Multiple Copies Of Chromosome Research Articles

Partial cytological diploidization of neoautotetraploid meiosis by induced cross-over rate reduction

Polyploids, which arise from whole-genome duplication events, have contributed to genome evolution throughout eukaryotes. Among plants, novel features of neopolyploids include traits that can be evolutionarily or agriculturally beneficial, such as increased abiotic stress tolerance. Thus, in addition to being interesting from an evolutionary perspective, genome duplication is also increasingly recognized as a promising crop improvement tool. However, newly formed (neo)polyploids commonly suffer from fertility problems, which have been attributed to abnormal associations among the multiple homologous chromosome copies during meiosis (multivalents). Here, we test the long-standing hypothesis that reducing meiotic cross-over number may be sufficient to limit multivalent formation, favoring diploid-like bivalent associations (cytological diploidization). To do so, we developed Arabidopsis thaliana lines with low cross-over rates by combining mutations for HEI10 and TAF4b. Double mutants showed a reduction of ~33% in cross-over numbers in diploids without compromising meiotic stability. Neopolyploids derived from the double mutant show a cross-over rate reduction of about 40% relative to wild-type neotetraploids, and groups of four homologs indeed formed fewer multivalents and more bivalents. However, we also show that the reduction in multivalents comes with the cost of a slightly increased frequency of univalents and that it does not rescue neopolyploid fertility. Thus, while our results do show that reducing cross-over rates can reduce multivalent frequency in neopolyploids, they also emphasize that there are additional factors affecting both meiotic stability and neopolyploid fertility that will need to be considered in solving the neopolyploid fertility challenge.

ODP469 Coexistence of two thyroid malignancies with Hurthle cell and Papillary thyroid carcinoma

Abstract Background The simultaneous presentation of mul tiple thyroid malignancies is a rare occurrence with each type of thyroid cancer having different cellular origins, histopathological appearance, and clinical course. Collision tumors representtwo histologically different tumor types occurring at the same anatomic region. When diagnosed within 6 months of the original they are also referred to as synchronous tumors. Papillary thyroid carcinoma (PTC) is the most common (80-85%) differentiated thyroid carcinomas (DTC), followed by follicular thyroid carcinoma (FTC) (10-15%), and Hurthle cell carcinoma (3–5%). We report a rare case of a patient with dual thyroid malignancy containing both PTC and Hurthle cell carcinoma in different thyroid lobes. Case presentation Patient is a 50 year old female with past medical history of hypertension and family history of thyroid disease in her sister and cousins, who presented with thyroid gland enlargement. Patient denied any hyper/hypothyroidism or compressive symptoms. Labs showed normal TSH and free T4. Thyroid ultrasound revealed a heterogeneous nodule with microcalcification in the right thyroid lobe and three well circumscribed left mid pole nodules. FNA thyroid biopsy of the right thyroid nodule demonstrated many Hurthle cells with uniform nuclei. The genomic classification identified multiple chromosomal copy number alterations consistent with a clonal neoplasm with intermediate-high risk for cancer, although no point mutations or gene fusions were found. Patient initially underwent right hemi-thyroidectomy and isthmectomy. Pathology confirmed two well circumscribed angioinvasive Hurthle cell carcinoma measuring 3.4×2.1×2. 0 cm and 1.6×1.5×1.4 cm with multiple foci of capsular invasion oncocytic features along with a 0.2 cm papillary thyroid microcarcinoma without extrathyroidal extension. After three weeks, patient proceeded with completion left thyroidectomy and showed a 1.2×1.1×1. 0 cm well circumscribed, partially encapsulated nodule, consistent with Papillary thyroid carcinoma (PTC). Lymph nodes were negative for metastasis. Patient received whole body I-131 scan and radioiodine ablation therapy. She was subsequently placed on Levothyroxine for thyroid hormone replacement and remained euthyroid without cancer recurrence. Conclusion Collision tumors account for 1% of thyroid malignancies with the most commonly reported combination being PTC and medullary thyroid carcinoma. Interestingly, this case presented with a rare finding of collision and synchronous tumors in the form of PTC and Hurthle cell carcinoma which was known to associate with increased chromosomal copy number instead of point genetic mutation. Previous literature have described collision effect, stem cell, hostage, and single progenitor theories in an attempt to explain the origin of these phenomena with possible molecular mechanisms. Clinicians should be aware that the dual thyroid malignancy with different histogenetic origin may co-exist in the same patient. The therapeutic strategy should be tailored based on each thyroid cancer variant with correlated biological behavior, clinical and genetic features. Presentation: No date and time listed

The Heterocyst-Specific Small RNA NsiR1 Regulates the Commitment to Differentiation in Nostoc.

