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Transposable elements in South American populations of
Drosophila simulans
Elgion Lucio da Silva Loreto, Arnaldo Zaha, Vera Lucia da Silva Valente
To cite this version:
Original
article
Transposable
elements
in
South
American
populations
of
Drosophila
simulans
Elgion
Lucio
da Silva Loreto
Arnaldo
Zaha
Vera
Lucia da
Silva Valente
a
Departamento
deBiologia,
Universidade Federal de Santa
Maria,
SantaMaria, RS,
Brazilb
Departamento
deBiotecnologia,
Universidade Federal do Rio Grande do
Sul,
PortoAlegre, RS,
Brazil°
Departamento
deGenética,
Caixa Postal 15053, Universidade Federal do Rio Grande doSul,
CEP
91501-970,
PortoAlegre, RS,
Brazil(Received
20 June 1997;accepted
27January
1997)
Abstract - This
study investigated
the occurrence of fourtransposable
elements(mariner,
gypsy, hobo and
412)
in South Americanpopulations
ofDrosophila
simulans. Thegenomic
hybridization
patterns of 12 differentpopulations
were determinedby
Southern blotanalyses.
Eventhough
a low number of marinercopies
wasobserved,
eachpopulation
presented
a characteristichybridization
pattern,suggesting
that the element is active.The number of gypsy
copies
was alsolow,
but allpopulations
bore a similarhybridization
pattern. In this paper we describe the occurrence of an almost
gypsy-free strain,
which had not yet been found for D. simulans nor D.melanogaster.
In the case ofhobo,
we didnot detect the
1.1-kb-long
deleted hobo element in any of the South Americanpopulations
while this element is present in all strainsoriginating
from othergeographical
sites thatwere
analysed
up to now,suggesting
that the hobo element may have veryrecently
invadedthe genome of South American D. simulans
populations.
The412
elementpresented
some
population-specific
band patterns,indicating
that this element may have sometransposition activity
in the differentpopulations. ©
Inra/Elsevier,
ParisDrosophila simulans
/
transposable element/
mariner/
hobo/
gypsy/
412*
Correspondence
andreprints
E-mail:valente@ifl.if.ufrgs.br
Résumé - Éléments transposables dans des populations sud-américaines de Drosophila simulans. Cette étude recherche la
présence
de quatre élémentstransposables
(mariner,
gypsy, hobo et
412)
au sein depopulations
sud-américaines deDrosophila
simulans. LeBlot. Bien que seul un
petit
nombre decopies
de marinerait étéobservé, chaque population
présentait
unprofil d’hybridation caractéristique,
cequi suggère
que l’élément était actif. Le nombre decopies
de l’élément gypsy s’est avéré trèspetit ;
avec unprofil d’hybridation
similaire dans toutes les
lignées,
cequi indique
que l’élément était inactif dans lespopu-lations étudiées. L’une des
lignées
necomportait pratiquement
pas d’élément gypsy, cequi
n’était encorejamais
arrivé chez D. simulans ni D.melanogaster.
L’élément hobode
1,1
kb n’a été détecté dans aucunelignée d’Amérique
du Sud alorsqu’il
existe danstoutes les
lignées, originaires
d’autres sitesgéographiques, analysées jusqu’à présent.
Ces informations en accord avec les données de lalittérature, suggèrent
que l’élément hobo n’apeut-être
envahi que récemment legénome
despopulations
de D. simulans enAmérique
du Sud. Pour l’élément l,12, les
populations
étudiées ont montré des bandesspécifiques,
ce
qui suggère
que cet élément peut avoir des activités detransposition
dans legénome
de ces
mouches. @
Inra/Elsevier,
ParisDrosophila simulans
/
élément transposable/
mariner/
hobo/
gypsy1. INTRODUCTION
Transposable
elements(TEs)
havealready
been shown to compose asignificant
fraction of the genome of a widevariety
oforganisms
[1].
