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drives cells into mitosis in a dosage-dependent fashion (Russell and

Nurse, 1 9 8 6 ). The fact th a t cells arrest just before mitosis in the

c d c 2 5 -2 2 mutant has proved to be very useful in synchronization

experiments. A culture blocked in G2 by shifting to the restrictive

temperature, generates a synchronous population in which all the

cells will enter mitosis at the same time once the block is released

(Booher e t a/., 1 9 8 9 ). Loss of cdc25+ function can be compensated by

dominant mutations of cdc2+ {such as cdc2-3w ) or by an equal loss

in function of the wee 7 + gene (Fantes, 1 9 7 9 ).

- w e e 7+ and m ik 1+.

In contrast to the role of cdc25+ as a dosage-dependent

inducer of mitosis, wee 7+ is best described as a dosage-dependent

inhibitor of mitosis. Cells in which wee 7+function has been lost

enter mitosis early and with a reduced size compared to the wild

type (Nurse, 1 9 7 5 ). Interestingly, in the double mutant w e e 7 -

5 0 /c d c 2 5 -2 2 , cells at the restrictive tem perature entered mitosis

(unlike cd c 2 5 -2 2 m utants) and divided, producing daughter cells that

Chapter 1 : General Introduction 17

observation provided the first evidence th at weeT+ is an antagonist

of cdc25+. In contrast, when the w e e ! - 5 0 mutation was combined

with the c d c Z -3 w mutation, cells underwent “m itotic catastrophe",

entering mitosis at an extremely small size and before chromosome

division had been completed. Using this double mutant, Russell and

Nurse (1 9 8 7 a ) cloned the wee 7+ gene by complementation and

showed it to encode a protein kinase of 107 kDa. The dosage-

dependent manner by which p i Q7weei inhibits mitosis is clearly

demonstrated by its overproduction; the greater the level of protein

produced, the greater the increase in cell length (Russell and Nurse,

1987a).

In addition to wee7+, a second gene, called mik1+, has been

found to have a role in the inhibition of mitosis in fission yeast.

m ik1+ was discovered as a multicopy suppressor of the strain carrying the double mutation c d c 2 -3 w /w e e 1 -5 0 . Cells deleted for

m ik1+ do not display a w ee phenotype, showing it to be non-

essential. However, overexpression of m ik1+ does cause cell

elongation. As with the overexpression of wee 7+ this e ffect is

cumulative and indicates th at the two proteins have a parallel

function and partial redundancy. This premise is substantiated by

the fact that a /^ m i k l/ w e e l - 5 0 strain has a wee phenotype at the

permissive temperature, but undergoes mitotic catastrophe at the

restrictive tem perature (Lundgren e t a/., 19 9 1 ).

- n im 1 + .

(hence the name new I nducer of mitosis) and also because it could

suppress the c d c 2 5 -2 2 mutation (Russell and Nurse, 1 9 8 7 b ). nim1+

encodes a 50 kDa protein kinase whose role as an inhibitor of the

w eel kinase can be demonstrated by a number of experimental

observations. Firstly, in addition to its capacity to suppress cdc25-

22, nim1+ can also suppress a cdc25+ deletion. Second, if nim1+ is overexpressed, cells become shorter and if nim1+ is deleted, cells

are longer. Third, the double mutant An/m 7/w e e 7 -5 0 has an identical

phenotype to the w e e l-5 0 strain (Russell and Nurse 1 9 8 7 b ). Finally,

overproduction of niml protein reverses the arrest of cells

overproducing w e e l, but overproducing niml in a strain devoid of

w e e l, does not have the additive effect seen when cdc25 is

overproduced in the same strain (Russell and Nurse 1 9 8 7 a ).

- su c1+ .

One final gene that has been found to interact with cdc2+ is

suc1+. When present on a high copy number plasmid, sue 7+ can rescue certain cdc2+ mutations and its overexpression forces cells to enter

mitosis at an increased size (Flayles e t al., 1986a; 1 9 8 6 b ). Deletion

of the suc1+ gene is lethal (Moreno e t a/., 1 9 8 9 ), arresting cells at

the end of mitosis (i.e. chromosomes are condensed and a mitotic

spindle is evident) with elevated levels o f p34cdc2/p63cdci3 activity

(Basi and Draetta, 1 9 9 5 b ). Immunoprécipitation of the gene product,

p l3 s u c i, shows that at least in vivo it is associated with p 3 4 cdc2

(Brizuela e t a!., 1 9 8 7 ). Homologues of the suc1+ gene have been

Chapter 1: General Introduction 19

higher eukaryotes {CksHsI and 2; Richardson e t a/., 1 9 9 0 ). The exact

role these small proteins play is still unclear, but recent

crystallisation of CksHsI suggests they may be involved in

structural organization of CDK/cyclin hexamers (Bourne e t a/.,

1996).

d) Genetic model o f p 3 4 cdc2 reg ulatio n by phosphorylation

in S. pombe.

When all the data from the studies on genes that interact with

cdc2+ were correlated, a model for the control of the G 2/M transition in S. pombe was proposed (figure 1.2). The two genes

cdc2+ and c d c l3+ hold the key to mitotic entry through the

association of their respective gene products. The state of p 3 4 cdc2

as either an inactive or active kinase is carefully balanced by the

inhibitory phosphorylation of the m ik 1+ and wee1+ gene products and

by the activating dephosphorylation of the cdc25+ gene product. The

degree to which w eel can negatively regulate p34cdc2 is further

controlled by the inhibitory action of n im l.

In addition to cdc25, a second protein tyrosine phosphatase has

been implicated in the induction of mitosis in S. pombe. This 33 kDa

protein, encoded by the pyp3+ gene, is thought to act co-operatively

with cdc25 in the dephosphorylation of Tyr 1 5 of p 3 4 cdc2 (Millar e t

n i m 1