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RESEARCH
Spherically symmetric empty space and
its dual in general relativity
Naresh Dadhich
Inter University Centre for Astronomy & Astrophysics, P.O. Box 4,
Pune 411 007, India
In the spirit of the Newtonian theory, we characterize
spherically symmetric empty space in general relativity
(GR) in terms of energy density measured by a static observer and convergence density experienced by null and
time-like congruences. It turns out that the space surrounding a static particle is entirely specified by the vanishing of energy and null convergence density. The
electrograv-dual1 to this condition would be the vanishing
of time-like and null convergence density which gives the
dual-vacuum
solution
representing
a Schwarzschild black hole with global monopole charge2
or with a cloud of string dust3. Here the duality1 is defined
by interchange of active and passive electric parts of the
Riemann curvature, which amounts to interchange of the
Ricci and Einstein tensors. This effective characterization
of stationary vacuum works for the Schwarzschild and
NUT solutions. The most remarkable feature of the effective characterization of empty space is that it leads to new
dual
spaces
and
the method can also be applied to lower and higher dimensions.
THE Newtonian gravitational field equation is given by
∇2φ = 4πGρ and empty space is characterized by ρ = 0. It is
well-known that the measure of energy is an ambiguous
issue in GR primarily because of the inherent difficulty of
non-localizability of gravitational field energy. However
there is no difficulty in defining various kinds of energy
density, signifying different aspects. The analogue of the
Newtonian matter density is the energy density measured
by a static observer and defined by ρ = Tabu au b, u au a = 1,
where u a = ((g)1/2, 0, 0, 0) and Tab is the matter–stress
tensor of non-gravitational matter field. Then there is
the convergence density experienced by time-like and
null particle congruences in the Raychaudhuri equation4.
They are defined as the time-like convergence density,
ρt = (Tab – 12Tgab)u au b and the null convergence density
ρn = Tabvavb, vava = 0, va = (1, (– g 11/g 00)1/2, 0, 0). The energy
density ρ refers to all kinds of energy other than the gravitational field energy, while the time-like and null convergence
densities act as active gravitational charge densities. For a
perfect
fluid
they
are
given
by
ρt =
1 (ρ + 3p) and ρ = ρ + p. It is important to recognize that
n
2
these three densities represent different aspects of energy
distribution and its gravitational linkage. They would thus
in general not be equal. Obviously all the three can never be
e-mail: nkd@iucaa.ernet.in
1118
equal unless space is flat. However ρ = ρt implies
vanishing of scalar curvature (radiation), ρ = ρn indicates
vanishing of pressure (dust) and ρt = ρn gives ρ = p (stiff
fluid). It may be noted that the weak field and slow
motion limit of the Einstein non-empty space equation is
∇2φ = 4πGρ, while its limit in weak field and relativistic motion is ∇2φ = 8πGρt.
In the following, we shall always refer ρ and ρt relative to
a static observer, and ρn to radial null geodesic. This does,
however, bring in a particular choice for the time-like and
null vectors but the choice is well motivated by the physics
of the situation. The radial direction is picked up by the 4acceleration of the time-like particle, identifying the direction of gravitational force, and so is the static observer for
measure of energy and time-like convergence densities.
The main question we wish to address in this note is, can
we characterize empty space solely in terms of these densities?
The answer is yes for the space surrounding a static particle. This may in general be true for an isolated particle with
some additional conditions which would specify the additional physical character of the problem. It is clear that any
specification of empty space must involve density relative
to both time-like and null particles. That means ρn must
vanish in any case and in addition one or both of ρ and ρt
must vanish. Of course there should be no energy flux,
Pc = h ac Tabu b = 0, h ac = g ac – u au c . It turns out that for
spherical symmetry the effective equation for vacuum is
ρ = ρn = Pc = 0, the solution of which would imply ρt = 0 and
vanishing of the all Ricci components. Thus vanishing of
energy and its flux, and null convergence density is sufficient to characterize empty space for spherical symmetry as
these conditions completely determine the unique Schwarzschild solution. The effective vacuum equation is less restrictive than the vanishing of the entire Ricci tensor.
What actually happens is, for the spherically symmetric
metric in the curvature coordinates, Pc = 0 and ρn = 0 lead to
R01 = 0 and R00 = R11 which imply g 00 = f (r) = – g 11, and then
ρ = 0 means R22 = 0 which integrates to give the Schwarzschild solution completely with g 00 = 1 – 2GM/r (we have
set c = 1). Thus instead of Rab = 0, the less restrictive effective equation ρ = ρn = Pc = 0 also equivalently characterizes
empty space for a static particle. It is a covariant statement
relative
to
a
static
observer
and
in
the curvature coordinates it takes the form R00 = R11,
R22 = 0 = R01.
Since there are three kinds of density, which could vanish
with two at a time in three different ways, it is then natural
to ask what would the other two cases give
rise to?
The first thing that comes to mind is to replace ρ by ρt in
the effective equation to write ρt = 0 = ρn = Pc , which would
imply G00 = G11, G22 = 0 = G01. That is replacing Ricci by Einstein, which represents a duality relation between the two.
Remarkably this duality transformation is implied
at a more fundamental level by interchange of the active and
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
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passive electric parts of the Riemann curvature1. (Active
and passive electric parts of the Riemann curvature are defined by the double (one for each 2-form) projection of the
Riemann tensor and its double (both left and right) dual on
a time-like unit vector, and dual is the usual Hodge dual,
.) That is, interchange of active
* Rabcd = 1/2εabmn
mn
(Eab = Racbdu c u d ) and passive (Rcd
= * R* acbdu c u d ) electric
~
parts implies interchange of the Ricci and
E ab Einstein tensors
because
contraction
of
Riemann
gives
Ricci
while that of its double dual gives Einstein tensor. We have
defined the electrogravity duality transformation1
by interchange of the active and passive electric parts,
Hab → Hab. Under this duality transformation it
~
E
isabclear
↔ Ethat
,
ρ
↔ ρt, ρn → ρn, Pc → Pc .
ab
Then the condition ρt = ρn = Pc = 0 is electrograv-dual to
the effective empty space equation given above, and its
solution would give rise to the space dual to empty space. It
can be easily verified that it integrates out to give
the general solution given by g 00 = – g 11 = 1 – 8πGη2 –
2GM/r, where η is a constant. This is an asymptotically
non-flat non-empty space which reduces to the Schwarzschild empty space for η = 0. At large r, the stresses it produces accord precisely to that of a global monopole of core
mass M and η indicating the scale of symmetry breaking2.
Alternatively it can exactly for all r represent a Schwarzschild black hole sitting in a cloud of string dust3. It is remarkable that here it arises as dual to empty space, i.e. dual
to the Schwarzschild black hole1. A global monopole is
supposed to be produced when global symmetry O(3) is
spontaneously broken into U(1) in phase transition in the
early Universe. The physical properties of this space have
been investigated5 and it turns out that the
basic character of the field remains almost the same
except for scaling of the Schwarzschild’s values for the
black hole temperature, the light bending and the perihelion
advance6. The difference between the Schwarzschild solution and its dual can be demonstrated as
follows. Both the solutions have g 00 = – g 11 = 1 + 2φ with
∇2φ = 0, which would have the general solution φ = k –
M/r. The Schwarzschild solution has k = 0, while the dual
does not. This is the only essential difference between the
two. It is this constant, which is physically trivial in the
Newtonian theory, that brings in the global monopole
charge, a topological defect.
Let us also consider the remaining possibility,
ρ = ρt = Pc = 0 which would in terms of the Ricci components imply R = 0, R00 = 0. This integrates out to give the
general solution, g 00 = (k +
1 − 2 GM / r ) 2g,11 = – (1 –
–1
2 GM/r) , where k is a constant. It is an asymptotically flat non-empty space with the stresses given
by
1
T1 =
2 kGM/r 3
k + 1 − 2GM /r
2
= −2T2 .
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
Obviously, these stresses cannot correspond to any
physically acceptable matter field because ρ = 0. On
the other hand, the space-time unlike the dual solution remains asymptotically flat. It will admit a static surface only if
k <0
at
rs =
2GM/(1 – k 2).
However
r ≥ 2GM
always for g 00 to be real. The region lying between rs and
2GM would define an ergosphere where negative energy
orbits can, as for the Kerr black hole, occur. The Penrose
process 7 can be set up to extract out the contribution of k
only if it is negative. However we do not know the physical
source for k.
On the other hand, when k > 0, there occurs no horizon
and it can represent a wormhole8 of throat radius r = 2GM. It
is remarkable that it has the basic character of a wormhole
which needs to be further investigated. Pursuing on this
track, we are presently working out a viable wormhole
model9.
This space is certainly empty relative to time-like particles
as both ρ and ρt vanish but not so for photons as ρn ≠ 0. At
the least, it can be viewed as an asymptotic flatness preserving perturbation to the Schwarzschild field.
Further it is also possible to characterize the Reissner–
Nordström solution of a charged black hole by ρ = ρt, ρn =
Pc = 0, and the de Sitter (Λ-vacuum) space by ρ + ρt = 0,
ρn = Pc = 0.
In
the
Ricci
components,
the
former would translate into R = 0, R00 = R11. This is clearly
invariant under the duality transformation. It is a non-empty
space with trace-free stress tensor. The de Sitter space is
given by ρ + ρt = Pc = 0, which implies Rab = Λg ab. Of course
under the duality transformation the sign of Λ would
change indicating that the de Sitter and anti de Sitter are
dual of each other.
The next question is, could other empty space solutions
representing isolated sources be characterized similarly?
It turns out that it is possible to characterize the
NUT solution and its dual10,11 in a similar manner. However
an additional condition would come from the gravomagnetic
monopole12 character of the field. The most difficult and
challenging problem would be to bring the Kerr solution in
line. That is an open question and would
engage us for some time in the future. The crux of the matter
is to identify the additional condition corresponding to gravomagnetic character of the field and solving the resulting
equations. Once that is achieved, our new characterization
of vacuum would cover all the interesting cases.
In conclusion, we would like to say that it is always
illuminating and insightful to understand the relativistic
situations in terms of the familiar Newtonian concepts and
constructs. Relating empty space to the absence of energy
and convergence density is undoubtedly physically very
appealing and intuitively soothing. The most remarkable
aspect of this way of looking at empty space is that it gives
rise, in a natural manner, to the new spaces dual to the corresponding empty spaces. The dual spaces only
differ from the original vacuum spaces by inclusion of a
topological defect, a global monopole charge.
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Note that the characterization of empty space and its dual
is by the covariant equations. Earlier the dual spacetimes1,13
were obtained by modifying the vacuum equation, so as to
break the invariance relative to the electrogravity duality
transformation, in a rather ad-hoc manner. Now the effective
vacuum equation has the direct physical meaning in terms
of the energy and convergence density. This characterization could as well be applied in lower and higher dimensions
to find new dual spaces. For example, in three-dimensional
gravity the dual space represents a new class of black hole
spaces 14 with a string dust matter field. For higher dimensions, the method would simply go through without any
change for n-dimensional spherically symmetric space and
the dual space would represent a corresponding Schwarzschild black hole with a global monopole charge. It can be
further shown that a global monopole field in the Kaluza–
Klein space can be constructed similarly15 as dual to the vacuum
solution16.
It is thus an interesting characterization of empty space
which leads to new spaces dual to corresponding empty
spaces.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Dadhich, N., Mod. Phys. Lett. A, 1999, 14, 337.
Barriola, M. and Vilenkin, A., Phys. Rev. Lett.,1989, 63, 341.
Letelier, P. S., Phys. Rev. D, 1979, 20, 1294.
Raychaudhuri, A. K., Phys. Rev., 1955, 90, 1123.
Harari, D. and Lousto, C., Phys. Rev. D, 1990, 42, 2626.
Dadhich, N., Narayan, K. and Yajnik, U., Pramana, 1998, 50,
307.
Penrose, R., Riv. Nuovo Cimento, 1969, 1, 252.
Visser, M., Lorentzian Wormholes: From Einstein to Hawking,
American Institute of Physics, 1995.
Mukherjee, S. and Dadhich, N., (under preparation).
Dadhich, N. and Nouri-Zonoz, M., (under preparation).
Nouri-Zonoz, M., Dadhich, N. and Lynden- Bell, D., Class.
Quantum Gravit., 1999, 16, 1021.
Lynden-Bell, D. and Nouri-Zonoz, M., Rev. Mod. Phys., 1998,
70, 427.
Dadhich, N., in Black Holes, Gravitational Radiation and the
Universe (eds Iyer, B. R. and Bhawal, B.), Kluwer, Dordrecht,
1999, p. 171.
Bose, S., Dadhich, N. and Kar, S., Phys. Lett. B, 2000, 477, 451.
Dadhich, N., Patel, L. K. and Tikekar, R., Mod. Phys. Lett. A,
1999, 14, 2721.
Banerjee, A., Chatterjee, S. and Sen, A. A., Class. Quantum
Gravit., 1996, 13, 3141.
ACKNOWLEDGEMENT.
comments.
I thank the referee for his constructive
Received 6 December 1999; revised accepted 4 March 2000
Comparative efficacy of Ayush-64 vs
chloroquine in vivax malaria
Neena Valecha†,‡, C. Usha Devi†, Hema Joshi†,
V. K. Shahi*, V. P. Sharma and Shiv Lal**
†
Malaria Research Centre, 22 Sham Nath Marg, Delhi 110 054, India
*Central Council for Research in Ayurveda and Siddha, Anusandhan
Bhavan, 61–65 Institutional Area, Opp. D-Block, Janakpuri,
Delhi 110 058, India
**National Anti Malaria Programme, 22 Sham Nath Marg,
Delhi 110 054, India
A phase II prospective comparative randomized clinical
trial was conducted in patients of vivax malaria to compare
the efficacy of Ayush-64 vs chloroquine. Ayush-64, a
herbal formulation patented by Council of Ayurveda and
Siddha was compared with chloroquine. Patients received
an oral dose of either 1 g Ayush-64, three times a day for
5–7 days or a total dose of 1500 mg chloroquine over 3
days. Peripheral smears were examined everyday for 3
days or till they were negative and then weekly up to 28
days.
