Autoradiographic Evidence For The In Situ Synthesis Of Chloroplast And Mitochondrial Rna

1968 ◽  
Vol 3 (3) ◽  
pp. 327-340
Author(s):  
SARAH P. GIBBS
Keyword(s):  
Chloroplast Development ◽  
Unit Area ◽  
Mitochondrial Rna ◽  
Cell Component ◽  
Electron Microscope Autoradiography ◽  
Mitochondrial Ribosomes ◽  
Ochromonas Danica ◽  
Microscope Autoradiography ◽  
Cytoplasmic Ribosomes

The rate of appearance of labelled RNA in the chloroplast and mitochondria as compared with the rate in the remaining cytoplasm was studied in the unicellular flagellate, Ochromonas danica, by electron-microscope autoradiography. Greening cells were labelled with uridine-5,6[3H] for a short (30 min) and a long (2 h) interval and the concentration of label, expressed as grains/unit area, determined for each cell component. The data demonstrate that there is the expected lag in the labelling of the cytoplasm proper, but no apparent lag in the labelling of the chloroplast and mitochondria. This observation, combined with the fact that after the short labelling time the chloroplast and mitochondria have a much heavier concentration of labelled RNA than the surrounding cytoplasm, indicates that most, if not all, chloroplast and mitochondrial RNA is synthesized in situ. The three kinds of ribosomes present in the cell are distinctly different in size. The mitochondrial ribosomes measure 150-170 Å in diameter, the chloroplast ribosomes average 170-200 Å in diameter, whereas the cytoplasmic ribosomes are 210-230 Å in diameter in glutaraldehyde-osmium-fixed cells. During chloroplast development in the light, the number of chloroplast ribosomes increases approximately tenfold.

scholarly journals EFFECTS OF CHLORAMPHENICOL ON CHLOROPLAST AND MITOCHONDRIAL ULTRASTRUCTURE IN OCHROMONAS DANICA

1972 ◽  
Vol 52 (3) ◽  
pp. 598-614 ◽  
Author(s):  
Heidi Smith-Johannsen ◽  
Sarah P. Gibbs
Keyword(s):  
Growth Rate ◽  
Ribosomal Proteins ◽  
Chloroplast Development ◽  
Chlorophyll Synthesis ◽  
Chloroplast Envelope ◽  
Mitochondrial Ultrastructure ◽  
Mitochondrial Ribosomes ◽  
Ochromonas Danica ◽  
Progressive Loss ◽  
Cytoplasmic Ribosomes

The effect of chloramphenicol (CAP) on cell division and organelle ultrastructure was studied during light-induced chloroplast development in the Chrysophyte alga, Ochromonas danica. Since the growth rate of the CAP-treated cells is the same as that of the control cells for the first 12 hr in the light, CAP is presumed to be acting during that interval solely by inhibiting protein synthesis on chloroplast and mitochondrial ribosomes. CAP markedly inhibits chloroplast growth and differentiation. During the first 12 hr in the light, chlorophyll synthesis is inhibited by 93%, the formation of new thylakoid membranes is reduced by 91%, and the synthesis of chloroplast ribosomes is inhibited by 81%. Other chloroplast-associated abnormalities which occur during the first 12 hr and become more pronounced with extended CAP treatment are the presence of prolamellar bodies and of abnormal stacks of thylakoids, the proliferation of the perinuclear reticulum, and the accumulation of dense granular material between the chloroplast envelope and the chloroplast endoplasmic reticulum. CAP also causes a progressive loss of the mitochondrial cristae, which is paralleled by a decline in the growth rate of the cells, but it has no effect on the synthesis of mitochondrial ribosomes. We postulate that one or more chloroplast ribosomal proteins are synthesized on chloroplast ribosomes, whereas mitochondrial ribosomal proteins are synthesized on cytoplasmic ribosomes.

