The methods used in these longer periods of dark fixation of C1402 are the same as those described in detail in a previous communication (15). A specially constructed apparatus permits excised leaves from B. calycinum to be exposed to C1402 in total darkness. Approximately one gm of young leaves taken from the apex of the plants immediately before use was placed in the apparatus, C1402 generated from 4 to 5 mg of BaC'403 (specific activity 0.0282 mc/mg) was admitted, and the reaction terminated at the desired time by homogenization in boiling 80 % ethanol. The extract was filtered, extracted with Skellysolv A (Pentane), and the ethanolic extract concentrated to a volume of 2.0 ml under reduced pressure. Two dimensional chromatography of an aliquot (0.1 to 0.2 ml) of the concentrated extract in 80 % phenol-20 % water (w/w) in the first direction and (79) butanol-(19) acetic acid-(50) water (v/v/v) in the second, was used to separate the compounds. Radio-autographs were made by exposing "no-screen" x-ray film to the chromatogram. Identification of radioactive compounds was made by elution and subsequent cochromatography with known compounds. The activity of each compound was measured directly on the paper with an end window Geiger tube. Derivatives of the a-keto acids were made by disrupting the tissue with 30 ml of 5 N H2SO4, adding 30 ml of 5 N H2SO4 saturated with 2,4-dinitrophenyl hydrazine, and forming the-hydrazone derivatives as described by Ranson (personal communication). The mixture was allowed to stand for one hour at room temperature and then filtered. The filtrate was extracted three times with 25-ml portions of ethyl acetate. The ethyl acetate was then extracted three times with 25-ml portions of 10 % Na2CO3. The Na2CO3 solution was acidified to pH 1.0 with cold 5 N H2SO4 and the hydrazones extracted into ethyl acetate. The ethyl acetate fraction was dried over anhydrous Na2SO4 and then concentrated to 2.0 ml under reduced pressure.The hydrazones were separated by paper chromatography by the method of Isherwood and Cruickshank (9), and were eluted and identified by cochromatography with authentic derivatives of the keto-acids.
The place occupied by the photosynthetic mechanism of energy conversion in the stream of biochemical evolution has been the subject of considerable speculation (van Niel, 1949;Blum, 1937). It is clear, however, that in order to establish such a place, a more or less definite notion of the nature of the entire evolutionary stream is necessary. In recent years the idea that the stream is flowing in the direction of the loss of synthetic biochemical abilities has gained a special prominence, particularly as a result of laboratory experimentation as well as the observation of naturally occurring systems (Beadle, 1945;Tatum, 1946;Knight, 1950). Such a view quite clearly places the photosynthetic mechanism very near the beginning. However, a variety of very powerful arguments have been adduced (Oparin, 1938;Haldane, 1934) against the sudden appearance of the very complex, completely autotrophic organism containing extremely involved biosynthetic sequences in an essentially inorganic world.A mechanism has been proposed (Horowitz, 1945) for the evolution of these very involved biosynthetic sequences leading to complex compounds by the gradual acquisition of synthetic abilities. Such a view of the evolutionary stream would, of course, place the photosynthetic energy converting system near the top or the end rather than at the bottom of the scale or near the beginning of the stream. It seemed to us not unreasonable to suppose that the entire evolutionary stream consists of both types of change, the one involving increasing biosynthetic abilities being the earlier leg, leading ultimately to completely autotrophic organisms, and the other involving the loss of biosynthetic abilities being a second leg and becoming the major direction comparatively later on the evolutionary time scale.The fossil record that is left to us is, of course, of very recent origin on such a scale. Presumably we have no direct geological record of the events on the rising leg of such a stream. It is, however, conceivable that some heterotrophic organisms alive today might represent stages on the rising leg, and the problem becomes the one of seeking to find criteria to determine whether or not such an organism represents a precursor to an autotroph or a product of the loss of synthetic abilities from an autotroph.
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