scholarly journals A SIR-independent role for cohesin in subtelomeric silencing and organization

2019 ◽  
Vol 116 (12) ◽  
pp. 5659-5664 ◽  
Author(s):  
Deepash Kothiwal ◽  
Shikha Laloraya
Keyword(s):  
Dna Binding ◽  
Nuclear Envelope ◽  
Budding Yeast ◽  
Genetic Interactions ◽  
Limited Extent ◽  
Binding Region ◽  
Chromosome Architecture ◽  
Independent Mechanism ◽  
Sir Complex ◽  
Mutant Cells

Cohesin is a key determinant of chromosome architecture due to its DNA binding and tethering ability. Cohesin binds near centromeres and chromosome arms and also close to telomeres, but its role near telomeres remains elusive. In budding yeast, transcription within 20 kb of telomeres is repressed, in part by the histone-modifying silent information regulator (SIR) complex. However, extensive subtelomeric repressed domains lie outside the SIR-binding region, but the mechanism of silencing in these regions remains poorly understood. Here, we report a role for cohesin in subtelomeric silencing that extends even beyond the zone of SIR binding. Clusters of subtelomeric genes were preferentially derepressed in a cohesin mutant, whereas SIR binding was unaltered. Genetic interactions with known telomere silencing factors indicate that cohesin operates independent of the SIR-mediated pathway for telomeric silencing. Mutant cells exhibited Mpk1-dependent Sir3 hyperphosphorylation that contributes to subtelomeric derepression to a limited extent. Compaction of subtelomeric domains and tethering to the nuclear envelope were impaired in mutant cells. Our findings provide evidence for a unique SIR-independent mechanism of subtelomeric repression mediated by cohesin.

Nuclear Envelope Dynamics During Mitosis

1988 ◽  
Vol 46 ◽  
pp. 224-225
Author(s):  
Brian Burke
Keyword(s):  
Nuclear Envelope ◽  
Membrane Vesicles ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Animal Cells ◽  
Interphase Chromosome ◽  
Lamin B ◽  
Chromosome Architecture ◽  
Complex Membrane ◽  
Pore Complexes

The nuclear envelope is a complex membrane structure that forms the boundary of the nuclear compartment in eukaryotes. It regulates the passage of macromolecules between the two compartments and may be important for organizing interphase chromosome architecture. In interphase animal cells it forms a remarkably stable structure consisting of a double membrane ouerlying a protein meshwork or lamina and penetrated by nuclear pore complexes. The latter form the channels for nucleocytoplasmic exchange of macromolecules, At the onset of mitosis, however, it rapidly disassembles, the membranes fragment to yield small vesicles and the lamina, which is composed of predominantly three polypeptides, lamins R, B and C (MW approx. 74, 68 and 65 kDa respectiuely), breaks down. Lamins B and C are dispersed as monomers throughout the mitotic cytoplasm, while lamin B remains associated with the nuclear membrane vesicles.

scholarly journals Far3 and Five Interacting Proteins Prevent Premature Recovery from Pheromone Arrest in the Budding Yeast Saccharomyces cerevisiae

2003 ◽  
Vol 23 (5) ◽  
pp. 1750-1763 ◽  
Author(s):  
Hilary A. Kemp ◽  
George F. Sprague,
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Open Reading Frames ◽  
Velocity Sedimentation ◽  
Interacting Proteins ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cycle Arrest ◽  
Mutant Cells ◽  
Two Hybrid ◽  
Reading Frames

ABSTRACT In budding yeast, diffusible mating pheromones initiate a signaling pathway that culminates in several responses, including cell cycle arrest. Only a handful of genes required for the interface between pheromone response and the cell cycle have been identified, among them FAR1 and FAR3; of these, only FAR1 has been extensively characterized. In an effort to learn about the mechanism by which Far3 acts, we used the two-hybrid method to identify interacting proteins. We identified five previously uncharacterized open reading frames, dubbed FAR7, FAR8, FAR9, FAR10, and FAR11, that cause a far3-like pheromone arrest defect when disrupted. Using two-hybrid and coimmunoprecipitation analysis, we found that all six Far proteins interact with each other. Moreover, velocity sedimentation experiments suggest that Far3 and Far7 to Far11 form a complex. The phenotype of a sextuple far3far7-far11 mutant is no more severe than any single mutant. Thus, FAR3 and FAR7 to FAR11 all participate in the same pathway leading to G1 arrest. These mutants initially arrest in response to pheromone but resume budding after 10 h. Under these conditions, wild-type cells fail to resume budding even after several days whereas far1 mutant cells resume budding within 1 h. We conclude that the FAR3-dependent arrest pathway is functionally distinct from that which employs FAR1.

