Investigation of stalk formation, frequency of dividing cells and gene expression in periphyton mats dominated by Didymosphenia geminata

Background: Metabolic alterations provide substrates that influence chromatin structure to regulate gene expression that determines cell function in health and disease. Heightened proliferation of smooth muscle cells (SMC) leading to the formation of a neointima is a feature of pulmonary arterial hypertension (PAH) and systemic vascular disease. Increased glycolysis is linked to the proliferative phenotype of these SMC. Methods: RNA Sequencing was applied to pulmonary arterial (PA) SMC from PAH patients with and without a BMPR2 mutation vs. control PASMC to uncover genes required for their heightened proliferation and glycolytic metabolism. Assessment of differentially expressed genes established metabolism as a major pathway, and the most highly upregulated metabolic gene in PAH PASMC was aldehyde dehydrogenase family 1 member 3 ( ALDH1A3 ), an enzyme previously linked to glycolysis and proliferation in cancer cells and systemic vascular SMC. We determined if these functions are ALDH1A3-dependent in PAH PASMC, and if ALDH1A3 is required for the development of pulmonary hypertension in a transgenic mouse. Nuclear localization of ALDH1A3 in PAH PASMC led us to determine whether and how this enzyme coordinately regulates gene expression and metabolism in PAH PASMC. Results: ALDH1A3 mRNA and protein were increased in PAH vs control PASMC, and ALDH1A3 was required for their highly proliferative and glycolytic properties. Mice with Aldh1a3 deleted in SMC did not develop hypoxia-induced PA muscularization or pulmonary hypertension. Nuclear ALDH1A3 converted acetaldehyde to acetate to produce acetyl-CoA to acetylate H3K27, marking active enhancers. This allowed for chromatin modification at nuclear factor Y (NFY)A binding sites via the acetyltransferase KAT2B and permitted NFY mediated transcription of cell cycle and metabolic genes that is required for ALDH1A3-dependent proliferation and glycolysis. Loss of BMPR2 in PAH SMC with or without a mutation upregulated ALDH1A3, and transcription of NFYA and ALDH1A3 in PAH PASMC was β-catenin dependent. Conclusions: Our studies have uncovered a metabolic-transcriptional axis explaining how dividing cells use ALDH1A3 to coordinate their energy needs with the epigenetic and transcriptional regulation of genes required for SMC proliferation. They suggest that selectively disrupting the pivotal role of ALDH1A3 in PAH SMC, but not EC, is an important therapeutic consideration.

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The 3’tsRNAs are aminoacylated: Implications for their biogenesis

PLoS Genetics ◽

10.1371/journal.pgen.1009675 ◽

2021 ◽

Vol 17 (7) ◽

pp. e1009675

Author(s):

Ziwei Liu ◽

Hak Kyun Kim ◽

Jianpeng Xu ◽

Yuqing Jing ◽

Mark A. Kay

Keyword(s):

Gene Expression ◽

Hepatocellular Carcinoma ◽

Small Rnas ◽

Ribosome Biogenesis ◽

Trna Synthetase ◽

Fine Tuning ◽

Enzymatic Method ◽

Dividing Cells ◽

Transcriptional Gene Regulation ◽

Induced Apoptosis

Emerging evidence indicates that tRNA-derived small RNAs (tsRNAs) are involved in fine-tuning gene expression and become dysregulated in various cancers. We recently showed that the 22nt LeuCAG tsRNA from the 3´ end of tRNALeu is required for efficient translation of a ribosomal protein mRNA and ribosome biogenesis. Inactivation of this 3´tsRNA induced apoptosis in rapidly dividing cells and suppressed the growth of a patient-derived orthotopic hepatocellular carcinoma in mice. The mechanism involved in the generation of the 3´tsRNAs remains elusive and it is unclear if the 3´-ends of 3´tsRNAs are aminoacylated. Here we report an enzymatic method utilizing exonuclease T to determine the 3´charging status of tRNAs and tsRNAs. Our results showed that the LeuCAG 3´tsRNA, and two other 3´tsRNAs are fully aminoacylated. When the leucyl-tRNA synthetase (LARS1) was inhibited, there was no change in the total tRNALeu concentration but a reduction in both the charged tRNALeu and LeuCAG 3´tsRNA, suggesting the 3´tsRNAs are fully charged and originated solely from the charged mature tRNA. Altering LARS1 expression or the expression of various tRNALeu mutants were also shown to affect the generation of the LeuCAG 3´tsRNA further suggesting they are created in a highly regulated process. The fact that the 3´tsRNAs are aminoacylated and their production is regulated provides additional insights into their importance in post-transcriptional gene regulation that includes coordinating the production of the protein synthetic machinery.

