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At least 631 records · Page 35Linked to original sources
PubMed1997

Human mitochondrial enoyl-CoA hydratase gene (ECHS1): structural organization and assignment to chromosome 10q26.2-q26.3.

The second step in mitochondrial fatty acid beta-oxidation is catalyzed by short chain enoyl-CoA hydratase (ECHS1; EC 4.2.1.17). Inherited disorders of this pathway of energy metabolism present clinical and laboratory features resembling sudden infant death syndrome and Reye-like syndrome. To investigate the role of ECHS1 further, the gene structure was analyzed and its chromosomal locus determined. A fragment of rat liver ECHS1 cDNA was employed for isolation and characterization of two overlapping genomic clones encompassing the entire human ECHS1 gene. The gene, approximately 11 kb, is composed of eight exons, with exons I and VIII containing the 5'- and 3'-untranslated regions, respectively. Two major transcription start sites, located 62 and 63 bp upstream of the translation initiation codon, were mapped by primer extension analysis. The immediate 5'-flanking region of the ECHS1 gene is GC-rich and contains several copies of the SP1 binding motive but no typical TATA or CAAT boxes are apparent. Alu repeat elements have been identified within the region -1052/-770 relative to the cap site and in intron 7. The human ECHS1 gene locus was assigned to chromosome 10q26.2-q26.3 by fluorescence in situ hybridization.

Animals↗
PubMed1998

The human cystathionine beta-synthase (CBS) gene: complete sequence, alternative splicing, and polymorphisms.

Cystathionine beta-synthase [CBS; l-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] catalyzes the first committed step of transsulfuration and is the enzyme deficient in classical homocystinuria. In this report, we describe the molecular cloning and the complete nucleotide sequence of the human CBS gene. We report a total of 28,046 nucleotides of sequence, which, in addition to the CBS gene, contains approximately 5 kb of the 5' flanking region. The human CBS gene contains 23 exons ranging from 42 to 209 bp. The 5' UTR is formed by 1 of 5 alternatively used exons and 1 invariably present exon, while the 3' UTR is encoded by exons 16 and 17. We also describe the identification of two alternatively used promoter regions that are GC rich (approximately 80%) and contain numerous putative binding sites for Sp1, Ap1, Ap2, and c-myb, but lack the classical TATA box. The CBS locus contains an unusually high number of Alu repeats, which may predispose this gene to deleterious rearrangements. Additionally, we report on a number of DNA sequence repeats that are polymorphic in North American and European Caucasians.

Alternative Splicing↗
PubMed2002

Methylation patterns of the human beta-glucuronidase gene locus: boundaries of methylation and general implications for frequent point mutations at CpG dinucleotides.

Methylation of CpG islands spanning promoter regions is associated with control of gene expression, although it is unclear what mechanisms define the boundaries between methylated and unmethylated regions in the genome. Methylation of genomic DNA in mammals also affects the frequency of inherited diseases by predisposing them to CpG mutations. To gain insight into these issues, we investigated patterns of cytosine methylation on almost the entire beta-glucuronidase gene (GUSB) from normal leukocyte DNAs by bisulfite genomic sequencing. We mapped the boundaries of methylation that flank the 5'- and 3'-ends of the CpG island region, and correlated methylation status with transitional mutations at CpG sites. GenBank sequence analyses showed that the CpG island of human GUSB is juxtaposed with multiple Alu repeats and also includes multiple Sp1 sites upstream and downstream of the transcription start, which has been suggested to prevent CpG islands from becoming methylated. We show that cytosine methylation is extensive across the entire gene except for CpG sites in the proximal promoter region, exon 1, and part of intron 1; the unmethylated CpG island is embedded between densely methylated flanking regions containing multiple Alu repeats; a sharp boundary separates the methylated and unmethylated regions of the 5'-flank of the CpG island, but a gradual change in methylation density over 1.0 kb is observed in the 3'-flank of the CpG island; boundaries of the 5'-end and 3'-end of the CpG island contain multiple Sp1 sites in addition to Alu repeats; methylation in both strands is symmetrical except at the boundary regions between methylated and unmethylated regions; and nonmethylation of exon 1 correlates with the absence of transitional mutations at CpG sites in exon 1.