Approximately
10%
of the D.
melanogaster
genome consists of 50 different families of TEs[13].
The amount of middlerepetitive
DNA sequences in D. simulans was estimated to be about one third thatpresent
in itssibling
species
D.melanogaster
!10).
It is believed that amajor portion
of thisrepetitive
DNA iscomposed
of TEs!26).
Virtually
all families of TEs that have been cloned from D.melanogaster
are alsorepresented
in the genome of D. simulans!6).
Theonly
exception
to the similar TEcomposition
between the twospecies
is the presence of the Pfamily,
which isonly
found in D.melanogaster
and mariner which isonly
found in D. simulans.Considering
thatvirtually
the same TE families arepresent
in bothspecies
and that D. simulans hasonly
one third the totalrepetitive
DNApresent
in D.melanogaster,
the mean number of elementcopies
perfamily
should be lower in D. simulans[17,
20,
23].
These last authors detected
significant
differences in copy number of TEs between thosespecies by
in situhybridization.
Studies
covering
the whole distribution range of D. simulans should make itpossible
to better understand the differences in the number ofcopies
of TEs between thisspecies
and D.melanogaster,
in addition toproviding
important
information on theevolutionary history
of TEs. In anattempt
to contribute to the resolution of thisquestion,
weinvestigated
the occurrence of four differenttransposable
element families(mariner,
hobo,
gypsy and412),
by
means of Southern blotanalyses,
in 12 D. simulanspopulations originating
from different locations within the American continent.2.MATERIALS AND METHODS
2.1.
Fly
stocksThe
following
strains of D. simulans wereemployed
in thepresent
study:
1)
PeruLima, Peru;
2)
Salvador - collected inSalvador,
Bahia -Brazil,
in1995;
3)
RJ -collected in the National Park ofTijuca,
Rio de Janeiro -Brazil,
in1995;
4)
SBC -collected in Sao Bernardo doCampo,
Sdo Paulo -Brazil,
in1993;
5)
MGS - collected inEldorado,
Mato Grosso do Sul -Brazil,
in1994;
6)
Maquin6 -
collected inMaquin6,
Rio Grande do Sul -Brazil,
in1994;
7)
Goethe - collected in PortoAlegre,
Rio Grande do Sul -Brazil,
in1991;
8)
dpp-like -
derived from aspontaneous
mutant
originating
from in ahypermutable
wild strain of D. simulans collected in PortoAlegre,
Rio Grande do Sul -Brazil,
in1990;
9)
yellow -
derived from aspontaneous
mutant of theyellow
locus encountered in apopulation
sample
fromItapua,
Rio Grande do Sul -Brazil,
in1982;
10)
Camobi - collected in SantaMaria,
Rio Grande do Sul -Brazil,
in1994;
11)
Montevideo - collected inMontevideo,
Uruguay,
in1991;
12)
1093(obtained
from CaltechCenter) -
collectedin
Islamorada,
Florida - USA.2.2. Southern blots
Genomic DNA was
prepared
from 40-50 adultflies,
according
to Jowett[16].
DNAsamples
(approximately
5 vg from eachstrain)
werecompletely digested
with restrictionendonucleases,
submitted toelectrophoresis
on 0.8%
agarosegels,
transferred tonylon
membranes andhybridized
to nick-translated DNAprobes
(labeled
with32
P-a-dATP)
in the presence of 50%
formamide at 42 °C. Each filter was washed three times with 0.2X SSC and 0.5%
SDS for 20 min at 42 °C.A 0.9-kb
fragment
obtainedby
NheI/PvuII
digestion
of Mos 1plasmid
DNA[19]
was used as aprobe
for theanalysis
of the mariner TEfamily.
As aprobe
for the gypsyelement,
weemployed
thepGGHS
plasmid,
which contains acomplete
gypsy element(9).
Members of the hobofamily
wereprobed
with thepHX4 plasmid,
which contains a XhoI 2.6-kbfragment
of hobo element of D.melanogaster.