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Developmental compartments in the larval trachea of Drosophila

eLife ◽

10.7554/elife.08666 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 9

Author(s):

Prashanth R Rao ◽

Li Lin ◽

Hai Huang ◽

Arjun Guha ◽

Sougata Roy ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Notch Signaling ◽

Clonal Analysis ◽

Border Cells ◽

Tracheal System ◽

Dorsal Branch ◽

Dividing Cells ◽

Organizational Feature ◽

Third Instar Larvae

The Drosophila tracheal system is a branched tubular network that forms in the embryo by a post-mitotic program of morphogenesis. In third instar larvae (L3), cells constituting the second tracheal metamere (Tr2) reenter the cell cycle. Clonal analysis of L3 Tr2 revealed that dividing cells in the dorsal trunk, dorsal branch and transverse connective branches respect lineage restriction boundaries near branch junctions. These boundaries corresponded to domains of gene expression, for example where cells expressing Spalt, Delta and Serrate in the dorsal trunk meet vein–expressing cells in the dorsal branch or transverse connective. Notch signaling was activated to one side of these borders and was required for the identity, specializations and segregation of border cells. These findings suggest that Tr2 is comprised of developmental compartments and that developmental compartments are an organizational feature relevant to branched tubular networks.

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Mapping Organism Expression Levels at Cellular Resolution in Developing Drosophila

Microscopy and Microanalysis ◽

10.1017/s143192760002612x ◽

2001 ◽

Vol 7 (S2) ◽

pp. 10-11

Author(s):

David W. Knowles ◽

Mark D. Biggin ◽

Stephen Richards ◽

Damir Sudar

Keyword(s):

Gene Expression ◽

Optical Imaging ◽

Imaging Techniques ◽

Single Layer ◽

Specific Gene ◽

Expression Levels ◽

Cellular Resolution ◽

Dividing Cells ◽

Total Dna ◽

Drosophila Embryos

Sequence specific transcription factors are the predominant regulators of animal gene expression controlling nearly all biological processes. We are developing novel quantitative optical imaging techniques to map gene expression levels at cellular and sub-cellular resolution within an entire organism. Pregastrula Drosophila embryos have been chosen because these embryos allow high resolution 3D optical imaging since they comprise a single layer of dividing cells surrounding a yolk sac. in addition, the transcription network controlling gene expression is well characterized in early Drosophila embryos[1], and is being further dissected by a multi-laboratory collaboration, the Berkeley Collaboration in Drosophila Genomics, which encompasses this work.Embryos at different stages of development are labeled for total DNA and specific gene products using different fluorophors and imaged in 3D with confocal microscopy (Figure 1). Intensity-based segmentation of the total DNA image[2] produces a nuclear mask which defines the nuclear boundaries, their location and the number of cells within the embryo (Figure 2). Presently, dilation of the nuclear volumes into their nearest-neighbours[3] is used to estimate the boundary of the cell (Figure 3) and superposition of these images produces a morphological mask defining each cell and its nucleus.