The results of the study showed that at day 28, only 23 of
47 (48.9%) patients in the Ayush group and all the 41 in
the chloroquine group were cured (p < 0.05). Even in these
23 patients in the Ayush group parasite clearance time was
longer than chloroquine (3.16 vs 1.5 days). Both regimens
were generally well tolerated. In conclusion, Ayush-64 in a
dose of 1 g three times a day for 5–7 days is not as effective
for treatment of vivax malaria, as standard chloroquine
therapy.
M ALARIA is a major health problem in India. The annual
incidence was between 2 and 3 million during the last decade1. The problem of drug resistance and treatment failures
in P. falciparum2 and recently in P. vivax3,4
malaria using chloroquine has focused interest on new
drugs/drug combinations/indigenous drugs or remedies.
Ayush-64 is a combination of four Ayurvedic drugs
namely Alstonia scholaris R. Br. (aqueous extract of the
bark – 1 part), Picrorhiza kurroa Royle (aqueous extract of
the rhizome – 1 part), Swertia chirata Buch-Ham (aqueous
extract of the whole plant – 1 part) and Caesalpinia crista
Linn (fine powder of seed pulp – 3 parts)5. The ingredients
after mixing are formulated into tablets of 500 mg each. The
drug/formulation is patented and registered by Central
Council for Research in Ayurveda and Siddha (CCRAS) and
was reported to cure malaria5,6. Since the studies were conducted more than 15 years ago and susceptibility of parasites to drugs keeps changing, it was considered essential
to evaluate the efficacy of the drug before introduction in
the national programme. Moreover, in these studies, patients were included on the basis of clinical diagnosis of
malaria and assessment criteria were not uniform5, therefore
‡
1120
For correspondence. (e-mail: valecha@del3.vsnl.net.in)
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
RESEARCH COMMUNICATIONS
the present comparative study was designed to confirm the
antimalarial activity of Ayush-64.
The study was conducted at Malaria Research Centre
(MRC), Delhi. The clinics at MRC and the National Anti
Malaria Programme (NAMP) were selected for the study.
Patients from 4 to 5 peri-urban villages and resettlement
colonies visit these clinics. They belong to the low socioeconomic status. It was a prospective comparative, open
and randomized study. The randomization was done by
using a simple random sampling table. Proven cases of vivax malaria seeking treatment at the clinics of MRC and
NAMP were asked to volunteer for the study if they were
18–60 years of age, had asexual parasitaemia < 50,000/µl,
were febrile or had recent history of fever within the last
48 h. Pregnant and lactating females and G-6 PD deficient
subjects were not included in the study. Patients who had
taken antimalarials within 7 days prior to enrollment in the
study were also not included.
The study was approved by an ethical committee of
MRC. Patients satisfying the inclusion criteria were
enrolled after obtaining an informed written consent.
Patients were randomly allocated to receive one of the two
treatment regimens. One group received a total dose of
1500 mg chloroquine over 3 days followed by 15 mg primaquine daily for 5 days as standard therapy for vivax malaria.
The other group received Ayush-64, 2 tablets of 500 mg
each three times a day for 5 days. The patients in the Ayush
group who did not respond, as evidenced by the presence
of parasites in the peripheral smear, were given treatment for
further 2 days and labelled as treatment failures if they remained positive. This dose was as per recommendation by
NAMP and in accordance with the doses used in earlier
studies 5,6.
Patients were observed for at least 2 h after drug administration for vomiting. They were then followed-up as outpatients on day 1, 2, 3, 7, 14, 21 and 28 for clinical and parasitological cure. All the patients who became positive after
smear being negative at day 7 were labelled as recrudescenes. Haematology and blood chemistry were done on
day 0 and day 7. A slide was considered negative only after
200 fields had been examined without finding a parasite. The
antimalarial efficacy in both groups was compared using
parasite clearance time (PCT), cure rate at day 28 and recrudescence rate as parameters. The technician involved in
investigations was not aware of the treatment regimen of
the patients. The statistical method used for comparing PCT
was the t-test and the z-test was used for cure rate and recrudescence rate in the two groups.
Fifty-four cases of P. vivax were enrolled in the Ayush-64
group and fifty in the chloroquine group. Seven cases in the
former (age 19–50 years) and nine in the latter (age 18–40
years) group were lost to follow-up before they had completed the 28-day observation period. Although the dropout rate was similar in both groups, the reasons were different. In chloroquine group the drop-out was because patients left the study area after being smear negative and the
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
28-day follow-up could not be completed. In the Ayush
group, 4 cases dropped out as they did not want to continue therapy after the third day while 3 left the study area.
Patients randomized to the Ayush-64 and chloroquine
groups had comparable baseline characteristics with respect
to age, sex, duration of illness, initial parasite counts, clinical symptoms, etc. (Table 1).
In the chloroquine group (n = 41) mean parasite clearance
time was 1.5 ± 0.5 days compared to 3.16 ± 2.4 days in the
Ayush-64
group
(n = 31
including
recrudescence cases). The values were significantly different
with p < 0.05 (t-test). Similarly cure rate of 100% vs 48.9% in
the chloroquine and Ayush groups, respectively was significantly different (p < 0.05) from each other. Recrudescence rate in the chloroquine group was 0% as against
17% in the Ayush group (p < 0.05).
In addition, clinical recovery was also slow in the Ayush
group and general acceptability of the drug was poor. The
patients complained about frequency and duration of dosing and size and quality of the tablet formulation. The
course of parasitaemia in the two treatment groups is shown
in Figure 1. Out of the 16 cases who were labelled as treatment failures in the Ayush group, three had to be referred to
hospital due to deterioration of clinical condition and all
responded to chloroquine therapy.
Both treatment regimens were generally well tolerated.
There was no significant difference in pre- and posttreatment haematological or biochemical findings in both
treatment groups. One patient of the Ayush group had generalized oedema and proteinuria but causality relationship
could not be established since the patient was lost to follow-up. Three patients in the Ayush group complained of
gastrointestinal disturbances, viz. nausea and diarrhoea.
In the recent past efforts are being made to revive
indigenous systems of medicine. Ayush-64 has been previously shown to be effective as an antimalarial in animal experiments and clinical studies conducted by the CCRAS5,6.
However, in experimental studies in rodent models, increase
in survival time and not the parasite clearance was observed
in the dose of 3 g per day for 4 days. In clinical studies an
efficacy of 72–90% in vivax malaria was reported with the
Table 1.
Curative efficacy of Ayush-64 and chloroquine
Treatment groups
Parameter
Ayush
Chloroquine
Total cases enrolled
Age
Parasitaemia on day 0
Dose of drug used
54 (46 M + 8 F)
29.40 ± 9.04 years
0.25 ± 0.26%
1 g tds × 5–7 days
Total cases completing 28 days
Cure rate
Recrudescence rate
Parasite clearance
time
47
50 (35 M + 15 F)
30.46 ± 10.25 years
0.31 ± 0.32%
1.5 g (base) over 3
days
41
48.9%
17%
3.16 ± 2.4 days
100%
0
1.5 ± 0.5 days
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RESEARCH COMMUNICATIONS
Figure 1. Course of parasitaemia in the two treatment groups. Data are arithmetic mean of
parasitaemia.
dose of 3 g/day for 4–5 days. However, some of the cases
were included on the basis of clinical diagnosis only.
Similarly Sharma et al.6 have shown the efficacy of
Ayush-64 with variable dose and duration of treatment to
be 70%. In all the studies the total dose of drug has varied
from 9 to 37 g. The frequency of administration varied from
patient to patient. However, the total daily dose was not
more than 3 g in any case.
In the present study a uniform dose schedule of 3 g per
day for 5 days (15 g) extending to 7 days if needed (21 g)
has been used.
A cure rate of less than 50% has been observed with
Ayush-64 as against 100% with chloroquine. The latter drug
was administered once a day only for 3 days and is therefore more acceptable and cost effective. It is evident from
the present study that Ayush-64 does not have specific
schizonticidal
or
gametocytocidal
activity.
Our
results are supported by lack of antimalarial action in rodent
and simian models even at dose of 3 g/kg (ref. 7). One of the
ingredients of Ayush-64, i.e. Alstonia scholaris has also
been shown to be devoid of specific antiplasmodial action8.
The drug does not appear promising as a primary drug for
treatment of malaria in the present dose regimen. In addition
to increasing morbidity, slow parasite clearance poses a
threat of continuing transmission of the disease. And this,
1122
therefore, is unlikely to be an alternative for chloroquine in
vivax malaria. Considering the poor efficacy of the drug and
risk of complications in falciparum malaria, further study in
P. falciparum cases was not conducted.
1. Sharma, V. P., Indian J. Med. Res., 1996, 103, 26–45.
2. Sharma, R. S., Sharma, G. K. and Dhillon, G. P. S., in Epidemiology and Control of Malaria in India, 1996, pp. 87–93.
3. Garg, M., Gopinathan, N., Bodhe, P. and Kshirsagar, N. A., Trans.
Soc. Trop. Med. Hyg., 1995, 89, 656–657.
4. Dua, V. K., Kar, P. K. and Sharma, V. P., Trop. Med. Intl Health,
1996, 1, 816–819.
5. Anon, Report on Ayush-64, Central Council for Research in Ayurveda and Siddha, Government of India, 1987, pp. 1–128.
6. Sharma, K. D., Kapoor, M. L., Vaidya, S. P. and Sharma, L. K., J.
Res. Ayurveda Siddha, 1981, 2, 309–326.
7. Kazim, M., Puri, S. K., Dutta, G. P. and Narasimham, M. V. V. L.,
Indian J. Malariol., 1991, 28, 255–258.
8. Gandhi, M. and Vinayak, V. K., J. Ethnophamacol., 1990, 1, 51–
57.
ACKNOWLEDGEMENTS. We thank Mr A. R. Kotnala, Mrs P.
Singhal, Mr Kesari Prasad, Mrs Alka Kapoor, Mrs Lalita Gupta and
Mr Bhopal Singh for technical assistance.
Received 6 December 1999; revised accepted 4 March 2000
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
RESEARCH COMMUNICATIONS
Correlating dynamics to conformational properties: An analysis of atomic
displacement parameters (B-values)
in high-resolution protein structures
S. Parthasarathy and M. R. N. Murthy*
Molecular Biophysics Unit, Indian Institute of Science,
Bangalore 560 012, India
Atomic displacement parameters (ADPs) obtained by high
resolution X-ray diffraction studies on single crystals of
proteins represent the mean square displacement of atoms
about their mean position. The relationship between the
flexibility of the protein molecule and its conformation
could be examined by a careful analysis of these ADPs.
This communication presents the results of such a statistical analysis. It is shown that the ADPs are related to side
chain conformations and are low for energetically favourable rotamers. Examination of the dependence of ADPs on
non-planar distortions of the peptide geometry as represented by ω angle reveals that the parameters depend on
the direction of non-planar distortion. Those conformations
with ω larger than the ideal trans geometry (180°–190°)
are more flexible when compared to those with ω < 180°
(170° –180°). The average ADP of a peptide unit depends
weakly on the Ramachandran angles at the corresponding
Cα
α atom. Thus, the flexibility of different segments of the
polypeptide appears to be sensitive to the conformation of
the side chains as well as the main chain.
A DVANCES in high brilliance X-ray sources as well as computational procedures for the refinement of proteins at high
resolution have provided accurate Atomic Displacement
Parameters (B-values) for several proteins1,2. These structures have been analysed with reference to the atomic interactions responsible for protein stability, evolution, function,
molecular recognition and principles that govern oligomerization. The information on protein flexibility and mobility
available in terms of the ADPs obtained by protein highresolution structure refinement, in contrast, has not been
exploited to the same extent3–5. ADPs (or B-factors or temperature factors) deposited in the Protein Data Bank correspond to B = 8π2 ⟨u 2⟩, where ⟨u 2⟩ is the average of the mean
square atomic displacements (Å2) along the three coordinate
axes and is given by (u 2x + u 2y + u 2z )/3. These parameters represent the flexibility of protein atoms and are important factors controlling protein function and stability. The atomic
displacement parameters determined for different proteins
show large variations from one structure to another due to
the differences in the static disorder and are also influenced
by refinement procedures6. However, it has been shown
that the frequency distribution of the B-factors expressed in
units of standard deviation about their mean value at the Cα
atoms (B′) are similar in protein structures7. This distribution
could be accurately described as a superposition of two
Gaussian functions. The frequency distribution for different
amino acids could be used to deduce flexibility indices that
are helpful in predicting mobile segments in proteins3,4,7. In
this communication, we examine the dependence of ADPs
on conformation of both the main chain and the side chain:
(a) χ1 representing the side chain rotamer, (b) ω representing the peptide bond geometry, and (c) Ramachandran angles φ and ϕ representing main chain conformation.
Generally, energetically unfavourable conformations are
associated with high flexibility.
The representative list of protein structures8 determined
by single crystal X-ray diffraction methods and available in
the Protein Data Bank9 (November 1996 release) was chosen
for the analysis. None of these structures has
sequence identity greater than 25%. The resolution is better
than 2.0 Å and the R factor is less than 0.2 for all the selected structures. The code numbers of the selected proteins are listed in Table 1.
