Identification of common fragile sites in chromosomes of 2 species of bat (Chiroptera, Mammalia)

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Identification of common fragile sites in chromosomes of

2 species of bat (Chiroptera, Mammalia)

E Morielle-Versute, M Varella-Garcia

To cite this version:

Original

article

Identification

of

common

fragile

sites

in chromosomes

of

2

_species

of bat

(Chiroptera, Mammalia)

E

Morielle-Versute

M

Varella-Garcia

1

Department of Zoology;

2

Department of Biology,

Institute

of Biosciences, UNESP,

PO Box 1,!6, Sdo Jos6 do Rio Preto 15054000,

SP,

Brazil

(Received

14 October 1992;

accepted

2 December

₁₉₉₃₎

Summary - In the

_karyotypes

of the bat

species

Molossus ater and M

_molossus,

spontaneous and

_{bromodeoxyuridine}

_(BrdU)-

or

aphidicolin

(APC)-sensitive

fragile

sites

were located. Four chromosome

_regions

harbored APC-sensitive

_fragile

sites:

_lq9

and

_8q4

in both M ater and M

_{molossus, 3q3}

in

_{M ater,}

and

lp7

in M molossus. The

fragile

sites in

lq9

and

_8q4

were also observed without induction in M molossus. BrdU-sensitive

_fragile

sites were not detected.

_Despite

observations in several other

species,

the

_fragile

sites detected in Molossus are not coincident with the

_breakpoints

involved in the chromosome

rearrangements

occurring

in the evolution of 7

_species

of the Molossidae

_family.

fragile site

_/

chromosome

_/

bat

_/

_{bromodeoxyuridine} induction

_/

_aphidicolin induction

Résumé - Identification de sites chromosomiques fragiles communs à 2 _espèces de chauve-souris.

_L’analyse

de la

_{fragilité chromosomique spontanée}

ou induite _par

bromodéoxyuridine

(BrdU)

et

_{aphidicholine}

_(APC),

réalisée sur le _caryotype de

_{2 espèces}

de

_{chauve-souris,}

Molossus ater et M

_molossus,

a _permis

_{d’identifier 4 sites fragiles}

induits

par APC:

1 q9

et

_8q4

chez M ater et M

_{molossus, 3q3}

chez M ater et

_{1 p7}

chez M molossus. Les sites

_fragiles

en

_{1 q9}

et 8q4 ont aussi été observés chez M molossus sans induction.

Les sites

_{fragiles repérés}

dans ces

_espèces

ne coincident _pas avec les _points de cassure

impliqués

dans les

_{réarrangements chromosomiques}

qui ont eu lieu au cours de l’évolution

de 7

_espèces

de la

_famille

des Molossidae.

INTRODUCTION

Fragile

sites are

_{specific points}

on chromosomes which are

_expressed

as

non-randomly

distributed gaps and breaks when chromosomes are

exposed

to

_specific

agents

or culture conditions

(Berger

et

_al,

_1985).

The induction of

_fragile

site

expression

is

_generally

related to imbalance of

deoxyribonucleotide pools

during

G

1

and S

_phases

_{following thymidylate}

stress

_(Yan

et

_al,

₁₉₈₈₎

or treatment with the

_{thymidine analogue bromodeoxyuridine}

_{(BrdU) (Sutherland}

et

_al,

_1985).

Expression

of

_fragile

sites can also be induced at

_{high frequencies by}

inhibitors

of DNA semiconservative and

_{repair synthesis,}

_{including aphidicolin}

_(Glover

et

_al,

1984),

arabinofuranosyl

cytosine,

and

arabinofuranosyl

adenosine

_(Leonard

et

al,

1988).

Although

the

_{biological significance}

of

fragile

sites remains

_unclear,

_they

have attracted attention since the rare

_fragile

site in

_Xq27.3

and a

_type

of X-linked mental retardation in humans were associated

_(Sutherland

and

Hecht,

1985).