ABSTRACTHeterocysts are specialized cells that filamentous cyanobacteria differentiate for the fixation of atmospheric nitrogen when other nitrogen sources are not available. Heterocyst differentiation at semiregular intervals along the filaments requires complex structural and metabolic changes that are under the control of the master transcriptional regulator HetR. NsiR1 (nitrogen stress-induced RNA 1) is a HetR-dependent noncoding RNA that is expressed from multiple chromosomal copies, some identical, some slightly divergent in sequence, specifically in heterocysts from very early stages of differentiation. We have previously shown that NsiR1 inhibits translation of the overlapping hetF mRNA by an antisense mechanism. Here, we identify alr3234, a hetP-like gene involved in the regulation of commitment (point of no return) to heterocyst differentiation, as a target of NsiR1. A strain overexpressing one of the identical copies of NsiR1 commits to heterocyst development earlier than the wild type. The posttranscriptional regulation exerted by NsiR1 on the expression of two genes involved in heterocyst differentiation and commitment, hetF and alr3234, adds a new level of complexity to the network of transcriptional regulation and protein-protein interactions that participate in heterocyst differentiation.IMPORTANCE Heterocysts are nitrogen-fixing specialized cells that appear at semiregular intervals along cyanobacterial filaments upon nitrogen starvation. The differentiation and patterning of heterocysts is a model for the study of cell differentiation in multicellular prokaryotes. The regulation of differentiation, which is only partially understood, includes transcriptional changes, factor diffusion between cells, and protein-protein interactions. This work describes the identification of a novel target for NsiR1, a small RNA (sRNA) encoded in multiple slightly divergent copies, and shows how different copies of “sibling” sRNAs regulate the expression of different targets involved in one of the few examples of a differentiation process in prokaryotes.

CRISPR‐based knock‐in mutagenesis of the pioneer transcription factor FOXA1: optimization of strategies for multi‐allelic proteins in cancer cells

Precise genome engineering of living cells has been revolutionized by the introduction of the highly specific and easily programmable properties of the clustered regularly interspaced short palindromic repeats (CRISPR) technology. This has greatly accelerated research into human health and has facilitated the discovery of novel therapeutics. CRISPR‐Cas9 is most widely employed for its ability to inactivate or knockout specific genes, but can be also used to introduce subtle site‐specific substitutions of DNA sequences that can lead to changes in the amino acid composition of proteins. Despite the proven success of CRISPR‐based knock‐in strategies of genes in typical diploid cells (i.e., cells containing two sets of chromosomes), precise editing of cancer cells, that typically have unstable genomes and multiple copies of chromosomes, is more challenging and not adequately addressed in the literature. Herein, we detail our methodology for replacing endogenous proteins with intended knock‐in mutants in polyploid cancer cells and discuss our experimental design, screening strategy, and facile allele frequency estimation methodology. As proof of principle, we performed genome editing of specific amino acids within the pioneer transcription factor FOXA1, a critical component of estrogen and androgen receptor signaling, in MCF‐7 breast cancer cells. We confirm mutant FOXA1 protein expression and intended amino acid substitutions via western blotting and mass spectrometry. In addition, we show that mutant allele frequency estimation is easily achieved by topoisomerase‐based cloning combined with allele‐specific PCR, which we later confirmed by next‐generation RNA‐sequencing. Typically, there are 4 ‐ 5 copies (alleles) of FOXA1 in breast cancer cells, making the editing of this protein inherently challenging. As a result, most studies that focus on FOXA1 mutants rely on ectopic overexpression of FOXA1 from a plasmid. Therefore, we provide an optimized methodology for replacing endogenous wild‐type FOXA1 with precise knock‐in mutants to enable the systematic analysis of its molecular mechanisms within the appropriate physiological context.

Cyanobacterial multi-copy chromosomes and their replication.