For each of the selected structures, the B-values at the
Cα positions were replaced by a normal variate as B′-factor,
B′ = (Bi – ⟨B⟩)/σ(B), where ⟨B⟩ = ΣBi/N, where Bi is the Bvalue associated with Cα of the ith residue and N is the
total number of residues in the protein, and σ2(B) = Σ(Bi –
⟨B⟩)2/N. Normalization of B-values is required to compare Bvalues of different protein structures.
The χ1 (N–Cα–Cβ–Cγ for most amino acids, Oγ for
Ser and Cγ1 for Ile) torsional angle for each side chain was
computed as the angle between the planes containing N–
Cα–Cβ and Cα–Cβ–Cγ atoms, respectively. Following the
usual convention, the angle was taken as the clockwise
rotation of the N–Cα vector, looking down the Cα–Cβ direction, required to bring it to overlap with the Cβ–Cγ vector. The rotamer conformation was represented as gauche –
for – 120°< χ1 < 0°, gauche + for 0° < χ1 < 120°, and trans for
120°< χ1 < 240°. Frequency distribution of χ1 was sampled at
10° intervals. Similarly, the ω angle representing the deviation of the peptide geometry from a planar configuration
Table 1.
131L
1BAM
1CSE
1EDE
1IAE
1MOL
1PDA
1RSY
1TAH
1XNB
2CCY
2GST
2PHY
3CLA
3TGI
8ABP
PDB codes for the high-resolution structures selected
for the analysis
153L
1BP2
1CSH
1FKJ
1ISC
1NFP
1PGS
1SAT
1TCA
2ACQ
2CDV
2HMZ
2POR
3COX
4ENL
8FAB
1AMP
1CCR
1DAA
1FNC
1KPT
1NHK
1PHG
1SBP
1THV
2ALP
2CPL
2HTS
2PRK
3DFR
4GCR
9RNT
1ARB
1CHD
1DTS
1GOF
1LCP
1NAR
1PTX
1SNC
1TRY
2AYH
2CTC
2MNR
2SIL
3GRS
4FGF
1ARV
1CHM
1DUP
1GPR
1LEN
1OVA
1REC
1SRI
1TTB
2AZA
2END
2NAC
2TGL
3PTE
5RUB
1ATL
1CNS
1DYR
1HMT
1LTS
1PBE
1REG
1TAG
1TYS
2CBA
2ER7
2OLB
3CHY
3SIC
5TIM
*For correspondence. (e-mail: mrn@mbu.iisc.ernet.in)
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
1123
RESEARCH COMMUNICATIONS
was evaluated as the torsional angle Cαi–C′i –Ni+1–Cαi+1
(where i and i + 1 represent consecutive residues). 0° and
180° in ω correspond to ideal cis and trans peptide geometries, respectively. Sampling was at 1° intervals for statistical analysis. Ramchandran angles φ and ϕ representing the
polypeptide conformation at each Cα atom were calculated
as angles C′i–1–Ni–Cαi–C′i and Ni–Cαi–C′i –Ni+1, respectively.
Frequencies were counted in 60° bins of φ and ϕ.
The mean value of B′-factors corresponding to each rotamer and residue type was evaluated considering all the
proteins. Similarly, the variation of mean B′-factors was
monitored as a function of χ1 angle by grouping side chains
into bins of 10°. Residues were also grouped in to different
bins in the Ramachandran plot10. Even for fairly large bins,
the population in certain regions of the Ramachandran map
is sparse and hence the estimation of the mean B′-factors
for these regions will not be statistically significant. An
interval
of
60°
in
φ
as
well
as
ϕ
was found to provide sufficient number of residues in the
bins corresponding to the allowed and partially allowed
regions of the map.
Figure 1 shows the frequency distribution of side chain
rotamers and their mean B′-factors for structures used in
this study. The χ1 distribution of side chain rotamers in the
representative structures follows features that have been
noted earlier11. For most residues, the preferred rotamers are
gauche – and trans with somewhat less frequency in the
gauche + . However, for amino acids branched at Cβ, the rotamer is either not well-defined or well determined. As these
residues form only a small fragment of all the
a
Figure 1. Frequency distribution of χ1 values and dependence of
mean B-factor on χ1 .
b
Figure 2. Frequency distribution of peptide non-planar distortion
as represented by the dihedral angle ω.
1124
Figure 3. a, Variation of mean B′-factors of the main (. . .) and
the side chain (– – –) of the Cαatom preceding the peptide bond as a
function of peptide plane distortion ω. b, Variation of mean B′factors of the main (. . .) and the side chain (– – –) of the Cα atom
following the peptide bond as a function of peptide plane distortion
ω.
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
RESEARCH COMMUNICATIONS
residues, they have not been excluded or treated independently in the present analysis. The peaks corresponding to
the preferred rotamers have widths at half maximum of ~ 25°.
Although this spread might depend on the resolution at
which the structures have been determined as well as the
constraints used in refinement, the distribution shown in
Figure 1 is typical11. The plot also shows the mean B′factors for these conformational states. It should be noted
that the frequency in bins mid way between preferred rotamers are low and hence the estimates of the mean B′factors have large errors. The frequencies for intervals close
to the preferred torsion angles are large and hence the estimated B′-factors are reliable. The B′-factors have clear minima at the preferred conformations of the side chains. It
might also be noted that the most favoured torsion angle
representing the gauche – form is less than 300°. The minimum in B-factor curve also occurs at this value. Similar observations have been made earlier11–13.
Figure 2 illustrates the frequency distribution of the ω
angle, representing out of plane deviation of the trans peptide unit about the C′–N bond and Figures 3 a and b show
the variation of the mean B′-factor as a function of ω for
Table 2.
both the preceding and succeeding residues, respectively.
It is clear that the highest frequency occurs, not for the
ideal trans geometry, but for ω= 179°. This deviation from
the planarity of the peptide unit in proteins has previously
been observed14. The distribution is nearly symmetrical
about the preferred angle. On the contrary, the mean B′factor appears to monotonically increase with increasing ω
in the range 170°–190°. It implies that the peptide group
becomes more flexible when ω increases from the value corresponding to the ideal trans geometry. The feature of
larger B′-factors associated with ω> 180° are similar to the
trend observed for rotamers – conformations with lower
occupancy are associated with larger B′-factors. However,
B′-factors continue to decrease when ω changes to values
lower than 180°, although the peptide changes to a higher
energy state, as reflected by lower frequency of occurrence.
Ramakrishnan and Balasubramanian15 have shown that the
area allowed in the Ramachandran diagram for non-glycyl
residues reduces with increasing ω, in the range 170°– 190°.
Therefore, the reduction of B′-factor for non-planar distortions with ω< 180° might be due to the fact that the energy
corresponding to the peptide geometry is determined by the
Frequency of residues in bins of Ramachandran map and respective average
B′-factors in brackets
4378 (– 0.19)
709 (0.04)
309 (0.20)
167 (0.32)
36 (0.35)
244 (0.00)
6220
1652
1253
9611
110
243
(– 0.06) 733 (0.23)
(– 0.06)
55 (0.94)
(0.32)
16 (0.65)
(– 0.09) 3382 (0.12)
(0.42)
64 (0.83)
(0.21)
15 (0.59)
a
16
47
453
20
28
75
(0.85)
(0.79)
(0.23)
(0.33)
(0.64)
(0.17)
138
39
722
348
34
177
(0.25)
(0.46)
(0.38)
(0.56)
(1.65)
(0.17)
91 (0.25)
11 (1.87)
13 (0.00)
22 (0.26)
9 (0.8)
114 (0.02)
b
Figure 4.
Variation of the B′-factor as a function of (a) φ and (b) ϕ.
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
1125
RESEARCH COMMUNICATIONS
partial double bond character of the C′–N bond, while the
flexibility might be related to the allowed area (lack of close
contacts) of the Ramachandran diagram for the corresponding ω. Similar trends are observed for the side chain
and both the preceding and succeeding residues of the peptide bond.
Table 2 lists the frequencies of residues occurring in different bins of the Ramchandran plot sampled at 60° intervals
in the structures selected for analysis. The numbers are
consistent with the well-known features of the
Ramachandran map16. The largest occupancy is found for
the helical regions in the third quadrant and for strand regions in the second quadrant of the map. The B′-factors
associated with the atoms Ni, Ciα, C′i, Ni+1 of all residues
falling in the bins were averaged. The mean B′-factor of all
Cα atoms was subtracted from this average. The values
shown in Table 2 correspond to these. It is evident
that the mean B′-factor is low for all cells where the
occupancy is high. Since the frequency of occurrence
depends on the energy corresponding to the particular cell,
the flexibility is linked to the energy as observed
for χ1. Similar observations were made for the B′-factors
associated with the Cα atom alone. Thus the occurrence of
a particular pair of Ramachandran angle has the
effect of influencing the flexibility of the local segment
of the polypeptide. Figure 4 a illustrates the frequency and
the mean B′-factor as a function of φ (including all ϕ), while
Figure 4 b shows the same parameters as functions of ϕ (including all φ). The correlation between
high frequency and low flexibility is evident in these
plots also.
Availability of a large number of well-determined threedimensional structures of proteins makes possible studies
on protein structure, function, evolution and dynamics.
Preliminary examination of the ADPs determined during the
course
of
high
resolution
X-ray
structure
determination of a set of unique proteins carried out in this
communication clearly demonstrates that flexibility of the
protein chain is dependent on conformation. Both main
chain and side chain conformations appear to affect the
mobility of residues. Residue mobility is minimum when the
1126
side chain confirms to the most preferred rotamer. On the
contrary, the mobility of the backbone depends asymmetrically on the departure of the peptide plane from ideal trans
geometry. For non-planar distortions resulting from increasing ω, the flexibility increases. Peptides with ω< 180°
tend to be less flexible although the distortions are not in
the direction of reducing energy. This might be a consequence of a larger area of Ramachandran diagram allowed
for peptides with ω< 180°. Flexibility of the polypeptide is
also found to depend on the Ramachandran angles, partially
allowed and disallowed regions correspond to larger B′factors. Thus, the local flexibility of polypeptides appears to
be influenced by main chain as well as side chain conformations.
1. Dauter, Z., Lamzin, V. S. and Wilson, K. S., Curr. Opin. Struct.
Biol., 1997, 7, 681–688.
2. Merrit, E. A., Acta Crystallogr. D, 1999, 55, 1109–1117.
3. Vihinen, M., Torkkila, E. and Riikonen, P., Proteins: Struct.
Funct. Genet., 1994, 19, 141–149.
4. Karplus, P. A. and Schulz, G. E., Naturwissenschaften, 1985, 72,
212–213.
5. Carugo, O. and Argos, P., Acta Crystallogr. D, 1999, 55, 473–
478.
6. Parthasarathy, S. and Murthy, M. R. N., Acta Crystallogr. D,
1999, 55, 173–180.
7. Parthasarathy, S. and Murthy, M. R. N., Protein Sci., 1997, 6,
2561–2567; 1998, 7, 525.
8. Hobohm U. and Sander C., Protein Sci., 1994, 3, 522 –524.
9. Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F.
Jr. and Brice, M. D., J. Mol. Biol., 1977, 112, 535–542.
10. Ramachandran, G. N. and Sasisekharan, V., Adv. Prot. Chem.,
1968, 23, 283–437.
11. Thanki, N., Umrania, Y., Thronton, J. M. and Goodfellow, J.
M., J. Mol. Biol., 1991, 221, 669–691.
12. Dunbrack, R. L. Jr. and Karplus, M., J. Mol. Biol., 1993, 230,
543–574.
13. Carugo, O. and Argos, P., Protein Eng., 1997, 10, 777–787.
14. MacArthur, M. W. and Thornton, J. M., J. Mol. Biol., 1996,
264, 1180–1195.
15. Ramakrishnan, C. and Balasubramanian, R., Int. J. Pept. Protein
Res., 1972, 4, 79–90.
16. Ramachandran, G. N., Ramakrishnan C. and Sasisekharan, V., J.
Mol. Biol., 1963, 7, 95–99.
Received 14 October 1999; revised accepted 9 February 2000
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
RESEARCH COMMUNICATIONS
The plasmodia of Physarum polycephalum, an elegant system to
demonstrate the importance of
genome integrity in traversing the
G2/M checkpoint of the cell cycle
P. R. Jayasree†, P. R. Manish Kumar†,* and
R. Vimala Nair**
†
Department of Biotechnology, University of Calicut,
Calicut 673 635, India
**19/2084/A17 Express Tower, P. V. Sami Road, Calicut 673 002,
India
In this communication we show the usefulness of the plasmodia of Physarum polycephalum to demonstrate important concepts in cell cycle regulation, such as the need for
an undamaged chromosomal DNA to allow the transition
from late G2 to prophase (G2/M checkpoint). In Physarum,
chromosomal DNA synthesis is confined approximately to
the first one-third of the cell cycle which comprises the S
phase, and rDNA and mitochondrial DNA are synthesized
throughout the cycle except during mitosis and early S
phase.
Taking
advantage of these facts, DNA of the plasmodia was substituted with 5-bromo-2′′ -deoxyuridine at a concentration
where it had no effect on DNA synthesis per se during different phases of the cell cycle before exposure to UV during late G2. A strong synergistic effect between BrdU and
UV,
in
terms
of
mitotic
delay,
was
observed only in plasmodia treated with the base analogue
during the early S phase, thereby showing that the integrity of chromosomal DNA is an important prerequisite for
the initiation of mitosis. It is suggested that this system is
ideal for molecular level studies on this aspect of checkpoint operation.