The 2.6-kbfragment
was removed from acomplete
element contained in thepHLFl plasmid
[4]
and subcloned into theBluescript
plasmid.
Theprobe
used to detect412,
was an element four HindIII-EcoRIfragment
fromcDml,l2
with a totallength
of 4.4-kb was used[27].
3. RESULTS
3.1. Southern blot
analyses
3.1.1. Mariner
family
When the DNAs from D. simulans
populations
of diversegeographical
origins
werehybridized
to the mariner elementprobe,
we observedhybridization
patterns
that were characteristic to each one of them. The mean number ofhybridizing
bands variedaccording
to the restriction enzyme used. Forinstance,
when D. simulans DNA wasdigested
withSall,
we saw, on average, 9.0 t 4.1hybridizing
bands(see
figure
1A).
Theyellow
strainpresented
thelargest
number ofhybridization
bandsdigested
with this enzyme. The number ofcopies
per genome should have beenequal
toapproximately
half the number of observed bands.Enzymes
that do nothave internal sites in the mariner element sequence would
yield
a smaller number ofbands,
which shouldcorrespond
to theapproximate
number ofcopies
of the element. When DNA wasdigested
withXhoI,
forexample,
the number ofhybridizing
bands was on average 4.4 f 1.5. Onceagain,
theyellow
strain was the one with thelargest
number of bands(a
total ofnine)
which were not seen in thisSouthern,
but have been observed in otherexperiments
(data
notshown).
Maquin6
and 1093 were the onesshowing
the smallest number(only
threehybridization
bands)
( figure 1 B).
3.1.2. Hobo
family
The
cleavage
sites for XhoI in the hobo element are close to the inverted terminalrepeats,
anddigestion
with this enzymeyields
a2.6-kb-long fragment
when acomplete
hobo copy ispresent,
and smaller bands if deleted elements arepresent.
Allstrains
analysed
possessed
the 2.6-kbband, indicating
the occurrence ofcomplete
elements(figure !).
Boussy
and Daniels[5]
have shown the occurrence of a 0.7-kb band(corresponding
to a deleted element very common in D.simulans)
in themajority
of the strainsthey
analysed.
In thepresent
study,
we detected this bandin the North American strains
(1093),
but we did not find it in any of the South Americanpopulations.
Bands
greater
than 2.6kb,
as seen infigure
2,
were also encountered in otherspecies
of themelanogaster subgroup.
According
toBoussy
and Daniels[5]
the occurrence of such bands can beexplained by
the presence of other TEs whichcarry sequences related to hobo. Hobo elements
bearing
a deleted or altered XhoI site are alsointerpreted
as old sequences of hobo localized in heterochromatin.3.1.3.
Gypsy family
Hybridizing
the membranes to the gypsy elementprobe
resulted in a small num-ber ofhybridization
bands,
with an average of 7.8 ! 2.8 bands. Ahighly
conservedhybridization
pattern
was observedthroughout
the D. simulanspopulations.
When thefly
DNA wasdigested
with Sall orEcoRI,
themajority
of the bands was com-mon to almost allpopulations -
few bands werespecific
to eachpopulation
( figure
3).
Based on the known sequence of D.melanogaster’s
gypsy, this element should have asingle cleavage
site for EcoRI and none for Sall.Therefore,
if we assume the same element structure for gypsy of D.simulans,
wemight
haveexpected
to see differenthybridization
bands among the different studiedpopulations
in the case of the occurrence of insertion sitepolymorphism.
The
yellow
strain was animportant exception.
It seemed to bear very fewcopies
of gypsy in its genome, as can be seen in
figure
3A(lane
8).
It should be noted that the Southern membrane shown infigure 3A
is the same as the one used infigure
3.1.4. 412
family
The
412
element of D.melanogaster
carries four XhoI restriction sites(27!.