NGS: next-generation sequencing; Repli-seq: replication sequencing; BrdU: 5-bromo-2'-deoxyuridine; TK: thymidine kinase; GCSI: GC skew index; PET: photosynthetic electron transport; RET: respiration electron transport; Cyt b6f complex: cytochrome b6f complex; PQ: plastoquinone; PC: plastocyanin.

OR21-02 Impact of Nodule Size on the Probability of Hurthle Cell Carcinoma and Other Cancers in Thyroid Nodules with Multiple Chromosomal Copy Number Alterations

Fine-needle aspiration (FNA) of thyroid nodules yields indeterminate cytological diagnosis in ~20% of cases, confounding patient management. This includes Hurthle cell nodules, which typically yield Bethesda IV and III cytology. Chromosomal copy number alterations (CNA) are known to occur in thyroid tumors, particularly in Hurthle cell carcinomas (HCC) as well as in other typically follicular-patterned tumors including papillary thyroid carcinomas (PTC) and poorly differentiated thyroid carcinomas (PDTC). The aim of this study was to evaluate thyroid nodules tested positive for CNA but negative for all other genomic alterations using ThyroSeq v3 NGS assay in order to establish the probability of cancer in these nodules and find whether it is influenced by the pattern of CNA and nodule size. We evaluated 111 nodules with multiple CNA detected by ThyroSeq in FNA samples and available surgical pathology outcome. Of those, 69 (62%) nodules showed CNA changes consistent with genome near-haploidization (GNH) whereas 42 (38%) nodules had multiple chromosomal losses and gains (CLG). Nodule size ranged from 0.5-10.2 cm; cytology was Bethesda III in 54%, Bethesda IV in 43%, and Bethesda V-VI in 3% of cases, with Hurthle cells mentioned in the cytology report in 64% of cases. On surgical pathology, 38 (34%) of these nodules were malignant (including 24 HCC, 8 PTC, and 5 oncocytic PDTC) and 73 (66%) were benign (including 43 Hurthle cell and 18 follicular adenomas). No significant difference was observed in probability of malignancy between the two patterns of CNA (p=0.41). However, a significant correlation between the nodule size and probability of cancer was found (p=0.006). In specific CNA groups, correlation between cancer and nodule size remained significant for nodules with GNH pattern (P=0.0002), but not with CLG pattern (p=0.449). Specifically, cancer probability in nodules with GNH pattern and <2 cm in size was 14% (all cancers minimally-invasive), 2.0-2.9 cm was 33%, 3.0-3.9 cm was 50%, 4-4.9 cm was 67%, and ≥5 cm was 80%. Among high-risk cancers (widely-invasive or angioinvasive HCC, PDTC), all 10 tumors had the GNH pattern (p=0.01) and average nodule size of 4.9 cm (range, 2.1-8.5 cm). These findings suggest that CNA of both types are frequently found in Hurthle cell tumors, and probability of cancer in nodules with CNA and no other mutations increases with larger nodule size. This may help to refine the pre-operative assessment of cancer probability and risk of more aggressive disease and offer more tailored management to these patients.

Derived alleles of two axis proteins affect meiotic traits in autotetraploid Arabidopsis arenosa

Polyploidy, which results from whole genome duplication (WGD), has shaped the long-term evolution of eukaryotic genomes in all kingdoms. Polyploidy is also implicated in adaptation, domestication, and speciation. Yet when WGD newly occurs, the resulting neopolyploids face numerous challenges. A particularly pernicious problem is the segregation of multiple chromosome copies in meiosis. Evolution can overcome this challenge, likely through modification of chromosome pairing and recombination to prevent deleterious multivalent chromosome associations, but the molecular basis of this remains mysterious. We study mechanisms underlying evolutionary stabilization of polyploid meiosis using Arabidopsis arenosa, a relative of A. thaliana with natural diploid and meiotically stable autotetraploid populations. Here we investigate the effects of ancestral (diploid) versus derived (tetraploid) alleles of two genes, ASY1 and ASY3, that were among several meiosis genes under selection in the tetraploid lineage. These genes encode interacting proteins critical for formation of meiotic chromosome axes, long linear multiprotein structures that form along sister chromatids in meiosis and are essential for recombination, chromosome segregation, and fertility. We show that derived alleles of both genes are associated with changes in meiosis, including reduced formation of multichromosome associations, reduced axis length, and a tendency to more rod-shaped bivalents in metaphase I. Thus, we conclude that ASY1 and ASY3 are components of a larger multigenic solution to polyploid meiosis in which individual genes have subtle effects. Our results are relevant for understanding polyploid evolution and more generally for understanding how meiotic traits can evolve when faced with challenges.

