Identification of a new gene encoding pericentromeric dodeca-satellite binding protein in Drosophila melanogaster.
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Dodeca-satellite (CCCGTACTCGGT)n is a type of tandemly repeated DNA sequence located in the pericentromeric region of the third chromosome of Drosophila melanogaster and that cross-hybridizes with DNA from other species such as Arabidopsis, mouse and human. This evolutionary conservation suggests that dodeca-satellite might play an important role in the centromeric function. Therefore, the aim of our research was the isolament of genes encoding proteins that might help stabilize these DNA structures, in vivo. To identify D. melanogaster sequence DNAs encoding dodeca-satellite binding proteins, we used the in vivo yeast assay, known as 'one-hybrid system'. Here, we identified a novel gene sequence that encoded pericentromeric dodeca-satellite binding protein and described its sequence characteristics. (+info)
Genetic fingerprinting in mouthwashes of patients after allogeneic bone marrow transplantation.
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Detection of chimerism by PCR analysis of short tandem repeats (STR) in blood samples of patients who received allogeneic bone marrow transplantation (BMT) has proved to be an important method for early detection of relapse. The prerequisite for this type of analysis is knowledge of donor and recipient pretransplantation genotypes. In some cases, recipient cells from time points prior to BMT are not available and the pretransplant fingerprint cannot be determined. As BM recipients only alter their genotype in blood cells, we attempted to identify patient's pretransplantation genotypes after transplantation in mouthwash samples that contain easily accessible epithelial cells. Of 17 patients who had undergone BMT between one week and 45 months prior to analysis, DNA was isolated from mouthwash cell pellets or from epithelial cells obtained from mouthwashes. PCR analysis of STR loci in the von Willebrand and the tyrosine hydroxylase genes were performed. Even though the mouthwash cell pellets contained about 75% epithelial cells (presumably of recipient origin) and only about 25% leukocytes (presumably of donor origin), three of five patients showed donor genotype and only two patients exhibited chimeric DNA patterns, when cellular DNA was obtained by boiling of mouthwash cell pellets. Following phenol/chloroform extraction, eight of 10 DNA samples exhibited a chimeric pattern, while two of 10 DNAs showed only donor genotype. Of three patients, epithelial cells were attached to magnetic beads prior to DNA isolation. Even this DNA contained donor and recipient material. From our results it appears that blood cells serve as preferential DNA source in mouthwash samples and cannot be removed by epithelial cell separation. (+info)
Type III collagen deficiency in saccular intracranial aneurysms. Defect in gene regulation?
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BACKGROUND AND PURPOSE: We sought to determine whether there are mutations in the COL3A1 gene in patients with saccular intracranial aneurysms with a type III collagen deficiency and whether there is an association between a marker in the COL3A1 gene and saccular intracranial aneurysms. One of the heritable factors possibly involved in the pathogenesis of saccular intracranial aneurysms is a reduced production of type III collagen, demonstrated earlier by protein studies. METHODS: We analyzed the type III collagen gene in a group of 41 consecutive patients with an intracranial aneurysm, of whom 6 patients had shown a reduced production of type III collagen in cultured diploid fibroblasts from a skin biopsy. RESULTS: No mutations could be demonstrated in the COL3A1 gene, especially not in the globular N- and C-terminal regions. A null allele was excluded in 25 patients, including 1 patient with a decreased type III collagen production. No differences were found between 41 patients and 41 controls in allele frequencies of a DNA tandem repeat polymorphism located in the COL3A1 gene. CONCLUSIONS: It is concluded that the COL3A1 gene is not directly involved in the pathogenesis of most of intracranial aneurysms. The reduced type III collagen production in cultured fibroblasts found in some patients with an intracranial aneurysm is not explained by the present study and needs further exploration. (+info)
Mapping of a new SGBS locus to chromosome Xp22 in a family with a severe form of Simpson-Golabi-Behmel syndrome.
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Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked overgrowth syndrome with associated visceral and skeletal abnormalities. Alterations in the glypican-3 gene (GPC3), which is located on Xq26, have been implicated in the etiology of relatively milder cases of this disorder. Not all individuals with SGBS have demonstrated disruptions of the GPC3 locus, which raises the possibility that other loci on the X chromosome could be responsible for some cases of this syndrome. We have previously described a large family with a severe form of SGBS that is characterized by multiple anomalies, hydrops fetalis, and death within the first 8 wk of life. Using 25 simple tandem-repeat polymorphism markers spanning the X chromosome, we have localized the gene for this disorder to an approximately 6-Mb region of Xp22, with a maximum LOD score of 3.31 and with LOD scores <-2.0 for all of Xq. These results demonstrate that neither the GPC3 gene nor other genes on Xq26 are responsible for all cases of SGBS and that a second SGBS locus resides on Xp22. (+info)
Abetalipoproteinemia caused by maternal isodisomy of chromosome 4q containing an intron 9 splice acceptor mutation in the microsomal triglyceride transfer protein gene.
