The molecular basis of oa-thalassaemia in Thailand - Europe PMC

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May 18, 1984 - The molecular basis of a-thalassaemia has been established in. 48 Thai subjects with Hb H disease and 15 with the Hb Bart's hydrops fetalis ...
The EMIBO Journal vol.3 no.8 pp.1813- 1818, 1984

The molecular basis of oa-thalassaemia in Thailand

P. Winichagoon1, D.R. Higgs, S.E.Y. Goodbourn, J.B. Clegg, D.J. Weatherall and P. Wasil MRC Molecular Haematology Unit, Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, UK, and 'Department of Medicine, Siriraj Hospital, Bangkok, Thailand Communicated by D. J. Weatherall

The molecular basis of a-thalassaemia has been established in 48 Thai subjects with Hb H disease and 15 with the Hb Bart's hydrops fetalis syndrome. This study has shown that in this population there are at least 18 different types of chromosome carrying seven independent a-thalassaemia mutations one of which is a novel deletion removing the entire a-globin gene complex. Although there are a limited number of athalassaemia determinants in the Thai population, there is a remarkable degree of variation in the genetic markers which flank them. These markers may be of value in establishing the evolutionary history of the a-thalassaemias. Key words: a-thalassaemia/genetic recombination/molecular evolution Introduction The human a-globin gene complex on chromosome 16 (Diesseroth et al., 1977) consists of two a genes (al and a2) separated from the embryonic a-like (O) gene by two pseudogenes (O'a and Ok) arranged in the order 5' t-i¢-ta-a2-al 3' (Lauer et al., 1980). The globin chains derived from the two a genes are identical (Foldi et al., 1980) and are produced in approximately equal amounts (Hollan et al., 1972; Liebhaber and Kan, 1982). Disorders which result in reduced a-chain synthesis (a-thalassaemia) are common and result in either diminished (a +-thalassaemia) or absent (aO-thalassaemia) a chain production from the affected chromosome. Heterozygotes for a +- and ax-thalassaemia show minor haematological abnormalities but are clinically unaffected while compound heterozygotes for a°- and a +-thalassaemia have Hb H disease, a moderately severe haemolytic anaemia. Homozygotes for a0-thalassaemia have a lethal condition known as the Hb Bart's hydrops fetalis syndrome (reviewed in Higgs and Weatherall, 1983). Molecular analysis has shown that a-thalassaemia is caused by a large variety of genetic defects. +-Thalassaemia can result either from deletions which remove one of the genes (- a3.7 and a4-2, where the superscript indicates the size of the deletion in kb) (Orkin et al., 1979; Embury et al., 1979, 1980) or a number of non-deletion defects (written as aaT) (Clegg et al., 1971; Orkin et al.. 1979; Higgs et al., 1981b,1983; Goossens et al., 1982). a0-Thalassaemia results from deletions which involve both a genes (-SEA, ..MED, (a)20.5 and (a)5.2 where the superscripts SEA and MED defects from Southeast refer to previously characterised Asia and Mediterranean subjects, and the superscripts 20.5 a

a

a0

IRL Press Limited, Oxford, England.

