BritishJourml ofllaetnatology, 1980, 46, 341-349.

Annotation

MAPPING OF THE HUMAN GLOBIN GENES

The mRNA isolated from reticulocytes largely codes for the globin chains of the red cell. It is easily isolated, and because of its relative purity globin mRNA was one the first mammalian ‘messenger’ molecules to be studied. When radioactively labelled, using enzymatic methods, it can be used as a gene-specific probe to count globin gene numbers (Williamson, 1976). However, the globin mRNA, even isolated from reticulocytes, is still contaminated with low levels of other mRNAs, and naturally this globin mRNA is a mixture of molecules, coding for mainly a- and Ij-globin with a small proportion coding for 6- and y-globin. The techniques of ‘genetic engineering’ have allowed completely pure globin gene sequences to be obtained. Any piece of DNA can now be inserted into a bacterial virus, purified by isolating a single colony (‘cloning’) and amplified in culture to provide milligrams of pure, single gene sequence (for review see Biochemistry of Genetic Engineering, ed. by Garland Lci Williamson, Biochemical Society Symposium no. 42, 1979).

Recently, two groups reported the isolation of recombinant plasmids containing SI-, p- and y-globin gene sequences, made by copying globin mRNA into DNA (Wilson et al, 1978; Little et al, 1978). Others have cloned the DNA fragments coding for globin from the genome itself. These contain sequences, both around and within the genes, not present in the mRNA. The recombinant molecules have allowed the structure of globin genes to be closely examined, even as far as the base sequence of the DNA itselfl

Gene mapping has been used to examine the globin genes as part of the total cellular DNA, rather than as isolated, recombinant molecules. It has further allowed the gross structure of the globin genes from patients with thalassaemia, sickle-cell disease and other haemoglobino- pathies to be investigated. Not only does this allow some conclusions to be drawn about the nature of these diseases, it extends our knowledge of the mechanism of the switch from 7- to p-globin which occurs around birth, and provides a relatively straight-forward tool for antenatal diagnosis of some of these diseases.

Gene Mapping Although the DNA in a human cell is sufficient to code for around a million proteins, there

are probably only lo4 structural genes. There is a 300-fold excess of DNA over that required to code for cellular protein constituents. The genes are arranged with segments of this non-coding DNA interspersed among them. Gene mapping examines both the coding DNA and the non-coding sequences surrounding it.

Gene mapping relies on the action of bacterial restriction enzymes. These enzymes recognize and cleave DNA at short, specific sequences, four to six base pairs in length, known as restriction sites. The enzymes are named according to the species and strain of bacterium from which they are isolated, thus the third restriction enzyme to be isolated from Huemophilus

Correspondence: Dr I. J. Jackson, Biochemistry Department, St Mary’s Hospital Medical School, Paddington, London, W2 1PG.

0007-1048/80/1100341$02.00 0 1980 Blackwell Scientific Publications

341

342 Annotation

injluenzae, serotype d, is known as Hind 111. A battery of restriction enzymes is available from different bacterial species, each cutting at a different site. Treatment of total cellular DNA with a restriction enzyme generates a distinct set of lo5 to 10‘ fragments per genome, ranging in size from a few hundreds to tens of thousands of base pairs. (Sizes are usually referred to in kilobases (kb), where one kilobase equals loo0 base pairs.) Electrophoresis of these restriction fragments in an agarose gel separates them according to size, the smallest fragments migrating fastest. The DNA can then be transferred, by blotting, onto a sheet of nitrocellulose, while retaining the size profile of the fragments (Southern, 1975).

The position of globin-gene bearing fragments within this smear of DNA can be deter- mined by using DNA hybridization. This involves using a radioactive DNA copy of the globin gene (usually a recombinant) to base pair to the fragments. This complementary DNA will only recognize globin gene fragments, which can then be visualized by autorndiography. Hence the size of a globin-gene bearing fragment can be determined.

By using several different restriction enzymes, singly or in pairs, the relative positions of their cleavage sites around the gene can be determined. The location of the gene within this ‘map’ can be found by using restriction enzymes known to cut within the coding sequence of the gene. We are thus able to describe how the DNA around the globin genes is arranged.

Normal H u m a n Globin Genes The globin genes are clustered on two chromosomes. O n chromosome 11 are the 6-, p-, y-

and E-globin genes whilst on chromosome 16 are the two cr-globin and probably the c-globin genes (Deisseroth et al, 1977, 1978).

An interesting feature to emerge rapidly from the mapping was the direct confirmation of the very close physical linkage of related globin genes. Several lines of evidence showed this, primarily enzymes which give only a single restriction fragment containing two or more genes. The maps so far elucidated are shown in Fig 1.