3' Flanking Region↗
PubMedDate not supplied

Mutations in the lysyl hydroxylase 1 gene that result in enzyme deficiency and the clinical phenotype of Ehlers-Danlos syndrome type VI.

The Ehlers-Danlos syndromes are a heterogeneous group of inherited connective tissue disorders that are characterized by joint hypermobility and skin fragility and hyperextensibility. Patients with the autosomal recessive type VI variant of the Ehlers-Danlos syndromes (EDS VI), also classified as the kyphoscoliotic type, are clinically characterized by neonatal kyphoscoliosis, generalized joint laxity, skin fragility, and severe muscle hypotonia at birth. Biochemically, this has been attributed to a deficiency of lysyl hydroxylase (LH), an important posttranslational modifying enzyme in collagen biosynthesis. This enzyme hydroxylates specific lysine residues in the collagen molecule to form hydroxylysines which have two important functions. The residues serve as attachment sites for galactose and glucosylgalactose and they also act as precursors of the crosslinking process that gives collagen its tensile strength. At least 20 different mutations have been identified in the LH1 gene (the originally described form) that contribute to LH deficiency and the clinical characteristics of EDS VI. Two of these mutations, a large duplication of exons 10-16, arising from a homologous recombination of intronic Alu sequences, and a nonsense mutation, Y511X, in exon 14 of the LH1 gene, have been identified in five or more unrelated patients. Both mutations appear to have originated from a single ancestral gene. Alternative processing pathways involving alternate splicing and mRNA degradation, which reduce the effect of the mutant allele and restore partial activity of the enzyme, have been identified. A second class of EDS VI has been proposed in which patients have the clinical phenotype of EDS VI but their levels of LH activity are normal. The biochemical basis for this form of EDS VI is currently unknown.

Alternative Splicing↗
PubMed2000

Transduction of human PBMC-derived dendritic cells and macrophages by an HIV-1-based lentiviral vector system.

Professional antigen-presenting cells, such as dendritic cells (DCs) and macrophages, are target cells for gene therapy of infectious disease and cancer. However, transduction of DCs and macrophages has proved difficult by most currently available gene transfer methods. Several recent studies have shown that lentiviral vector systems can efficiently transduce many nondividing and differentiated cell types. In this study, we examined the gene transfer to DCs and macrophages using a lentiviral vector system. Human DCs were propagated from the adherent fraction of peripheral blood mononuclear cells (PBMCs) by culture in medium containing GM-CSF, IL-4, and TNF-alpha. Human macrophages were propagated from adherent PBMCs in medium containing GM-CSF. High titers of a replication-defective vesicular stomatitis virus glycoprotein G pseudotyped HIV-1-based vector encoding the enhanced yellow fluorescent protein were produced. In immature DCs (culture days 3 and 5), transduction efficiencies of 25 to 35% were achieved at a multiplicity of infection of 100. However, the transduction efficiency was decreased in more mature DCs (culture day 8 or later). Furthermore, monocyte-derived macrophages were also transduced by the lentiviral vector system. In addition, Alu-LTR PCR demonstrated the integration of the HIV-1 provirus into the cellular genome of the transduced DCs and macrophages. Allogeneic mixed lymphocyte reactions revealed similar antigen-presenting functions of untransduced and lentivirally transduced DCs. Thus, the results of this study demonstrate that both PBMC-derived DCs and macrophages can be transduced by lentiviral vectors.

Alu Elements↗
PubMed1986

Molecular evolution of the carbonic anhydrase genes: calculation of divergence time for mouse carbonic anhydrase I and II.

A cDNA clone in pBR322 that cross-hybridizes with a mouse carbonic anhydrase form II (CAII) probe has been sequenced and identified as mouse carbonic anhydrase form I (CAI). The 1224-base-pair clone encodes the entire 260-amino-acid protein and appears to contain an Alu-like element in the 3' untranslated region. The deduced amino acid sequence exhibits 77% homology to human CAI and contains 17 of the 20 residues that are considered unique to and invariant for all mammalian CAI isozymes. The results of a detailed comparison of the nucleic acid sequences spanning the coding regions of mouse CAI and rabbit CAI have been used to calibrate an evolutionary clock for the carbonic anhydrases (CAs). These data have been applied to a comparison of the mouse CAI and CAII nucleic acid sequences to calculate the divergence time between the two genes. The divergence-time calculation provides the first estimation of the evolutionary relationship between CAs based entirely on nucleotide sequence comparison.

