phase, where it associates with Cdk2 (Pines and Hunter, 1990;

Tsai e t a/., 1 9 9 1 ). Much evidence suggests that cyclin A/Cdk2

complexes are essential for DNA replication. For example, three

groups have described experiments where cells microinjected with

anti-cyclin A antibodies are unable to replicate their DNA (Girard a t

a/., 1991; Pagano a t a/., 1 9 9 2 ; Zindy a t a/., 1 9 9 2 ), and ectopic

expression of cyclin A in mammalian fibroblasts can promote S-

phase entry (Rosenberg a t a/., 1 9 9 5 ). However, it is generally

accepted th at cyclin A /C dk2 does not initiate S-phase, but rather

acts to maintain its progression once started by the cyclin E/Cdk2

complex (see next section, 4 .1 .3 ). The cyclin A/Cdk2 complex has

additionally been linked to adhesion-dependent cell proliferation

(Guadagno a t a/., 1 9 9 3 ). A stable cell line of NRK (Normal Rat Kidney)

cells expressing ectopic cyclin A are able to grow in suspension,

whilst the parental cell line depend upon anchorage for growth and

arrest in late Gl expressing cyclin E but not cyclin A. This

Chapter 4: Cyclin A in Fission Yeast 119

ectopically expressed, cells are unable to grow in suspension. One

interpretation of this experiment is that a requirement for cyclin A

transcription is a signal in late Gl or early S-phase from surface

adhesion molecules (Guadagno e t a/., 19 9 3 ).

A fte r cells have entered G2, cyclin A no longer binds Cdk2 and

instead can be seen complexed with Cdc2 (Pagano e t al., 19 9 2 ). The

exact functions of cyclin A /C dc2 remain unclear, although work on

Xenopus egg extracts reveals one potential role at the end o f G2 may lie in the reorganisation of the cytoskeleton prior to mitosis (Verde

e t a/., 1 9 9 2 ). Interestingly, the kinase activities of cyclin A/Cdc2 and cyclin B/Cdc2, which initially seem to significantly overlap (see

figure 4.1 a), are actually separated both temporally and spatially.

Regarding the timing of kinase activation, whereas the cyclin

A /C dc2 complex is active over a large part of early and mid G2, the

rapidly forming cyclin B/Cdc2 complex is kept inactive by inhibitory

phosphorylations until the onset of mitosis (see section 1.3.2b ). A t

this point differences in the destruction boxes of the two cyclins

means th at whilst cyclin A is rapidly degraded, the presence of

cyclin B until anaphase, maintains the activity of cyclin B

associated-Cdc2 kinase (Pines and Hunter, 1 9 9 0 ). On the other hand,

the spatial separation o f cyclin A and cyclin B associated-Cdc2

kinase activity results from their different subcellular

localisations: cyclin A is nuclear (Pines and Hunter, 1 9 9 1 , 1 9 9 4 )

whilst cyclins B1 and B2 are cytoplasmic, only undergoing

translocation to the nucleus following the onset of M-phase (Pines

4 .1 .3 : Cvclin A in te ra c ts w ith tra n sc rip tio n fa c to rs .

During its S-phase role cyclin A, like cyclin E, binds to the Rb-

related protein p i 0 7 and with the transcription factor E2F when

associated with Cdk2 (Mudryj e t al., 1991; Devoto e t a/., 1992;

Pagano e t a!., 1992; Lees e t a!., 1992; Shirodkar e t a/., 1 9 9 2 ). At

present, it is not entirely clear what the effect of cyclin binding is

on these proteins. However, with much data suggesting the E2F

family o f factors stimulate the transcription of genes involved in

DNA replication and the observation that like Rb, pi 0 7 inhibits E2F

transcriptional activation (Zhu e t a/., 1 9 9 3 ), one interpretation of

cyclin control of S-phase is described as follows: formation of a

c y c lin /p 1 0 7 /E 2 F complex inactivates p i 0 7 and allows associated

E2F to be freed for transcription of genes essential to replication of

DNA, such as ribonucleotide reductase and DNA polymerase a. During

this process the cyclin E /p 1 0 7 /E 2 F complex accumulates in late Gl

and peaks in early S-phase (Lees e t a/., 1 9 9 2 ). It then disappears

with the destruction of cyclin E in mid to late S-phase, and is

replaced by another complex consisting of cyclin A, p i 0 7 and E2F.

4 .1 .4 : The pREP vectors o f 5. pombe.

The pREP family of shuttle vectors allow the regulatable

expression of proteins in fission yeast. They contain the n m tl

thiamine-repressible prom oter (Maundrell, 1 9 9 0 ), which as its name

Chapter 4: Cyclin A in Fission Yeast 121

thiamine is present in the growth media (Maundrell, 1 9 9 0 , 1993;

Basi e t a/., 1 9 9 3 ). The family consists of pREPl, 41 and 81 which

correspond to high, medium and low levels of protein expression

respectively. In addition there is a pREP42 vector (with an

equivalent level of expression to pREP41), which differs to the other

members in th at it carries the URA4 marker instead of the LEU2 gene

as a means of selecting transformed cells (Basi e t a/., 1 9 9 3 ).

4 .1 .5 : Aims.

The aim of the following work is to characterize the effects of expressing human cyclin A in 5. pombe, which has no known

homologue of this protein. It should be interesting to see if any of

the usual cell cycle phenotypes, such as small wee cells or long cdc

cells are generated and, if so, whether they are a result of foreign

cyclin A expression alone, or due to cyclin A disrupting the normal

association of p 3 4 cdc2 with its native cyclin partners. Since in S.

p o m b e p63cdci 3 is linked with microtubule stability (Booher and

Beach, 1 9 8 8 ), and a G2 role for cyclin A/Cdc2 in Xenopus may involve

cytoskeletal reorganisation (Verde e t a/., 1 9 9 2 ), any disruption

observed in the normal pattern o f microtubules will be of particular