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Localization of the human α-globin gene cluster to the short arm of chromosome 16 (16p12–16pter) by hybridization in situ
(1981) 156, 269-278
.J. ;Vol. Biol.
Localization of the Human a-Globin Gene Cluster to the Short Arm of Chromosome 16 (16p12-16pter) by Hybridization in situ I’. K.-IKTOS’.
S. MALCOLM’. C. MURPHY~
’ Department
Roy01 Hospital (Krcrived
ANI) M. A. FERGUSON-SMITH’
of Biochem.istry
‘Institute of Medical Genetics for Sick Children,, Yorkhill. Glasyou~. U.K.
12 November
1981, trod in revised form 21 IIecember
1981)
The human n-globin gene cluster has been assigned previously t’o chromosome IS using a molecular hybridization assay of somatic cell hybrids. We have carried out a regional assignment to the upper half of the short arm of chromosome 16 in the in situ of a-globinspecific probes to fixed region lBpl2-pter by hybridizat.ion
metaphase chromosomes. Suitable hybridization
probes, of sufficient length to give
a detectable signal, were prepared free from highly repetitive sequences, by the isolation of restriction enzyme fragments from genomic recombinant clones covering the 5. $I and a-globin gene regions of the chromosome. A total of 52 x lo3 bases of hybridized sequence was used to detect the cr-globin gene cluster. The criteria of chromosome specificit’y, overall efficiency of hybridization and regional localization were used to establish the localization. These criteria have been applied previously to the mapping of the fi-globin gene cluster.
1. Introduction Several techniques are available for finding the regional chromosomal localization of a gene (McKusick, 1980). Where suitable t,ranslocation break-points covering the whole chromosome are available, somatic cell fusion techniques have been of great, use. The analysis of hybrid cells either depends on the production of protein in a fused human/rodent cell line or, when a cloned gene probe is available, the gene may be detect,ed directly by nucleic acid hybridization, without any need for expression of the gene product. Hybridization in situ to chromosome spreads has the advantage that normal chromosome preparations from dividing lymphocytes are used and a rather precise regional localization can be obtained. This method has been used to localize a number of repetitive gene families in man where the target, site for hybridization is >50 kbt; for example, the 28+ 18 S ribosomal genes i Abbreviations
used: kb. lOa bases; cRNA. complementary
RNA.
269 ~H122-2R36/X2/1(~26!~~10
$03.00/O
C 1982 Academia Press Inc. (London)
Ltd.
270
P. 5
BARTON
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W BE
a2
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H
H
3’ I
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psstI
Pst
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H
I
H Xba H I kb
FIG. 1. Arrangement of the human a-globin-like genes. 1. Restriction map of the a-globin gene cluster. 2. Regions of homologous DNA sequence. 3. DNA contained in the genornic subclones. () Regions gelpurified as an a-specific probe ; (-f-j-j) re g ions containing highly repetitive DNA. Restriction enzyme sites. B. BarnHI; Bg, BgZII: H, HindIII: Pst. PstI; S. Sac1 (=SstI): Xba, XbnI.
(Henderson et al.. 1972; Evans et al.. 1974). the 5 S ribosomal genes (Steffenson et al., 1974; Fenttel et al., 1979) and the histone genes (Chandler et a,l., 1979). Recently, the method has been extended to the detection of structural genes present in only a few copies. The sensitivity was increased either by using recombinant DNA probes that form networks at the site of hybridization (Malcolm ~1 al.. 1977; Gerhard et al., 1981: Harper et al.. 1981; Harper & Saunders. 1981). or by using hybridization probes made from genomic recombinants that hybridize to adjacent and itttervettittg sequences as well as to the structural gene (Malcolm et al., 1981a). It has been shown by the work of Deisseroth et al. (1977) and Diesseroth & Hendrick (1978), using somatic cell hybrids. that the human aglobin genes are found on chromosome 16. In order to localize the a-globin genes more precisely we have applied the method of hybridization in situ to normal chromosome spreads. Fine-structure mapping of the humatt J-like globin genes (Lauer et al.. 1980) has revealed a closely linked multigene family containing the genes for the two adult n-globin chains. the embryonic forms of the a-globin chain and also non-functional pseudo-3 genes (see Fig. 1); the entire region of over 18 kb has been isolated in overlapping recombittattt clones. As both the B and p-globin gene clusters are interspersed with highly repeated DNA sequences (Lauer et al.. 1980; Fritsch et al., 1980; Malcolm et al., 1981b: Coggins et al., 1980; Adams et al.. 1980), which would interfere with hybridization. suitable restriction fragments, free of repetitive DNA, must be prepared for use in hybridization experiments in situ. This approach has been used recently to confirm the localization of the human p-globin gene cluster to the short arm of chromosome 11 close to the centromere (Malcolm et al.. 1981a). In that case a region of 4500 base-pairs including the /?-gene was used as the template for synthesis of [3H]cRNA to be used as a hybridization probe. For the localization of the a-globin gene clust,er we have used a mixture of probes prepared from the Iglobin region making a total of 5200 bp of hybridizing sequence.
,r-GLOBIN
GENE
ON SHORT
ARM
OF CHROMOSOME
16
271
The realization that single base changes in DNA that give rise to restrict.ion enzyme site polymorphisms (Kan & Dozy, 1978) can be used as genetic markers (Botstein et al., 1980) has made the need for precise regional assignment of welldefined genes such as the ir-globin genes more pressing. The establishment of a large number of such polymorphisms linked to the cu-globin cluster will rapidly provide genetic markers for a relatively large and well-defined region of the human genome (Higgs et al.. 1981).