An Ultrastructural Autoradiographic Study of the Incorporation of [35S]Cystine in the Wool Fibre Cortex

1973 ◽  
Vol 13 (3) ◽  
pp. 811-819
Author(s):  
R. E. CHAPMAN ◽  
R. T. GEMMELL
Keyword(s):  
Unit Area ◽  
Autoradiographic Study ◽  
Wool Fibre ◽  
Cross Sectional ◽  
Electron Microscope Autoradiography ◽  
Merino Sheep ◽  
Microscope Autoradiography ◽  
Cystine Content ◽  
Chemical Studies ◽  
Filament Bundle

Previous autoradiographic and chemical studies gave incompatible results as regards the incorporation of cystine into and cystine content of the 2 cortical segments of the wool fibre. In this study the incorporation of [35S]cystine into the wool fibre cortex was therefore re-examined by electron-microscope autoradiography. Skin samples were taken from a Merino sheep 1 h and 5 h after intradermal injections of L-[35S]cystine. At both times there was very little incorporation of 35S in the follicle bulbs. By 5 h incorporation occurred distally from the suprabulbar region throughout the zone of macrofibril (filament bundle) formation. More 35S was incorporated per unit area in the paracortex than in the orthocortex, and at the level of maximal uptake near the middle of this zone there was about a 2-fold difference per unit area. However, when the relative cross-sectional areas of the cortical segments were also considered, the actual amount of 35S incorporated at this level was slightly greater in the orthocortex than in the paracortex. These differences in the incorporation of [35S]cystine by the cortical segments agreed with previous results from chemical studies on cortical fractions separated from wool.

The Measurement of Fluorophosphate-Reactive Esterase Sites in the Liver of Barbiturate Treated Rats Using Quantitative E.M. Autoradiography

1976 ◽  
Vol 34 ◽  
pp. 102-103
Author(s):  
G. C. Budd
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Untreated Control ◽  
Unit Area ◽  
Smooth Endoplasmic Reticulum ◽  
Electron Microscope Autoradiography ◽  
Microscope Autoradiography ◽  
Sodium Phenobarbital ◽  
And Control

Male 100 gm Holtzman rats each received 100 mg/Kg of sodium phenobarbital intraperitoneally per day for 4 days. On the fourth day samples of fresh and glutaraldehyde fixed liver were reacted with 10-4 molar tritium labeled diisopropylfluorophosphate (3H-DFP) to permit the measurement of fluorophosphatereactive (FPR) esterase sites using quantitative electron microscope autoradiography (EMARG). The distribution and measured concentrations of FPR sites in the liver of barbiturate treated rats were compared with similar measurements obtained with untreated control rats. Measurements of the density of developed autoradiographic grains (grains/unit area) revealed that in both the treated and control preparations, the FPR sites were concentrated in the rough and smooth endoplasmic reticulum and associated ground cytoplasm of the hepatocytes (RER and SER respectively).

Incorporation of Cholesterol into Peripheral Nerve Myelin

1972 ◽  
Vol 30 ◽  
pp. 334-335
Author(s):  
Frank A. Rawlins
Keyword(s):  
Electron Microscope ◽  
Sciatic Nerve ◽  
Electron Microscope Autoradiography ◽  
Myelin Sheaths ◽  
Microscope Autoradiography ◽  
Grain Distribution ◽  
Sciatic Nerves ◽  
Autoradiographic Analysis ◽  
Old Mice ◽  
Short Times

Several speculations exist as to the site of incorporation of preformed molecules into myelin. The possibility that an autoradiographic analysis of cholesterol-1,2-H3 incorporation at very short times after injection might shed some light in the solution of that problem led to the present experiment.Cholesterol-1,2-H3 was injected intraperitoneally into 24 tenday old mice. The animals were then sacrificed at 10,20,30,40,60,90,120 and 180 min after the injection and the sciatic nerves were processed for electron microscope autoradiography. To analyze the grain distribution in the autoradiograms of cross and longitudinal sections from each sciatic nerve myelin sheaths were subdivided into three compartments named: outer 1/3, middle 1/3 and inner 1/3 compartments.It was found that twenty min. after the injection of cholesterol -1.2-H3 (Figs. 1 and 2), 55% of the total number of grains (t.n.g) found in myelin were within the outer 1/3 compartment, 9% were within the middle 1/3 and 36% within the inner 1/3 compartment

The effects of spectinomycin and ethidium bromide on the synthesis of organelle rRNA and on ultrastructure in Ochromonas danica

