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Linear density gradients in isopycnic centrifugation technique for selecting synchronous daughter cells from asynchronous Chlorella cultures
Ribulose 1,5-bisphosphate (RuBP) carboxylase and phosphoribulokinase, enzymes in the reductive pentose-phosphate cycle, were measured in cell-free extracts of Prochloran didemni. The partial purification and characterization of RuBP carboxylase were described. Prochloron RuBP carboxylase, when purified by isopycnic centrifugation in reoriented linear 0.2 to 0.8 M sucrose gradients, sedimented to a position which corresponded to that of the 520,000-dalton spinach enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the Prochloron enzyme was composed of large and small subunits (MW = 57,500 and 18,800). Though results established that the enzymes RuBP carboxylase and phosphoribulokinase were present in levels comparable to other CO2-fixing microorganisms, it was suggested that other enzymes in the Calvin cycle limit growth or that additional enzymic insufficiencies exist.
To date, 14 C tracer studies using accelerator mass spectrometry (AMS) have not yet resolved lipid-soluble analytes into individual lipoprotein density subclasses. The objective of this work was to develop a reliable method for lipoprotein separation and quantitative recovery for biokinetic modeling purposes. The novel method developed provides the means for use of small volumes (10–200 µL) of frozen plasma as a starting material for continuous isopycnic lipoprotein separation within a carbon- and pH-stable analyte matrix, which, following post-separation fraction clean up, created samples suitable for highly accurate 14 C/ 12 C isotope ratio determinations by AMS. Manual aspiration achieved 99.2 ± 0.41% recovery of [5- 14 CH 3 ]-(2R, 4'R, 8'R)-α-tocopherol contained within 25 µL plasma recovered in triacylglycerol rich lipoproteins (TRL = Chylomicrons + VLDL), LDL, HDL, and infranatant (INF) from each of 10 different sampling times for one male and one female subject, n = 20 total samples. Small sample volumes of previously frozen plasma and high analyte recoveries make this an attractive method for AMS studies using newer, smaller footprint AMS equipment to develop genuine tracer analyses of lipophilic nutrients or compounds in all human age ranges.
Strain PS of Methanococcus voltae (a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (voltae transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the M. voltae cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 x 10(-5) (BES resistance) to a maximum of 10(-3) (histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the AluI restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of M. voltae.