Project description:The complete 1,751,377-bp sequence of the genome of the thermophilic archaeon Methanobacterium thermoautotrophicum deltaH has been determined by a whole-genome shotgun sequencing approach. A total of 1,855 open reading frames (ORFs) have been identified that appear to encode polypeptides, 844 (46%) of which have been assigned putative functions based on their similarities to database sequences with assigned functions. A total of 514 (28%) of the ORF-encoded polypeptides are related to sequences with unknown functions, and 496 (27%) have little or no homology to sequences in public databases. Comparisons with Eucarya-, Bacteria-, and Archaea-specific databases reveal that 1,013 of the putative gene products (54%) are most similar to polypeptide sequences described previously for other organisms in the domain Archaea. Comparisons with the Methanococcus jannaschii genome data underline the extensive divergence that has occurred between these two methanogens; only 352 (19%) of M. thermoautotrophicum ORFs encode sequences that are >50% identical to M. jannaschii polypeptides, and there is little conservation in the relative locations of orthologous genes. When the M. thermoautotrophicum ORFs are compared to sequences from only the eucaryal and bacterial domains, 786 (42%) are more similar to bacterial sequences and 241 (13%) are more similar to eucaryal sequences. The bacterial domain-like gene products include the majority of those predicted to be involved in cofactor and small molecule biosyntheses, intermediary metabolism, transport, nitrogen fixation, regulatory functions, and interactions with the environment. Most proteins predicted to be involved in DNA metabolism, transcription, and translation are more similar to eucaryal sequences. Gene structure and organization have features that are typical of the Bacteria, including genes that encode polypeptides closely related to eucaryal proteins. There are 24 polypeptides that could form two-component sensor kinase-response regulator systems and homologs of the bacterial Hsp70-response proteins DnaK and DnaJ, which are notably absent in M. jannaschii. DNA replication initiation and chromosome packaging in M. thermoautotrophicum are predicted to have eucaryal features, based on the presence of two Cdc6 homologs and three histones; however, the presence of an ftsZ gene indicates a bacterial type of cell division initiation. The DNA polymerases include an X-family repair type and an unusual archaeal B type formed by two separate polypeptides. The DNA-dependent RNA polymerase (RNAP) subunits A', A", B', B" and H are encoded in a typical archaeal RNAP operon, although a second A' subunit-encoding gene is present at a remote location. There are two rRNA operons, and 39 tRNA genes are dispersed around the genome, although most of these occur in clusters. Three of the tRNA genes have introns, including the tRNAPro (GGG) gene, which contains a second intron at an unprecedented location. There is no selenocysteinyl-tRNA gene nor evidence for classically organized IS elements, prophages, or plasmids. The genome contains one intein and two extended repeats (3.6 and 8.6 kb) that are members of a family with 18 representatives in the M. jannaschii genome.
Project description:Reprogramming of the gamete into a developmentally competent embryo identity is a fundamental aspect of preimplantation development. One of the most important processes of this reprogramming is the transcriptional awakening during embryonic genome activation (EGA), which robustly occurs in fertilized embryos but is defective in most somatic cell nuclear transfer (SCNT) embryos. However, little is known about the genome-wide underlying chromatin landscape during EGA in SCNT embryos and how it differs from a fertilized embryo. By profiling open chromatin genome-wide in both types of bovine embryos, we find that SCNT embryos fail to reprogram a subset of the EGA gene targets that are normally activated in fertilized embryos. Importantly, a small number of transcription factor (TF) motifs explain most chromatin regions that fail to open in SCNT embryos suggesting that over-expression of a limited number of TFs may provide more robust reprogramming. One such TF, the zygotically-expressed bovine gene DUXC which is a homologue of EGA factors DUX/DUX4 in mouse/human, is alone capable of activating ~84% of all EGA transcripts that fail to activate normally in SCNT embryos. Additionally, single-cell chromatin profiling revealed low intra-embryo heterogeneity but high inter-embryo heterogeneity in SCNT embryos and an uncoupling of cell division and open chromatin reprogramming during EGA. Surprisingly, our data also indicate that transcriptional defects may arise downstream of promoter chromatin opening in SCNT embryos, suggesting additional mechanistic insights into how and why transcription at EGA is dysregulated. We anticipate that our work will lead to altered SCNT protocols to increase the developmental competency of bovine SCNT embryos.