Molecular cloning and partial sequence characterization of the duplicated amylase genes from Drosophila erecta

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Molecular cloning and partial sequence characterization

of the duplicated amylase genes from Drosophila erecta

L Bally-Cuif, V Payant, S Abukashawa, Bf Benkel, Da Hickey

To cite this version:

Original

article

Molecular

_cloning

and

_partial

_sequence

characterization of the

_duplicated

amylase

genes from

Drosophila

erecta

L

_Bally-Cuif,

V

_Payant,

S Abukashawa BF Benkel DA

_Hickey

Department of Biology University of Ottawa, Ottawa, Ontario KlN 6N5, Canada

(Received

16 _{May 1989; accepted} 15 November

₁₉₈₉₎

Summary - Previous studies have shown that the a-amylase gene is duplicated in each of the 8 species

comprising

the _{Drosophila melanogaster species}

_subgroup.

In this _study,

a _genomic

library

in

phage

lambda was _prepared from the DNA of 1 _species member of

this _subgroup, D erecta. _{This gene library} was screened with an _{amylase-specific probe}

from the _{closely-related species} D _{melanogaster.} One of the _{cross-hybridizing phage} clones

was _isolated, insert

_fragments

were sub-cloned into _{plasmid vectors,} and the

_identity

of

the cloned locus was verified _by DNA _sequencing. The results confirm that this _species

contains _{2 very} similar _copies of the

_{amylase-coding}

_sequence, as does D _{medanogaster.}

Within the _{amylase-coding region,} there is a 3% sequence divergence between the 2 species;

this value rises to 6% if we consider _only silent sites within the

_coding

_{region. Sequence}

divergence

between D erecta and D _melanogaster in the _non-coding

_intergenic

_region is

approximately 15%.

These results constitute the first molecular sequence data available for protein-coding

genes of D erecta. The data set allows us to calculate an estimated _divergence time of more than 10 million _years between the 2 _species. This is consistent with the results of

previous

phylogenetic

studies.

Drosophila erecta _/ a-amylase / molecular evolution _/ gene conversion

Résumé - _Clonage et caractérisation d’une _{séquence partielle} des _{gènes dupliqués} de _l’amylase chez Drosophila erecta - Des études antérieures ont montré que le

gène

a-amylase était _dupliqué chez chacune des 8 _espèces du sous-groupe

Drosophila melanogaster.

Dans la présente étude, une banque génomique a été préparée dans le phage lambda à _partir de l’une des espèces membres du sous-groupe; D erecta. Cette banque a été criblée avec une _sonde, provenant de _l’espèce voisine D _{melanogaster,} et contenant les

séquences codant pour

l’amylase.

Un des clones

hybridant

à la sonde a été _isolé, et

ses _fragments de restriction sous-clonés dans des _plasmides. La nature des _gènes a été

vérifiée par séquençage. Les résultats obtenus confirment que D erecta contient, comme

D

_{melanogaster,}

2 _copies très semblables de la _région codante du _{gène a-amylase.} Dans

cette

_région,

la _divergence de _séquence entre les _{2 espèces} est de

_3%,

et atteint 6% si l’on

ne considère _que les _positions silencieuses des codons. Dans la région intergénique

(non

codante),

la _divergence est d’environ 15%. Ces résultats constituent les _premières données de _{séquence pour} des _gènes codants _pour une _protéine chez D erecta. Ils nous permettent

*

d’estimer un _temps de _divergence de _plus de 10 millions d’années entre les L _espèces, ce

qui est _compatible avec les résultats des études _{phylogénétiques} antérieures.

Drosophila erecta _{/ !Y-amylase /} évolution moléculaire _/ conversion _génique

INTRODUCTION

Drosophila a-amylase

genes have

already

been

extensively

studied

(Levy

et

al, 1985;

Gemmil et

_al,

_1986;

Doane et

_al,

_1987;

_Langley

et

_al,

_1988;

_Hickey

et

_al,

_1989b),

and

_they

have

_proved

to be

_particular

_interest,

_mainly

because of their numerous encoded

_allozymes

_(Singh

et

_{al, 1982;}

Dainou et

_al,

_1987),

their

_complex

_pattern

of

_{regulation, including glucose repressibility}

_(Benkel

and

_Hickey,

_1987),

and their

duplicated

structure. Within natural

_populations

of D

_{medanogaster,}

the gene is

duplicated

(Levy

et

_{al, 1985;}

Gemmill et

_al,

_1986),

with the 2

_copies,

each 1.4 kb

in

_{length, being separated by approximately}

4.8 kb of sequence.

