Functional Characterisation of Bile Metagenome: Study of Metagenomic Dark Matter

Gallbladder metagenome involves a wide range of unidentified sequences comprising the so-called metagenomic dark matter. Therefore, this study aimed to characterise three gallbladder metagenomes and a fosmid library with an emphasis on metagenomic dark matter fraction. For this purpose, a novel data analysis strategy based on the combination of remote homology and molecular modelling has been proposed. According to the results obtained, several protein functional domains were annotated in the metagenomic dark matter fraction including acetyltransferases, outer membrane transporter proteins, membrane assembly factors, DNA repair and recombination proteins and response regulator phosphatases. In addition, one deacetylase involved in mycothiol biosynthesis was found in the metagenomic dark matter fraction of the fosmid library. This enzyme may exert a protective effect in Actinobacteria against bile components exposure, in agreement with the presence of multiple antibiotic and multidrug resistance genes. Potential mechanisms of action of this novel deacetylase were elucidated by molecular simulations, highlighting the role of histidine and aspartic acid residues. Computational pipelines presented in this work may be of special interest to discover novel microbial enzymes which had not been previously characterised.

Epigenetic Dysregulation of Mammalian Male Meiosis Caused by Interference of Recombination and Synapsis

Meiosis involves a series of specific chromosome events, namely homologous synapsis, recombination, and segregation. Disruption of either recombination or synapsis in mammals results in the interruption of meiosis progression during the first meiotic prophase. This is usually accompanied by a defective transcriptional inactivation of the X and Y chromosomes, which triggers a meiosis breakdown in many mutant models. However, epigenetic changes and transcriptional regulation are also expected to affect autosomes. In this work, we studied the dynamics of epigenetic markers related to chromatin silencing, transcriptional regulation, and meiotic sex chromosome inactivation throughout meiosis in knockout mice for genes encoding for recombination proteins SPO11, DMC1, HOP2 and MLH1, and the synaptonemal complex proteins SYCP1 and SYCP3. These models are defective in recombination and/or synapsis and promote apoptosis at different stages of progression. Our results indicate that impairment of recombination and synapsis alter the dynamics and localization pattern of epigenetic marks, as well as the transcriptional regulation of both autosomes and sex chromosomes throughout prophase-I progression. We also observed that the morphological progression of spermatocytes throughout meiosis and the dynamics of epigenetic marks are processes that can be desynchronized upon synapsis or recombination alteration. Moreover, we detected an overlap of early and late epigenetic signatures in most mutants, indicating that the normal epigenetic transitions are disrupted. This can alter the transcriptional shift that occurs in spermatocytes in mid prophase-I and suggest that the epigenetic regulation of sex chromosomes, but also of autosomes, is an important factor in the impairment of meiosis progression in mammals.

Aging Negatively Impacts DNA Repair and Bivalent Formation in the C. elegans Germ Line

Defects in crossover (CO) formation during meiosis are a leading cause of birth defects, embryonic lethality, and infertility. In a wide range of species, maternal aging increases aneuploidy and decreases oocyte quality. In C. elegans which produce oocytes throughout the first half of adulthood, aging both decreases oocytes quality and increases meiotic errors. Phenotypes of mutations in genes encoding double-strand break (DSB)-associated proteins get more severe with maternal age suggesting that early meiosis reflects a particularly sensitive node during reproductive aging in the worm. We observed that aging has a direct effect on the integrity of C. elegans meiotic CO formation, as observed by an increase of univalent chromosomes and fusions at diakinesis, with a considerable increase starting at 4 days. We also characterize the possible causes for the age-related changes in CO formation by analyzing both steady-state levels and kinetics of the ssDNA binding proteins RPA-1 and RAD-51. Profound reductions in numbers of both RPA-1 and RAD-51 foci suggests that both DSB formation and early meiotic repair are compromised in aging worms. Using laser microirradiation and γ-irradiation to induce exogenous damage, we show specifically that recruitment of these homologous recombination proteins is altered. Repair defects can be seen in two-and-one-half day-old adults making the loss of germline repair capacity among the earliest aging phenotypes in the worm.

