scholarly journals Synthetic chromosome fusion: effects on genome structure and function

2018 ◽  
Author(s):  
Jingchuan Luo ◽  
Guillaume Mercy ◽  
Luis A. Vale-Silva ◽  
Xiaoji Sun ◽  
Neta Agmon ◽  
...  
Keyword(s):  
Genome Structure ◽  
Three Dimensional ◽  
Wild Type ◽  
3D Interaction ◽  
Chromosome Fusion ◽  
Size Dependent ◽  
Varying Length ◽  
And Function ◽  
Chromosome I ◽  
Wild Type Yeast

SUMMARYAs part of the Synthetic Yeast 2.0 (Sc2.0) project, we designed and synthesized synthetic chromosome I. The total length of synI is ~21.4% shorter than wild-type chromosome I, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns of potential instability and karyotype imbalance. We used a variation of a previously developed, robust CRISPR-Cas9 method to fuse chromosome I to other chromosome arms of varying length: chrIXR (84 kb), chrIIIR (202 kb) and chrIVR (1 Mb). All fusion chromosome strains grew like wild-type so we decided to attach synI to synIII. Through the investigation of three-dimensional structures of fusion chromosome strains, unexpected loops and twisted structures were formed in chrIII-I and chrIX-III-I fusion chromosomes, which depend on silencing protein Sir3. These results suggest a previously unappreciated 3D interaction between HMR and the adjacent telomere. We used these fusion chromosomes to show that axial element Red1 binding in meiosis is not strictly chromosome size dependent even though Red1 binding is enriched on the three smallest chromosomes in wild-type yeast, and we discovered an unexpected role for centromeres in Red1 binding patterns.

scholarly journals Cohesin Releasing Factor WAPL Regulates Genome Structure and Function of Mature T Cells

2021 ◽  
Author(s):  
Yaping Sun ◽  
Gabrielle A. Dotson ◽  
Lindsey A. Muir ◽  
Scott Ronquist ◽  
Katherine Oravecz-Wilson ◽  
...  
Keyword(s):  
T Cells ◽  
Hematopoietic Cell Transplantation ◽  
Genome Structure ◽  
3D Structure ◽  
Three Dimensional ◽  
Structure And Function ◽  
Cell Functions ◽  
And Function

ABSTRACTThe cohesin complex modulates gene expression and cellular functions by shaping three-dimensional (3D) organization of chromatin. WAPL, cohesin’s DNA releasing factor, regulates 3D chromatin architecture. The 3D genome structure and its relevance to mature T cell functions is not well understood. We show that in vivo lymphopenic expansion, and allo-antigen driven proliferation, alters the 3D structure and function of the genome in mature T cells. Conditional deletion of Wapl in T cells reduced long-range genomic interactions, altered chromatin A/B compartments and the topologically associating domains (TAD) of the chromatin in T cells at baseline. Comparison of chromatin structure in normal and WAPL-deficient T cells after lymphopenic and allo-antigen driven stimulation revealed reduced loop extensions with changes in cell cycling genes. WAPL-mediated changes in 3D architecture of chromatin regulated activation, cycling and proliferation of T cells in vitro and in vivo. Finally, WAPL-deficient T cells caused reduced severity of graft-versus-host disease following experimental allogeneic hematopoietic cell transplantation. These data collectively characterize 3D genomic architecture of T cells in vivo and demonstrate biological and clinical implications for its disruption by cohesin releasing factor WAPL.

scholarly journals Three-dimensional Organization of Basal Bodies from Wild-Type and δ-Tubulin Deletion Strains of Chlamydomonas reinhardtii

2003 ◽  
Vol 14 (7) ◽  
pp. 2999-3012 ◽  
Author(s):  
Eileen T. O'Toole ◽  
Thomas H. Giddings ◽  
J. Richard McIntosh ◽  
Susan K. Dutcher
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Basal Body ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Specimen Preparation ◽  
Basal Bodies ◽  
Wild Type ◽  
Tubulin Isoforms ◽  
Striated Fiber ◽  
And Function