Furthermore,

several

_findings

have correlated

_fragile

sites with chromosomal

rearrangements

in cancer

(Le

_Beau,

_1986;

De

_{Braekeleer, 1987;}

Mir6 et

_al,

_1987),

_infertility

in humans

(Schlegelberger

et

al,

1989),

breakpoints

involved in chromosomal evolution of

primates

(Mir6

et

_al,

_1987),

and

_preservation

_{of syntenic}

_{groups in} mammals

_(Djalali

et

_{al, 1987;}

_Threadgill

and

Womack,

1989).

More than 100

_fragile

sites have been identified in human

_chromosomes,

all

classified

_by

their band

_location,

_gene

_{symbol, population}

_frequencies,

and mode of induction

_(Mir6

et

_{al, 1987;}

Hecht et

_al,

_1990).

BrdU-sensitive

_fragile

sites have

also been described in Chinese hamsters

_(Hsu

and

_Sommers,

1961;

Lin et

_al,

_1984),

cactus mice

_(Schneider

et

_al,

_1980),

cattle

_(Di

Berardino et

_al,

_1983),

and reindeer

(Gripenberg

et

_al,

_1991).

BrdU-

_and/or

folate-sensitive

_fragile

sites were

_recently

reported

in the horse

karyotype

(R

o

nne, 1992).

Aphidicolin

(APC)-sensitive

fragile

sites have been detected in the chromosomes of mice

_(Djalali

et

_{al, 1987;}

Elder and

Robinson,

1989;

McAllister and

_Greenbaum,

_1991),

rats

_(Robinson

and

Elder,

1987),

dogs

(Wurster-Hill

et

_{al, 1988;}

Stone et

al, 1991a,

1991b),

pigs

(Riggs

and

Chrisman,

1991),

and rabbits

_(Poulsen

and

_Ronne,

_1991).

Folate-sensitive

_fragile

sites were detected in the Persian vole

_(Djalali

et

_al,

_1985),

the mouse

(Sanz

et

_al,

1986),

cattle

_(Uchida

et

_al,

_1986),

and in the Indian mole rat

_(Tewari

et

_al,

_1987).

To determine the

_potential

_phylogenetic

_implications

of chromosomal

_fragility

in the evolution of

bats,

common BrdU- and APC-sensitive

_fragile

sites in the

karyotype

of 2

_species

of the

_family

Molossidae

_{(Chiroptera, Mammalia)}

were

examined.

MATERIALS AND METHODS

Primary

cultures of fibroblasts were derived from

_explants

of ears from a total of 9 animals of the

species

Molossus ater and 8 from Molossus molossus. The cultures were established and maintained in

_Eagles’

minimum essential medium

(MEM)

supplemented

with

20%

fetal calf serum,

L-glutamine, penicillin

and

streptomycin.

BrdU

_{(20 gM)}

and APC

_{(0.02 gM)}

were added to cultures 26 h before harvest. In

was

_performed

with concurrent control cultures. Colchicine

₍₄

x

10-

4

M)

was added to the cultures 30 min before harvest. Cells were

_exposed

to

0.8%

sodium citrate for 30

_min,

fixed in

_{methanol/acetic}

acid

_3:1,

_dropped

onto wet

_slides,

and air-dried. Slides were

_{homogeneously}

stained with

2%

Giemsa and around 100

metaphases

from coded slides of treated and untreated cultures of each animal were scored for

breaks,

gaps, and

rearrangements.

After identification of the

lesion,

the slides were destained and GTG

banding

(G-band

after

_trypsin

and

_giemsa

_treatment)

was used to

_identify

the exact localization of the aberrations.

To determine the presence of a

fragile

site,

2 criteria were considered:

(i)

the

occurrence of at least

2%

lesions at a

_given

chromosome

_region

in cells submitted to a certain culture condition in at least 2 animals of the same

_species;

and

(ii)

the

homozygous

expression

of a lesion. A

_{chi-squared analysis}

of the distribution of anomalies was

_performed

to determined whether their

frequencies

were

equally

distributed in treatments and controls.

RESULTS

The

diploid

number of chromosomes in M ater and M molossus is 2n = 48 and their

karyotypes

have similar

_morphology

and G-band

pattern

(fig 1).

The

_frequencies

of

spontaneous,

BrdU- and APC-induced lesions in bat chromosomes are

_given

in table I. These lesions manifested themselves as either

nonstaining

gaps, chromatid or chromosome

breaks,

or deletions.

