Hum Genet (1991) 86:515-518

�9 Springer-Verlag 1991

The human placental protein 14 (PP14) gene is localized on chromosome 9q34

Nguyen Van Cong 1, Christian Vaisse 2, Marie-Sylvie Gross 1, Rima Slim 1, Edwin Milgrom 2, and Alain Bernheim 1

1Laboratoire de Cytog6n6tique et de G6ndtique Oncologiques, UA 1158 CNRS, Institut Gustave Roussy, 39, Rue Camille Desmoulins, F-94805 Villejuif Cedex, France zINSERM U. 135, CHU Kremlin-Bic~tre, 78, Avenue du G6ndral Leclerc, F-94275 Le Kremlin-Bic~tre, France

Received July 2, 1990

Summary. PP14 protein (placental protein 14) is abun- dantly secreted by the human endometr ium under the influence of progesterone. H u m a n PP14 is homologous to ~-lactoglobulin, the main component of equine, bovine, and ovine milk whey. A genomic PP14 probe (PP14G1) was used for the chromosome assignment of the PP14 gene. Somatic hybrid cells enabled PP14G1 to be assigned to chromosome 9. In situ hybridization further refined this assignment to 9q34. The localization of the PP14 gene in the region of the A B O locus is consistent with the linkage described in bovines between beta-lactoglob- ulin and the J blood group (homologous to the human A B O group).

Introduction

Placental protein 14 (PP14) is quantitatively the major protein secreted by the human endometr ium during the late luteal phase of the menstrual cycle and the first tri- mester of pregnancy (Bell at al. 1985). PP14 is immuno- logically indistinguishable f rom pregnancy-associated endometr ial alpha-2-globulin (alpha-2-PEG) (Bell and Bohn 1986), progestagen endometrial protein (PEG) (Joshi 1983; Julkunen et al. 1986a) and alpha-uterine protein (AUP) (Sutcliffe et al. 1982), which appear to be different names for a single protein. Protein (Hutala et al. 1987) and cDNA sequence (Julkunen et al. 1988) ana- lysis have clearly indicated that PP14 is homologous to [3- lactoglobulin, the major component of equine, ovine and bovine whey. In human, however, PP14 is absent from milk but is secreted by the glandular epithelium of the endometr ium (Julkunen et al. 1986b). PP14 is believed to have an important immunopressive activity during im- plantation and early placentation. PP14 has also been found in the circulation and has therefore been proposed as a marker for endometrial function (Julkunen et al. 1986c; Bolton et al. 1987).

Complete sequencing of the PP14 gene indicates that it encompasses 5.05 kb divided into 7 exons and 6 introns

Offprint requests to: v. C. Nguyen

(Vaisse et al. 1990). We report here the chromosome as- signment of the PP14 gene by somatic hybrid cell analy- sis and by in situ hybridization with a PP14 genomic probe (PP14G1) containing exons 1, 2 and 3.

Materials and methods

Parental and hybrid cells

The parental rodent cell lines with selected markers were the mouse cell lines CL1D L/TK-, CLID LA/TK-HPRT-, and the hamster cell lines V79/HPRT-, CH/HPRT-. The parental human cell lines were normal cultured fibroblasts, strain 106, 46,XY, and the fol- lowing cultured fibroblasts possessing a balanced reciprocal trans- location: strain 34, 46,Y,t(X;2)(p22.3;q32.1); strain BL, 46X,t(X;2) (p22.3;q32.1); strain 56, 46,X,t(X;5)(q21;qll); and strain 53, 46, XX,t(2;17) (q14;q21). Twenty five human-rodent hybrids were used in this study (Table 1). Seven human-mouse hybrids (M1-M7) and 18 human-hamster hybrids (H1-H18) were derived from a pri- mary panel described by Nguyen et al, (1986). The human-mouse hybrids consisted of two 53-CLID L hybrids (M1, M2) and five 56- CL1D LA hybrids (M3-M7). The human-hamster hybrids consist- ed of one 106-V79 hybrid (H1), one t06-CH hybrid (H2), seven BL-CH hybrids (H3-Hg), two 56-CH hybrids (H10, Hll) and seven 34-CH hybrids (H12-H18). About twelve R-banded mitoses (Dutrillaux and Lejeune 1971) were analyzed for each hybrid cell line.