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Regulation of Fruit Growth and Fruit Size in Apple

HortScience ◽

10.21273/hortsci.40.4.1097a ◽

2005 ◽

Vol 40 (4) ◽

pp. 1097A-1097

Author(s):

Anish Malladi ◽

Peter Goldsbrough ◽

Peter Hirst

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cell Division ◽

Fruit Development ◽

Fruit Size ◽

Cell Number ◽

Rt Pcr ◽

Cell Production ◽

Golden Delicious ◽

Dividing Cells

Fruit development in apple cultivars varying in their ultimate fruit size was analyzed using cytology, flow cytometry (FCM), and semi-quantitative RT-PCR. Fruit size variation across cultivars was largely explained by variation in cell number. The cell division phase lasted for less than 30 days in all varieties, less than previously believed. A distinct overlap between the cell division and cell expansion phases was present. Analysis of the relative cell production rate (rCPR) showed a major peak about 10 days after full bloom (DAFB) after which it declined. Comparison of the rCPR across varieties suggested distinct patterns of cell production with `Gala' having a low but sustained rCPR, `Pixy Crunch' a short but high rCPR, and `Golden Delicious' having a high and sustained rCPR. FCM analysis also showed similar patterns with a peak in the proportion of dividing cells about 10 DAFB followed by a decline. To further understand regulation of cell number, four cell cycle related genes were cloned from `Gala'. Cyclin Dependent Kinase B (CDK B) and Cyclin B were found to be highly cell division phase specific in their expression. Analysis of gene expression by semi-quantitative RT-PCR indicated peak expression of these two genes at 5-10 DAFB, consistent with the peaks in rCPR and proportion of dividing cells. Comparison of gene expression across the varieties showed higher peak expression of the above genes in the larger-fruited `Golden Delicious' than in the smaller-fruited `Gala.' This study provides novel insight into the regulation of fruit development in apple and also suggests a role for the cell cycle genes in fruit size regulation.

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Actin and tubulin in Tetrahymena

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o85-058 ◽

1985 ◽

Vol 63 (6) ◽

pp. 389-396 ◽

Cited By ~ 4

Author(s):

E. Jane Mitchell ◽

Selma Zimmerman ◽

Arthur M. Zimmerman

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Tetrahymena Pyriformis ◽

Model System ◽

Cytoskeletal Proteins ◽

Actin Gene ◽

Ciliated Protozoan ◽

Dividing Cells ◽

And Control

Tubulin and actin are cytoskeletal proteins known to play a major role in dividing cells. Tetrahymena pyriformis, a ciliated protozoan, was used as a model system for investigating tubulin synthesis during cilia regeneration and during the cell cycle. Until recently the identification of actin in Tetrahymena has been controversial. In this report evidence for the presence of actin in Tetrahymena is reviewed and control of actin gene expression during the cell cycle is discussed.

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Engineered phenotype patterns in microbial populations

10.1101/575068 ◽

2019 ◽

Author(s):

Philip Bittihn ◽

Andriy Didovyk ◽

Lev S. Tsimring ◽

Jeff Hasty

Keyword(s):

Gene Expression ◽

Synthetic Biology ◽

Expression Patterns ◽

Local Environment ◽

Heterogeneous Environments ◽

Gene Circuits ◽

Sensor Module ◽

Dividing Cells ◽

Actuator Module ◽

And Control

AbstractRapid advances in cellular engineering1,2have positioned synthetic biology to address therapeutic3,4and industrial5problems, but a significant obstacle is the myriad of unanticipated cellular responses in heterogeneous environments such as the gut6,7, solid tumors8,9, bioreactors10or soil11. Complex interactions between the environment and cells often arise through non-uniform nutrient availability, which can generatebidirectionalcoupling as cells both adjust to and modify their local environment through different growth phenotypes across a colony.12,13While spatial sensing14and gene expression patterns15–17have been explored under homogeneous conditions, the mutual interaction between gene circuits, growth phenotype, and the environment remains a challenge for synthetic biology. Here, we design gene circuits which sense and control spatiotemporal phenotype patterns in a model system of heterogeneous microcolonies containing both growing and dormant bacteria. We implement pattern control by coupling different downstream modules to a tunable sensor module that leveragesE. coli⁉sstress response and is activated upon growth arrest. One is an actuator module that slows growth and thereby creates an environmental negative feedback via nutrient diffusion. We build a computational model of this system to understand the interplay between gene regulation, population dynamics, and chemical transport, which predicts oscillations in both growth and gene expression. Experimentally, this circuit indeed generates robust cycling between growth and dormancy in the interior of the colony. We also use the stress sensor to drive an inducible gating module that enables selective gene expression in non-dividing cells. The ‘stress-gated lysis circuit’ derived from this module radically alters the growth pattern through elimination of the dormant phenotype upon a chemical cue. Our results establish a strategy to leverage and control the presence of distinct microbial growth phenotypes for synthetic biology applications in complex environments.

