Preliminary studies on the gene map of cattle

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Preliminary studies on the gene map of cattle

Lv Monteagudo, Mt Tejedor, Mv Arruga

To cite this version:

Preliminary

studies

on

the

_gene

_map

of cattle

LV

_Monteagudo,

MT

_Tejedor

MV

_Arruga

Facultad de Veterinaria, Universidad de

Zaragoza,

Laboratorio de

_{Citogen6tica, Departamento}

de

Anatomia,

Embriologia

y

Genetica,

C/Miguel

Servet,

177, 50013

Zaragoza, Spain

(Proceedings

of the 9th

European Colloquium

on

Cytogenetics

of Domestic

Animals;

Toulouse-Auzeville,

10-13

_July

₁₉₉₀₎

gene map

/

cattle

INTRODUCTION

Gene

_mapping

has become one of the most

_interesting

fields in

_genetics

research.

However,

data available on _{animal gene}

_mapping

are still _very

few,

as

compared

with

_present

_knowledge

_{of the human gene} map.

During

the last two

_decades,

somatic cell

_{hybridization}

has _proven to be one of the most useful tools _{for gene}

mapping

studies both in the human and in animal

_species

_{(Pontecorvo, 1975).}

In this

_report,

we

_present

our

_preliminary

results in cattle gene

_mapping,

obtained

using

this

_methodology.

MATERIALS AND METHODS

Somatic cell

_{hybridization}

Somatic cell

hybridization

was

_performed

as described

_by

Pontecorvo

(1975).

The

line

_wg3hcl2

_(a

Chinese hamster cell line

_negative

for

_hypoxanthine

phosphoribosyl-transferase

_(HPRT);

Echard et

al,

1984)

and fibroblasts obtained from one cow

were used for this _purpose.

Hypoxanthine-aminopterine-thymidine

(HAT)

culture

medium was used to

_prevent

_growth

of HPRT- cells. Ouabain was added to the medium at a final concentration of

10-

7

M,

to

_prevent

_growth

of the unfused cattle fibroblasts.

Karyotype analysis

Karyotyping

and cellular extract

_analysis

for

_isozyme

characterization were carried

out at the same culture passage; colcemid

(GIBCO)

was added 3 or 3

_1/2

h before

harvesting,

at a final concentration of 0.05

_!g/ml.

Cells were then removed from

the culture

_flask,

underwent a 20 min

hypotonic

treatment with 0.075 M KCI and

Isozyme

characterization

Cells were removed from the culture flask

_{by trypsinization.}

As soon as cells

started to

_detach,

_they

were diluted in Hanks’ balanced salt solution

_(HBSS).

Sometimes,

10%

fetal calf serum was added to HBSS to

_prevent

_trypsin

_degradation

of enzymes. After

centrifugation,

cells were diluted in Shaws’ buffer and

_subjected

to three

_{freezing-thawing}

_cycles

before

_being

sonicated.

_Finally,

_samples

were

centrifuged

and the

_supernatants

_(containing

the

_enzymes)

were stored in

_liquid

nitrogen

until

_{analyzed. Electrophoresis}

of the

samples,

followed

by

specific

staining

was used to characterize the cattle

_{isozymes expressed}

in the

_hybrid

clones. The references for tank buffers and

_{staining procedures}

are as follows: AK:

_adenylate

kinase

_{(Heuertz, 1981);}

PGD:

_{phosphogluconate dehydrogenase}

_{(Heuertz, 1981);}

FH: fumarate

_hydratase

_(Harris

and

_Hopkinson,

_1976);

MANA: a-mannosidase

(Harris

and

_Hopkinson,

_1976);

ACP: acid

_phosphatase:

buffers

_according

to Benne

(1990,

personal

communication)

and

_{staining procedure}

described

_by

Harris and

Hopkinson

(1976);

LDHA and LDHB: lactate

_{dehydrogenase}

A and B

_(Van

Someren

et

_al,

_1974);

_PGM1,

PGM2 and PGM3:

_{phosphoglucomutase}

_{(Heuertz, 1981);}

NP: nucleoside

_{phosphorylase:}

buffers used

_by

Womack and Moll

₍₁₉₈₆₎

and

_staining

procedure

described

_{by Ansay}

and Hanset

_(1972);

PEPC:

_peptidase

C: buffers and

staining

method of Van Someren et al

_(1974).