Preparation of cell extracts for enzyme analysis

Cells were washed three times in phosphate buffered saline and the final pellet was homogenized in distilled water. The suspension was then frozen and thawed three times. Analysis for adenylate kinase 1 (AK1), a well-known chromosome 9 marker (HGM10 1989), was performed according to Meera Kahn (1971).

Probes

The PP14G1 probe is a 2.9-kb EcoRI-HindlII genomic fragment inserted in the Bluescript plasmid. Out of the seven exons of the PP14 gene, exons, 1, 2 and 3 are present in this probe (Vaisse et al. 1990) which is devoided of repetitive sequences.

Southern blot

DNA samples from 25 human-rodent hybrids, from their human and rodent parents, and from human cell lines containing one to four copies of X or Y chromosomes were digested with the restric-

516

tion enzyme BamHI. The DNA fragments were separated by elec- trophoresis and transferred onto nylon membranes. Hybridization was performed under stringent conditions with the nick translation 32p-labled PP14G1 probe (specific activity 5 x 10Scpm/gg DNA), The membranes were then washed in 1 x SSC (1 x SSC = 150raM NaCII15mM sodium citrate, pH 7.0), 0.1% SDS, for 10min at 20~ and 10 rain at 65~

In situ hybridization

Phytohemagglutinin-stimulated human peripheral blood lympho- cytes were cultured at 37~ for 72 h in 199 medium supplemented with 20% fetal calf serum and 2 mM L-glutamine: 5-bromodeoxy- uridine (30 gg/ml medium) and colchicine(150 ng/ml medium) were added 6h and lh, respectively, before harvesting. Cells were treated with 0.075M KC1, fixed for 20min in ethanol/acetic acid (3:1), stored in this fixative at -20~ for 10 days, and then spread onto cold wet slides. Over the following 48 h, the slides were treated with 20 IA 100 I~g/ml RNase and denatured at 70~ in 70% form- amide prior to hybridization (Mattei et al. 1985). Labeling of the PP14G1 probe was performed b~r nick translation using three tritiated nucleotides (3H)dTTP, ('H)dCTP, and (3H)dATP, and dGTP (Zabel et al. 1983; Mattei et al. 1985). The DNA, labeled to a specific activity of 2 x 107 cpm/gg, was hybridized at the final concentrations of 1000 ng/ml, 500 ng/ml and 100 ng/ml at 42~ for 18 h. The slides were then washed extensively (Mattei et al. 1985) and dipped into LM-1 photographic emulsion (Amersham). Auto- radiography was performed in light-tight boxes at 4~ for 5-10 days. Slides were first stained with buffered Giemsa solution and the metaphases with a low background (1-5 silver grains) were selected. Silver grains were scored if they were located either on the contour of or above a chromosome and the metaphases were photographed. Banding and heterochromatin were revealed using a DAPI (diamino-2-phenyl-indole) staining under UV light (355 nm absorption wavelength, 450 nm emission wavelength) and the metaphases were photographed again. The Q bands and the hetero- chromatin regions of chromosomes 1, 9, 16 and Y were strongly fluorescent.

R e s u l t s

Somatic cell analysis by the Southern technique

DNAs from 25 man-rodent hybrids were BamHI-digested and probed with human PP14G1. In man, two major (18- kb, l l .5 -kb) and two minor (7-kb, 2-kb) D N A fragments were recognized. Two major (5.6-kb, 4.4-kb) and four minor (>30-kb , 15-kb, 8-kb 4.2-kb) D N A fragments were found in mouse, whereas in hamster, there are one major (3.2-kb) and five minor bands (25-kb, 13.5-kb, 7.6-kb, 7-kb, 2.3-kb) (Fig. 1). Except for the human 7-kb BamHI PP14 sequence that comigrates with the 7-kb hamster BamHI sequence, the other human BamHI PP14 sequences were identifiable in the hybrid somatic cell DNAs. The results are summarized in Table 1.

