Mapping of genes belonging to the halothane linkage group in pigs using in situ hybridization

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Submitted on 1 Jan 1991

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Mapping of genes belonging to the halothane linkage

group in pigs using in situ hybridization

Bp Chowdhary, I Harbitz, W Davies, I Gustavsson

To cite this version:

Mapping

of

_genes

_belonging

to

the

halothane

_linkage

_group

in

_{pigs using}

in

situ

_{hybridization}

BP

_Chowdhary

₁

I

Harbitz

2

W

Davies

2

I

Gustavsson

1

Swedish

_{University of}

_{Agricultural Sciences,}

Department of

Animal

_Breeding

and

_{Genetics, I!PPsala, Sweden;}

2

Norwegian College of Veterinary Medicine, Department of Biochemistry, Oslo, Norway

(Proceedings

of the 9th

_European

Colloquium

on

Cytogenetics

of Domestic

Animals;

Toulouse-Auzeville,

10-13

_{July 1990)}

gene mapping

/

in situ hybridization

/

glucose phosphate isomerase

_{(GPI) /}

6-phosphogluconate dehydrogenase

(PGD) /

calcium release channel

_{(CRC) /}

_pig

INTRODUCTION

In

_pigs,

the halothane

_(HAL)

_linkage

group, which

comprises

the loci for

_glucose

phosphate

isomerase

_(GPI),

_HAL,

_{the blood groups} S and

_H,

_{a-l-glycoprotein}

(A1BG)

and

_{6-phosphogluconate dehydrogenase}

_(PGD),

has been

_intensively

stud-ied

_(see

review

_by

Archibald and

_Imlah,

_1985).

The HAL locus is

_important

with

respect to the

_{genetic predisposition}

of

_pigs

to

_{develop porcine}

stress

_syndrome

(PSS).

Based on

_{physiological evidence,}

the calcium release channel

(CRC)

of the

sarcoplasmic

reticulum has been

_proposed

as a candidate for the defective

_protein

in

_{malignant hyperthermia}

_(MH).

_Recently,

the CRC _{gene -} referred to as the

ryanodine

receptor

(RYR) -

has been found to be

_tightly

linked to the MH _{gene in} humans

_(NIacLennan

et

al,

1990).

The present

study

was undertaken to sublocalize

two known member loci of the HAL

_linkage

group, viz GPI and

PGD,

along

with the localization of the CRC _{gene in}

_pigs.

MATERIALS AND METHODS

The

_probes

used were:

1)

a

_{porcine genomic GPI-specific}

DNA

_fragment,

2)

a

porcine

PGD cDNA and

₃₎

a

_porcine

CRC CDNA. The

probes

were

radioactively

labeled

_using

the

_{random-priming}

method

_(Feinberg

and

_Vogelstein,

_1983),

modi-fied for tritium

_labeling

_(Lin

et

_al,

_1985).

The

_{specific activity}

of the

probes

ranged

Chromosome

_preparations

were obtained from

_{pokeweed-stimulated pig}

lympho-cytes

that had been cultured for 72 h.

_{5-Bromodeoxyuridine}

was added

(200 pg/ml

of

_medium)

for the final 7 h of the culture. The chromosomes were identified

_using

the

_RBA-banding

_technique

_(Dutrillaux

et

_al,

₁₉₇₃₎

before

_{hybridization,}

and the

GTG-banding

technique

(Popescu

et

_al,

₁₉₈₅₎

after

_{hybridization.}

The

_technique

of in situ

_{hybridization}

was

_essentially

the same as that described

_{by Harper}

and Saunders

₍₁₉₈₁₎

with some modifications. The concentration of the DNA

_probe

in the

_{hybridization}

mixture was

100 pg/ml (3 ng/slide).

The slides were

exposed

for

18-25

days

in

_light-tight

boxes with dessicator at

+4°C.

The

_grains

scored were

plotted

on the

_idiogram

of G-banded

_pig

chromosomes

(Committee

for the

Stan-dardized

_Karyotype

of the Domestic

_Pig,

_1988),

and the results were

_analyzed

_using

standard statistical

_procedures.

Chromosome measurements

_according

to Lin et al

(1980)

were used. The

_x

₂

_-test

was used for statistical

_analysis.

RESULTS

For the GPI _gene, a total of 124

_metaphases

₍₆₇

RBA-banded and 57

_GTG-banded)

from one

_{hybridization}

_experiment

were

_analyzed

and the

_{grains plotted}

as shown

in

_figure

1a. Of the total 646

_grains

_scored,

163

_(25%)

were located on chromosome

6

_(P

<

_0.001).

_Analysis

of the

_grain

distribution on this chromosome revealed that

about 126

_(77%)

_grains

were clustered on the

_p12-q21

_segment,

and 106

(65%)

on

the

cent-q21

segment.

The latter area forms less than

1%

of the total genome. On

this

basis,

there is a

high

probability

that the GPI _gene is located in this

_segment

(P

<

_0.001).

There was a clear

_predominance

of

_grains

on the _q arm near the

centromere with the

_peak

_clustering

on the

_q12

band which could be

_proposed

as

the

_probable

site of the GPI _gene.

