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/ 42
waldvVHo
Cell Division
SEEKING IMMORTALITY
Eero of mont al of he since one
svt yer saya ugh onan eed by
meaning tah wilelangr Sd tog ot
meals sy gt che comaged oy wor and
ory ae cottcu apache nek
stem cells (as described in Chapter “). In contrast, other
Sihinuavodyawowenaonglvedand drt
rognwtroncalsanduc vomits ces ey
rovers Tis nso roel are
Coors sours hr ‘los ctor of
sasrethe 5 cols fw nwo hv bon
marvel
Rents for mamol wea nou ty qettbinteatvommsgeanitapane
CHAPTER OUTLINE Hl
TA] hecrioee TAS Goecltee lg caper 1411 Wogan ad opt
14.3 EXPERIMENTAL PATHWAYS: 14.6 Overview of M Phase: Mitosis and 14.13 The Stages of Meiosis
‘The Discovery and ‘Cytokinesis 14.14 THE HUMAN PERSPECTIVE:
Characterization 14.7 Prophase Meiotic Nondlsjunction and ts
144 comlattmcetoyte 149 towne 1415 Sante fern erg
539 ‘540 regenerate entire mbs, Clusters of stem cells are activated
at the wound site andl undergo a series of division and
differentiation to give rise to new tissue, such as skin, muscle,
and blood vessels
Pethaps the most remarkable regenerative ability belongs to
smal flatworms of the Planaria genus. As demonstrated in
numerous teaching laboratories around the werld, 2 planat=
ian can be sliced and diced into numerous small piaces,
‘each of which will eventually form a complate worm,
Research has shown that a mass of stem cells, called a
blastema, migrate to the sites of injury and coordinate the
14.1 The Cell Cycle
“The process by which new cells aise from other living cells called
cell division, For a multicellular organism, such as a human or an
‘oak tree, countless divisions of a single-celled zygote produce an
‘organism of astonishing cellular complexity and organization. Cell
division docs not stop with the formation of the mature orgeniem
boat continues in certain Ussuee throughout life. Millions of eels
residing within the marzow of your bones or the lining of your intes-
tinal tact ate undergoing division at this very moment. This enor
‘mous output of cells is needed to replace cells that have aged or died
‘Although cll division occurs in all organisms ittakes place very
differently in prokaryotes and eukaryotes, We will estrit discussion
to the eukaryotic version. Two distinct types of eukaryotic cell division
Intergase
6
el grows and
‘ares cut oral
mmetsaln:
| eames dite
and prepares
Termtoss
destruction of older tissues and the formation of new tissue.
+0 reform what has been lost, The blastema is thought
+0 "know" what to build based on a complex system
of signals and interactions that is currently not wall
understood.
Like stem calls, cancer cells are also well known for their
ability to underge numerous cell divisions rapialy, but often
with dire consequences. Understanding the mechanisms of
call division and how the cell eyele is regulated is key to
potentially hameszing the power of regenerative therapeu
tics, as well as to keeping cancer in check.
willbe discussed inthis chapter. Mitosis leads to production of cells
that are genetically identical to their parent, whereas meiosis leads to
production of cells with halfthe genetic content of the parent. Mitosis
serves as the bass for producing new cells, meiosis asthe basis for
producing new sexually reproducing organiems. Together, these two
{ypes of cell division form the inks inthe chain between parents and
thei offspring and, in a broader sense, between living species and
the earliest eukaryotic life forme present on Earth,
Phases of the Cell Cycle
Im a population of dividing cells, whether inside the body or in a
culture dish, each cell passes through a series of defined stages, which
constites the cell eyele (FIGURE 14-1). The cell cycle canbe divided
FIGURE 14.1 An overview of the oukarytic call cyte This agri ofthe cll cle
ingests the stages tvough which 3 call szres rom one dvsion tothe nes. The cll cyl ic
vied into two major phases: M phase and interphase. M ghase neues the successive events
cof mites ana cytokines. terphase s vied int G, 5, and Gz phases, wih S phase beng
saquvalent to he perod of DNA syshess The ivsion of rterprase nta tives eaparate phater
based onthe timing of DNA sythexe was st propace in 1953 by Alma Howard and Stepnen
Pele of Hammersmith Hosptal, Londen, based onthe experiments on plant meristem cll, {nto to major phates based on cellular activities readily vse with
alight microscope: M phase and interphase. M phase includes (1)
the process of mitosis, daring which duplicated chromosome are
separated into two nuclei, and (2) eytokinesis, during which the
entice cll divides into two daughter cell. Interphase, the period
between cel divisions isa time when the cll grows and engage in
diverse metalic activities. Whereas M phase wally lasts only an
hour or so in mammalian cells, interphase may extend for day,
wosks or longer depending onthe cll ype and the conditions.
