Identification of aphidicolin-induced fragile sites in domestic pig chromosomes

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Identification of aphidicolin-induced fragile sites in

domestic pig chromosomes

Pk Riggs, Cl Chrisman

To cite this version:

Identification

of

_{aphidicolin-induced}

fragile

sites in domestic

_pig

chromosomes

PK

_Riggs,

CL

_{Chrisman†}

Pv,rdue

_{University, Cytogenetics Laboratories, Departement of}

Animal

Sciences,

West

_Lafayette,

IN _47907, USA

(Proceedings

of the 9th

_{European Colloquium}

on

_Cytogenetics

of Domestic

_Animals;

Toulouse-Auzeville,

10-13

_July

₁₉₉₀₎

chromosome

/ fragile sites

/ pig

INTRODUCTION

Fragile

sites are

specific

chromosomal loci _prone to

_breakage

which are observed as

gaps, breaks or

_{rearrangements}

under

_appropriate

in vitro culture conditions. More than 100

_fragile

sites have been identified in human chromosomes and classified

according

to their mode of induction and

_{population frequency}

_(Sutherland

and

Ledbetter,

1989).

All

_fragile

_sites,

_by

_definition,

are inducible in

_culture,

and induction results in their

_expression

or elevates the

_proportion

of cells in which

fragile

sites are observed if

they

are

_{’spontaneously’ expressed}

_(Sutherland

and

Hecht,

1985).

Fragile

sites are also identified as rare

(occurring

in less than

1%

of

the

_population)

or common.

One useful

_{fragile site-inducing}

_agent

is

_aphidicolin

_(APC).

This chemical is a selective inhibitor of DNA

_{polymerase-a,}

the

_{major eukaryotic}

_polymerase

which adds

_{deoxynucleotide monophosphates}

to the 3’-end of a DNA

_primer,

_during

both

_replicative

and excision

_repair

DNA

_synthesis

_(Fry

and

_Loeb,

_1986).

At low

concentrations

_{(0.2 ttM),}

APC has been used to

induce

fragile

sites in human

lymphocyte

cultures without

_{significantly decreasing}

mitotic

_yield

_(Glover

et

al,

1984).

The

_significance

of

_fragile

sites remains

_unclear,

_although

numerous

hypothe-ses have been

_suggested

_{linking fragile}

sites to cancer chromosome

breakpoints

and

_{protooncogene}

locations

_{(LeBeau, 1988),}

_reciprocal

translocation

_breakpoints

(Riggs

and

_Chrisman,

_1989),

chromosome

_{rearrangements}

_resulting

in

_spontaneous

abortion and

_infertility

_{(Schlegelberger}

et

_al,

₁₉₈₉₎

and

_evolutionary

preservation

of

_syntenic

groups

(Threadgill

and

Womack,

1989).

Chromosome

_aberrations,

es-pecially

reciprocal translocations,

are of economic

_importance

in the domestic

_pig

because

_{they usually}

result in

_{significantly}

reduced

_fertility

_(reviewed

_by

_Popescu

et

_al,

_1984).

Although

extensively

documented in human

chromosomes,

the

study

of

_fragile

sites has been

_neglected

in domestic animals. Since

_fragile

sites are distributed

non-randomly,

and it has been

_suggested

that

_reciprocal

translocations are distributed

non-randomly

across the

_pig

_genome

_(Fries

and

_Stranzinger,

_1982),

this

investiga-tion was undertaken to

_study

common

fragile

sites in

_pig

chromosomes. The initial

objectives

of the

_project

were 3-fold:

1)

establish

protocols

for

studying fragile

sites

in chromosomes of domestic

animals,

specifically

the

_pig;

₂₎

_identify

band

loca-tions of

_fragile

sites in

_{pig chromosomes;}

₃₎

determine whether a correlation exists

between common

_fragile

site locations and

_reciprocal

translocation

_breakpoints.

MATERIALS AND METHODS

Peripheral

blood was collected via the anterior vena cavae of 2 male and 2 female

purebred

Duroc

_pigs,

and 7 male and 8 female 3- or

_4-way

crossbred

_pigs

( Yorkshire

x Landrace x

_{Hampshire ;}

Yorkshire x Chester White x

_{Hamsphire ;}

Yorkshire x Landrace x

_Harnpsiaire

x Duroc ; Yorksiaire x Chester White

x

_Hampshire

x Duroc

_).

Standard cultures were established with RP1VII-1640

(Gibco),

10%

fetal bovine serum

(Gibco),

2%

phytohemagglutinin-M

(Gibco),

1%

1-glutamine,

0.3%

sodium

_heparin,

0.5%

gentamicin

sulfate and

5%

whole blood.

Cultures were _grown in

5%

CO

z

in air at 37°C for 64.5 h.

_Aphidicolin

_(Sigma)

was dissolved in ethanol and diluted with saline. Cultures received APC or the carrier solution 24 h

_prior

to harvest.

To establish

_{dose-response}

curves and determine the

_optimal

APC

concentra-tion for

_study,

50 solid-stained

_metaphases

_per culture were scored for _gaps, breaks and

_{rearrangements}

in both crossbred and Duroc

_pigs.

Chromosome

_preparations

from controls and cultures that received 0.2

_p,M

APC were

trypsin-Giemsa

(GTG-)

banded,

and band locations

_according

to the international nomenclature

(Commit-tee for the Standardized

_Karyotype

of the Domestic

_Pig,

₁₉₈₈₎

were

assigned

to observed gaps, breaks and rearrangements.