2. Materials (a) Preparation
and Methods
of restriction
fragments
R.estric:t~ion enzyme digests were carried out under conditions recommended by the in an O+V& agarose with low manufacturers. Digested DNA (10 to 30 pg) was separated gelling temperature (Miles) gel using a buffer containing 40 mmTris (pH 7.7), 40 mm NaOAc, 1 mmEDTA. To recover DNA, gels were stained with ethidium bromide, visualized under 360 nm ultraviolet light and the DNA bands cut out from the gel. The agarose was melted at 65”C, cooled to 37”C, shaken with an equal volume of phenol and centrifuged at 10,000 revs/min for 10 min. The aqueous phase was extracted with chloroform and the DNA precipitated by addition of @l vol. 2 M-Na acetate and 2.5 vol. 95% ethanol. DNA yields wefe >700,. (lo) Detection The method
used was exactly
of repetitive sequences by hybridization “P-labelled human DXA as described (c) cHSd
by Fritsch
using
et al. (1980).
preparation
13H]cRNA was made from gel-purified x-specific DNA by transcription using Escherichia coli RNA polymerase. Transcription reactions were performed in 30 ~M-[~H]ATP, [ 3H]CTP 13H ]UTP (Amersham Radiochemicals), 200 PM-GTP (Boehringer), 150 mmKC1. 10 mMM&l,. 15 mmM&l,, 40 mw-Tris (pH 7.9). 1 mw-dithiothreitol, @4 mmNa,HPO,, @l mm EDTA, 25Opg DNA/ml, 150 units E. coli RNA polymerase/ml. After incubation at 37°C for 3 to 4 h DNAase I (Worthington) was added to 200 pg/ml, and incubating was continued at 37°C for 30 min. This was followed by addition of 50 pg yeast transfer RNA (Sigma) and an equal volume of @3 M-NaCl, @2% sodium dodecyl sulphate. Samples were extracted with an equal volume of phenol/chloroform (1 : 1) and [‘H]cRNA separated from unincorporated nucleotides by passage through Sephadex G50 in 40 mw-Tris. HCI (7.6). Pooled fractions were desalted by passage through a second Sephadex G50 column in distilled water and dried under a vacuum. (d) Hybridization
in situ
Chromosome preparations fixed to glass slides were made from short-term lymphocyte cultures as previously described (Ferguson-Smith, 1974). Prior t,o hybridization, chromosomes were banded by immersion in 1 9, Lipsol detergent solution for 15 s (Stephen. 1977), followed by Giemsa stain. Chromosome spreads were photographed for karyotype analysis. Slides were treated with 100 pg RNAase A/ml (Sigma) in 2 x SSC! (SSC is 0.15 MNaCl, 0015 iv-sodium citrate) for 30 min at 37”C, washed thoroughly in 2 x SSC and dehydrated by immersion in 50%, 759,, 95% and 100% ethanol. Chromosomes were denatured in SOoi, formamide, 0.1 mmEDTA. 5 mM-HEPES (pH 7.0), at 55°C for 15 min, washed in 2 x SSC and dehydrated as above. 13H]cRNA prepared from the gel-purified a-
272
P. HARTOS
ET
AL
globirt recombinant DIVAS and dissolved in hybridization buffer (500,, (v/v) formatnide, 0% M-NaCl, 1 mM-EDTA, 5 mM-HEXES, pH 7%) was added to prewarmed slides at 43”C, such that each slide received 20 to 30 ng [ 3H]cRNA in 5 ~1 of hybridization buffer, covered with a cover-slip (previously washed in 1 M-HCI) and incubated at 43°C for 18 h. After incubation, unhybridized cRNA was removed by treatment with 20 pg RNAase A/ml (Sigma) in 2 x SSC at’ 37°C for 30 min, followed by extensive washing in 2 x WC at 4°C. After dehydration through ethanol, slides were dipped in Ilford KZ nuclear research emulsion diluted 1 : 1 with water. Autoradiographs were exposed for 42 days at 4X”, stained in Giemsa and the chromosome spreads that, had been photographed were relocated for grain
counting.
3. Results
TINA for use as hybridization probes was isolated by restriction enzyme digestion from plasmids previously sub-cloned into pBR322 from AHaG and XHaG2 recombinants (which cover the entire T-globin gene cluster) (Lauer et nl., 1980) (see Fig. 1). The cluster contains two adult n-globin genes (01~and a2), a non-functional a gene (@t) and one of the two etnbryonic <-globin genes (
kb
4-3
l-9 l-2
FIG:. 2. Southern blot hybridization to detect repetitive DNA in plasmid recombinant pBR{. (a) Ethidium bromide stained. (b) Autoradiograph after hybridization. Track 1: pBR( DNA digested with EcoRI and BglII giving 2 fragments. (i) A 6.6 kb fragment containing the c-gene, surrounding DNA and the vector pBR322. (ii) A 2 kb fragment, of genomic DNA located 1 kb 5’ of the 5 gene. Track 2: hKcoRI/RnmHI marker DSA. Track 3: Plasmid RI 3.2 digested with EcoRI and XbaI giving three fragments. (i) A 4.3 kb fragment of pBR322 DNA. (ii) A 1.2 kb fragment extending 3’ of the Xba site located downstream from the 8.globin gene : known to contain a number of the highly repetitive Alu family of repetitive DSA sequences (Coggins et (II.. 1980: Malcolm et al.. 1981). (iii) A I.9 kb fragment from adjacent to the @-globin gene containing single-copv DNA.
