Sequential GTG-RBA banding pattern in prometaphase chromosomes of cattle (Bos taurus L)

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Sequential GTG-RBA banding pattern in prometaphase

chromosomes of cattle (Bos taurus L)

D Di Berardino, Mb Lioi, D Matassino

To cite this version:

Original

article

Sequential

GTG-RBA

_banding

_pattern

in

_prometaphase

chromosomes

of

cattle

(Bos

taurus

_L)

D

Di

Berardino

MB Lioi

2

D Matassino

1

Universita,

_degli

Studi di

Napoli, Dipartimento

di Scienza

della Produzione

Animale, 80055, Portici,

Naples;

2

Universita,

_degli

Studi della

Basilicata,

Istituto di Produzione

Animale,

Facoltti di

_Agraria,

85100

_{Potenza, Italy}

(Received

14 November

_{1990; accepted}

17

_{April 1991)}

Summary - A

_sequential

GTG-RBA

_{banding procedure,}

performed

for the first time on

the same

_prometaphase

chromosomes of

cattle,

is

presented

with the aim of

establishing

correlations between G and R bands. The results of the _present

_{investigation}

contributed

to the establishment of new standard GTG and RBA-banded

karyotypes

at

_prometaphase

level,

useful for the

precise

identification of chromosomal

abnormalities,

comparative

cytogenetics

and gene

mapping

in the

species

Bos taurus L.

sequential banding

/

GTG-bands

_/

RBA-bands

_/

standardization

_/

cattle

Résumé - Une nouvelle technique de mise en évidence séquentielle des bandes G

et R sur les chromosomes prométaphasiques du bovin

_(Bos

taurus

_L).

Une nouvelle

technique

de mise en évidence

séquentielle

de bandes G et R est

_{présentée pour la première}

fois

sur les chromosomes

prométaphasiques

du bovin dans le but d’établir des corrélations

entre les bandes G et R. Les résultats de cette étude ont contribué à l’établissement de

nouveaux _caryotypes standards en bandes G et R au niveau

prométaphasique.

Cette étude est utile pour

l’identification précise

des anomalies

chromosomiques,

pour les études de

cytogénétique comparée

et

_{pour la}

localisation des

_gènes

de Bos taurus.

bandes _{séquentielles}

_/

bandes G

_/

bandes R

_/

standardisation

_/

bovin

*

INTRODUCTION

The

_Reading

conference on the standardization of banded

_karyotypes

of domestic

animals

_(Ford

et

al,

1980),

undoubtedly

one of the most

_important

_steps

in the

history

of animal

_{cytogenetics, provided}

for the first time the ’standard’ G-banded

karyotype

of cattle

_(Bos

taurus

_L)

as well as that of other domestic

_{species. Despite}

its excellent

_quality,

the bovine standard G-banded

_karyotype

soon revealed some

limitations for chromosome

_{identification,}

due

_mainly

to the

_degree

of contraction of the chromosomes used for the standard

_and,

_secondarily,

to an intrinsic feature

of bovine

_chromosomes,

common to all the

_Bovidae,

ie

_G-negative

centromeres and telomeres.

These

_problems

have made it _necessary to

_improve

the

_Reading

standard

_by

using

less contracted chromosomes and to

_adopt

alternative

_banding

_techniques

such as RBA and

_QFQ

for standardization of new

karyotypes

which could be used more

_{widely by}

the scientific

_community.

This _paper

_reports

the results of the

_sequential

GTG-RBA

_{banding technique}

performed

for the first time on the same

_prometaphase

chromosomes of cattle in order to establish correlation between the GTG and the RBA bands. These results

provided

an

_important

_part

of the material used for the discussions at the 2nd International Conference for the Standardization of Banded

_Karyotypes

in Domestic

Animals,

held at

_{Jouy-en-Josas}

_(France)

the 22-26th of

_May,

1989

_{(ISCNDA, 1989).}

MATERIAL AND METHODS

Peripheral blood,

drawn from the

_jugular

vein of _{5 young} bulls of the Italian Friesian

_breed,

was cultured at 37.5° C for 72 h in RPMI 1640 medium

_(Flow,

Dutch

_{modification)}

_supplemented

with

10%

fetal calf serum

_(Gibco),

0.1%

L-glutamine

and 0.1 ml of

_{pokeweed mitogen}

_(Gibco).

6.5 h before

_harvesting

_cells,

5’-bromodeoxy-uridine (BUdR, Sigma)

at a final concentration of 20

pg/ml

and 5.5 h later colcemid solution

_(Gibco,

final concentration of 0.03

_{pg /ml)}

were added. In order to facilitate the

_spreading

of the

_prometaphase

chromosomes the cell

suspension

was

_subjected

to a

_stronger

_hypotonic

shock than usual

_(0.05

M

_KCI)

at 37.5° C for 20 min and fixed with methanol-acetic acid solution

_(3:1)

for 1

_h,

centrifuged,

fixed

_again

and left

_overnight

in the

_{refrigerator.}

Air-dried slides were

prepared.

Sequential

GTG-RBA

_banding

_procedure

The air-dried

slides,

3-5-d

_old,

were treated for GTG

_{banding according}

to Lin et

al

_(1977);

soon after the Giemsa

_staining,

the slides were flooded with

phosphate

buffer

_(pH

=

7),

covered with _a

_coverslip

and examined with _a Leitz Dialux under

bright

field

_optics.

