The accuracy of statistical methods for estimation of haplotype frequencies: an example from the CD4 locus.
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Haplotype analysis has become increasingly important for the study of human disease as well as for reconstruction of human population histories. Computer programs have been developed to estimate haplotype frequencies statistically from marker phenotypes in unrelated individuals. However, there currently are few empirical reports on the accuracy of statistical estimates that must infer linkage phase. We have analyzed haplotypes at the CD4 locus on chromosome 12 that consist of a short tandem-repeat polymorphism and an Alu insertion/deletion polymorphism located 9.8 kb apart, in 398 individuals from 10 geographically diverse sub-Saharan African populations. Haplotype frequency estimates obtained using gene counting based on molecularly haplotyped (phase-known) data were compared with haplotype frequency estimates obtained using the expectation-maximization algorithm. We show that the estimated frequencies of common haplotypes do not differ significantly with the use of phase-known versus phase-unknown data. However, rare haplotypes are occasionally miscalled when their presence/absence must be inferred. Thus, for those research questions for which the common haplotypes are most important, frequency estimates based on the phase-unknown marker-typing results from unrelated individuals will be sufficient. However, in cases where knowledge of rare haplotypes is critical, molecular haplotyping will be necessary to determine linkage phase unambiguously. (+info)
Complete genomic sequence of the human ABCA1 gene: analysis of the human and mouse ATP-binding cassette A promoter.
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The ABCA1 gene, a member of the ATP-binding cassette A (ABCA1) transporter superfamily, encodes a membrane protein that facilitates the cellular efflux of cholesterol and phospholipids. Mutations in ABCA1 lead to familial high density lipoprotein deficiency and Tangier disease. We report the complete human ABCA1 gene sequence, including 1,453 bp of the promoter, 146,581 bp of introns and exons, and 1 kb of the 3' flanking region. The ABCA1 gene spans 149 kb and comprises 50 exons. Sixty-two repetitive Alu sequences were identified in introns 1-49. The transcription start site is 315 bp upstream of a newly identified initiation methionine codon and encodes an ORF of 6,783 bp. Thus, the ABCA1 protein is comprised of 2,261 aa. Analysis of the 1,453 bp 5' upstream of the transcriptional start site reveals multiple binding sites for transcription factors with roles in lipid metabolism. Comparative analysis of the mouse and human ABCA1 promoter sequences identified specific regulatory elements, which are evolutionarily conserved. The human ABCA1 promoter fragment -200 to -80 bp that contains binding motifs for SP1, SP3, E-box, and AP1 modulates cellular cholesterol and cAMP regulation of ABCA1 gene expression. These combined findings provide insights into ABCA1-mediated regulation of cellular cholesterol metabolism and will facilitate the identification of new pharmacologic agents for the treatment of atherosclerosis in humans. (+info)
The most frequent constitutional translocation in humans, the t(11;22)(q23;q11) is due to a highly specific alu-mediated recombination.
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The t(11;22) is the most common recurrent non-Robertsonian constitutional translocation in humans, having been reported in more than 160 unrelated families. Balanced carriers are at risk of having offspring with the derivative 22 syndrome owing to 3:1 meiotic non-disjunction event. Clinical features of the der(22) syndrome include mental retardation, craniofacial abnormalities and congenital heart defects. The breakpoints for the t(11;22) translocation have been mapped to specific Alu repeats on chromosomes 11 and 22, indicating that this event is due to an Alu-Alu recombination. Remarkably, in five samples derived from individuals with no apparent common ancestry the der(11) and der(22) breakpoints appear to be almost identical at the genomic sequence level. The small number of base differences between the samples indicates some variation in the position of the breakpoints, although this appears to be quite limited. Indeed, the der(11) breakpoints are all located within a region of just 32 bp and the der(22) breakpoints within 21 bp. If, as suggested by current data, the widespread occurrence of this translocation is due to multiple independent events, our results suggest that this particular Alu-Alu recombination is subject to an unprecedented degree of selection. (+info)
Inverted Alu repeats unstable in yeast are excluded from the human genome.
