Changes in native alcohol dehydrogenase activity ofDrosophila upon treatment with guanidine hydrochloride, urea and heat

Abstract

Fifty-two isochromosomal lines ofDrosophila melanogaster were examined for the existence of additional genetic variations in ADH activity subsequent to treatment With guanidine hydrochloride,
urea and heat. A wealth of hidden variation was discovered among and within the Mexican populations of the insect after treatment
with the denaturants.

Full text

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J. Biosci., Vol. 3 Number 3, September 1981, pp. 275-284. © Printed in India.






Changes in native alcohol dehydrogenase activity of Drosophila
upon treatment with guanidine hydrochloride, urea and heat*

GURBACHAN S. MIGLANI** and FRANKLIN R.AMPY
Department of Zoology, Howard University, Washington D.C. 20059 USA

MS received 23 February 1981

Abstract. Fifty-two isochromosomal lines of Drosophila melanogaster were examined for the
existence of additional genetic variations in ADH activity subsequent to treatment With
guanidine hydrochloride, urea and heat. A wealth of hidden variation was discovered among
and within the Mexican populations of the insect after treatment with the denaturants.

Keywords. Drosophila melanogaster; alcohol dehydrogenase; guanidine hydrochloride; urea; heat

Introduction

The existence of clines in the frequency of alcohol dehydrogenase (Adh) alleles in
Drosophila under various environmental conditions offers impressive evidence to
support the hypothesis of natural selection affecting enzyme polymorphism. The
lines of evidence include reports from Russia (Grossman et al.,1970), United States
(Johnson and Burrows, 1976; Johnson and Schaffer, 1973; Vigue and Johnson,
1973; Voelker et al., 1977) and Mexico (Pipkin et al., 1973; 1975; 1976). Pipkin et
al., (1976) suggested that the cline of Adh alleles depended on selection and gene
exchange between Mexican populations of D. melanogaster from low and high
temperature regions. The above relationships between the gene product and the
environmental factors were mainly based on the genetic variation detected by the
use of electrophoretic techniques. A variety of techniques revealed additional
genetic variation in Drosophila (Bernstein et al., 1973; Coyne, 1976; Coyne and
Felton, 1977; Gibson, 1970; McDowel and Parkash 1976; Milkman, 1976;
Sampsell, 1977; Singh et al., 1976). The number of variants that were observed in a
population depended on methodologies used, intensity of effort and the
geographical diversity of the strains examined (Koehn and Eanes, 1979).

Recently a statistical analysis revealed cryptic variation in the activity of alcohol
dehydrogenase (ADHI and ADH II) in 12 Adh1/Adh1 and 40 Adh11/Adh11 isochro-
mosomal lines extracted from 16 Mexican populations of D. melanogaster (Miglani,


* Dedicated to the late Dr. Sarah B. Pipkin of Howard University.
** Present address: Department of Genetics, Punjab Agricultural University, Ludhiana 141 004.

Abbreviations used: ADH, alcohol dehydrogenase; GuHCl, guanidine hydrochloride; CV, Co-efficient
of variation; X, mean; SD, standard deviation; SF, Standard error.

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1980). The present paper reports the existence of additional genetic variation in
ADH activity in the 52 isochromosomal lines subsequent to treatment with various
denaturants.

Materials and methods

The sixteen populations of D. melanogaster used in construction of isochromosomal
lines were: Puebla (Pu), San Louis Potosi (SLP), Tahuacan (Teh), Saltillo (Sa),
Actalan (At), Oaxaca (Ox), Cuatla (Cu), Orizaba (Oz), Cordoba (Co), Yanga (Ya),
Palenque (Pe), Acayuacan (Ac), Aleman (Al), Coatzocoalcos (Cz), Merida (Me)
and Rio Papalopan (Rio). The isochromosomal lines of D. melanogaster used in
the present investigation were described elsewhere (Miglani, 1980). Flies used for
ADH activity were cultured at 25°C. Within 24 h after emergence, forty males
were chosen at random and allowed to develop for six days at which time, D.
melanogaster ADH shows peak activity (Ursprung et al., 1968). The flies were
weighed (Sartorium micro-balance) and hand ground in 2 ml of 0.05 Μ phosphate
buffer (pH 7.5). This homogenate was centrifuged at 27,000 g for 30 min at 4°C
in a Sorvall RC-2B centrifuge. The supernatant, referred to as crude extract was
immediately used for ADH activity measurements (Miglani, 1980).