The number of aberrations in BrdU-treated and untreated

_(control)

cultures of

M ater and M molossus was

_low,

but BrdU-treated cells were

_{significantly}

more

damaged

than controls

_(x

₂

₌

_8.9;

1

_df;

P <

_0.05).

_Only

3.6%

of the cells in the BrdU-treated cultures and

0.6%

of cells in the control cultures showed chromosome

lesions in

M ater,

with a total of 20 and 3

events,

respectively.

In M molossus

3.8%

of the cells in the control culture and

4.8%

of BrdU-treated cells showed chromosome

and breaks was variable but

_they

occurred in the euchromatic chromosome arms. Chromatid _gap was the most

_frequent

event.

Four chromosome bands exhibited lesions in at least

2%

of the cells in the BrdU-treated cultures:

_lq5

and

_lq9

in

_{M ater; 1q13}

and

8q4

in M molossus. M molossus

bands were considered to harbor

_fragile

sites since the aberrations were not observed in the

_homozygous

conditions or in more than one animal of the same

_species.

The APC treatment was more effective in the induction of chromosomal aberra-tions than BrdU:

9.2%

of the cells

_presented

a total of 50 anomalies in

_{M ater;}

11.5%

of the cells exhibited a total of 75 aberrations in M molossus

_{(table I).}

More than one lesion or the

_homozygous

_expression

of a

_given

aberration occurred in a number of cells. In these

tests,

the most

_frequent

_type

of aberration was the chromosome gap. The

chi-squared

analysis

detected

significantly

more

_damaged

chromosomes in the APC-treated than in the control cultures

_(x

₂

=

20.0;

1

df;

P _<

0.001).

Fourteen

_regions

in the euchromatic arms in which such lesions occurred were identified in at least

2%

of the cells:

_{lp7, lq5, lq9, 1q13, 3q3, 4q3-4, 5q8, 7q3-4,}

8q4, 8q5-6, lOq3-4, 20q2

and

_Xq4-6

in the APC-treated cultures and

_1q13-15

in the control cultures

_{(fig 2A).}

_{However, only}

4 of these 14

_regions

fulfilled the criteria to be

_qualified

as

_{harboring fragile}

sites

_(fig

_2B):

_lq9

and

_8q4

in both M ater and M

_{molossus, lp7}

in M

_Molossus,

and

_3q3

in M ater. The

_fragile

sites in

_lq9

and

8q4

were also observed without induction in M molossus. The

_highest

_expression

rate

_(8%)

was achieved

_{by 8q4. Furthermore,}

an interindividual variation in the

frequencies

of

_expression

of the

_fragile

sites was observed in all of the 4

_bands,

as well as an

_{interspecific}

variation observed in

_lq9

and

_8q4.

It is

_important

to

_emphasize

that the 5 bands referred to above as

_presenting

lesions in the test with BrdU

_(lp7,

_{lq5, lq9, 1q13}

and

_8q4)

are included in the

14 identified in APC treatment and 3

_(1p7,

_lq9,

and

_8q4)

are included in the 4 that

harbored

_fragile

sites.

DISCUSSION

The mechanisms of

_expression

of the BrdU-sensitive

_fragile

site are not

_totally

understood. The

_chronology

of the events after _{exposure to} this chemical indicates that it acts

_during

the late

_S-phase

and affects late

_replicating

_regions

_(Sutherland

et

al, 1984,

1985).

An increased

_frequency

of _gaps and breaks in the chromosomes of M ater and M molossus was observed when the

_thymidine

_analogue

BrdU was

incorporated. However,

the

frequencies

and conditions in which these alterations were

_expressed

did not fit the criteria for

_{qualification}

of the affected

_region

as

harboring fragile

sites. These

_findings

may be related to the

_period

of _exposure to BrdU

₍₂₆

_h).

_Although

_exposure to BrdU for 18-26 h has been used for

_experiments

with human

_lymphocytes

and several mammalian fibroblasts

_(Schneider

et

_{al, 1980;}

Lin et

_{al, 1984;}

Fundia and

_Larripa,

_1989),

the

_highest

_expression

of common BrdU-sensitive

_fragile

sites in human

_lymphocytes

was achieved after 4-12 h of treatment

(Sutherland

et

al, 1984,

1985).