Improved methods of specimen preparation and dual-axis electron tomography have been used to study the structure and organization of basal bodies in the unicellular alga Chlamydomonas reinhardtii. Novel structures have been found in both wild type and strains with mutations that affect specific tubulin isoforms. Previous studies have shown that strains lacking δ-tubulin fail to assemble the C-tubule of the basal body. Tomographic reconstructions of basal bodies from the δ-tubulin deletion mutant uni3-1 have confirmed that basal bodies contain mostly doublet microtubules. Our methods now show that the stellate fibers, which are present only in the transition zone of wild-type cells, repeat within the core of uni3-1 basal bodies. The distal striated fiber is incomplete in this mutant, rootlet microtubules can be misplaced, and multiflagellate cells have been observed. A suppressor of uni3-1, designated tua2-6, contains a mutation in α-tubulin. tua2-6; uni3-1 cells build both flagella, yet they retain defects in basal body structure and in rootlet microtubule positioning. These data suggest that the presence of specific tubulin isoforms in Chlamydomonas directly affects the assembly and function of both basal bodies and basal body-associated structures.

A survey of proteins encoded by non-synonymous single nucleotide polymorphisms reveals a significant fraction with altered stability and activity

2009 ◽  
Vol 424 (1) ◽  
pp. 15-26 ◽  
Author(s):  
Abdellah Allali-Hassani ◽  
Gregory A. Wasney ◽  
Irene Chau ◽  
Bum Soo Hong ◽  
Guillermo Senisterra ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Coding Region ◽  
Wild Type Enzyme ◽  
Single Nucleotide ◽  
The Stability ◽  
And Function

On average, each human gene has approximately four SNPs (single nucleotide polymorphisms) in the coding region, half of which are nsSNPs (non-synonymous SNPs) or missense SNPs. Current attention is focused on those that are known to perturb function and are strongly linked to disease. However, the vast majority of SNPs have not been investigated for the possibility of causing disease. We set out to assess the fraction of nsSNPs that encode proteins that have altered stability and activity, for this class of variants would be candidates to perturb cellular function. We tested the thermostability and, where possible, the catalytic activity for the most common variant (wild-type) and minor variants (total of 46 SNPs) for 16 human enzymes for which the three-dimensional structures were known. There were significant differences in the stability of almost half of the variants (48%) compared with their wild-type counterparts. The catalytic efficiency of approx. 14 variants was significantly altered, including several variants of human PKM2 (pyruvate kinase muscle 2). Two PKM2 variants, S437Y and E28K, also exhibited changes in their allosteric regulation compared with the wild-type enzyme. The high proportion of nsSNPs that affect protein stability and function, albeit subtly, underscores the need for experimental analysis of the diverse human proteome.

scholarly journals Site-Directed Mutagenesis of Myoglobin for Studies of Their Interaction with Iron(III) by Multi-Spectroscopic Techniques

2013 ◽  
Vol 2013 ◽  
pp. 1-11
Author(s):  
Qian Tang ◽  
Xiao-Jun Peng ◽  
Hong-Yu Cao ◽  
Yan-Jie Yang ◽  
Jing Ma ◽  
...  
Keyword(s):  
Amino Acids ◽  
Metal Ion ◽  
Binding Capacity ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Spectroscopic Techniques ◽  
Wild Type ◽  
Bimolecular Quenching ◽  
And Function ◽  
Binding Distance

In order to investigate how the amino acids on the surface of myoglobin molecule influence myoglobin's structure and function, a variety of spectroscopy techniques were applied in the study of the interaction between Fe(III) and myoglobin (wild type and its mutants, D44K, D60K, and K56D). The results demonstrate that Fe(III) can quench the fluorescence of wild type and mutants of myoglobin, and the quenching mechanisms are static quenching. It is found that the binding distance between Fe(III) and myoglobin mutants gets smaller, the binding capacity increases by the values of binding constant and the bimolecular quenching constant as well as the binding distance. Those data also indicate that the metal ion Fe(III) can interact strongly with myoglobin mutants. The three-dimensional conformation change after surface amino acids are replaced is detected by the UV absorption spectroscopy and fluorescence spectroscopy, which make mutants become more dynamic and change its function and interaction with Fe(III) strongly.

scholarly journals Establishment of heterochromatin in domain-size-dependent bursts

