scholarly journals ScII: an abundant chromosome scaffold protein is a member of a family of putative ATPases with an unusual predicted tertiary structure.

1994 ◽  
Vol 127 (2) ◽  
pp. 303-318 ◽  
Author(s):  
N Saitoh ◽  
I G Goldberg ◽  
E R Wood ◽  
W C Earnshaw
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Topoisomerase Ii ◽  
Tertiary Structure ◽  
Coiled Coil ◽  
Scaffold Protein ◽  
The Other ◽  
Mitotic Chromosomes ◽  
Chromosome Scaffold

Here, we describe the cloning and characterization of ScII, the second most abundant protein after topoisomerase II, of the chromosome scaffold fraction to be identified. ScII is structurally related to a protein, Smc1p, previously found to be required for accurate chromosome segregation in Saccharomyces cerevisiae. ScII and the other members of the emerging family of SMC1-like proteins are likely to be novel ATPases, with NTP-binding A and B sites separated by two lengthy regions predicted to form an alpha-helical coiled-coil. Analysis of the ScII B site predicted that ScII might use ATP by a mechanism similar to the bacterial recN DNA repair and recombination enzyme. ScII is a mitosis-specific scaffold protein that colocalizes with topoisomerase II in mitotic chromosomes. However, ScII appears not to be associated with the interphase nuclear matrix. ScII might thus play a role in mitotic processes such as chromosome condensation or sister chromatid disjunction, both of which have been previously shown to involve topoisomerase II.

scholarly journals Localization of topoisomerase II in mitotic chromosomes.

1985 ◽  
Vol 100 (5) ◽  
pp. 1716-1725 ◽  
Author(s):  
W C Earnshaw ◽  
M M Heck
Keyword(s):  
Present Article ◽  
Immunoelectron Microscopy ◽  
Polyclonal Antibody ◽  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Experimental Protocol ◽  
Mitotic Chromosomes ◽  
Restricted Domains ◽  
Loop Domains ◽  
Chromosome Scaffold

In the preceding article we described a polyclonal antibody that recognizes cSc-1, a major polypeptide component of the chicken mitotic chromosome scaffold. This polypeptide was shown to be chicken topoisomerase II. In the experiments described in the present article we use indirect immunofluorescence and immunoelectron microscopy to examine the distribution of topoisomerase II within intact chromosomes. We also describe a simple experimental protocol that differentiates antigens that are interspersed along the chromatin fiber from those that occupy restricted domains within the chromosome. These experiments indicate that the distribution of the enzyme appears to be independent of the bulk chromatin. Our data suggest that topoisomerase II is bound to the bases of the radial loop domains of mitotic chromosomes.

scholarly journals The Unstable F-box Protein p58-Ctf13 Forms the Structural Core of the CBF3 Kinetochore Complex

1999 ◽  
Vol 145 (5) ◽  
pp. 933-950 ◽  
Author(s):  
Iain D. Russell ◽  
Adam S. Grancell ◽  
Peter K. Sorger
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Properties ◽  
Chromosome Segregation ◽  
Half Life ◽  
Degradation Pathway ◽  
The Other ◽  
Yeast Cells ◽  
Centromeric Dna ◽  
Structural And Functional Properties ◽  
F Box Protein

Kinetochores are smaller and more accessible experimentally in budding yeast than in any other eukaryote. Believing that simple and complex kinetochores have important structural and functional properties in common, we characterized the structure of CBF3, the essential centromere-binding complex that initiates kinetochore formation in Saccharomyces cerevisiae. We find that the four subunits of CBF3 are multimeric in solution: p23Skp1 and p58Ctf13 form a heterodimer, and p64Cep3 and p110Ndc10 form homodimers. Subcomplexes involving p58 and each of the other CBF3 subunits can assemble in the absence of centromeric DNA. In these subcomplexes, p58 appears to function as a structural core mediating stable interactions among other CBF3 proteins. p58 has a short half-life in yeast, being subject to ubiquitin-dependent proteolysis, but we find that it is much more stable following association with p64. We propose that p23Skp1-p58-p64 complexes constitute the primary pool of active p58 in yeast cells. These complexes can either dissociate, reexposing p58 to the degradation pathway, or can bind to p110 and centromeric DNA, forming a functional CBF3 complex in which p58 is fully protected from degradation. This pathway may constitute an editing mechanism preventing the formation of ectopic kinetochores and ensuring the fidelity of chromosome segregation.

scholarly journals Amino Acid Sequence Studies on the β-Chain of Human Fibrinogen

