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Chromosome Structure in Ciliated Protozoans

D. M. PRESCOTT AND K. G. MURTI
Department of Molecular, Cellular and Developmental Biology, University of Colorado,
Boulder, Colorado 80302

The ciliated protozoa contain two kinds of by an appropriate number of nuclear divisions. The
nuclei: a micronucleus and a macronucleus. The macronucleus develops singly and then splits in
micronucleus possesses clearly visible chromosomes, two when the cell resumes reproduction. The two
is diploid, divides by mitosis, and synthesizes only a macronnclei present during interphase of the cell
trace of RNA. The macronucleus contains no cycle fuse just prior to amitotic division, and each
discernible chromosomes, has many times the daughter macronucleus splits into two just after
diploid amount of DNA, divides amitotically, and cell division is completed.
provides virtually all of the RNA needed to run the After conjugation in Stylonychia, a micronucleus
vegetative life of the cell. is converted into a macronucleus by the course of
During conjugation in ciliates, the micronucleus changes in DNA content shown in Figure 2
undergoes meiosis, the two cells exchange haploid (Ammermann, 1965). During the first period of
micronuclei, and then separate. An exchanged DNA synthesis, lasting about 40hr, polytene
micronucleus fuses with a resident, haploid micro- chromosomes are formed (Fig. 3). None of the bands
nucleus to produce a new diploid micronucleus in has been observed to undergo puffing, and attempts
both exconjugant cells. The new micronucleus then to demonstrate RNA synthesis in the polytene
divides by mitosis without division of the cell. Ac- chromosomes have been unsuccessful.
companying these events, the old macronucleus When the polytene chromosomes reach full
starts to disintegrate and eventually disappears
completely. A new macronucleus grows from one of
the new micronuclei, the principal event being the
production of the large amount of DNA charac-
teristic of the macronucleus. When formation of the
new macronucleus is complete, the ciliate resumes
its vegetative life of cell growth and reproduction.
In some types of ciliates, e.g., holotrichous
organisms such as Tetrahymena and Paramecium,
the macronucleus is formed by means of an uninter-
rupted series of DNA replications. Genetically, the
completed macronucleus appears to be a highly
multiplied version of the micronucleus (Allen and
Gibson, 1972), although the macronucleus divides
amitotically and no chromosomes are distinguish-
able.
In the group of ciliates known as hypotrichs, the
development of the macronucleus from a micro-
nucleus follows a completely different course. We
describe here the development of the macronucleus
in the hypotrich Stylonychia and present an
explanation of chromosome structure based on this
work. Most of the data have already been reported
elsewhere (Ammermann, 1964, 1965, 1968, 1969,
1971; Bostock and Prescott, 1972; Kloetzel, 1970;
Murti, 1973; Murti et al., 1972; Prescott et al., 1971;
Prescott et al., 1973).
Stylonychia in the vegetative state has four Figure 1. A Stylonyehia cell stained by the Feulgen
micronuclei and two macronuclei (Fig. 1). This technique and counterstained with fast green. The macro-
nuclei (ma) and micronuclei (mi) are visible. Other dense
nuclear complement is achieved after conjugation spots in the cell are food vacuoles that stain with fast green.