RESULTS AND DISCUSSION

We have now obtained a

panel

of 31

hybrid

clones. So

far,

14 clones have been

Previously,

PGD was found to

_belong

to the bovine U1

syntenic

group, while

PGM1 was located in the U6

_syntenic

_group

_(Echard

et

_{al, 1982;}

Heuertz and

Hors-Cayla,

1981;

Womack and

_{Moll, 1986; Womack,}

_1990).

FH and PEPC were

not

_previously

studied

_(fig

1 shows the

_{electrophoretic}

_enzyme

_pattern

obtained for

FH).

Our data

_suggest

that the human

_syntenic

_group

_{including FH,}

_PGD,

PGM1

and PEPC

_(Van

Someren et

_al,

₁₉₇₄₎

is not conserved in cattle.

_PGD,

PGM1 and FH cannot be considered as

being

_syntenic

in

_light

of the

_preliminary

results shown

in table I.

REFERENCES

Ansay M,

Hanset R

₍₁₉₇₂₎

Purine nucleoside

_{phosphorylase}

_(NP)

of bovine

_{erythrocytes:}

genetic

control

_{of electrophoretic}

variants. Anim Blood

_Groups

Biochem Genet

_3,

219-227 Echard

G,

Gellin

_J,

Benne

_F,

Gillois M

₍₁₉₈₂₎

_Progress

in _gene

_mapping

of

rabbits,

cattle and

_pigs

_using

somatic cell

_{hybridization.}

In: Proc 5th

_{European Colloquium}

on

Cytogenetics

in Domestic Animals

_{(Milan) (Succi}

_G,

_ed),

237-252

Echard

G,

Gellin J, Gillois M

_(1984).

Localisation des

_{genes MPI, PKM2,}

NP sur le chromosome 3 du porc

(Sus

scrofa

L)

et

_{analyse cytog6n6tique}

d’une

_lign6e

de hamster chinois issue de la DON

_(wg3h).

G6n6t S61 Evol _16, 261-270

Harris

_{H, Hopkinson}

DA

₍₁₉₇₆₎

Handbook

_{of Enzyme Electrophoresis}

in Human Genetics. North

_Holland,

Amsterdam

Heuertz S

₍₁₉₈₁₎

Carte

_genetique

des bovins par les

techniques d’hybridation

cellulaire. Doctoral thesis. Universite de Paris-Sud

Orsay,

Paris XI

Heuertz

_S,

_Hors-Cayla

MC

₍₁₉₈₁₎

Cattle _gene

_mapping

_by

somatic cell

_{hybridization:}

study

of 17 enzyme markers.

_Cytogenet

Cell Genet

_30,

137-145

Pontecorvo G

₍₁₉₇₅₎

Production of mammalian somatic cell

hybrids by

means of

polyethy-lene

_glycol

treatment. Somatic Cell Genet 1, 397-400

Van Someren

_H,

Van

_{Henegouwen HB,}

Westerveld

_A,

Bootsma D

₍₁₉₇₄₎

_Synteny

of the human loci for fumarate

_hydratase

and UDPG

_{pyrophosphorylase}

with chromosome 1

markers in somatic cell

_hybrids.

_Cytogenet

Cell Genet

_13,

551-557

Womack JE

₍₁₉₉₀₎

Gene map of the cow

(Bos taurus).

In: Genetic

_Maps.

vol 5

(O’Brien

SJ,

ed)

Cold

_Spring

Harbor

_Laboratory,

Cold

_Spring

Harbor, NY,

4121-4125

Womack

_JE,

Moll YD

₍₁₉₈₆₎

Gene _map of the cow: conservation of

_linkage

with mouse