IN eukaryotic cells, progression of cell cycle through different phases (G1, S, G2 and M) is controlled by a cell cycle
control system. This system comprises brakes – the check
points – that can stop the cell cycle at two specific points –
one in G1 before entry into S phase (G1/S checkpoint) and
another in late G2, at the entry into mitosis (G2/M checkpoint). The checkpoints are signal transduction pathways
whose effectors interact with cyclin/Cdk complexes to block
cell cycle progression. A large number of studies have been
carried out in yeast and mammalian cells to understand the
molecular mechanisms operating at these points1–7.
Although the importance of chromosomal DNA in initiating mitosis8,9, and that of G2 phase as a period of surveillance to monitor the state of the nuclear DNA, before
chromosomes are allowed to participate in mitosis and another cycle of reproduction, was envisaged since some
years10, there has been a renewed interest now in under-
*For corresondence. (e-mail: prm@unical.ac.in)
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
standing the mode of communication of chromosomal DNA
with the signals which allow the cells to traverse the G2/M
checkpoint. In the case of severe damage, G2 phase is also a
time to activate programmed cell death11–13.
Entry into mitosis requires at least two events: activation
of a mitosis promoting protein called Cdc2 by the Cdc25
phosphatase; and accumulation of active Cdc2 in the nucleus. The DNA damage checkpoint seems to block both
these processes via pathways dependent on p53, a tumour
suppressor protein, firstly by maintaining Cdc25 phosphatase in a phosphorylated form by protein kinase Chk1, followed by the binding of various 14-3-3 proteins14 and the
resultant export of this phosphatase out of the nucleus,
thus preventing the Cdc2–cyclinB complex from getting
activated. The other pathway operates via 14-3-3σ protein
which binds Cdc2–cyclin complex and sequesters it in the
cytoplasm. There is evidence for the involvement of yet
other factors and there could also be some amount of redundancy in the mechanisms operating at this checkpoint
and further investigations are required to elucidate them15,16.
Recent studies on cell-free extracts of Xenopus eggs which
analysed the role of protein kinase Chk1 in checkpoint control show the similarity of this system with that of yeast and
mammals14. There is also the proposal by Lydall and Weinert13, that damaged DNA could structurally inhibit mitosis,
i.e. if condensed chromatin was a structural prerequisite for
entry into mitosis, and damaged DNA could not be efficiently condensed.
Different types of perturbers have been used to analyse
cell cycle regulation in the synchronously mitotic plasmodia
of Physarum polycephalum17. It is known that UV irradiation during G2 inhibits RNA and protein syntheses18, and
interferes with the organization of microtubular organizing
centres19,20 in this organism. Irradiation also leads to long
mitotic delays18,21. It had also been observed that UVirradiation causes decondensation of chromosomes in the
plasmodia22. The higher susceptibility of S phase to UV in
the plasmodia is also known21. Therefore, to single out the
mitotic delaying effect of UV at the level of the integrity of
chromosomal DNA per se, it is necessary to irradiate the
plasmodia during very late G2, when chromosomal DNA
synthesis is already completed and the chromosomes are
condensed. It would also be ideal to use plasmodia whose
DNA has already been sensitized to UV such that any effect
is amplified.
The analogue of thymidine, 5-bromo-2′-deoxyuridine
[BrdU] is readily incorporated into the DNA of the cells
including that of the plasmodia of Physarum23. In the present experiments, it was thought desirable to accentuate the
effect of UV on DNA by substituting it with this analogue,
as BrdU incorporated DNA is known to be highly sensitive
to radiation24. Along with BrdU, 5-fluorodeoxyuridine
(FudR) and uridine (UR) were also supplied in the ratio
8 : 1 : 20, as was done earlier by McCormick et al.25. They
had shown that FudR given in this ratio to the plasmodia of
Physarum along with BrdU, would increase the incorpora1127
RESEARCH COMMUNICATIONS
tion of BrdU, into the DNA, because of FudR’s known ability to block thymidine synthesis. UR in the above proportion was sufficient to prevent a decrease in the rate of RNA
synthesis, caused by a breakdown of FudR to 5-fluorouracil
and the consequent inhibition of UR biosynthesis. In the
plasmodia of Physarum the chromosomal DNA synthesis is
confined to the first one-third of the cell cycle which comprises
the
S
phase
(the
G1
being absent). The cycle time varies from 8 to 12 h and the
replication of the other DNA species (ribosomal DNA and
mitochondrial DNA) takes place throughout the cycle except during mitosis and the early part of S phase26,27. In order to distinguish between the different DNA types, BrdU
treatment was carried out at different phases of
the cell cycle in surface plasmodia prepared as described
below.
The McArdle strain (M3C) of P. polycephalum was
grown as microplasmodia in shaken cultures in the dark at
24°C on a semi-defined medium (SDM)28. Mitotically synchronous surface (macro) plasmodia were prepared by the
fusion of microplasmodia (0.4 ml packed suspension) on
Whatman No. 40 filter paper29 and grown in petri dishes
supplied with SDM. Thereafter at regular intervals, synchronous postfusion mitoses (PFM) occur in these plasmodia.
Mitotic delay studies were carried out after irradiating
(dose: 1400 Jm–2), both the BrdU-substituted (at different
phases of the cell cycle) and unsubstituted plasmodial sectors at late G2 prior to the third PFM (details are given in
legend to Figure 1), using a Philips 15 W germicidal lamp
which had earlier been calibrated (dose rate: 7.18 Jm–2 s –1) by
actinometry30, and which emits approximately 90% of its UV
energy at 2537 Å. DNA synthesis was evaluated by the radioisotope dilution technique of Devi and Guttes31 (details
are given in the legend to Figure 2), in the case of plasmodia
substituted with BrdU during early S phase, where alone
enhanced mitotic delay was observed.
The synergistic effect of UV and BrdU on mitotic delay
was observed only in early S phase analogue-substituted
category of plasmodia, in spite of increasing the treatment
doses to 20 µg ml–1 during late S phase or to 40 µg ml–1
thereafter (Figure 1). Since mitochondrial and nucleolar
DNA synthesis is seen throughout the cell cycle except
during early S phase and mitosis26,27, the mitotic delay data
(Figure 1) clearly indicate that BrdU incorporated into the
chromosomal DNA of Physarum during the S phase of the
cell
cycle
is
the
cause
for
the
observed
enhanced delay in the UV-irradiated system. The incorporated BrdU as such also causes some delays, particularly in
those plasmodia treated with this analogue during early S
phase.
The extra sensitivity seen in the plasmodia treated with
BrdU during the early part of the S phase (significantly more
than that seen with late S phase treatments) is understandable in view of the reported master-initiation of
majority of the chromosomal replicons towards the early
1128
part of the S phase and the increasing distances between
regions in actual replication at later times in the S phase in
the plasmodia of Physarum23. In fact, it is known that approximately 80% of the nuclear DNA of the plasmodia is
replicated during the first 1 h 30 min of the S phase
and much of the remaining 20% during the latter part of the
S phase26. This way a higher rate of incorporation of BrdU
into the chromosomal DNA is expected during early S
phase.
Figure 1. Effect of late G2, UV-irradiation on the mitotic cycle of
BrdU substituted (at different phases of the cell cycle) plasmodia of
Physarum polycephalum.
Each of the plasmodium in a set of sister plasmodia, made from
pooled microplasmodial suspension, was cut into 4 sectors at the
metaphase of the second PFM, and immediately after this, two of
the sectors (sectors are synchronous with respect to each other) were
pulse-treated for 2 h, during early S phase, with BrdU (5 µg ml–1 of
SDM). After the BrdU pulse and serial changes in fresh SDM to wash
off the unincorporated analogue, both the control and the BrdUsubstituted sectors were grown on fresh SDM till late G2. At approximately 30 min before their respective third PFM, one control
(BrdU unsubstituted) and one BrdU-substituted sector from each
plasmodium were UV-irradiated. The time of mitosis in all the sectors was noted and the mitotic delay due to the perturbations (i.e.
BrdU alone, UV alone and BrdU + UV) was calculated with respect to
their respective control sector (untreated and unirradiated).
Keeping the basic scheme of the experiment as above, 2 h BrdU
pulse treatment was given during other phases of the cell cycle (late
S, early G2 and late G2), followed by irradiation in late G2. In all
these cases the mitotic delay due to the perturbations was calculated
with respect to the respective control. Since 5 µg ml–1 of BrdU was
found to have no mitotic delaying effect when supplied during late S
and G2 phases, still higher concentrations (20 and 40 µg ml–1 ) were
used for these phases.
Values obtained from two plasmodia of each category have been
averaged out and plotted in the bar diagram (Figure 1). Inset shows a
projection of the values from four different plasmodia of the early S
phase category, to highlight the extra sensitivity obtained here.
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
ng early S
olycephan of BrdU
the points
h with 3 Hle; BARC,
re washed
plasmodia.
dium, and
or 2 h on
of the secthe third
etermining
The diluDNA synwe have
DNA/104
nthesis, as
not possiDNA was
al.36 , and
filters and
, POPOP
ed out and
s. Samples
after secd (c′) the
RESEARCH COMMUNICATIONS
The enhancement of the G2-phase, UV-induced mitotic
delay, because of S phase incorporation of Brdu into the
chromosomal DNA of the plasmodia, is in agreement with
the known extra vulnerability of BrdU-substituted DNA to
UV32, apparently because of the production of DNA strand
breaks and also DNA–DNA and DNA–protein crosslinkings in such systems33,34. Although G2-phase UVirradiation as such is highly inhibitory to RNA and protein
synthesis 18, it is known from earlier studies that additional
inhibitions were not observed because of BrdU incorporation35. At the concentration (5 µg ml–1) used to obtain extra
mitotic delay, DNA synthesis was also not affected (Figure
2). As noted earlier, there was no enhancement in late G2,
UV-induced mitotic delay, when BrdU treatment was carried
out during the other phases of the cell cycle (Figure 1). The
results of the present experiments clearly show that UVinduced alterations of chromosomal DNA, as late as G2prophase transition period, can inhibit and postpone mitosis. This could well be because of the need for a signal
based on the status of chromosomal DNA (undamaged) to
start the process of mitosis8,9,12,13. It is already known that
the DNA synthetic phase in the plasmodia is highly susceptible to UV-irradiation and maximum mitotic delay is obtained when irradiated during early S phase21. However, in
this study we have been able to show the importance of the
integrity of chromosomal DNA as such, in initiating the
process of mitosis in this organism, which is quite independent of DNA synthesis. The idea behind these experiments was to show the elegance of the experimental system
for demonstrations of this nature, and to suggest that the
natural
synchrony
obtained here could be utilized in future molecular level investigations on checkpoints linking chromosomal DNA to
G2/M transitions.
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20. Kumar, P. R. M. and Nair, V. R., Biomed Lett., 1993, 48, 137–
143.
21. Devi, V. R., Guttes, E. and Guttes, S., Exp. Cell Res., 1968, 50,
589–598.
22. Jayasree, P. R. and Nair, V. R., J. Biosci., 1991, 16, 1–7.
23. Funderud, S., Andreassen, R. and Haugli, F., Nucleic Acids Res.,
1978, 5, 3303–3313.
24. Kinsella, T. J., Mitchell, J. B., Russo, A., Aiken, M., Morstyn,
G., Hsu, S. M., Rowland, J. and Glatstein, E., J. Clin. Oncol.,
1984, 2, 1144–1150.
25. McCormick, J. J., Marks, C. and Rusch, H. P., Biochim. Biophys.
Acta, 1972, 287, 246–255.
26. Pierron, G., in The Molecular Biology of Physarum polycephalum (eds Dove, W. F. et al.), Plenum Press, New York, 1986,
pp. 67–77.
27. Evans, T. E., in Cell Biology of Physarum and Didymium (eds
Aldrich, H. C. and Daniel, J. W.), Academic Press, New York,
1982, vol. 2, pp. 371–386.
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(ed. Presscott. D. M.), Academic Press, New York, 1964, vol. 1,
pp. 9–41.
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Presscott. D. M.), Academic Press, New York, 1964, vol. 1, pp.
43–54.
30. Jagger, J. (ed.), Introduction to Research in Ultraviolet Photobiology, Prentice Hall, New Jersey, 1967.
31. Devi, V. R. and Guttes, E., Radiat. Res., 1972, 51, 410–430.
32. Murray, V. and Martin, R. F., Nucleic Acids Res., 1989, 17,
2675–2691.
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33. Smith, D. W. and Hanawalt, P. C. (eds), Molecular Photobiology: Inactivation and Recovery, Academic Press, New York,
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ACKNOWLEDGEMENTS. We thank Dr M. V. Joseph, Head and
Co-ordinator, Department of Biotechnology, Prof. U. V. K. Mohammed, Head of the Department of Zoology, University of Calicut,
and Prof. P. A. Wahid, Radiotracer Laboratory, Horticultural College, Thrissur for permission to use their departmental facilities for
this work. Financial assistance to P.R.J. by the Council of Scientific
and Industrial Research, Govt. of India is gratefully acknowledged.
