< 1990 S. Karger AG. Basel 0301 0171 90 0533 0140 S 2.75 0

Cytogenet Cell Genet 53:140-143 (1990)

Chromosome regional mapping for the human thyroid stimulating hormone p subunit (TSH B) gene T. T okino,1 Y. Hayashizaki.1 K.-I. T ak a ta,1M .C. Yoshidac and K. M atsu b ara1 Institute for Molecular and Cellular Biology. Osaka University. Suita, and Chromosome Research Unit. Faculty of Science. Hokkaido University. Sapporo(Japan)

Abstract. The human thyroid stimulating hormone P subunit (TSHB) gene, located on chromosome 1. was studied to determine its subregional location by in situ hybridization and Southern blot analysis of human x mouse hybrid cells. The results allowed

localization of TSHB to the proximal portion of lp22. which is in the region of localization of the linkage group including amylase (AMY), nerve growth factor p subunit (NGI B). and NRAS. which are conserved in humans and rodents.

Thyroid stimulating hormone (TSH) is a pituitary glycoprotein hormone which acts at the thyroid gland and induces secretion of thyroid hormone. It is closely related to other glycoprotein hor­ mones. including luteinizing hormone (LH). follicle stimulating hormone (FSH). and chorionic gonadotropin (CG), all of which are heterodimeric molecules consisting of u and P subunits (Pierce and Parsons. 1981). The a subunit is encoded by a single gene (Fiddes and Goodman. 1981) and is shared in common among these hormones, whereas the P subunit is encoded by an in­ dividual. specific gene (Talmadge et ah. 1984: Hayashizaki ct al.. 1985: Watkins et al.. 1987: Tatsumi et al.. 1988). Recent studies revealed that the expression levels of these p subunit genes are controlled at the transcriptional level (G urrand Kourides. 1983) and the subunits are encoded by separate genes and mRNAs (Tal­ madge et al., 1984; Hayashizaki et al.. 1985: Watkins et al.. 1985; Tatsumi et al., 1988). The u gene, the LHB-CGB cluster, and FSHB are separately located on chromosomes 6 (ql2-*q21 or p23->p21.1). 19q 13.3, and 11 pi 3. respectively (Naylor ctaL 1983: Watkins et al.. 1987; Tatsumi et al.. 1988). We have located the TSHB gene on chromosome 1 using a chromosome sorting tech­ nique (Fukushige et al.. 1986). Recently. Dracopoli et al. (1986) found that TSHB mapped to 1p22. based on analysis of somatic cell hybrids. In the mouse, the Tshb subunit gene is localized on chromosome 3. and it is in a syntenic linkage group with Amy. Ngfb, and Nras (Kourides etal.. 1984). In this communication, we offer proof for the localization of the human TSHB to 1p22 based on in situ hybridization and confirmed the assignment of TSHB near AMY, NGFB, and NRAS. These are located on 1p21 (Cook and Hamerton. 1979: Hamerton el al., 1984). Ip22.1 or 1p 13

(Franckeet al., 1983: Zabeletal., 1983: Muenke et al.. 1984. 1985: Middleton-Priceet al., 1987; O'Connell et al.. 1987). lp22or 1p 13 (Davisetal.. 1983: McBrideetal.. 1983: Muenkeetal., 1984.1985; Petlenati et al.. 1985; Popescu et al.. 1985: Middleton-Price et al.. 1987).

Received 6 June 1989; accepted X December 1989 Request Reprints from Dr. Tokashi Tokino. Institute for Molecular and Cellular Biology. Osaka University 1 3. Yamada-oka. Suita 535 (Japan).

In situ hybridization. Metaphase spreads were obtained from phytohemagglutinin-stimulated peripheral blood lymphocytes from a normal male as described elsewhere (Yoshida et al.. 1986). Before hybridization, the chromosome preparations were pretreated with RNase A (100 gg ml in 2 x SSOand denatured in 70% fortnannde. 0.3 M sodium chloride. 0.03 M sodium citrate at 70 C for 2 min. The probe was labeled with 11-dCTP to a specific

Fig. I. Normal human lymphocyte chromosomes I from different metaphase spreads at the 300-band stage after in situ hybridization with the TSHB probe, photographed sequentially with incident fluorescent light (upper) and with a combination of incident fluorescent and transmitted visible light (lower) for detecting silver grains. Arrows indicate the positions of individual grains.

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Supported by Ministry of Education, Science, and Culture o f Japan grants to T.T., Y.H.. and K.M. T.T. and Y.H. hold Special Postdoctoral Fellowships from the Japan Society for Promotion o f Sciences.