2021 ◽  
Vol 118 (15) ◽  
pp. e2022887118
Author(s):  
Jan Fabio Nickels ◽  
Ashleigh Katrine Edwards ◽  
Sebastian Jespersen Charlton ◽  
Amanda Møller Mortensen ◽  
Sif Christine Lykke Hougaard ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Dynamic Properties ◽  
Three Dimensional ◽  
Domain Size ◽  
Wild Type ◽  
Independent Manner ◽  
Type Region ◽  
Size Dependent ◽  
Evolutionarily Conserved ◽  
Highly Correlated

Methylation of histone H3K9 is a hallmark of epigenetic silencing in eukaryotes. Nucleosome modifications often rely on positive feedback where enzymes are recruited by modified nucleosomes. A combination of local and global feedbacks has been proposed to account for some dynamic properties of heterochromatin, but the range at which the global feedbacks operate and the exact mode of heterochromatin propagation are not known. We investigated these questions in fission yeast. Guided by mathematical modeling, we incrementally increased the size of the mating-type region and profiled heterochromatin establishment over time. We observed exponential decays in the proportion of cells with active reporters, with rates that decreased with domain size. Establishment periods varied from a few generations in wild type to >200 generations in the longest region examined, and highly correlated silencing of two reporters located outside the nucleation center was observed. On a chromatin level, this indicates that individual regions are silenced in sudden bursts. Mathematical modeling accounts for these bursts if heterochromatic nucleosomes facilitate a deacetylation or methylation reaction at long range, in a distance-independent manner. A likely effector of three-dimensional interactions is the evolutionarily conserved Swi6HP1 H3K9me reader, indicating the bursting behavior might be a general mode of heterochromatin propagation.

scholarly journals Topologically associating domains and their role in the evolution of genome structure and function in Drosophila

2020 ◽  
Author(s):  
Yi Liao ◽  
Xinwen Zhang ◽  
Mahul Chakraborty ◽  
J.J. Emerson
Keyword(s):  
Genome Structure ◽  
Three Dimensional ◽  
Genomic Analysis ◽  
Purifying Selection ◽  
Drosophila Species ◽  
Comparative Genomic ◽  
Topologically Associating Domains ◽  
Transcript Structure ◽  
And Function ◽  
Long Genes

Topologically associating domains (TADs) were recently identified as fundamental units of three-dimensional eukaryotic genomic organization, though our knowledge of the influence of TADs on genome evolution remains preliminary. To study the molecular evolution of TADs in Drosophila species, we constructed a new reference-grade genome assembly and accompanying high-resolution TAD map for D. pseudoobscura. Comparison of D. pseudoobscura and D. melanogaster, which are separated by ~49 million years of divergence, showed that ~30-40% of their genomes retain conserved TADs. Comparative genomic analysis of 17 Drosophila species revealed that chromosomal rearrangement breakpoints are enriched at TAD boundaries but depleted within TADs. Additionally, genes within conserved TADs exhibit lower expression divergence than those located in nonconserved TADs. Furthermore, we found that a substantial proportion of long genes (>50 kbp) in D. melanogaster (42%) and D. pseudoobscura (26%) constitute their own TADs, implying transcript structure may be one of the deterministic factors for TAD formation. Using structural variants (SVs) identified from 14 D. melanogaster strains, its 3 closest sibling species from the D. simulans species complex, and two obscura clade species, we uncovered evidence of selection acting on SVs at TAD boundaries, but with the nature of selection differing between SV types. Deletions are depleted at TAD boundaries in both divergent and polymorphic SVs, suggesting purifying selection, whereas divergent tandem duplications are enriched at TAD boundaries relative to polymorphism, suggesting they are adaptive. Our findings highlight how important TADs are in shaping the acquisition and retention of structural mutations that fundamentally alter genome organization.

scholarly journals The Three-Dimensional Structure of Human Interphase Chromosomes Is Related to the Transcriptome Map

2007 ◽  
Vol 27 (12) ◽  
pp. 4475-4487 ◽  
Author(s):  
Sandra Goetze ◽  
Julio Mateos-Langerak ◽  
Hinco J. Gierman ◽  
Wim de Leeuw ◽  
Osdilly Giromus ◽  
...  
Keyword(s):  
Gene Expression ◽  
Structural Parameters ◽  
Expression Patterns ◽  
Genome Structure ◽  
Three Dimensional ◽  
Chromosome Territory ◽  
Dimensional Structure ◽  
Compact Structure ◽  
General Aspects ◽  
And Function