1980 ◽  
Vol 43 (1) ◽  
pp. 119-136
Author(s):  
H. Smith-Johannsen ◽  
D. Fromson ◽  
S.P. Gibbs
Keyword(s):  
Electron Microscopy ◽  
Ethidium Bromide ◽  
Ribosomal Proteins ◽  
Specific Inhibitor ◽  
Chloroplast Ultrastructure ◽  
Normal Amount ◽  
Chloroplast Membranes ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Rrna ◽  
Ochromonas Danica

The effects of 24-h exposure to spectinomycin (100 microgram/ml) and ethidium bromide (1 microgram/ml) on the accumulation of chloroplast and mitochondrial rRNAs and on organelle ultrastructure were studied in greening cells of Ochromonas danica. Cells treated with ethidium bromide for 24 h divide at the same rate as controls but contain less than one third the normal amount of mitochondrial rRNA. Ultrastructural observations showed that these cells contain only 10% the number of mitochondrial ribosomes found in controls as well as fewer mitochondrial cristae. Ethidium bromide has no effect on chloroplast ultrastructure in Ochromonas. Greening cells treated with spectinomycin grow at close to control rates but contain 30–40% less chloroplast rRNA than do controls. Electron microscopy showed that spectinomycin disrupts the organization of chloroplast membranes and reduces the number of chloroplast ribosomes by 30%. Under these conditions, spectinomycin has no effect on mitochondrial rRNA or ultrastructure. Since spectinomycin is a specific inhibitor of translation on 70S ribosomes, these results are consistent with the possibility that at least some chloroplast ribosomal proteins are synthesized in the chloroplast of Ochromonas.

The route of entry of cytoplasmically synthesized proteins into chloroplasts of algae possessing chloroplast ER

1979 ◽  
Vol 35 (1) ◽  
pp. 253-266
Author(s):  
S.P. Gibbs
Keyword(s):  
Protein Synthesis ◽  
Nuclear Envelope ◽  
Outer Membrane ◽  
Chloroplast Envelope ◽  
Inhibit Protein Synthesis ◽  
Ochromonas Danica ◽  
Plastid Proteins ◽  
Regular Network ◽  
Cytoplasmic Ribosomes ◽  
Plastid Ribosomes

In 8 classes of algae, namely the Cryptophyceae, Raphidophyceae, Haptophyceae, Chrysophyceae, Bacillariophyceae, Xanthophyceae, Eustigmatophyceae and Phaeophyceae, the chloroplasts, in addition to being surrounded by a double-membraned chloroplast envelope, are also enclosed by a cisterna of endoplasmic reticulum called the chloroplast ER. Often this ER cisterna is continuous with the outher membrane of the nuclear envelope in such a manner that the nuclear envelope forms a part of the ER sac enclosing the chloroplast. In all these classes of algae except the Cryptophyceae, a regular network of tubules and vesicles, named the periplastidal reticulum, is present at a specific location between the chloroplast envelope and the chloroplast ER. In the Cryptophyceae, scattered vesicles are found between the chloroplast envelope and the chloroplast ER. Ribosomes which have been shown to be arranged to polysomes are found on the outer membrane of the chloroplast ER. It is proposed that nuclear-coded proteins which are destined for the chloroplast are synthesized on these polysomes, passing during synthesis into the lumen of the ER cisterna. Vesicles containing these proteins then pinch off the chloroplast ER and form the periplastidal reticulum. Vesicles containing these proteins then pinch off the chloroplast ER and form the periplastidal reticulum. Vesicles then fuse with the outer membrane of the chloroplast envelope thereby delivering their contents to the lumen of the chloroplast envelope. Proteins then cross the inner membrane of the chloroplast envelope in an as yet unknown manner. Experimental evidence for this hypothesis comes from studies on Ochromonas danica using chloramphenicol and spectinomycin, which inhibit protein synthesis on plastid ribosomes, and cycloheximide, which inhibits protein synthesis on cytoplasmic ribosomes. In cells of Ochromonas exposed to chloramphenicol or spectinomycin, the periplastidal reticulum proliferates markedly becoming several layers thick. Presumably this build up of periplastidal reticulum occurs because the transport of cytoplasmically synthesized plastid proteins is slowed down when protein synthesis in the chloroplast is inhibited. Conversely, when cells of Ochromonas are treated with cycloheximide, there is a reduction in the amount of periplastidal reticulum presumably because there are no cytoplasmically synthesized proteins to be transported into the chloroplast.

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