In the man-rodent hybrids, a very strong positive cor- relation was observed between the 18-kb, 11.5-kb and 2- kb BamHI PP14 bands, A K 1 and chromosome 9. Seven hybrids were positive and sixteen hybrids were negative for these five markers. All the human chromosomes, with the exception of chromosome 9 were represented in these negative hybrids. The exclusion criterion (chromo- some present/PP14 D N A sequences absent) indicated the location of the 18-kb, 11.5-kb and 2-kb BamHI PP14 sequences on chromosome 9. Two hybrids were discor- dant in having the PP14 18-kb, 15.5-kb and 2-kb BamHI bands, being positive for AK1, but lacking chromosome

Fig. 1. Detection of PP14 sequences by hybridization to BamHI-di- gested DNAs. Lane 1 mouse DNA; lanes 2, 3 human-mouse hy- brids negative for PP14 sequences; lanes 4, 5, 6 human DNAs; lanes' 7, 9, 10 human-hamster hybrids positive for PP14 sequences (18kb, 11.5 kb, 2kb); lane 8 human-hamster hybrid negative for PP14 sequences (18 kb, 11.5 kb, 2 kb); lane 11 hamster DNA

9 as shown by cytogenetics. These results could be ex- plained by chromosome breakage with the retention of a chromosome 9 fragment bearing these PP14 sequences and AK1 (a chromosome 9 reference marker) . The lack of the minor 7-kb BamHI PP14 band was correlated with the absence of chromosome 9 in all the man-mouse hybrids in which chromosome Y was also absent. The theoretical possible assignment of 7-kb BamHI PP14 to chromo- some Y could be disregarded since no dosage effect was observed with human cells containing from zero to four Y chromosomes (data not shown). These different ob- servations indicated the localization of the 7-kb BamHI PP14 sequence on chromosome 9.

In situ hybridization

Some 81 grains were found associated with chromosomes in the 30 mitoses analyzed. The average number of grains was 2.7 per cell. Chromosome 9 showed 31 grains (38%) and of these, 94% (29 grains) were clustered on a small 9qter region. Chromosome 9 was identified by its size and centromeric region, and especially by its hetero-

chromat in region, which appeared strongly fluorescent with D A P I coloration (Fig. 2). The small 9qter region on which 29 silver grains were clustered represents less than 9% of the length of chromosome 9. This region is in- cluded in the 9q34 band, which represents 12.5% of this chromosome. A histogram with the distribution of silver grains on chromosome 9 is shown in Fig. 3. The different results obtained indicate that the PP14 gene is localized on 9q34.

Table 1. Analysis of human chromosomes and enzyme marker (AK1) in man-rodent hybrids negative and positive for human PP14 BarnHI sequences. On the left, 25 independent man-rodent somatic cell hybrids are arranged according to the presence or ab- sence of the human PP14 BamHI sequences (18 kb, 11.5 kb, 2 kb).

517

The human chromosome content of each hybrid is indicated in the second column. Chromosomes detected in less than 30% of hybrid cells are given in brackets; Xa, active X; Xi, inactive X. The col- umns on the right show the data concerning different PP14 BamHI sequences and the AK1 analysis

Man-rodent hybrids

Human chromosome PP14 BamHI sequences (kb)

18 11.5 2 7

AK1

Hybrids PP14 - $1 $2 $3 $4

$5 $6 $7

H1 H2 H3 H4 H6 H8 H10 H13 H17

Hybrids PP14 + H5 H7 H9 H11 H12 H14 H15 H16 H18

1,(2),3,4,5,7,8,11,(14),17,(20),2q-,17q+ 3,6,(8),12,(13),(14),17,19,20,21 2,3,4,5,6,7,11,(15),(16),17,19,20,21,22,Xi,(Xq+)(5q-) (I),2,3,5,6,7,8,10,(11),12,13,16,17,18,19,20,21,22,Xi 2,3,(4),5,7,8,12,13,14,16,17,18,20,21,22,5q- 2,3,6,7,11,12,17,(21),Xi,Xq + , 5q - 1,2,4,5,7,8,10,11,12,15,16,17,18,19,20,21,22,(Xi),Xq +