and 5.2 refer to the size of the deletions in two less common a0 defects) (Pressley et al., 1980a,1980b; Orkin and Michelson, 1980; Nicholls et al., 1984). Provisional surveys in several populations have indicated that there are marked differences in the relative frequencies of these defects in regions where a-thalassaemia is prevalent (reviewed by Weatherall and Clegg, 1981). To characterise the types of c-thalassaemia defect and their relative frequency more completely in a Southeast Asian population we have determined the molecular basis of athalassaemia in patients with Hb H disease or Hb Bart's hydrops fetalis from Thailand (Wasi et al., 1974). In addition, we have studied the association of these defects with a polymorphic, hypervariable region of DNA (HVR) which is closely linked to the a-globin genes (Higgs et al., 1981a; Goodbourn et al., 1983). There is a remarkable degree of genetic heterogeneity at this locus which may be of value in determining the evolutionary history of a-thalassaemia in this region. Results Forty-eight patients with Hb H disease and 15 with the Hb Bart's hydrops fetalis syndrome were randomly identified from cases seen at the Division of Haematology, Siriraj Hospital, Bangkok, using standard haematological procedures (Dacie and Lewis, 1975). a +-Thalassaemia defects Normal DNA (aa/aa), when digested with BamHI and hybridised with an a-specific probe (A in Figure 1), produces a 14-kb fragment (Table I and Figure 1). DNA from 39 patients with Hb H disease had a shortened BamHI a-specific fragment (10.5 kb) characteristic of a single gene deletion which results in an a +-thalassaemia haplotype (- a) (Table I). This haplotype may arise by unequal, homologous recombination between the duplicated DNA sequences which include the a globin genes (Orkin et al., 1979; Lauer et al., 1980; Embury et al., 1980). Two different arrangements have been observed: one which involves a deletion of 3.7 kb of DNA and another which removes 4.2 kb (Embury et al., 1980). To characterise these haplotypes further, DNA was digested with BgIII which distinguishes between deletions of 3.7 or 4.2 kb; the - t3.7 and - a4(2 haplotypes being associated with 16-kb and 7.4-kb BgllI a-specific fragments respectively (Table I and Figure 1). We observed 36 haplotypes of the - a3M7 and three of the - a4 2 type. In one case, the remaining a gene in a - ac42 defect also carried the - a74 Asp--His (Hb Q) mutation (Lie-Injo et al., 1979; Higgs et al., 1980). Of the remaining nine patients with Hb H disease, the BamHI and BglII digested DNA produced only the normal caspecific fragments, suggesting that the a + haplotypes in these cases were of the non-deletion type. In eight of these, Hb Constant Spring, a chain termination mutant resulting in the 1813

P.Winichagoon et al. 1

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Fig. 1. Restriction enzyme maps of the common ac-thalassaemia defects in Thailand (see also Table I). ZI is a PvuII/HincII, ¢-specific probe obtained as previously described (Pressley et al., 1980b) and A is the a-specific plasmid JW1I1 (Wilson et al., 1978). Hypervariable regions (Higgs et al., 1981a) are indicated thus \AA Solid bars beneath each map indicate the sizes (kb) of BamHI and BglII a-specific fragments observed. The sizes of various c-specific BgIII hypervariable fragments shown in the figure, are given in Table I. .

Table 1. Summary of a+-thalassaemia haplotypes from the 48

-

a37 a4.2

cases of Hb H disease

Number of

a

haplotypes

size of a-specific fragments (kb)

observed

BamHI

BgIII

36 2

10.5 10.5

16.0 7.4

probe (A in Figure 1)

a4-2 (Hb Q) acsca

1

10.5

8

14.0

aaT

1

14.0

7.4

12.6,7.4 12.6,7.4

r probe (ZI in Figure 1) Size of interzeta hypervariable fragments (kb)a

Bglll (numbers of haplotypes)

10.5(13); 1 1 .3(2); 11.3(1); 11.3(6); 11.3(1);

11.3(17);

12.0(5);

12.0(1);

triple r gene (1)

aFor simplicity only the size of the hypervariable fragment is tabulated. Other predicted BglII ir-specific fragments

production of an elongated chain in very low amounts (Clegg et al. 1971) was demonstrated in the patient's peripheral blood, indicating that the haplotype was aCsa. The remaining case in which no abnormality in the patient's Hb or DNA was demonstrated, can be considered as an unclassified non-deletion a thalassaemia (aaT). Linkage of +-thalassaemia defects to the polymorphic, interzeta HVR Previous analysis has revealed a highly variable region of DNA, located between the r and At genes (Higgs et al., 198 la; Goodbourn et al., 1983). Since this region is polymorphic it provides a useful genetic marker for the gene complex. Using the enzyme BglII and probe ZI (Figure 1) which 1814 a

a

a

were

single r gene (1)

also observed in Southern blots.

hybridises to both the r and AD genes, it is possible to identify at least three different haplotypes in the Thai population on the basis of the length of the interzeta HVR; these are 10.5 kb, 11.3 kb and 12.0 kb (Table I and Figure 1). Of the 36 - a37 haplotypes, 13 were linked to the 10.5-kb fragment, 17 to the 11.3-kb fragments and five to the 12.0-kb fragment. The remaining - a3.7 haplotype was on a chromosome with only one zeta gene as previously reported (Winichagoon et al., 1982). The three - a4.2 haplotypes were all associated with an 11.3-kb fragment. The aCsa haplotypes were found in association with 11.3-kb (six cases), 12.0 kb (one case) and a chromosome bearing triplicated zeta genes (Winichagoon et al., 1982) (Table I and Figure 1).