1977 ◽  
Author(s):  
K. Watt ◽  
D. Goldbaum ◽  
B. A. Cottrell ◽  
T. Takagi ◽  
R. F. Doolittle
Keyword(s):  
Disulfide Bonds ◽  
Coiled Coil ◽  
Enzymatic Digestion ◽  
The Other ◽  
Human Fibrinogen ◽  
Parent Molecule ◽  
General Arrangement ◽  
The One ◽  
Β Chain

The β-chain of human fibrinogen contains 480 ± 15 residues, sixteen of which are methionines. In this vein, we have isolated and characterized all seventeen cyanogen bromide peptides. The arrangement of most of these fragments has been achieved by the identification of key overlap peptides derived from enzymatic digestion of β-chains, on the one hand, and a characterization of β-chain fragments isolated from fragments D and E, on the other. In some cases the alignment is based only on homologies with the α- and/or γ-chains, and in a few instances some definite ambiguities still exist. For the most part, however, the general arrangement is in hand. Of particular interest is the plasmin-sensitive segment which is a part of the inter-domainal connection, which in turn we believe is a three-stranded coiled-coil punctuated by an unusual arrangement of disulfide bonds. The positioning of the other cysteine residues in the β-chain is also of considerable interest, since it sheds light on the extent of connections between portions of the three non-identical chains in the parent molecule.

scholarly journals Identification and characterization of mutations affecting sporulation in Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 120 (4) ◽  
pp. 899-907
Author(s):  
L M Smith ◽  
L G Robbins ◽  
A Kennedy ◽  
P T Magee
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Analysis ◽  
Dna Synthesis ◽  
The Other ◽  
Negative Regulators ◽  
Identification And Characterization

Abstract Mutations affecting the synthesis of the sporulation amyloglucosidase were isolated in a homothallic strain of Saccharomyces cerevisiae, SCMS7-1. Two were found, both of which were deficient in sporulation at 34 degrees. One, SL484, sporulated to 50% normal levels at 30 degrees but less than 5% at 34 degrees or 22 degrees. The other, SL641, failed to sporulate at any temperature. Both mutants were blocked before premeiotic DNA synthesis, and both complemented spo1, spo3, and spo7. Genetic analysis of the mutation in SL484 indicated linkage to TRP5 and placed the gene 10 map units from TRP5 on chromosome VII. A plasmid containing an insert which complements the mutation in SL484 fails to complement SL641. We therefore conclude that these two mutations are in separate genes and we propose to call these genes SPO17 and SPO18. These two genes are (with SPO7, SPO8, and SPO9) among the earliest identified in the sporulation pathway and may interact directly with the positive and negative regulators RME and IME.

scholarly journals Molecular Characterization of the Extracellular Domain of Human Junctional Adhesion Proteins

2021 ◽  
Vol 22 (7) ◽  
pp. 3482
Author(s):  
Christopher Mendoza ◽  
Sai Harsha Nagidi ◽  
Dario Mizrachi
Keyword(s):  
Tertiary Structure ◽  
Extracellular Domain ◽  
The Other ◽  
Immunoglobulin Superfamily ◽  
Adhesive Properties ◽  
The Family ◽  
Heterotypic Interactions ◽  
Hydrophobic Nature ◽  
And Function

The junction adhesion molecule (JAM) family of proteins play central roles in the tight junction (TJ) structure and function. In contrast to claudins (CLDN) and occludin (OCLN), the other membrane proteins of the TJ, whose structure is that of a 4α-helix bundle, JAMs are members of the immunoglobulin superfamily. The JAM family is composed of four members: A, B, C and 4. The crystal structure of the extracellular domain of JAM-A continues to be used as a template to model the secondary and tertiary structure of the other members of the family. In this article, we have expressed the extracellular domains of JAMs fused with maltose-binding protein (MBP). This strategy enabled the work presented here, since JAM-B, JAM-C and JAM4 are more difficult targets due to their more hydrophobic nature. Our results indicate that each member of the JAM family has a unique tertiary structure in spite of having similar secondary structures. Surface plasmon resonance (SPR) revealed that heterotypic interactions among JAM family members can be greatly favored compared to homotypic interactions. We employ the well characterized epithelial cadherin (E-CAD) as a means to evaluate the adhesive properties of JAMs. We present strong evidence that suggests that homotypic or heterotypic interactions among JAMs are stronger than that of E-CADs.

scholarly journals Smc5-Smc6-Dependent Removal of Cohesin from Mitotic Chromosomes