Although M phase isthe period when the contents of eel are
acually divided, numerous preparations for an upcoming mitosis
‘occur daring interphase, including replication of the cells DNA.
(One might guess that a cell engages in replication throughout inter
phase, However, staiee in the easly 1950s on asynchronous culares
(Ge, cultures whose cells are randomly distributed throughout the
celleycle) showed that thisisnotthe case Asdeseribed in Chapter 13,
DNA replication ean be monitored bythe incorporation of ['HIchy
aidine into nevely synthesized DNA. If H]thymidine given to a
culture of cells for a short period (eg, 30 minutes) and a sample of
the cll population eed, dried onto aside, and examined by aito-
sadiograpy, only fraction ofthe cells are found to have radioactive
vices. Among cell that were engaged in mitosis atthe time of fxa
tion (a evidenced by their compacted chromosomes) none ie found
to have a radioactively beled nucleus. These moti cells have
unlabeled chromosomes because they were not engaged in DNA
replication during the labeling period
fishing i allowed to contin for one or two hours befor the
cellsar sampled, here ae still no cells with labeled mitotic chromo
somes (FIGURE 162), We can condlude from these results that there
{sa definite period of time between the end of DNA synthesis and
the begining of M phase, This perio is termed G, (for second ga)
The duration of Gis revealed as one continues to take samples of
cells fom the culvure until labeled mitotic chromoromes are
observed. The first eels whose mitotic chromosomes are labeled
smust have been atthe lst stages of DNA synthesis atthe start ofthe
incubation with H]thymidine. The length of time between the stat
ofthe abeng period and the appearance of cell with labeled mitotic
figures corresponds othe duration of G,
Percentage of beled mitoses
Hours ater Styne acon
FIGURE 14.2 Experimental results demonstrating tha
‘curs during a defined perio of the cell cycle HeLa cls were cured
for 30 rsinues in meaiom containing PHRhymaine and hen ineuoated
{chased for various ties in unlabeled medium before being fined ane
prepared for ateradography Ea
‘wore in tosis athe time they were xed, an the perce
‘rteli cols whose chromosomes were labeled wat plotted as shown,
Source: From a study by R, Baserge and F Wiel
DNA replication occuts during a period ofthe cell cycle termed 541
S phase. § phase is alzo the period when the cll sythesizs the
additonal bistones that ibe needed as the cell doubles the number
of nudeosomes init chromosomes (ee Figre 15.2). The length of
S phase can be determined dtectly. In an asynchronous culture, the
percentage of cells engaged in a particelac activity is an approximate
rncarure ofthe percentage of time that this activity occupies in the
lives of els. Thus, ifwe know the length ofthe entre ell cyl, the
length of § phase can be calculated directly from the percentage of
the cells whose nucle are radioactively labeled during a brief pulse
with PHlthymiine. Sala, the length of M phase can be calcu
Inte from the percentage of cells inthe population that are seen tobe
“engaged in mitosis or cytokinesis. When one adds up the periods of
1G, +S + M,stis apparent that theres an additional period inthe el
«yee yet to be accounted for. This other phase, termed G (for fst
2p) isthe period following mitosis and preceding DNA syathesis
Cell Cycles in Vivo
‘One ofthe properties thet distinguishes various (ypes of cele within
‘a mullicelllar plant or animal is their capacity to grow and divide,
‘We can recognize three broad categories of cells
1. Calls, such as neurons, muscle cells, or red blood cells, that are
highly specialized and lack the ability to divide, Once these cells
have differentiated, they zemain in that sate unt they de.
2. Celle that normally do not divide but can be induced to begin
DNA synthesis and divide when given an appropriate stimulus.
Included in this group aze liver eels, which can be induced to
liferate by the surgical removal of par ofthe liver, and lym:
phocytes, which can be induced to proliferate by interaction with
sn appropriate antigen,
3, Cells that normally possess a relatively high level of mitotic
activity. Included inthis category ae stem cell of various adult
issues, such as hematopoietic stem cells that give rise to red and
‘white blood cells (Figure 17.6) and stem cellsatthe base of mumer-
‘ous epithelia that line the body cavities and the body surface
(Bigure 7.1). The relatively wnspecialized cells of apical meri
stems located near the tips of plant roots and stems also exhibit
rapid and continual cell division, ters cells have an important
property that is not shared by most cll; they are able to divide
‘seymmetricall, An asymmetric cell division is one in which
the two daughter cells have diferent sizes, components, or fates.