Amino Acid Sequence↗
PubMed2006

Molecular characterization of large deletions in the von Hippel-Lindau (VHL) gene by quantitative real-time PCR: the hypothesis of an alu-mediated mechanism underlying VHL gene rearrangements.

INTRODUCTION: Mutations of the von Hippel-Lindau (VHL) gene are responsible for VHL disease. This is a familial autosomal-dominant syndrome, predisposing to the development of benign and malignant tumors, including CNS and retinal hemangioblastomas, pheochromocytomas, and clear cell renal carcinomas. At least 30% of the disease-causing mutations in the VHL gene involve large alterations. Identification of these mutations is not possible using PCR-based mutational scanning methods. Quantitative Southern blot analysis has been traditionally employed for the detection of complete or partial deletions and more complex rearrangements of the gene. METHODS: An alternative quantitative method was developed using a combination of quantitative Southern blot analysis and real-time PCR. With this approach, we studied 24 large VHL gene alterations to determine the exact nature of the mutations and to possibly characterize the boundaries of the deleted regions. RESULTS: This combined molecular approach showed that all the VHL alterations studied were due to deletions, from which the position in the gene could be more precisely mapped. One of the samples that was completely characterized was found to carry an intragenic 2.2kb deletion with both 5' and 3' breakpoints located within Alu-repeat sequences. CONCLUSION: This is the first report on the molecular analysis of large VHL alterations. The results of our study and the complete characterization of a large deletion lead to the hypothesis that an Alu-mediated mechanism may be responsible for the common occurrence of large alterations in the VHL gene.

Alu Elements↗
PubMed1999

Vitamin D binding protein gene in male osteoporosis: association of plasma DBP and bone mineral density with (TAAA)(n)-Alu polymorphism in DBP.

Vitamin D binding protein (DBP) is a major carrier protein for the vitamin D metabolites, but may also play an important role in osteoclast differentiation. Polymorphisms of the DBP gene have been reported, including (TAAA)(n)-Alu repeat polymorphisms downstream of intron 8. We have examined the relationship between polymorphisms of the DBP gene and bone mineral density (BMD) and vertebral fractures in a group of 26 men with vertebral fractures but no underlying secondary cause of osteoporosis (median age 64, ages 27-72 years) and 21 male control subjects (median age 65, ages 40-77 years). There was no apparent effect of DBP phenotype on BMD, but there was a relationship between certain genotypes of (TAAA)(n)-Alu repeats and reduced BMD and vertebral fracture. Lumbar spine and femoral neck BMD were significantly lower in men with 10/8 genotype than 10/10 genotype (P < 0.05). Furthermore, the predominant genotype in men with vertebral fractures was 10/8, whereas the most common genotype in control subjects was 10/10 (odds ratio 56; 95% confidence interval 7-445). Plasma DBP was higher in men with 10/8 genotype than those with 10/10 genotype (P < 0.05), and patients with vertebral fractures were found to have higher levels than control subjects (P < 0.0005). Although our study is small because of the relative rarity of idiopathic osteoporosis in men, the results suggest that (TAAA)(n)-Alu polymorphism may have an important effect on plasma levels of DBP, bone density and fracture risk in men.

Adult↗
PubMed2006

The distribution of L1 and Alu retroelements in relation to GC content on human sex chromosomes is consistent with the ectopic recombination model.

