Nuclear Transplantation in Amoebae. I. - Journal of Cell Science

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448 Lorch and Danielli—Nuclear Transplantation in Amoebae. I Cultures may be maintained indefinitely in this manner. Like Dawson (1928) we found that the 'depression periods' described by Taylor (1924) do not occur if a plentiful food supply is maintained and dense bacterial growth avoided. Methods of examination The paraffin chamber (Commandon and de Fonbrune, 1938) was found most useful for observing active amoebae over protracted periods of time. The amoebae were placed in very shallow hanging drops on clean coverslips. They remain active in the drops for days but do not divide unless food organisms are supplied and the fluid is renewed periodically. To study the pattern of locomotion of the two species camera lucida drawings of the outlines of active amoebae were made, each animal being drawn at 1 -minute intervals for 4-5 minutes. The significance of these outline drawings was estimated by the following method: unlabelled sheets of drawings, each representing 4-6 consecutive sketches of one amoeba of either species, were mixed in batches of about 20 and sorted by various observers. The observers were provided with type specimens considered to be typical A. proteus (P) and A. discoides (D), and asked to sort the drawings into 4 groups: typical P, like P, like D and typical D. The intermediate groups 'like P' and 'like D' were always included because the sorting method was also used to detect intermediate types in amoebae with transplanted nuclei (Lorch and Danielli, 1953). In order to compare the nuclear sizes, measurements of the longest diameter of the nuclei (which are discoid in both species) were made with an ocular scale. Some measurements of the total lengths of amoebae when spread out were also made. The cytoplasmic inclusions were studied under an oil immersion objective by transmitted light. Phase contrast was not found to be advantageous. Dividing amoebae are easily recognized in culture dishes as they are roughly spherical and covered with small pseudopods. Such 'division spheres' were also studied in the paraffin chamber. In order to get a sufficient number of dividing amoebae at a given time, culture dishes were placed in the refrigerator (4° C.) overnight, and brought back to room temperature in the morning (Dawson, Kessler, and Silberstein, 1935, 1937)- About 6-7 hours after removal from the refrigerator many amoebae entered mitosis. Fixed and stained preparations of active amoebae and of division stages were made by Doljansky(i954). RESULTS General appearance When comparing amoebae it is important to do so under identical or at least closely similar conditions. An 'old' starved Amoeba proteus resembles an 'old' starved Amoeba discoides much more than it resembles an active 'young' specimen of its own species. (The term 'old' is used throughout this paper to denote an amoeba which has not divided for several days. A 'young' amoeba is one resulting from a recent division.) In well fed cultures the amoebae divide,
Larch and Danielli—Nuclear Transplantation in Amoebae. I 449 on the average, every 36 hours. No significant difference in division-rate was noted between the two species under optimum conditions, but A. discoides appears to be less affected by adverse conditions, and is therefore easier to culture. Amoebae from rapidly increasing cultures present the following features: in the culture dish they are firmly attached to the glass or to moulds and are not dislodged by gentle shaking of the dish. They either crawl on a surface or have a rosette-like appearance. The latter is the most common shape wherever food organisms are very plentiful. The amoeba then remains sessile in one spot and feeds by extending short, blunt pseudopods, rather like the partly contracted tentacles of a sea anemone. Such amoebae are always packed with food vacuoles. The more rapidly the animals are dividing, the smaller they tend to be. The daughter cells do not move apart but remain attached side by side. In this way whole colonies of 'rosettes' are formed, usually as a ring round a mouldy wheat grain, at the distance of optimum density of food organisms. Aggregates of amoebae in culture dishes always seem to arise in this way, i.e. by multiplication of amoebae which do not move away, and not by chemotactic migration towards the food source. If such a 'rosette* amoeba was picked out and placed in Chalkley's medium in a dish or on a coverslip, it did not at first stream about. But as the food vacuoles discharged, the amoeba became more active, and after about 24 hours in a dish it had assumed a stellate appearance with pseudopods radiating in all directions. On a coverslip it crept about incessantly and put out many pseudopods. If the amoeba was now returned to a dense culture of ciliates it began feeding at once and soon returned to the rosette shape which later changed into a division sphere. If starvation was continued for days the amoeba became sluggish and opaque, owing to the increased number of crystals and 'spheres' in the cytoplasm. At this stage A. discoides sometimes assumed a clavate shape —monopodal with a clear cap at the anterior end. This shape was only very rarely seen in A. proteus cultures. Underfed and starved amoebae are generally not attached to any surface and float about in the culture dish. Comparisons between the two species regarding cytoplasmic inclusions, mode of pseudopod formation and general pattern of locomotion were made on 'active' amoebae, i.e. on animals which were neither overfed (and hence sessile) nor deprived of food for more than 24 hours. Multi-nucleate amoebae, which are seen occasionally in cultures, were not used. Specimens were also compared during the first few hours after division. The general impression gained after prolonged observation of both species was that A. discoides tends to put out a greater number of pseudopods than A. proteus. This impression led us to a closer analysis of the shapes assumed during active movement, by making camera lucida drawings of single amoebae. The majority of the sheets of outline drawings, each sheet representing 4 to 6 consecutive drawings of a single active amoeba, were readily sorted by independent observers, whether these were familiar with amoebae or not, into two categories which represented the two species. Examples of outline drawings are given in fig. 1. As described under 'Methods' the observers were supplied with two type-
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Nuclear Transplantation in Amoebae. I. - Journal of Cell Science

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