‘The asymmetric division of a stem cell produces one daughter
cell that remains an uncommitted stem cell like ils parent and
another daughter cell that has taken a step toward becoming &
ieceotiated cll ofthat tissue. In other words, asyramettc di
sone allow stem cell to engage in both self-renewal and the f
‘mation of differentiated true cell, Some types of nonstem cells
can also engage in arymmetric cell divisions, a ilustrated by the
formation of oveytes and polar bodies in Figure 14416.
Cell cyles can range in length from as short as 30 minutes in @
cleaving frog embryo, whose cell cycles lack both G, and G, phases,
to several months in slowiy growing tissues, such asthe mammalian
liver. Many cells in the body are said to be quiescent, which meant
that they are ina state that will not lead thet to an upcoming cell
Alivision, but they retain the capability to divide if conditions should
change, With afew notable exceptions, cells that have stopped divid-
ing are arrested in a stage preceding the initiation of DNA synthesis.
P3199 04 + LPL 542
(Quiescent cells are often described as being in the Gi state to distin-
{guich them from the typical G,-phace eels that may soon enter
S phase, A cell must receive a growth-promoting signal to proceed
from Gy into Gy phase and thus reenter the cell cyele
EV PI
41. Whatis the cal cycle? What are the stages ofthe cal
cycle? How doas the cal cycle vary among different
‘ypes of eels?
2. Describe how [thymidine anc autoradiography
can be used to determine the length of the various
periods of the eal cycle
14.2 Regulation of the Cell Cycle
‘The study ofthe cell cycle is not only important in basic cell biology,
bbatit also has enormous practical implication in combating cancer, 2
sense that results from a breakdown in cells ability to regulate its
‘vn division. In 1970, a series of ell fusion experiments carried out
bby Potu Rao and Robert fohnson ofthe University of Colorado helped
‘open the door to understanding how the cell cycle is regulated
‘Rao and Johnson wanted to know whether the cytoplasm of ells
contains regulatory factors that act cell cycle activites, They
approached the question by fusing mammalian cll that were in di
ferent stages ofthe cell cycle. In one experiment, they fused mitotic
cells with cells other stages ofthe cell cyce, The mitotic cel always
induced compaction ofthe chromatin in the nucleus of the nonmi-
totic cell (FIGURE 14.3). Ifa G,-phase and an M-phase cell were fased,
the chromatin ofthe G,-phase nucleus underwent premature chroma-
somal compaction to form a set of elongeted compacted chromo-
somes (Figure 1430). Ifa Gy-phase and M-phase cell were fused, the
G, chromosomes also underwent premature chromosome compaction,
Dut unlike those of « G, nucleus, the compacted G, chromosomes
‘were visibly doubled, reflecting the fact that replication had already
occurred (Figure 14.3). If « mitotic cell was fused with an S-phase
cell, the S-phase chromatin also became compacted (Figure 1436)
However, replicating DNA is especially sensitive to damage, 0 that
compaction in the S-phase nucleus led to the formation of “pulver-
ied” chromosomal fragments rather than intact, compacted chromo
somes, The results ofthese experiments suggested that the cytoplasm,
ofa mitotic ell contained diffusible factors that could induce mitosis
ina nonmitotc (ie, interphase) cell This finding suggested that the
transition from G, to M was under positive control thats, the tansi-
tion was induced by the presence of some stimulatory agent,
‘While the cell fasion experiments revealed the existence of fc
tors that regulated the cell eyele, they provided no information about
‘the biochemical properties ofthese factors, Insight into the nature of
the agen that promote entry ofa cell nto mitosis (or meiosis) were
first guined in a series of experiments on the oocytes and eazly
embryos offroge and invertebrates. These experiments ae described
sn the Experimental Pathways in Section 14.3. To summarize here,
was shown that entry ofa cel into M phase is initiated by a protein
called maturation-promoting factor (MPF), MPF consists of two sub
‘units: (1) a subunit with kinase activity that transfers phosphate groups
from ATP to specific serine and threonine residues of specific protein
substrates and (2) a regulatory subunit called cylin. The term cyclin
‘was coined because the concentration ofthis regulatory protein rece
and falls in a predictable pattern with each cell cyele (FIGURE 14).