x-GLOBIN
GENE
ON SHORT
ARM
OF CHROMOSOME
16
273
within the a-globin-like gene cluster (Lauer et al.. 1980). Such sequences can be identified within plasmid molecules by an adaptation of the Southern procedure (Fritsch et nl., 1980), in which radioactively labelled human genomic DNA is used probe. Only sequences present many times in the human genomr as a hybridization will be detected upon autoradiography. The clones pBR[, p&t1 and pBRa1 used in the a-globin mapping experiments were analysed using this technique. Figure 2 shows the results of such an experiment: pBR< digested with EcoRI and &$I1 gives two fragments. The smaller 2 kb fragment of insert DNA located 1 kb 5’ Tao the 5 gene shows strong hybridization to 32P-labelled total huma’n DNA. indicating the presence of highly repetitive DNA. The larger 6.6 kb fragment containing the 5 gene coding sequence, the surrounding genomic DNA and the vector pHR322 shows no hybridization. Control tracks containing known highly repetitive or single-copy DNA from around the /3-globin gene cluster were also included. In similar experiments pBRn1 was digested with Hind111 and EcoRI giving a 6 kb non-repetitive fragment including 2 kb of genomic human insert DNA and a 2 kb fragment from the human genome containing repetitive DNA. pSst1 digested with BumHI and HilldIII gave an 8.25 kb fragment containing repetitive sequences and a 1.6 kb fragment of non-repetitive DNA including 1.05 kb of human DNA. Three highly repeated sequences were identified within the sub-clones tested and the regions in which they are found are marked on Figure 1. The restriction enzyme digests for preparation of probes were chosen to give the maximum separation on preparative gels between fragments containing the repeats and fragments to be used for hybridization. These are marked on Figure 1. (b) Hybridization
in situ
[‘H JcRNA (spec. act. 1.7 x 10’ disints/min per pg) was prepared from the three plasmid-derived gel-purified DNA regions described, by transcription with E. coli RNA polymerase. Previous experiments using ribosomal, globin and immunoglobulin recombinants have shown that both inserted and plasmid-derived sequences are transcribed (apparently randomly) under the conditions used here. The DNA fragments contain 2.6. 2 and 1.05 kb of a-globin-specific DNA but the extent of the gene duplication around the two 3c-globin genes (Lauer et al.. 1980) is such that t.here are long regions ( >2 kb) flanking the coding sequences that are common to both at and a2 genes and which will. therefore, cross-hybridize. The probes prepared from both pBR’*l and p&t1 contain part of these duplicated regions and [3H]cRNA prepared from them will hybridize to 3.54 and 2.88 kb, respectively. Three sets of slides for hybridization in situ were set up using L3H]cRNA transcripts as follows : (1) pBRnl-derived probe (total target size 3.5 kb) (2) pBRcv1 +pBR[-derived probes (total target size 6.14 kb) (3) pBR%l +pSstl-derived probes (total target size 5.8 kb). Hybridization was to pre-banded and photographed chromosomes to allow unequivocal chromosome identification for grain counting after hybridization (Malcolm et c&l., 1981a). Autoradiographs were exposed for 42 days and a total of
274
P. KARTOK
E7’ AI,
I 2 3 4 5 6 7 8 9 IO II 1213141516171819202122XY Chromosome number
PIG. 3. Hybridization in situ (i). The total number of grains counted ou each chromosome from 146 karyotyped cells divided by the relative length of each chromosome (“6 haploid genome length : FergusmSmith. 1974). Broken line shows the average value.
146 prephotographed karyotypes frotn seven slides were scored for silver grains occurring over chromosomes. Data front each slide were analysed individually and the results pooled.
Figure 3 shows t.he distribut.ion of grains for all 146 kayotypes over the genome. To allow for the fact that. chromosomes of different lengths will contain different numbers of grains due to random background the results are presented as grains/unit chromosome length where each unit lengt,h is I(/, of the gettome (Ferguson-Smith. 1974). Chromosome 16 scored the highest grain level and gave a value + 2.8 standard deviations from the mean (I’
wGLOBIN
GENE
ON
SHORT
ARM
OF
CHROMOSOME
275
16
Y
-12ot I 234
, , , , , , , , , , , , , , , , , , , , , , 56 78910111213141516171819202122XY Chromosome number
FIG. 4. Hybridization in situ (ii). Distribution ofgrains over the genome. Number each chromosomecompared tothenumberexpectedforachromosomeofthatlengthifthetotalgraincount were distributed proportionately over the genome.
(d) IKstribution
of gmins
along
chromosome
ofgrains
occurring
on
16
The distribution of grains along the length of chromosome I6 was determined in each experiment. Chromosome 16 has an arm ratio of 1.51 and therefore divides conveniently into five equal divisions (two in the short arm. three in the long arm). In each set of slides examined there was an excess of grains in the upper half of the short arm (corresponding to band positions 16pter-p12). Pooled data are shown in Figure 5. Virtually all the extra grains found on chromosome 16, compared to the number expected if grains were random over the genome (represented by the broken line), are found in the same segment of the chromosome.
200 r-l g b B L IOO2 ----------_---E 2
----------
Flct. 5. Hybridization in&u (iii). Distribution ofgrains along chromosome 16. p, short arm : q, long arm : arrow marks the position of the centromere. Broken line indicates the number of grains expected along
chromosome lti.fro~naproportionaldistributionofthetotalgraincount.distributedevenl~overitslength.
276
P. BARTON
Chromosome
h’T
AL.
number
FIG. 6. Hybridization in S&L (iv). Regional distribution of grains over the entire genome. Data from 15 complete kargotypes : values are given as grains per unit length (derived from the total grain count in each division divided by the relative length of that division).
In order t,o ensure that the grain distribution over the rest of the genome did not show any other regions of local high concentrations of grain. 15 karyotypes were fully analysed. Each chromosome was divided into convenient divisions, of approximately equal length, and the grain counts in each division were scored. To correct for differences in division size each grain count was divided by the relative length of that division. The data are shown in Figure 6. There is some fluctuation due to the relatively small sample size but. the short arm of chromosome 16 shows a significantly higher grain count than any other division of the genome (+4-8 s.I). from the mean value. I’ < 106).