The

best

G-banded

_prometaphase

_spreads

were selected and

photographed

with a Kodak microfilm 1454. After

_{microphotography,}

the

_coverslip

was

_removed,

the slide was destained

_gently

in

30%

ethanol for 10

_min,

washed

in distilled

_water,

air-dried and stained with an _{acridine orange solution}

_(0.2%

in

phosphate

buffer)

for 15

_min,

washed

_again

in

_{tap water,}

mounted in the same

spreads

previously

examined for GTG

_banding

were relocated and

_photographed

again

for RBA

_banding

with the same Kodak microfilm 1454. Kodabrome F2M and

F3M _papers were used for

_printing

GTG and RBA banded

_prometaphase

spreads,

respectively.

RESULTS

Figure

1

_(A

and

_B)

_{shows, respectively,}

a GTG-banded

prometaphase spread

of

cattle

_(2n

=

60,

XY)

and the same

_{spread sequentially}

stained for

_RBA-banding.

In order to

_verify

the

correspondence

between the

_Reading

standard and the

prometaphase

G-banding

pattern, individual chromosomes from

_figure

1A were

arranged,

side

_{by side,}

with the G-banded chromosomes of the

_Reading

standard,

as shown in

figure

2. From this

figure

it is

possible

to

_verify

the

_great

_advantage

and usefulness of

_{using prometaphase}

instead of

_{metaphase chromosomes, especially}

for the identification of the smallest autosomes

_ranking

from

pairs

No 21-29. All the G-banded

_prometaphase

chromosomes fit very well to the

_Reading

standard,

thus

providing

more information for a definite characterization and identification of

in-dividual chromosomes of the

_{species. However,}

because of chromosome contraction of the G-banded cattle chromosomes

_{reported by}

the

_Reading

_standard,

it is

_quite

difficult to

_distinguish

_among the chromosomes Nos

_25,

27 and

_{29; hence,}

it is

not

_fully

evident that the actual

_prometaphase

_pairs

_correspond

to the ones of the

Reading

standard.

In order to examine in detail the correlation between the GTG and the RBA

banding

pattern

of individual

prometaphase

chromosomes of

cattle,

figure

3

_(A,

B and

_C)

was

_prepared

in which the GTG-banded

_prometaphase

chromosomes from

_figure

lA

_(b

and

_d)

are

_compared

with the

sequentially

stained RBA-banded

chromosomes

_(a

and

_e)

and with the ’direct’ RBA banded chromosomes

_(c).

From this

_figure

it is

_possible

to

_verify

the correct

_{correspondence}

between G and R bands

in almost all of the

_chromosomes,

_including

the X and Y sex chromosomes.

DISCUSSION

The

_sequential

GTG-RBA

_{banding procedure, performed}

for the first time on

chromosome of

_cattle,

is suitable for a

_specific

characterization of individual

chromosomes of this

_species.

Previous contributions on the RBA

_banding

_pattern

in cattle chromosomes

_(Popescu,

_1975;

Gustavsson and

_Hagelthorn,

_1976;

De Giovanni et

_al,

_{1979; 1988;}

Di Berardino et

_{al, 1979, 1983, 1985a, 1985b;}

Di Berardino and

Iannuzzi,

1982)

reported karyotypes

which were

_based,

as far as

possible,

on the

Reading

G-banded standard

karyotype,

but a direct correlation

between G and R bands has not so far been

reported. Recently,

a G- and

_R-banding

comparison

of cattle

_prometaphase

chromosomes

_{arranged according}

to the

_Reading

system

has been

_reported

_{(Iannuzzi, 1990)}

but without use of a

_sequential

G-R

banding

procedure.

The

_present

_{investigation}

was carried out in order to make correlations between G and R bands on the same chromosome

_preparation,

thus

providing

the _necessary

information for the definition of new standard GTG and RBA banded

_karyotypes

In the

_{sequential procedure reported here,}

the BUdR

_{incorporation}

_necessary to

achieve the RBA

_banding

did not seem to affect the

_G-banding

_pattern.

Also the

trypsin

treatment used for

_GTG-banding

did not

_{produce significant}

effects on the

k

c1

xyotypes

of other domestic

_species

for which extensive

_cytogenetic

material is

already

available.

Other _ways

_using

G- and R-banded marker

_chromosomes,

such as centric

_fusions,

translocations and nucleolus

_organizer

chromosomes of

_Bovidae,

as well as the biarmed chromosomes of related

_species,

could

_provide

additional information for

a. definitive characterization of the

_banding

_pattern

of individual chromosomes of

the

_species

Bos taurus L.

The results of the present paper contributed to the definition of the ’standard’ GTG and RB A-banded

_karyotypes

and

_idiograms

of cattle at the

_prometaphase

level

_(ISCNDA,

₁₉₈₉₎

useful for

_{precise description}

and identification of numerical

as well as structural chromosomal

aberrations, comparative cytogenetics

and _gene

mapping.

Financial

_support

for this

_study

was obtained from the National Research

Council

_(CNR)

of

_{Rome, Italy.}

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