Identification of flagellar proteins that initiate the activation of sperm motility in vivo.

Protein phosphorylation appears to be a necessary step in the intracellular signaling pathway that initiates the activation of sperm motility. Activation of live immotile sea urchin sperm produced rapid, time-dependent increased phosphorylation on proteins of 32, 45, 130, and 500 kDa. Fractionation of immotile and motile sperm indicated that these motility-related phosphoproteins are associated with flagella. These proteins showed greater phosphorylation in the flagellar fraction from motile sperm, suggesting that subcellular boundaries are in place to keep protein kinases and their substrates spatially separated. Solubility properties suggest that these proteins are the heavy chain and smaller subunits of sea urchin sperm dynein which are phosphorylated in vivo to initiate activation of motility. This also suggests that phosphorylation of only these few proteins, out of the nearly 100 phosphorylations known to occur in the basic axoneme, appears to be associated with the early signaling pathways of motility activation in intact sperm.

A method for preparation, storage, and activation of large populations of immotile sea urchin sperm.

Reversible protein phosphorylation is associated with initiation and modulation of sperm flagellar motility. Many studies aimed at examining the signal transduction mechanisms underlying the expression of motility have relied on detergent-permeabilized sperm reactivated with exogenous 32P-ATP. However, the reactivation conditions allow variable levels of motility to be expressed and phosphorylation of many proteins that appear to be unrelated to sperm motility. Thus, identification of the few relevant proteins is difficult. We have developed a method to collect and keep sperm immotile until reactivated for analysis to normal motility levels. Artificial sea water (ASW) buffered with 5 mM 2-[N-morpholino]ethanesulfonic acid at pH 6.0 and containing 50 mM KCl allows collection and storage of immotile sea urchin sperm for up to 96 h at 4-5 degrees C. Motility under these conditions is essentially zero, but sperm is rapidly reactivated to normal motility by diluting with ASW to standard pH (8.0) and KCl concentration (10 mM).

Colonizing populations of Candida albicans are clonal in origin but undergo microevolution through C1 fragment reorganization as demonstrated by DNA fingerprinting and C1 sequencing.

The genetic homogeneity of nine commensal and infecting populations of Candida albicans has been assessed by fingerprinting multiple isolates from each population by Southern blot hybridization first with the Ca3 probe and then with the 0.98-kb C1 fragment of the Ca3 probe. The isolates from each population were highly related, demonstrating the clonal origin of each population, but each population contained minor variants, demonstrating microevolution. Variation in each case was limited to bands of the Ca3 fingerprint pattern which hybridized with the 0.98-kb C1 fragment. The C1 fragment was therefore sequenced and demonstrated to contain an RPS repetitive element. The C1 fragment also contained part or all of a true end of the RPS element. These results, therefore, demonstrate that most colonizing C. albicans populations in nonimmuno-suppressed patients are clonal, that microevolution can be detected in every colonizing population by C1 hybridization, and that C1 contains the repeat RPS element.