The Ultimate (Mis)match: When DNA Meets RNA

RNA-containing structures, including ribonucleotide insertions, DNA:RNA hybrids and R-loops, have recently emerged as critical players in the maintenance of genome integrity. Strikingly, different enzymatic activities classically involved in genome maintenance contribute to their generation, their processing into genotoxic or repair intermediates, or their removal. Here we review how this substrate promiscuity can account for the detrimental and beneficial impacts of RNA insertions during genome metabolism. We summarize how in vivo and in vitro experiments support the contribution of DNA polymerases and homologous recombination proteins in the formation of RNA-containing structures, and we discuss the role of DNA repair enzymes in their removal. The diversity of pathways that are thus affected by RNA insertions likely reflects the ancestral function of RNA molecules in genome maintenance and transmission.

CRISPY-BRED and CRISPY-BRIP: efficient bacteriophage engineering

Genome engineering of bacteriophages provides opportunities for precise genetic dissection and for numerous phage applications including therapy. However, few methods are available for facile construction of unmarked precise deletions, insertions, gene replacements and point mutations in bacteriophages for most bacterial hosts. Here we describe CRISPY-BRED and CRISPY-BRIP, methods for efficient and precise engineering of phages in Mycobacterium species, with applicability to phages of a variety of other hosts. This recombineering approach uses phage-derived recombination proteins and Streptococcus thermophilus CRISPR-Cas9.

Distal Bias of Meiotic Crossovers in Hexaploid Bread Wheat Reflects Spatio-Temporal Asymmetry of the Meiotic Program

Meiotic recombination generates genetic variation and provides physical links between homologous chromosomes (crossovers) essential for accurate segregation. In cereals the distribution of crossovers, cytologically evident as chiasmata, is biased toward the distal regions of chromosomes. This creates a bottleneck for plant breeders in the development of varieties with improved agronomic traits, as genes situated in the interstitial and centromere proximal regions of chromosomes rarely recombine. Recent advances in wheat genomics and genome engineering combined with well-developed wheat cytogenetics offer new opportunities to manipulate recombination and unlock genetic variation. As a basis for these investigations we have carried out a detailed analysis of meiotic progression in hexaploid wheat (Triticum aestivum) using immunolocalization of chromosome axis, synaptonemal complex and recombination proteins. 5-Bromo-2′-deoxyuridine (BrdU) labeling was used to determine the chronology of key events in relation to DNA replication. Axis morphogenesis, synapsis and recombination initiation were found to be spatio-temporally coordinated, beginning in the gene-dense distal chromosomal regions and later occurring in the interstitial/proximal regions. Moreover, meiotic progression in the distal regions was coordinated with the conserved chromatin cycles that are a feature of meiosis. This mirroring of the chiasma bias was also evident in the distribution of the gene-associated histone marks, H3K4me3 and H3K27me3; the repeat-associated mark, H3K27me1; and H3K9me3. We believe that this study provides a cytogenetic framework for functional studies and ongoing initiatives to manipulate recombination in the wheat genome.

λ Recombineering Used to Engineer the Genome of Phage T7

Bacteriophage T7 and T7-like bacteriophages are valuable genetic models for lytic phage biology that have heretofore been intractable with in vivo genetic engineering methods. This manuscript describes that the presence of λ Red recombination proteins makes in vivo recombineering of T7 possible, so that single base changes and whole gene replacements on the T7 genome can be made. Red recombination functions also increase the efficiency of T7 genome DNA transfection of cells by ~100-fold. Likewise, Red function enables two other T7-like bacteriophages that do not normally propagate in E. coli to be recovered following genome transfection. These results constitute major technical advances in the speed and efficiency of bacteriophage T7 engineering and will aid in the rapid development of new phage variants for a variety of applications.