Analysis of function of the pair-rule genes hairy, even-skipped and fushi tarazu in mosaic Drosophila embryos

Development ◽  
1989 ◽  
Vol 107 (4) ◽  
pp. 847-853 ◽  
Author(s):  
P.A. Lawrence ◽  
P. Johnston
Keyword(s):  
Genetic Interactions ◽  
Nuclear Transplantation ◽  
Fushi Tarazu ◽  
Segmentation Gene ◽  
Hairy Cells ◽  
Analysis Of Function ◽  
Mutant Cells ◽  
Pair Rule ◽  
Drosophila Embryos

We report the first attempt of its kind to study genetic interactions using young Drosophila embryos that are mosaic for wildtype and mutant cells. Using nuclear transplantation we make mosaic embryos in which a patch of cells lacks a particular segmentation gene, A. With antibodies, we than look at the expression of another gene that is known to be downstream of gene A, with respect to the cells in the patch. We have examples of patches of hairy cells (where we monitor the effect on fushi tarazu (ftz) expression), even-skipped (monitoring ftz) and ftz (monitoring engrailed and Ultrabithorax). Our main finding is that the dependence of engrailed expression on the ftz gene is strictly cell-autonomous. This result goes some way towards explaining the dependence of Ultrabithorax expression on ftz, a dependence we show to be locally cell-autonomous within parts of parasegments 6 and 8 but non autonomous within parasegment 7.

A second c-myb protein is translated from an alternatively spliced mRNA expressed from normal and 5'-disrupted myb loci

1989 ◽  
Vol 9 (12) ◽  
pp. 5456-5463 ◽  
Author(s):  
G L Shen-Ong ◽  
B Lüscher ◽  
R N Eisenman
Keyword(s):  
Dna Binding ◽  
Nuclear Dna ◽  
Dna Binding Protein ◽  
Major Protein ◽  
Tumor Cell Lines ◽  
Binding Region ◽  
Large Intron ◽  
Alternatively Spliced ◽  
Myb Genes ◽  
Cryptic Splice Sites

The major protein encoded by the c-myb oncogene in many species has been identified as an unstable, nuclear DNA-binding protein with an apparent molecular mass of 75 to 80 kilodaltons (p75c-myb). Recently, an alternatively spliced form of c-myb-encoded mRNA has been identified in murine cells containing either normal or rearranged c-myb genes. This mRNA includes a new exon, termed E6A, formed through use of cryptic splice sites located in the large intron between c-myb exons vE6 and vE7. E6A is predicted to contribute an internal 121-residue in-frame insertion into a region C terminal of the DNA-binding domain the c-myb-encoded protein. Here we report the identification of an 85-kilodalton (p85c-myb-E6A) protein as the translation product of the alternatively spliced E6A c-myb mRNA. This protein as well as p75c-myb were precipitated with anti-Myb antibodies raised against the conserved DNA-binding region of c-Myb. Proteolytic mapping studies showed that the two proteins are highly related but not identical. However, only the p85 protein reacted with an antiserum prepared against the E6A region expressed in bacteria, demonstrating that p85 but not p75 contains E6A sequences. In addition, the mobilities of both p85 and p75 were increased in myeloid tumor cell lines containing proviral integrations upstream of the 5' coding exons of v-myb, indicating that both proteins are truncated forms of c-Myb expressed from the same disrupted allele. p75c-myb and p85c-myb-E6A were indistinguishable with respect to nuclear localization and protein half-life. Furthermore, both forms of Myb were synthesized continuously throughout the cell cycle in 70Z ore-B cells. The contribution of the E6A domain to c-myb function remains to be elucidated.

scholarly journals Mitochondrial Histone-Like DNA-Binding Proteins Are Essential for Normal Cell Growth and Mitochondrial Function in Crithidia fasciculata

2004 ◽  
Vol 3 (2) ◽  
pp. 518-526 ◽  
Author(s):  
Nuraly K. Avliyakulov ◽  
Julius Lukeš ◽  
Dan S. Ray
Keyword(s):  
Cell Growth ◽  
Dna Binding ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Mrna Levels ◽  
Chromosomal Dna ◽  
Double Knockout ◽  
Crithidia Fasciculata ◽  
Sequence Encoding ◽  
Mutant Cells

ABSTRACT The Crithidia fasciculata KAP2 and KAP3 proteins are closely related kinetoplast-specific histone-like DNA-binding proteins. The KAP2 and KAP3 genes are 46% identical and are arranged in tandem on the chromosomal DNA. Disruption of both alleles of either gene alone shows no detectable phenotype. However, replacement of both copies of the sequence encoding the entire KAP2 and KAP3 locus increases maxicircle mRNA levels two- to fourfold. These double-knockout cells are viable but grow extremely slowly, have reduced respiration and very abnormal cell morphologies, and accumulate numerous large vacuoles. The extreme phenotype of these mutant cells suggests an important role for the KAP2 and KAP3 proteins in mitochondrial metabolism and cell growth.

Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis

1986 ◽  
Vol 6 (4) ◽  
pp. 1218-1227
Author(s):  
L Naumovski ◽  
E C Friedberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Excision Repair ◽  
Random Mutagenesis ◽  
Nucleotide Binding ◽  
Yeast Cells ◽  
Wild Type ◽  
Binding Region ◽  
Site Specific ◽  
Essential Function

The RAD3 gene of Saccharomyces cerevisiae, which is involved in excision repair of DNA and is essential for cell viability, was mutagenized by site-specific and random mutagenesis. Site-specific mutagenesis was targeted to two regions near the 5' and 3' ends of the coding region, selected on the basis of amino acid sequence homology with known nucleotide binding and with known specific DNA-binding proteins, respectively. Two mutations in the putative nucleotide-binding region and one in the putative DNA-binding region inactivate the excision repair function of the gene, but not the essential function. A gene encoding two tandem mutations in the putative DNA-binding region is defective in both excision repair and essential functions of RAD3. Seven plasmids were isolated following random mutagenesis with hydroxylamine. Mutations in six of these plasmids were identified by gap repair of mutant plasmids from the chromosome of strains with previously mapped rad3 mutations, followed by DNA sequencing. Three of these contain missense mutations which inactivate only the excision repair function. The other three carry nonsense mutations which inactivate both the excision repair and essential functions. Collectively our results indicate that the RAD3 excision repair function is more sensitive to inactivation than is the essential function. Overexpression of wild-type Rad3 protein and a number of rad3 mutant proteins did not affect the UV resistance of wild-type yeast cells. However, overexpression of Rad3-2 protein rendered wild-type cells partially UV sensitive, indicating that excess Rad3-2 protein is dominant to the wild-type form. These and other results suggest that Rad3-2 protein retains its affinity for damaged DNA or other substrates, but is not catalytically active in excision repair.

Physical and genetic interactions between Alx4 and Cart1

Development ◽  
1999 ◽  
Vol 126 (2) ◽  
pp. 359-369 ◽  
Author(s):  
S. Qu ◽  
S.C. Tucker ◽  
Q. Zhao ◽  
B. deCrombrugghe ◽  
R. Wisdom
Keyword(s):  
Dna Binding ◽  
Double Mutant ◽  
Biochemical Characterization ◽  
Gene Dosage ◽  
Genetic Relationships ◽  
Branchial Arch ◽  
Genetic Interactions ◽  
Limb Bud ◽  
High Affinity

Alx4 and Cart1 are closely related members of the family of transcription factors that contain the paired-type homeodomain. In contrast to other types of homeodomains, the paired-type homeodomain has been shown to mediate high-affinity sequence-specific DNA binding to palindromic elements as either homodimers or as heterodimers with other family members. Alx4 and Cart1 are co-expressed at several sites during development, including the craniofacial mesenchyme, the mesenchymal derivatives of neural crest cells in the first branchial arch and the limb bud mesenchyme. Because of the molecular similarity and overlapping expression pattern, we have analyzed the functional and genetic relationships between Alx4 and Cart1. The two proteins have similar DNA-binding activity in vitro and can form DNA-binding heterodimers; furthermore, they activate transcription of reporter genes that contain high-affinity DNA-binding sites in cell culture in a similar manner. Therefore, at least by these criteria, the two proteins are functionally redundant. Analysis of double mutant animals reveals several genetic interactions. First, mutation of Cart1 exacerbates Alx4-dependent polydactyly in a manner that is dependent on gene dosage. Second, there are complex genetic interactions in the craniofacial region that reveal a role for both genes in the fusion of the nasal cartilages and proper patterning of the mandible, as well as other craniofacial structures. Third, double mutant mice show a split sternum that is not detected in mice with any other genotype. Interpreted in the context of the biochemical characterization, the genetic analysis suggests that Alx4 and Cart1 are indeed functionally redundant, and reveal both unique and redundant functions for these genes in development.

scholarly journals N-terminal regions of Mps1 kinase determine functional bifurcation

2010 ◽  
Vol 189 (1) ◽  
pp. 41-56 ◽  
Author(s):  
Yasuhiro Araki ◽  
Linda Gombos ◽  
Suellen P.S. Migueleti ◽  
Lavanya Sivashanmugam ◽  
Claude Antony ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Chemical Biology ◽  
Molecular Level ◽  
Budding Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Deletion Analysis ◽  
Checkpoint Activation ◽  
Pole Body ◽  
N Terminus

Mps1 is a conserved kinase that in budding yeast functions in duplication of the spindle pole body (SPB), spindle checkpoint activation, and kinetochore biorientation. The identity of Mps1 targets and the subdomains that convey specificity remain largely unexplored. Using a novel combination of systematic deletion analysis and chemical biology, we identified two regions within the N terminus of Mps1 that are essential for either SPB duplication or kinetochore biorientation. Suppression analysis of the MPS1 mutants defective in SPB duplication and biochemical enrichment of Mps1 identified the essential SPB components Spc29 and the yeast centrin Cdc31 as Mps1 targets in SPB duplication. Our data suggest that phosphorylation of Spc29 by Mps1 in G1/S recruits the Mps2–Bbp1 complex to the newly formed SPB to facilitate its insertion into the nuclear envelope. Mps1 phosphorylation of Cdc31 at the conserved T110 residue controls substrate binding to Kar1 protein. These findings explain the multiple SPB duplication defects of mps1 mutants on a molecular level.

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