Received 2 August 1999; revised accepted 13 January 2000
Agrobacterium-mediated genetic
transformation and regeneration
of transgenic plants from cotyledon
explants of groundnut (Arachis hypogaea L.) via somatic embryogenesis
P. Venkatachalam†, N. Geetha, Abha Khandelwal,
M. S. Shaila and G. Lakshmi Sita*
Department of Microbiology and Cell Biology, Indian Institute of
Science, Bangalore 560 012, India
†
Rubber Research Institute of India, Kottayam 686 009, India
An efficient transformation protocol was developed for
groundnut (Arachis hypogaea L.) plants. Precultured cotyledons were co-cultured with Agrobacterium tumefaciens
strain LBA 4404 harbouring the binary vector pBI121
containing the uidA (GUS) and nptII genes for 2 days and
cultured
on
an
embryo
induction medium containing 0.5 mg/l NAA, 5.0 mg/l BAP,
75 µ g/ml kanamycin and 300 µ g/ml cefotaxime. The putatively transformed embryos were transferred to the medium with reduced kanamycin (50 µ g/ml) for further
development. Prolific shoots developed from these embryos
on a MS medium containing 0.5 mg/l BAP and 50 µ g/ml
kanamycin with a transformation efficiency of 47%. The
elongated kanamycin-resistant shoots were subsequently
rooted on the MS medium supplemented with 1.0 mg/l IBA.
The transgenic plants were later established in plastic
cups. A strong GUS activity was detected in the putatively
transformed plants by histochemical assay. Transformation
was confirmed by PCR analyses. Integration of T-DNA into
nuclear genome of transgenic plants was further confirmed by Southern hybridization with nptII gene probe. A
large number of transgenic plants were obtained in this
study. This protocol allows effective transformation and
quick regeneration via embryogenesis.
GROUNDNUT or peanut (Arachis hypogaea L.) is one of the
principal economic oilseed legumes and is largely cultivated
in many tropical and subtropical regions of the world. The
seeds are mostly used to supply vegetable oil, carbohydrates and proteins for human as well as animal consumption. Crop improvement by conventional breeding in this
important oilseed crop is not as rapid as envisaged to meet the demands of increasing population, especially in seed quality improvement and developing virusand insect-resistant varieties. There is an urgent need to
improve several commercially grown varieties in India and
elsewhere. Tools of genetic engineering can be exploited as
an additional method for introduction of agronomically useful traits into established cultivars. Major seed proteins of
groundnut as well as of other leguminous crop species, are
deficient in the essential sulphur containing amino acid
*For correspondence. (e-mail: sitagl@mcbl.iisc.ernet.in)
1130
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
RESEARCH COMMUNICATIONS
methionine. Efforts are already made in this direction1. There
are a few reports on transformation in some varieties of
groundnut with marker genes2–4 as well as one or two desirable genes like 2S albumin gene1 and Bt gene5. However, to
accomplish
this
and
other
genetic engineering objectives, an efficient gene delivery to
isolated tissues and the subsequent regeneration of transformed plants must still be solved for commercially important, generally recalcitrant, groundnut cultivars1.
Although the transfer of foreign genes into the genome
of groundnut calli was previously achieved with hypocotyl
explants and other seedling explants, only a few of these
reported the production of whole transgenic plants. This
was mainly due to difficulties in transforming groundnut
cells at sufficiently high frequencies. Although there are
several approaches of developing transgenic plants, somatic embryogenesis has great potential in terms of high proliferation rates. Genetic transformation through somatic embryogenesis has not yet been reported in this oilseed crop.
For successful introduction of desirable traits into the extensively cultivated varieties, efficient regeneration protocols as well as a gene delivery system need to be developed
either by Agrobacterium-mediated gene transfer or the
bombardment method. In this paper, we report successful
transformation of groundnut by somatic embryogenesis
with uidA and nptII genes.
Seeds of A. hypogaea L. cv. TMV-2 were obtained from
the Agricultural College and Research Station, Tamil Nadu
Agricultural University, Tiruchirapalli, India.
Seeds were surface disinfected with 5% Bavistin for
15 min, thoroughly washed with running tap water. Then
they were sterilized with 0.1% (W/V) aqueous mercuric chloride solution for 7 min and washed with sterilized distilled
water. Twenty seeds per plate were germinated aseptically in
90 × 15 mm petri dishes containing 40 ml of seed germination
medium
composed
of
the
MS
basal
medium6 consisting of 3% (W/V) sucrose and 0.8% (W/V)
agar. Seeds and all in vitro plant materials were incubated at
25 ± 2°C under a 16 h photoperiod. Light was provided by
cool white fluorescent lamps with an intensity of 60 µE m–
2 –1
s . Cotyledon explants from 7-day-old seedlings were separated and used as explants for transformation experiments.
Cotyledon explants were cultured on the embryo induction
medium which consisted of the MS medium supplemented
with 0.5 mg/l NAA and 1–10.0 mg/l BAP (MS1).
LBA 4404 strain of A. tumefaciens harbouring a binary
plasmid pBI121 was used as the vector system for transfor-
mation. The vector map is given in Figure 1. Bacteria were
maintained on LB (ref. 7) agar plates (1% W/V tryptone,
0.5% W/V yeast extract and 1% W/V sodium chloride, pH
7.0) containing 50 µg/ml kanamycin and 25 µg/ml rifampicin.
For inoculation, one single colony was grown overnight on
liquid LB at 28°C with appropriate antibiotics.
The explants were precultured for 2 days on the embryo
induction medium prior to co-cultivation with bacterial culture collected at late log phase (A600 0.6). The cotyledons
(300 explants) were gently shaken in the bacterial suspension for about 10 min and blotted dry on a sterile filter paper.
Afterwards,
they
were
transferred
to
the
medium and co-cultivated under the same condition of the
preculture period (16 h photoperiod of 60 µE m–2 s –1) for 2
days at 25 ± 2°C. After co-culture, the explants were washed
in the MS liquid medium, blotted dry on a sterile filter paper
and
transferred
to
the
embryo
induction
medium (MS1) with antibiotics (75 µg/ml kanamycin and
300 µg/ml cefotaxime). Three subcultures are usually needed
for the elimination of escapes. Later, the concentration
of kanamycin was reduced to 50 µg/ml and completely devoid of cefotaxime.
After 4 weeks, the growing embryos were excised from
the primary explant and subcultured into a fresh embryo
proliferation medium containing 0.5 mg/l BAP and 50 µg/ml
kanamycin (MS2). The green healthy shoots from the embryos were subjected to 2–3 more passages of selection by
repeated excision of buds and their exposure to selective
elongation medium (MS2). Healthy and elongated shoots
were rooted in the MS medium containing 1.0 mg/l IBA but
without kanamycin to facilitate rooting (MS3). Untransformed embryos do not withstand more than 25 µg/ml
kanamycin. All experiments were carried out in ten replicates
and the experiments were repeated at least three times,
keeping all the parameters unchanged.
The β-glucuronidase (GUS) histochemical assay was
used as a rapid way to detect the presence of the uidA gene
(GUS) in the putative transformants as described by Jefferson et al.8 using leaf segments in regenerated shoots from
explants.
Presence of GUS and nptII was confirmed by PCR
amplification of the uidA and nptII genes using two specific
primer sequences. Plant DNA for PCR analysis was isolated
as described by Edwards et al.9. Specific primers
for gus (uidA) primer sequences (5′–3′) were TTC GCG TCG
GCA TCC GCT CAG TGG CA and GCG GAC GGG TAT CCG
GTT CGT TGG CA. The nptII primer sequences (5′–3′) were
Figure 1. Diagrammatic representation of the pBI121 vector. The NOS-P, nopaline synthase
gene promoter and NOS-T, nopaline synthase gene terminator signal control the expression of
the nptII gene.
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
1131
RESEARCH COMMUNICATIONS
GAG GCT ATT CGG CTA TGA CTG and ATC GGG AGG
GGC GAT ACC GTA. Each PCR reaction was performed in
25 µl (total volume) of reaction mixture consisting of 10X
reaction buffer, 150 ng DNA, 200 mM dNTPs, 25 mM MgCl2,
100 ng of each primer DNA and 1 unit of Taq DNA polymerase. PCR was carried out in a thermal cycler under the following conditions: 94°C for 4 min as preheating, then 35
cycles of 94°C denaturing for 1 min, 58°C annealing for
1 min and 72°C extension for 1 min and another 10 min at
72°C final extension for uidA gene detection, 94°C denaturing for 4 min as preheating, then 35 cycles of 94°C denaturing for 1 min, 55°C annealing for 1 min and 72°C synthesis
for 1 min and 10 min at 72°C final extension for nptII gene
detection. Amplified DNA fragments were electrophoresed
on 1.2% agarose gel and detected by ethidium bromide
staining, photographed under ultraviolet light.
Total genomic DNA was isolated from fresh leaves of
transformed and untransformed (control) plants using the
CTAB (cetyltrimethylammonium bromide) method. Southern
blot hybridization analysis was carried out using standard
procedures7,10. Ten µg of total genomic DNA from transgenic plants and untransformed control plant was digested
with HindIII. 2 µg of plasmid DNA was digested with SmaI
and SacI to release the gus fragment which was used to
prepare the probe.
We have standardized plant regeneration in groundnut
earlier by somatic embryogenesis11. After 3 weeks of culture, green multiple somatic embryos formed along the cut
surface of the distal end of the cotyledons on the MS
medium containing 0.5 mg/l NAA and 1–10.0 mg/l BAP.
Among the various concentrations of BAP used, BAP
5.0 mg/l and NAA 0.5 mg/l (MS1) were found to be the best
for maximum frequency of embryo formation (data not
shown). This combination was routinely used for
the transformation experiments. Each explant produced 20–
30 somatic embryos (Figure 2 a), in some instances up to 60
embryos could be seen. Early separation shows the bipolar
nature of the embryos (Figure 2 b) and these germinate with
proper tap root if removed early (Figure 2 c). Histological
observations also confirmed the nature of the bipolar embryos. If somatic embryos are not separated from the original cotyledon explants at an early stage, they tend to
develop into profuse shoots (Figure 2 d ) without root development. Embryos have to be separated for proper tap
root development. However, separation of the embryos from
clusters originating from the cotyledon explants was impossible without damaging the embryos as they fused with
each other and to parent cotyledon explants, and have no
discernible radicles. It was easier to allow the embryos to
develop shoots for subsequent rooting by transferring to a
medium with reduced BAP (MS2). This has been reported
for groundnut12 as well as eastern redbud13 wherein multiple
shoots
developed
from
the
somatic embryos. Single shoots can be excised from shoot
clumps for rooting (MS3) to develop individual plants.
Similar observations have been made with other legumes
1132
like Albizzia lebbeck14 and rosewood15. In general, somatic
embryo morphology affects germination and plant conversion percentage in groundnut12,16. Multiple shoot formation
from somatic embryos could be an alternative method of
regeneration, especially if the somatic embryos are abnormal
or there is a low percentage of germination.
Kanamycin sensitivity of cotyledon explants was assessed prior to Agrobacterium transformation, to determine
the concentration of kanamycin needed for effective growth
of transgenic plants. At 50 µg/ml, kanamycin caused chlorosis and eventual necrosis in all explants by the end of the
fourth week. Concentrations of 75 µg/ml and 100 µg/ml
kanamycin completely inhibited embryo formation. Kanamycin concentration of 125 µg/ml and higher caused all explants to become necrotic by the third week of culture.
Higher concentrations of 125 µg/ml kanamycin even killed
almost all the cotyledon explants. In all previous reports of
Agrobacterium-mediated transformation of groundnut,
kanamycin
concentration
of
50–100 µg/ml was used in both the selection and regeneration medium2,3,17. In the present study, slightly higher kanamycin concentration (75 µg/ml) was used for the initial selection of transformants to prevent possible escapes.
Subsequently, lower kanamycin concentration of 50 µg/ml
was used to enhance the embryo proliferation. According to
Pena et al.18, application of higher concentrations of the
selection agent is the best way to eliminate the untransformed cells or organized tissues.
One of the critical factors in achieving high frequencies
of transformation in groundnut is related to preculture of
explants on the embryo induction medium prior to cocultivation. Cotyledon explants were precultured for 0, 1, 2,
3, 4 and 5 days and transformation frequency was calculated by their embryo-forming ability in the selection medium (data not shown). When cotyledon explants were
precultured on the regeneration medium for 2 days, the hypersensitivity response was reduced compared to cocultivation with Agrobacterium without preculture. Preculture of the explants in the regeneration medium for 2 days
prior to co-cultivation was found to be optimum for efficient
transformation.
After selection through a two-day preculture, healthy
cotyledons were infected and co-cultivated with A. tumefaciens LBA4404 harbouring pBI121 vector. During the initial
two days on the non-selective embryo induction medium, all
co-cultivated and control explant materials retained a
healthy green colour. After transfer to the embryo induction
medium with 75 µg/ml kanamycin and 300 µg/ml cefotaxime
(selection medium) control explants and some of the cocultivated explants became completely necrotic within 3–4
weeks. In contrast, control cotyledon explants maintained
on
non-selective
embryo
induction medium (without kanamycin) exhibited embryogenesis (86.6%) by the end of the fourth week in culture.
Embryos formed directly around the distal end of the cotyledons without intervening callus and the maximum transCURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
Figure 2. Different developmental stages of groundnut transgenic plants. a, Induction of embryos; b, Single bipolar embryo; c, Plantlet derived from embryo with typical tap root; d, Regeneration and shoot formation from the transformed embryos on selection medium; e, Rooted
transformed plantlet; f, Well-developed transformed plantlet established in soil; and g, Leaves from transgenic plants showing GUS activity
(blue colour).
