Biomedical subjects

Jorja G Henikoff

Publications and source records attributed to Jorja G Henikoff.

10 recordsLinked to original sources

PubMed2006

High-throughput TILLING for Arabidopsis.

Targeting induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. In this chapter, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.

Arabidopsis↗

PubMed2005

Chromatin and siRNA pathways cooperate to maintain DNA methylation of small transposable elements in Arabidopsis.

BACKGROUND: DNA methylation occurs at preferred sites in eukaryotes. In Arabidopsis, DNA cytosine methylation is maintained by three subfamilies of methyltransferases with distinct substrate specificities and different modes of action. Targeting of cytosine methylation at selected loci has been found to sometimes involve histone H3 methylation and small interfering (si)RNAs. However, the relationship between different cytosine methylation pathways and their preferred targets is not known. RESULTS: We used a microarray-based profiling method to explore the involvement of Arabidopsis CMT3 and DRM DNA methyltransferases, a histone H3 lysine-9 methyltransferase (KYP) and an Argonaute-related siRNA silencing component (AGO4) in methylating target loci. We found that KYP targets are also CMT3 targets, suggesting that histone methylation maintains CNG methylation genome-wide. CMT3 and KYP targets show similar proximal distributions that correspond to the overall distribution of transposable elements of all types, whereas DRM targets are distributed more distally along the chromosome. We find an inverse relationship between element size and loss of methylation in ago4 and drm mutants. CONCLUSION: We conclude that the targets of both DNA methylation and histone H3K9 methylation pathways are transposable elements genome-wide, irrespective of element type and position. Our findings also suggest that RNA-directed DNA methylation is required to silence isolated elements that may be too small to be maintained in a silent state by a chromatin-based mechanism alone. Thus, parallel pathways would be needed to maintain silencing of transposable elements.

Arabidopsis↗

PubMed2005

Genome-scale profiling of histone H3.3 replacement patterns.

Histones of multicellular organisms are assembled into chromatin primarily during DNA replication. When chromatin assembly occurs at other times, the histone H3.3 variant replaces canonical H3. Here we introduce a new strategy for profiling epigenetic patterns on the basis of H3.3 replacement, using microarrays covering roughly one-third of the Drosophila melanogaster genome at 100-bp resolution. We identified patterns of H3.3 replacement over active genes and transposons. H3.3 replacement occurred prominently at sites of abundant RNA polymerase II and methylated H3 Lys4 throughout the genome and was enhanced on the dosage-compensated male X chromosome. Active genes were depleted of histones at promoters and were enriched in H3.3 from upstream to downstream of transcription units. We propose that deposition and inheritance of actively modified H3.3 in regulatory regions maintains transcriptionally active chromatin.

Animals↗

PubMed2005

DNA methylation profiling identifies CG methylation clusters in Arabidopsis genes.

Cytosine DNA methylation in vertebrates is widespread, but methylation in plants is found almost exclusively at transposable elements and repetitive DNA. Within regions of methylation, methylcytosines are typically found in CG, CNG, and asymmetric contexts. CG sites are maintained by a plant homolog of mammalian Dnmt1 acting on hemi-methylated DNA after replication. Methylation of CNG and asymmetric sites appears to be maintained at each cell cycle by other mechanisms. We report a new type of DNA methylation in Arabidopsis, dense CG methylation clusters found at scattered sites throughout the genome. These clusters lack non-CG methylation and are preferentially found in genes, although they are relatively deficient toward the 5' end. CG methylation clusters are present in lines derived from different accessions and in mutants that eliminate de novo methylation, indicating that CG methylation clusters are stably maintained at specific sites. Because 5-methylcytosine is mutagenic, the appearance of CG methylation clusters over evolutionary time predicts a genome-wide deficiency of CG dinucleotides and an excess of C(A/T)G trinucleotides within transcribed regions. This is exactly what we find, implying that CG methylation clusters have contributed profoundly to plant gene evolution. We suggest that CG methylation clusters silence cryptic promoters that arise sporadically within transcription units.

Arabidopsis↗

PubMed2004

Suboptimal pharmacotherapeutic management of chronic stable angina in the primary care setting.

PURPOSE: To assess the adequacy of symptomatic treatment for chronic stable angina among primary care patients who receive care from the Department of Veterans Affairs (VA). METHODS: We conducted a cross-sectional analysis involving 7038 veterans with self-reported coronary heart disease who completed the Seattle Angina Questionnaire and who had made a primary care visit to one of seven VA general internal medicine clinics between May 1997 and January 2000. The main outcome measures included the anginal frequency scale of the questionnaire and receipt of prescriptions for antianginal medication in three classes (beta-blockers, calcium antagonists, and long-acting nitrates). RESULTS: Seventy percent of the patients experienced angina or took sublingual nitroglycerin preparations two or fewer times per week. Of the 30% of patients with more frequent symptoms, 22% were receiving no antianginal medications and 33% were receiving only one class of antianginal medication. Of the patients with frequent angina who were prescribed medications, 18% were taking no medications at the recommended therapeutic dose and 50% were receiving only one class of antianginal medication at the recommended therapeutic dose. CONCLUSION: A substantial proportion of patients with frequent episodes of chronic stable angina appeared to be receiving an inadequate antianginal regimen in terms of number of agents and dosages. While numerous studies have described inadequate treatment of asymptomatic conditions and risk factors, our results suggest similar deficiencies in addressing a serious and eminently treatable symptomatic problem.

