Adaptive significance of amylase polymorphism in Drosophila. Analysis of the association between tissue-specific expression and specific activity in AmyS or AmyF genotypes of Drosophila subobscura

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Adaptive significance of amylase polymorphism in

Drosophila. Analysis of the association between

tissue-specific expression and specific activity in AmyS

or AmyF genotypes of Drosophila subobscura

Tatjana Terzić, Mirjana Milanović, Mile Ivanović, Marina

Stamenković-Radak, Marko Andjelković

To cite this version:

Note

Adaptive

significance

of

_amylase

polymorphism

in

_Drosophila.

Analysis

of the association between

tissue-specific

expression

and

_specific

activity

in

_Amy

_S

or

Amy

F

genotypes

of

_Drosophila

subobscura

Tatjana

Terzi&jadnr;

_Mirjana

Milanovi&jadnr;

Mile

Ivanovi&jadnr;

b

Marina

Stamenkovi&jadnr;-Radak

Marko

_{Andjelkovi&jadnr;}

a

Department of

_Genetics,

Institute for

Biological Research,

29, novembra 142,

11000

_{Belgrade, Serbia, Yugoslavia}

b

Maize

Research Institute ’Zemun

_Polje’,

Slobodana

Baji6a

1,

11081

_{Belgrade, Serbia, Yugoslavia}

c

Faculty

of

_{Biology, University}

of

_Belgrade,

Akademski _trg 16,

11000

Belgrade, Serbia, Yugoslavia

(Received

5

_{January 1998;}

_accepted

2 December

₁₉₉₈₎

Abstract - The

_{phenotypic variability}

at the level of the

_{specific activity}

of

a-amylases

and their

_{tissue-specific}

_expression in the

_midgut

of adult

Drosophila

subobscura

flies, homozygous

for the

_Amy

_s

or

Amy

F

allele,

was

analysed.

The

re-sults indicate a

_homogeneous

distribution of the

_phenotypes

with a different numbers

of

a-amylase activity regions

in the adult

midgut

between the lines

homozygous

for

A!nys

and

_Amy

_F

alleles. The mean number of

a-amylase midgut

activity differs

sig-nificantly only

between the groups of lines

homozygous

for

A!ays,

with the

specific

activity

of the enzyme above the average, and the groups of

Amy

F

homozygote

with

a

_{significantly}

lower mean

_{specific activity}

of

_amylase.

The

_analysis

_suggests the

exis-tence of

_compensation

between the number of active

_regions

and the

_{specific activity}

of

_a-amylase

within

_Amy

_s

and

_Amy

_F

lines.

_©

_{Inra/Elsevier,}

Paris

Drosophila

/ amylase

/ tissue-specific expressions

/ specific

activity of the enzymes / polymorphisms

*

Correspondence

and

reprints

Résumé - Signification adaptative du _{polymorphisme} de l’amylase chez

Droso-phila. Analyse de l’association entre l’expression tissulaire et l’activité spécifique

des _génotypes

Amys

et

Amy

F

chez _Drosophila subobscura. La variabilité

phéno-typique de l’activité

amylasique

dans l’intestin _moyen de

_Drosophila

subobscura a été

analysée

dans des

_{lignées homozygotes pour}

l’allèle

_Amy

ou

_Amy

_F

_.

Dans les deux

lignées

on observe les mêmes

_phénotypes

_comportant un nombre variable de

_régions

où

_l’amylase

est

_{exprimée. Globalement,}

l’activité

amylasique

est

_{significativement}

différente entre les

_{lignées homozygotes pour}

_Amys,

activité

spécifique supérieure

à la valeur moyenne, et

Amy

F

,

valeur inférieure à la valeur moyenne.

L’analyse

suggère

l’existence d’une _compensation entre le nombre de

_régions

actives et l’activité

enzymatique spécifique

dans ces

_{lignées. ©}

_{Inra/Elsevier,}

Paris

Drosophila

/ amylase ex

/ expression tissulaire

/ enzyme

/ polymorphisme

1. INTRODUCTION

Besides structural gene

polymorphism, analyses

of enzyme

systems

in

eu-karyotes

reveal the existence of

_polymorphism

in

_{tissue-specific}

_enzyme

expres-sion. Various kinds of

_regulatory

_genes have different effects on

_{tissue-specific,}

developmental

and

_{quantitative expression}

of the enzymes coded from

struc-tural _genes. Because differences in

_{morphological,}

biochemical and

physiologi-cal

_{characteristics,}

as well as differences between

_species,

which _appear

_despite

similarities in the

_protein

_structure,

_originate

from

_changes

in the

_polygenic

complex

of

_regulatory

_genes, examination of their

_variability

is of

_importance.

a-Amylase

in

_Drosophila,

active in the

_midgut

and

_hemolymph,

is a

well-known model suitable for

_analysing

the

_adaptations

of

_organisms

to different environmental

conditions,

and for

examining

the

_{general biological}

significance

of

_genetic

_diversity

in natural

_populations

of different

_organisms.

_a-Amylase

polymorphism

includes both the

_variability

of the structural

_Amy

locus and the

variability

of

_{tissue-specific expression}

_[9].