2009 ◽  
Vol 29 (16) ◽  
pp. 4363-4375 ◽  
Author(s):  
Emily A. Outwin ◽  
Anja Irmisch ◽  
Johanne M. Murray ◽  
Matthew J. O'Connell
Keyword(s):  
Dna Damage ◽  
Chromosome Segregation ◽  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Chromosome Structure ◽  
Synthetic Lethality ◽  
Mitotic Chromosomes ◽  
Interphase Chromosome ◽  
Chromosome Arm ◽  
Null Mutants

ABSTRACT The function of the essential cohesin-related Smc5-Smc6 complex has remained elusive, though hypomorphic mutants have defects late in recombination, in checkpoint maintenance, and in chromosome segregation. Recombination and checkpoints are not essential for viability, and Smc5-Smc6-null mutants die in lethal mitoses. This suggests that the chromosome segregation defects may be the source of lethality in irradiated Smc5-Smc6 hypomorphs. We show that in smc6 mutants, following DNA damage in interphase, chromosome arm segregation fails due to an aberrant persistence of cohesin, which is normally removed by the Separase-independent pathway. This postanaphase persistence of cohesin is not dependent on DNA damage, since the synthetic lethality of smc6 hypomorphs with a topoisomerase II mutant, defective in mitotic chromosome structure, is also due to the retention of cohesin on undamaged chromosome arms. In both cases, Separase overexpression bypasses the defect and restores cell viability, showing that defective cohesin removal is a major determinant of the mitotic lethality of Smc5-Smc6 mutants.

scholarly journals Characterization of messenger-like ribonucleic acid from Saccharomyces cerevisiae by the use of chromatography on methylated albumin–kieselguhr

1970 ◽  
Vol 119 (4) ◽  
pp. 699-706 ◽  
Author(s):  
Roger Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Carbon Source ◽  
Ribosomal Rna ◽  
Sodium Dodecyl ◽  
Nucleotide Composition ◽  
The Other ◽  
Dilute Solution ◽  
A Minor

Chromatography on methylated albumin–kieselguhr of RNA from Saccharomyces cerevisiae was used to separate stable RNA from a tenaciously bound DNA-like RNA fraction. The tenaciously bound RNA, which was eluted with a dilute solution of sodium dodecyl sulphate, was characterized as messenger-like RNA by its sedimentation behaviour, nucleotide composition, lack of methylated bases and labelling kinetics. Chromatography of purified ribosomal RNA indicated a minor contamination of the tenaciously bound fraction with ribosomal RNA. On the other hand, a large portion of pulse-labelled polyribosomal RNA from protoplasts of Saccharomyces cerevisiae was tenaciously bound to the columns. The `chase' of isotopic label from the messenger-like RNA was found to be retarded during inhibition of protein synthesis both by cycloheximide and by starvation for a carbon source.

A very rapid in situ Kikuchi technique to determine relative crystal misorientation

1988 ◽  
Vol 46 ◽  
pp. 830-831
Author(s):  
J. I. Bennetch
Keyword(s):  
Electron Beam ◽  
Analytical Techniques ◽  
Superplastic Forming ◽  
The Other ◽  
Kikuchi Pattern ◽  
Kikuchi Line ◽  
Line Analysis

In a recent study of the superplastic forming (SPF) behavior of certain Al-Li-X alloys, the relative misorientation between adjacent (sub)grains proved to be an important parameter. It is well established that the most accurate way to determine misorientation across boundaries is by Kikuchi line analysis. However, the SPF study required the characterization of a large number of (sub)grains in each sample to be statistically meaningful, a very time-consuming task even for comparatively rapid Kikuchi analytical techniques.In order to circumvent this problem, an alternate, even more rapid in-situ Kikuchi technique was devised, eliminating the need for the developing of negatives and any subsequent measurements on photographic plates. All that is required is a double tilt low backlash goniometer capable of tilting ± 45° in one axis and ± 30° in the other axis. The procedure is as follows. While viewing the microscope screen, one merely tilts the specimen until a standard recognizable reference Kikuchi pattern is centered, making sure, at the same time, that the focused electron beam remains on the (sub)grain in question.

Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

1982 ◽  
Vol 47 (03) ◽  
pp. 197-202 ◽  
Author(s):  
Kurt Huber ◽  
Johannes Kirchheimer ◽  
Bernd R Binder
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Gel Filtration ◽  
Chain Structure ◽  
The Other ◽  
Single Chain ◽  
Apparent Homogeneity ◽  
Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

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