609
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610 PRESCOTT AND MURTI

160 of radioautographic grains decreased uniformly

over all of the vesicles during the period of DNA
J40
degradation (Fig. 5a,b,c). Thus, we conclude that
120 the reduction in DNA content is achieved by degra-
I00
dation of most of the DNA in all of the vesicles and
therefore in all of the bands of the polytene
8(3 chromosomes. Whether the DNA that is retained is
60
[v derived from interband or band regions is not
known.
40 From buoyant-density profiles and melting
20
curves of DNA we have concluded that the degrada-
tion of DNA constitutes reduction in the complexity
I I i i i i i i
0
0 I0 20 30 40 SO 60 I00 of nucleotide sequences within the developing
HOURS macronucleus. The buoyant-density profile of
micronuclear DNA centrifuged to equilibrium in
Figure 2. Changes in the DNA content that occur during
the development of a macronucleus from micronucleus. CsC1 is complex (Fig. 6). Four density components
Initial DNA synthesis results in the formation of polytene are discernible. Computer resolution of the profile
chromosomes. Next, the polybene chromosomes are cut up
band-by-band, each band becomes enclosed in a vesicle,
into separate density components suggests the
and then most of the DNA in each vesicle is degraded. presence of a fifth, small density component on the
Finally, the vesicles breakdown, and the remaining DNA light side of the profile. Macronuclcar DNA centri-
from each vesicle is replicated many times to produce a
mature macronucleus. Modified, with permission, from fuged in CsC1 equilibrates as a single density com-
Ammermann, 1965. ponent with a peak value of 1.701 gm/cm -3 (Fig.
6). In micronuclear DNA, the component with
this density, presumably representing the nucleo-
development, they become transected by membra- tide sequences to be retained in the formation of the
neous partitions that form in every interband
(Fig. 4a). The membraneous partitions are not
unit membranes. They appear to form from mate-
rial, probably protein, that first accumulates just
inside the nuclear envelope. The membraneous
partitions quickly develop into vesicles, such that
each of the thosands of bands becomes enclosed in
a separate vesicle (Fig. 4b, c). I f the macronucleus
is isolated and broken open at this stage, the vesicles
disperse freely, demonstrating that each vesicle is
a physically separate unit (Ammcrmann, 1971).
Thus, the developing macronucleus is converted
from a bag of polytene chromosomes to a tightly
packed bag of vesicles, each vesicle containing the
material from a single band plus portions of the
two adjacent interbands of a polytene chromosome.
Ammermann (pers. commun.) has estimated the
total number of bands (vesicles) to be about 10,000.
As the vesicles are formed, the developing macro-
nucleus enters a stage of DNA destruction.
Ammermann (1971) estimated by microspectro-
photometry that about 93 ~ of the DNA is degraded
during the vesicle stage (Fig. 2), and we subse-
quently obtained approximately the same result.
The reduction in DNA could be accounted for by
complete degradation of all of the DNA in most of
the vesicles or by degradation of most of the DNA
in all of the vesicles. The question was answered in Figure 3. Polytene chromosomes from the developing
the following experiment. Cells were labeled with macronueleus ofa hypotriehous ciliate, Euplotes. Formalin-
[aH]thymidine and were then induced to conjugate. acetic-acid squash preparation. Photographed at 1000 kv
with an AEI-EM-7 electron microscope, l l , 0 0 0 •
Exconjugants in the vesicle stage were studied by (Courtesy of Dr. Hans Ris and Dr. J o h n Ruffolo, Depart-
electron microscopy radioautography. The number ment of Zoology, University of Wisconsin.)
 Downloaded from symposium.cshlp.org on June 26, 2016 - Published by Cold Spring Harbor Laboratory Press
CHROMOSOME STRUCTURE IN CILIATES
maeronueleus, constitutes approximately 1 0 ~ of
the total micronuclear DNA.
In agreement with the buoyant-density results,
the melting curve for micronuclear DNA shows the
presence of multiple components, whereas macro-
nuclear DNA melts as a single component (Fig. 7).
The difference between micronuclear and macro-
nuclear DNAs is also reflected by the renaturation
kinetics of heat-denatured DNA. Macronuclear
DNA renatures as if it were a single component (no
differential repetitiousness of nucleotide sequences)
with a 89Cot about 15 times greater than E. coll.
The single renaturation curve obtained so far for
micronuclear DNA suggests the presence of some
differentially repeated sequences. The 89 value
estimated for micronuclear DNA is not very
accurate because the renaturation curve is rather