The latter

_type

of

_variability

is

_{represented by}

the number and

_position

of the

_amylase

_{activity regions}

in the

_midgut

_[5].

Inter- and

_{intrapopulation variability}

exists both for the number and

_position

of the active

_regions

in adult

_midgut

_{[3, 12].}

At the

phenotypic

level the

_Amy

locus

_variability

is associated with the

_specific

_activity

of the enzyme

a-amylase.

Physicochemical

conditions for the

_{optimal activity}

of

a-amylase

are

_{species-specific}

[8].

The

_{present report}

_gives

an

_analysis

of the

_{phenotypic variability}

of

geno-types

homozygous

for the

_Amy

_s

and

_Amy

_F

allele of the

_Amy

locus at the level of

_{tissue-specific}

_expression,

as reflected in the number of active

_midgut

_regions

and the

_specific

_activity

of

_amylase

in

_Drosophila

subobscura adults.

2. MATERIALS AND METHODS

Drosophila

subobscura lines

_homozygous

for the

_A!rcys

_(S)

or

A!nyF

(F)

al-leles,

inbred for 20

_generations

in

_optimal

_laboratory

conditions en _masse, were taken for dissection of the

_midgut

and for the

_specific

_enzyme

_activity

_assay. Determination of the

_specific

_activity

of

_a-amylase

was carried out

_according

to

the method described

_{by Noelting}

and Bernfeld

_[11].

_Midgut

dissection and

The results were

_analysed

for each line of

_Drosophila

subobscura

_homozygous

for

_Amy

_s

and

_Amy

_F

_.

_Midgut

dissection was

_performed

with 12 to 15 flies

per

line,

and

a-amylase activity

pattern

was

analysed

with 50 flies in three

replicates

per line.

The

_digestive

function of the

_a-amylase

enzyme is

present

in the AMG

(anterior)

and PMG

_(posterior)

_parts

of the

_Drosophila

_{midgut owing}

to

suitable

pH

values in those

parts.

The

_a-amylase

_activity

can be detected in a

maximum of three AMG

_regions

and two PMG

_regions.

Parametric tests

_(chi-square

_!k2!

and

_Student’s)

and

_{non-parametric}

tests

(Mann-Whitney,

Kruskal-Wallis

_analysis

of variance and

_correlation)

were used for the

_analysis

of the results. In this _way, the

_variability

in the number of active

_regions

and the

specific activity

of the enzyme, as

_parameters,

were

analysed

within and between the

_Amy

_s

and

_Amy

_F

_genotypes.

Line

_grouping

was

performed

according

to deviations outside ± 2 standard errors

(SE)

from the mean value of the observed

_parameter.

In this _way, three

_categories

of lines were made for the number of active

_regions

and three for the

_specific

_activity

of the enzyme.

3. RESULTS

Results of the

_analysis

of 37 lines

_homozygous

for the

_Amy

_s

allele and 19 lines

_homozygous

for the

_Amy

_F

allele with

_respect

to the

_{phenotypic variability}

of the total number of active

_{midgut regions}

are shown in table I.

_According

to

the

_previous

results

_[2],

there is no difference between the sexes in their MAP

variability,

so the data for sexes are

_pooled

in this

_analysis.

On _average, lines

_homozygous

for the

_Amy

_F

allele have more active

_regions

(3.577::1: 0.109)

than the _group of

_S/S

lines

_{(3.318::1: 0.134).}

It is indicative that for the

_S/S

_genotype

the most abundant

_phenotypes

_(29.4

_%)

are the ones with three active

_regions,

while

F/F

_genotypes

have 32.2

%

flies with five active

_regions.

In the lines of both

_genotypes

flies with

_only

one active

_region

Homogeneity

is found in the distributions of

_phenotypes

with various num-bers of active

_regions

between the _groups of

_S/S

and

_F/F

lines

_(x

₂

=

8.614,

df =

_4,

P _>

0.05),

although

the differences between

genotypes

homozygous

for either the S or F allele are not

_{statistically}

_significant

for the average number

of active

regions

(t

=

1.500,

df =

52,

P _>

0.05).

Regarding

the

specific activity

of

a-amylase,

the group of lines

homozygous

for the S allele shows a

_higher

_activity

(3.292 ± 0.154)

than the _group of lines

homozygous

for the F allele

_{(3.042 ± 0.241).}

_However,

the mean

_{specific activity}

values do not differ

_{significantly}

between these

_genotypes

_(t

=

_0.910,

df =

54,

P >

_0.05).

Differences between the lines characterised

_{by extremely low, extremely}

_high,

or moderate _average values for the number of active

regions

and

specific

_activity

are considered for additional

_analysis

of the association between the

_phenotypic

variabilities caused

by

the

_polymorphism

of the structural

_and/or

_regulatory

components

of the

a-amylase

gene-enzyme

system

in

_Drosophila

subobscura

(tables

II and

_III).

