My laboratory has been been studying the physiological and genetic control of events in the cell cycle and sexual pathway of the ciliate, Paramecium. The cell cycle is the central developmental sequence for unicellular eukaryotes. From the vegetative cell cycle, cells are able to enter alternative pathways leading, for example, to mating or stationary phase. Our approach has been to use gene mutations as physiological probes for analysis of cell cycle control processes. It has been possible to elucidate the major cell cycle control points and the requirements for progression through each. These control functions govern commitment to the vegetative replication pathway (as opposed to alternative processes such as meiosis), control of the timing of initiation of DNA synthesis and commitment of cells to division at the end of the cell cycle.
The unique feature of the cell cycle in Paramecium is that the first major control point (functionally similar to "start" in yeasts) is located about 90 minutes prior to division in the preceding cell cycle, rather than at the end of the G1 interval as in most eukaryotes. Recently we have begun to examine the sexual pathway which can be entered in two ways: via cell interaction leading to mating (conjugation) or via an endogenous processing leading to autogamy (self-fertilization). Autogamy is entered from the same point in the cell cycle at which cells enter the vegetative replication pathway and can be studied in synchronous cell samples. Analysis of conditions required to induce autogamy has made it possible to isolate and analyse gene mutations affecting macronuclear differentiation which occurs in the latter part of the sexual pathway. For example, analysis of the phenocritical period of a mutant blocking macronuclear development shows that the gene product is required during a very brief interval coinciding with the initial determination and differentiation of the macronucleus.
The cdc2 kinases family of protein kinases(cdk) are the major cell cycle regulators in eukaryotes. Paramecim has two major cdk proteins, a major class with molecular weight of 36 D and a minor clas with mass of 34kD. The 36kD class of kinases does not bind to years p13 suc1 proetin as do most cdc2 molecules. In contrast the 34 kD molecules do. These two classes of molecules also differ in their peak activity periods within the cell cycle. The p36 kinase is active during the period of macronuclear DNA synthesis and has an activity peak with its highest level at the time of initiation of macronuclear DNA synthesis.The 34 kD enzyme has a sharp activity peak that coincides with the point of committment to cell division.
At present full sequences have been established for two different but very closely related 36kD molecules and partial sequences for another class of cdk (p34?) hasve been obtained. We are examining the role of cyclin-like regulatory peptides in regulation of the activity of these enzymes. Evidence to date suggests that both types of Paramecium cdk have at least some enzymatic activity as momomers.
The cc1 cell cycle mutation has bben one of the chief tools that we have used over the years in analysis of hte cell cycle. recently we have obtained a set of second-sit suppressor of this gene. A further locus (cc3) has been identified that is involved in cell cycle regulation. There are two different alleles. one of these is suppressed by some of the cc1 suppressors, and the other is not.
Link to present grant proposal