RESEARCH COMMUNICATIONS
formation frequency was 47% (Table 1). The embryos were
maintained by regular subculturing on the MS1
medium at 3-week intervals. After 3 passages on the MS1
medium, explants with somatic embryos were subcultured
onto the MS2 medium for proliferation of shoots. On the
other hand, controls did not require more than 4–6 weeks
for transfer into the MS2 medium for proliferation. After
prolonged culture period on the same medium, multiple
shoots were regenerated from embryos on the MS2
medium (Figure 2 d ). Similar results were also reported in
eggplant19. Shoots developed from an individual embryo
were sliced and subcultured on the MS medium with a low
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
level of BAP (0.2 mg/l) containing 50 µg/ml kanamycin for
proliferation. The kanamycin concentration was lowered to
50 µg/ml at the third subculture to reduce antibiotic stress
on the developing shoot buds. They were multiplied in vitro in the presence of kanamycin. Most of the transgenic
clones appeared morphologically normal in comparison with
the untransformed plants. The putative transformed shoots
which attained 2–3 cm in length were excised and then
transferred
for
rooting
to
the
MS3
medium (Figure 2 e). Fifty plants were selected and these
were transferred to the soil. Figure 2 f shows the well- established transgenic plant. The survival rate was
90–95%.
1133
RESEARCH COMMUNICATIONS
The transformation efficiency obtained in this study is as
high as that reported earlier using groundnut leaf tissue2,3.
Eapen and George2 observed an average of 6.7% of shoot
regeneration on the selection medium containing 50 µg/ml
kanamycin. Cheng et al.3 reported that the frequency of
transformed fertile plants was 0.2 to 0.3% of the leaf explants inoculated. In the previous reports, transformation
efficiency was low, as the explants were co-cultivated without preculturing. In the present study, an important factor
which enhanced the transformation efficiency of groundnut
was the 2-day preculture of the explants, which probably
served to reduce wound stress and increased the number of
competent cells at the wound site. A similar result was also
reported in other species by Akama et al.20 and Muthukumar et al.21. The results indicated that the efficiency of
shoot regeneration was dependent on the explant type. Recent success in the production of transgenic plants using
Agrobacterium in cowpea21 and chickpea22 was based on
explants like hypocotyls, epicotyls and cotyledons. These
results suggest that under the experimental conditions used
in the present study cells of cotyledons may havecomeence
or ‘shoot induction signals’ and that the competence may
viously by Eapen and George2 and Cheng et al.3 in groundnut.
Presence of the uidA and nptII genes in the plant
genome was confirmed by PCR analysis with specific primers using DNA from leaves. PCR analysis revealed that
0.53 kb uidA and 0.8 kb nptII DNA fragments amplified from
genomic DNA of all putative transgenic plants. However, no
uidA and nptII PCR products were seen for untransformed
control plants. Figure 3 a shows that all the samples of
transgenic plants (lanes 4–9) gave the predicted size of
DNA fragment of 0.53 kb of uidA gene. No band was detected in the DNA sample from an untransformed control
plant (lane 3). The 0.53 kb DNA fragment was also amplified
from the pBI121 plasmid as positive control (lane 2). DNA
products with the expected size of 0.8 kb were amplified
from total genomic DNAs of the putative transgenic plants
(Figure 3 b, lanes 5–11). These DNA fragments were not
Table 1. Transformation frequency of putatively transformed
embryos from groundnut cotyledon explants on MS medium containing
NAA (0.5 mg/l), BAP (5.0 mg/l), kanamycin (75 µg/ml) and
cefotaxime (300 µg/ml)
Experiment
Control*
Control**
Expt. 1
Expt. 2
Expt. 3
Expt. 4
Expt. 5
No. of
explants
co-cultured
No. of
explants
responded
Frequency
of embryos
Average no.
of embryos/
cotyledon
75
86
45
36
51
39
40
65
0
17
15
24
17
15
86.6±4.3
00.0±0.0
37.7±2.6
41.6±3.8
47.0±4.2
43.5±3.6
37.5±3.3
65.0±3.7
00.0±0.0
52.0±3.4
48.0±3.1
56.0±4.7
32.0±2.8
41.0±3.9
*No co-cultivation, non-selective medium (without kanamycin).
**No co-cultivation, selection medium (with 75 µg/ml of kanamycin).
vary in different groundnut explants.
The regenerated embryos or leaves were subjected to in
situ GUS assay. The expression of uidA gene was verified
by histochemical staining of the leaf of the transgenic
plants. The nptII positive regenerants showed the typical
indigo blue colouration of X-Gluc treatment, while the negative ones did not. Also, more than 45% of the regenerants
were GUS positives. Young leaves were more densely
stained than other tissue of the plant (Figure 2 g). This
seems to be a typical expression pattern of the CaMV 35S
promoter regulated uidA gene in young tissues 8. Leaf tissues from untransformed plants did not show GUS activity.
These results and the extensive GUS expression in tissues,
clearly demonstrate the stability of the inserted genes in the
transformed plants. GUS expression was also reported pre1134
Figure 3. PCR analysis of transgenic shoots by amplication of the
uidA (GUS) and nptII genes from total plant DNA extracts. a, Detection of the uidA (GUS) gene. Lane 1, DNA size marker; lane 2,
Plasmid PBI121 (positive control); lane 3, Untransformed plant
(negative control); lanes 4–9, Transformed plants; and b, Detection
of the nptII gene. Lane 1, DNA size marker; lanes 2, 4, Untransformed plants (negative control); lane 3, Plasmid pBI121 (positive
control); lanes 5–11, Transformed plants.
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
RESEARCH COMMUNICATIONS
Figure 4. Southern blot analysis of transgenic groundnut plants
previously identified by PCR analysis. Total genomic DNA (10 µg)
digested with HindII was hybridized with random primed α32P labelled
GUS probes. Lane M, Labelled DNA size marker; lanes 1–10, Genomic DNA from independent lines of transformed plants.
detected in the untransformed control plant (Figure 3 b,
lanes 2, 4). As a positive control, the nptII gene products
were also amplified from the pBI121 plasmid (Figure 3 b
Lane 3). Based on the resistance to kanamycin and PCR
detection, presence of both uidA and nptII genes was confirmed in the transgenic groundnut plants. In the previous
reports on transgenic groundnut plants obtained by microprojectile bombardment1,23 and Agrobacterium-mediated
transformation2,24,25 foreign gene products were not analysed by the PCR method.
The integration of the gus reporter gene into the transformed plant genomic DNA was further confirmed by
Southern blot hybridization analysis. The genomic DNA
from randomly selected GUS positive transgenics and one
untransformed control plant was digested with HindIII
which cuts at only one site located between the uidA (GUS)
and nptII genes in the T-DNA insert of pBI121 plasmid. The
gus gene was used as a probe. The gus DNA probe hybridized to DNA from transgenic plants (Figure 4) showed integration at different positions ranging from 2.5 kb to 23 kb.
Few lines have shown single integration (lanes 1–4) and
others have shown multiple integration (lanes 5–10). The
DNA from the untransformed control plant did not show
any signal (data not shown); the result indicated that the
gus gene was integrated into the groundnut genome. A
similar type of multiple copies of gene integration with the
groundnut genome was reported by Ozias-Akins et al.26 and
Cheng et al.3.
With the exception of the report by McKently et al.17,
transformation frequencies for groundnut appear to be substantially lower in other reports of transformation. In this
study protocol modification in the form of kanamycin concentration and preculture treatment substantially enhanced
the transformation efficiency in groundnut and may be a
major reason for the higher frequency obtained in this study
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
compared to the other reports on Agrobacterium-mediated
transformation
of
this
species.
The
results presented also demonstrate a simple and highly efficient system for producing peanut somatic embryos from
cotyledon explants. The system does not require maintenance of embryogenic callus or apical meristem excision as
in most previous procedures on genetic transformation of
peanut, which are labour-intensive techniques. Moreover, in
many cases the use of these explant materials results in the
production of sterile plants and/or somaclonal variants. In
the present study fertile plants were obtained. In addition,
transformation was done via Agrobacterium and not by
particle bombardment as was done in most of the previous
reports which is an expensive method compared to Agrobacterium-mediated transformation. The protocol can be
used efficiently for the introduction of more desirable genes
and
is
currently
being used for producing transgenic plants with desirable
genes.
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RESEARCH COMMUNICATIONS
23. Schnall, J. A. and Weissinger, A. K., Plant Cell Rep., 1993, 12,
316–319.
24. Mansur, E. A., Lacorte, C., deFreitas, V. G., deOliveria, D. E.,
Timmerman, B. and Cordeiro, A. R., Plant Sci., 1993, 89, 93–
99.
25. Lacorte, C., Mansur, E. A., Timmerman, B. and Cordeiro, A. R.,
Plant Cell Rep., 1991, 10, 354–357.
26. Ozias-Akins, P., Schnall, J. A., Anderson, W. F., Singsit, C.,
Clemente, T. E., Adang, M. J. and Weissinger, A. K., Plant Sci.,
1993, 93, 185–194.
ACKNOWLEDGEMENT. P. V. is grateful to Department of Biotechnology DBT, Govt. of India, New Delhi for the financial assistance in the form of post-doctoral fellowship.
Received 14 October 1999; revised accepted 29 February 2000
Blinding induces copulatory activity
among inactive males of South Indian
gerbils
Biju B. Thomas and Mathew M. Oommen*
Department of Zoology, University of Kerala, Kariavattom,
Thiruvananthapuram 695 581, India
The effect of blinding on reproductive behavioural physiology of male South Indian gerbils (Tatera indica cuvieri)
was assessed. Blinding of reproductively inactive adult
males resulted in 60% of them exhibiting sexual activity.
Reproductive behavioural variables studied, such as intromission frequency, thrust frequency, ejaculation frequency
and postejaculatory copulation frequency were exhibited to
the level of reproductively active males. Such reproductive
activation was also reflected in the epididymal sperm count.
These studies reveal that blinding (constant darkness) may
have a stimulatory effect in the reproduction of the tropical
rodent T. indica cuvieri.
A MONG mammals, photoperiodic cues determine the periods of sexual activity and inactivity especially in temperate
species1,2. Several investigations have been carried out to
establish the influence of photoperiodic changes on reproduction. In hamsters, exposure to short photoperiod resulted in gonadal regression and loss of sexual behaviour3,4.
Constant
darkness
(blinding)
also
influences
reproduction of male rodents. According to Hard and Larsson 5, though mating was not fully impaired, the intervals
between ejaculations were shorter for blinded male laboratory rats. In laboratory rats whose visual function was destroyed neonatally, a deficit in copulatory behavioural
variables coupled with a decreased androgen level were
observed6. Lack of responsiveness of the reproductive sys-
tem to photoperiodic changes has been reported for male
laboratory rats7 and prairie voles8. Among tropical rodents
gonadal regression consequent to short photoperiod has
been reported for Indian palm squirrel Funambulus pennanti 9. Another tropical rodent, the cane mouse (Zygodontomys brevicauda), however, was not responsive to
variation in photoperiod with regard to the weight of testis
and seminal vesicle10. Sinhasane and Joshi11 reported that in
Indian desert gerbil, Meriones hurrianae, constant light
resulted in reproductive inhibition whereas constant darkness was stimulatory.
The South Indian gerbil Tatera indica cuvieri is a burrow
dwelling tropical rodent with adult males and females occupying separate closed burrows12. They were found to be
breeding throughout the year13. Our field observations
show that the animal is nocturnal with foraging habits during the night after the onset of darkness. The male
reproductive behaviour pattern of this rodent has been described14. The mating pattern consists of a courtship fighting (upright boxing), several mountings by the male, and
intromissions. Ejaculation takes place during one of the
intromissions. Typically a male exhibits one to four ejaculations during a complete mating period, and following the
final ejaculation the male continued the copulatory activity
without any ejaculation. However about 50% of the males
were found to be reproductively inactive and did not exhibit
these mating sequences15. Though photoperiodic alterations do influence the reproductive phenomenon of rodents, the response depends on the experimental schedule
and the species employed. Hence further investigations on
the male reproductive mechanism of this rodent is worth
undertaking. As a first step we carried out the present study
by assessing the impact of blinding on the reproduction of
adult reproductively inactive male gerbils.
Gerbils were collected regularly from the field throughout
the year, brought and acclimatized to the laboratory conditions under light/dark cycle of 12 : 12 h. Adult animals (165–
185 g) were selected from among a stock of healthy ones.
Only those males (n = 8) which did not copulate in several
mating tests (range 4–6) were employed for blinding experiments. These reproductively inactive males did not show
any mating behaviour other than random mounting attempts. Animals were housed individually in polypropylene
cages provided with bedding of wood shavings and paper
bits. They were maintained on laboratory rat feed and water
ad libitum.
Blinding (constant darkness) was effected according to
the method described by Thomas and Oommen16. For this,
0.2–0.3 ml of absolute alcohol (Merck, Germany) was administered into the vitreous chamber of the eye under mild
etherization. This volume ensured a rapid and
uniform distribution of the alcohol within the vitreous
chamber under a pressure, with the excess flowing out.
Following this the iris turned white, and an antiseptic eye
ointment was applied to prevent any possible infection.
*For correspondence.
1136
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
RESEARCH COMMUNICATIONS
Table 1.
Reproductive behavioural variables of reproductively inactive, blinded and reproductively active males of Tatera indica cuvieri
Courtship
frequency
Courtship
duration
Inactive males before
blinding (n = 9)
3.7 ± 0.9
50.3 ± 11.6
511.1 ± 218.6
0.4 ± 0.2
0.9 ± 0.4
Blinded males (n = 8)
2.6 ± 1.4 NS
55.1 ± 15.5 NS
299.5 ± 171.2 NS
6.2 ± 0.6*
23.8 ± 1.0*
2.8 ± 0.2**
6.8 ± 2.3 NS
Reproductively active
males (n = 7)
5.0 ± 0.8
76.1 ± 15.2
414.5 ± 126.2
4.6 ± 0.3
19.6 ± 1.2
2.3 ± 0.2
9.1 ± 1.6
Group
Mount
latency
Intromission
frequency
Thrust
frequency
Ejaculation
frequency
0
Postejaculatory
copulation frequency
0
Values are means ± SE; NS, Not significant (Wilcoxon–Mann–Whitney test, two-tailed).