‘When the cyclin concentration islow, the kinase lacks the cyclin sub-
uit and, asa result is inactive. When the cyclin concentration rises,
‘the kinase is activated, causing the cell to enter M phase. These results
=
ot,
- pe
« a? «
1% beg i
a
S
6, chromosomes
fe
FIGURE 14.3 Experimental demonstration that calls contain factors that stimulate enty into mitosis. The photographs show the results ofthe fusion
ofan Nephase HeLa cel ih at kangaroo PUK? eel hat had bean i (a Gi ahase, (8S phase, ore) G, phe
3 the time of ell fasion. As described in
‘he text, the evomatin of the G phase and Grphase PiK2 cells undergoes premature carmpactn, whereas tat of the S-phase cel Becomes pulverized,
‘The elongates entomstide ofthe Gr-onas callin ear8 doubled in comparsn wth thore oF he Gs
Source From Kal Speting ans Potu N. Rao, Humangeneti 23:297, 1978, Wh kind permission of Sprnger Science ~ Business Media toss
Vitoss Mitosis
shemation et
‘center
suggested that (1) progression of cells into mitosis depends on an
tenzyme whose sole activity is to phosphorylate other proteins, and
(2) the activity ofthis enzyme is controlled by a subunit whose com:
centration varie from one stage ofthe cll eyele to another,
thanges in MPF trase
concentrations of ecine that conto the elative actly ofthe MPF kinase
Sovice: From A.W, Muay and MW. Kischne, Seiance 246:616, 1989, copyright 1989,
BAS, Scence by Motes Xing, reproduced wth parson of Amarin Assocation forthe
‘Advancement of Science in he format reure na Bool/Textok vs Copyright Clearance
FIGURE 14.4 Fluctuation of cyclin and MPF levels during the call eel. This saving
epics the eles enanges that occur during ety fog development when matic divions|
occur reply and synetvonousl i al cl ofthe embryo. The top vacing shows the
n periods of mitre sed intershaee, the mide acing show he eels
yan the ower taeng shows the eycleal eranges inthe
REVIEW PAA
1. What isthe effect of fusing a Gy-phase cell wth one
inM; of fusing a G,- or S-phase cell with one in M?
2. How does the activity of MPF vary throughout the cell
cycle? How is this correlated with the concentration
cof cyclins? How does the eyclin concentration affect
MPF activity?
14.3 EXPERIMENTAL PATHWAYS
The Discovery and Characterization of MPF
As an amphibian oocyte nears the end of cogenesis, the lige
rucleus (called a germinal vesile) moves toward the periphery of
‘thecal. In subsequent stops, the ruclear envelope disassernles, the
compactes chromosomes bacome aligned along a metaphase plate
rear one end the animal poe} ofthe oocyte, and the call undergoes
“ha fist meiotic divsion to produce a large secondary oocyte and a
«mall polar body. The processes of germinal vesicle breakdown and
‘rst meiotic eivision are referred to as maturation and canbe incuced
in fly grown oocytes by teatmant wth the staid hormone proges
terona. The fstsignof maturation n the hormone-treatad amphibian
‘0¢yte is seen 13-18 hours folowing progesterone treatment the
‘germinal vesicle roves near the oocyte surface. Germinal vesicle
Breakdown #200 fllons, and the oocyte reaches metaphace of the
second meiotic division by about 36 hours afer hormone treatment
Progesterone induces maturation only ifits applied to the external
medium surounding the o2eyt; ifthe hormone is injected ico the
0¢yt9, the ao¢yte shows no response.’ It appears thatthe hormone:
acts at the call surface to ager secondary cnanges nthe etoplasm
FIGURE 1. Change of activity of he maturation-prometing factor in
the o02yteconhsrn af Rona pipiens dung the course of maturation
td eal development. Ornate: te at frequency of nduced
‘maturation to volume of injected eoplasn, The higher the ratio, the
more efectva she extoplaem nl nanaers of njeced eyoplem
Abscissa: age ofthe donors (nous ater adem nistation of
progesterone!