The molecular basis of cr-thaassaemia in Thailand

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Fig. 2. Restriction map of the common (--SEA') defect compared with a normal (aa) gene complex. The symbols (+) and (-) indicate the presence or absence of a polymorphic Sacl restriction site, (0) denotes the BgII site described in the text.AA indicates the hypervariable regions (Higgs et al., 1981a) in the normal complex, and --- denotes the 3' end of the __SEm defect. The deletion breakpoint is indicated III on the map of the SEA defect and the corresponding region from the normal chromosome is shown in detail above, with the limits of the breakpoint shown thus --. An additional variable segment of DNA is present in the first intervening sequence of the ,6 gene (Proudfoot et al., 1982).

Table I. Sizes of r and i/ -specific fragments in the --_SE deletions __SEA I-VII

BamHI EcoRI BglII HpaI HindII

KpnI BglII/EcoRI BglII/BamHI BglII/HindIII BgllI/HpaI Sacl SacI/BgIII

5.9

5.8b 10.5 11.7 13.5 11.0 3.8 1.5 4.8 5.8

6.0,1.5d 1.0

I

II

III

IV

V

VI

VII

-17-20 15 17c

-17-20 -15 11.0 -13

-17-20 14.7 9.8 12.5

-17-20 14.2 9.5 11.7

-17-20 13.7 9.0 11.3

-17-20 13.0 8.8 10.7

-17-20 12.7 8.4 10.4

-

10.5a

-13 17-20 >23 8.5 10.5 10.0 8.5

1.8,8.0" 7.3

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-

-

-

-

-

-

-

-

9.0 11.0

8.2 9.8

7.7 9.5

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-

9.0 1.8,8.6 7.7

-

-

1.8,7.6 7.1

1.8,7.1 6.7

7.3 9.0 -

7.3

6.8 8.8

6.2 8.4

-

-

-

-

1.8,6.4

1.8,6.1

6.4

6.1

aCannot be resolved from c-specific fragment. bEcoRI hybridised with 150-bp SmaI fragment (Z3 in Figure 2). 'Did not hybridise with 150-bp SmaI (Z3) in 10 cases of a°-thalassaemia. dSacI hybridised with 450-bp SmaI fragment (Z2 in Figure 2) in 18 cases of a°-thalassaemia.

ac°-Thalassaemia defects When DNA from subjects with the Hb Bart's hydrops fetalis syndrome was analysed with an a-specific probe no fragments were detected, indicating that in all cases the four a genes were deleted (--I--) (Table I and Figure 2). Furthermore, blot hybridisation studies using probe Zl on DNA digested with BamHI, BgIIl and EcoRI (Table II) showed that in 69 a0 thalassaemia haplotypes, from cases of Hb H disease and the Bart's hydrops fetalis syndrome, the patterns of i-specific fragments were identical to those seen in

previously characterised homozygotes for the --SEA haplotype (Pressley et al., 1980b and Table II). To map the 5' breakpoint in the --SEA deletion accurately, we examined DNA from several individuals with the Hb Bart's hydrops fetalis syndrome in further detail using a variety of enzyme combinations and probes (Table II). Probe Z2 derived from a normal haplotype aa (Figure 2) hybridised to Sacl fragments containing the r and &6 gene from the SEA defect, indicating that at least part of this 450-bp SmaI fragment which was used as the probe (Z2) must be present in 1815

P.Winichagoon et al.

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Fig. 3. Restriction enzyme maps of the _SEA variants I and VI compared with a normal complex (ca). The symbols (+) and (-) indicate the presence or absence of a polymorphic Sacl restriction site.M indicates hypervariable regions of DNA. The variable region 3' to the breakpoint of the __SEA defect is not necessarily that which is normally present 3' to the complex. Probes VZ (V-zeta) and Zl were prepared as previously described (Higgs et al., 1981a; Pressley et al., 1980b).

t35

2,. 0.