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The nearly one million ALU: repeats in human chromosomes are a potential threat to genome integrity. ALU:s form dense clusters where they frequently appear as inverted repeats, a sequence motif known to cause DNA rearrangements in model organisms. Using a yeast recombination system, we found that inverted ALU: pairs can be strong initiators of genetic instability. The highly recombinagenic potential of inverted ALU: pairs was dependent on the distance between the repeats and the level of sequence divergence. Even inverted ALU:s that were 86% homologous could efficiently stimulate recombination when separated by <20 bp. This stimulation was independent of mismatch repair. Mutations in the DNA metabolic genes RAD27 (FEN1), POL3 (polymerase delta) and MMS19 destabilized widely separated and diverged inverted ALU:s. Having defined factors affecting inverted ALU: repeat stability in yeast, we analyzed the distribution of ALU: pairs in the human genome. Closely spaced, highly homologous inverted ALU:s are rare, suggesting that they are unstable in humans. ALU: pairs were identified that are potential sites of genetic change. (+info)
K562 cells implicate increased chromatin accessibility in Alu transcriptional activation.
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Alu repeats in K562 cells are unusually hypomethylated and far more actively transcribed than those in other human cell lines and somatic tissues. Also, the level of Alu RNA in K562 cells is relatively insensitive to cell stresses, namely heat shock, adenovirus infection and treatment with cycloheximide, which increase the abundance of Alu RNA in HeLa and 293 cells. Recent advances in understanding the interactions between DNA methylation, transcriptional activation and chromatin conformation reveal reasons for the constitutively high level of Alu expression in K562 cells. Methylation represses transcription of transiently transfected Alu templates in all cell lines tested but cell stresses do not relieve this repression suggesting that they activate Alu transcription through another pathway. A relatively large fraction of the Alus within K562 chromatin is accessible to restriction enzyme cleavage and cell stresses increase the chromatin accessibility of Alus in HeLa and 293 cells. Cell stress evidently activates Alu transcription by rapidly remodeling chromatin to recruit additional templates. (+info)
Transduction of human PBMC-derived dendritic cells and macrophages by an HIV-1-based lentiviral vector system.
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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. (+info)
Recovery and potential utility of YACs as circular YACs/BACs.
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A method has been established to convert pYAC4-based linear yeast artificial chromosomes (YACs) into circular chromosomes that can also be propagated in Escherichia coli cells as bacterial artificial chromosomes (BACs). The circularization is based on use of a vector that contains a yeast dominant selectable marker (G418R), a BAC cassette and short targeting sequences adjacent to the edges of the insert in the pYAC4 vector. When it is introduced into yeast, the vector recombines with the YAC target sequences to form a circular molecule, retaining the insert but discarding most of the sequences of the YAC telomeric arms. YACs up to 670 kb can be efficiently circularized using this vector. Re-isolation of megabase-size YAC inserts as a set of overlapping circular YAC/BACs, based on the use of an Alu-containing targeting vector, is also described. We have shown that circular DNA molecules up to 250 kb can be efficiently and accurately transferred into E.coli cells by electroporation. Larger circular DNAs cannot be moved into bacterial cells, but can be purified away from linear yeast chromosomes. We propose that the described system for generation of circular YAC derivatives can facilitate sequencing as well as functional analysis of genomic regions. (+info)
Short tandem-repeat polymorphism/alu haplotype variation at the PLAT locus: implications for modern human origins.
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Two dinucleotide short tandem-repeat polymorphisms (STRPs) and a polymorphic Alu element spanning a 22-kb region of the PLAT locus on chromosome 8p12-q11.2 were typed in 1,287-1,420 individuals originating from 30 geographically diverse human populations, as well as in 29 great apes. These data were analyzed as haplotypes consisting of each of the dinucleotide repeats and the flanking Alu insertion/deletion polymorphism. The global pattern of STRP/Alu haplotype variation and linkage disequilibrium (LD) is informative for the reconstruction of human evolutionary history. Sub-Saharan African populations have high levels of haplotype diversity within and between populations, relative to non-Africans, and have highly divergent patterns of LD. Non-African populations have both a subset of the haplotype diversity present in Africa and a distinct pattern of LD. The pattern of haplotype variation and LD observed at the PLAT locus suggests a recent common ancestry of non-African populations, from a small population originating in eastern Africa. These data indicate that, throughout much of modern human history, sub-Saharan Africa has maintained both a large effective population size and a high level of population substructure. Additionally, Papua New Guinean and Micronesian populations have rare haplotypes observed otherwise only in African populations, suggesting ancient gene flow from Africa into Papua New Guinea, as well as gene flow between Melanesian and Micronesian populations. (+info)