Adaptive laboratory evolution of a genome-reduced Escherichia coli.

Synthetic biology aims to design and construct bacterial genomes harboring the minimum number of genes required for self-replicable life. However, the genome-reduced bacteria often show impaired growth under laboratory conditions that cannot be understood based on the removed genes. The unexpected phenotypes highlight our limited understanding of bacterial genomes. Here, we deploy adaptive laboratory evolution (ALE) to re-optimize growth performance of a genome-reduced strain. The basis for suboptimal growth is the imbalanced metabolism that is rewired during ALE. The metabolic rewiring is globally orchestrated by mutations in rpoD altering promoter binding of RNA polymerase. Lastly, the evolved strain has no translational buffering capacity, enabling effective translation of abundant mRNAs. Multi-omic analysis of the evolved strain reveals transcriptome- and translatome-wide remodeling that orchestrate metabolism and growth. These results reveal that failure of prediction may not be associated with understanding individual genes, but rather from insufficient understanding of the strain's systems biology

한국인 다발골수종 환자의 유전체변이 분석

학위논문(박사) -- 서울대학교대학원 : 의과대학 의학과, 2021.8. 이누리.To investigate the prognostic value of gene variants and copy number variations (CNVs) in patients with newly diagnosed multiple myeloma (NDMM), an integrative genomic analysis was performed using conventional cytogenetics, fluorescent in situ hybridization, and whole-exome sequencing. Sixty-seven patients with NDMM exhibiting more than 60% plasma cells in the bone marrow aspirate were enrolled in the study. Whole-exome sequencing was performed on bone marrow nucleated cells. Mutation and CNV analyses were performed using the CNVkit and Nexus Copy Number software. Eighty-three driver gene mutations were detected in 63 patients with NDMM. The median number of mutations per patient was 2.0 (95% confidence interval [CI] = 2.0–3.0, range = 0–8). MAML2 and BHLHE41 mutations were associated with decreased survival. CNVs were detected in 56 patients (72.7%; 56/67). The median number of CNVs per patient was 6.0 (95% CI = 5.7–7.0; range = 0–16). Among the CNVs, 1q gain, 6p gain, 6q loss, 8p loss, and 13q loss were associated with decreased survival. Additionally, 1q gain and 6p gain were independent adverse prognostic factors. Increased numbers of CNVs and driver gene mutations were associated with poor clinical outcomes. Cluster analysis revealed that patients with the highest number of driver mutations along with 1q gain, 6p gain, and 13q loss exhibited the poorest prognosis. In addition to the known prognostic factors, the integrated analysis of genetic variations and CNVs could contribute to prognostic stratification of patients with NDMM.이번 연구는 처음 진단된 다발골수종 환자에서 유전자 변이 및 구조적 변이의 예후를 분석하기 위해 기존의 세포유전학, 형광접합보인자법 및 전체엑솜염기서열분석을 이용하여 통합적인 유전체 분석을 수행하였다. 골수흡인액에서 60% 이상의 형질 세포를 보이는 다발골수종 환자 67명을 대상으로 골수 유핵 세포에서 전체엑솜염기서열분석을 수행하였다. 체세포 돌연변이 및 구조적변이 분석에는 두 가지의 응용 프로그램(CNVkit 및 Nexus Copy Number)이 이용되었다. 63명의 다발골수종 환자에서 83개의 유발 유전자 돌연변이가 발견되었다. 환자당 체세포 돌연변이 수의 중앙값은 2.0이었다(95 % 신뢰 구간 = 2.0–3.0, 범위 = 0–8). MAML2 및 BHLHE41 돌연변이는 짧은 생존률과 관련이 있었다. 구조적 변이는 56 명의 환자 (72.7%; 56/67)에서 발견되었다. 환자 당 구조적 변이 수의 중앙값은 6.0 (95 % CI = 5.7 – 7.0, 범위 = 0 – 16)이었다. 구조적 변이 중 1q 증폭, 6p 증폭, 6q 결실, 8p 결실 및 13q 결실은 생존 감소와 관련이 있었다. 또한 1q 증폭과 6p 증폭은 독립적인 불량한 예후 인자였다. 한 환자에서 보이는 높은 빈도의 구조적 변이와 유발 유전자 돌연변이는 불량한 임상 예후와 상관관계가 높았다. 클러스터링 분석을 통해 1q 증폭, 6p 증폭 및 13q 결실과 함께 가장 높은 빈도의 유발 유전자 돌연변이를 가진 환자가 가장 나쁜 예후를 나타냄을 알 수 있었다. 다발골수종 기존의 알려진 예후인자와 함께, 체세포 돌연변이와 구조적변이의 통합 분석은 환자의 보다 정확한 예후 예측 및 분류에 있어 의미가 높을 것으로 기대된다.1. Introduction 1 2. Materials and Methods. 4 2.1. Patients 4 2.2. DNA extraction and exome sequencing. 5 2.3. Bioinformatic evaluation of sequencing data 6 2.4. Copy number analysis . 9 2.5. Statistical analysis 11 3. Results 12 3.1. Characteristics of the study population 12 3.2. Identification of CNVs in MM 14 3.3. Analysis of selected driver gene mutations. 18 3.4. Correlation analysis of genomic variants. 26 3.5. Prognostic impact of CNVs and somatic mutations. 29 3.6. Factors affecting OS in patients with hyperdiploid NDMM. 40 3.7. OS of clusters classified based on K-means analysis 42 4. Discussions. 45 References. 52 Abstract in Korean 61박