Specific binding of DnaA to the DnaA box motif in the cyanobacterium Synechococcus elongatus PCC 7942.

In bacterial DNA replication, the initiator protein DnaA binds to the multiple DnaA box sequences located at oriC to facilitate the unwinding of duplex DNA strands. The cyanobacterium Synechococcus elongatus PCC 7942, which contains multiple chromosomal copies per cell, has DnaA box (like sequences around the oriC region, which is located upstream of dnaN. We previously observed the binding of DnaA around the oriC region; however, the DNA-binding specificity of DnaA to DnaA box sequences has not been examined. Here, we analyzed the binding specificity of DnaA protein to the DnaA box in S. elongatus by using bio-layer interferometry (BLI), a method for monitoring intermolecular interactions. We observed that recombinant DnaA protein recognized specifically the DnaA box sequence TTTTCCACA in vitro. In addition, DNA binding activity was significantly increased by R328H mutation of DnaA. This is the first report to characterize DnaA binding to the DnaA box sequence in cyanobacteria.

No Zones Left Behind: Democratic Hepatocytes Contribute to Liver Homeostasis and Repair.

Despite minimal turnover, liver cells possess immense regenerative capacity. Some studies suggest existence of a hepatocyte subset with such unique capabilities. However, in the current issue of Cell Stem Cell, three independent studies (Chen etal., 2020; Matsumoto etal., 2020, and Sun etal., 2020) demonstrate an equitable homeostatic and reparative potential of all hepatocytes, irrespective of their lobular location or ploidy status.

MPC-14 BRAF V600E MUTANT OLIGODENDROGLIOMA-LIKE TUMORS WITH CHROMOSOMAL INSTABILITY IN ADOLESCENTS AND YOUNG ADULTS

We performed genome-wide methylation analysis on 136 pediatric low grade gliomas, identifying a unique cluster consisting of oligodendroglioma-like BRAF V600E mutant tumors with Recurrent gain of Chromosome 7 and loss of Chromosome 10 (OLIVER). Hierarchical clustering and t-stochastic neighbor embedding analyses cluster them with previously described pediatric-type low grade gliomas, separate from adult gliomas. OLIVERS exhibit distinct clinical behavior as temporal lobe lesions in adolescents and young adults, prolonged history of seizures and all are alive with no recurrence (follow-up 3.2 to 13.2 years). Morphogically, all showed oligodendroglioma-like features, including round nuclei with perinuclear halos, a chicken-wire pattern of branching capillaries and microcalcification. None showed astrocytic features or characteristics suggestive of high-grade tumors including necrosis or mitotic figures. All tumors harbored multiple chromosomal copy number abnormalities (more than 10 chromosomes per OLIVER), but none showed 1p/19q co-deletion or IDH1 mutation. Interestingly, one tumor showed a TERT promoter mutation. Although the series is small, OLIVER may represent a new category of IDH wild-type low grade gliomas which may be confused with molecular GBM. Further, they highlight the heterogeneity of IDH wild-type gliomas and the relatively indolent behavior of pediatric-type gliomas.

BRAF V600E mutant oligodendroglioma-like tumors with chromosomal instability in adolescents and young adults.