4. Discussion Hybridization in situ to fixed chromosome preparations from human lymphocytes is rapidly becoming a useful method for mapping human structural genes (Malcolm et (11.. lQ8ln,b; Gerhard ef (~2.. 1981; Harper et al.. 1981), and has several advantages over standard somatic cell techniques. No complex and timeconsuming cell culture is required and protein production and characterization are not necessary. The location of a gene can be narrowed down to a comparatively small region. about, 20% of the average size of chromosome (lojO of the genome), without using any special translocations or cell-fragmentation techniques. Although fluorescence-activated chromosome sorting has been used to exploit’ translocations to map the /3-globin gene cluster (Lebo et ul., 1979). this requires elaborate and expensive equipment that is not readily available at present. When a cloned DNA is available for use as a hybridization probe two methods of hybridization in situ can be used successfully to localize a gene sequence. In the first, nick-translated plasmid DNA is used and the number of site-specific autoradiographic grains is increased by disintegrations from annealed plasmid DNA molecules, which form loose networks at the hybridized locus. This is particularly effective when loo/, dextran sulphate is included in the hybridization buffer (Wahl et al., 1979; Gerhard et al.. 1981: Harper & Saunders. 1981) and “‘1 is
x-GLOBIN
GENE
ON SHORT
ARM
OF CHROMOSOME
16
2X
used as the source of radioactivity (Gerhard et al., 1981). However, there are also the disadvantages that the optimal probe concentration must be determined carefully to avoid excess background, the resolution may be decreased by the plasmid networks and the high energy of disintegration of the iodine, and there is no way of determining how the observed graining level compares with the expected efficiency of hybridization and detection. Using 3H-labelled probes, Harper 8 Saunders (1981) were able to localize a cloned fragment only 14.9 kb long and present in one or two copies. All these difficulties are overcome by using genomic [ 3H]cRNA transcripts of DNA containing sequences adjacent to and intervening in t,he gene-coding region (Malcolm et ul., 1981~). Here the major problem that arises is the presence of ubiquitous highly repeated sequences (Jelinek et al.. 1980: Deininger & Schmid. 1978) found interspersed between struct.ural genes. such that they occur in the majority of clones in a total human genomic library (Lawn et 01.. 1979: Tashima et al., 1981). One such family of repeats, the Alu family, has been analysed in detail around both the fl-globin and a-globin gene clusters (Fritsch et al.. 1980: (‘oggins et al., 1980: Lauer ef al., 1980; Malcolm et al., 1981b). These repeats may be located in restriction fragments of genomic DNA clones by using radioactively labelled total human DNA as a hybridization probe (Fritsch et nl.. 1980) and we have separated fragments from them by gel purification. Polymorphic variations in DNA sequence have been found around both the ,5globin (Jeffreys, 1979) and a-globin (Higgs et al.. 1981) gene clusters. In the case of the /Sglobin genes only point mut’at,ions have been detected (Jeffreys, 1979) but around the n-globin gene clusters two highly variable areas due to DNA rearrangements have been observed (Higgs et al.. 1981). Both the polymorphic, areas are outside the regions used by us t,o map the a-globin gene locus. Our finding of the a-globin genes in the region 16~12 + pter is compatible with the finding (Wainscoat et al., 1981) in a male infant who had trisomy of the short arm of chromosome 16 distal to the 16~12 of an unbalanced globin chain synthesis X/~OIM 1.6. The large number of potential genetic markers detected as restriction enzyme site polymorphisms now makes it feasible to construct a detailed linkage map for the human genome and makes it of particular significance to link these markers to classic genetic loci. such as the a-globin genes, and to chromosome regions defined cytogenetically.
This work was supported providing x-globin clones.
by the Medical
Research
Council.
We thank
Dr Tom Maniatis
for
REFERENCES Adams.
J. W., Kaufman,
R. E., Kretschner,
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A. W. (1980).
Sucl. ilcids Res. 8, 6113-6128. Botstein, D., White, R. L., Skolnick, M. & Davis, R. W. (1980). Amer. .I. Human Genet. 32. 314-331. Chandler, M. E., Kedes, L. H., Cohn, R. H. & Yunis, J. J. (1979). Science, 205, 908-910. Coggins, L. W., Grindley, G. J., Vass, K. J., Salter, A. A.. Montague, P.. Htinson, M. A. & Paul. cJ. (1980). Nucl. Acids Res. 8, 3319-3333.
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Deininger, P. L. & Schmid, C. W. (1978). J. ,+101. Biol. 106, 773-790. Deisseroth, A. & Hendrick. D. (1978). Cell, 15, 55-63. Deisseroth, A., Nienhuis, A., Turner, P., Velze, R., French. W. F., Ruddle, F., Lawrence, J.. Creagen, R. 8: Kucherlapti, R. (1977). Cell. 12, 205-218. Evans. H. J., Buckland, R. A. & Pardue, M. L. (1974). Chromosoma, 48, 405-426. Fennel. S. J., Malcolm, S., Williamson. R. & FergusoIl-Smith. M. A. (1979). J. Med. Genet.
16, 246-253. Fergusoll-Smith, M. A. (1974). La ricerca Clin. Lab. 4, 297-335. Fritsch. E. F., Lawn, R. M. & Maniatis, T. (1980). Cell. 19. 959-972. Gerhard, D. S., Kawasaki, E. S., Bancroft, P. (‘. & Szabo. P. (1981). Pror. ,Vut. ilcad. &i.. U.S.A. 78, 3755-3759. Harper, M. E. & Saunders, G. F. (1981). Chromosoma, 83. 431-439. Harper. M. E., Ullrich. A. & Saunders, G. F. (1981). Proc. Nat. =frxxd. Sci., U.H.A 78. 44584460. Henderson, A. S., Warburt,on, D. & Atwood, K. (‘. (1972). Proc. Sat. dcud. Sri.. cT.S’.d. 69.
