Freeze-fracture architecture and polypeptide composition of thylakoid membranes from euploid Ricinus cells

1981 ◽  
Vol 52 (1) ◽  
pp. 167-181
Author(s):  
M.P. Timko ◽  
R.E. Triemer ◽  
A.C. Vasconcelos
Keyword(s):  
Ricinus Communis ◽  
Relative Proportion ◽  
Freeze Fracture ◽  
Nuclear Genome ◽  
Electron Microscopic Examination ◽  
Thylakoid Membranes ◽  
Polypeptide Composition ◽  
Electron Microscopic ◽  
Size Category ◽  
Ploidy Levels

The freeze-fracture architecture and polypeptide composition of thylakoid membranes of euploid cells of Ricinus communis L. were examined. Electron microscopic examination of the chloroplasts of 1N 2N and 4N cells revealed little variation in the size of chloroplasts, lamellar structure and internal organization of plastids, despite increases in plastid numbers per cell observed to accompany the increase in nuclear ploidy. Thylakoid membranes from euploid cells were also similar in their freeze-fracture morphology. Two basic types of intramembranous particles were observed on the fracture faces of thylakoid membranes of euploid cells. The endoplasmic fracture (EF) face of experimentally unstacked thylakoid membranes of 1N, 2N and 4N cells contain 2 size categories of particles (115-121 A and 164–166 A), whereas the protoplasmic fracture (PF) face of these membranes contain a single size category of particles (85-88 A). The distribution and size of the EF- and PF-face particles were found to be similar among membranes from cells of the 3 ploidy levels. Analysis of the polypeptide composition of thylakoid membranes from 1N, 2N and 4N cells revealed no difference in the relative proportion of the constituent polypeptides of these membranes. The possible factors involved in the regulation of the development of thylakoid structure and composition in the presence of altered nuclear genome size are discussed.

scholarly journals Saccharomyces cerevisiae cdc2 mutants fail to replicate approximately one-third of their nuclear genome.

1983 ◽  
Vol 3 (6) ◽  
pp. 1000-1012 ◽  
Author(s):  
M N Conrad ◽  
C S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Nuclear Genome ◽  
Electron Microscopic Examination ◽  
Temperature Sensitive ◽  
Chromosomal Dna ◽  
Electron Microscopic ◽  
Dna Strands ◽  
Total Dna ◽  
Random Fraction

Chromosomal DNA replication was examined in temperature-sensitive mutants of Saccharomyces cerevisiae defective in a gene required for the completion of S phase at the nonpermissive temperature, 37 degrees C. Based on incorporation of radioactive precursors and density transfer experiments, strains carrying three different alleles of cdc2 failed to replicate approximately one-third of their nuclear genome at 37 degrees C. Whole-cell autoradiography experiments demonstrated that 93 to 96% of the cells synthesized DNA at 37 degrees C. Therefore, all cells failed to replicate part of their genome. DNA isolated from terminally arrested cells was of normal size as measured on neutral and alkaline sucrose gradients, suggesting that partially replicated DNA molecules do not accumulate and that DNA strands are ligated properly in cdc2 mutants. In addition, electron microscopic examination of the equivalent of more than one genome's DNA from arrested cells failed to reveal any partially replicated molecules. The sequences which failed to replicate at 37 degrees C were not highly specific; eight different cloned sequences replicated to the same extent as total DNA. The 2-microns plasmid DNA and rDNA replicated significantly less well than total DNA, but approximately one-half of these sequences replicated at 37 degrees C. These observations suggest that cdc2 mutants are defective in an aspect of initiation of DNA replication common to all chromosomes such that a random fraction of the chromosomes fail to initiate replication at 37 degrees C, but that once initiated, replication proceeds normally.

scholarly journals An improved method for freeze-fracture radioautography of tissues and cells, as applied to duodenal epithelium and thymic lymphocytes.

1984 ◽  
Vol 32 (1) ◽  
pp. 17-29 ◽  
Author(s):  
F W Kan ◽  
B M Kopriwa ◽  
C P Leblond
Keyword(s):  
Freeze Fracture ◽  
Electron Microscopic Examination ◽  
Improved Method ◽  
Electron Microscopic ◽  
Carbon Replica ◽  
Radiation Sources ◽  
Freeze Dried ◽  
Duodenal Epithelium ◽  
Tissue Specimens ◽  
Silver Grains