Adrenergic beta-Antagonists↗

PubMed2003

CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design.

We have developed a new primer design strategy for PCR amplification of distantly related gene sequences based on consensus-degenerate hybrid oligonucleotide primers (CODEHOPs). An interactive program has been written to design CODEHOP PCR primers from conserved blocks of amino acids within multiply-aligned protein sequences. Each CODEHOP consists of a pool of related primers containing all possible nucleotide sequences encoding 3-4 highly conserved amino acids within a 3' degenerate core. A longer 5' non-degenerate clamp region contains the most probable nucleotide predicted for each flanking codon. CODEHOPs are used in PCR amplification to isolate distantly related sequences encoding the conserved amino acid sequence. The primer design software and the CODEHOP PCR strategy have been utilized for the identification and characterization of new gene orthologs and paralogs in different plant, animal and bacterial species. In addition, this approach has been successful in identifying new pathogen species. The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) is linked to BlockMaker and the Multiple Alignment Processor within the Blocks Database World Wide Web (http://blocks.fhcrc.org).

Amino Acid Sequence↗

PubMed2003

Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis.

Chemical mutagenesis has been the workhorse of traditional genetics, but it has not been possible to determine underlying rates or distributions of mutations from phenotypic screens. However, reverse-genetic screens can be used to provide an unbiased ascertainment of mutation statistics. Here we report a comprehensive analysis of approximately 1900 ethyl methanesulfonate (EMS)-induced mutations in 192 Arabidopsis thaliana target genes from a large-scale TILLING reverse-genetic project, about two orders of magnitude larger than previous such efforts. From this large data set, we are able to draw strong inferences about the occurrence and randomness of chemically induced mutations. We provide evidence that we have detected the large majority of mutations in the regions screened and confirm the robustness of the high-throughput TILLING method; therefore, any deviations from randomness can be attributed to selectional or mutational biases. Overall, we detect twice as many heterozygotes as homozygotes, as expected; however, for mutations that are predicted to truncate an encoded protein, we detect a ratio of 3.6:1, indicating selection against homozygous deleterious mutations. As expected for alkylation of guanine by EMS, >99% of mutations are G/C-to-A/T transitions. A nearest-neighbor bias around the mutated base pair suggests that mismatch repair counteracts alkylation damage.

Arabidopsis↗

PubMed2003

Large-scale discovery of induced point mutations with high-throughput TILLING.

TILLING (Targeting Induced Local Lesions in Genomes) is a general reverse-genetic strategy that provides an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and low-cost discovery of induced point mutations in populations of chemically mutagenized individuals. As chemical mutagenesis is widely applicable and mutation detection for TILLING is dependent only on sufficient yield of PCR products, TILLING can be applied to most organisms. We have developed TILLING as a service to the Arabidopsis community known as the Arabidopsis TILLING Project (ATP). Our goal is to rapidly deliver allelic series of ethylmethanesulfonate-induced mutations in target 1-kb loci requested by the international research community. In the first year of public operation, ATP has discovered, sequenced, and delivered >1000 mutations in >100 genes ordered by Arabidopsis researchers. The tools and methodologies described here can be adapted to create similar facilities for other organisms.

Alkylating Agents↗

PubMed2003

High-throughput TILLING for functional genomics.

Targeting-induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. Here, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.

DNA, Plant↗

PubMed2002

Genome-wide profiling of DNA methylation reveals transposon targets of CHROMOMETHYLASE3.

DNA methylation has been implicated in a variety of epigenetic processes, and abnormal methylation patterns have been seen in tumors. Analysis of methylation patterns has traditionally been conducted either by using Southern analysis after cleavage with methyl-sensitive restriction endonucleases or by bisulfite sequencing. However, neither method is practical for analyzing more than a few genes. Here, we describe a simple technique for genome-wide mapping of DNA methylation patterns. Fragmentation by a methyl-sensitive restriction endonuclease is followed by size fractionation and hybridization to microarrays. We demonstrate the utility of this method by characterizing methylation patterns in Arabidopsis methylation mutants. This analysis reveals that CHROMOMETHYLASE3 (CMT3), which was previously shown to maintain CpXpG methylation, preferentially methylates transposons, even when they are present as single copies within the genome. Methylation profiling has potential applications in disease research and diagnostic screening.

Arabidopsis↗