3394-3398. Higgs. D. R.. Goodbourn, S. E. Y., Wainscoat. J. S., Clegg. ,J. B. & Weatherall. D. ,J. (1981). Nucl. Acids Rcs. 9, 4213-4224. .Jeffreys, A. J. (1979). Cell. 18. l-10. ,Jelinek, W. R., Toomey. T. P., Leinwand, L., Duncan. (1. H.. Biro, P. A., Chouldary, P. V.. Weissman, S. M., Rubin, C. M.. Houck. C. M., Deininger. P. L. & Schmid, C. W. (1980). Proc. Sat. Acad. Ski.. U..B.il. 77, 1398-1482. Kan. Y. W’:. Br Dozy, A. M. (1978). Proc. Xut. dcud. S’ci., IT~A’.=~.75, 5631-5635. Lauer. ,J., Shen, C. K. J. & Maniatis. T. (1980). Cell. 20, 319-130. Lawn. R. M., Fritsch, E. F.. Parker. R. V., Blake. G. & Maniatis. T. (1979). Cell. 15, 11577 1174. Lebo, R. I’., Carrano, A. V., Burkhart-Schultz, K., Dozy, A. M., Loh-Chung, I,. 8: Kan, Y. W. (1979). Proc. Sat. Acad. Sri.. L’..A.d. 76, 3804-5808. Malcolm, S., Williamson. R., Boyd. E. & Ferguson-Smith, M. A. (1977). Cytoqenet. Cell Oen,et.
19, 256G261. Malcolm, S.. Barton. P., Murphy. C. & Fergusoll-Smith. M. A. (1981a). dnn. Human Genet. 45, 135-141. Malcolm, S., Barton, P. & Ferguson-Smith, M. A. (1981b). Human &net. 547, 388-393. McKusick, V. A. (1980). J. Hered. 71, 370-391. Steffenson, D. M., Duffey. P. & Prensky. W. (1974). Sufurr (London,), 252, 741-743. Stephen, G. S. (1977). Genetica. 47. 115-116. Tashima, M.. Calabretta, B.. Torelli, G., Scofield, M., Maize], A. & Saunders, G. F. (1981). Proc. Xut. Acud. Sri., U.S.A. 78, 1508-1512. Wahl, G., Stern, M. & Stark, G. K. (1979). I’roc. Sat. dcud. AX., U.AS.A. 76, 3683-3687. Wainscoat, .J. S.. Kanavakis, E., Weatherall, D. .J., \Valker, ,J., Holmes-Seidle. M.. Bobrow. M. & Donnison. A. B. (1981). Lancet. II, 301b1.
Edited by 1’. (‘htrmbon
.J. ;Vol. Biol.
Localization of the Human a-Globin Gene Cluster to the Short Arm of Chromosome 16 (16p12-16pter) by Hybridization in situ I’. K.-IKTOS’.
S. MALCOLM’. C. MURPHY~
’ Department
Roy01 Hospital (Krcrived
ANI) M. A. FERGUSON-SMITH’
of Biochem.istry
‘Institute of Medical Genetics for Sick Children,, Yorkhill. Glasyou~. U.K.
12 November
1981, trod in revised form 21 IIecember
1981)
The human n-globin gene cluster has been assigned previously t’o chromosome IS using a molecular hybridization assay of somatic cell hybrids. We have carried out a regional assignment to the upper half of the short arm of chromosome 16 in the in situ of a-globinspecific probes to fixed region lBpl2-pter by hybridizat.ion
metaphase chromosomes. Suitable hybridization
probes, of sufficient length to give
a detectable signal, were prepared free from highly repetitive sequences, by the isolation of restriction enzyme fragments from genomic recombinant clones covering the 5. $I and a-globin gene regions of the chromosome. A total of 52 x lo3 bases of hybridized sequence was used to detect the cr-globin gene cluster. The criteria of chromosome specificit’y, overall efficiency of hybridization and regional localization were used to establish the localization. These criteria have been applied previously to the mapping of the fi-globin gene cluster.
1. Introduction Several techniques are available for finding the regional chromosomal localization of a gene (McKusick, 1980). Where suitable t,ranslocation break-points covering the whole chromosome are available, somatic cell fusion techniques have been of great, use. The analysis of hybrid cells either depends on the production of protein in a fused human/rodent cell line or, when a cloned gene probe is available, the gene may be detect,ed directly by nucleic acid hybridization, without any need for expression of the gene product. Hybridization in situ to chromosome spreads has the advantage that normal chromosome preparations from dividing lymphocytes are used and a rather precise regional localization can be obtained. This method has been used to localize a number of repetitive gene families in man where the target, site for hybridization is >50 kbt; for example, the 28+ 18 S ribosomal genes i Abbreviations
used: kb. lOa bases; cRNA. complementary
RNA.
269 ~H122-2R36/X2/1(~26!~~10
$03.00/O
C 1982 Academia Press Inc. (London)
Ltd.
270
P. 5
BARTON
ET
AI,
W BE
a2
al
H
H
3’ I
’
I
j
psstI
Pst
/ PM4
H
I
H Xba H I kb
FIG. 1. Arrangement of the human a-globin-like genes. 1. Restriction map of the a-globin gene cluster. 2. Regions of homologous DNA sequence. 3. DNA contained in the genornic subclones. () Regions gelpurified as an a-specific probe ; (-f-j-j) re g ions containing highly repetitive DNA. Restriction enzyme sites. B. BarnHI; Bg, BgZII: H, HindIII: Pst. PstI; S. Sac1 (=SstI): Xba, XbnI.