For the PGD _gene, a total of 180 GTG-banded

_metaphases

from two

hybridiza-tion

_experiments

were

analyzed,

and the

_{grains plotted}

as shown in

_figure

lb. In

all,

1052

_grains

were

scored,

of which 235

(22%)

were located on chromosome 6

(P

<

_0.001).

Of the total number of

_grains

on this

_chromosome,

21

(9%)

were

present

on the p arm and 214

_(91%)

on the q arm. On the _{q arm,} the

_grains

_mainly

clustered on the

_6q25-q27

_segment,

which had a total of 117

_grains.

This

_segment

forms

_{approximately}

1%

of the total _genome and had

11%

of the total number of

grains.

On this

_basis,

the

_probability

that the PGD _{gene is} located in the

_6q25-q27

segment

is

_{highly significant}

_(P

<

_0.001).

For the CRC _gene, a total of 208 GTG-banded

_metaphases

from one

hybridiza-tion

_experiment

were

_analyzed

and the

_{grains plotted}

as shown in

_figure

lc. In

all,

1456

_grains

were

_scored,

of which 298

(20%)

were located on chromosome 6

(P

<

_0.001).

Of the total number of

_grains

on this

_chromosome,

35

(12%)

were

present

on the _p arm and 263

(88%)

on the _q arm. On the _q arm, the

grains mainly

clustered on the

_cent-q21

_segment,

which had a total of 192

_grains.

This

_segment

forms

_{approximately}

1%

of _{the total genome and had}

13%

of the total number of

grains.

Indications from the

_grain

distribution

are that the gene for CRC is located

in the

_pll-q21

_segment

of chromosome

_6,

with a very

high probability

of it

being

present

on the _q arm in the

_cent-q21

_segment

(P

<

_0.001).

The

_{signal peaked}

on

DISCUSSION

The in situ

_{hybridization}

results thus confirm that the HAL

_linkage

_group is located on chromosome 6 in

_pigs,

and that the CRC locus is within this

_complex.

Interestingly,

the

_signal

of the CRC

_peaked

on the same

_{segment (6ql2)}

as that of

the GPI gene. Various studies have shown

that,

of the known

_loci,

GPI is located closest to the HAL locus

_(see

Archibald and

_{Imlah, 1985).}

The localization of the CRC gene at almost the same

_{site, together}

with the accumulated evidence of

possible

involvement of this _{gene in}

_causing

NIH both in humans and

_pigs

is a

powerful

argument

that the basic defect in MH results from a mutation in the CRC

gene.

However,

conclusive

proof

of this

hypothesis

will

require

the identification of

a mutation in the CRC _gene

_exclusively

found in

_pigs,

a mutation that

directly

affects the

_regulation

of the calcium release process.

REFERENCES

Archibald

AL,

Imlah P

₍₁₉₈₅₎

The halothane

_sensitivity

locus and its

_{linkage relationships.}

Anim Blood

_Groups

Biochem Genet 16, 253-263

Committee for the Standardized

_Karyotype

of the Domestic

_Pig

₍₁₉₈₈₎

Standard

kary-otype of the domestic

_pig.

Hereditas 109, 151-157

Dutrillaux B, Laurent

_C,

Couturier

J,

Lejeune

J

₍₁₉₇₃₎

Coloration des chromosomes humains _par I’acridine _orange

_apr6s

_{traitement par} le

5-bromodesoxyuridine.

CR Acad Sci

_(Paris)

_276,

3179-3181

Feinberg AP, Vogelstein

B

₍₁₉₈₃₎

A

_technique

for

_{radiolabeling}

DNA restriction endonu-clease

_fragments

to

_{high specific activity.}

Anal Biochem

_132,

6-13

Harper ME,

Saunders GF

₍₁₉₈₁₎

Localization of

_single

copy DNA sequences on G-banded

human chromosomes

_by

in situ

_{hybridization.}

Chromosoma

_(Berlin)

83, 431-439

Lin

_CC,

Biederman BM, Jamro IIK, Hawthorne

_AB,

Church RB

₍₁₉₈₀₎

Porcine

_(Sus

scrofa

domestica)

chromosome identification and

_suggested

nomenclature. Can J Genet

CytoL 22,

103-116

Lin

_{CC, Draper}

_PN, DeBraekeleer M

₍₁₉₈₅₎

_{High-resolution}

chromosomal localization of the

_$-gene

of the human

_/3-globin

_gene

_{complex by}

in situ

_{hybridization.}

_Cytogenet

Cell

Genet 39, 269-274

MacLennan

_DH,

Duff C,

Zorzato

_{F, Fujii J, Phillips NI,}

Korneluk

RG,

Frodis

_W,

Britt

BA,

Worton RG

₍₁₉₉₀₎

Ryanodine

receptor gene is a candidate for

predisposition

to

_malignant

hyperthermia.

Nature

_343,

559-561

Popescu NC, Amsbaugh SC,

Swan

DC,

DiPaolo JA

₍₁₉₈₅₎

Induction of chromosome