Guanidine hydrochloride (GuHCl) and urea treatment

To a 1 ml cuvette containing 0.89 ml of a 0.7M GuHCl or 1.0 Μ urea solution
(prepared daily in 0.05 Μ phosphate buffer, pH 7.5) 0.05 ml of the crude extract was
added. This mixture was kept in the Gilford recording spectrophotometer
(maintained at 30 ± 1°C) for 40 s after which 0.05 ml of NAD+ (27.6 mg
NAD+/ml deionized water) and 0.01 ml of 2-butanol were added. The reaction
mixture was vortexed and. conversion of NAD+ to NADH was recorded spectro-
photometrically for 2 min. Six replicates were run for each set of homogenates as
previously described by Miglani (1980).

Heat treatment

To a 1 ml cuvette containing 0.89 ml of 0.05 Μ phosphate buffer (pH 7.5)
maintained at 45°C, 0.05 ml of crude extract was added. The mixture was
vortexed and incubated for 15 min at 45°C. The heat treatment was terminated by
adding cold (4°C) 0.05 ml of NAD+ solution (27.6 mg NAD+/ml deionized water)
and 0.01 ml of 2-butanol. The mixture was again vortexed and ADH activity
remaining after treatment was determined as described above.

The native (prior to various treatments) ADH activity for each isochromosomal
line was compared with its ADH activity after treatment with denaturants or heat.

Statistical analysis

Mean (X), standard deviation(s) and coefficient of variation (CV) values for native
ADH activity (data reported by Miglani, 1980) and ADH activity after treatment
with denaturants (present paper) were calculated only for those populations in
which it was possible to extract three or more isochromosomal lines. The
differences in the mean and CV values were analysed by standard statistical
procedures (Zar, 1974).
Miglani and Ampy

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In order to separate AdhI/Adh1 and AdhII/AdhII isochromosomal lines which
differ significantly in terms of the percent ADH activity remaining after treatment
with the denaturants, the data were analyzed following the method described by
Zar (1974).

Results

A differential decrease in ADH activity was observed after treatment with GuHCl,
urea and heat among the AdhI/AdhI (table 1) and the AdhII/AdhII (table 2) strains
of D. melanogaster. Among all of the 12 AdhI/AdhI lines (table 3), the mean
native ADH activity was significantly lowered after treatment with GuHCl urea
and heat. The CV values were significantly higher in all the 12 AdhI/AdhI lines
for ADH activity after treatment with GuHCl and urea. However, the CV for the
three lines of the Orizaba population significantly increased after heat treatment
whereas the 5 lines of the Pueola population did not differ significantly in CV
values after treatment.

Table 1. Comparison of ADH activity of isochromosomal lines of phenotype AdhI/AdhI
from D, melanogaster.


The ADH activity is defined as nmol of NADH produced/ml/min/mg of body weight. The
crude extract was treated with 0.7 Μ GuHCl or 1 Μ urea for 40 s. The heat treatment was for
15 min at 45°C.

a the numbers in parentheses refer to percentage ADH activity remaining after treatment with
GuHCl or urea or heat. The ADH activity of untreated extracts was taken as 100% (for
absolute values refer to Miglani, 1980).

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Miglani and Ampy
Table 2. Comparison of ADH activity of isochromosomal lines of genotype AdhII/AdhII
from D. melanogaster.


a Values in parentheses represent per cent activity.

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Table 3. Statistical evaluation of the data for the isochromosomal lines of genetype
AdhI/Adh1


*Significant p<0.05
** Significant p<0.01
*** Significant p<0.001

X: Mean
SD: Standard deviation
CV: Coefficient of variation



While considering all AdhII/AdhII lines collectively, a significant decrease in
ADH activity was observed after treatment with GuHCl, urea and heat (table 4).
However, when we considered individual populations, there existed no significant
decrease in mean ADH activity after treatment with urea in Actalan and Merida,
and after heat treatment in Merida. There was a significant increase in the CV
values for ADH activity in all AdhII/AdhII lines (collectively) after treatment with
urea and heat with the exception of Actalan population which showed a significant
decrease in the CV value (table 4). A significant decrease in the CV value was
observed for all AdhII/AdhII lines after treatment with GuHCl


Analysis of data given in tables 1 and 2 [on the percent ADH activity subsequent
to treatment with 0.7 Μ GuHCl, 1.0 Μ urea or heat (45°C) for isochromosomal
lines raised at 25°C] indicated that the lines having residual activity differing by
60% were statistically different. This established for us, a criterion to designate
lines with 80% or greater residual ADH activity as "resistant" and those with 20% or

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Miglani and Ampy
Table 4. Statistical evaluation of the data for the isochromosomal lines of genotype
AdhII/AdhII.