Sovece:¥ Masui and C. L. Maker, Journal Exp Zoology 177-142,
1971 Reprinted with Permistion rom Jahn Wiley & Sons
Publishers, he
ofthe oocyte that lad to gorminal vesicle breakciown and the other
changes associated with maturation
"Ta learn more about tha nature of the cytoplasmic change
that was responsible for wiegering maturation, Yoshie Mazi of
the Universiy of Toronto and Clement Marker’ of Yale University
bagan a seras of experiments in which they removed cytoplasm
from jzlated frog oocytes a various stages folowing progesterone
tveatment and injected 40-89 nano ters (of the donor eyronlasen
inso fully grown, immature oocytes that had not bash teatad wth
‘he hormone. Thay feund that cytaplasm taken from oocytes during
the frst 12 hours following progesterone twatment had litle or no
effect on recipient oocytes After this perod, however, the ytoelasr
sgaines the ability to nduce maturation in the recipient oocyte. The
tytoplasm from the donor oocyte was maximally fective about 20,
hours ater progesterone weatment, ands efectveness declined by
40 hours (FIGURE 1). However, cytoplasm taken from early embryos
continued to show same abil to induce oocyte maturation. Masui
and Markert refered {0 the eytoplasmie substancess) that induce
riperocere
‘Memes jauowuedry » EPL 544
uoysna a0 + #1 Bad
maturation inrecipiont oocytes as “maturation promoting factor”
‘Which bacame known as MPF
Because was assumed that MPF was involved spacifcallyin
triggering oocyte maturation, relatively litle interest as paid at
first te the substance ofits possile mechanism of action. Then in
1978, William Waeeerman ang Dannie Smth of Puraus University
publ shed report on the behavior of MPF during early amph dian
evelopment It had been assumed that MPF activity present in
early embryos was simply a residue of activity shat had bean pro
ont nthe oocyte. But Wascerman and Smith aiecovered that MPF
Sctvity undergoes dramatic fuctuations in cleaving eggs that cor
relate with changes inthe call eyle. twas found, for example, that
cytoplasm takan from cloaving frog eggs within 20-60 minutos
ater fertilization contains litle oF no detectable MPF activity 3s
esayed by injection into immature oocytes (FIGURE 2), However,
if cytoplasm is taken from an egg at 90 minutes aftr fertilization,
MPF activity ean again be demonstrated, MPF activiy roaches 3
peak at 120 minutes after feral zation and starts to decline again
32 150 minutas (Figure 2) At the time the eggs undergo thai fist
fytokines' at 180 minutes, no activity is detected in the eggr.
“Then, as the second cleavage cycle gets underway, MPF activity
once again reappears, reaching & peak at 225 minutes oostfersl-
izavon, and then declining again toa vay low lave, Similar results
were found in Xenopus eggs, except that the fuetuations ip MPF
Setvity eccur more rapidly than in Rana and corralate with the
more rape rat of cleavage divisions inthe early Xenopus embryo.
‘Thus, MPF actly disappears anc reappears in both amphibian
pacias on a time scale that corslates with the length of the cel
cycle. Inbeth species, she peak of MPF activity corascands tothe
{ime of nuclear membrane breakdown and the nity ofthe cals
inte mitosis, Trese findings suggested that MPF does more than
siraplycontrel the tme of aocyte maturation and, in fac, may play
key role in equating the col cycle of cividing coll,
é 60
§ ao /\,
201804000
TIME POSTFERTILIZATION (min)
VL \ / \
FIGURE 2 Cycling of MPF activity in ‘ertaed 8 pipiens ogg
Ordinate: percent recipient eoeytes undergoing germinal vescle
bretkdown in response to 80 nanclter of cytoplasm fom fetid
1998 Abscias time ater frtzation when the Rena egg cytoplasm
‘wae aesayed for MPF sesviy rome indeatetme of lavage svions
Sounce: WJ. Wasserman and LD. Srsth, Journal Cal Biology 78:25,
1978. The Journal of Cell Biology by Rockefeller Isitte; American
Society ‘or Cell Biology Copyright 1978. Reproduced wih permission of
Rockefller University Presein the format rpuelsh in textbook a
Copyright Clearance Center
It became apparent about this same time that MPF activity
's not limited to amphibian eggs and aacytes but is present
In a wide variety of organisms. It was found, for example, that
mammalian cells growing in culture also possess MPF acivily as
assayed by the abilty of mammalian call extacts to induce ger
‘minal vesicle breakciowm when injected into amphibian oocytes *
MPF activiy of mammalian calle uctaates withthe coll cycle, as
‘does in dividing amehibian sags. Extracts from cultured Hola
calls prepared from eadly Gate Gy, or S-phase coll ack MPF
Sctivty (FIGURE 2) MPF appears in early Gz, risas dramaticaly in
late G,, and reaches a peak in mitoss
Another slamant of he machinery that regulates the call ycla
was dscovered in stusies on sea urenin emaryos. Sea urchin oggs
ar favorite subjects for studios of cel division because the marie
visions fellowing ferilization occur rapialy and are separated by
ign predictable time intervals t'sea urchin age are ‘erzed in
seawater containing an inhibitor of protein synthoss, the oggs fall
10 undergo the fist mtotc evsion, aresting at a stage prior to
chromosome comeaction and breaiciown of te nuclear arvelope.