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Uniparental disomy (UPD), a rare inheritance of 2 copies of a single chromosome homolog or a region of a chromosome from one parent, can result in various autosomal recessive diseases. Abetalipoproteinemia (ABL) is a rare autosomal recessive deficiency of apoB-containing lipoproteins caused by a microsomal triglyceride transfer protein (MTP) deficiency. In this study, we describe a patient with ABL inherited as a homozygous intron 9 splice acceptor G(-1)-to-A mutation of the transfer protein gene. This mutation alters the splicing of the mRNA, resulting in a 36 amino acids, in-frame deletion of sequence encoded by exon 10. We analyzed chromosome 4, including MTP gene (4q22-24), using short tandem repeat markers. The proband has only his mother's genes in chromosome 4q spanning a 150-centimorgan region; ie, segmental maternal isodisomy 4q21-35, probably due to mitotic recombination. Nonpaternity between the proband and his father was excluded using 6 polymorphic markers from different chromosomes (paternity probability, 0.999). Maternal isodisomy (maternal UPD 4q) was the basis for homozygosity of the MTP gene mutation in this patient. (+info)
Long range cooperative interactions regulate the initiation of replication in the Tetrahymena thermophila rDNA minichromosome.
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The Tetrahymena thermophila rDNA exists as a 21 kb palindromic minichromosome with two initiation sites for replication in each half palindrome. These sites localize to the imperfect, repeated 430 bp segments that include the nucleosome-free domains 1 and 2 (D1 and D2). To determine if the D1 and D2 segments act independently or in concert to control initiation, stable DNA transformation assays were performed. Single domain derivatives of the plasmid prD1 failed to support autonomous replication in Tetrahymena. Instead, such constructs propagated exclusively by integration into endogenous rDNA minichromosomes and displayed weak origin activity as detected by 2D gel electrophoresis. D1/D1 and D2/D2 derivatives also transformed Tetrahymena poorly, showing similar replication defects. Hence, the D1 and D2 segments are functionally non-redundant and cooperate rather than compete to control initiation. The observed replication defect was greatly reduced in a plasmid derivative that undergoes palindrome formation in Tetrahymena, suggesting that a compensatory mechanism overcomes this replication block. Finally, using a transient replication assay, we present evidence that phylogenetically-conserved type I elements directly regulate DNA replication. Taken together, our data support a model in which cooperative interactions between dispersed elements coordinately control the initiation of DNA replication. (+info)
Direct identification of NH...N hydrogen bonds in non-canonical base pairs of RNA by NMR spectroscopy.
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It is shown that the recently developed quantitative J(NN)HNN-COSY experiment can be used for the direct identification of hydrogen bonds in non-canonical base pairs in RNA. Scalar(2h)J(NN)couplings across NH.N hydrogen bonds are observed in imino hydrogen bonded GA base pairs of the hpGA RNA molecule, which contains a tandem GA mismatch, and in the reverse Hoogsteen AU base pairs of the E-loop of Escherichia coli 5S rRNA. These scalar couplings correlate the imino donor(15)N nucleus of guanine or uridine with the acceptor N1 or N7 nucleus of adenine. The values of the corresponding(2h)J(NN)coupling constants are similar in size to those observed in Watson-Crick base pairs. The reverse Hoogsteen base pairs could be directly detected for the E-loop of E.coli 5S rRNA both in the free form and in a complex with the ribosomal protein L25. This supports the notion that the E-loop is a pre-folded RNA recognition site that is not subject to significant induced conformational changes. Since Watson-Crick GC and AU base pairs are also readily detected the HNN-COSY experiment provides a useful and sensitive tool for the rapid identification of RNA secondary structure elements. (+info)
Centromeres from telomeres? The centromeric region of the Y chromosome of Drosophila melanogaster contains a tandem array of telomeric HeT-A- and TART-related sequences.
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Cytological and cytogenetic studies have previously defined the region needed for centromeric function in the Y chromosome of Drosophila melanogaster. We have identified a YAC clone that originated from this region. Molecular analysis of the YAC and genomic DNAs has allowed the description of a satellite DNA made of telomeric HeT-A- and TART-derived sequences and the construction of a long-range physical map of the heterochromatic region h18. Sequences within the YAC clone are conserved in the centromeric region of the sibling species Drosophila simulans. That telomere-derived DNA now forms part of the centromeric region of the Y chromosome could indicate a telomeric origin of this centromere. The existence of common determinants for the function of both centromeres and telomeres is discussed. (+info)