We performed genome-wide methylation analysis on 136 pediatric low-grade gliomas, identifying a unique cluster consisting of three tumors with oligodendroglioma-like histology, BRAF p.V600E mutations and recurrent whole chromosome gains of 7 and loss of 10. Morphologically, all showed similar features, including a diffusely infiltrative glioma composed of round nuclei with perinuclear halos, a chicken-wire pattern of branching capillaries and microcalcification. None showed astrocytic features or characteristics suggestive of high-grade tumors including necrosis or mitotic figures. All tumors harbored multiple chromosomal copy number abnormalities (>10 chromosomes altered), but none showed 1p/19q co-deletion or IDH1 p.R132H mutation. Hierarchical clustering and t-stochastic neighbor embedding analyses from DNA methylation data cluster them more closely to previously described pediatric-type low-grade gliomas and separate from adult gliomas. These tumors exhibit distinct clinical features; they are temporal lobe lesions occurring in adolescents and young adults with a prolonged history of seizures and all are alive with no recurrence (follow-up 3.2 to 13.2years). We encountered another young adult case with quite similar pathological appearance and molecular status except for TERT promoter mutation. Although the series is small, these may represent a new category of IDH wild-type low-grade gliomas which may be confused with "molecular GBM." Further, they highlight the heterogeneity of IDH wild-type gliomas and the relatively indolent behavior of "pediatric-type" gliomas.

Classic Chromophobe Renal Cell Carcinoma Incur a Larger Number of Chromosomal Losses Than Seen in the Eosinophilic Subtype.

Chromophobe renal cell carcinoma (chRCC) is a renal tumor subtype with a good prognosis, characterized by multiple chromosomal copy number variations (CNV). The World Health Organization (WHO) chRCC classification guidelines define a classic and an eosinophilic variant. Large cells with reticular cytoplasm and prominent cell membranes (pale cells) are characteristic for classic chRCC. Classic and eosinophilic variants were defined in 42 Swiss chRCCs, 119 Japanese chRCCs and in whole-slide digital images of 66 chRCCs from the Cancer Genome Atlas (TCGA) kidney chromophobe (KICH) dataset. 32 of 42 (76.2%) Swiss chRCCs, 90 of 119 (75.6%) Japanese chRCCs and 53 of 66 (80.3%) TCGA-KICH were classic chRCCs. There was no survival difference between eosinophilic and classic chRCC in all three cohorts. To identify a genotype/phenotype correlation, we performed a genome-wide CNV analysis using Affymetrix OncoScan® CNV Assay (Affymetrix/Thermo Fisher Scientific, Waltham, MA, USA) in 33 Swiss chRCCs. TCGA-KICH subtypes were compared with TCGA CNV data. In the combined Swiss and TCGA-KICH cohorts, losses of chromosome 1, 2, 6, 10, 13, and 17 were significantly more frequent in classic chRCC (p < 0.05, each), suggesting that classic chRCC are characterized by higher chromosomal instability. This molecular difference justifies the definition of two chRCC variants. Absence of pale cells could be used as main histological criterion to define the eosinophilic variant of chRCC.

Mucinous Tubular and Spindle-Cell Carcinoma of the Kidney: Clinical Features, Genomic Profiles, and Treatment Outcomes.

Mucinous tubular and spindle-cell carcinoma (MTSCC) is a rare kidney cancer subtype with limited cases reported in the literature. We report on outcomes of 25 patients with this variant who were managed at our institution. The institution database was queried, and clinical data extracted for patients with MTSCC. Molecular features examined included next-generation sequencing with Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets and allele-specific copy number analysis using the Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing (FACETS) algorithm in a subset of patients. All patients underwent primary tumor-directed therapy (nephrectomy= 23, cryoablation= 2). Metastases were diagnosed in 6 patients (24%), 3 (12%) of whom had de novo metastatic disease. Five of 6 patients with metastatic disease had high-grade histological features compared with 0 of 19 nonmetastatic patients (83% vs. 0%; P< .001, Fisher exact test). Three-year overall survival from diagnosis was 84.8% (95% confidence interval, 59.6-94.9) with a median follow-up time of 3.9 years (range, 1 month to 10.3 years). Three deaths occurred, all from metastatic disease. Four patients received systemic therapy with time to treatment failure≤6 months across different agents with the exception of 1 patient with prolonged response with sunitinib treatment (30.6 months). The most frequent molecular alterations were neurofibromin 2 mutations (n= 2; 40%), germline alterations (n= 2; 40%) including checkpoint kinase 2 and BRCA2 DNA repair associated mutations, multiple chromosomal copy number losses, and mismatch repair deficiency in 1 patient. MTSCC is characterized by localized tumors treated successfully with primary tumor-directed therapy. However, patients with high-grade histological features were more likely to develop metastatic disease with limited responses to standard therapies.