ABSTRACT The three-dimensional (3D) organization of the chromosomal fiber in the human interphase nucleus is an important but poorly understood aspect of gene regulation. Here we quantitatively analyze and compare the 3D structures of two types of genomic domains as defined by the human transcriptome map. While ridges are gene dense and show high expression levels, antiridges, on the other hand, are gene poor and carry genes that are expressed at low levels. We show that ridges are in general less condensed, more irregularly shaped, and located more closely to the nuclear center than antiridges. Six human cell lines that display different gene expression patterns and karyotypes share these structural parameters of chromatin. This shows that the chromatin structures of these two types of genomic domains are largely independent of tissue-specific variations in gene expression and differentiation state. Moreover, we show that there is remarkably little intermingling of chromatin from different parts of the same chromosome in a chromosome territory, neither from adjacent nor from distant parts. This suggests that the chromosomal fiber has a compact structure that sterically suppresses intermingling. Together, our results reveal novel general aspects of 3D chromosome architecture that are related to genome structure and function.

Conformation of Ribosomes from Escherichia Coli

1974 ◽  
Vol 32 ◽  
pp. 210-211
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Binding Sites ◽  
Ribosomal Proteins ◽  
Fluorescent Dyes ◽  
Protein Biosynthesis ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Dimensional Structure ◽  
Complete Understanding ◽  
And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

Structure and function of neural networks in the retina

1988 ◽  
Vol 46 ◽  
pp. 26-27
Author(s):  
Peter Sterling
Keyword(s):  
Ganglion Cells ◽  
Three Dimensional ◽  
Structure And Function ◽  
Synaptic Connections ◽  
Visual Axis ◽  
One Dimensional ◽  
Cat Retina ◽  
Ultrathin Sections ◽  
And Function ◽  
Insight Into

The synaptic connections in cat retina that link photoreceptors to ganglion cells have been analyzed quantitatively. Our approach has been to prepare serial, ultrathin sections and photograph en montage at low magnification (˜2000X) in the electron microscope. Six series, 100-300 sections long, have been prepared over the last decade. They derive from different cats but always from the same region of retina, about one degree from the center of the visual axis. The material has been analyzed by reconstructing adjacent neurons in each array and then identifying systematically the synaptic connections between arrays. Most reconstructions were done manually by tracing the outlines of processes in successive sections onto acetate sheets aligned on a cartoonist's jig. The tracings were then digitized, stacked by computer, and printed with the hidden lines removed. The results have provided rather than the usual one-dimensional account of pathways, a three-dimensional account of circuits. From this has emerged insight into the functional architecture.

Structural Role of rRNAs on the Ribosomal Subunits from E. Coli by Scanning Transmission Electron Microscopy

1981 ◽  
Vol 39 ◽  
pp. 424-425
Author(s):  
M. Boublik ◽  
N. Robakis ◽  
J.S. Wall
Keyword(s):  
Three Dimensional ◽  
Uranyl Acetate ◽  
Dimensional Structure ◽  
Electron Microscopic ◽  
Ribosomal Subunits ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
Scanning Transmission ◽  
And Function

The three-dimensional structure and function of biological supramolecular complexes are, in general, determined and stabilized by conformation and interactions of their macromolecular components. In the case of ribosomes, it has been suggested that one of the functions of ribosomal RNAs is to act as a scaffold maintaining the shape of the ribosomal subunits. In order to investigate this question, we have conducted a comparative TEM and STEM study of the structure of the small 30S subunit of E. coli and its 16S RNA.The conventional electron microscopic imaging of nucleic acids is performed by spreading them in the presence of protein or detergent; the particles are contrasted by electron dense solution (uranyl acetate) or by shadowing with metal (tungsten). By using the STEM on freeze-dried specimens we have avoided the shearing forces of the spreading, and minimized both the collapse of rRNA due to air drying and the loss of resolution due to staining or shadowing. Figure 1, is a conventional (TEM) electron micrograph of 30S E. coli subunits contrasted with uranyl acetate.

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