Determination of molecular weights and frictional ratios of macromolecules in impure systems: Aggregation of urease

Siegel, Lewis M.; Monty, Kenneth J.

doi:10.1016/0006-291x(65)90152-x

Cited by 91 publications

(29 citation statements)

References 14 publications

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“…From the experiments with wild-type cells, we estimated the Sla1-Las17 complex has a Stokes radius of 224 ± 6 Å, and from sedimentation velocity experiments (Figure 1G), we estimated a sedimentation coefficient of 8.1 ± 0.5 S. The native molecular mass of the Sla1-Las17 complex was calculated from the Stokes radius and sedimentation coefficient as 822 ± 28 kDa (Siegel and Monty, 1965). A similar analysis for the well-known heterotetrameric AP-2 complex revealed a native molecular mass of 212 ± 5 kDa, while its theoretical molecular mass (one copy each of the Apl1, Apl3, Apm4, and Aps2 subunits) is 286 kDa (unpublished data).…”

Section: Resultsmentioning

confidence: 99%

SLAC, a complex between Sla1 and Las17, regulates actin polymerization during clathrin-mediated endocytosis

Feliciano

Pietro

2012

MBoC

111

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Section: Resultsmentioning

confidence: 99%

SLAC, a complex between Sla1 and Las17, regulates actin polymerization during clathrin-mediated endocytosis

Feliciano

Pietro

2012

MBoC

111

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“…The molecular mass of a native protein or a protein complex can be determined accurately by an equation derived by Siegel and Monty, which combines the Stokes radius (a hydrodynamic radius of a molecule freely tumbling in solution) calculated from size exclusion chromatography with the sedimentation coefficient determined by separation in sucrose gradient (35). Based on the elution profile of protein standards from size exclusion chromatography we calculated the Stokes radius of LSH to be 4.94 nm.…”

Section: Resultsmentioning

confidence: 99%

“…The sedimentation coefficient of LSH was derived from the plotted standards. Calculations to determine the molecular weight of native LSH were applied as described previously (35) 3 ), and is the partial specific volume of protein (used 0.725 cm 3 /g). Plasmids and reporter assays.…”

Section: Methodsmentioning

confidence: 99%

LSH Cooperates with DNA Methyltransferases To Repress Transcription

Myant

Stancheva

2008

Molecular and Cellular Biology

122

129

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LSH, a protein related to the SNF2 family of chromatin-remodeling ATPases, is required for efficient DNA methylation in mammals. How LSH functions to support DNA methylation and whether it associates with a large protein complex containing DNA methyltransferase (DNMT) enzymes is currently unclear. Here we show that, unlike many other chromatin-remodeling ATPases, native LSH is present mostly as a monomeric protein in nuclear extracts of mammalian cells and cannot be detected in a large multisubunit complex. However, when targeted to a promoter of a reporter gene, LSH acts as an efficient transcriptional repressor. Using this as an assay to identify proteins that are required for LSH-mediated repression we found that LSH cooperates with the DNMTs DNMT1 and DNMT3B and with the histone deacetylases (HDACs) HDAC1 and HDAC2 to silence transcription. We show that transcriptional repression by LSH and interactions with HDACs are lost in DNMT1 and DNMT3B knockout cells but that the enzymatic activities of DNMTs are not required for LSH-mediated silencing. Our data suggest that LSH serves as a recruiting factor for DNMTs and HDACs to establish transcriptionally repressive chromatin which is perhaps further stabilized by DNA methylation at targeted loci.In vertebrate genomes, DNA methylation patterns are established during gametogenesis, embryo development, and cell differentiation by enzymes of the DNA cytosine methyltransferase family, which includes the maintenance DNA methyltransferase (DNMT) DNMT1 and the de novo methyltransferases DNMT3A and DNMT3B (1,24,45). DNMT1 binds to PCNA and functions primarily during S phase to restore fully methylated CpGs on hemimethylated daughter DNA strands generated during DNA replication (3, 4). DNMT3A and -3B are able to methylate unmethylated DNA and in mouse embryogenesis are required during gastrulation, when DNA methylation patterns are established in differentiating cell lineages of the embryo (24). Mice lacking DNMT proteins die early during embryogenesis and display aberrant expression of retrotransposons and various imprinted and nonimprinted genes (14,18,24). Genetic studies with plants and mammals have revealed that additional factors besides DNMTs are required for the establishment of DNA methylation patterns in vivo. Loss-of-function mutations in SNF2 family-related putative chromatin-remodeling ATPases such as the Arabidopsis thaliana DDM1 (decrease in DNA methylation 1) protein and its murine homolog Lsh (lymphoid-specific helicase) lead to dramatic hypomethylation of the genome in Arabidopsis thaliana and mice, respectively (5, 16). Unlike animals that are null for DNMTs, Lsh-deficient mice develop to term but die soon after birth with symptoms of renal failure (5). Interestingly, mice expressing a hypomorph allele of Lsh with targeted disruption of the SNF2 domain survive much longer and display modest hypomethylation of DNA and premature aging (39). Collectively, these studies indicate that the low levels of DNA methylation (ϳ30 to 35% of the wild-type level) in Lsh...

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“…The native molecular weight and shape (frictional coefficient) of protein complexes was calculated using established equations (Siegel and Monty, 1965; Harding and Colfen, 1995; Schuyler and Pellman, 2002). …”

Section: Methodsmentioning

confidence: 99%

Bod1, a novel kinetochore protein required for chromosome biorientation

Porter¹,

McClelland

Khoudoli³

et al. 2007

The Journal of Cell Biology

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We have combined the proteomic analysis of Xenopus laevis in vitro–assembled chromosomes with RNA interference and live cell imaging in HeLa cells to identify novel factors required for proper chromosome segregation. The first of these is Bod1, a protein conserved throughout metazoans that associates with a large macromolecular complex and localizes with kinetochores and spindle poles during mitosis. Small interfering RNA depletion of Bod1 in HeLa cells produces elongated mitotic spindles with severe biorientation defects. Bod1-depleted cells form syntelic attachments that can oscillate and generate enough force to separate sister kinetochores, suggesting that microtubule–kinetochore interactions were intact. Releasing Bod1-depleted cells from a monastrol block increases the frequency of syntelic attachments and the number of cells displaying biorientation defects. Bod1 depletion does not affect the activity or localization of Aurora B but does cause mislocalization of the microtubule depolymerase mitotic centromere- associated kinesin and prevents its efficient phosphorylation by Aurora B. Therefore, Bod1 is a novel kinetochore protein that is required for the detection or resolution of syntelic attachments in mitotic spindles.

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Determination of molecular weights and frictional ratios of macromolecules in impure systems: Aggregation of urease

Cited by 91 publications

References 14 publications

SLAC, a complex between Sla1 and Las17, regulates actin polymerization during clathrin-mediated endocytosis

SLAC, a complex between Sla1 and Las17, regulates actin polymerization during clathrin-mediated endocytosis

LSH Cooperates with DNA Methyltransferases To Repress Transcription

Bod1, a novel kinetochore protein required for chromosome biorientation

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