We thusexpected
to obtain bandscorresponding
to2.6,
2.1 and 0.9-kb in size in the presence of acomplete
copy ofl,12.
As shown infigure 4A,
all theanalysed
populations
carried theexpected
bands,
in addition togreater-sized bands,
some of which werespecific
for eachpopulation.
The 1.4- and 0.6-kb
fragments expected
with an EcoRIdigestion
were found in allpopulations,
in addition togreater-sized
bands,
some of which werespecific
for eachpopulation
(figure
4B).
4. DISCUSSION
Capy
et al.(8!,
Maruyama
and Hartl[18]
and Giraud andCapy
[14]
haveanalysed
several D. sim!alanspopulations by
SouthernBlot,
and their results showed that the number of marinercopies
per genome varies from 0 to 15. Ourpresent
findings
on the number of marinercopies occurring
in the genome of D.simulans,
as inferred from the number of bands encountered in the Southern blot assays, were very much inagreement
with values found inprevious
studies about the occurrence of mariner in strains of thisspecies
from otherorigins.
to strains from diverse
geographical
origins,
indicating
that at least some of them arelong-existing
insertion sites and may be fixed within thespecies.
Giraud andCapy
[14]
described threebanding
patterns
in mariner Southern blotanalysis:
i)
populations
from differentgeographical
origins
which share the same threebands,
these
populations
having
fewbands;
ii)
populations
with morebands,
some of these common to all the lines of apopulation;
iii)
populations
with few bands different amongpopulations.
In thepresent
study,
we did not find anyhybridization
bands common to the different
populations;
eachpopulation
showed a characteristichybridization profile,
inagreement
with Giraud andCapy’s
pattern
3.Laboratory populations
showgreater
variability
in mariner copy number than naturalpopulations
(15!.
This factmight
explain
thehigh
copy numberoccurring
inour
yellow
strain,
as thispopulation
has beenkept
in thelaboratory
since 1982. All South American strainsemployed
in thepresent
study
wererecently
collected in thewild,
except
for theyellow
anddpp-like
strains,
and none of them were mariner-free. Thisapparent
non-existence of mariner-freepopulations
amongrecently
collected ones is alsosupported by
thefindings
ofCapy
et al.[8]
and Giraud andCapy
(14!.
Periquet
et al.[21]
recorded the fact that almost all naturalpopulations
of D.melanogaster
possesscopies
of a1.5-kb-long internally
deleted hobo element.They
called this element Th. These authorssuggest
that the accumulation of these Th elements in the genomemight
have someregulatory
role in hobotrans-position.
Such a role has beensuggested
for the KP element of the Pfamily
ofwere found in both
species.
In several D.melanogaster
strainsthey
observed the occurrence of the 1.5-kb(Th)
element. In D.simulans,
only
four strains(one
fromPeru;
one fromColombia;
and one of unknownorigin)
did not have thehybridization
band that indicates the presence of acomplete
element. The three South American strains also did not bear the 0.7-kb band thatcorresponds
to aninternally
deleted element of 1.1 kb. All otherstrains, originating
from NorthAmerica,
Australia and SouthAfrica,
as well as the one with unknownorigin,
showed a verystrong
hybridization signal corresponding
to the 0.7-kb band.In the
present
study,
we found two D. simulans strains with very weak hybridiza-tionsignals
for the 2.6-kb band(the
existence of whichrepresents
the occurrence of acomplete
element),
indicating
that these strains carry fewcopies
of the hobo element. Such strains have beenkept
in thelaboratory
for alonger period
of time(14
and 6years)
than all the otherpopulations,
which wererecently
collected innature and have
strong
hybridization signals
for the 2.6-kb band.Furthermore,
the 0.7-kb band(corresponding
to aninternally
deletedelement)
was encounteredonly
in the two North Americanpopulations;
all the South Americanpopulations
are devoid of this element.Considering
ourfindings
of few hobo sequences in olderfly
stocks and the results obtainedby Boussy
and Daniels[5],
as awhole,
we maysuggest
that hobo has beenrecently
scattered in the genome of South Americanpopulations
of D. simulans. Anotherargument
in favor of thispossible
event is the reduced number ofcopies
of deleted elements in the South Americanpopulations,
most of
all,
the absence of the 1.1-kb elements which are so common topopulations
of other
geographical origins.