The results obtained indicate that a

_{statistically significant}

difference in the

the group of

S/S

lines,

whose

_specific

_{activities range} within ± 2 SE of the

mean, and the group of

F/F

lines which is at least + 2 SE below the mean

(U

=

7.00,

P <

_0.05),

as well as between the _groups of lines of both

_genotypes

within ± 2 SE of the mean

(U

=

24.00,

P _<

0.05).

The

_analysis

of

_intergroup

differences in the number of active

_regions

for the six _groups formed

_according

to

_{amylase-specific}

_activity

confirmed that the difference is not

_significant

_(H

=

_8.424,

P _>

0.05).

It also confirmed the

previously

obtained

_results,

i.e. the

_equivalence

in the distribution of the number of active

_regions

between

_S/S

and

_F/F

lines

_grouped

in three

categories.

When the _groups are formed

according

to the number of active

_regions,

the same test shows no

_{statistically significant intergroup}

difference in the

variability

of the _enzyme

_specific

_activity

of either

_genotype

_(H

=

5.727,

P >

_0.05).

Analyses

of the association between the number of active

_regions

and the

enzyme-specific

activity through compensation

of the _enzyme

_quantity

in

_S/S

and

_F/F

_genotypes,

carried out

_by

_Spearman

and Pearson tests of correlation on all

_categories,

indicate

_{statistically non-significant negative}

correlations in five

cases. Such correlations are found

_mostly

in the _{groups in} which the number of active

_regions

or _enzyme

_{specific activity}

fall below and within the mean value

±2 2 SE.

Correlation tests

_applied

to the

_ungrouped

lines of

_S/S

or

_F/F

_genotype,

in-dicate

_possible

_compensation

of

_deficiency

or excess of the _enzyme

_by

the

corre-spondingly higher

or lower number of active

_regions,

_respectively

_(negative

cor-relation without statistical

_{significance;}

rpearson =

_-0.139,

rgp

earman = -0.178

for the

_S/S

genotype;

for the

_F/F

genotype,

rPearso&dquo; _

-0.279,

rspearman

- -0.309).

4. DISCUSSION

The association between the

_genetic

determination and the

_phenotypic

func-tionality

of the gene-enzyme

system

is still obscure. The functional

relation-ship

between the structural and

regulatory

genes, realised

through complex

and

_{multiple interactions,}

leads to _many

_hypotheses

in the

_{interpretation}

of the

_experimental

data.

The structural and

_regulatory

_variability

of

_a-amylase

_represents

one of the

most

_{frequently analysed}

_gene-enzyme

_systems

in

_Drosophila

_(see

_[9]).

The

analysis

of the _enzyme

_activity

_represents

a model for

_{distinguishing}

between

the effects of the structural and

_regulatory

genes involved in the control of a

_particular

_gene-enzyme

_system.

According

to _many

_authors,

the

_variability

of

_regulatory

_{genes may} have an

evolutionary role,

even more

_important

than the structural gene

polymorphism

[7, 10].

Tissue-expressed polymorphism

in

Drosophila

adult

_midgut

_exemplifies

a

_specific

determination of

_regulation

[1, 12].

The absence of differences at the level of the mean

tissue-specific expression

and

_{specific amylase activity}

between two

_Amy

_genotypes

_{observed, along}

with the _presence of differences _among

_{particular categories,}

_may indicate the

The

_analysis

of

_Drosophila

subobscura

_{amylase activity}

shows that the individuals

_homozygous

for the S allele

_generally

have a

_{higher amylase activity}

than those

_homozygous

for the F

_allele,

as

_reported

for other

_Drosophila

species

[13].

Immunoelectrophoretic

data reveal that different levels of

_amylase

activity

are a _consequence of different

_quantities

of

_amylase

_protein,

which is

directly

related to the

_regulation

at the

_{transcriptional}

level

_[6].

The existence of different biochemical

_phenotypes

in

_Drosophila

subobscura may be the result

of the

_genetic

_variability

in the structural

_and/or

_regulatory

_genes

_responsible

for the

_synthesis

and

_expression

of

_cr-amylase.

In the work

_by

Doane

_[4],

a clear absence of

dependence

was found

be-tween the total

_amylase

_activity

and the distribution of the active

_regions

in

Drosophila melanogaster.

In the

_present

_paper the

_negative

_(although

non-significant)

correlation between the number of active

_regions

and

_specific

amy-lase

_activity

within each of the

genotypes,

as well as between certain

_categories,

suggest

a

_compensatory

effect between these two

_phenotype

_expressions.

The differences in the mean number of

_a-amylase

active

_regions

in adult

midgut

between the lines

_homozygous

for the

_Amy

_s

allele with above _average

specific amylase activity

and lines

_homozygous

for the

_Amy

_F

allele with

significantly

lower _enzyme

_activity

_may indicate additional

_{genetic variability}

within the

_Amy

locus. This

_possibility

adds to the

_complexity

of

_studying

the

degree

of the functional

_significance

of different

types

of

_{genetic polymorphism}

in

_adaptation

_processes.

ACKNOWLEDGEMENTS

We wish to thank _anonymous referees for the valuable comments on the _manuscript

and Ms

_Bojana

Jelisavcic for excellant technical assistance.

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_W.W.,

Genetic

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