Hum Genet (1990) 85 : 130-132

�9 Springer-Verlag 1990

Regional chromosomal assignment of the human mineralocorticoid receptor gene to 4q31.I Norma Morrison 1, Stephen B.Harrap 2, Jeffrey L. Arriza 3, Elizabeth Boyd I, and J. Michael Connor I

1Duncan Guthrie Institute of Medical Genetics, Yorkhill, Glasgow G3 8SJ, UK 2MRC Blood Pressure Unit, Western Infirmary, Glasgow Gll 6NT, UK 3Gene Expression Laboratory, The Salk Institute for Biological Studies, P.O. Box 85800, San Diego, CA 92138, USA

Received August 14, 1989

Summary. The gene for human mineralocorticoid recep- tor (hMR), previously mapped to chromosome 4, has been further localized to 4q31.1 by in situ hybridization using a biotinylated 3.75 kb human cDNA clone encod- ing the primary amino acid sequence of hMR as a probe. Preliminary comparative mapping studies in orangutan (Pongo pygmaeus) suggest localization of the probe to the long arm of chromosome 3.

Introduction

Mineralocorticoids exert important influences on blood pressure by affecting the homeostasis of body fluids and electrolytes via actions on secretory epithelia of the in- testine and kidney. These effects are presumably medi- ated through the mineralocorticoid receptor. In addi- tion, it is now clear that glucocorticoids may also interact with the mineralocorticoid receptor (Baxter and Tyrell 1981).

Offprint requests to." J. M. Connor

The human mineralocorticoid receptor gene was re- cently cloned (Arriza et al. 1987) and its localization as- signed to chromosome 4. The present study was de- signed to define more precisely the regional chromosom- al assignment of the human mineralocorticoid receptor gene.

Materials and methods

DNA probe

The cDNA clone phMR3750 was used as a probe for the hMR gene. The clone is derived from lambda hkl0 (Arriza et al. 1987) and contains nucleotides 1 to 3750 inserted into pGEM 4, a pBR322 based Amp R vector.

Chromosome preparation

Using standard cytogenetic methods, metaphase chromosome spreads were obtained from PHA-stimulated lymphocyte cultures from five human males of normal karyotype. For confirmation of the initial result, chromosome preparations were obtained from a

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Fig. 1. Diagram of t(4;11)(q31.1;q25) Fig.2a, h. Human metaphase chromosome spread pre- and post- hybridization with sites of hybridization arrowed

131

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Fig. 3. Signal distribution over a the normal male karyotype, b the translocation line karyotype

lymphoblastoid cell line from an individual with the unbalanced karyotype 46,XX,-ll,+der(ll)t(4;ll)(q31.1;q25)pat. This girl, investigated at birth because of multiple congenital anomalies, was the index case in the family. The translocation is shown diagram- matically in Fig. 1. Metaphase chromosome spreads were also pre- pared from male orangutan skin fibroblasts obtained from the American Type Culture Collection. The metaphases were banded, photographed and destained prior to hybridization as previously described (Garson et al. 1987; Boyd et al. 1989).

In situ hybridization The probe was labelled with biotin-ll-dUTP and used at a concen- tration of 2.5-5ng/1~1 for in situ hybridization according to the method described by Gars0n et al. (1987). The distribution of hy- bridization in normal human cells was analysed statistically using the chi-square test.

Resul t s

In total, 87 metaphases f rom five normal males were scored following hybr idizat ion (an example is given in Fig. 2) and the posit ions of 352 hybridizat ion signals re- co rded as shown in Fig. 3a. Of these signals, 74 (21.0%) were located on c h r o m o s o m e 4 and 56 (15.9%) located be tween 4q28 and 4q31 with a peak at 4q31.1. The dis- t r ibut ion of the signal on the normal h u m a n chromo- some 4 is shown in Fig. 4. The hybr id iza t ion observed at 4q28-q31 was found to be highly significant (P < 0.005). The presence of hybridizat ion signal on the derivative c h r o m o s o m e 11 (Figs. 3b, 5) refines this localization. The results strongly suggest that the h M R locus is located

132

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22 �9 23 2~ 25 26 0 0

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2e 31.1 31.2 31.3 32 33 3~ 35

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Fig. 4. Signal distribution over human chromosome 4

Fig. 5a, b. Partial metaphase from translocation line pre- and post- hybridization with sites of hybridization on normal 4s and deriva- tive 11 arrowed

Discussion

Arriza et al. (1987) used a series of rodent-human so- matic cell hybrids to assign the human mineralocorticoid receptor to human chromosome 4. The work described here supports this assignment and indicates a regional localization to 4q31.1. This is immediately adjacent to the sub-band 4q31.2, which Fan et al. (1989) have re- cently identified as the mineralocorticoid receptor locus using isotopic in situ hybridization.