(Henderson et al.. 1972; Evans et al.. 1974). the 5 S ribosomal genes (Steffenson et al., 1974; Fenttel et al., 1979) and the histone genes (Chandler et a,l., 1979). Recently, the method has been extended to the detection of structural genes present in only a few copies. The sensitivity was increased either by using recombinant DNA probes that form networks at the site of hybridization (Malcolm ~1 al.. 1977; Gerhard et al., 1981: Harper et al.. 1981; Harper & Saunders. 1981). or by using hybridization probes made from genomic recombinants that hybridize to adjacent and itttervettittg sequences as well as to the structural gene (Malcolm et al., 1981a). It has been shown by the work of Deisseroth et al. (1977) and Diesseroth & Hendrick (1978), using somatic cell hybrids. that the human aglobin genes are found on chromosome 16. In order to localize the a-globin genes more precisely we have applied the method of hybridization in situ to normal chromosome spreads. Fine-structure mapping of the humatt J-like globin genes (Lauer et al.. 1980) has revealed a closely linked multigene family containing the genes for the two adult n-globin chains. the embryonic forms of the a-globin chain and also non-functional pseudo-3 genes (see Fig. 1); the entire region of over 18 kb has been isolated in overlapping recombittattt clones. As both the B and p-globin gene clusters are interspersed with highly repeated DNA sequences (Lauer et al.. 1980; Fritsch et al., 1980; Malcolm et al., 1981b: Coggins et al., 1980; Adams et al.. 1980), which would interfere with hybridization. suitable restriction fragments, free of repetitive DNA, must be prepared for use in hybridization experiments in situ. This approach has been used recently to confirm the localization of the human p-globin gene cluster to the short arm of chromosome 11 close to the centromere (Malcolm et al.. 1981a). In that case a region of 4500 base-pairs including the /?-gene was used as the template for synthesis of [3H]cRNA to be used as a hybridization probe. For the localization of the a-globin gene clust,er we have used a mixture of probes prepared from the Iglobin region making a total of 5200 bp of hybridizing sequence.
,r-GLOBIN
GENE
ON SHORT
ARM
OF CHROMOSOME
16
271
The realization that single base changes in DNA that give rise to restrict.ion enzyme site polymorphisms (Kan & Dozy, 1978) can be used as genetic markers (Botstein et al., 1980) has made the need for precise regional assignment of welldefined genes such as the ir-globin genes more pressing. The establishment of a large number of such polymorphisms linked to the cu-globin cluster will rapidly provide genetic markers for a relatively large and well-defined region of the human genome (Higgs et al.. 1981).
2. Materials (a) Preparation
and Methods
of restriction
fragments
R.estric:t~ion enzyme digests were carried out under conditions recommended by the in an O+V& agarose with low manufacturers. Digested DNA (10 to 30 pg) was separated gelling temperature (Miles) gel using a buffer containing 40 mmTris (pH 7.7), 40 mm NaOAc, 1 mmEDTA. To recover DNA, gels were stained with ethidium bromide, visualized under 360 nm ultraviolet light and the DNA bands cut out from the gel. The agarose was melted at 65”C, cooled to 37”C, shaken with an equal volume of phenol and centrifuged at 10,000 revs/min for 10 min. The aqueous phase was extracted with chloroform and the DNA precipitated by addition of @l vol. 2 M-Na acetate and 2.5 vol. 95% ethanol. DNA yields wefe >700,. (lo) Detection The method
used was exactly
of repetitive sequences by hybridization “P-labelled human DXA as described (c) cHSd
by Fritsch
using
et al. (1980).
preparation
13H]cRNA was made from gel-purified x-specific DNA by transcription using Escherichia coli RNA polymerase. Transcription reactions were performed in 30 ~M-[~H]ATP, [ 3H]CTP 13H ]UTP (Amersham Radiochemicals), 200 PM-GTP (Boehringer), 150 mmKC1. 10 mMM&l,. 15 mmM&l,, 40 mw-Tris (pH 7.9). 1 mw-dithiothreitol, @4 mmNa,HPO,, @l mm EDTA, 25Opg DNA/ml, 150 units E. coli RNA polymerase/ml. After incubation at 37°C for 3 to 4 h DNAase I (Worthington) was added to 200 pg/ml, and incubating was continued at 37°C for 30 min. This was followed by addition of 50 pg yeast transfer RNA (Sigma) and an equal volume of @3 M-NaCl, @2% sodium dodecyl sulphate. Samples were extracted with an equal volume of phenol/chloroform (1 : 1) and [‘H]cRNA separated from unincorporated nucleotides by passage through Sephadex G50 in 40 mw-Tris. HCI (7.6). Pooled fractions were desalted by passage through a second Sephadex G50 column in distilled water and dried under a vacuum. (d) Hybridization
in situ
Chromosome preparations fixed to glass slides were made from short-term lymphocyte cultures as previously described (Ferguson-Smith, 1974). Prior t,o hybridization, chromosomes were banded by immersion in 1 9, Lipsol detergent solution for 15 s (Stephen. 1977), followed by Giemsa stain. Chromosome spreads were photographed for karyotype analysis. Slides were treated with 100 pg RNAase A/ml (Sigma) in 2 x SSC! (SSC is 0.15 MNaCl, 0015 iv-sodium citrate) for 30 min at 37”C, washed thoroughly in 2 x SSC and dehydrated by immersion in 50%, 759,, 95% and 100% ethanol. Chromosomes were denatured in SOoi, formamide, 0.1 mmEDTA. 5 mM-HEPES (pH 7.0), at 55°C for 15 min, washed in 2 x SSC and dehydrated as above. 13H]cRNA prepared from the gel-purified a-
272
P. HARTOS
ET
AL
globirt recombinant DIVAS and dissolved in hybridization buffer (500,, (v/v) formatnide, 0% M-NaCl, 1 mM-EDTA, 5 mM-HEXES, pH 7%) was added to prewarmed slides at 43”C, such that each slide received 20 to 30 ng [ 3H]cRNA in 5 ~1 of hybridization buffer, covered with a cover-slip (previously washed in 1 M-HCI) and incubated at 43°C for 18 h. After incubation, unhybridized cRNA was removed by treatment with 20 pg RNAase A/ml (Sigma) in 2 x SSC at’ 37°C for 30 min, followed by extensive washing in 2 x WC at 4°C. After dehydration through ethanol, slides were dipped in Ilford KZ nuclear research emulsion diluted 1 : 1 with water. Autoradiographs were exposed for 42 days at 4X”, stained in Giemsa and the chromosome spreads that, had been photographed were relocated for grain
counting.