5,10,15,19,20,21,22,Xa 6,8,11,12,20,(21),(22),Xa 3,(4),6,(8),(13),14,15,16,(18),19,Xp + 4,5,6,(8),10,11,13,14,19,21,(Xi),Xp + 4,(8),11,12,18,19,Xi,Xp + 3,5,6,(10),11,13,15,(18),(20),21,22,Xi,Xp + 8,14,15,16,19,21,Xi (17),21,Xp + 3,4,8,14,(16),(19),20,Xp +,Y

1,3,4,6,9,11,12,13,14,15,16,18,19,20,21,22,Xi,Xp + 4,5,6,7,(8),11,12,13,14,15,16,18,20,(21),22,(Xi),Xp + 1,4,5,6,7,9,11,16, (18), 19,20,21,22,Xi 1 ~ (4) ,5,6,8,9,12,18,19,22 ,Xq + 3,5,8,9,11,12,13,14,16,19,20,(2l) 6,(7),(8),10,11,12,14,(16),20,21,22,Xp + 1,3,4,5,8,9,11,12,13,14,(15),18,19,20,Xp + 1,(3),6,8,9,(10),11,12,13,15,16,18,21,Xp + 4,6,8, (9), 10,12,13, (14), (18),(20),21 ,Xp +

m

- - m

m

_ m

m m

_ m

m m

m m

m m

m m

+ + + + + + + + + + + + + + + + + + + + + + + + + + +

Fig. 2a, b. Two partial metaphases showing the specific hybridization site for the PP14 on chromosome 9. a Giemsa-stained chromo- somes with silver grains; b the same chromo- some after DAPI coloration

518

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Fig. 3. Diagram of distribution of hybridization grains on human chromosome 9. PP14 grains are clustered on 9q34. The nomencla- ture used for this diagram follows the international recommenda- tion (ISCN 1981)

D i s c u s s i o n

Somatic cell hybrids, have allowed us to assign unam- bignously the PP14 gene to ch romosome 9. We prefer- red, for the in situ hybridizat ion, to use the very simple D A P I colorat ion technique definitively to identify chro- mosome 9. The localization of the PP14 gene was con- f i rmed on c h r o m o s o m e 9 with a regional assignment on 9q34-qter with 94% of the grains clustered on the telo- meric region of the long arm.

The A B O locus is located on 9q34 ( H G M 1 0 1989). The localization of the PP14 gene on the same band is consistent with the l inkage (0 = 4 - 2 0 cM) described in bovines (Hines et al. 1969; Larson 1971) be tween IMacto- globulin (homologous to the h u m a n PP14) and the J b lood group (homologous to the h u m a n A B O blood group) (Neimann-Sorensen et al. 1954). It is thus proba- ble that PP14 and A B O are l inked in man.

Othe r genes of interest are located in the same band and consti tute a linkage group: A B L (Abelson murine leukemia viral oncogene homolog) involved in the ac- quired t(9;22) t ranslocat ion found in C M L (chronic my- elocytic leukemia) and acute leukemias ( H u m a n Gene Mapping 10 1989), AK1, A L A D (amino levulinate delta- dehydratase) , ASS (arginiosuccinate synthetase) , D B H (dopamine beta-hydroxylase), GSN (gelsolin), NPS1 (nail patella syndrome 1), WS1 (Waardenburg syndrome 1) ( H u m a n Gene Mapping 10 1989). Fur ther studies will be necessary to determine their order relative to PP14.

Acknowledgements. We are grateful to M. F. Maman for typing the manuscript, to V. Hurteloup for helpful comments and to G. Merault for the photography.

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