Biomedical subjects

Douglas L Chalker

Publications and source records attributed to Douglas L Chalker.

4 recordsLinked to original sources

PubMed2006

The germ line limited M element of Tetrahymena is targeted for elimination from the somatic genome by a homology-dependent mechanism.

A RNA interference (RNAi) like mechanism is involved in elimination of thousands of DNA segments from the developing somatic macronucleus of Tetrahymena, yet how specific internal eliminated sequences (IESs) are recognized remains to be fully elucidated. To define requirements for DNA rearrangement, we performed mutagenesis of the M element, a well-studied IES. While sequences within the macronucleus-retained DNA are known to determine the excision boundaries, we show that sequences internal to these boundaries are required to promote this IES's rearrangement. However, this element does not contain any specific sequence required in cis as removal of its entire left or right side was insufficient to abolish all rearrangement. Instead, rearrangement efficiency correlated with the overall size of the M element sequence within a given construct, with a lower limit of nearly 300 bp. Also, the observed minimal region necessary to epigenetically block excision supports this size limit. Truncated M element constructs that exhibited impaired rearrangement still showed full transcriptional activity, which suggests that their defect was due to inefficient recognition. This study indicates that IESs are targeted for elimination upon their recognition by homologous small RNAs and further supports the idea that DNA elimination is a RNAi-related mechanism involved in genome surveillance.

Animals↗

PubMed2005

Genome rearrangements: mother knows best!

In Paramecium, developmentally programmed genome rearrangements can be altered by the presence of homologous sequences within the maternal somatic nucleus. Newly identified RNA-binding proteins appear to mediate the transfer of homologous sequence information from the maternal to the developing somatic nucleus, facilitating epigenetic regulation of this large-scale genome reorganization.

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PubMed2005

Germ line transcripts are processed by a Dicer-like protein that is essential for developmentally programmed genome rearrangements of Tetrahymena thermophila.

Abundant approximately 28-nucleotide RNAs that are thought to direct histone H3 lysine 9 (H3K9) methylation and promote the elimination of nearly 15 Mbp of DNA from the developing somatic genome are generated during Tetrahymena thermophila conjugation. To identify the protein(s) that generates these small RNAs, we studied three Dicer-related genes encoded within the Tetrahymena genome, two that contain both RNase III and RNA helicase motifs, Dicer 1 (DCR1) and DCR2, and a third that lacks the helicase domain, Dicer-like 1 (DCL1). DCL1 is expressed upon the initiation of conjugation, and the protein localizes to meiotic micronuclei when bidirectional germ line transcription occurs and small RNAs begin to accumulate. Cells in which we disrupted the DCL1 gene (DeltaDCL1) grew normally and initiated conjugation as wild-type cells but arrested near the end of development and eventually died, unable to resume vegetative growth. These DeltaDCL1 cells failed to generate the abundant small RNAs but instead accumulated germ line-limited transcripts. Together, our findings demonstrate that these transcripts are the precursors of the small RNAs and that DCL1 performs RNA processing within the micronucleus. Postconjugation DeltaDCL1 cells die without eliminating the germ line-limited DNA sequences from their newly formed somatic macronuclei, a result that shows that this Dicer-related gene is required for programmed DNA rearrangements. Surprisingly, DeltaDCL1 cells were not deficient in overall H3K9 methylation, but this modification was not enriched on germ line-limited sequences as it is in wild-type cells, which clearly demonstrates that these small RNAs are essential for its targeting to specific loci.

Animals↗

PubMed2004

Communication between parental and developing genomes during tetrahymena nuclear differentiation is likely mediated by homologous RNAs.

Approximately 6000 DNA elements, totaling nearly 15 Mb, are coordinately excised from the developing somatic genome of Tetrahymena thermophila. An RNA interference (RNAi)-related mechanism has been implicated in the targeting of these germline-limited sequences for chromatin modification and subsequent DNA rearrangement. The excision of individual DNA segments can be inhibited if the homologous sequence is placed within the parental somatic nucleus, indicating that communication occurs between the parental and developing genomes. To determine how the DNA content of one nucleus is communicated to the other, we assessed DNA rearrangement occurring in wild-type cells that were mated to cells that contained the normally germline-limited M element within their somatic nuclei. M-element rearrangement was blocked in the wild-type cell even when no genetic exchange occurred between mating partners, a finding that is inconsistent with any genetic imprinting models. This inhibition by the parental somatic nucleus was rapidly established between 5 and 6 hr of conjugation, near or shortly after the time that zygotic nuclei are formed. M-element small RNAs (sRNAs) that are believed to direct its rearrangement were found to rapidly accumulate during the first few hours of conjugation before stabilizing to a low, steady-state level. The period between 5 and 6 hr during which sRNA levels stabilize correlates with the time after which the parental genome can block DNA rearrangement. These data lead us to suggest that homologous sRNAs serve as mediators to communicate sequence-specific information between the parental and developing genomes, thereby regulating genome-wide DNA rearrangement, and that these sRNAs can be effectively compared to the somatic genome of both parents.

Animals↗