Hereditas I 17: 231 -236 (1992)

Chromosomal localization of the hormone sensitive lipase ( LIPE) and insulin receptor (INSR) genes in pigs F. GU1, I . HARBITZ2, B. P. CHOWDHARY', M. BOSNES2, and I. GUSTAVSSON'

Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala,

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

Gu, F., HARBITZ, I . , CHOWDHARY, B. P., BOSNES, M. and GUSTAVSSON. I. 1992. Chromosomal localization of the hormone sensitive lipase (LIPE) and insulin receptor (INSR) genes in pigs - Hervditas 117 231-236. Lund, Sweden. ISSN 0018-0661. Received April 8, 1992. Accepted June 29 1992

Using rat hormone sensitive lipase (LIPE) and human insulin receptor (INSR) cDNA probes, the LIPE gene was assigned to chromosome 6pllLq21 and the INSR gene to chromosome 2 q l l L q 2 1 in pigs by in situ hybridization. In humans, these two genes are located on the q and p arms of chromosome 19. respectively. The present results provide the first in situ hybridization mapping data for porcine chromosome 2.

Ingemar Guslaosson, Department of Animal Breeding and Genetics, Swedish University Uf Agricultural Sciences. Box 7023, S - 750 07 Uppsala 7, Sweden

Hormone sensitive lipase (LIPE) is one of the key enzymes in the mobilization of free fatty acids from adipose tissue and is acutely controlled through reversible phosphorylation by cate- cholamines and insulin. LIPE cDNA from rat adipocytes has been cloned and sequenced (HOLM et al. I988a). This cDNA was used as a probe to localize the human LIPE gene to chromosome 19cent-q13.3 by hybridization to DNA from a panel of mouse-human somatic cell hybrids (HOLM et al. 1988b).

Insulin receptor (INSR) is an integral membrane glycoprotein which consists of two a-subunits and two fi-subunits linked by disulphide bonds. This receptor exhibits insulin-dependent tyrosine kinase and autophosphorylation activity (VAN OBBER- GHEN et al. 1983). Isolation and cloning of INSR cDNA from human placenta have been reported by EBINA et al. (1985) and ULLRICH et al. (1985). The INSR gene was assigned to chromosome 19~13.2-13.3 in humans (YANG-FENG et al. 1985) and is considered as a highly informative marker for linkage studies of this chromosomal region (ELBEIN et al. 1986).

Previous studies demonstrated that homologous genes are present on chromosomes 6 and 19q in pigs and humans, respectively ( CHOWDHARY et al. 1989; HARBITZ et al. 1990; YERLE et al. 1990b). However, no information is available concerning the presence of this synteny homology across the centromere in the two species. In humans, the

INSR and LIPE genes are located on the p and q arms of chromosome 19, respectively. Hence, these two loci were chosen for localization on pig chro- mosomes in the present study, in order to ascertain the extent of segment homology in pigs and hu- mans. These comparative mapping data could be valuable in understanding the evolutionary rear- rangements of the genomes in both species.

Material and methods Probe preparation

A rat LIPE cDNA of 3.2 kb, covering the entire coding region, was used as a probe (HOLM et at. 1988a). The INSR probe used was a human cDNA (ATCC 57492) of 4.1 kb coding from amino acid 294 through the 3' untranslated and poly A region (ULLRICH et al. 1985). Tritium labelling of the probes was performed by the random priming method modified by LIN et al. (1985) to a specific activity of 4 x lo8 dpm/pg DNA.

Chromosome preparation

Chromosome preparations were made from pe- ripheral lymphocytes of 2 Swedish Hampshire boars. Heparinized blood, 0.5-1.0 ml, was cultured for 72 h at 38°C in 10 ml RPMI 1640 medium containing 20 % fetal calf serum and 0.1 ml poke-

332 I- GC E l \ L H~redirus I I 7 (1992)

weed mitogen. In order to obtain metaphase chro- mosomes. 0.1 ml colcemid was added to the cul- ture 30min before harvest. Using the standard techniques. the metaphase chromosomes were spread on clean slides and then stored frozen until use.

h i situ h~.hridi:crtion

The in situ hybridization technique was used as described by MAKINEN et a]. (1989) and CHOWD- HARY et al. (1989). The probe concentration was 100 ng,'ml ( 3 ngdide) in the hybridization solu- tion. The slides were developed after 18- 21 days of exposure at 1 4 . C .

