Manipulating the 3D organization of the largest synthetic yeast chromosome

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes. </p

Cell wall protein variation, break-induced replication, and subtelomere dynamics in Candida glabrata.

Candida glabrata is an opportunistic pathogen of humans, responsible for up to 30% of disseminated candidiasis. Adherence of C. glabrata to host cells is mediated by adhesin-like proteins (ALPs), about half of which are encoded in the subtelomeres. We performed a de novo assembly of two C. glabrata strains, BG2 and BG3993, using long single-molecule real-time (SMRT) reads, and constructed high-quality telomere-to-telomere assemblies of all 13 chromosomes to assess differences between C. glabrata strains. We documented variation between strains, and in agreement with earlier studies, found high (~0.5%-1%) frequencies of SNVs across the genome, including within subtelomeric regions. We documented changes in ALP gene structure and complement: there are large length differences in ALP genes in different strains, resulting from copy number variation in tandem repeats. We compared strains to characterize chromosome rearrangement events including within the poorly characterized subtelomeric regions. We show that rearrangements within the subtelomere regions all affect ALP-encoding genes, and 14/16 involve just the most terminal ALP gene. We present evidence that these rearrangements are mediated by break-induced replication. This study highlights the constrained nature of subtelomeric changes impacting ALP gene complement and subtelomere structure

RADOM, an Efficient <i>In Vivo</i> Method for Assembling Designed DNA Fragments up to 10 kb Long in <i>Saccharomyces cerevisiae</i>

We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method via homologous recombination in Saccharomyces cerevisiae, which combines assembly in yeasto with blue/white screening in Escherichia coli. We show that RADOM can successfully assemble ∼3 and ∼10 kb DNA fragments that are highly similar to the yeast genome rapidly and accurately. This method was tested in the Build-A-Genome course by undergraduate students, where 125 ∼3 kb "minichunks" from the synthetic yeast genome project Sc2.0 were assembled. Here, 122 out of 125 minichunks achieved insertions with correct sizes, and 102 minichunks were sequenced verified. As this method reduces the time-consuming and labor-intensive efforts of yeast assembly by improving the screening efficiency for correct assemblies, it may find routine applications in the construction of DNA fragments, especially in hierarchical assembly projects

Polo-like kinase-1 as a potential prognostic marker of prostate cancer utilizing ORIEN data.

384 Background: Polo-Like Kinase-1(PLK1) is one of the key regulators in G2/M cell cycle. Preclinical studies have shown that PLK1 might be involved in treatment resistance in prostate cancer (PCa). PLK1 overexpression by immunohistochemistry is associated with higher Gleason grade and thus PLK1 could play a role in PCa tumorigenesis. In this study, we utilized a different methodology by using RNA-seq PLK1 expression and correlated with clinical outcomes. Methods: Patients with all stages of prostate adenocarcinoma diagnosed between 2014 and 2020 from nine participating members of the Oncology Research Information Exchange Network (ORIEN) were included in the study. Tumor RNA-seq was analyzed for PLK1 expression and associated with clinical data obtained from ORIEN AVATAR. PLK1 expression was stratified into high expression (defined as ≥75th percentile of PLK1 mRNA expression) vs. low expression (≤75th percentile). Univariate and multivariate Cox proportional hazard model were used to compare overall survival between patients with low vs high PLK1 expression adjusting for age, race, PSA and stage. Results: A total of 452 tumors were evaluated for PLK1 expression. Of those, 241 (53.3%) had high PLK1 and 211 (46.7%) had low PLK1 expression. Median age at diagnosis for high and low PLK1 expression was 64.5 and 63.2 years respectively. The majority of patients were Caucasian in both groups. In high PLK1 group, 93 (65%) had localized high risk/very high risk disease, 27 (19%) had localized intermediate risk, 20 (14%) had metastatic vs. 97 (63%), 38 (25%) and 18(12%) respectively in low PLK1 group. Gleason ≥ 8 and PSA &lt;20 were seen slightly more in high PLK1 compared to low PLK1; 40% vs 33% and 96% vs.94% respectively. The median follow-up time was 2.74 years in low PLK1 group and 2.71 years in high PLK1 group. There was no difference between PLK1 low vs. high expression for age, race, stage, PSA and Gleason grade. There was no difference in overall survival between low PLK1 and high PLK1 expression by univariate analysis (p=0.86; HR 1.09) or after adjusting for multivariate analysis (p=0.85; HR 1.12). Conclusions: In our study, we did not find RNA-seq PLK1 expression as a potential prognostic marker for prostate cancer. There was no survival difference between low PLK1 vs. high PLK1 expression. </jats:p

subCULTron - Cultural development as a tool in underwater robotics

This paper presents the research done in the field of robotic cultural evolution in challenging real world environments. We hereby present these efforts, as part of project subCULTron, where we will create an artificial society of three cooperating sub-cultures of robotic agents operating in a challenging real-world habitat. We introduce the novel concept of “cultural learning”, which will allow a swarm of agents to locally adapt to a complex environment and exchange the information about this adaptation with other subgroups of agents. Main task of the presented robotic system is autonomous environmental monitoring including self organised task allocation and organisation of swarm movement processes. One main focus of the project is on the development and implementation of bio-inspired controllers, as well as novel bio-inspired sensor systems, communication principles, energy harvesting and morphological designs. The main scientific objective is to enable and study the emergence of a collective long-term autonomous cognitive system in which information survives the operational lifetime of individuals, allowing cross-generation learning of the society by self-optimising.SCOPUS: cp.kinfo:eu-repo/semantics/publishe