complex, but it is clear that micronuclear DNA as
a whole renatures several times more slowly than
macronuclear DNA.
About 30 hours after the destruction of DNA has
been completed, the conserved DNA in the de-
veloping macronucleus begins a series of multiple
replications (Fig. 2). Three rounds of DNA synthesis
occur by means of the replication bands that are
typical for the cell cycle of Stylonychia, Euplotes,
Oxytricha, and other hypotrichous ciliates. Finally,
the macronucleus attains the mature DNA content
(the two macronuclei contain several hundred times
more DNA than a diploid micronucleus), and
vegetative cell reproduction is resumed.
From the events occurring during macronuclear
development, it may be predicted that the DNA in
the macronucleus should be of low molecular
weight. DNA prepared from isolated m~cronuclei
sediments in a neutral sucrose gradient with an S
value of about 10 (Fig. 8). Included in Figure 8
are the optical-density profiles for 26 S and 17 S
ribosomal RNA and 4-5 S RNA. The DNA had been
labeled with [aH]thymidine to identify the DNA
peak. The labeled macronuclei were isolated after
DNA synthesis had stopped, and the cells had
become arrested in the G 1 stage; so the sedimenta-
tion profile is not complicated by replicating DNA.
Micronuclear DNA (not included in Fig. 8)
sediments with S values ranging from about 30 to
greater than 100, depending on the care taken to
avoid shearing during preparation. Thus, macro-
nuclear DNA is quite low in molecular weight and
micronuclear DNA has high molecular weight (cf.
Fig. 9a and 9b). A variety of experiments have been
done (Prescott et al., 1971) to prove that the low
molecular weight nature of macronuclear DNA is
not due to degradation during DNA preparation.
The curve defined by [aH]thymidine counts shows
that macronuclear DNA consists of two compo-
nents: most of the DNA sediments at 10 S, but a
611
small amount sediments at about 14 S, indicating a
slightly higher molecular weight. The presence of
two size classes of macronuclear DNA has been con-
firmed with measurements of DNA molecules in the
electron microscope. The histogram in Figure 10
shows that the distribution of lengths for several
hundred molecules is bimodal. Most of the mole-
cules form a size class with a range of 0.2 to 1.6 #m,
with an average length of 0.75 #m. The minor size
class consists of DNA pieces 2.0 to 2.2 #m in length.
These two size classes, with average molecular
weights of about 1.6 • l0 s and 4.2 • l0 s daltons,
presumably correspond to the 10-S and 14-S
fractions seen in sucrose gradients.
We have begun to characterize macronuclear
DNA in several ways. When macronuclear DNA is
heated in the presence of formaldehyde, to the point
at which hyperchromicity just begins to appear,
every piece of DNA melts at one end (Fig. 11). The
same result has been achieved by partial denatura-
tion with high pH. In no case is melting ever ob-
served at both ends of a molecule. This shows that
every piece of macronuclear DNA has a short region
of relatively higher AT content at one end. In addi-
tion, a single molecule of RNA polymerase (ob-
tained from Bacillus subtilis) binds at one end of
each piece of macronuclear DNA (Fig. 12) (Murti et
al., 1972). The binding was done in the presence of
purine deoxynucleoside triphosphates and the
absence of pyrimidine deoxynucleoside triphos-
phates to prevent transcription. Whether the end
that binds RNA polymerase is the AT-rich end has
not been determined. We do not know whether the
RNA polymerase of B. subtilis identifies the natural
binding site of RNA polymerase to macronuclear
DNA in Stylonychia. RNA polymerase from B. sub-
tilis and E. coli transcribe native macronuclear
DNA more rapidly than they transcribe DNA of
bacteriophage T4.
Stylonychia synthesizes a ribosomal precursor
molecule of 34 S that is subsequently processed into
a 26-S and a 17-S molecule of rRNA. The 34-S
precursor has a molecular weight of about 2 • l0 s
daltons. The synthesis of such a molecule requires a
template of duplex DNA of at least 4 • l0 s daltons.
Sucrose gradient analysis (Fig. 9) and electron
microscope measurements (Fig. 10) have shown a
minor class of macronuclear DNA molecules with an
average molecular weight of 4 • 10~ daltons or
slightly larger. Hybridization experiments have
confirmed that the genes for rRNA are present in
the minor class of DNA molecules. Ribosomal RNA
from Stylonychia was hybridized with 10 S DNA
and 14 S DNA from the macronucleus and with
DNA from E. coll. The results (Fig. 13) show slight
binding of ribosomal RNA to 10 S DNA and E. coli
DNA, but pronounced binding to 14 S DNA. The
 Downloaded from symposium.cshlp.org on June 26, 2016 - Published by Cold Spring Harbor Laboratory Press