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897. A Class I FIV Integrase Mutant Reveals Short-Term Gene Expression from Unintegrated Lentiviral Vector DNA in Non-Dividing Cells

Molecular Therapy ◽

10.1016/s1525-0016(16)43727-5 ◽

2002 ◽

Vol 5 (5) ◽

pp. S292-S293

Keyword(s):

Gene Expression ◽

Lentiviral Vector ◽

Class I ◽

Short Term ◽

Dividing Cells ◽

Vector Dna

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Gene Expression Profiling in Quiescent Stem Cells from Normal and Chronic Myeloid Leukaemia Patients.

Blood ◽

10.1182/blood.v104.11.2962.2962 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2962-2962

Author(s):

Susan M. Graham ◽

Gerry J. Graham ◽

Tessa L. Holyoake

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Stem Cell ◽

Chronic Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Stem Cell Marker ◽

Quiescent Cells ◽

Dividing Cells ◽

Quiescent Stem Cells

Abstract Earlier studies have shown that Ph+ quiescent cells exist in chronic myeloid leukaemia (CML) (Blood (1999)94:2056) and we have previously shown that these cells are primitive in that they express the stem cell marker CD34. We have also shown that quiescent CML stem cells are insensitive to the effects of imatinib (IM Novartis Pharma) (Blood (2002) 99:319) and may present a possible source for relapse. This quiescent population therefore represents a potentially significant clinical problem and thus studies aimed at developing methods for eradicating this population are timely. In an effort to identify molecular markers of this population that may allow it to be specifically targeted during therapy, we have set out to investigate the transcriptional differences between quiescent and cycling stem cells. To this end, we have used specific stem cell enrichment and sorting protocols. Leukapheresis products from CML patients (N=5) in chronic phase at diagnosis and mobilised peripheral blood from allogeneic donors (N=3), were selected for CD34+ cells. Hoechst 33342 and Pyronin Y were used to discriminate the quiescent (G0) cells identified as Hoechstlo/Pyroninlo from the cycling cells. In combination with propidium iodide for dead cell exclusion we were able to sort 4–9x105 viable, quiescent stem cells and 4–11x106 cycling cells, which were processed for microarrays. Affymetrix gene chips (U133A) were used for the analysis and the data obtained was analysed using GeneSpring. Number of Genes Changed in Each Comparison 3 Fold 4 Fold 5 Fold CML G0 V CML Div 37 21 10 Norm G0 V Norm Div 188 92 47 CML G0 V Norm G0 168 85 49 CML Div V Norm Div 49 27 8 Initial analysis indicates that the greatest differences in gene expression are between the normal quiescent cells (G0) and normal dividing cells (Div) and between the normal quiescent cells and CML quiescent cells. A large percentage of the genes differentially expressed between the quiescent and cycling normal cells encode regulators of the cell cycle confirming the success of the sorting strategy for quiescent and cycling cells A selection of Genes Up-Regulated in Normal Cycling Cells Compared to G0 Gene Fold Up-regulation PCNA 3 CDC2 8 CCNB2 5 CCN1 3.5 CDC20 6 CDC25A 3.5 MCM5 3 In addition, many of the genes identified in our analysis are consistent with other published expression profiles for haemopoietic cells. Curiously, we have identified unanticipated changes in expression of cell cycle genes in the CML quiescent cells, which merit further investigation. We have also identified a number of unexpected genes as being more than 5 fold changed in the quiescent cells compared to dividing cells for both normal and CML samples. Specifically, there is a large cohort of genes preferentially expressed in quiescent normal or CML cells, which encode members of the chemokine family of proteins. Work is ongoing to establish the relevance, if any, of these genes to stem cell quiescence.

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