The distribution of Alu and L1 retroelements in the human genome changes with their age. Active retroelements target AT-rich regions, but their frequency increases in GC- and gene-rich regions of the genome with increasing age of the insertions. Currently there is no consensus on the mechanism generating this pattern. In this paper we test the hypothesis that selection against deleterious deletions caused by ectopic recombination between repeats is the main cause of the inhomogeneous distribution of L1s and Alus, by means of a detailed analysis of the GC distribution of the repeats on the sex chromosomes. We show that (1) unlike on the autosomes and X chromosome, L1s do not accumulate on the Y chromosome in GC-rich regions, whereas Alus accumulate there to a minor extent; (2) on the Y chromosome Alu and L1 densities are positively correlated, unlike the negative correlation on other chromosomes; and (3) in gene-poor regions of chromosome 4 and X, the distribution of Alus and L1s does not shift toward GC-rich regions. In addition, we show that although local GC content of long L1 insertions is lower than average, their selective loss from recombining chromosomes is not the main cause of the enrichment of ancient L1s in GC-rich regions. The results support the hypothesis that ectopic recombination causes the shift of Alu and L1 distributions toward the gene-rich regions of the genome.

Alu Elements↗
PubMed2000

Duplication and diversification of the apolipoprotein CI (APOCI) genomic segment in association with retroelements.

We have previously shown that several multicopy gene families within the major histocompatibility complex (MHC) arose from a process of segmental duplication. It has also been observed that retroelements play a role in generating diversity within these duplicated segments. The objective of this study was to compare the genomic organization of a gene duplication within another multicopy gene family outside the MHC. Using new continuous genomic sequence encompassing the APOE-CII gene cluster, we show that APOCI and its pseudogene, APOCI', are contained within large duplicated segments which include sequences from the hepatic control region (HCR). Flanking Alu sequences are observed at both ends of the duplicated unit, suggesting a possible role in the integration of these segments. As observed previously within the MHC, the major differences between the segments are the insertion of sequences (approximately 200-1000 bp in length), consisting predominantly of Alu sequences. Ancestral retroelements also contribute to the generation of sequence diversity between the segments, especially within the 3' poly(A) tract of Alu sequences. The exonic and regulatory sequences of the APOCI and HCR loci show limited sequence diversity, with exon 3 being an exception. Finally, the typing of pre- and postduplication Alus from both segments indicates an estimated time of duplication of approximately 37 million years ago (mya), some time prior to the separation of Old and New World monkeys.

Alu Elements↗
PubMed2002

Alu polymorphism within the MICB gene and association with HLA-B alleles.

We describe the finding of an Alu repeat dimorphism within the first intron of the MICB gene. The frequencies of the two AluyMICB alleles, AluyMICB*0(absence of insertion) and AluyMICB*1(presence of insertion), and their associations with the highly polymorphic HLA-B locus were determined for 51 human cell lines and for 109 and 200 Caucasians and northeastern Thais, respectively. Analysis of the AluyMICB and HLA-B allelic relationships revealed that AluyMICB*1 occurred at relatively low gene frequency (0.118-0.157) [corrected] but was strongly associated with HLA-B17 (HLA-B57,HLA-B58) and HLA-B13. The AluyMICB locus provides a useful dimorphic marker for investigations on the level of linkage disequilibrium between MICB, MICA, and HLA-B loci.

Alleles↗
PubMed2004

The distribution of polymorphic Alu insertions within the MHC class I HLA-B7 and HLA-B57 haplotypes.

There are five polymorphic Alu insertion (POALIN) loci within the major histocompatibility complex (MHC) class I region that have been strongly associated with HLA class I alleles, such as HLA-A1, HLA-A2 and HLA-B57. In order to assess the variability and frequency of POALIN distribution within two common HLA-B haplotypes, we detected the presence of the MHC class I POALIN by PCR in a panel of 15 individuals with HLA-B57 and 47 homozygous individuals with 7.1 AH (HLA-B7, -Cw7, -A3) obtained from the Australian Bone Marrow Donor Registry, and also from four families (25 individuals) containing the HLA-B57 allele. Only two of the 47 HLA-B7 genotypes had a detectable POALIN, whereas all of the HLA-B57 genotypes had at least one or more POALINs present, confirming that certain MHC class I haplotypes are relatively POALIN-free and others are POALIN-enriched. Six distinct HLA-B57 haplotypes, based on differences at the HLA-A locus and three of five POALIN loci, were identified that appear to have evolved by different mechanisms, including either by shuffling different combinations of conserved alpha and beta blocks or by recombination events involving two or more previously generated HLA-B57 haplotypes.

Alleles↗
PubMed1999

Gorilla RH-like genes and antigens.