Figure 4. Electron mierographs of portions of developing macronuclei that are in the stage of DNA degradation. Mem-
braneous material is present between the polytene chromosomes and in the interband regions of the individual polytene
chromosomes (a). Subsequently, each band and portions of the two adjacent bands of the polytene chromosome become
enclosed by membrane, thus forming a large number of vesicles (b) in the macronucleus. At the end of DNA-degradation
stage, the vesicles contain very little chromatin (c). 10,200 •
 Downloaded from symposium.cshlp.org on June 26, 2016 - Published by Cold Spring Harbor Laboratory Press

F i g u r e 5. Electron microscope radioautographs of portions of [aH]thymidine-labeled, developing maeronuclei. Early

polytene chromosomes (banding p a t t e r n is not evident) are heavily labeled (a), Recenty formed vesicles show less label (b),
and the later vesicles (c) show far less label. (a) 15,000 • ; (b) 10,500 • ; (c) 10,000•
 Downloaded from symposium.cshlp.org on June 26, 2016 - Published by Cold Spring Harbor Laboratory Press

614 PRESCOTT AND MURTI

G~O0 0 small amount of binding to 10 S DNA probably

~ONI ~ I~
represents contamination of 10 S DNA with 14 S
DNA, since the two size classes are difficult to
separate cleanly in a sucrose gradient. About 7 ~o of
the 14 S DNA consists of genes coding for rRNA.
We believe that the pieces of DNA isolated from
the macronucleus represent individual genes. The
macro
re main fraction of DNA has an average length of
0.75 #m, which is sufficient to code for 750 amino
acids. This represents an unusually large polypep-
tide, but it is likely that a significant portion of each
Figure 6. Buoyant.density profiles in CsCI for micro- piece of DNA serves for functions other than amino
nuclear DNA and macronuclear DNA. Micronuclear DNA acid coding, e.g., control regions for transcription
consists of at least four density components and macro- and replication. We cannot, however, exclude the
nuclear DNA contains only one.
14
possibility that the larger pieces of DNA code for
two to several polypeptides.
Finally, the pieces of macronuclear DNA code for
all of the functions necessary for ceil maintenance,
cell growth, and cell division. This is known from
two observations. First, the macronucleus produces
more than 99 ~o of the R N A in the cell, and the
micronucleus synthesizes no more than a trace.
Second, Stylonychia occasionally loses its micro-
"6 ~ 12
nucleus, for reasons that we do not know. Such

;i; amicronucleated organisms grow and divide ex-

tensively, although they tend to die out eventually,

/
perhaps because they are incapable of successful
conjugation.
These observations on Stylonychia have led us to
/ propose the model of the chromosome and the ex-
planation of chromosome processing during macro-
nuclear development shown in Figure 14. Successive
genes within the chromosome are separated from
9 ~. I I [
65 70 75 SO 85 90 95 one another by long stretches of "nongene" or
Temperoture, (~ "spacer" DNA. The "spacers" are presumed to ac-
Figure 7. Melting curves for mieronuelear DNA (O) and count for more than 90 ~ of the DNA duplex within
maeronuelear DNA (t). Macronuclear DNA shows a sharp a chromosome. This is similar to the spacer-gene ar-
transition in hyperchromieity, indicating the presence of a
single component. Micronuclear DNA melts with a complex rangement of cistrons generally established for
pattern, indicating the presence of several components. rRNA. Since the transection of the polytene
1750
chromosome occurs in interband regions, we assume
3Hfhyrflidine- ~ *~ that each DNA piece in each vesicle consists initially
1500 26S .
of a small gene portion and a large spacer portion.
f25C The subsequent loss of most of the DNA in each
vesicle is presumed to reflect the degradation of the
f7S 10
NolO0( "spacer" portion of each molecule. As yet we know
nothing about the deoxyribonucleases that are re-
t
u 750 45 sponsible for transection of the polytene chromo-
somes and the subsequent degradation of DNA
within the vesicles.
We have assumed in the chromosome model for
25C
Stylonychia in Figure 14 that each band of a poly-
tene chromosome contains a single kind of gene.
O-
The assumption is based on what is known about
Froctic41 numb~
polytene chromosomes in Diptera, and particularly
Figure 8. Sucrose gradient sedimentation of nucleic acids
from isolated macronuclci. The absorbance profile shows on the work on Drosophila (summarized by Judd et
ribosomal RNA (26 and 17 S), 4 S RNA, and DNA (10 S). al., 1972). The role of "spacer" DNA is unknown,
The cells were labeled with [att]thymidine for several but conceivably it could have some function in
generations and all radioactivity is contained in the 10-S
peak plus a small 14-S shoulder. crossing-over during meiosis.
 Downloaded from symposium.cshlp.org on June 26, 2016 - Published by Cold Spring Harbor Laboratory Press

Figure 9. (a) Electron mierograph of macronuclear DNA (22,400 • (b) Electron micrograph of mieronuclear DNA
(9000 • ).