An improved method has been devised for the localization of radioactive substances to either one of the leaflets of cellular membranes. After tissue specimens are freeze-fractured and covered with a platinum-carbon replica, they are freeze-dried to allow coating with radioautographic emulsion at room temperature. After exposure at 4 degrees C and development, the emulsion is protected by layers of carbon and grease before the tissue underlying the replica is dissolved in sodium hypochlorite. The grease is removed in Freon 14 and the replica with its emulsion cover is mounted on a specimen grid for electron microscopic examination. The accuracy of radioactivity localization was demonstrated using 3H-thymidine-labeled liver by finding silver grains over the same sites after freeze-fracture as after thin section radioautography. Tests with 3H-methacrylate revealed that the interposition of a platinum-carbon replica decreased the radioautographic reaction by over 80%; hence, the need for long exposure. Only 67% of the silver grains came from radiation sources located beyond the upper 0.05 micron of the specimen and, therefore, the emulsion could be affected by radiation sources located not only within membrane leaflets but also in nearby cytoplasm. Thus, when 3H-fucose was injected into rats to locate newly formed glycoproteins within intestinal epithelium membranes, some of the silver grains found over E and P faces might be produced by radiation coming from the adjacent cytoplasm. To localize label within membrane leaflets in the absence of radiation sources in the cytoplasm, lymphocyte suspensions were incubated with 3H-concanavalin A at 0 degrees C. The plasmalemma radioactivity was then restricted to the two membrane leaflets, with 87-93% of the silver grains on the E leaflet and 7-13% on the P leaflet. It appears that, under these conditions, the technique provides adequate localization of radioactivity to the leaflets of the cell membrane.

Rhodopsin Particles in the Photoreceptor Membrane of an Insect

1976 ◽  
Vol 31 (11-12) ◽  
pp. 763-763 ◽  
Author(s):  
C. Bruce Boschek ◽  
Kurt Hamdorf
Keyword(s):  
Microscopic Examination ◽  
Visual Pigment ◽  
Particle Density ◽  
Substantial Reduction ◽  
Freeze Fracture ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Deficient Diet ◽  
Photoreceptor Membrane ◽  
Fracture Electron

Abstract Rhodopsin, Insect Photoreceptor, Freeze Fracture Electron-microscopic examination of freeze-fractured fly retinae has revealed the presence of particles, 80 to 100 Å in diameter, on the photoreceptor membrane. Flies which were raised on a vitamin-A deficient diet show a substantial reduction in the density of such particles. The reduction in particle density is in agreement with the reduction in visual-pigment concentration as measured spectrophotometrically for these flies. These results suggest that the particles are identical with molecules of the visual pigment, rhodopsin.

A Freeze-Fracture Technique for Examining Human Stratum Corneum

1972 ◽  
Vol 30 ◽  
pp. 298-299
Author(s):  
Daniel P. Hannon
Keyword(s):  
Electron Microscopy ◽  
Sample Preparation ◽  
Stratum Corneum ◽  
Microscopic Examination ◽  
Freeze Fracture ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Human Stratum Corneum ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

Human stratum corneum is a difficult tissue to process for electron microscopic examination. Most of the difficulty is due to the inability of the chemical fixative to adequately preserve all of the constituents of this tissue. As a result, during sectioning, breaks occur between cells creating voids which preclude any studies of these areas. Also, because the fixative is usually in an aqueous medium, studies of hydrated or dehydrated states are impossible. However, these problems can be overcome by using an alternative method of sample preparation, freeze-fracture electron microscopy.Samples of human skin both untreated and soaked for 20 minutes in 20% glycerin were cut into strips 1 mm wide by 2 mm long and placed in specially made copper discs with a 1 mm deep central cup. Freezing was accomplished by immersion in liquid Freon 22.

Saccharomyces cerevisiae cdc2 mutants fail to replicate approximately one-third of their nuclear genome

1983 ◽  
Vol 3 (6) ◽  
pp. 1000-1012
Author(s):  
M N Conrad ◽  
C S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Nuclear Genome ◽  
Electron Microscopic Examination ◽  
Temperature Sensitive ◽  
Chromosomal Dna ◽  
Electron Microscopic ◽  
Dna Strands ◽  
Total Dna ◽  
Random Fraction

Chromosomal DNA replication was examined in temperature-sensitive mutants of Saccharomyces cerevisiae defective in a gene required for the completion of S phase at the nonpermissive temperature, 37 degrees C. Based on incorporation of radioactive precursors and density transfer experiments, strains carrying three different alleles of cdc2 failed to replicate approximately one-third of their nuclear genome at 37 degrees C. Whole-cell autoradiography experiments demonstrated that 93 to 96% of the cells synthesized DNA at 37 degrees C. Therefore, all cells failed to replicate part of their genome. DNA isolated from terminally arrested cells was of normal size as measured on neutral and alkaline sucrose gradients, suggesting that partially replicated DNA molecules do not accumulate and that DNA strands are ligated properly in cdc2 mutants. In addition, electron microscopic examination of the equivalent of more than one genome's DNA from arrested cells failed to reveal any partially replicated molecules. The sequences which failed to replicate at 37 degrees C were not highly specific; eight different cloned sequences replicated to the same extent as total DNA. The 2-microns plasmid DNA and rDNA replicated significantly less well than total DNA, but approximately one-half of these sequences replicated at 37 degrees C. These observations suggest that cdc2 mutants are defective in an aspect of initiation of DNA replication common to all chromosomes such that a random fraction of the chromosomes fail to initiate replication at 37 degrees C, but that once initiated, replication proceeds normally.