3. Results
TINA for use as hybridization probes was isolated by restriction enzyme digestion from plasmids previously sub-cloned into pBR322 from AHaG and XHaG2 recombinants (which cover the entire T-globin gene cluster) (Lauer et nl., 1980) (see Fig. 1). The cluster contains two adult n-globin genes (01~and a2), a non-functional a gene (@t) and one of the two etnbryonic <-globin genes (
kb
4-3
l-9 l-2
FIG:. 2. Southern blot hybridization to detect repetitive DNA in plasmid recombinant pBR{. (a) Ethidium bromide stained. (b) Autoradiograph after hybridization. Track 1: pBR( DNA digested with EcoRI and BglII giving 2 fragments. (i) A 6.6 kb fragment containing the c-gene, surrounding DNA and the vector pBR322. (ii) A 2 kb fragment, of genomic DNA located 1 kb 5’ of the 5 gene. Track 2: hKcoRI/RnmHI marker DSA. Track 3: Plasmid RI 3.2 digested with EcoRI and XbaI giving three fragments. (i) A 4.3 kb fragment of pBR322 DNA. (ii) A 1.2 kb fragment extending 3’ of the Xba site located downstream from the 8.globin gene : known to contain a number of the highly repetitive Alu family of repetitive DSA sequences (Coggins et (II.. 1980: Malcolm et al.. 1981). (iii) A I.9 kb fragment from adjacent to the @-globin gene containing single-copv DNA.
x-GLOBIN
GENE
ON SHORT
ARM
OF CHROMOSOME
16
273
within the a-globin-like gene cluster (Lauer et al.. 1980). Such sequences can be identified within plasmid molecules by an adaptation of the Southern procedure (Fritsch et nl., 1980), in which radioactively labelled human genomic DNA is used probe. Only sequences present many times in the human genomr as a hybridization will be detected upon autoradiography. The clones pBR[, p&t1 and pBRa1 used in the a-globin mapping experiments were analysed using this technique. Figure 2 shows the results of such an experiment: pBR< digested with EcoRI and &$I1 gives two fragments. The smaller 2 kb fragment of insert DNA located 1 kb 5’ Tao the 5 gene shows strong hybridization to 32P-labelled total huma’n DNA. indicating the presence of highly repetitive DNA. The larger 6.6 kb fragment containing the 5 gene coding sequence, the surrounding genomic DNA and the vector pHR322 shows no hybridization. Control tracks containing known highly repetitive or single-copy DNA from around the /3-globin gene cluster were also included. In similar experiments pBRn1 was digested with Hind111 and EcoRI giving a 6 kb non-repetitive fragment including 2 kb of genomic human insert DNA and a 2 kb fragment from the human genome containing repetitive DNA. pSst1 digested with BumHI and HilldIII gave an 8.25 kb fragment containing repetitive sequences and a 1.6 kb fragment of non-repetitive DNA including 1.05 kb of human DNA. Three highly repeated sequences were identified within the sub-clones tested and the regions in which they are found are marked on Figure 1. The restriction enzyme digests for preparation of probes were chosen to give the maximum separation on preparative gels between fragments containing the repeats and fragments to be used for hybridization. These are marked on Figure 1. (b) Hybridization
in situ
[‘H JcRNA (spec. act. 1.7 x 10’ disints/min per pg) was prepared from the three plasmid-derived gel-purified DNA regions described, by transcription with E. coli RNA polymerase. Previous experiments using ribosomal, globin and immunoglobulin recombinants have shown that both inserted and plasmid-derived sequences are transcribed (apparently randomly) under the conditions used here. The DNA fragments contain 2.6. 2 and 1.05 kb of a-globin-specific DNA but the extent of the gene duplication around the two 3c-globin genes (Lauer et al.. 1980) is such that t.here are long regions ( >2 kb) flanking the coding sequences that are common to both at and a2 genes and which will. therefore, cross-hybridize. The probes prepared from both pBR’*l and p&t1 contain part of these duplicated regions and [3H]cRNA prepared from them will hybridize to 3.54 and 2.88 kb, respectively. Three sets of slides for hybridization in situ were set up using L3H]cRNA transcripts as follows : (1) pBRnl-derived probe (total target size 3.5 kb) (2) pBRcv1 +pBR[-derived probes (total target size 6.14 kb) (3) pBR%l +pSstl-derived probes (total target size 5.8 kb). Hybridization was to pre-banded and photographed chromosomes to allow unequivocal chromosome identification for grain counting after hybridization (Malcolm et c&l., 1981a). Autoradiographs were exposed for 42 days and a total of
274
P. KARTOK
E7’ AI,
I 2 3 4 5 6 7 8 9 IO II 1213141516171819202122XY Chromosome number
PIG. 3. Hybridization in situ (i). The total number of grains counted ou each chromosome from 146 karyotyped cells divided by the relative length of each chromosome (“6 haploid genome length : FergusmSmith. 1974). Broken line shows the average value.
146 prephotographed karyotypes frotn seven slides were scored for silver grains occurring over chromosomes. Data front each slide were analysed individually and the results pooled.
Figure 3 shows t.he distribut.ion of grains for all 146 kayotypes over the genome. To allow for the fact that. chromosomes of different lengths will contain different numbers of grains due to random background the results are presented as grains/unit chromosome length where each unit lengt,h is I(/, of the gettome (Ferguson-Smith. 1974). Chromosome 16 scored the highest grain level and gave a value + 2.8 standard deviations from the mean (I’
wGLOBIN
GENE
ON
SHORT
ARM
OF
CHROMOSOME
275
16
Y
-12ot I 234
, , , , , , , , , , , , , , , , , , , , , , 56 78910111213141516171819202122XY Chromosome number
FIG. 4. Hybridization in situ (ii). Distribution ofgrains over the genome. Number each chromosomecompared tothenumberexpectedforachromosomeofthatlengthifthetotalgraincount were distributed proportionately over the genome.