RESULTS AND DISCUSSION

Aphidicolin

induced gaps, breaks and

rearrangements

in

_pig

chromosomes when added to

_lymphocyte

cultures 24 h before harvest. Induction of chromosome aberrations was

_dependent

_upon the APC

_{concentration,}

and _response was similar to that demonstrated for human chromosomes

by

Glover et al

_(1984).

_Gaps,

breaks and

_{rearrangements}

induced

_by

0.2

_{pM APC.}

were

analyzed

from 345

metaphase

plates

from 7 individuals. A total of 345 aberrations were observed at 94 different band locations.

_Only

3 breaks were observed in 350

_metaphases

from control cultures.

So-called common

_fragile

sites were identified

by x2 analysis.

Based on a

or more

_breakage

events was

significantly damaged

(x

2

,

1

df >

4.41 with Yates’

correction; P

<

_0.05).

Band locations

_{of fragile}

sites with 4 or more

_breakage

events are listed in table I. Of 94 different

breakpoints,

21 locations were

significantly

damaged.

Breakpoints

of 29

reciprocal

translocations

₍₄₃

different

_breakpoints)

_reported

in the literature were

_compared

to APC-induced chomosome

_breakpoints.

Twenty-nine locations were identified as both translocation and APC-induced

breakpoints.

X

2

analysis

indicated that APC-induced

_breakage

and

_reciprocal

translocation

breakpoints

were not

_independent

₍

_X

₂

_,12

_df

=

_57.30;

P _<

0.001).

The

_primary

_objective

of this

_study

was to establish

_protocols

for

_{studying fragile}

sites in

_pig

chromosomes and

_identify

the common APC-induced

_fragile

sites in the

pig

genome. At 0.2

fL

M

concentration,

as in

_previous

human

fragile

site

studies,

APC was useful for

_inducing

non-random gaps, breaks and

_{rearrangements, ie,}

fragile

sites.

_Twenty-one

_fragile

sites located on 8 chromosomes

(1,

_{4, 6,}

_10,

_11,

_13,

17,

X)

were identified. The

_relationship

of

_fragile

sites to

_reciprocal

translocation

breakpoints

was also examined.

Breakage

was induced in 29 bands which had been

previously

identified as translocation

_{breakpoints. Preliminary analysis}

indicated that the two events were not

_{independent. Stronger}

conclusions cannot be drawn at

this

_{time, however,}

because no data are available

concerning

the actual

population

frequency

of de novo

(or

_{environmentally}

induced)

translocation incidents.

Also,

translocations which arise at some

_fragile

sites may result in

inappropriate

gene

expression

and be lethal. These translocations would

_probably

never be observed. This

study

was

designed

as a

_preliminary

examination of chromosomal

fragile

sites in domestic animals. A better

_{understanding}

of the nature of

_fragile

sites should

_provide

_insight

for clinical and research

_{investigations}

on mechanisms

REFERENCES

Committee for the Standardized

Karyotype

of the Domestic

_Pig

₍₁₉₈₈₎

Standard

kary-otype of the domestic

_pig.

Hereditas

_109,

151-157

Fries

R,

Stranzinger

G

₍₁₉₈₂₎

Chromosomal mutations in

_pigs

derived from

X-irradiated semen.

Cytogen,et

Cell Genet

34,

55-66

Fry M,

Loeb LA

₍₁₉₈₆₎

Animal Cell DNA

Polymerases.

CRC

_Press,

Boca

_Raton,

FL Glover TW,

Berger C, Coyle J,

Echo B

₍₁₉₈₄₎

DNA

polymerase-a

inhibition

_{by aphidicolin}

induces gaps and breaks at common

_fragile

sites in human chromosomes. Hum Genet

_67,

136-142

LeBeau MM

₍₁₉₈₈₎

Chromosomal

_fragile

sites and

_{cancer-specific breakpoints -}

a

moder-ating

viewpoint. Cancer Genet

Cytogenet

31, 55-61

Popescu

CP,

Bonneau M, Tixier

_M,

Bahri

_I,

Boscher J

₍₁₉₈₄₎

_Reciprocal

translocations in

_pigs.

J Hered 75, 448-452 .

Riggs PK,

Chrisman CL

₍₁₉₈₉₎

Preliminary analyses

of

_{aphidicolin-induced fragile}

sites in

pig

chromosomes. Sixth North American

Colloquium

on

Cytogenetics of Domestic

Animals.

West

_{Lafayette, Indiana,}

abstr 4

Schlegelberger

B,

Gripp K,

Grote W

₍₁₉₈₉₎

Common

_fragile

sites in

_couples

with recurrent spontaneous abortions. Am J Med Genet 32, 45-51

Sutherland

GR,

Hecht F

₍₁₉₈₅₎

_Fragile

Sites on Human Chromosomes. Oxford

University

Press,

New York

Sutherland

GR,

Ledbetter DH

₍₁₉₈₉₎

_Report

of the Committee on

_{Fragile Sites,}

Human Gene

_Mapping

10.

_Cytogenet

Cell Genet

_51,

324-332

Threadgill

DW,

Womack JE

₍₁₉₈₉₎

_{Syntenic assignment}

of HSA 9 and HSA 12

_homologs

in the bovine.

_Preliminary

evidence for the role of

fragile

sites in mammalian genome