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9.5. 0.O

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a

haplotypes described above (Tables I and II). In each of these cases the region 5' to the breakpoint was identical to the common --SEA defect (Figure 3). However, fragments which span the breakpoint differed in size from the common --;A

_.6

_

.w

_

Fig. 4. BgllI/BamHI-digested DNA hybridised with probe ZI from individuals with the following a"° haplotypes (1) __SEA1 and SEAII, (2) __SEAI, (3) __SEA1 and SEAV, (4) --SEAI,, (5) -S>EAIV, (6) SEAVI' Only the &rspecific fragments from the region spanning the breakpoint are seen on the autoradiograph. Fragments from the r gene (2.5 kb) and {D gene of the normal chromosome (3.0 kb) were run off this gel.

defect (Figure 4). Furthermore, for each digest (BamHI, BglII, EcoRI, HpaI and Sacl) the size variations from the usual pattern suggested that they were due to length differences in the variants, 3' to the breakpoint, rather than a series of entirely different rearrangements. This was confirmed by comparing the relationship of various restriction sites (BamHI, EcoRI, HindIII, HpaI and Sacl) to a fixed point (BglII) close to the 5' junction of the breakpoint (indicated by * in Figure 2). In each variant, the group of restriction sites on the 3' side of the breakpoint maintained the same order relative to each other while they all varied by the same amount relative to the fixed BglII site (Figure 3). These six variants of the --SEA defect are subsequently referred to as --SEA II to SEA VII, the common defect being --SEA I (Table II). Linkage of the seven --SEA thalassaemia variants to the polymorphic interzeta HVR All of the --SEA/ defects (I VII) were linked to 10.5-kb interzeta BglII fragments, suggesting that the polymorphic interzeta HVR was the same in all of these chromosomes. Using the enzyme SacI and probe VZ (Figure 3) it is possible to improve the resolution of the different HVRs and to determine the presence (+) or absence (-) of a polymorphic Sacl restriction site in the interzeta region. Using this improved level of resolution, in all cases the size of the HVR was identical and the polymorphic Sacd restriction site was absent (-) (Figure 3). Furthermore the first intron of the {D gene, which is also known to be polymorphic (Goodbourn et al., 1983; Chapman and Wilson, 1983), was of the same size in all of these -SEA defects, as judged by the size of a Sacd fragment (1.85 kb) which spans this region (Figure 2). A novel a0-thalassaemia defect In the subject with Hb Q-H disease, the pattern of BamHI -

the ca-thalassaemia haplotype. However, an adjacent 150-bp SmaI fragment from the haplotype (Z3 in Figure 2) hybridised to EcoRI fragments containing the ¢ but not the O6 gene from the -SEA defect (Table II), showing that this 150-bp region is deleted from the mutant chromosome. These results indicate that the breakpoint lies close to the junction of these two SmaI fragments which have been used as probes, probably within the third exon of the {D gene. Using various single and double digests including the enzyme BglII it was possible to reconstruct a provisional restriction map of the 3' component of this deletion (Figure 2 and Table II). Variation at the 3' end of the --SEA defect The restriction maps from seven haplotypes (two from patients with the Hb Bart's hydrops syndrome and five from subjects with Hb H disease) differed from that seen in the 69 aa

1816

The molecular basis of a-thaassacmia in Thailand

ci

EcoRI

1

2

Bgl'

BamHI

3

1

2

3

1

2

;

EcoRI

Hind III

1

2

1

3

2 3

rn:HI

.

3

Hind III

Bgl II

2

3

1

2

3

I' ... _

1 7_ 1/.~~~~~~~~~ 1 1

_

-NP

-p

.__

10

W0V

8 6

5

_

--

-

4W

Om p

IFlg. 5. Various restriction enzyme digests on DNA from an individual homozygous for -_sA (1), an individual (HWaL in Higgs et al., 1980) with -SEA/ a42Q (3), and the subject with Hb Q-H disease in this study (2). While all fragments associated with the C4y2 haplotype are present in (2) none of those from the __SEA haplotype can be identified in this subject. Since no abnormal or r-specific bands are seen in (2) it seems most likely that the entire a-complex is deleted from the ca0 chromosome of this individual. -

