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The blastoderm stage of Drosophila embryogenesis is a time of crucial transitions in RNA transcription, the cell cycle and segment determination. We have previously identified three loci encoding RNAs specific to this stage (Roark et al., Dev. Biol. 109, 476-488, 1985). We present here the complete nucleotide sequence of one of these loci, bsg25D, which encodes a 2.7 kb blastoderm-specific RNA. The primary structure of this RNA, and that of an overlapping 4.5 kb RNA, has been determined. The amino acid sequence of the predicted bsg25D protein has been compared to the NBRF protein database. Structural similarities between domains in the bsg25D, fos, and tropomyosin proteins, and their possible significance for early embryogenesis are discussed.

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Zone sedimentation through sucrose gradients was used for preparing Rhizobium bacteroids from lupin nodules and for separating them into slowly and rapidly sedimenting fractions.The purity of the bacteroids was established by electron microscopy and by enzyme assays, and they were shown to contain a CO-insensitive cytochrome c oxidase.Bacteroids sedimented more rapidly than broth-cultured Rhizoblum bacteria, and bacteroids from old nodules sedimented more rapidly than bacteroids from young nodules.There appear to be at least two forms of bacteroid in old nodules: slowly sedimenting bacteroids with moderate colony-forming ability resembling the bacteroids found in young nodules, and rapidly sedimenting bacteroids with much lower colony-forming ability.Rhizobium bacteroids have usually been prepared from homogenized nodule tissue by differential centrifugation (5,7,8,10). Although such procedures give good separations of soluble plant proteins from bacteroids, the complete removal of plant membrane fragments and particulate enzymes such as Cyt c oxidase from the bacteroid fraction is difficult, and has led in some cases to the use of techniques such as detergent treatment (7) that would be expected to have detrimental effects on bacteroid viability.In this paper we report the use of sucrose gradient sedimentation to obtain clean bacteroid fractions and to demonstrate changes in the sedimentation rate of bacteroids during lupin nodule development. MATERIALS AND METHODSBlue lupins (Lupinus angustifolius L. cv. Bitter Blue, inoculated with Rhizobium strain NZP 2257) were grown under controlled environment conditions (9). Growth of Rhizobium NZP 2257 in broth cultures without mannitol, preparation of sterile and nonsterile nodule homogenates, bacteroid counts, and bacteroid colony formation assays on media containing 0.055 M mannitol were as previously described (10).Sucrose gradient centrifugation was carried out using linear 20-ml gradients of 12 to 30% (w/v) sucrose in 50 mm tris-HCl, pH 7.4, equilibrated at 0 to 5 C before use. All centrifugations were at about 2,000g for 15 min at 0 to 5 C, using the HB4 swinging bucket rotor of a Sorvall RC-2 centrifuge. For preparing total bacteroids, up to 5 ml of filtered nodule homogenate (in 0.5 M mannitol, 50 mm tris-HCI, pH 7.4) was applied to each gradient, and after centrifugation the bacteroid zone was detected by eye and removed from the top with a Pasteur pipette. The bacteroids were then rinsed twice by centrifuging for 5 min at 6,000g and resuspending in mannitol-tris buffer. For resolving different classes of bacteroids, 1 ml of bacteroid suspension or filtered nodule homogenate was applied to each 20-ml gradient, and after centrifugation the tubes were punctured with a hot needle and 1-ml fractions collected from the bottom. The bacteroids were located by absorbance measurements at 600 nm. Comparisons of sedimentation rate were made only between gradients matched volume prepared in the same batch and centrifuged simultaneously.For sterile gradient work al...