('hrotmscmre hriririirig t r r d urur/j,si.\

After Ihc metaphase chromosomes with silver grains had been photographed. the preparations were G-banded using the trypsin EDTA technique (POPESCI et al. 1985) with some modifications. P!ie preparations were destained in 95 O/i, ethanol Tor 1.5 niin. 1% HCI in ethanol for 35 sec. and I00 'Q methanol for 1.5 min. air-dried and im- nxrseci i n '1 coplin jar. containing 0.02 9," trypsin O,i) l2 " 0 EDTA in Hank's salt balanced solution kxithout C'a' and Mg'* (pH 6.8). for 2 min. The dicies ~ e i c \+ashed twice with distilled water. dehy- drated i n I00 " b methanol for 3 min. and air-dried. The slides \vere then immersed in the same trqpsin rc~rlution lhr 40 sec- 1 min. washed twice with dis- til!ed uati ' r and stained for 10- 15 min with 35 O/O

ii rishi's wlution t Merck) i n S6renseii.s phosphate huKcr ipH 6.8). The grains scored here plotted on an idiogram of G-bandcd pig chromosomes arranged according to the standard karlotype (Conimittec for Standardized Karyotype of the Doinestic Pig 1988). The rcsults were analysed using standard statistical procedure (%'-test).

Results 1-ocalization of the LIPE gene

.Anal!\is o f thc silver grain distribution was done in I??, metaphase cells from two experiments. The g w n s scorcd werc plotted on the idiograni of the s m d a r d G-banded pig chromosomes as presented in Fig. I . Totally, 374 grains were counted. and 78 (20.9 Yo) grains were observed on chromosome 6. The statistical evaluation revealed that the signal

for this chromosome was highly significant ( P < 0.001). Further, analysis of the grain distribu- tion on this chromosome indicated that 48 (61.5 YO) grains were clustered on the pll-q21 bands of this chromosome, which is the most likely location of the LIPE gene in pigs. Representative Giemsa stained metaphase chromosomes showing the specific hybridization site and the same metaphase chromosomes after G-banding are pre- sented in Fig. 2A and B, respectively.

Localization of the INSR gene

Totally. 125 metaphase cells from two experiments were analysed. Among the 502 grains counted, 102 (20.3 oh) grains were observed on chromosome 2. which gave a highly significant deviation ( P < 0.001) compared with other chromosomes. On chromosome 2.49 (48.0 YO) grains were plotted on the q l 1 ---q21 bands. which indicated the proba- ble location of the INSR gene in pigs. The silver grain distribution plotted on the standard G- banded pig chromosomes is shown in Fig. 3. Metaphase chromosomes. Giemsa stained after au- toradiography. showing the silver grains and the same metaphase chromosomes G-banded. are prc- sented in Fig. 4A and B, respectively.

Discussion Using in situ hybridization. we mapped the LIPE gene to porcine chromosome 6pl lLq21. Similar observations have recently been made ( J . GELLIN. personal communication) for the localization of this gene in pigs. Previous studies have dcmon- strated that 4 genes. i.e.. apolipoprotein E (APOE) ( Y F R L F et al. 1990b). calcium release channel (CRC) ( H ~ K H I T L et al. 1990). glucosephosphate

DA\ . IFS et al. 1988) and transforming growth fac- tor /I (TGFB-I) ( Y E R L F et al. 1990a) are respec- ti\ely located on the c e n t ~ q 2 1 , pl1 -q21. p12-q21 and cent-q?l regions of this chromosome in pigs. In humans. these 4 genes are also located together with the LIPE gene on the q arm of chromosome 19 (ROPERS and PETRICAK-VANCE 1990) (Fig. 5 ) . The present localization of the LIPE gene, thus, not only provides more mapping information on chromosome 6 in pigs but also indicates the pres- ence of an additional gene common with human chromosome 19q. Localization of more genes from this region of the human chromosome to pig chro-

isomerase ( G P I ) (CHOWDHARY et d . 1989:

Heredifus 117 (IYYZ) LOCALIZATION OF LIPE A N D I N S K GENES I N PIGS 233

I O C

13 14 15 16 17

5

55 18

Fig. 1. Histogram showing the grain distribution in 129 pig metaphase cells hybridized with the LIPE probe. A highly significant amount of grains is found on chromosome 6pl1 -q21.

Fig. 2A and B. (A) Representative pig metaphase chromosomes Giemsa-stained after autoradiography, showing specific hybridization (arrow) on chromosome 6. (B) The same metaphase chromosomes G-banded after in situ hybridization.

mosomes could be useful in defining the extent of localization was to ascertain if, like the q arm, segment homology for these chromosomes, be- homologous genes are present also on the p arm of tween the two species. chromosome 19 in humans and chromosome 6 in

INSR is the other gene chromosomally localized pigs. However, the results of the present study in the present study. The primary interest for this demonstrated that the INSR gene maps to chro-

'34 F o t t 7 4 L

1 2 3 4 5

X "

Fig. 3. The distributioii of sii\er grains i iv r i i 175 pip inetaphase cells iybr id ixd n i th thc lNSR probe S p w t i c labelling \\a> ohserLcd o n chrorntisoiiie 2

Fig. 4A arid B. ( A ) Representatlie Gimisa-stained pip metaphase chromosomes. Arrows show specific hriclizaiii~n o n chromosome 2ql1 q 2 l . (B) The same mrtaphase chromosomes after subsequent G-banding.

momiie ?pl 1 q2l in pigs This indicates that the maps in humans and mice indicate the prcsence of g ims piesent on the human chromosome 19 are homologous genes between human chromosome found o n ,it ien\t t w o different chromosomes. i e . 19q and mouse chromosome 7 (DAVISSON et dl

2 dnd 6. in pigs (Fig 5 ) A similar condition hdS 1991) However. the genes present on the p arm dlw been obsened in mice Comparison of gene (including the INSR gene) of human chromosome

H u r d r r u ~ 117 (1992) LOCALIZATION OF LIPE A N D INSR GENES I N PIGS 235

11 3

13 2

' 3 1

' 3 1

11 2

1 3 3

1 1 4

I N S R ' I

GP I

APOE 1 U 19

HUMAN

2

P I G Fig. 5. Comparative status of human chromosome 19 (HSA19) loci mapped in pigs. The localization of the porcine INSR and LIPE genes indicates that HSA19 loci are present on pig chromosomes 2 and 6.

19 are not found on mouse chromosome 7 but, instead, on other chromosomes, e.g., chromosomes 8, 9, and 17. Thus, on the basis of the INSR localization in the present study, it can be pro- posed that the homology observed between human chromosome 19 and porcine chromosome 6 proba- bly also does not extend beyond the centromere on the p arm of this human chromosome. However, more mapping data in pigs is needed to substanti- ate these observations.

Up to date, only the lactate dehydrogenase A (LDHA) gene has been assigned to porcine chro- mosome 2 (RYTTMAN et al. 1986). This localiza- tion is inconsistent as the gene has also been mapped to chromosome 4 in pigs (FORSTER and HECHT 1984). The present results thus provide the first in situ hybridization mapping data for porcine chromosome 2. Mapping of more loci to this chro- mosome will be helpful in improving the knowl- edge of comparative genome evolution in humans and pigs.

Acknowledgements. -We would like to thank Dr Cecilia Holm for a kind gift of the rat LIPE cDNA, and Ms Gudrun Wieslan- der for secretarial assistance. The project was financed by a grant from the Swedish Council for Forestry and Agricultural Research and the Nordic Contact Organ for Agricultural Research.

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