It has been previously shown that most of the human IgG monoclonal D-specific antibodies define a polymorphism in the gorilla consisting of two phenotypes: Dgor-positive and Dgor-negative. By quantitative indirect immunofluorescence assay and quantitative immunoblotting it was evaluated that the number of Dgor antigenic sites per gorilla red cell varies from a level equivalent to that observed for human RhD-positive cells to a level eight times higher. By immunoblotting with a rabbit reagent specific for the carboxylic end of human Rh-polypeptides it was demonstrated that RBCs from all gorillas, whatever their Dgor phenotype, possess 33000 relative molecular mass Rh-like polypeptides. The expression of the Dgor antigen was shown to be associated with the presence of three polymorphic bands defined by Southern blot using a human exon 4 RHCE probe, and to a length polymorphism of gorilla intron 3 evidenced by polymerase chain reaction. By contrast, the expression of the Dgor antigen was not associated to the length polymorphism of gorilla intron 4 which is related to the presence or absence of an Alu-Sx element in intron 4, paralleling the situation observed in human. These results confirmed the presence in the gorilla genome of at least two RH-like genes, one of which being responsible for Dgor polymorphism. The phylogenesis of the human and gorilla RH genes is discussed in light of the comparison of intron 4 sequences.

Animals↗
PubMed2003

Association study between Alzheimer's disease and genes involved in Abeta biosynthesis, aggregation and degradation: suggestive results with BACE1.

BACKGROUND: Amyloid beta-peptide (Abeta) biosynthesis, aggregation and degradation constitute three important steps to consider in the study of pathological mechanisms involved in Alzheimer's disease (AD). Several proteins have been suggested as involved in each of these processes: proteolytic cleavage of the amyloid precursor protein by the beta-site APP cleaving enzyme (BACE), increased amyloid fibril formation by the activity of the acetylcholinesterase (ACHE gene), and degradation of Abeta aggregates by the plasmin system have been exhaustively documented. METHODS: A case-control design was used to evaluate the possible association between candidate genes involved in these three processes and AD. We analysed three polymorphisms located at the BACE1 gene, one polymorphism at the ACHE gene, and two variants located at the tissue plasminogen activator and plasminogen activator inhibitor-1 (genes TPA and PAI- 1, respectively), both part of the plasmin system. RESULTS: We found an association between BACE1 exon 5 GG genotype and AD (age-and gender-adjusted odds ratio = 2.14, P =0.014). Although a similar association was reported previously by Nowotny and collaborators only in subjects carrying the epsilon4-allele of the apolipoprotein E gene (APOE), we did not detect this effect. However,when we combined our results with those previously reported, a clear increase of the risk to develop AD appeared in subjects carrying both the BACE1 exon 5 GG genotype and the APOE epsilon4-allele (crude OR = 2.2, P = 0.004). CONCLUSION: These data suggest a possible genetic relation between BACE1 and AD.

3' Untranslated Regions↗
PubMed2003

Experimental rat model for therapeutic retinal pigment epithelium transplantation--unequivocal microscopic identification of human donor cells by in situ hybridisation of human-specific Alu sequences.

Transplantation of retinal pigment epithelial (RPE) cells is discussed as a possible therapeutic approach for retinal degeneration. Xenogeneic transplantation of human RPE cells in animal models has been studied extensively. Various methods have been used to identify the graft cells, but these methods interfere with cell behaviour so that the monitored physiological post-transplantation course may be influenced. In the present study, we applied a method for an unequivocal identification of the graft cells without interfering cell metabolism or behaviour using in situ hybridisation (ISH) of human specific Alu sequences. Visualisation of the strong extended nuclear signal of Alu sequences was much easier than that of the small nuclear signals of donor-specific sex chromosome probes. With Alu probe, even single graft cells can be identified and their development can be observed in short-term and long-term studies. With this procedure, we could prove that donor cells were injected correctly into the subretinal space by a special injection technique that we developed previously. In combination with immunohistochemistry, donor cells could be clearly discriminated from macrophages, which contained phagocytosed donor cell fragments. Application of these ISH methods for species-specific identification was valuable for follow-up-studies of RPE transplantation.

Alu Elements↗