Electron microscopic studies on ultrathin frozen sections of lactating mouse mammary gland

1978 ◽  
Vol 36 (2) ◽  
pp. 46-47
Author(s):  
Jan Zarzycki ◽  
Joseph Szroeder
Keyword(s):  
Mammary Gland ◽  
Protein Denaturation ◽  
Chemical Constituents ◽  
Freeze Substitution ◽  
Electron Microscopic Examination ◽  
Mouse Mammary Gland ◽  
Electron Microscopic ◽  
Preparation Methods ◽  
Microscopic Studies ◽  
Ultrathin Frozen Sections

The mammary gland ultrastructure in various functional states is the object of our investigations. The material prepared for electron microscopic examination by the conventional chemical methods has several limitations, the most important are the protein denaturation processes and the loss of large amounts of chemical constituents from the cells. In relevance to this,one can't be sure about a degree the observed images are adequate to the realy ultrastructure of a living cell. To avoid the disadvantages of the chemical preparation methods,some autors worked out alternative physical methods based on tissue freezing / freeze-drying, freeze-substitution, freeze-eatching techniqs/; actually the technique of cryoultraraicrotomy,i,e.cutting ultrathin sections from deep frozen specimens is assented as a complete alternative method. According to the limitations of the routine plastic embbeding methods we were interested to analize the mammary gland ultrastructure during lactation by the cryoultramicrotomy method.

Freeze-Fracture Replication in the Ultrastructure of Blue-Green Algae

1967 ◽  
Vol 25 ◽  
pp. 74-75
Author(s):  
L. V. Leak
Keyword(s):  
Cell Wall ◽  
Electron Microscope ◽  
Green Algae ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Electron Microscopic ◽  
Photosynthetic Membranes ◽  
Blue Green Algae ◽  
Cell Biol ◽  
Cellular Components

Electron microscopic observations of freeze-fracture replicas of Anabaena cells obtained by the procedures described by Bullivant and Ames (J. Cell Biol., 1966) indicate that the frozen cells are fractured in many different planes. This fracturing or cleaving along various planes allows one to gain a three dimensional relation of the cellular components as a result of such a manipulation. When replicas that are obtained by the freeze-fracture method are observed in the electron microscope, cross fractures of the cell wall and membranes that comprise the photosynthetic lamellae are apparent as demonstrated in Figures 1 & 2.A large portion of the Anabaena cell is composed of undulating layers of cytoplasm that are bounded by unit membranes that comprise the photosynthetic membranes. The adjoining layers of cytoplasm are closely apposed to each other to form the photosynthetic lamellae. Occassionally the adjacent layers of cytoplasm are separated by an interspace that may vary in widths of up to several 100 mu to form intralamellar vesicles.

A Comparative Study of Fractographic Replicas

1974 ◽  
Vol 32 ◽  
pp. 566-567
Author(s):  
Loren Anderson ◽  
Pat Pizzo ◽  
Glen Haydon
Keyword(s):  
Electron Microscopy ◽  
Electron Microscopic Examination ◽  
Direct Examination ◽  
Electron Microscopic ◽  
Reliable Technique ◽  
Service Failures ◽  
Transmission Electron ◽  
Mode Of Failure ◽  
Scanning Electron Microscopic ◽  
Scanning Electron

Transmission electron microscopy of replicas has long been used to study the fracture surfaces of components which fail in service. Recently, the scanning electron microscope (SEM) has gained popularity because it allows direct examination of the fracture surface. However, the somewhat lower resolution of the SEM coupled with a restriction on the sample size has served to limit the use of this instrument in investigating in-service failures. It is the intent of this paper to show that scanning electron microscopic examination of conventional negative replicas can be a convenient and reliable technique for determining mode of failure.

Novel methods for demonstrating osseointegration of hydroxylapatite-coated titanium hip prostheses

1991 ◽  
Vol 49 ◽  
pp. 34-35 ◽  
Author(s):  
P. Frayssinet ◽  
J. Hanker ◽  
D. Hardy ◽  
B. Giammara
Keyword(s):  
Hip Prosthesis ◽  
Ethanol Concentration ◽  
Bone Matrix ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Hip Prostheses ◽  
Cellular Inclusions ◽  
Microscopic Studies ◽  
Diamond Saw ◽  
Plasma Sprayed

Prostheses implanted in hard tissues cannot be processed for electron microscopic examination or microanalysis in the same way as those in other tissues. For these reasons, we have developed methods allowing light and electron microscopic studies as well as microanalysis of the interface between bone and a metal biomaterial coated by plasma-sprayed hydroxylapatite(HA) ceramic.An HA-coated titanium hip prosthesis (Corail, Landos, France), which had been implanted for two years, was removed after death (unrelated to the orthopaedic problem). After fixation it was dehydrated in solutions of increasing ethanol concentration prior to embedment in polymethylmethacrylate(PMMA). Transverse femur sections were obtained with a diamond saw and the sections then carefully ground to a thickness of 200 microns. Plastic-embedded sections were stained for calcium with a silver methenamine modification of the von Kossa method for calcium staining and coated by carbon. They have been examined by back-scatter SEM on an ISI-SS60 operated at 25 KV. EDAX has been done on cellular inclusions and extracellular bone matrix.

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