INCORPORATION OF NUCLEOTIDES INTO NUCLEI OF FIXED CELLS BY DNA POLYMERASE

The enzyme calf thymus polymerase requires denatured or single-stranded DNA as a primer for DNA synthesis and is inactive on native DNA preparations. The enzyme and tritium-labeled deoxyribonucleoside triphosphates were incubated with alcohol-fixed and Carnoy-fixed tissue preparations to see if primer DNA could be found in several types of cells undergoing DNA synthesis. In all cases, low-pH controls were prepared for comparison. Priming activity was not found in nuclei that had been fixed in alcohol. Priming activity was found in cell nuclei that had been fixed with an acid fixative or had been treated at a low pH prior to treatment with the enzyme reaction mixture.     

THE INCORPORATION OF TRITIUM FROM THYMIDINE INTO PROTEINS OF THE MOUSE

Tritium from methyl-H³-thymidine was found to be incorporated into proteins in mice. This incorporation in the mouse as a whole represented between 1 and 10% of the injected tritium. Tritiated water was not an intermediate. Transmethylation reactions are proposed as a means whereby certain amino acids might have acquired the tritium from thymidine at some stage of its catabolism. The initial (2 hr) ratios of DNA to protein tritium activities per milligram of wet tissue ranged from 5 in two tissues of low DNA synthetic activity (pancreas, liver) to 35 to 40 in two tissues of high DNA synthetic activity (spleen, small intestine). Labeled nuclear protein was coincident with labeled DNA in nuclei, where it constituted less than 2.5% of the total tritium. The significance of the findings is discussed.     

IDENTIFICATION OF THE ADRENOCORTICOTROPHIN-PRODUCING CELLS IN THE RAT HYPOPHYSIS BY AUTORADIOGRAPHY

The relative rates of protein (hormone) synthesis and secretion by the various cell types in the anterior hypophysis of the rat have been studied by means of autoradiography. Normal and adrenalectomized male rats were injected with tritiated glycine and their hypophyses removed and fixed at 20, 40, and 90 minutes and 15 hours after injection. Autoradiograms of the hypophysial sections were prepared and autoradiographic grains were counted in the film overlying the cytoplasm of individual cells. With the aid of this method, a unique cell type was identified in the hypophyses of adrenalectomized rats. This cell is morphologically distinct from "gonadectomy cells," "thyroidectomy cells," and from previously described normal cell types, and is therefore designated as the "adrenalectomy cell." Among the 7 cell types differentiated in this study, the "adrenalectomy cell" had the highest tritium content and, in addition, at the time intervals studied this cell had the fastest rate of appearance and disappearance of protein tritium. This autoradiographic evidence of rapid protein (or polypeptide) turnover following adrenalectomy indicates that the "adrenalectomy cell" is the site of adrenocorticotrophin production in the adrenalectomized rat. Further autoradiographic and cytological evidence is presented which suggests that the "adrenalectomy cells" may be derived from chromophobes, and that a portion of the "large chromophobes" as defined in this study may be the site of adrenocorticotrophin production in the normal rat.     

RADIOAUTOGRAPHIC STUDY OF IN VIVO AND IN VITRO INCORPORATION OF FUCOSE-3H INTO THYROGLOBULIN BY RAT THYROID FOLLICULAR CELLS