The Integrative Conjugative Element (ICE) of Mycoplasma agalactiae: Key Elements Involved in Horizontal Dissemination and Influence of Coresident ICEs.

ABSTRACTThe discovery of integrative conjugative elements (ICEs) in wall-less mycoplasmas and the demonstration of their role in massive gene flows within and across species have shed new light on the evolution of these minimal bacteria. Of these, the ICE of the ruminant pathogen Mycoplasma agalactiae (ICEA) represents a prototype and belongs to a new clade of the Mutator-like superfamily that has no preferential insertion site and often occurs as multiple chromosomal copies. Here, functional genomics and mating experiments were combined to address ICEA functions and define the minimal ICEA chassis conferring conjugative properties to M. agalactiae. Data further indicated a complex interaction among coresident ICEAs, since the minimal ICEA structure was influenced by the occurrence of additional ICEA copies that can trans-complement conjugation-deficient ICEAs. However, this cooperative behavior was limited to the CDS14 surface lipoprotein, which is constitutively expressed by coresident ICEAs, and did not extend to other ICEA proteins, including the cis-acting DDE recombinase and components of the mating channel whose expression was detected only sporadically. Remarkably, conjugation-deficient mutants containing a single ICEA copy knocked out in cds14 can be complemented by neighboring cells expressing CDS14. This result, together with those revealing the conservation of CDS14 functions in closely related species, may suggest a way for mycoplasma ICEs to extend their interaction outside their chromosomal environment. Overall, this report provides a first model of conjugative transfer in mycoplasmas and offers valuable insights into understanding horizontal gene transfer in this highly adaptive and diverse group of minimal bacteria.

Direct Visualization of the Multicopy Chromosomes in Cyanobacterium Synechococcus elongatus PCC 7942.

Cyanobacteria are prokaryotic organisms that carry out oxygenic photosynthesis. The fresh water cyanobacterium Synechococcus elongatus PCC 7942 is a model organism for the study of photosynthesis and gene regulation, and for biotechnological applications. Besides several freshwater cyanobacteria, S. elongatus 7942 also contains multiple chromosomal copies per cell at all stages of its cell cycle. Here, we describe a method for the direct visualization of multicopy chromosomes in S. elongatus 7942 by fluorescence in situ hybridization (FISH).

Condensins promote chromosome individualization and segregation during mitosis, meiosis, and amitosis in Tetrahymena thermophila.

Condensin is a protein complex with diverse functions in chromatin packaging and chromosome condensation and segregation. We studied condensin in the evolutionarily distant protist model Tetrahymena, which features noncanonical nuclear organization and divisions. In Tetrahymena, the germline and soma are partitioned into two different nuclei within a single cell. Consistent with their functional specializations in sexual reproduction and gene expression, condensins of the germline nucleus and the polyploid somatic nucleus are composed of different subunits. Mitosis and meiosis of the germline nucleus and amitotic division of the somatic nucleus are all dependent on condensins. In condensin-depleted cells, a chromosome condensation defect was most striking at meiotic metaphase, when Tetrahymena chromosomes are normally most densely packaged. Live imaging of meiotic divisions in condensin-depleted cells showed repeated nuclear stretching and contraction as the chromosomes failed to separate. Condensin depletion also fundamentally altered chromosome arrangement in the polyploid somatic nucleus: multiple copies of homologous chromosomes tended to cluster, consistent with a previous model of condensin suppressing default somatic pairing. We propose that failure to form discrete chromosome territories is the common cause of the defects observed in the absence of condensins.

C-terminal lysine repeats in Streptomyces topoisomerase I stabilize the enzyme-DNA complex and confer high enzyme processivity.

