Tracing of cell lineage in embryonic development of Phallusia mammillata (ascidia) by vital staining of mitochondria

Vital staining of mitochondria with a fluorescent dye 3,3′-diethyloxacarbocyanine was used to follow cell lineage in embryos of Phallusia mammillata. The results agree in general with the plan established by Conklin in 1905. Strong fluorescence migrated after fertilization similarly to the pigment of the “yellow crescent” in Styela. Later, fluorescence segregated into muscle cell primordia, but not into mesenchyme cells. An animal hemisphere cell, b 8.17 also exhibited strong fluorescence and joined a group of muscle primordia, very likely becoming a muscle cell itself. In the tadpole, all the tail muscle cells were fluorescent. Fluorescence was also noticed in nerve cell primordia of the vegetal hemisphere, particularly in the cell A 8.16 whose descendants appeared to become part of the sensory vesicle which was strongly fluorescent in the tadpole. The usefulness of this type of vital staining in following cell lineage of colorless embryos is stressed.     

Initiation of meiosis and sporulation in Saccharomyces cerevisiae does not require a decrease in cyclic AMP.

Meiosis and sporulation of Saccharomyces cerevisiae are initiated in a guanine auxotroph by guanine deprivation (E. Bautz Freese, Z. Olempska-Beer, A. Hartig, and E. Freese, Dev. Biol. 102:438-451, 1984). We used this condition to examine a hypothesis (K. Matsumoto, I. Uno, and T. Ishikawa, Cell 32:417-423, 1983) that initiation of meiosis requires a low level of cAMP. We found that, after guanine deprivation, the intracellular concentration of cAMP transiently decreased not more than 20% and not at all if the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was added to the medium. Under these conditions, at least 76% of the cells sporulated in the absence of IBMX, and almost 100% sporulated in its presence. The sporulating cells continually excreted cAMP and utilized the gluconeogenic carbon source. The cells failed to sporulate efficiently and to form four-spored asci if simultaneously deprived of guanine and carbon. After guanine deprivation in glucose medium, sporulation remained suppressed and intracellular cAMP was unchanged. We conclude that, under conditions of guanine starvation, cAMP deficiency is not required for initiation of meiosis and sporulation, cAMP is produced in excess and excreted to the medium, the cells sporulate better if the cAMP concentration is increased by addition of IBMX, the cells require a gluconeogenic carbon source for complete and efficient sporulation, and suppression of sporulation by glucose is not mediated by cAMP.     

Sequence homologies of glucose-dehydrogenases of Bacillus megaterium and Bacillus subtilis

The sequence homologies of the glucose dehydrogenase subunits of B. megaterium and B. subtilis are compared. From the known B. megaterium aminoacid sequence and the base sequence of the cloned B. subtilis structural gene we predict the B. megaterium structural glucose dehydrogenase gene. Assuming the minimal mutational changes to convert one gene into the other 23 transitions, 30 transversions, 1 inversion, 3 insertion-deletions, but no frameshifts are postulated necessary to interconvert the structural genes. The homology of both enzyme subunits of 85% reflects the close evolutionary distance between B. subtilis and B. megaterium.     

Monovalent cations enable cell wall turnover of the turnover-deficient lyt-15 mutant of Bacillus subtilis.

A lyt-15 mutant reported to be unable to turn over the cell wall exhibited the same rate of wall turnover as the standard strain if the medium contained 0.2 M NaCl, which did not affect growth. Cell wall autolysis was also optimal at 0.2 M NaCl.     

Rates of peptidoglycan turnover and cell growth of Bacillus subtilis are correlated

Peptidoglycan turnover was measured by the decrease of trichloroacetic acid-precipitable label in cells labeled with N-acetyl-D-[14C]glucosamine. The rate of turnover was reduced strongly by the inhibition of RNA or protein synthesis and weakly by the inhibition of lipid, peptidoglycan, or DNA synthesis. It increased with the growth rate (which was controlled by the concentration of oxomethylvalerate limiting the intracellular isoleucine supply) to the same degree in stringent (rel+) and isogenic relaxed (relA) strains. In these and all other strains tested, the turnover rate (k) increased with the growth rate (g) according to the equation, k = 0.70 X g1.38, even when the growth rate was systematically altered by changes in the temperature or in the composition of the medium.     

Explanation for the apparent inefficiency of reduced nicotinamide adenine dinucleotide in energizing amino acid transport in membrane vesicles

Lineweaver-Burk plots of reduced nicotinamide adenine dinucleotide (NADH) oxidation by membrane preparations from Bacillus subtilis are biphasic, with two K(m) values for NADH. The higher K(m) corresponds to the only K(m) observed for NADH oxidation by whole cells, whereas the lower K(m) corresponds to that observed with open cell envelopes. Membrane preparations apparently contain a small fraction of open or inverted vesicles which is responsible for the low K(m) reaction, whereas entry of NADH into the larger portion of closed, normally oriented vesicles is rate limiting and responsible for the high K(m) reaction. In contrast, the oxidation of l-alpha-glycerol-phosphate (glycerol-P) by membrane preparations shows only one K(m) that corresponds to that of glycerol-P oxidation by whole cells or lysates. Since glycerol-P dehydrogenase (NAD independent) has the same K(m), this enzyme reaction rather than entry of glycerol-P into vesicles represents the rate-limiting step for glycerol-phosphate oxidation. The K(m) for amino acid uptake by vesicles in the presence of NADH corresponds to the high K(m) for NADH oxidation, indicating that NADH energizes transport only if it enters closed, normally oriented vesicles. Studies with rotenone and proteolytic enzymes support this interpretation. The apparent efficiency of NADH in energizing uptake seems to be lower than that of glycerol-P because, under the experimental conditions usually employed, open or inverted vesicles that do not participate in amino acid uptake are responsible for the major portion of NADH oxidation. When the results are corrected for this effect, the efficiency of NADH is essentially the same as that of l-alpha-glycerol-P.