The incorporation of fucose-³H in rat thyroid follicles was studied by radioautography in the light and electron microscopes to determine the site of fucose incorporation into the carbohydrate side chains of thyroglobulin, and to follow the migration of thyroglobulin once it had been labeled with fucose-³H. Radioautographs were examined quantitatively in vivo at several times after injection of fucose-³H into rats, and in vitro following pulse-labeling of thyroid lobes in medium containing fucose-³H. At 3–5 min following fucose-³H administration in vivo, 85% of the silver grains were localized over the Golgi apparatus of thyroid follicular cells. By 20 min, silver grains appeared over apical vesicles, and by 1 hr over the colloid. At 4 hr, nearly all of the silver grains had migrated out of the cells into the colloid. Analysis of the changes in concentration of label with time showed that radioactivity over the Golgi apparatus increased for about 20 min and then decreased, while that over apical vesicles increased to reach a maximum at 35 min. Later, the concentration of label over the apical vesicles decreased, while that over the colloid increased. Similar results were obtained in vitro. It is concluded that fucose, which is located at the end of some of the carbohydrate side chains, is incorporated into thyroglobulin within the Golgi apparatus of thyroid follicular cells, thereby indicating that some of these side chains are completed there. Furthermore, the kinetic analysis demonstrates that apical vesicles are the secretion granules which transport thyroglobulin from the Golgi apparatus to the apex of the cell and release it into the colloid.     

INCORPORATION OF KINETIN INTO SALVINIA

The uptake of kinetin-8¹⁴C in the aquatic fern, Salvinia, wascorrelated with an inhibition of stem elongation and floatingleaf expansion. The kinetin-inhibition depended on intact moleculesof kinetin which are incorporated mostly in a supernatant fractioncontaining soluble non-polynucleotides and RNA. Recovery frominhibition occurred when kinetin was depleted from the medium.That portion of the plant which was produced after recoverydid not contain significant levels of kinetin-8¹⁴C. (Received April 18, 1967; )     

INCORPORATION OF TRITIATED CYTIDINE INTO RIBONUCLEIC ACID BY ISOLATED PEA NUCLEI

A new method for the isolation of the nuclei from plant tissue has been devised. This method consists in passing the plant tissue through a set of spring-loaded, counter-rotating rollers and collecting the liberated nuclei in sucrose solution containing calcium ions. A semi-automatic machine which couples the rollers with a special tissue chopping device has been constructed. It has been shown that nuclei obtained in this way actively incorporate cytidine-H³ into RNA. This incorporation is increased in the presence of nucleoside triphosphates and an energy-regenerating system.     

TIMING OF INCORPORATION OF TRITIATED NUCLEOSIDES INTO DNA AND RNA OF EMBRYONIC CELLS OF RANA PIPIENS

The incorporation of tritiated nucleosides into DNA and RNA has been examined in partially synchronized cells of Rana pipiens embryos at the neurula and tailbud stages. Tritiated thymidine and deoxyguanosine are incorporated into the DNA in two maxima, or waves, during the S phase at both stages. More DNA replicates in the early maximum at the neurula stage than at the tailbud stage. A comparison of the degree of incorporation of labelled deoxyguanosine to labelled thymidine into DNA suggests that earlier replicating DNA at both stages may be GC-rich compared to later replicating DNA. The incorporation of tritiated uridine into RNA during the S phase also differs between the neurula and tailbud stages. Pulse and continuous label experiments indicate that at the neurula stage the highest rate of RNA synthesis occurs late in the S phase whereas at the tailbud stage the higher rate of RNA synthesis has shifted to an interval earlier in the S phase.     

QUANTITATIVE TRITIUM AUTORADIOGRAPHY OF MAMMALIAN CHROMOSOMES : I. The Basic Method

A technique has been developed that allows repeated autoradiographs to be made of the isotope distribution in the chromosomes of a single cell. A series of 10 separate autoradiographs were made of a Chinese hamster diploid male metaphase cell which had been labeled with tritiated thymidine during the first 15 minutes of its DNA synthesis period in the previous interphase. Each autoradiograph had low grain densities above the chromosomes so that quantitation was feasible. The separate autoradiographs were photographically combined into a single composite in which grain images were converted to lines oriented at right angles to the chromosome axis. The line densities were then measured with a recording microdensitometer to yield graphs reflecting the isotope distribution along each chromosome. The area under each graph was directly proportional to the total number of grains counted above the corresponding chromosome in the 10 separate autoradiographs. The distribution of isotope along the chromosomes was different for each chromosome, and in some cases homologs also differed in their early labeling patterns.     

