Unique Pattern of Gene Expression in the Erythroid Precursor Cells

Erythroid cells were fractionated by preformed Percoll density gradient from livers of 12.5 day old mouse fetuses. With combination of lysing of mature erythroid cells, the CFU-E (colony forming unit of erythroid) was enriched as high as 30% pure. The mRNA levels of the rt-genes previously cloned as genes expressed in the reticulocytes are estimated in the fractionated erythroid cells. These rt-genes show a drastic change in expression during erythroid differentiation; Their expression was not detectable at the CFU-E cell stage. But it reached to maximum at the polychromatic erythroblast (stage I) and then decreases with maturation. The result suggests that mRNA synthesis of these rt-genes may be induced after the stimulation of erythropoietin.     

Protein Synthesis during Photocontrolled Progression of the Cell Cycle in Single-celled Protonemata of the Fern Adiantum Capillus-veneris

Protein synthesis during photoinduced, synchronous progression of the cell cycle in single-celled protonemata of the fern Adiantum capillus-veneris was studied by tracer techniques. Nuclei of the protonemata were labelled with ³H-thymidine during spore germination so that the amount of ³H incorporated into the TCA-insoluble fraction of the cells could be used as a measure of the cell number in each sample. The rate of the incorporation of ¹⁴C-amino acids into TCA-insoluble materials was not significantly varied at different stages of the cell cycle or by treatment with blue light. Extracts of cells labelled with ³⁵S-methionine at various times after the transfer from red light condition (G₀) to darkness (G₁ to S) were analyzed by two-dimensional gel electrophoresis. At least 3 of about 200 spots showed significant changes in intensity on fluorograms. Spot A (molecular weight 20,000, isoelectric point 6.3) was detectable only in early G₁, whereas spot B (molecular weight 19,500, isoelectric point 6.3) was found only in the late G₁ and S phases. When the cells were exposed to blue light before the dark incubation, the times of disappearance of spot A and appearance of spot B were advanced depending upon the progression of the cell cycle but not upon the clock time.     

Reactivity of a Monoclonal Antibody Raised against Human Leukemic Cells to Embryonic and Adult Tissues of the Mouse and Teratocarcinomas

C-9-1, a monoclonal IgM antibody raised against human null cell acute lymphocytic leukemia cells reacted with restricted regions of embryonic and adult tissues of the mouse. The antigen positive sites in the embryos included embryonic ectoderm, visceral endoderm, trophoblastic cells invading the maternal decidua of 5∼7-day embryos, primordial germ cells of 10∼12-day embryos, epithelium of nasal chamber, the bronchus, Mullerian duct, epididymis and bladder of 12∼17-day embryos. In the adult mice, C-9-1 antigen was detected in renal tubules, a part of stomach, bladder, endometrium and epididymal sperm. Embryonal carcinoma cells, but not endodermal cells of teratocarcinoma expressed the antigen. Thus, C-9-1 antigen showed distribution similar to SSEA-1. However, C-9-1 antigen was not detected in preimplantation embryos, nor in oviduct, both of which are positive for SSEA-1.     

Factors controlling rates of nonphotochemical transformation of Pisum phytochrome in vitro

Nonphotochemical transformations of the far-red absorbing formof phytochrome, such as its decay or dark reversion to Pr, werestudied with solutions obtained from etiolated pea epicotyltissues at various steps of purification. At pH 7.8, the rateof dark Pfr reversion became significantly faster after thecrude extract was purified by gel filtration, but that of wellpurified solutions was quite low. Decay of Pfr was not seenduring any purification step at an alkaline pH, but it occurredin the acidic range of pH even in the presence of sulfhydrylcompounds. The rate of Pfr reversion was also influenced bypH; it increased with an increasing pH. Dark reversion of Pisum Pfr was confirmed to proceed in a short,rapid initial phase followed by a slow phase. (Received September 9, 1970; )     

Reactivation of the Hill reaction of Tris-washed chloroplasts

The Hill reaction of chloroplasts was inhibited by washing themwith 0.8 M Tris buffer. This inhibition was further promotedby adding ferricyanide in the washing medium. When a reducingreagent, such as the 2,6-dichlorophenol indophenol (DCPIP)-ascorbatesystem or the hydroquinone (HQJ-ascorbate system, had been addedto the Tris buffer, Hill reaction activity was unaffected. Hill reaction activity of Tris-washed chloroplasts recoveredup to 70% of the initial level by re-washing the chloroplastswith a preparation medium containing theabove reducing reagents. Photobleaching of carotenoid and chlorophyll is characteristicof Tris-washed chloroplasts. However, reactivated chloroplastsshowed no photobleaching as in the case with intact chloroplasts. (Received April 20, 1970; )     

DIRECT ISOLATION OF NATIVE THIN FILAMENTS FROM EMBRYONIC MUSCLE CELLS*

A method is presented for the release of “native” thin filaments from 13-day old embryonic chick muscle without tryptic digestion or desoxycholate (DOC) solubilization of Z bands. The isolated filaments were 50–60 Å in diameter, of variable length, and formed “arrowhead-like” complexes with heavy meromyosin (HMM). In addition, the filaments interacted with purified myosin to form actomyosin as effectively as action extracted from an acetone powder of muscle. The Mg⁺⁺-dependent ATPase activity and extent of superprecipitation of the synthetic actomyosin required a low concentration of Ca⁺⁺, strongly suggesting the presence of troponin and tropomyosin on the thin filaments isolated from muscle at this stage of embryogenesis. The native thin filaments were more sensitive to trypsin than synthetic F-actin prepared from an acetone powder based on measurements of flow birefrengence, viscosity and the ability to activate myosin ATPase.     