*Blinded males vs inactive males before blinding, p < 0.05.
**Blinded males vs reproductively active males, p < 0.05.
Standard male copulatory behavioural variables were
measured 12 days after blinding, employing healthy adult
females chosen from the main stock. For this, a receptive
female showing natural oestrus cycle was introduced into
the male cage and observations were recorded till the end of
the entire copulatory activity including the postejaculatory
copulations, or 60 min without any intromissions. Each male
was given three pre-experimental and three experimental
mating tests. Data on epididymal sperm count were taken by
sacrificing the males after 90 days of experiment. For this the
epididymis were dissected out, finely minced and lightly
homogenized in a 10 ml medium (20 ml Triton-X-100 in
100 ml saline) for one minute to obtain a complete sperm
suspension, and centrifuged at 1000 rpm for one minute to
separate the tissue debris. Sperm count in the supernatant
was determined using a counting chamber. Statistical methods employed include Student’s t-test and Wilcoxon–
Mann–Whitney test 17.
Blinding resulted in sexual activity being induced among
the formerly reproductively inactive adult males. A considerable proportion of such males (5/8) exhibited ejaculation.
The reproductive behavioural variables of the blinded males
were more or less equal to that of the reproductively active
males (Table 1). This impact was also discernible at the level
of the reproductive system as evidenced by an increase in
the epididymal sperm count (Figure 1).
The reproductively inactive males, on blinding, attained
active reproductive status. Reproductive behavioural variables such as intromission frequency, thrust frequency and
ejaculation frequency exhibited by the blinded males were
comparable to that of the reproductively active males suggesting that the blinded males were capable of inducing
pregnancy. This is further evidenced from the postejaculatory copulation frequency since postejaculatory
copulation is reported to have a major role in inducing
pregnancy in this species14. Blinding also leads to an
increase in the epididymal sperm count in them. Considering
the fact that photic alterations can bring about changes in
the androgen sensitivity of the brain18, the stimulatory effect
of
blinding
can
be
ascribed
to
be
androgen-related.
These responses of T. indica cuvieri to blinding is worth
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
Figure 1. Effect of blinding on the epididymal sperm count (Student’s t-test).
mentioning, as constant darkness in several instances resulted in deficit in copulatory behaviour and
gonadal regression of other rodents6,19,20. Increased exposure to darkness also brought about deficit in reproductive
behaviour and delayed sexual maturity3,21,22. These suppressive effects on reproduction by increased darkness is attributed to an antigonadal effect of the pineal gland and its
secretion, melatonin23. Comparable to our observations,
increase in reproductive organ weight and epididymal sperm
count consequent to darkness is reported for another Indian
gerbil
M.
hurrianae
for
which
the
pineal hormone, melatonin, is ascribed a role11. Since the
action of the pineal gland among mammals is closely
related with the photoperiodic history of the concerned
species24, the present stimulatory effect of blinding can also
be pineal-mediated.
1137
RESEARCH COMMUNICATIONS
1. Nelson, R. J., An Introduction to Behavioural Endocrinology,
Sinauer Associates Inc. Publishers, Sunderland, 1995.
2. Pevet, P., Comparative Physiology of Environmental Adaptations, S. Karger, Basel, 1987, vol. 3.
3. Pospichal, M. W., Karp, J. D. and Powers, J. B., Physiol. Behav.,
1991, 49, 417–422.
4. Schilatt, S., Niklowitz, P., Hoffman, K. and Nieschlag, E., Biol.
Reprod., 1993, 49, 243–250.
5. Hard, E. and Larsson, K., J. Comp. Physiol. Psychol., 1968, 66,
805–807.
6. Hsu, C., Huang, H-T., Hsu, H-K., Yu, J. Y-L. and Peng, M-T., J.
Pineal Res., 1990, 8,107–114.
7. Wallen, E. P., DeRosch, M. A., Thebert, A., Losee-Olson, S. and
Turek, F. W., Biol. Reprod., 1987, 37, 22–27.
8. Nelson, R. J., Biol. Reprod., 1985, 33, 418–422.
9. Haldar, C. and Pandey, R., Indian J. Exp. Biol., 1988, 26, 516–
519.
10. Bronson, F. H. and Heidman, P. D., Biol. Reprod. 1992, 46,
246–250.
11. Sinhasane, S. and Joshi, B. N., Indian J. Exp. Biol., 1997, 35,
719–721.
12. Thomas, B. B. and Oommen, M. M., J. Bombay Nat. Hist. Soc.,
1997, 94, 149–151.
13. Thomas, B. B., Ph D thesis, University of Kerala, 1997.
Large palaeolakes in Kaveri basin in
Mysore Plateau: Late Quaternary fault
reactivation
14. Thomas, B. B. and Oommen, M. M., Acta Theriol., 1998, 43,
263–270.
15. Thomas, B. B. and Oommen, M. M., Mammalia, 1999, 63
(in press).
16. Thomas, B. B. and Oommen, M. M., Indian J. Exp. Biol., 1999,
37, 1201–1204.
17. Siegel, S. and Castellan, N. J. Jr., Nonparametric Statistics for the
Behavioural Sciences, McGraw-Hill, Singapore, 1988.
18. Hoffmann, K., in Hand Book of Behavioral Neurobiology (ed.
Aschoff, E. J.), Plenum Press, New York, 1981, vol. 4, pp. 449–
473.
19. Morin, L. P., Physiol. Behav., 1981, 27, 89–94.
20. Vaughan, M. K., Vaughan, G. M., Blask, D. E. and Reiter, R. J.,
Experientia, 1976, 32,1341.
21. Cherry, J. A., Physiol. Behav., 1987, 39, 521–526.
22. Honrado, G. I., Bird, M. and Fleming, A. S., Physiol. Behav.,
1991, 49, 277–287.
23. Bartness, T. J. and Goldman, B. D., Experientia, 1989, 45, 939–
945.
24. Ebling, F. J. and Foster, D. L., Experientia, 1987, 45, 946–954.
Received 20 December 1999; revised accepted 29 February 2000
ity of faults are responsible for the evolution of the > 1200 m
high Biligirirangan–Mahadeswaramalai Ranges in the east
and > 1800 m Central Sahyadri mountain in the west. These
high mountains bordering the Mysore Plateau enclose the
basin of the Kaveri, an antecedent river of
K. S. Valdiya*,† and G. Rajagopalan**
*Geodynamics Unit, Jawaharlal Nehru Centre for Advanced Scientific
Research, Bangalore 560 064, India
**Birbal Sahni Institute of Palaeobotany, Lucknow 226 007, India
Late Quaternary movements along the faults that demarcate tectonic boundaries of the N-S trending
terranes of Biligirirangan and Closepet Granite in the
Southern Indian Shield caused ponding of the River Kaveri
and its tributaries Suvarnavati, Kabini and Shimsha of the
Mysore Plateau. The resulting large lakes stretched tens
of kilometres upstream in their valleys. Approximately
10 m thick black carbonaceous clay, characterized by prolific calcareous nodules including rhyzocretions in the top
horizon, and covered locally by overbank fine sediments in
floodways, represents the lakes that came into existence in
the Late Pleistocene and lasted until Late Holocene. Subsequent fault movements were responsible for vertical displacement and attenuation of the lacustrine sediments. Ongoing movement is indicated by stream ponding in some
segments and accelerated erosion of the elevated blocks.
THE much-faulted Southern Indian Shield (Figure 1) has
time and again witnessed reactivation of faults of Precambrian antiquity1. Neotectonic activities on the multiplic-
†
For correspondence. (e-mail: valdiya@jncasr.ac.in)
1138
Figure 1. Much faulted Southern Indian Shield1 showing the faults
and shear zones that demarcate boundaries of Precambrian terranes.
The profile across the Southern Indian Shield is after Vaidyanadhan7 .
The Kaveri basin lies between the BR and central Sahyadri.
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
RESEARCH COMMUNICATIONS
considerable maturity which breaks forcibly through the
mountain barrier of the Biligirirangan–Mahadeswaramalai
Ranges2–9. The Mysore Plateau is drained by the rivers that
are strongly controlled by faults and shear zones10, as evident from the remarkably straight courses and abrupt deflections.
Neotectonic movements on the N-S as well as NE-SW
oriented fault caused blockade of streams, culminating in
the formation of lakes now represented by black carbonaceous clay riddled with calcareous concretions in the
upper part such as seen at Manchenabele in the Arkavati
River Valley, at Uchchivalase in the Antargange Hole and at
Vaddarakuppe in the Rayatmala stream (Figure 1), southsoutheast of Bangalore8.
The first author’s fieldwork in the Kaveri basin encompassing the talukas of Maddur, Kollegal, Chamarajanagar
Figure 2. Upstream of the active Kollegal–Iggaturu fault the Kaveri and its tributaries are characterized by nearly 10 m thick accumulation
of black carbonaceous clay deposited in the lakes resulting from stream blockade. Lakes were formed also in the Shimsha valley and its tributary Hulu Halla resulting from movements on faults that cross the streams (localities from where samples were collected for dating are shown
in boxes).
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
1139
RESEARCH COMMUNICATIONS
and T. Narasipura demonstrated that what has been described9 as ‘black soils in the form of horizontal sea-like surface almost devoid of elevation contrast and indicative of
their formation in shallow water environment’ actually represents lacustrine carbonaceous black clay resulting from
ponding of the River Kaveri and its tributaries
Suvarnavati, Kabini and Shimsha. The black clay grades
downwards and laterally into greyish-brown clay and mud.
Near stream channels the black clay is underlain–overlain
by as well as interfinger with fluvial silt and fine sand. The
top layer of the lacustrine clay is characterized by prolific
calcareous concretionary nodules, including rhyzocretions.
Interestingly, a large part of this region of sedimentary mantle had earlier been depicted as the basin in the Mysore
Plateau11. The present work shows that the lacustrine deposit is confined only to the floodways of
the rivers (Figure 2). The black carbonaceous clay transitionally overlie fluvial gravel and sand in channels but
rests directly on weathered countryrocks in wide valley
flanks. Finer sediments brought by recurrent floods cover
the lacustrine deposits as overbank alluvial sediments
closer to the channels. Likewise at the foot of the uplifted
hills eroded material – sheet wash – brought by hill torrents
conceal the lake clay. The lacustrine deposits form remarkably flat stretches of uniform elevation in the otherwise undulating terrain of the Mysore Plateau (Figure 3 a). These
are being intensely used for paddy and sugarcane cropping.
The Biligirirangan Range is demarcated by a series of N-S
trending faults registering oblique-slip displacement8. One
of these faults that passes by Kollegal and Iggaturu (Figure
2) demarcates the downstream limits of the deposits of black
clay filling the valleys of the Kaveri and its tributaries. A
linear series of ponds follows the fault line. Apart from the
abrupt termination of the black lacustrine clay and sharp
juxtaposition against brown alluvial coarser sediments,
there is marked deflection of all the rivers as they cross the
fault line. For example, the east-flowing Shimsha suddenly
runs southward at Iggaturu, the Hulu Halla swerves south
near Hadli, the Kaveri swings north-west of Saraguru and
near Hebsur (ESE of Chamarajanagar) and the Suvarnavati
deflects northward (Figure 2). Furthermore, there is accentuation of gully erosion and channel entrenchment on the
block east of the fault. Clearly the Kollegal–Iggaturu fault is
active and has caused uplift of the downstream eastern
block. The fault movements must have resulted in the
blockade of rivers and streams and consequent formation of
Figure 3. a, Flat stretch of black carbonaceous lacustrine clay succession near Nanjangud in the Kaveri Valley; b, Rayatmala stream at Vaddarakuppe has eroded its bank and exposed the full succession of the lake deposit. The basement gneisses overlain by fluvial gravel is succeeded
by 7 m thick succession of black clay. The top of the clay sequence is characterized by calcareous concretions; c, Closer view of the Vaddarakuppe palaeolake succession comprising predominant black clay resting on gneisses and grading upward into greyish-brown clay. The top is
characterized
by
calcareous concretions in black clay. Samples were collected from this succession at a fixed interval of ~ one metre.
1140
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
RESEARCH COMMUNICATIONS
lakes. The lakes stretch far upstream – 8 km up to ESE of
Chamarajanagar in the Suvarnavati, 14 km up to west of
Nanjangud in the Kabini, 25 km up to NW of T. Narasipura
in the Kaveri (Figures 2 and 3 a), up to Malavalli in the Hulu
Halla, and 10 km up to north of Maddur in the Shimsha
(Figure 2). The thickness of the black clay varies from less
than 2 m in the Shimsha valley to 9–15 m in the Suvarnavati
valley.
Similarly, the uplifts of the south-eastern block along the
NE-SW trending active faults resulted in the impoundment
in the two tributary streams of the Arkavati River –
the Antaragange and Rayatmala – giving rise to lacustrine
deposition at Uchchivalase and Vaddarakuppe, respectively
(Figures 1, 3 b, c and 4).
Radiocarbon dating of the palaeolake sediments has been
done by the second author. Samples of black clay rich in
organic matter were collected at intervals of one metre from
a nearly vertical section cut by the Rayatmala at Vaddarakuppe (Figures 3 b and 4). Radiocarbon dating of carbonaceous clay gives reliable ages if the organic matter
content is more than 2–3%. Intensely cultivated and irrigated almost round the year for cash crops, the lake sediments are in the disturbed condition. The sediment samples
were
treated
with
10%
HCl
solution
to
remove the carbonates. CO2 was obtained from organic matter in the sediment by dry combustion using a standard
procedure. Sample CO2 was synthesized to C2H2 in a carbide
reaction vessel supplied by Phonon Corporation, UK and
Figure 4. Antaragange and Rayatmala streams – tributaries of the
Arkavati River – were ponded due to movements on the NE-SW
oriented faults. Later movements on these and N-S faults uplifted and
truncated the lacustrine deposits.