Gene expression in Leishmania is regulated predominantly by gene dosage

ABSTRACT Leishmania tropica, a unicellular eukaryotic parasite present in North and East Africa, the Middle East, and the Indian subcontinent, has been linked to large outbreaks of cutaneous leishmaniasis in displaced populations in Iraq, Jordan, and Syria. Here, we report the genome sequence of this pathogen and 7,863 identified protein-coding genes, and we show that the majority of clinical isolates possess high levels of allelic diversity, genetic admixture, heterozygosity, and extensive aneuploidy. By utilizing paired genome-wide high-throughput DNA sequencing (DNA-seq) with RNA-seq, we found that gene dosage, at the level of individual genes or chromosomal “somy” (a general term covering disomy, trisomy, tetrasomy, etc.), accounted for greater than 85% of total gene expression variation in genes with a 2-fold or greater change in expression. High gene copy number variation (CNV) among membrane-bound transporters, a class of proteins previously implicated in drug resistance, was found for the most highly differentially expressed genes. Our results suggest that gene dosage is an adaptive trait that confers phenotypic plasticity among natural Leishmania populations by rapid down- or upregulation of transporter proteins to limit the effects of environmental stresses, such as drug selection. IMPORTANCE Leishmania is a genus of unicellular eukaryotic parasites that is responsible for a spectrum of human diseases that range from cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL) to life-threatening visceral leishmaniasis (VL). Developmental and strain-specific gene expression is largely thought to be due to mRNA message stability or posttranscriptional regulatory networks for this species, whose genome is organized into polycistronic gene clusters in the absence of promoter-mediated regulation of transcription initiation of nuclear genes. Genetic hybridization has been demonstrated to yield dramatic structural genomic variation, but whether such changes in gene dosage impact gene expression has not been formally investigated. Here we show that the predominant mechanism determining transcript abundance differences (>85%) in Leishmania tropica is that of gene dosage at the level of individual genes or chromosomal somy

Molecularly defined diffuse leptomeningeal glioneuronal tumor (DLGNT) comprises two subgroups with distinct clinical and genetic features

Diffuse leptomeningeal glioneuronal tumors (DLGNT) represent rare CNS neoplasms which have been included in the 2016 update of the WHO classification. The wide spectrum of histopathological and radiological features can make this enigmatic tumor entity difficult to diagnose. In recent years, large-scale genomic and epigenomic analyses have afforded insight into key genetic alterations occurring in multiple types of brain tumors and provide unbiased, complementary tools to improve diagnostic accuracy. Through genome-wide DNA methylation screening of > 25,000 tumors, we discovered a molecularly distinct class comprising 30 tumors, mostly diagnosed histologically as DLGNTs. Copy-number profiles derived from the methylation arrays revealed unifying characteristics, including loss of chromosomal arm 1p in all cases. Furthermore, this molecular DLGNT class can be subdivided into two subgroups [DLGNT methylation class (MC)-1 and DLGNT methylation class (MC)-2], with all DLGNT-MC-2 additionally displaying a gain of chromosomal arm 1q. Co-deletion of 1p/19q, commonly seen in IDH-mutant oligodendroglioma, was frequently observed in DLGNT, especially in DLGNT-MC-1 cases. Both subgroups also had recurrent genetic alterations leading to an aberrant MAPK/ERK pathway, with KIAA1549:BRAF fusion being the most frequent event. Other alterations included fusions of NTRK1/2/3 and TRIM33:RAF1, adding up to an MAPK/ERK pathway activation identified in 80% of cases. In the DLGNT-MC-1 group, age at diagnosis was significantly lower (median 5 vs 14 years, p < 0.01) and clinical course less aggressive (5-year OS 100, vs 43% in DLGNT-MC-2). Our study proposes an additional molecular layer to the current histopathological classification of DLGNT, of particular use for cases without typical morphological or radiological characteristics, such as diffuse growth and radiologic leptomeningeal dissemination. Recurrent 1p deletion and MAPK/ERK pathway activation represent diagnostic biomarkers and therapeutic targets, respectively—laying the foundation for future clinical trials with, e.g., MEK inhibitors that may improve the clinical outcome of patients with DLGNT