Mutational pattern and frequency of induced nucleotide changes in mouse ENU mutagenesis

<p>Abstract</p> <p>Background</p> <p>With the advent of sequence-based approaches in the mutagenesis studies, it is now possible to directly evaluate the genome-wide pattern of experimentally induced DNA sequence changes for a diverse array of organisms. To gain a more comprehensive understanding of the mutational bias inherent in mouse ENU mutagenesis, this study describes a detailed evaluation of the induced mutational pattern obtained from a sequence-based screen of ENU-mutagenized mice.</p> <p>Results</p> <p>Based on a large-scale screening data, we derive the sequence-based estimates of the nucleotide-specific pattern and frequency of ENU-induced base replacement mutation in the mouse germline, which are then combined with the pattern of codon usage in the mouse coding sequences to infer the spectrum of amino acid changes obtained by ENU mutagenesis. We detect a statistically significant difference between the mutational patterns in phenotype- versus sequence-based screens, which presumably reflects differential phenotypic effects caused by different amino acid replacements. We also demonstrate that the mutations exhibit strong strand asymmetry, and that this imbalance is generated by transcription, most likely as a by-product of transcription-coupled DNA repair in the germline.</p> <p>Conclusion</p> <p>The results clearly illustrate the biased nature of ENU-induced mutations. We expect that a precise understanding of the mutational pattern and frequency of induced nucleotide changes would be of practical importance when designing sequence-based screening strategies to generate mutant mouse strains harboring amino acid variants at specific loci. More generally, by enhancing the collection of experimentally induced mutations in unambiguously defined genomic regions, sequence-based mutagenesis studies will further illuminate the molecular basis of mutagenic and repair mechanisms that preferentially produce a certain class of mutational changes over others.</p

Srs2 and RecQ homologs cooperate in mei-3-mediated homologous recombination repair of Neurospora crassa

Homologous recombination and post-replication repair facilitate restart of stalled or collapsed replication forks. The SRS2 gene of Saccharomyces cerevisiae encodes a 3′–5′ DNA helicase that functions both in homologous recombination repair and in post-replication repair. This study identifies and characterizes the SRS2 homolog in Neurospora crassa, which we call mus-50. A knockout mutant of N.crassa, mus-50, is sensitive to several DNA-damaging agents and genetic analyses indicate that it is epistatic with mei-3 (RAD51 homolog), mus-11 (RAD52 homolog), mus-48 (RAD55 homolog) and mus-49 (RAD57 homolog), suggesting a role for mus-50 in homologous recombination repair. However, epistasis evidence has presented that MUS50 does not participate in post-replication repair in N.crassa. Also, the N.crassa mus-25 (RAD54 homolog) mus-50 double mutant is viable, which is in contrast to the lethal phenotype of the equivalent rad54 srs2 mutant in S.cerevisiae. Tetrad analysis revealed that mus-50 in combination with mutations in two RecQ homologs, qde-3 and recQ2, is lethal, and this lethality is suppressed by mutation in mei-3, mus-11 or mus-25. Evidence is also presented for the two independent pathways for recovery from camptothecin-induced replication fork arrest: one pathway is dependent on QDE3 and MUS50 and the other pathway is dependent on MUS25 and RECQ2

A series of ENU-induced single-base substitutions in a long-range cis-element altering Sonic hedgehog expression in the developing mouse limb bud

AbstractMammal–fish-conserved-sequence 1 (MFCS1) is a highly conserved sequence that acts as a limb-specific cis-acting regulator of Sonic hedgehog (Shh) expression, residing 1 Mb away from the Shh coding sequence in mouse. Using gene-driven screening of an ENU-mutagenized mouse archive, we obtained mice with three new point mutations in MFCS1: M101116, M101117, and M101192. Phenotype analysis revealed that M101116 mice exhibit preaxial polydactyly and ectopic Shh expression at the anterior margin of the limb buds like a previously identified mutant, M100081. In contrast, M101117 and M101192 show no marked abnormalities in limb morphology. Furthermore, transgenic analysis revealed that the M101116 and M100081 sequences drive ectopic reporter gene expression at the anterior margin of the limb bud, in addition to the normal posterior expression. Such ectopic expression was not observed in the embryos carrying a reporter transgene driven by M101117. These results suggest that M101116 and M100081 affect the negative regulatory activity of MFCS1, which suppresses anterior Shh expression in developing limb buds. Thus, this study shows that gene-driven screening for ENU-induced mutations is an effective approach for exploring the function of conserved, noncoding sequences and potential cis-regulatory elements