STIMULATED INCORPORATION OF AMINO ACIDS INTO PROTEINS OF SYNAPTOSOMAL FRACTIONS INDUCED BY DEPOLARIZING TREATMENTS

Abstract— Rat cortical synaptosome preparations incorporated l -amino acids into protein at a linear rate over 30–60 min. Synaptosomes showed large increases in incorporation after treatment with electrical pulses, veratrine or K⁺. This was inhibited (controls, 73%; electrically stimulated 58%) by cycloheximide and by chloramphenicol (28 and 44% respectively). Omission of Ca²⁺ from the medium had no effect, but the absence of Na⁺ greatly diminished the stimulus-induced increase in incorporation due to pulses. Electrical pulses, veratrine and K⁺ (in order of effectiveness), all showed their greatest proportional effects on amino acid incorporation into the Triton-X-100 insoluble portions of the synaptosomal membrane: the 'junctional complex', soluble in SDS, and the residue. Tetrodotoxin (1 μ m ), although ineffective when potassium was the stimulus, prevented or reduced the respiratory response, K⁺ loss and differential amino acid release, as well as the amino acid incorporation into protein observed with pulses and veratrine.
C-6 glioma cells showed no stimulus-dependent increase in incorporation. Preliminary data, of gel electrophoretic analysis, on the proteins labelled under our conditions is presented.     

细胞电穿孔导入DNA的动态特征

本文以Ｅ．ＣｏｌｉＨＢ１０１为模型细胞,研究了不同幅度和时间常数的ＲＣ放电脉冲条件下,触发细胞穿孔,导入长度分别为４２２和１６８０ｎｍ的超螺旋长丝形质粒ｐＵＣ－１８和ｐＣＰ１０,及精胺缩合后直径为８８ｎｍ的复曲面体的质粒ｐＣＰ１０,测定了相应的细胞转化率。实验结果显示了细胞电穿孔导入ＤＮＡ的动态特征。     

FUCOSE INCORPORATION INTO GLYCOPROTEINS OF MOUSE BRAIN

—Radioactive fucose was incorporated into glycoproteins of brain in vivo. After intracerebral administration of this precursor, radioactive glycoproteins were the sole detectable product. The glycoproteins formed appeared to have a slow turnover but this was due, at least in part, to re-utilization of fucose released from degraded glycoproteins. Incorporation of fucose into glycoproteins differed from that of glucosamine, since a much smaller proportion of the radioactive fucose was incorporated into soluble glycoproteins. Fucose was rapidly incorporated into glycoproteins of nerve endings, although there was relatively little incorporation into glycoproteins associated with the soluble component of the nerve-ending fraction. As found in previous studies with glucosamine, soluble glycoproteins of nerve endings turned over relatively rapidly. Pretreatment with acetoxycycloheximide markedly inhibited incorporation of fucose into glycoproteins of brain. In contrast to the results with glucosamine, comparable inhibition was observed for fucose in all subcellular fractions of brain including the particulate and soluble components derived from the nerve-ending fraction.     

THE INCORPORATION OF RADIOACTIVE PROLINE INTO CULTURED CELLS : Interpretations Based On Radioautography and Electron Microscopy

Cultured carrot explants, stimulated to grow rapidly in a medium containing coconut milk, were labeled with radioactive proline. After an initial period of absorption (8 hr for proline-³H; 24 hr for proline-¹⁴C) the tissue was allowed to grow for a further period of 6 days in a similar medium free from the radioactivity. Samples were prepared for electron microscopy and radioautography at the end of the absorption period and also after the further growth. The distribution of the products from the radioactive proline in the cells is shown by high-resolution radioautography and is rendered quantitative for the different regions of the cells. The results show that the combined label, which was present in the form of proline and the hydroxyproline derived from it, was all in the protoplasm, not in the cell walls. Any combined label that appeared to be over the cell walls is shown to be due to scatter from adjacent cytoplasmic sites. Initially the radioactivity was concentrated in nuclei, even more so in nucleoli, but it subsequently appeared throughout the ground cytoplasm and was also concentrated in the plastids. The significance of these observations for the general concept of a plant cell wall protein and for the special problem of growth induction in otherwise quiescent cells is discussed.     