* Significant p<0.05
** Signifcant p<0.01
*** Significant p<0.001

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less residual ADH activity as 'susceptible'. All the isochromosomal lines resistant
or susceptible to the action of the denaturants used will, here onwards, be referred
to as 'cryptic variant' lines. Thirtyfive cryptic variant lines selected according to
the above criterion are listed in table 5.

Table 5. Cryptic varient lines resistant/susceptible to the action of GuHCl, urea and heat.




Discussion

Ursprung and Carlin (1968), using 6 Μ GuHCl and 10 Μ urea, found that the
activity of ADH was completely lost as indicated by the absence of isozyme bands
after electrophoresis. Therefore, the concentrations used by these investigators
would have been inappropriate for the present investigation whose purpose was to
detect cryptic variation in ADH activity after treatment with GuHCl,urea and heat.
The concentration of GuHCl (0.7 M), urea (1.0 M) and temperature (45°C) used in
this study, were, on the average, found to be statistically effective in reducing ADH
activity (table 3 and 4).

A positive correlation was found between native ADH activity for AdhI /AdhI
lines and ADH activity after treatment with urea (Miglani and Ampy, unpublished).
These studies also reported a positive correlation between native ADH activity for
AdhII/AdhII lines and ADH activity after treatment with GuHCl, urea and heat.

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These relationships suggested that the decrease in ADH activity was possibly
dependent on the levels of native ADH activity of the strain. In other words,
strains exhibiting a high.native ADH activity, on the average, showed a high
residual ADH activity after treatment with the denaturants.

Heat stability is affected by a number of genetic factors (Milkman, 1976).
Electrophoretically-cryptic allelic differences exist which were expressed as
differences in thermostability (Milkman and Pruss, 1975). The present investiga-
tion provided additional data showing the presence of cryptic polymorphism for
Drosophila ADE. activity after treatment with GuHCl, urea and heat. The thermal
stability of ADH in D. melanogaster appeared to be controlled by closely linked
genes, if not by the same gene (Sampsell and Milkman, 1977) and on the basis of
heat denaturation studies it was further suggested from the magnitude and
regularity of differences between free energies of the activation (of the inactivatiorr
process) that each thermostability step stemmed from one additional Van der
Walls bond (Sampsell and Milkman, 1978).

Some investigators observed that homogenates from AdhII homozygotes were
more heat stable than homogenates from homozygotes of AdhI (Day et al., 1974;
Gibson, 1970), The present study showed that some AdhII/AdhII isochromosomal
lines were more heat stable than some AdhI/AdhI lines and vice versa (table 1 and
2). The degree of thermal stability did not seem to be diagnostic of the electro-
phoretic class of ADH (i.e. ADHI or ADHII) . Anderson et al. (1980) made similar
observations after heat treatment with D. melanogaster. The same pattern was
also observed with GuHCl and urea treatments in the present study. Thus some
AdhI/AdhI isochromosomal lines were more resistant to the action of GuHCl or
urea than some AdhI/AdhII lines and vice versa.

A significant increase in the CV values after treatment with GuHCl, urea and
heat was interpreted as evidence to support the discovery of additional genetic
variation in isochromosomal lines having approximately the same native ADH
activity. These observations clearly indicated
denaturants revealed an enormous amount of cryptic variation at a biochemical
level among and within the Mexican strains of D. melanogaster examined.

Johnson and Schaffer (1973) suggested that enzyme polymorphisms were often
associated with regulatory reactions in metabolism. McDonald and Avise (1976)
suggested that at least one of the major functions in the variable levels of enzyme
activity might perhaps be of an adaptive significance. Since the cryptic variant
lines reported (table 5) differ with respect to a biochemical property of ADH,
developmental studies on these thirty-five lines are in progress under variable
reaction conditions to examine the regulatory mechanisms involved in genetic
modulation of alcohol metabolism and to see if this variation has an adaptive
significance.

Miglani and Ampy
that treatment with certain
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