Materials and methods

141

Subchromosomal localization o f TSH B

••• •

•

P

• • •

•••

q • • • •

activity of2 x lOdptn pg with a nick translation kit (Boehringer Mannheim). In situ hybridization was performed as described by Harper and Saunders (1981) with a slight modification (Yoshida et al., 1986). After hybridization, the slides were washed twice in 50% formamide, 2 x SSC at 41 C for 15 min. twice in 30% formamide.O.I x SSC for 15 min each at 39 C. and once in 0.1 x SSC for 15 min al room temperature, followed by dehydration in 70%. 80%. and 95% ethanol for 5 min each. After being coated with emulsion (NR-M2. Konica Co.. Japan) for autoradiography, the slides were exposed for a week at 4 C and then developed and stained with quinacrinc mustard and Hoechst 33258 (Yosh­ ida et al.. 1975). Grains were plotted on a standard idiogram (ISCN. 1985). DNA probes. The isolation, characterization, and DNA sequencing of the TSHBgcnomic DNA have been described elsewhere (Hayashizaki et al.. 1985). For in situ hybridization and Southern blot analysis, a 4.4-kb llg I 11 fragment and a 3.2-kb fcoRl fragment of the TSHB gene were used, respectively. The blotting experiments were performed by the modified Southern hybridization as described elsewhere (Southern. 1975: Hayashizaki et al.. 1985). Hybrid cells. Human x mouse hybrid cells were provided by Dr. N. Shimi­ zu. Dr. S. Christie, and Dr. C. Bostock. as shown in Table 1. Relevant chro­ mosome karyotypes were examined by Gicmsa-11 staining. trypsin-Giemsa banding, and by isozyme analysis as reported previously (Koch et al.. 1978: Shimizu etal.. 1980: Lund et al.. 1983: McLellanet al.. 1985). Blotting filters, on which DNA from hybrid cells WIL-5. WIL-7. WIL-2. WIL-8. JSR-17S. DUM-13. NSL-16. DUA-3BSAGA. REW-8D, WIL-14. JSR-2. VTL-6, GAR-1. TSL-1. JWR-22H. XOL-9. and XOL-6 were present, were provided by Dr. T.B. Shows.

Results and discussion In situ hybridization. A 4.4-/?g/II fragment carrying the unique sequence that covers the downstream half of intron 1 through the 3' flanking region was used as a probe for in situ hybridization (Hayashizaki et al.. 1985). About 300 normal human peripheral

TSHB

+

-

+

-

A m y la s e

+

—

+

-

p21

NG FB

+

-

+

-

p 2 2 .l

1

2

3

b

4 Total

3.2kb

m -

2.2kb

Fig. 3. (a) Regional localization of TSH B to human chromosome Ip22 using a set of hybrids containing partially overlapping segments of this chromosome. Vertical bars represent the region of chromosome I retained in individual hy­ brids in which the normal homolog is missing. Scoring indicates the presence ( + ) or absence ( —) of TSHB in Southern analysis, (b) Southern analysis of £c«RI-digested DNAs of translocation-carrying hybrids, using the 4.4-kb fragment as probe. Lane I, JWR-22H; 2, XOL-9; 3, XOL-6; and 4, SK81-Wg3H-H4-7-13.

blood metaphase chromosomes at the 300-band stage were exam­ ined and a total of 522 grains were observed. Among these, 78 grains ( 15%) were located on chromosome 1, 27 (34%) being clus­ tered on the short arm region lp22 (P < 0.001). No secondary peaks of hybridization were seen on other chromosomes. This re­ sult is consistent with the previous assignments using a hybrid clone panel (Dracopoli et al., 1986) and chromosome sorting (Fukushige et al„ 1986).

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Fig. 2. Diagram of distribution of silver grains on chromosome 1 with a significant peak at band p22.

T okino Ilayashizaki T akata Yoshida M atsubara

142

Table I. Segregation ofTSHB in human x mouse cell hybrids Hybrid

WIL-5 W1L-7 WIL-2 WIL-8 JSR-I7S DUM-T3 NSL-16 DUA-3BSAGA REW-8D WIL-14 JSR-2 VTL-6 GAR-1 TSL-I TA-I TA-3 TA -4c TA-4d B4AI 17a D5BI B2B4 JWR-22H XOL-9 XOL-6 SK8l-W g3H-H4-7-l3 Concordant hybrids Discordant hvbrids % discordancy

TSHB gene

Chromosome ------------------------------------------------------------------------------------- -------------------------------------------------------------------------------2 18 19 20 2l 22 14 16 17 X 3 13 15 4 6 8 9 10 h 12 1 5 7