Furthermore,

fragile

sites have been identified in both

_lymphocyte

and fibroblast

_cultures,

but the cells in the latter appear

refractory

to their

_expression

_(Robinson

and

_Elder,

_1987).

_Hence,

the lower

_frequency

in the

expression

of the

_fragile

sites in bat fibroblasts _may be due to the

_{specific refractivity}

of this cell

type

as well as to a

_{susceptibility}

of

_lymphocytes.

(1991)

also confirmed that the aberrations induced

_by

this chemical were

_primarily

of the chromatid

type

and included gaps, breaks and

interchanges.

APC,

a

_{diterpenoid mycotoxin}

that inhibits

_alpha

DNA

_polymerase

associated with DNA

_replication,

induces _gaps and breaks at common

_fragile

sites in human

chromosomes

_(Glover

et

_al,

_1984),

either as chromosome or chromatid aberrations

(Murano

et

_al,

_1989).

The most

_frequent

_type

of aberration exhibited

_by

APC-treated cells in this

_study

was the chromosome _gap. The results _may reflect the

number of

_cycles

a cell had

_completed

after the introduction of APC into

_cultures,

and/or

even the

efficiency

of the

_repair

mechanisms.

It is

_interesting

to note that the

_fragility

observed in the

Xq4-6

was

displayed by

only

1 animal of the

_{species M ater,}

and so this

_region

was not

_qualified

as

_harboring

a

_fragile

site in this work.

_{Corresponding X-fragility}

has been observed in several

distantly

related mammalian

_species

_including

_{humans, horses,}

rats,

rabbits, pigs,

study

may then

correspond

to the

Xq22

fragility

observed in

humans, horses,

and rats

_(R

_o

_nne

et

_al,

_1993).

Since the

_species

_present

_{complete homology}

in their

_karyotypes,

the

interspe-cific variation was

_surprising.

Conservation of

5-azacytidine-sensitive fragile

sites was described in

_primates

_(Schmid

et

_al,

_1985),

as well as

_fragility

in bands shared

by

horses and humans

_{(Ronne, 1992).}

_Beyond

the

_{interspecific}

and interindividual variations observed in the number of

_regions

_{harboring fragile}

_sites,

individual vari-ation in the

_frequency

of cells

_expressing

the

_fragile

sites was also observed _among

positive specimens,

as

_{previously reported,}

for

_instance,

in rabbits

(Poulsen

and

Ronne,

1991)

and humans

_{(Craig-Holmes}

et

_al,

_1987).

Variation in the molecular nature of the

_fragile

sites could

_explain

variation in

_{expressivity,}

as

exemplified by

the human

_fragile

site in

_Xq27.3.

A

_highly

_polymorphic

CGG

_repeat

was discovered within the gene FMR-1

mapped

in this

region

and a somatic mosaicism was well

documented, indicating

mitotic

_instability

of alleles

_(Fu

et

_al,

_1991).

_Large

expan-sions of the

_{repeated region}

_{(250-4 000 repeats)}

are

_probably

more

_easily

detected

by cytogenetic analysis

than small

_expansions

_{(52-200 repeats).}

Despite

the observed association between the

_fragile

sites and the

_breakpoints

involved in chromosomal

_{rearrangements}

in several animal

_species

_(Djalali

et

_al,

1985;

Mir6 et

_{al, 1987;}

_Riggs

and

_Chrisman,

_1991),

our results did not show _any coincidence between the detected bands

_{harboring fragile}

sites in the

_species

of

Molossus and the

breakpoints

involved in chromosomal

_{rearrangements}

occurring

in the evolution of 7

_species

of the

_family

Molossidae

_{(Morielle-Versute,}

_1992).

However a more detailed

_study

is _necessary to

_verify

the

_{complete relationship}

between these 2

_phenomena

in bats.

ACKNOWLEDGMENTS

We are indebted to FAPESP and FUNDUNESP for

partial

financial support. The authors are

grateful

to VA Taddei for

helping

in

identifying

the

specimens

studied and to

J

Rodrigues

dos Santos and C Antonio N6bile for their excellent technical assistance.

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