers predominantly covering the critical region. Linkage analyses defined a locus for CSNB1 with flanking markers DXS8042 and DXS228, refining the interval to 2.5 cM in Xp11.4. In addition, two-point linkage analysis was carried out using the MLINK computer program. In agreement with meiotic breakpoints, lod scores of 3.0 and greater were obtained for markers located to the proximal site of the former 5 cM CSNB consensus interval. A large Dutch CSNB1 family was re-evaluated with markers from the Xp11.4 region, and supports the CSNB1 minimal interval found in the German family. Together with previous results from three unrelated families from Sweden, Sardinia and Great Britain, our results provide evidence of genetic homogeneity in the disorder. Subsequent mutation analyses in CSNB1 patients revealed no pathogenic sequence alterations in DFFRX and CASK genes, but retain candidates for other diseases mapping to that region.

Item does not contain fulltext Retinitis pigmentosa 3 (RP3) is a progressive retinal degeneration due to mutations in the X-linked RPGR gene. Transcription studies in human and mouse tissues have revealed ubiquitously expressed transcripts and also an exceptional high number of tissue-specific alternative splice variants. However, regulation of tissue-specific expression and splicing is unclear, but this is of particular interest as mutations in this ubiquitously expressed gene lead to severe retinal degeneration, while other tissues are unaffected. To elucidate the conservation pattern of RPGR and to identify additional tissue-specific exons and putative regulatory elements we performed comparative genomic sequencing of the human and mouse RPGR gene. Each of the genes spans a region of nearly 59 kb, and all previously identified exons are conserved between the two species. DNA sequence comparison identified 28 conserved sequence elements (CSEs) in introns, upstream of exon 1, within the promotor region, and downstream of the most 3' exon. Some of the intronic CSEs flank tissue-specific exons and therefore may represent important regulatory elements for alternative splicing. Comparative northern blot hybridization of ubiquitous and tissue-specific RPGR probes identified high molecular weight transcripts with similar expression patterns in both human and mouse. These transcripts range from 6 to 15 kb in size and suggest the presence of additional transcribed sequences within RPGR. Our cross-species sequence comparison enables us to define candidate regions that may explain these large transcripts and will therefore contribute to the understanding of RPGR expression and splicing.

Udgivelsesdato: 2000-Nov-15 Studies of chromosomal losses at 17p13 have suggested the presence of at least two distinct regions for tumor suppressor genes, the TP53 region at 17p13.1 and a more distal region at 17p13.3. Within the latter region, Hypermethylated in Cancer 1 (HIC1) is located, a likely candidate for a tumor suppressor gene that has also been suggested to play a role in the pathogenesis of Miller-Diecker syndrome (MDS). However, single-gene isolation efforts have retrieved additional genes from 17p13.3 that could play a role in tumorigenesis. This indicates that the full potential of this chromosomal region with respect to disease-related genes has not yet been exhausted and that there may exist still unknown genes that contribute to tumorigenesis or to the complex MDS phenotype. To provide a basis for the systematic isolation and evaluation of such genes, we established a physical map over 1.5 Mb of 17p13.3 and assigned 29 transcriptional units within this region.

Hailey-Hailey disease (HHD) is an autosomal dominant skin disorder characterized by suprabasal cell separation (acantholysis) of the epidermis. Previous genetic linkage studies localized the gene to a 5 cM interval on human chromosome 3q21. After reducing the disease critical region to

Large-scale genomic sequencing projects generally rely on random sequencing of shotgun clones, followed by different gap closing strategies. To reduce the overall effort and cost of those projects and to accelerate the sequencing throughput, we have developed an efficient, high throughput oligonucleotide fingerprinting protocol to select optimal shotgun clone sets prior to sequencing. Both computer simulations and experimental results, obtained from five PAC-derived shotgun libraries spanning 535 kb of the 17p11.2 region of the human genome, demonstrate that at least a 2-fold reduction in the number of sequence reads required to sequence an individual genomic clone (cosmid, PAC, etc.) can be achieved. Treatment of clone contigs with significant clone overlaps will allow an even greater reduction.

Tumour-specific splicing is known to contribute to cancer progression. In the case of the L1 cell adhesion molecule (L1CAM), which is expressed in many human tumours and often linked to bad prognosis, alternative splicing results in a full-length form (FL-L1CAM) and a splice variant lacking exons 2 and 27 (SV-L1CAM). It has not been elucidated so far whether SV-L1CAM, classically considered as tumour-associated, or whether FL-L1CAM is the metastasis-promoting isoform. Here, we show that both variants were expressed in human ovarian carcinoma and that exposure of tumour cells to pro-metastatic factors led to an exclusive increase of FL-L1CAM expression. Selective overexpression of one isoform in different tumour cells revealed that only FL-L1CAM promoted experimental lung and/or liver metastasis in mice. In addition, metastasis formation upon up-regulation of FL-L1CAM correlated with increased invasive potential and elevated Matrix metalloproteinase (MMP)-2 and -9 expression and activity in vitro as well as enhanced gelatinolytic activity in vivo. In conclusion, we identified FL-L1CAM as the metastasis-promoting isoform, thereby exemplifying that high expression of a so-called tumour-associated variant, here SV-L1CAM, is not per se equivalent to a decisive role of this isoform in tumour progression.