ABSTRACT
PURPOSE: Our aim was to compare the accuracy of family- or disease-specific targeted haplotyping and direct mutation-detection strategies with the accuracy of genome-wide mapping of the parental origin of each chromosome, or karyomapping, by single-nucleotide polymorphism genotyping of the parents, a close relative of known disease status, and the embryo cell(s) used for preimplantation genetic diagnosis of single-gene defects in a single cell or small numbers of cells biopsied from human embryos following in vitro fertilization. METHODS: Genomic DNA and whole-genome amplification products from embryo samples, which were previously diagnosed by targeted haplotyping, were genotyped for single-nucleotide polymorphisms genome-wide detection and retrospectively analyzed blind by karyomapping. RESULTS: Single-nucleotide polymorphism genotyping and karyomapping were successful in 213/218 (97.7%) samples from 44 preimplantation genetic diagnosis cycles for 25 single-gene defects with various modes of inheritance distributed widely across the genome. Karyomapping was concordant with targeted haplotyping in 208 (97.7%) samples, and the five nonconcordant samples were all in consanguineous regions with limited or inconsistent haplotyping results. CONCLUSION: Genome-wide karyomapping is highly accurate and facilitates analysis of the inheritance of almost any single-gene defect, or any combination of loci, at the single-cell level, greatly expanding the range of conditions for which preimplantation genetic diagnosis can be offered clinically without the need for customized test development.
Subject(s)
Chromosome Mapping/methods , Genotyping Techniques/methods , Karyotyping/methods , Preimplantation Diagnosis/methods , Blastocyst , Female , Genome, Human , Humans , In Vitro Techniques , Male , Parents , Polymorphism, Single Nucleotide , Reproducibility of Results , Retrospective StudiesABSTRACT
Genes encoding plastid ribosomal proteins are distributed between the nuclear and plastid genomes in higher plants, and coordination of their expression is likely to be required for functional plastid protein synthesis. A custom microarray has been used to examine the patterns of accumulation of transcripts from plastid and nuclear genes encoding plastid ribosomal proteins during seedling development in tobacco and Arabidopsis. The transcripts accumulate coordinately during early seedling development and show similar responses to light and to inhibitors, such as norflurazon and lincomycin, affecting plastid signaling. Computational analysis of the promoters of these genes revealed a shared initiator motif and common cis-elements characteristic of photosynthesis genes, specifically the GT-1 element, and the I-box. Analysis of the RPL27 gene of Arabidopsis thaliana indicated that transcription initiates from an initiator-like region. Deletion analysis of the RPL27 promoter in transgenic plants revealed that the identified shared cis-elements were not all required for wild-type expression patterns, and full developmental, light- and plastid-regulation can be conveyed by a region of the promoter from -235 to +1 relative to the transcription start site.
Subject(s)
Arabidopsis/metabolism , Cell Nucleus/metabolism , Gene Expression Regulation, Plant , Nicotiana/metabolism , Plastids/metabolism , Ribosomal Proteins/metabolism , Seedlings/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Light , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Plants, Genetically Modified , Promoter Regions, Genetic/genetics , Ribosomal Proteins/genetics , Seedlings/genetics , Seedlings/growth & development , Sequence Alignment , Signal Transduction , Time Factors , Nicotiana/genetics , Nicotiana/growth & development , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic/geneticsABSTRACT
The acetylation states of histones present on the upstream, promoter, coding or intronic regions of 88 tobacco genes were examined with chromatin immunoprecipitation (ChIP) experiments using antibodies that recognised acetylated histone H4. The DNA sequences enriched in the immunoprecipitates were amplified by ligation-mediated PCR, labelled with Cy-dUTP and hybridised to DNA microarrays. In green tobacco shoots, histone H4 acetylation was localised to 300-600-bp sequences in the promoters or coding regions of 31 genes, or occurred extensively over several kilobase-pair regions containing the upstream, promoter and/or coding regions of 25 genes. Genes associated with high histone H4 acetylation levels at promoters were actively expressed, whereas genes depleted in acetylated histone H4 were non-transcribed or expressed at very low levels, suggesting a correlation between histone H4 acetylation and gene activity. Trichostatin A (TA), an inhibitor of histone deacetylases (HDAs), did not alter histone H4 acetylation states globally but increased acetylation levels at specific tobacco sequences, suggesting that HDAs are targeted to particular nucleosomes. Genes that were upregulated by TA were associated with increased histone H4 acetylation at promoter or coding regions, indicating that acetylation of histones on coding regions may activate transcription. Increased histone H4 acetylation leading to elevated expression was observed on genes with diverse functions, suggesting that histone H4 acetylation is involved in regulation of many plant processes.