Streptomyces topoisomerase I (TopA) exhibits exceptionally high processivity. The enzyme, as other actinobacterial topoisomerases I, differs from its bacterial homologs in its C-terminal domain (CTD). Here, bioinformatics analyses established that the presence of lysine repeats is a characteristic feature of actinobacterial TopA CTDs. Streptomyces TopA contains the longest stretch of lysine repeats, which terminate with acidic amino acids. DNA-binding studies revealed that the lysine repeats stabilized the TopA–DNA complex, while single-molecule experiments showed that their elimination impaired enzyme processivity. Streptomyces coelicolor TopA processivity could not be restored by fusion of its N-terminal domain (NTD) with the Escherichia coli TopA CTD. The hybrid protein could not re-establish the distribution of multiple chromosomal copies in Streptomyces hyphae impaired by TopA depletion. We expected that the highest TopA processivity would be required during the growth of multigenomic sporogenic hyphae, and indeed, the elimination of lysine repeats from TopA disturbed sporulation. We speculate that the interaction of the lysine repeats with DNA allows the stabilization of the enzyme–DNA complex, which is additionally enhanced by acidic C-terminal amino acids. The complex stabilization, which may be particularly important for GC-rich chromosomes, enables high enzyme processivity. The high processivity of TopA allows rapid topological changes in multiple chromosomal copies during Streptomyces sporulation.

Aneuploidy Predicts Progression of Flat Low-Grade Dysplasia in Patients with Ulcerative Colitis

Aneuploidy, or the presence of multiple copies of chromosomes, has been associated with worse prognosis in ulcerative colitis. In the current

Evolutionary Engineering in Chemostat Cultures for Improved Maltotriose Fermentation Kinetics in Saccharomyces pastorianus Lager Brewing Yeast

The lager brewing yeast Saccharomyces pastorianus, an interspecies hybrid of S. eubayanus and S. cerevisiae, ferments maltotriose, maltose, sucrose, glucose and fructose in wort to ethanol and carbon dioxide. Complete and timely conversion (“attenuation”) of maltotriose by industrial S. pastorianus strains is a key requirement for process intensification. This study explores a new evolutionary engineering strategy for improving maltotriose fermentation kinetics. Prolonged carbon-limited, anaerobic chemostat cultivation of the reference strain S. pastorianus CBS1483 on a maltotriose-enriched sugar mixture was used to select for spontaneous mutants with improved affinity for maltotriose. Evolved populations exhibited an up to 5-fold lower residual maltotriose concentration and a higher ethanol concentration than the parental strain. Uptake studies with 14C-labeled sugars revealed an up to 4.75-fold higher transport capacity for maltotriose in evolved strains. In laboratory batch cultures on wort, evolved strains showed improved attenuation and higher ethanol concentrations. These improvements were also observed in pilot fermentations at 1,000-L scale with high-gravity wort. Although the evolved strain exhibited multiple chromosomal copy number changes, analysis of beer made from pilot fermentations showed no negative effects on flavor compound profiles. These results demonstrate the potential of evolutionary engineering for strain improvement of hybrid, alloploid brewing strains.

Unique Function of the Bacterial Chromosome Segregation Machinery in Apically Growing Streptomyces - Targeting the Chromosome to New Hyphal Tubes and its Anchorage at the Tips.

The coordination of chromosome segregation with cell growth is fundamental to the proliferation of any organism. In most unicellular bacteria, chromosome segregation is strictly coordinated with cell division and involves ParA that moves the ParB nucleoprotein complexes bi- or unidirectionally toward the cell pole(s). However, the chromosome organization in multiploid, apically extending and branching Streptomyces hyphae challenges the known mechanisms of bacterial chromosome segregation. The complex Streptomyces life cycle involves two stages: vegetative growth and sporulation. In the latter stage, multiple cell divisions accompanied by chromosome compaction and ParAB assisted segregation turn multigenomic hyphal cell into a chain of unigenomic spores. However, the requirement for active chromosome segregation is unclear in the absence of canonical cell division during vegetative growth except in the process of branch formation. The mechanism by which chromosomes are targeted to new hyphae in streptomycete vegetative growth has remained unknown until now. Here, we address the question of whether active chromosome segregation occurs at this stage. Applied for the first time in Streptomyces, labelling of the chromosomal replication initiation region (oriC) and time-lapse microscopy, revealed that in vegetative hyphae every copy of the chromosome is complexed with ParB, whereas ParA, through interaction with the apical protein complex (polarisome), tightly anchors only one chromosome at the hyphal tip. The anchor is maintained during replication, when ParA captures one of the daughter oriCs. During spore germination and branching, ParA targets one of the multiple chromosomal copies to the new hyphal tip, enabling efficient elongation of hyphal tube. Thus, our studies reveal a novel role for ParAB proteins during hyphal tip establishment and extension.