Physarum myxamoebae can be reversibly induced to become flagellates. Physarum flagellates contain a new form of tubulin, ca3, that is not found in nonflagellated cells. Evidence is presented that suggests that a3 tubulin arises through posttranslational modification of a preexisting a tubulin. Pulse-chase experiments showed that labeled a3 tubulin could be detected when flagellates formed after a chase. RNA was isolated from myxamoebae at different times after induction of flagellum formation. When this RNA was translated in vitro, the resulting products contained no 03 tubulin, also consistent with a3 being made by posttranslational modification. Levels of a and ,I tubulin RNA increased with the proportion of flagellates in the culture. These elevated tubulin RNA levels declined after the number of flagellates in the population achieved plateau values.Physarum polycephalum expresses multiple forms of tubulin through its life cycle. Two-dimensional (2D) gel electrophoresis of myxamoebal proteins resolves otl and 31 tubulin spots. When myxamoebae are induced to become flagellates, a new spot appears in the tubulin region of 2D gels of cellular proteins. Peptide mapping confirms that this spot is composed of an a tubulin. The appearance of this new tubulin, a3, is correlated with the presence of flagella (2).In vitro translations of total RNA from axenically grown myxamoebae, which do not flagellate, reveal no a3 tubulin in the protein products, as is the case for in vitro translations of RNA from Physarum plasmodia (2). This information suggests that there is not a constitutively expressed RNA for a3. These data, coupled with the relatively rapid appearance of a3 tubulin after induction of flagellation, have led Burland et al. (2) to suggest that a3 tubulin is made via a posttranslational modification of al tubulin. The flagellum-specific a tubulin of Chlamydomonas reinhardtii is made by posttranslational modification of an a tubulin (9), and it has also been reported that the flagellar a tubulin of the trypanosome Crithidia fasciculata is posttranslationally modified (17).Changes in levels of tubulin RNAs occur after deflagellation of Chlamydomonas reinhardtii (13, 18), deciliation of Tetrahymena pyriformis (6,10) potassium orthophosphate, 0.025% sodium chloride, 0.021% magnesium sulfate, 0.005% calcium chloride in distilled water, pH 5.0 with potassium hydroxide). A suspension of myxamoebae in basal salts solution was diluted to a starting density of ca. 5 x 105 myxamoebae per ml, and one-tenth of this volume of the concentrated E. coli was added. This suspension was transferred to glass or plastic petri plates to give about 1 ml of suspension per 6-cm2 surface area. All incubations were stationary at 26°C.In vivo labeling of polypeptides. Myxamoebal proteins were labeled by adding 50 ,uCi of [35S]methionine (SJ204;Amersham Corp., Arlington Heights, Ill.) per ml of liquid culture at the time of preparation of the culture. Myxamoebae grown by this method settle on the bottom of the plate so they must be resuspe...
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