_

_

_

-

-

4 -

+ -

+ + ♦ 4 -

+ 4 + 4 -

+ 4 +

4 4 44-

+ — —

+ -

+ 4 + — 4 -

-

+ 4 4 -

4 4+ + + + -

—

_

_

+ — + 4* + 4— 44-

+ — 444-

— 4t 4444+ 444t t t -

44444-

-

-

-

-

+ t 4-

44-

-

4-

4444-

21

24

il

14

18

0 0

9 36

14 56

II 44

7 28

+ + t t t t

—

+ + + + 4-

+ -

_

4444444+ + — 4* 4-

-

+ + -

4— t -

+ 444 44 4 4 4 -

-

-

-

-

_

_

_

—

_

4 + + + 4 — 4 4 — 4 4

4 + 4 4 4 4 4 4

4 4 4 4 4 4 + 4 + + 4

4 4 + t 4 4 4 4 4 -

4 4 4 4 4 4 • •

+ + 4 + + 4 4 4 4 + 4 + -

4 + + + + 4 -

—

+ • 4 4 4 4 —

+ + -

4 + -

4 4 + +

4 4 — + — — 4 4 4 .-

4 + 4

+ 4 -

+ -

4 4 4 4 4 4

4 4 4 4 4 -

16

17

17

13

7 29

8 32

12 48

+

4-

— -

+ 4 4 — 4

18

14

12

16

17

18

17

16

7 28

7 33

13 52

8 33

8 32

7 28

8 32

9

9

36

36

+ + + + 4 4 4 + + 4

4 4 4 4 4 4 4 4 — 4 + + -

— + + 4 + 4 4 -

4 + 4 t 4 4 • 4 -

4 4 4 — 4 4

— — 4 +

— 4 4 4 4 + -

— 4 4

— t t t t t

17

18

16

14

18

10

8 32

7 28

9 36

II 44

7 28

9 47

— 4 + -

Translocation

7:9 X. 15. I5:X 17:9

X:7.7:X X:7. 7:X X:7. 7:X 2:1 XI l:X

( + ) indicates the presence, ( - ) the absence o f the radioactive bands on the blots. Hybrid cells containing chromosome translocationsare excluded from the scorings, (t) indicates translocation involving the particular chromosome, leaving no intact chromosome JWR-22H has the 2;1 translocation (2pter-*q37:;lp2l -*pter) (6 of 30 metaphase spreads). XOL-9 has the X;l translocation (X ptcr— *q26: :lq 12— ♦qtcr) (2 o f 30 metaphase spreads). XOL-6 has the l;X translocation ( 1pier—♦q 12: :Xq26— ♦qter) ( 15 of 33 metaphase spreads). SK8l-Wg3H-H4-7-l3 has the translocation Ip22-*p36: :mouse chromosome.

Somatic hybrid cells. We attempted to assign the human TSHB using a somatic hybrid clone panel. A 3.2-kb fragment carrying the downstream half ofTSHB intron 2 through the 3' flanking region was used as a probe for Southern blot analysis. The results in Table 1 show that the gene is located on chromosome 1. in complete accordance with the in situ hybridization data. Additional analysis was conducted on a set of hybrids contain­ ing partially overlapping segments. Figure 3 shows that the TSH B locus is neither on the long arm of human chromosome I (1 q21—>qter) nor on the distal region of the short arm of chro­ mosome 1 (Ip22-*p36). In particular, since a hybrid cell. SK 81Wg3H-H4-7-13, contains the distal portion of Ip22. lack of the

signal with this cell indicates that TSHB is located on the proximal portion of lp22. In the mouse, Tshh is mapped in a region where Amy. Ngjh. and Nras are clustered. The site lp22 where the TSH B gene was located in man is also surrounded by the AMY. NGFB. and NR AS genes, showing that this syntenic region is conserved in both human and mouse.

Acknowledgements We thank Drs. N. Shimizu. C. Bostock, and S. Christie for providing the human x mouse hybrid cell lines, ard Dr.T. B. Shows for providing the blotting filters of human x mouse hybrid cell lines.

Cook PJL. Hamerton JL. Report o f the committee on the genetic constitution o f chromosome I. Fifth Interna­ tional W orkshop on Human Gene Mapping. Cytogenei Cell Genet 25:9 20 ( 1979).

Davis M. Malcolm S. Hall A. Marshall CJ: Localization of thehuman N-ras oncogene to chromosome tccn-p2l by in situ hybridization. EMBO J 2:2281-22X3 (1983) Dracopoli NC. Retting WJ. Whitfield G K , Darlington GJ.

Spender BA. Biedler J L. Old LJ. Kourides I A: Assign­ ment of the gene for the (3 subunit of thyroid-stimulat­ ing hormone to the short arm of human chromosome I Procnatl Acad Sci. USA 83:1822 1826 (1986).

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Subchromosomal localization o f TSH B

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