The observation that human chromosomes 11 and 12 carry genes for very similar products, for example L D H - A on 11 and LDH-B on 12, has led to the speculation that tetraploidization may have occurred at an early stage of human evolution (see Seuanez 1979, chapter 12). In this context and in view of the structural and functional kin- ship between the two receptor molecules (Arriza et al. 1987), it is interesting to note that the human glucocorti- coid receptor gene, at 5q31 (Francke and Foellmer 1989; Theriault et al. 1989), occupies a similar position on chromosome 5 to that of the mineralocorticoid receptor on chromosome 4.

Although the presence of hybridization sites on other orangutan chromosomes cannot be excluded, the provi- sional localization of hMR to PPY 3q21.1-q21.3, a re- gion homologous to H S A 4q31.1-q31.3 according to banding pat tern (ISCN 1985), represents further evi- dence of the homology between H S A 4 and PPY 3 sug- gested by other comparat ive mapping studies (Seuanez 1979).

Acknowledgement. We thank Mr. Alasdair McWhinnie who kindly amplified and purified the hMR probe.

Fig. 6. Partial orangutan metaphase, post-hybridization, with sites of hybridization on chromosome 3 arrowed

within chromosome region 4q31.1-q31.3 and probably at 4q31.1.

In the experiment with orangutan cells, 16.3% (35/ 214) of the signal in 53 cells was on chromosome PPY 3 and 7.9% (17/214) on PPY 3q21.1-q21.3 (as arrowed in Fig. 6) suggesting a localization of the probe to this re- gion. Unfortunately, complete analysis of these cells was not possible because of inadequate pre-banding quality; therefore, signals on other individual chromosomes were not counted.

References

Arriza JL, Weinberger C, Cerelli G, Glaser TM, Handelin BL, Housman DE, Evans RM (1987) Cloning of human mineralo- corticoid receptor complementary DNA: structural and func- tional kinship with the glucocorticoid receptor. Science 237: 268-275

Baxter JD, Tyrrell JB (1981) In: Felig P, Baxter JD, Broadus AE, Frohman LA (eds) Endocrinology and metabolism. McGraw- Hill, New York, pp 385-510

Boyd E, Theriault A, Goddard JP, Kalaitsidaki M, Spathas DH, Connor JM (1989) Chromosomal assignment of a glutamic acid

Glu transfer RNA (tRNA ) gene to lp36. Hum Genet 81:153-156 Fan Y-S, Eddy RL, Byers MG, Haley LL, Henry WM, Nowak N J,

Shows TB (1989) The human mineralocorticoid receptor gene (MLR) is located on chromosome 4 at q31.2. Cytogenet Cell Genet 52 : 83

Francke U, Foellmer BE (1989) The glucocorticoid receptor gene is in 5q-q32. Genomics 4 : 610-612

Garson JA, Berghe JA van den, Kemshead JT (1987) Novel non- isotopic in situ hybridization technique detects small (1 kb) unique sequences in routinely G-banded human chromosomes: fine mapping of N-myc and beta-NGF genes. Nucleic Acids Res 15 : 4761-4770

ISCN (1985) An International System for Human Cytogenetic No- menclature. Harnden DG, Klinger HP (eds) Published in col- laboration with Cytogenet Cell Genet. Karger, Basel, pp 95-107

Seuanez HN (1979) The phylogeny of human chromosomes. Sprin- ger, Berlin Heidelberg New York

Theriault A, Boyd E, Harrap SB, Hollenberg SM, Connor JM (1989) Regional chromosomal assignment of the human gluco- corticoid receptor gene to 5q31. Hum Genet 83 : 289-291