They

are _present in

_opposite

orientation

_(Levy

et

_al,

_1985;

Benkel et

al,

1987)

and are

divergently

transcribed

_(Boer

and

_Hickey,

_1986).

A

_simplified

map of this locus is

presented

in

Fig 1.

The number and _pattern of

_{electrophoretic}

variants within each

_species

and

in

_{interspecific hybrids}

_(Dainou

et

_al,

_1987),

_coupled

with the recent

_analyses

of restriction maps

(Payant

et

_al,

_1988),

have shown that the

_duplication

is not

restricted to D

_{melanogaster,}

but is also _present in the 7 other

_species

of the

melanogaster subgroup.

The

_a-amylase

_gene thus _appears as a small

_multigene

family, containing only

2 members. Because of the _presence of this

_duplication

in all the

_species

of the

_subgroup,

it can be considered as an

_{evolutionarily}

ancient trait.

The 2

_copies

of the

_a-amylase

_gene from a strain of D

_melanogaster

have

_already

been

_{partly sequenced}

_(Boer

and

_Hickey,

_1986),

and the 2

_coding

_regions

showed a

_{high degree}

of sequence

similarity.

This,

in addition to the close

_linkage

of the

2

_copies,

raised the

_possibility

of sequence recombinations between the 2

_copies

of the _gene, even

_though

their

_opposite

orientation is considered as a more stable arrangement than tandem

_duplication.

The construction of a restriction map for the

a-amylase

locus in each of the 8

_species

of the

_subgroup

_(Payant

et

_al,

₁₉₈₈₎

_proved

gene in both D erecta and D teissieri

(Fig 1).

If the mutation

eliminating

this Bam

HI site

_proved

to be the same in the 2

_copies

of each

_species,

this would support the

_hypothesis

of recombination events

_occuring

between the 2

copies

of the _gene within a locus.

We cloned both

_copies

of the

_amylase

gene from D erecta and

_sequenced

part of the

_{coding region}

_(containing

the Bam HI

_location)

for each _gene copy. We

then

_performed

_sequence

_comparisons

_both,

within the

_a-amylase

locus in the

D erecta

_strain,

and between the two

_species,

D erecta and D

_{melanogaster.}

This allowed us to assess _sequence

_similarity

between the _{2 gene}

_copies

in D erecta

and, also,

to

_quantify

the

_degree

of sequence of

divergence

between D erecta and

D

_{melanogaster.}

These _sequence data are discussed in relation to the results obtained in

_previous

_phylogenetic

studies.

MATERIAL AND METHODS

The D erecta strain

_220S,

collected in 1980 in the

_{Ivory Coast,}

West

_Africa,

and

provided by

Dr J

_David,

was used in the _present

_study.

Twelve flies of that strain were

sampled

and tested for their

a-amylase allozymic

_pattern

by polyacrylamide

gel electrophoresis. Having

verified the

_identity

of the

_strain,

_genomic

DNA was isolated from the

descendants

of these flies

_using

a

_procedure

described in detail elsewhere

_(Payant

et

_al,

_1988).

After

_digestion

of the DNA with Sau

_3A,

a

_genomic

library

was constructed in lambda

_phage

EMBL

3,

according

to the manufacturer’s instructions

_{(Stratagene).}

_Plaque

lifts were

prepared

as described

by

Benton and Davis

_(1977),

_{using nylon}

membranes

_(Biotrans;

ICN

_{Biomedicals).}

The membranes

were

_hybridized

to the

_pOR-M7

_{(coding region)}

D

_{melanogaster amylase}

cDNA

32

p-labelled

probe

(Benkel

et

_al,

_1987);

this

_probe

is indicated

_by

the bar labelled

pm7

in

_Fig

1. The filters were then washed under conditions where

only

the recombinant

_phages,

with at least 80% sequence

similarity

to the a

_amylase

_gene, would remain bound to the

_probe

_(for

detailed

_procedure,

see

Payant

et

al,

1988).