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
then to benzene as described by Gupta and Polach12. Overall recovery of 75–85% was obtained consistently in CO2 to
C6H6 conversion. The 14C activity in benzene (with butylPBD scintillator, 15 mg/ml C6H6) was counted in the ultra
high sensitivity liquid scintillation counting system,
QUANTULUS 1200 of LKB Wallac. The background and
NBS oxalic acid standard (RM-49) counting rates in the system for 3 ml benzene sample in teflon vial are
0.453 ± 0.014 cpm and 30.73 cpm, respectively with a figure
of merit of 8854. The dated samples give consistent chronology of 14C ages (Figure 5). Noting that the thickness of
various lacustrine successions in different valleys in the
Kaveri basin varies uniformly between 7 and 10 m, it is assumed that they are the products of nearly contemporaneous fault reactivation. Being intensely cultivated and
irrigated practically round the year for cash crops, the flat
stretches of palaeolake sediments are in highly disturbed
and contaminated conditions.
The Vaddarakuppe succession (Figure 3 b) provides an
ideal setting for sample collections and the newly dug canal
near Homma (Figure 2) provides good exposure of the upper
part of the succession for sampling and dating. The life of
the
Vaddarakuppe
palaeolake
spanned
the
period from ~ 26,000 ± 825 to 11,800 ± 825 yr BP. Significantly, the basal samples from the depth of 7 m below the
surface of the Uchchivalase palaeolake are dated
26,470 ± 900 yr BP, indicating that both the lakes came into
existence at nearly the same time. East of Kollegal in the
Kaveri Valley, the black clay sample collected from a depth
of 25 cm below the surface at Muguru gave a date of
8290 ± 140 yr BP, while the one taken from just below the
surface near Chelukavadi is dated 4420 ± 110 yr BP. In the
Suvarnavati valley, samples collected from depths of 20 and
Figure 5. Plotting of measured radiocarbon (14 C) dates of samples
against their depths (below surface) gives a straight line for the age of
the Vaddarakuppe lake.
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RESEARCH COMMUNICATIONS
140 cm at Homma (Figure 2) yield ages of 3540 ± 170 yr BP
and 7850 ± 140 yr BP, respectively. The Muguru and Chelukavadi dates of the Kaveri sediments fall in the isochron of
the Vaddarakuppe samples. From the calibrated age range it
seems
that
the
end
of
the
lacustrine phase in the Kaveri Valley was sometime
between 4860 and 5290 yr BP, and in the upper reaches of
the Suvarnavati around 2600 to 3900 yr BP. In the Arkavati
valley, the Manchenabele lake vanished around 1900 yr BP
and the Mosalehosahalli lake south-east of Hassan dried up
around 1300 yr BP.
It seems that these lakes did not dry up during the same
period. Although substantial draining out of the impoundments could have been caused by revival of movements on
the active faults, it was the growing aridity in the later Holocene that was responsible for the drying up of the lakes.
Presence in abundance of calcareous nodules including
rhyzocretions in the black clay succession implies growing
aridity in the climate as was the case in the dryland of western Rajasthan13. The Indian subcontinent had experienced a
spell of dryness beginning around 3500–4000 yr BP and
ending at about 2000 yr BP14–16. The lakes of the Kaveri basin dried up possibly during this period. Revival of movements on active faults must have helped in the draining out
of the impoundments as stated earlier in the article.
The expanse of the lacustrine clay deposit in the Kaveri
valley has been dismembered and vertically displaced by
the NNE-SSW trending fault that is traceable through Talakad (where the east-flowing Kaveri suddenly swings SSW)
(Figure 2), and then past Malavalli to the straight N–S
course of the Shimsha near Maddur. Not only is there a
difference in the elevation of the order of 15 m but also the
black lacustrine clay is sharply juxtaposed against brown
fluvial sediment west of Talakad. In the Hulu Halla and
Shimsha valleys, the elevation difference of the black clay
surface is 15 m where the eastern side was downfaulted.
Upstream of Maddur the black clay is considerably truncated, there being none on the eastern side.
Likewise in the Antargange and Rayatmala valleys later
movements have faulted up the black clay and exposed the
sheared gneisses of the floor at Sitalavadi.
Ponding of these and other streams in some segments cut
by the active faults suggests that the fault movements are
continuing in the present.
1. Ramakrishnan, M., in Geo Karnataka (eds Ravindra and Ranganathan, N.), Karnataka Assistant Geologists’ Association, Bangalore, 1994, pp. 6–35.
2. Radhakrishna, B. P., Bull. Mysore Geol. Assoc., 1952, 3, 1–56.
3. Radhakrishna, B. P., Bull. Indian Geophys. Union, 1966, 3, 95–
106.
4. Radhakrishna, B. P., J. Geol. Soc. India, 1992, 40, 1–12.
5. Radhakrishna, B. P., Curr. Sci., 1993, 64, 787–793.
6. Vaidyanadhan, R., J. Geol. Soc. India, 1971, 12, 14–23
7. Vaidyanadhan, R., in Exploration in the Tropics (ed. Jog, S. R.),
Geography Department, University of Poona, Pune, 1987, pp.
125–126.
8. Valdiya, K. S., J. Geol. Soc. India, 1998, 51, 139–166.
1142
9. Radhakrishna, B. P. and Vaidyanadhan, R., Geology of Karnataka, Geol. Soc. India, Bangalore, 1997, p. 353.
10. Jayananda, M. and Mahabaleswar, B., Proc. Indian Acad. Sci.
(Earth Planet. Sci.), 1991, 100, 31–36.
11. Demongeot, J., Z. Geomorphol., N.F., 1975, 19, 229–272.
12. Gupta and Polach, Radiocarbon Dating Practices at ANU, Austr.
Nat. Univ., Canberra, 1985.
13. Andrews, J. E. et al., Quat. Res., 1998, 50, 240–251.
14. Naidu, P. D., Curr. Sci., 1996, 71, 715–718.
15. Rajagopalan, Geeta, Sukumar, R., Ramesh, R., Pant, R. K. and
Rajagopalan, G., Curr. Sci., 1997, 72, 60–63.
16. Gasse, F., Fontes, J. Ch., VanCampo, E. and Wei, K., Palaeogeogr. Palaeoclimatol. Palaeoecol., 1996, 79–92.
ACKNOWLEDGEMENT. K.S.V. thanks S. G. Suresh and G. T.
Vijaykumar for providing enjoyable and helpful company during the
fieldwork.
Received 25 November 1999; revised accepted 9 February 2000
Occurrence of pteropods in a deep
eastern Arabian Sea core: Neotectonic
implications
A. D. Singh*,‡, R. Schiebel† and N. R. Nisha*
*Department of Marine Geology and Geophysics, School of Marine
Sciences, Cochin University of Science and Technology,
Cochin 682 016, India
†
Institute and Museum of Geology and Palaeontology, Tubingen
University, Tubingen, Germany
This paper reports pteropod shells (aragonitic) at 100, 200,
270–277 and 470 cm sediment depths in a core (EAST)
recovered from 3820 m deep water from the eastern Arabian Sea. Ages of the four stratigraphic levels showing
pteropod presence are estimated as 29, 52, 70–72 and
127 kyr.
In
normal
circumstances
microfaunal assemblages of this core are expected to be
devoid of pteropod shells because the site is situated far
below (~ 3.5 km) the Aragonite Compensation Depth.
Therefore, the recorded pteropod shells are exotic to the
location and may have been transported from the shallower
depths by the turbidity currents. The plausible reason for
the preservation of aragonitic shells at such greater depth
appears to be quick burial of pteropods resulting from
large-scale vigorous slumping triggered by neotectonic
activity.
PTEROPODS are marine holoplanktic micro-gastropods
having aragonitic shells. Living pteropods are abundant in
pelagic environment, but rarely preserved in deep-sea sediments because of their dissolution-prone shells. Well
preserved pteropod shells are limited to the marginal seas
and tropical to subtropical shallow areas and continental
margins1–6. In deep ocean, decreasing carbonate saturation
‡
For correspondence. (e-mail: arundeosingh61@hotmail.com)
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
RESEARCH COMMUNICATIONS
results in selective and substantial dissolution of the aragonitic shells7. The level of Aragonite Compensation Depth
(ACD) is shallower by about 3 km on an average, than Calcite Compensation Depth8. ACD in the eastern Arabian Sea
has been estimated approximately at 0.3 km water depth8.
Therefore, bottom sediments of the eastern Arabian Sea
below ACD are expected to be devoid of pteropod shells.
Hence, the occurrence of pteropod shells in deep-sea sediments is a rare and unusual event that is worth examining.
A 5.19 m long core (EAST) recovered from 3820 m deep
water of the eastern Arabian Sea (lat. 15°35.52′N, long.
68°35.09′E) was taken for the study (Figure 1 a). The main
characters of down-core variation in lithology are shown in
Figure 1 b. Core samples at 10 cm regular intervals were
used for the study. The qualitative and quantitative microfaunal analyses (planktic foraminifers and pteropods) were
carried out on 1 g of > 250 µm in size fraction. Ages of the
samples were estimated based on the stable oxygen isotope
(G. ruber) stratigraphy established by correlating δ 18O record of the core with time scale of Martinson et al.9 (Figure
2).
Pteropod shells were recorded from four stratigraphic
levels, viz. 100 cm, 200 cm (Stage 3), 270–277 cm (Stage 4/5)
and 470 cm (Stage 5). The number of specimens of aragonitic shells varies from 3562 to 6400 per gram. The assemblage
comprises Limacina inflata, L. trochiformis, Creseis
acicula, C. virgula, C. chierchiae, Clio convexa, Cavolina
a
Figure 1.
sp. and Dicaria quadridentata. C. acicula (an epipelagic
form) dominates the assemblage. The question arises as to
how the well-preserved aragonite shells occur
in core sediments approximately 3.5 km below the ACD.
There can be two probable reasons: (i) considerable deepening of the ACD during certain periods resulting in
favourable conditions for the aragonite preservation or
(ii) transportation of the pteropod shells from the shallow
areas. The first possibility is ruled out because the ACD
variation with amplitude of 3.5 km and that too for very
short interval seems to be impossible. Moreover, the available data on the fluctuations in the Aragonite Compensation Level during Quaternary do not reveal such a wide
range of variation. Therefore, the plausible cause appears to
be the transportation of pteropod shells from the shallow
areas to the deeper sites. Presence of shallow benthic foraminifers
along
with
the
pteropods
in
the
microfaunal assemblages supports this view. A significant
reduction in total planktic foraminifers noticed in the samples containing shallow microfaunal elements further suggests dilution of autochthonous sediment concentration
due to carbonate as well as non-carbonate input to the
deeper regions (Figure 2).
Deposition of bioclastic sediments consisting of aragonite component at greater depths (below ACD) has been
reported from the Columbus Basin10, Blake Basin11, Mediterranean Sea12 and Sargasso Sea13. Occurrence of pteropods
b
a, Location of the core (EAST) site; b, Core description (drawn from Meteor Cruise-33 Report, 1996).
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000
1143
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site. Earlier workers16,17 have suggested sediment transport
by slumping from the outer shelf-slope region of western
continental margin of India during the late Pleistocene.
Stackelberg18 has also reported slumping all along the central western continental margin of India.
Data on the pteropod occurrence in deep sedimentary
records are crucial for determination of ACD in the eastern
Arabian Sea. The study reveals that the depths to which
pteropods occur in surface sediments may not
always represent actual Aragonite Compensation Level.
Redeposition can introduce pteropod assemblages into
depths even far below the ACD. A detailed study on composition, chronology and source area of the pteropod turbidites based on more sediment cores will provide better
understanding of the depositional history of the eastern
Arabian Sea.
Figure 2. Stable oxygen isotope record of G. ruber (white
> 250 µm). Isotope stages are determined following Martinson et
al.9 , and total number of specimens of planktic foraminifers (p.f) and
pteropods (pt) in one gram of dry sediment (> 250 µm). T1, T2, T3
and T4 indicate turbidites in chronological order.
along with ooids was also observed in the late Pleistocene–
Holocene deep-sea cores from the continental rise/abyssal
plain of the Bay of Bengal14. In the Arabian Sea, ooid turbidites consisting of pteropods and shallow marine microfauna have been recorded from the deep western
continental margin of India15–17. Previous documentation of
turbidites from the eastern Arabian Sea was mainly limited
to the sedimentary records representing last glacial and
Holocene
periods.
The
present
study
enables to document four layers of pteropod turbidites (T1,
T2, T3 and T4) formed approximately during 127, 70–72, 52
and 29 kyr BP (Figure 2). Ooids were also found in turbiditic
layer T2 (Figure 1). The preservation of pteropods at such
greater depths can be possible if the rate of deposition of
bioclastic sediments is rapid. It appears that the large-scale
vigorous slumping triggered by neotectonic activity would
have resulted in quick burial of pteropod shells at present
1144
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ACKNOWLEDGEMENTS. We thank Dr H. Erlenkeuser, Kiel
University for isotope analysis. A.D.S. thanks Indian National Science Academy and Deutsche Forschunsgemeinschaft Germany for
the fellowship. This work was also partially supported by the Department of Ocean Development. We thank the anonymous reviewers for their constructive comments.
Received 4 September 1999; revised accepted 11 January 2000
CURRENT SCIENCE, VOL. 78, NO. 9, 10 MAY 2000