a

and BgIIl a-specific fragments indicated that the chromosome bearing the + mutation has only one a gene (-aQ) and the other chromosome has no genes (--). Although the expected genotype in this case was SEA/ a42 Q, comparison of this person's DNA with that of a previously documented individual (HWaL, in Higgs et al., 1980) with Hb Q-H disease (--SEA/- a4-2 Q) and a homozygote for the --SEA defect, failed to detect any of the rspecific fragments normally associated with the common SEA deletion (Figure 5). In fact, no abnormal a- or tspecific fragments associated with the a°-thalassaemia haplotype in this case could be identified (Figure 5). Similar observations were also made on a second subject with Hb CSH disease. The blot hybridisation data in either of these cases could be explained if the ax gene complexes on both chromsomes were identical ( a4_2 Q/ a4.2 Q, or acsa/aCsa). However, this would not be consistent with the observed phenotype unless a further event had inactivated one of the chromosomes in each case to give the expected a0/a +.-thalassaemia interaction. It seems more likely therefore that a less common a0-thalassaemia deletion which removes both a- and c-genes, thus producing no abnormal bands from this chromosome, is present in these individuals. a

a

-

Discussion Alpha-thalassaemia is probably the commonest human single gene disorder (reviewed in Higgs and Weatherall, 1983). Very little is known about the reasons for its high frequency, wide geographical distribution and molecular diversity. In particular, it is not clear to what extent these characteristics reflect a high frequency of mutation within the c-globin gene complex and independent selection in different areas, or a few mutations which have come under very strong selection in localised regions and then dispersed. As in many other populations, a +-thalassaemia in

Thailand is most frequently due to the deletion of a single a the - a3 7 haplotype being more prevalent than the a4.2. Since these deletions arise by misaligned cross-overs (Orkin et al., 1979; Embury et al., 1980; Lauer et al., 1980), a bias towards the a3C7 haplotype could reflect differences in the constraints on different misalignments (Trent et al., 1981). Alternatively, the relative fitness of the a37 and a4.2 haplotypes may differ. The three less common c + thalassaemias in this study (atcsa, aaT and ca thalassaemia in association with Hb Q) are all known to occur in Southeast Asia and the a74 Asp-His mutation (Hb Q) is on a chromosome with an a4_2 deletion. We have now observed three samples of this -aQ chain mutant, all in patients of Chinese ancestry and all of which occur in association with the - a4.2 defect, suggesting that this deletion has a single evolutionary origin. The origins of the common +-thalassaemias are less clear. Analysis of the interzeta HVRs of the a3-7 haplotypes showed that at least three genetically distinct chromosomes carry this defect; a fourth type of chromosome was identified in which the 13.7 defect is linked to a single zeta gene. Our data cannot distinguish whether this deletion has arisen on at least four separate occasions in the Thai population, or if it arose once and there has been subsequent rearrangement of DNA by inter or intrachromosomal recombination, giving rise to 'apparently' different chromosomes. Thus it is also possible that there has been equal, homologous crossing-over within the ca gene complex, in a similar manner to that seen in the ,B-globin complex (Antonarakis et al., 1982; Wainscoat et al., 1983), and that the a37 deletion has become linked to different zeta gene arrangements and interzeta HVRs which occur in both normal chromosomes and those with a-thalassaemia defects within each population. Unlike the - C3.7 deletion, which is common in many different populations, the --SEA deletion is found exclusively in Southeast Asians (including Thai, Chinese, Filipino and Viet1817 gene,

-

-

-

-

a

-

-

-

P.Winichagoon et al.