After elimination of the

_{radioactivity,}

the same

_procedure

was used to

_hybridize

the membrane to the CS1.4a D

_{melanogaster amylase}

₃₂

_p-labelled

_probe

_(Hickey

et

_al,

_1988;

and see

_Fig

_1).

Five clones

_hybridizing

to both

_probes

were identified.

Their DNA was isolated and

digested

with Sal 1. A southern blot

(Southern,

1975)

of the

_digested

DNAs was

made,

and

_probed

with the

_pOR-M7

and CS.4a

probes,

as described above. A 6.4 kb Sal

_{1 fragment hybridized}

to the 2

_probes.

Based on its

_length

and

_{hybridization}

_pattern, it was concluded that this

fragment

contained the

_{5-prime region}

of both

_copies

of the

_a-amylase

_gene. This 6.4 kb

_{fragment (see}

Fig

2)

was then

_{isolated, purified,}

and subcloned into the Sal site of the

_pUC-based

vector,

pIBI

21. The orientation of the subclones was determined

by

an Eco Rl

digestion.

Each subclone was then

_digested

with Sal 1 and Hind

_III,

and the Sal

1/Hind

III

_fragments

were further subcloned and

_inserted,

in both

_{orientations,}

into

_pIBI

24 and

_pIBI

25

_plasmid

vectors. The

_resulting

subclones are

_presented

in

_Fig

2

_(inserts

of 4 kb and 2.4

_kb,

_{respectively,}

for the

_proximal

and the distal

genes).

These subclones were used for

_sequencing.

se-quences were assembled and

analysed using

a sonic

digitizer

and the

Microgenie

(Beckman)

computer programs

(Queen

and

Korn,

1984).

RESULTS AND DISCUSSION

For both

_amylase

gene

copies

of D erecta, 500

bp

of

coding region

were

_sequenced,

along

with a short

_portion

(200

bp)

of the

non-coding intergenic region.

These

regions

are indicated in

_Fig

_2,

and the

_sequencing

results are

_presented

in

_Fig

3.

Sequence similarity

between the two D erecta _gene

_copies

We found that the 2 _gene

_copies

in D erecta were 100% similar for the

_region

studied. For

instance,

the mutation of the Bam HI site in D erecta is due to an identical nucleotide substitution in the _{2 gene}

_copies.

Several other mismatches

_separated

D

_medanogaster

and D erecta at the sequence

level,

but all of these

polymorphisms

were shared

_by

both gene

copies

of D erecta.

_Thus,

for that

region,

all the nucleotide substitutions that have occured since the D erecta and D

_{medarcogaster species}

divergence,

have been

_incorporated

into both _gene

_copies

of D erecta. Out of the 15 5

nucleotide substitutions that

_separated

D

_melanogaster

and D erecta in that

_region,

9 affected the 3rd base of a

_codon,

and did result in any amino-acid substitution

between the derived

_proteins.

It is worth

_noting

that even the silent substitutions were identical in both

copies

of the D erecta

coding

sequence.

Thus,

the

near-identity

of the two D erecta

_{coding regions}

cannot be

_{explained solely}

as result of

strong selection

_acting

in favour of the occurence of certain new amino acids in the

amylase

of D erecta

_(compared

to that of D

_{melanogaster),}

and

thus,

favouring

the

incorporation

of the

_{corresponding}

new nucleotides into both

_copies

of _{the gene.}

There are several

_{possible explanations}

for the conservation of _sequence

similarity

between the members of a _gene

family.

In this case, neither very recent

_duplication,

nor

_{retrotransposition}

are

plausible explanations,

since

(i),

as was mentioned

before,

the

_duplication

has been shown to be _very ancient

_(Dainou

et

_al,

_1987;

_Payant

et

al,

1988),

and

_(ii)

a

_comparison

of the _upstream

regions

of the 2 genes

copies

in D

melanogaster

(Boer

and

_Hickey,

₁₉₈₆₎

showed that the _sequence

_identity

exdented further than the

_{coding regions. Thus,}

the 2

_a-amylase

_gene

_copies

within these

Sequence

divergence

between D erecta and D

_melanogaster

An

_alignment

of the D erecta

_amylase

_sequences with the

_{proximal amylase}

of the D

_melanogaster

strain

_Oregon

R is

_presented

in

_Fig

3. The time of

_divergence

between the 2

_species,

D erecta and D

_{melanogaster,}

can be estimated from these data. The short _segment from the

_{intergenic region}

_(see

_Fig

2 and

_Fig

_3A)

shows

approximately

15%

divergence

between the 2

_species.