Non-cyclic photophosphorylation by spinach grana treated with 0.8 M Tris buffer

1. Photophosphorylation was measured with spinach grana sampleswashed by 0.8 M Tris buffer at pH 8.0, which no longer catalyzedthe ferricyanide and NADP HILL reactions with water as the electrondonor. The photophosphorylation with the reaction mixture containing2 10^–4 M 2,6-dichlorophenol indophenol (DCPI) plus above2 10^–3 M ascorbate as the electron donor system insteadof water under anaerobic conditions was, in the most part, dependenton the addition of both PPNR (a nonheme iron protein requisitefor photosynthetic pyridine nucleotide reduction ; spinach ferredoxin)and NADP as the electron acceptor system. However, when ascorbateconcentration only was lowered to 2 10^–4 M, the entirephotophosphorylation proceeded, even in the absence of the electronacceptor system. 2. When the NADP added in the reaction mixture had been reducedby glucose-6-phosphate and glucose-6-phosphate dehydrogenasebefore illumination, the photophosphorylation with 2 10^–4M DCPI plus 6.7 10^–3 M ascorbate decreased to aboutthe same rate as that obtained without NADP. 3. The time course for photophosphorylation in the presenceof NADP was consistent with the time course for the photoreductionof NADP: On the complete reduction of NADP, the photophosphorylationstopped. 4. In the presence of 6.7 10^–3 M dichloropheny 1.1,1-dimethylureaor 3 10^–4 M o-phenanthroline, non-cyclic photophosphorylationwith 2 10^–4 M DCPI plus 6.7 10^–3 M ascorbateas the electron donor system decreased to about half that ofthe control, and the remaining activities were hardly affectedeven at higher concentrations of both inhibitors. The P/2e^–ratios of non-cyclic photophosphorylation in the absence andpresence of ophenanthroline were 0.74 and 0.48, respectively. ¹Present address: Department of Biology, University of California,San Diego, La Jolla, California 92037, U. S. A.     

Cycloheximide Insensitive Amoebo-Flagellate Transformation in Starved Amoebae of Physarum polycephalum

The requirement of protein synthesis for amoebo-flagellate transformation of Physarum polycephalum was re-examined. When amoebae were grown on nutrient agar in association with live food bacteria and harvested in mid-exponential phase of growth, it took ca. 2 hours for half the cells to form flagella after suspension in phosphate buffer. The transformation was completely inhibited by 5 μg/ml cycloheximide. To the contrary, when the amoebae in mid-exponential phase were starved for 3 hr on non-nutrient agar and then suspended in phosphate buffer, the duration required for this process was shortened to ca. 8 min and it was not inhibited by up to 100 μg/ml cycloheximide. A similar result was obtained using bactobolin, another inhibitor of protein synthesis. When amoebae were starved on non-nutrient agar containing 5 μg/ml cycloheximide, however, the starvation effect described above was not observed. The results indicate that protein(s) necessary for the transformation might be synthesized during the starvation period, and that the amoebo-flagellate transformation may or may not require concomitant protein synthesis depending upon preculture conditions.     

POLYOL ACCUMULATION IN IN VITRO SYSTEMS OF BOMBYX EGGS

The mechanism of polyol accumulation in diapausing Bombyx eggs, conversion of [6-¹⁴C] glucose-6-phosphate into polyols and other neutral sugars was investigated in in vitro reaction systems. When a crude homogenate or a press juice of the eggs was incubated with [6-¹⁴C]glucose-6-P, the labelled trehalose, sorbitol and glycerol accumulated in the reaction mixture. In the press juice incubation system of developing eggs at day 1, ¹⁴C-sorbitol was detected in appreciable amounts, but it decreased rapidly with the development of the embryos. When the press juice was prepared from eggs in diapause, the formation of ¹⁴C-sorbitol was 3–5 times greater in eggs at early stages (day 2 to day 4) than in developing eggs.     

SPICULE FORMATION IN VITRO BY THE DESCENDANTS OF PRECOCIOUS MICROMERE FORMED AT THE 8-CELL STAGE OF SEA URCHIN EMBRYO*

The micromeres at the 16-cell stage of sea urchin embryo have already been endowed with a faculty to self-differentiate into spicule-forming cells (11). The present experiment was designed to test whether the factor(s) necessary for such self-differentiation had already been localized at the 8-cell stage in an area corresponding to the presumptive micromere region in Hemicentrotus pulcherrimus. Since the blastomeres at the 8-cell stage are all equal in size in normal embryo, unequal 3rd cleavage, by which small blastomeres are pinched off toward the vegetal pole (precocious micromeres), was experimentally induced either by treatment with 4NQO (4-nitroquinoline-1-oxide) at the 2-cell stage or by continuous culture in Ca-free sea water. The precocious micromeres were cultured in vitro in natural sea water containing horse serum. Descendants of the precocious micromeres formed spicules. In comparison their spicule formation with that by the descendants of the micromere of normal embryo, no differences were found regarding 1) time of initiation of spicule formation, 2) rate of growth of spicule, 3) size and shape of resultant spicule and 4) percentage of clones which formed spicule. The fact indicates that factor(s) indispensable for self-differentiation into spicule-forming cells have already been localized near the vegetal pole as early as the 8-cell stage.