Figure I0. Length distribution of maeronuelear DNA

observed in the electron microscope. Pieces over 2/zm are Figure 11. Electron micrograph of macronuclear DNA
believed to represent the small 14-S shoulder seen in subjected to partial thermal denaturation in the presence of
sucrose gradients. formaldehyde. Each piece shows a melted region at one end.
 Downloaded from symposium.cshlp.org on June 26, 2016 - Published by Cold Spring Harbor Laboratory Press

Figure 12. Electron micrograph of macronueloar DNA
complexed with RNA polymeraso from Bacillus subtilis.
Each piece of DNA binds an RNA polymerase molecule at
one end.
Figure 13. Hybridization of ribosomal RNA of Stylony-
chin with the 10-S ( ~ ) and 14-S ( 9 fractions of macro-
nuclear DNA and with DNA of Escherichia coli (@). The
ribosomal RNA complexes slightly with DNA of E. coli and
slightly more with the 10 S DNA of Stylonychia. Ribo-
somal RNA complexes to a much greater extent with the
14 S DNA fraction.

interband-~ bond
Figure 14. A scheme to explain the
polytene chromosome derivation of the gene-sized pieces of
: : ; = in the developing macro- DNA in the mature maeronucleus from
nucleus the chromosomes of the miaronucleus.
The micronueleus goes through several
"spacer" * cjene model rounds of DNA replication to produce
of the chromosome the polyteno chromosomes of the early
maeronuclear anlago. Each band of the
polytone chromosomes in the macro-
tronsectionincJ of DNA nuclear anlage is assumed to represent
<1, a single genetic locus. The polytene

@ @@ vesicle stage
chromosomes are transeeted between
successive bands as shown in Figure 4.
The polytenic copies of DNA in each
band become enclosed in a vesicle
(Fig. 4). Only one copy is shown in each

9 @@
e.
destruction of "spacer"
DNA
of the three vesicles in the drawing.
Most of the DNA in each vesicle (band)
is considered to consist of a "spacer"
t h a t separated the gene copy in one
band of a chromosome from the gene
destruction of vesicle
copy in the next band. Destruction of
, /
the "spacers" occurs in the vesicle stage
membranes and is assumed to account for the
degradation of 93% of the DNA in the
anlage. Only the structural genes with
control regions, accounting for 7% of
the original chromosomal DNA, are
preserved. Destruction of the vesicle
mature macronucleus membranes produces a macronuclear
anlage containing gene-sized pieces of
DNA. This DNA is replicated many
times to produce the DNA-rich, mature
macronueleus.
 Downloaded from symposium.cshlp.org on June 26, 2016 - Published by Cold Spring Harbor Laboratory Press

CHROMOSOME STRUCTURE IN CILIATES 617

Figure 15. E l e c t r o n m i e r o g r a p h of m i e r o n u e l e a r D N A d e n a t u r e d w i t h alkali a t p H 11.1. A t t h i s p H , e a c h piece of m a c r o -

n u c l e a r D N A s h o w s a d e n a t u r e d region a t one end. T h e d e n a t u r e d sites seen here (arrows) are believed to r e p r e s e n t t h e
d i s t r i b u t i o n of m a c r o n u c l e a r D N A s e q u e n c e s in t h e m i c r o n u c l e a r D N A . E a c h d e n a t u r e d site is s e p a r a t e d b y s t r e t c h e s of
D N A ( " s p a c e r " ) r a n g i n g in l e n g t h f r o m 0 . 5 / ~ m to over l 0 / ~ m . 45,000 •

We have tested the model in Figure 14 by map- separated, AT-rich regions in micronuclear DNA
ping single-stranded regions in micronuclear DNA consistent with the occurrence of long "spacers" of
denatured under conditions that melt only the ends various lengths between macronuclear DNA se-
of macronuclear DNA. In this way, we have at- quences (Fig. 15). The chromosome model might
tempted to locate macronuclear DNA sequences also be tested by binding RNA polymerase to
within micronuclear DNA using the AT-rich regions micronuclear DNA to map the locations of macro-
as markers. The electron micrographs show widely nuclear sequence. In addition, we hope to reanneal
 Downloaded from symposium.cshlp.org on June 26, 2016 - Published by Cold Spring Harbor Laboratory Press
618 PRESCOTT
macronuclear DNA with micronuclear DNA and
map the location of macronuelear sequences by
electron microscopy.
Acknowledgments
This work was supported by a grant #GM19199-
02 f r o m t h e N a t i o n a l I n s t i t u t e s o f H e a l t h a n d a
National Science Foundation grant #GB-32232 to
D r . D a v i d M. P r e s c o t t .
Portions of this paper are reproduced, with
p e r m i s s i o n , f r o m P r e s c o t t e t al., 1973.
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Downloaded from symposium.cshlp.org on June 26, 2016 - Published by Cold Spring Harbor Laboratory Press

Chromosome Structure in Ciliated Protozoans

D. M. Prescott and K. G. Murti

Cold Spring Harb Symp Quant Biol 1974 38: 609-618

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