Similar, each of the susequent mitotic divisions can alzo be
blocked ifn inhibitor of protein synthesis is added to the medium
ata time val bsfore the division would normally occur. Ths Snaing
hac suggested that one or more proteins must be synthesized cur
Ing each ofthe early call eyeles ithe onsiing mitotie division is to
‘secur Gut early stusies on clasving aa urchin egg files to raveal
‘the appearance of new species of proteins during this perio.
In 1983, Tim Hunt ang hs colleagues atthe Marine Biological
Laboratory at Wood: Hola reported on saveral proteins that
are symthasized in fortiized sea urchin eggs ut not unferiized
10995” To study these proteins further, thay incubated fertilized
299% in seawater containing [Sknsthionine and withdrew sarm-
ples at 10-minute intervals baginning a: 16 minutas afer fertlz3-
tion Crude pretein extracts were prepared from the samples anc
*%.VB0 pe 228 ng pte
& #
©
FIGURE 3 Maturaion-promoting activity of HeLa cell extracts during
ifort stages of the cal cycle. Because 228 ng of mitt proton
Induced germinal vesiele breakdown (GVBD} in 100 percent ofthe
cases, she percent acti for other phases ofthe cel ele was
normalzed te that amount of poten €, ea; M, ide, an te
Source: P'S Sunkara,D. A Wright, nd PN. Rao, roe Natl ead
Seienes USA 762801, 1979. subjacted ta polyacrylamide gal alecrophorasis, and tha labeled
pprateins were located autoradiograph ealy. Several prominant
bbands were labeled in ges ror ferlized egg exacts that were
not evdert in comparable excracts made from unferiizes eggs.
‘One ofthe bards that appeared strongly labeled at early stages
ater fetlzation virtually csappeated from the gellby 85 minutes
ater ferclzaton, suggesting that the protein hadbeen selectively
degraded. This same band than reappeared in gels from eggs
sampled at later times and disappeared once again in a sample
taken at 127 minutes after ferilzation. The fluctuations in the
amount of this protein are plated in FIGURE 4 (oro:ein band A)
‘together with the cleavage index, which indicates the time course
of the fst two cell divisions, The degradation of the protein
‘Secure t acout the same time thatthe calls unergo the fist and
second division. A similar protein was found in the eggs of tho
surf clam, another invertebrate whose eggs are widely studies
Hunt and colleagues namad the protein “cyclin” and noted the
vicng parallel in behavior botwoen the fuctustions in cyclin
levels in their investigation and MPF activity in the earlier studios
Subsequent studiae showed that there ware two distinct cyclins,
Aaand 8, which aro degrasiec at diffrant times during the call
cycle. Cyclin As degraded curing 25-8 minute period beginning
Just before the mataphase-anaphase ‘aston, and eylin B
Is degraded a few minutos after this transition. These studies
provided the fis indication of the importance of controlled pro:
teolsis (page 578) nthe regulation ofa major celular act vty
“The first clear link between cyclin and MPF as demon-
vatedby Joan Ruderman and ner colleagues atthe Woods Hele
Marine Bislogical Laboratory® In these studios, an mRINA encoa.
ing oyelin & wae transeribea in vita from 2 cloned DNA fragrant
that contained the entire cyclin A ceding sequence. The idant'y
lof this mRNA was verified by translating in vita and finding
that t encoded authentic clam cyclin A. When the syrtheticeychin
mRNA vias injected into Xenopus ooeytes, the cells underwent
{germinal vesicle breakdown and chromoseme compacton over
a me course not unlike that induced by progesterone treatment
(FIGURE 5) These results suggested tha the rie in yelin A which
‘curs normally during meiosis and mitass, has a direct role in
promoting entry into M phase. The amount ef cyclin A normally
‘rons rapidly and must resynthesized prior to the next vision
forthe cels cannot reenter M phase,
Bur what isthe relationship between cyclins and MPF? One
of the difficulties in answering ths question was tho use of ciffer
tent organisms. MPF had been stucied primariy in amphibians,
Sand cyclins nea urchins and clams, Evidence indicated hat frog
f0