If the deleted elements do accumulate in the genome as a function of time afterinvasion,
and ifthey
aretruly
involved in theregulation
of hoboactivity
!11!,
then wemight
suppose that hobo is active in the South Amer-icanpopulations
of D. simulans but notenough
time haselapsed
for a sufficient accumulation of deleted hobocopies
in their genomes.All D.
melanogaster
strains contain inactivecopies
of gypsy located at the samepositions
in thepericentromeric
heterochromatin[22].
Even non-relatedstrains,
whencompared by
Southern blotanalysis,
show very similarhybridization
patterns,
suggesting
that the gypsy element has invaded the genome of D.melanogaster
veryearly
in thespecies evolutionary history
[7].
Nevertheless,
a low copy number ofputative
active gypsy with no fixed sites were detectedby
in situhydridization
inD.
melanogaster
chromosomes arms[2, 23]
and in D. simulans[23].
The South American
populations
of D. simulanspresently
examined bear very similarhybridization
patterns,
in the same way as D.melanogaster
strainscarrying
few gypsycopies
do.According
to ourestimates,
asjudged by
the number of Southernhybridization
bands,
South Americanpopulations
of D. simulans also bear a reduced number of gypsycopies. However,
this Southernpattern
mayrepresent
the ancient inactivecopies
of gypsy in the heterochromatin. If there exist activecopies
of gypsy in a low number inpolymorphic
insertionssites,
these can be related to the weak bandsspecifically
detected in somepopulations
or thesecopies
were not able to be detectedby
the Southerntechnique,
since every individualpresents
onespecific
band.for 14 years. Stochastic loss
[12]
may be anexplanation
for theextremely
low number of gypsy sequences it carries. Thishypothesis
issupported by
the factthat all other studied strains that were derived from
recently
collectedpopulation
samples
do possess the gypsy element.The
412
element is aretrotransposon,
7.6 kb inlength,
with481-pb-long
LTRs(27].
Thiselement,
similar to otherretrotransposons,
hasapparently
beeninhabiting
the genome ofdrosophilids
for a verylong
period
of time. Vieira and Bi6mont[24,
25]
determined, by
in situhybridization
topolytene chromosomes,
the number ofcopies
of the41!
elementoccurring
in naturalpopulations
of D. simulans. Agradient
in copy number wasobserved, varying
from 20 inEurope,
to 3-9 inAfrica,
with the same tendencies between North and South America. Sometransposition
bursts were observed within certain localpopulations
that possess ahigh
copy number of412.
These authorssuggest
this element hasrecently
invaded the genome of D. simulanspopulations.
In the
present
study
we observed that all the studiedpopulations
carriedcopies
of anapparently
complete 412 element,
and that the EcoRI and XhoI sites were conserved in relation to the D.melanogaster
412
element. The occurrence of a few bands that were characteristic of each strain maysuggest
that this element is activein South American D. simulans
populations.
ACKNOWLEDGEMENTS
This research was
supported by
grants andfellowships
ofCNPq, CAPES, FINEP,
PROPESP-UFRGS and FAPERGS(grant
number931017.6).
We would like to expressour
gratitude
to Drs D.Hartl,
D. Dorsett, R. Blackman and P.Sniegowsky
forgifts
of TEclones;
to C.Rohde,
L. Basso and O. Crosa forcollecting
the flies strains and to J.Rodrigues
and L.P.Regner
forsuggestions.
We are alsograteful
to the two anonymous reviewers for their valuablesuggestions.
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