(d) IKstribution
of gmins
along
chromosome
ofgrains
occurring
on
16
The distribution of grains along the length of chromosome I6 was determined in each experiment. Chromosome 16 has an arm ratio of 1.51 and therefore divides conveniently into five equal divisions (two in the short arm. three in the long arm). In each set of slides examined there was an excess of grains in the upper half of the short arm (corresponding to band positions 16pter-p12). Pooled data are shown in Figure 5. Virtually all the extra grains found on chromosome 16, compared to the number expected if grains were random over the genome (represented by the broken line), are found in the same segment of the chromosome.
200 r-l g b B L IOO2 ----------_---E 2
----------
Flct. 5. Hybridization in&u (iii). Distribution ofgrains along chromosome 16. p, short arm : q, long arm : arrow marks the position of the centromere. Broken line indicates the number of grains expected along
chromosome lti.fro~naproportionaldistributionofthetotalgraincount.distributedevenl~overitslength.
276
P. BARTON
Chromosome
h’T
AL.
number
FIG. 6. Hybridization in S&L (iv). Regional distribution of grains over the entire genome. Data from 15 complete kargotypes : values are given as grains per unit length (derived from the total grain count in each division divided by the relative length of that division).
In order t,o ensure that the grain distribution over the rest of the genome did not show any other regions of local high concentrations of grain. 15 karyotypes were fully analysed. Each chromosome was divided into convenient divisions, of approximately equal length, and the grain counts in each division were scored. To correct for differences in division size each grain count was divided by the relative length of that division. The data are shown in Figure 6. There is some fluctuation due to the relatively small sample size but. the short arm of chromosome 16 shows a significantly higher grain count than any other division of the genome (+4-8 s.I). from the mean value. I’ < 106).
4. Discussion Hybridization in situ to fixed chromosome preparations from human lymphocytes is rapidly becoming a useful method for mapping human structural genes (Malcolm et (11.. lQ8ln,b; Gerhard ef (~2.. 1981; Harper et al.. 1981), and has several advantages over standard somatic cell techniques. No complex and timeconsuming cell culture is required and protein production and characterization are not necessary. The location of a gene can be narrowed down to a comparatively small region. about, 20% of the average size of chromosome (lojO of the genome), without using any special translocations or cell-fragmentation techniques. Although fluorescence-activated chromosome sorting has been used to exploit’ translocations to map the /3-globin gene cluster (Lebo et ul., 1979). this requires elaborate and expensive equipment that is not readily available at present. When a cloned DNA is available for use as a hybridization probe two methods of hybridization in situ can be used successfully to localize a gene sequence. In the first, nick-translated plasmid DNA is used and the number of site-specific autoradiographic grains is increased by disintegrations from annealed plasmid DNA molecules, which form loose networks at the hybridized locus. This is particularly effective when loo/, dextran sulphate is included in the hybridization buffer (Wahl et al., 1979; Gerhard et al.. 1981: Harper & Saunders. 1981) and “‘1 is
x-GLOBIN
GENE
ON SHORT
ARM
OF CHROMOSOME
16
2X
used as the source of radioactivity (Gerhard et al., 1981). However, there are also the disadvantages that the optimal probe concentration must be determined carefully to avoid excess background, the resolution may be decreased by the plasmid networks and the high energy of disintegration of the iodine, and there is no way of determining how the observed graining level compares with the expected efficiency of hybridization and detection. Using 3H-labelled probes, Harper 8 Saunders (1981) were able to localize a cloned fragment only 14.9 kb long and present in one or two copies. All these difficulties are overcome by using genomic [ 3H]cRNA transcripts of DNA containing sequences adjacent to and intervening in t,he gene-coding region (Malcolm et ul., 1981~). Here the major problem that arises is the presence of ubiquitous highly repeated sequences (Jelinek et al.. 1980: Deininger & Schmid. 1978) found interspersed between struct.ural genes. such that they occur in the majority of clones in a total human genomic library (Lawn et 01.. 1979: Tashima et al., 1981). One such family of repeats, the Alu family, has been analysed in detail around both the fl-globin and a-globin gene clusters (Fritsch et al.. 1980: (‘oggins et al., 1980: Lauer ef al., 1980; Malcolm et al., 1981b). These repeats may be located in restriction fragments of genomic DNA clones by using radioactively labelled total human DNA as a hybridization probe (Fritsch et nl.. 1980) and we have separated fragments from them by gel purification. Polymorphic variations in DNA sequence have been found around both the ,5globin (Jeffreys, 1979) and a-globin (Higgs et al.. 1981) gene clusters. In the case of the /Sglobin genes only point mut’at,ions have been detected (Jeffreys, 1979) but around the n-globin gene clusters two highly variable areas due to DNA rearrangements have been observed (Higgs et al.. 1981). Both the polymorphic, areas are outside the regions used by us t,o map the a-globin gene locus. Our finding of the a-globin genes in the region 16~12 + pter is compatible with the finding (Wainscoat et al., 1981) in a male infant who had trisomy of the short arm of chromosome 16 distal to the 16~12 of an unbalanced globin chain synthesis X/~OIM 1.6. The large number of potential genetic markers detected as restriction enzyme site polymorphisms now makes it feasible to construct a detailed linkage map for the human genome and makes it of particular significance to link these markers to classic genetic loci. such as the a-globin genes, and to chromosome regions defined cytogenetically.
This work was supported providing x-globin clones.
by the Medical
Research
Council.
We thank
Dr Tom Maniatis
for
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