_{This sequence is} not

_subject

to the constraints of natural selection

_acting

on its

_{coding capacity; therefore,}

it serves as a useful

_qualitative

indicator of the considerable amount of

_divergence

between the 2

_species

at the molecular level.

_Although

other members of the

_species

subgroup

have been

_subjected

to molecular

_analysis

_(eg,

Bodmer and

_Ashburner,

1984),

no

_previous

molecular studies have been made on D erecta.

’

As one

_might

_expect, the

coding

_region

showed

_considerably

less

_interspecies

divergence

than did the

_{non-coding intergenic region.}

The 500

_bp

of

_coding

_sequence

presented

here

_(see

_Fig

_3B),

shows 3.3% nucleotide

_divergence

between the 2

_species;

to

_give

a

_{statistically}

valid

_{approximation}

of the _percentage of nucleotide

_divergence

through

the whole

_{coding region.}

Many analyses

have

_already

been carried out to estimate the

_divergence

time

between the 8

_species

of the

_{medanogaster subgroup. Mainly}

based on

_allozymes

and the 2-dimensional

_gel

_{electrophoresis}

_{(Cariou, 1987),}

mitochondrial DNA

_(Solignac

et

at,

1986),

ribosomal DNA and histone _gene

_{family organization}

_(Coen

et

_at,

_1982),

DNA sequence data

(Bodmer

and

Ashburner,

1984)

and

polytene

chromosomes

banding

patterns

(see

Lemeunier et

_at,

_1986),

the D

_{melanogaster subgroup}

has been

_split

into 3

_complexes:

the D erecta - D orena

_complex,

the D

_{yakuba -}

D Teissieri

_complex,

and the D

_{melanogaster complex}

_(including

D

_{melanogaster,}

D

simulans,

D mau7itiana and D

_{sechedlia) (see}

Lachaise et

_at,

_1988;

Lemeunier et

at,

1986,

for a

review).

_{Moreover, mainly}

because of its members’

amylase

_allozyme

patterns, mitochondrial DNA _patterns and its

_inability

to interbreed with

_species

from the other

_complexes,

the D erecta - D orena

_complex

appears to be farthest apart from the 2

_others,

and the

_divergence

time has been estimated to be as

_high

as 15 million _years.

Since some of our data concern the

_{coding region}

of the gene,

they

can be used to confirm the above estimates

_of

_species

_divergence

times. As

_expected,

the level of

_divergence

found between D erecta and D

_melanogaster

_(3%)

is

_{significantly}

higher

than what was found between the

_a-amylase

_genes of different strains of D

_melanogaster

_{(approximately 1%),}

_indicating

a

_greater

_divergence

between the

2

_species

than within the latter

_(Boer

and

_Hickey,

_1986;

_Langley

et

_at,

_1988;

and our

_unpublished

data).

The _percentage

_divergence

between the 2

_species

at silent

sites

_(6.1%)

can be used to estimate time since the

_divergence

of D erecta and D

melanogaster.

If we use the rate of 5.6 x

10-

9

_changes

_per

_site,

_{per year,}

_proposed

by Hayashida

and

_Miyata

₍₁₉₈₃₎

for silent sites within exons, this percentage

gives

a

divergence

time of

_{approximately}

11 million years; the order of which is consistent

with the

previous

estimates of

_divergence

time. It should be

noted,

however,

that

this may be an underestimate of the real

_divergence

time. For

_instance,

as noted

above,

gene conversion can eliminate all

_{substitutions,}

_including

those at silent

_size,

between _gene

_copies

within a

_species,

and this would result in a _gross underestimates of the _age of the

_duplication

if we relied on sequence

divergence

at silent sites.

Although

gene conversion cannot, of course, occur between _sequences which are

separated

in different

_lineages,

other

_factors,

such as biased GC content

_(see

_Hickey

et

_at,

_1989b),

could reduce the observed rate of

_divergence

at silent

_sites;

this would

result in a lowered estimate of the

_divergence

time between

_species.

ACKNOWLEDGMENTS

This work was

_supported

_by

an

_Operating

Grant from NSERC Canada to DA

Hickey

and a Postdoctoral

_Fellowship

from

_{CNRS, France,}

to V

_Payant.

We are

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