Biology subjects

Murakami, H.

Publications and source records attributed to Murakami, H..

4 recordsLinked to original sources

bioRxiv2018

Multilayered mechanisms ensure that short chromosomes recombine in meiosis

To segregate accurately during meiosis, homologous chromosomes in most species must recombine. Very small chromosomes would risk missegregation if recombination were randomly distributed, so the double-strand breaks (DSBs) that initiate recombination are not haphazard. How this nonrandomness is controlled is not under-stood. Here we demonstrate that Saccharomyces cerevisiae integrates multiple, temporally distinct pathways to regulate chromosomal binding of pro-DSB factors Rec114 and Mer2, thereby controlling duration of a DSB-competent state. Homologous chromosome engagement regulates Rec114/Mer2 dissociation late in prophase, whereas replication timing and proximity to centromeres or telomeres influence timing and amount of Rec114/Mer2 accumulation early. A distinct early mechanism boosts Rec114/Mer2 binding quickly to high levels specifically on the shortest chromosomes, dependent on chromosome axis proteins and subject to selection pressure to maintain hyperrecombinogenic properties of these chromosomes. Thus, an organisms karyotype and its attendant risk of meiotic missegregation influence the shape and evolution of its recombination landscape.

genetics↗

bioRxiv2018

Potential Application of Bacteriophages in Enrichment Culture for Improved Prenatal Streptococcus agalactiae Screening

Vertical transmission of Streptococcus agalactiae can cause neonatal infections. A culture test in the late stage of pregnancy is used to screen for the presence of maternal S. agalactiae for intrapartum antibiotic prophylaxis. For the test, vaginal-rectal swab sampling is immediately followed by enrichment culture and bacterial identification. In some cases, Enterococcus faecalis competes with and overgrowths S. agalactiae in the enrichment culture. Consequently, the identification test occasionally yields false-negative results. Bacterial viruses, bacteriophages (phages), infect and kill specific host bacteria. In the current study, we explored the feasibility of using phages to minimize the undesirable E. faecalis outgrowth and facilitate S. agalactiae detection in an experimental setting. Phage mixture was prepared using three phages that specifically infect E. faecalis: phiEF24C, phiEF17H, and phiM1EF22. The mixture inhibited the growth of 86.7% (26/30) of E. faecalis strains tested in the enrichment broth. When single strains of E. faecalis and S. agalactiae were inoculated in the enrichment broth containing the phage mixture, bacterial growth was inhibited or facilitated, respectively. Further, several sets of S. agalactiae and E. faecalis strains were co-cultured, and bacteria were detected on chromogenic agar after the enrichment culture. S. agalactiae was dominant after plating a phage mixture-treated co-culture, while it was barely detected after plating the untreated co-culture. Considering these observations, the phage mixture can be employed in the S. agalactiae culture test to increase test accuracy.

microbiology↗

bioRxiv2017

Quantitative monitoring of multispecies fish environmental DNA using high-throughput sequencing

Effective ecosystem conservation and resource management require quantitative monitoring of biodiversity, including accurate descriptions of species composition and temporal variations of species abundance. Therefore, quantitative monitoring of biodiversity has been performed for many ecosystems, but it is often time- and effort-consuming and costly. Recent studies have shown that environmental DNA (eDNA), which is released to the environment from macro-organisms living in a habitat, contains information about species identity and abundance. Thus, analyzing eDNA would be a promising approach for more efficient biodiversity monitoring. In the present study, we added internal standard DNAs (i.e., known amounts of short DNA fragments from fish species that have never been observed in a sampling area) to eDNA samples, which were collected weekly from a coastal marine ecosystem in Maizuru-Bay, Kyoto, Japan (from April 2015 to March 2016), and performed metabarcoding analysis using Illumina MiSeq to simultaneously identify fish species and quantify fish eDNA copy numbers. A correction equation was obtained for each sample using the relationship between the number of sequence reads and the added amount of the standard DNAs, and this equation was used to estimate the copy numbers from the sequence reads of non-standard fish eDNA. The calculated copy numbers showed significant positive correlation with those determined by quantitative PCR, suggesting that eDNA metabarcoding with standard DNA enabled useful quantification of eDNA. Furthermore, for samples that show a high level of PCR inhibition, our method might allow more accurate quantification than qPCR because the correction equations generated using internal standard DNAs would include the effect of PCR inhibition. A single run of Illumina MiSeq produced > 70 quantitative fish eDNA time series in our study, showing that our method could contribute to more efficient and quantitative monitoring of biodiversity.

ecology↗

bioRxiv2017

Histone H3 threonine 11 phosphorylation is catalyzed directly by the meiosis-specific kinase Mek1 and provides a molecular readout of Mek1 activity in vivo

Saccharomyces cerevisiae Mek1 is a CHK2/Rad53-family kinase that regulates meiotic recombination and progression upon its activation in response to DNA double-strand breaks (DSBs). The full catalog of direct Mek1 phosphorylation targets remains unknown. Here, we show that phosphorylation of histone H3 on threonine 11 (H3 T11ph) is induced by meiotic DSBs in S. cerevisiae and Schizosaccharomyces pombe. Molecular genetic experiments in S. cerevisiae confirmed that Mek1 is required for H3 T11ph and revealed that phosphorylation is rapidly reversed when Mek1 kinase is no longer active. Reconstituting histone phosphorylation in vitro with recombinant proteins demonstrated that Mek1 directly catalyzes H3 T11 phosphorylation. Mutating H3 T11 to nonphosphorylatable residues conferred no detectable defects in otherwise unperturbed meiosis, although the mutations modestly reduced spore viability in certain strains where Rad51 is used for strand exchange in place of Dmc1. H3 T11ph is therefore mostly dispensable for Mek1 function. However, H3 T11ph provides an excellent marker of ongoing Mek1 kinase activity in vivo. Anti-H3 T11ph chromatin immunoprecipitation followed by deep sequencing demonstrated that H3 T11ph was highly enriched at presumed sites of attachment of chromatin to chromosome axes, gave a more modest signal along chromatin loops, and was present at still lower levels immediately adjacent to DSB hotspots. These localization patterns closely tracked the distribution of Red1 and Hop1, axis proteins required for Mek1 activation. These findings provide insight into the spatial disposition of Mek1 kinase activity and the higher order organization of recombining meiotic chromosomes.\n\nbioRxiv version 2 (June 2017)One major experimental change was incorporated into the revised manuscript: We repeated the anti-H3 T11ph ChIP-seq experiment on larger scale, including two meiotic time points from each of two wild type cultures and one time point from a spo11-Y135F mutant culture. To facilitate comparison of different samples, we used meiotic S. pombe cells as a spike-in control for all samples for both anti-H3 and anti-H3 T11ph ChIP-seq. Most conclusions described in the first bioRxiv submission were confirmed, but the improved datasets allowed us to derive more detailed information in particular about H3 T11ph patterns around DSB sites.\n\nbioRxiv version 3 (October 2017)The following experimental changes were incorporated, along with more minor changes in response to reviewer comments: O_LIWe added previously unpublished ChIP-seq data for Red1 protein, generated by Masaru Ito and Kunihiro Ohta, who have been added as coauthors.\nC_LIO_LIWe repeated key experiments with the H3-T11V single point mutant. No conclusions were changed relative to prior experiments with the H3-S10, T11V mutant.\nC_LIO_LIWe repeated the analysis of spore viability in a dmc1 rad54-T132A background using a more appropriate isogenic control, and recapitulated the original conclusion that the H3-T11V mutation modestly decreases spore viability in this sensitized background.\nC_LI

molecular biology↗