Analysis of endometrial microbiota by 16S ribosomal RNA gene sequencing among infertile patients: a single‐center pilot study

Abstract Purpose The present study aimed to analyze the endometrial and vaginal microbiome among a Japanese infertile population by sequencing and the impact of the endometrial and vaginal environment on implantation. Methods In total, 102 infertile (79 in vitro fertilization [IVF] and 23 non‐IVF) patients and seven healthy volunteers were recruited from August to December, 2017. Endometrial fluid and vaginal discharge samples for sequencing were collected by using an intrauterine insemination catheter. The bacterial status of the endometrium and vagina were analyzed. Results The Lactobacillus‐dominated microbiota (>90% Lactobacillus spp.) in the endometrium vs vagina was 38% (30/79) vs 44.3% (44/79) in the IVF patients, 73.9% (17/23) vs 73.9% (17/23) in the non‐IVF patients, and 85.7% (6/7) vs 85.7% (6/7) in the healthy volunteers. The percentage of endometrial Lactobacillus in the healthy volunteers was highly stable within the same menstrual cycle and even in the following cycle. The major taxonomies were Gardnerella, Streptococcus, Atopobium, Bifidobacterium, Sneathia, Prevotella, and Staphylococcus. Fifteen patients achieved pregnancy by a single vitrified‐warmed blastocyst transfer during this study; the median percentage of Lactobacillus in the pregnant women was 96.45 ± 33.61%. Conclusion A considerable percentage of non‐Lactobacillus‐dominated (NLD) microbiota was found in the endometrium of Japanese infertile women. Increasing the endometrial level of the Lactobacilli to >90% might favor the implantation outcome of NLD infertile patients

Differential Vaginal Microbiota Profiling in Lactic-Acid-Producing Bacteria between Infertile Women with and without Chronic Endometritis

Purpose: Chronic endometritis (CE) is an infectious and inflammatory disorder associated with infertility of unknown etiology, repeated implantation failure, and recurrent pregnancy loss. In the current clinical practice, intrauterine interventions such as endometrial biopsy/histopathologic examinations and/or hysteroscopy are required for the diagnosis of CE. In this study, we analyzed the microbiota in vaginal secretions (VS) as a potential prediction tool for CE in infertile women. Methods: Using next-generation sequencing analysis, we compared the VS and endometrial fluid (EF) microbiota in infertile women with (n = 20) or without CE (n = 103). Results: The detection rate of Streptococcus and Enterococcus as well as the bacterial abundance of Atopobium and Bifidobacterium in the VS microbiota was significantly lower in the CE group than in the non-CE group. Meanwhile, the detection rate and bacterial abundance of Lactobacillus in the EF and VS microbiota were at similar levels between the two groups. Conclusion: These findings suggest that VS microbiota in infertile women with CE is characterized by the reduction in Bifidobacterium and lactic-acid-producing bacteria other than Lactobacillus. Our results hold promise for the prediction of CE, not by somewhat interventional intrauterine procedures, but by less invasive VS sampling. TRIAL REGISTRATION NUMBER: UMIN000029449 (registration date 6 October 2017)

Characterization of Microbiota in Endometrial Fluid and Vaginal Secretions in Infertile Women with Repeated Implantation Failure

Studies suggest that persisting intrauterine bacterial infectious conditions such as chronic endometritis potentially impair the embryo implantation process. The microbial environment in the female reproductive tract, however, remains largely undetermined in infertile patients with a history of repeated implantation failure (RIF). Using next-generation sequencing, we aimed to characterize the microbiota in the endometrial fluid (EF) and vaginal secretions (VS) in women with RIF. Twenty-eight infertile women with a history of RIF and eighteen infertile women undergoing the first in vitro fertilization-embryo transfer attempt (the control group) were enrolled in the study. On days 6-8 in the luteal phase of the natural, oocyte-pickup, or hormone replacement cycle, the paired EF and VS samples were obtained separately. Extracted genomic DNA was pyrosequenced for the V4 region of 16S ribosomal RNA using a next-generation sequencer. The EF microbiota had higher α-diversity and broader bacterial species than the VS microbiota both in the RIF and control groups. The analysis of the UniFrac distance matrices between EF and VS also revealed significantly different clustering. Additionally, the EF microbiota, but not the VS microbiota, showed significant variation in community composition between the RIF group and the control group. Burkholderia species were not detected in the EF microbiota of any samples in the control group but were detectable in a quarter of the RIF group. To our best knowledge, this is the first study investigating the microbiota in the paired EF and VS samples in infertile women with RIF