ELECTRON MICROSCOPE AUTORADIOGRAPHY OF ERYTHROID CELLS USING RADIOACTIVE IRON

The "circle analysis" method of Williams (1969) and a new improved method employing hypothetical grains described in the previous paper have been used to analyze the distribution of autoradiographic grains over erythroid bone marrow cells labeled with radioactive iron, ⁵⁵Fe. The resolution obtainable with this isotope was determined by measuring the distribution of grains about a thin line source. This distribution was also used in calculation of the circle size for the Williams's analysis and the distances of hypothetical grains for the new method. The new method provides estimates for the amount of activity in the regions of condensed and extended nuclear chromatin and for the concentration of isotope at the junction between these two areas. The possible significance of activity in this junctional region is discussed.     

INHIBITION OF PROTEIN SYNTHESIS IN NONINFECTED L CELLS BY PARTIALLY PURIFIED INTERFERON PREPARATIONS

Partially purified interferon preparations, obtained from L-cell monolayers infected with Newcastle disease virus (NDV), were shown to inhibit protein synthesis in noninfected L cells. The incorporation of several amino acids-¹⁴C was equally sensitive to the pretreatment of the cells with the interferon preparation. Treatment of L-cell monolayers for 24 hr with 800 units of interferon resulted in a 50% decrease in amino acid incorporation. The degree of inhibition was found to be a function of the interferon concentration and the time of exposure of the cells to the partially purified preparations. No inhibitory effect was detected in medium obtained from noninfected cells and purified in an identical manner. The inhibitory effect was shown to be cell specific in that the partially purified interferon from L cells did not reduce amino acid incorporation in heterospecific cell lines. Heating the interferon preparations at 60°C destroyed their antiviral activity and their ability to inhibit valine-¹⁴C incorporation in L cells.     

EFFECT OF NOREPINEPHRINE ON 32P INCORPORATION INTO INDIVIDUAL PHOSPHATIDES IN SLICES FROM DIFFERENT AREAS OF THE GUINEA PIG BRAIN1

Abstract—

• 1 Incorporation of ³²P into phosphatidic acid was significantly increased in the presence of an appropriate concentration of norepinephrine, either 0.1 mm , or 1 mm , or both, in slices of guinea pig cerebellar cortex, olfactory bulbs, cerebral cortex, hypothalamus and thalamus; it was not significantly affected at either concentration of norepinephrine in slices of corpus striatum and the posterior and anterior colliculi.
• 2 Incorporation of ³²P into phosphatidylinositol was significantly increased in the presence of norepinephrine in a concentration of either 0.1 mm , or 1 mm , or both, in three of the areas studied—the olfactory bulbs, cerebral cortex, and thalamus.
• 3 There was no significant effect of 0.1 mm -norepinephrine on ³²P incorporation into phosphatidylcholine or phosphatidylethanolamine plus phosphatidylserine in any of the areas studied. Norepinephrine (1 mm ) significantly inhibited ³²P incorporation into phosphatidylcholine in cerebral cortex, hypothalamus, corpus striatum and anterior colliculus; ³²P incorporation into phosphatidylethanolamine plus phosphatidylserine was inhibited by 1 mm -norepinephrine in cerebral cortex, hypothalamus, and posterior and anterior colliculi.
• 4 In slices of cerebellar cortex, 0.1 mm and 1 mm -norpinephrine did not significantly affect either the specific activity or the level of nucleotide ～ P in the tissue.
• 5 The physiological significance of changes in phosphatide metabolism in response to neurotransmitters in the central nervous system is discussed.