namese). In view of this limited geographical distribution of the --SEA deletion, it was surprising to identify six variants of this lesion in the Thai population. However, in each case, the differences from the prototype --SEA deletion were 3' to the breakpoint and all the variants were identical at the 5' end of the complex (Figure 3). This deletion probably arose on a single chromosome, possibly by an illegitimate recombination event (Nicholls et al., 1984; Vanin et al., 1983; Tuan et al., 1983), and subsequent mutations have been superimposed, either within the 3' HVR or a similar region further downstream. Although the --SEA deletion accounts for the majority of a0-thalassaemia deletions in the Thai population we also observed two examples of a less common haplotype in which the a andr genes are apparently deleted. An ao-thalassaemia deletion which removes the entireca-globin gene complex has only been previously described in patients with the rare syndrome of congenital Hb H disease associated with mental retardation (Weatherall et al., 1981). There is no evidence for this condition in the two patients described here, and therefore it appears that these mutants represent a hitherto undescribed and uncommon deletion form of a0-thalassaemia. Previous characterisation of the humana-globin gene complex has shown that there is considerable genetic rearrangement in this cluster. This study has shown that in the Thai population there are at least 18 different types of chromosomes carrying independent a-thalassaemia mutations. The occurrence of these particular rearrangements from what is presumably an extremely large number of spontaneous mutations in this gene family probably results from a combination of chance and selection due to the associated red cell changes. The chance of a particular rearrangement being found at polymorphic frequencies will depend on several factors, in particular the rate at which it occurs. Unequal homologous recombination is common in many duplicated genes in vivo and in vitro; the high prevalence and wide geographical distribution of the - a3.7 deletion suggests that it may have arisen several times during evolution although there may also have been genetic rearrangements on the original chromosome which contained the deletion within particular populations. In contrast, all of thec0-thalassaemia deletions are found in relatively limited geographical regions and even within these areas are less frequent than the ca +-thalassaemia deletions. The localised geographical occurrence and structure of the --SEA deletion are consistent with a single origin in Southeast Asia, and subsequent rearrangement of the affected chromosome to produce the six variants found in this study. However, other factors must contribute to the frequency of these chromosomes within a population, including their relative fitness, time of origin during evolution, changes in size and movements of the population, and the rates of change in genetic markers linked to the deletions. Of these it may be possible to assess the latter (Chapman and Wilson, 1983) which, combined with the data from this type of study, may enable us to provide a more comprehensive evolutionary history of the a thalassaemias. Materials and methods All haematological studies and haemoglobin analyses were carried out using standard techniques (Dacie and Lewis, 1975), DNA from buffy coats, spleen or liver samples were prepared and used in blot hybridisation studies (Southern, 1975) after digestion with HindII, BamHI, BglI, EcoRI, KpnI, HpaI or Sacl either singly or in combination. Restriction fragments were subsequently identified by blot hybridisation using various 32P-labelled DNA

1818

probes (Figures1 and 2) and autoradiography, and Higgs, 1982).

as previously described (Old

Acknowledgements We are grateful to the Rockefeller Foundation for financial support of this project and to R.D. Nicholls and A.V.S. Hill for helpful comments on the manuscript. We are also very grateful to Dr. S. Fucharoen for help in collecting samples for this study. References Antonarakis,S.E., Boehm,C.D., Giardinia,P.J. and Kazzazian,H.H. (1982) Proc. Natl. Acad. Sci. USA, 79, 137-141. Chapman,B.S. and Wilson,A.C. (1983)J. Cell. Biochem., Suppl. 73, 0912. Clegg,J.B., Weatherall,D.J. and Milner,P.F. (1971) Nature, 234, 337-340. Dacie,J. and Lewis,S.M. (1975) Practial Haematology, published by Churchill Livingstone, London. Diesseroth,A., Nienhuis,A., Turner,P., Velez,R., French Anderson,W., Ruddle,F., Lawrence,J., Creagen,R. and Kucherlapati,R. (1977) Cell, 12, 205-218. Embury,S.H,. Lebo,R.V., Dozy,A.M. and Kan,Y.W. (1979) J. Clin. Invest., 63, 1307-1310. Embury,S.H,. Miller,J.A., Dozy,A.M., Kan,Y.W., Chan,V. and Todd,D. (1980) J. Clin. Invest., 66, 1319-1325. Cohen-Solal,M., Valentin,C., Blouquit,W., Hollan,S.R. and Rosa, Foldi,J., J. (1980) Eur. J. Biochem., 109, 463-470. Goodbourn,S.E.Y., Higgs,D.R., Clegg, L.B. and Weatherall,D.J. (1983)

Proc.

Natl. Acad. Sci.-USA, 80, 5022-5026.

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Received on 10 April 1984; revised on 18 May 1984