The duplicated a-globin genes are about 3-5 kb apart (Surrey et a f , 1978; Orkin, 1978), the two y-globin genes, ‘7 and (;?, are separated by about 3.5 kb (Little et al, 1979) and the 6-gene is approximately 5.5 kb from the related gene coding for &-globin, the minor adult b-like globin chain (Flavell et al , 1978). Linkage between the y-locus and the S/p locus has been determined, and these two loci are separated by about 14 kb (Tuan et al, 1979; Bernards et al , 1979a).

a a

Eg H 3’ 5’ ( a 1

! 3.5 kb ! GY A Y 6 P

X E E P E E P X 3’ 5’ Eg E E B E Bg ( b ) A L l i L 1 A L I I

13.5kb I I I , 13.5kb I 5.5kb I I FIG 1. Restriction enzyme maps around the globin genes. The orientations shown are with respect to the mRNAs transcribed from the globin genes. (a) The a-globit] locus. (b) The non-a-globin locus. The embryonic (c) globin genes are known to be located to the 5’-side of the Gy globin gene. B=BamH I, Bg=BglII, E=EcoRI, H=Hind 111, P=Pst I, X=Xba I.

A n n o tation 343

Whether the fact that the genes expressed at the same time (both r-genes, both y-genes or the 6- and P-genes) are closer together in the genome than those differentially expressed (*y/"y or 6/p) has any significance is not known. These mapping studies also confirmed the non-r gene order as S'-';yAy6P-3', as previously inferred from the structure of the fusion haemoglobins, Hb Lepore and Hb Kenya (Baglioni, 1962; Huisman et a l , 1972).

Another fascinating aspect to emerge from gene mapping is the demonstration of interven- ing sequences (IVS) which interrupt the coding region of globin and many other genes. Their presence can be deduced as follows. In the DNA copies of P- and y-globin mRNAs there are sites for the restriction enzymes EcoRI and BamHI, which can be shown to be 67 base pairs apart. O n mapping the genes in cellular DNA however, the sites are over 1000 base pairs apart, indicating that the mRNA present in the cytoplasm is not coded by a continuous piece of DNA, but is made up of a transcript which has been spliced together from separated regions of IINA. They are shown as gaps on Fig 1. These IVS have been the subject of much recent research and the analysis of isolated genes from several mammals, including man, reveals two IVS in the same relative position in each globin gene, CL, p, 6, (Van den Berg et al, 1978; Tilghman rt al , 1978; Smithies et a l , 1978; Lawn et al, 1978; Leder et a l , 1979).

Allelic Variations Any DNA sequence is subject to mutation. If the mutation occurs in a coding region the

result may be a mutant protein, as in the many haemoglobin variants. The position of restriction enzyme sites in the IINA of the a-, 8- and y-globin genes, and the amino acids encoded by that DNA, are known. These variants reveal individuals who would be expected to have a particular restriction site missing. Thus, EcoRI cleaves at the sequence GAATTC, which codes in the P-globin gene for amino acids 121 and 122 (lys, phc). This produces two p-specific fragments of 5.5 and 3.5 kb in length. Patient\ with H b 0-Arabia have a mutation changing p12, lys-glu, due to codon GAA having changed to AAA, which removes the EcoRI site. When the Hb-0-Arabia patients' DNA was cleaved with EcoRI only one P-globin gene fragment of 9.0 kb is seen, equal to the sum of the two normal fragments (Flavell et al, 1978). The method allows us to actually see in DNA term7 what had previously been inferred to have occurred from protein data.

gene of HbF Hull (121 glu-lys) and the absent Hind111 site of Hb J Brussais (a-90 Lys-Asn) (Little et al, in press). The single amino acid change which distinguishes ' y from "7 (136 ala-gly) causes a PstI site in the * y gene to be absent in the ' y gene (Poon P t a l , 1978). This allowed Little et al(1979) to distinguish between Ay and "7 in their map.

DNA outside the regions coding for proteins can also mutate. Indeed some of this DNA may not have any evolutionary constraints to select and maintain the exact DNA sequence. Gene mapping also examines a limited number of sequences outside the genes (those included in restriction sites) and examination of many individuals hasahown that these sequences do indeed vary from person to person (Jeffreys, 1979). A number of these polymorphisms are located in the IVS of the globin genes which indicates that the sequences of the IVS are not absolutely fixed by evolution. Other polymorphic differences have been found outside the genes, notably by using the enzyme HpaI.

The same principle applies in revealing the absence of an EcoRI site in the

344 Annotation

a- Thalassamia There are four a-globin genes in normal individuals. Lack of function of any number of

these genes results in a-thalassaemia of increasing severity. Three functional genes is a chnically silent carrier trait, whilst two genes functioning give rise to a mild thalassaemic trait. HbH disease is due to the loss of function of three out of four genes. If all four genes are non-functional the invariably perinatally fatal hydrops foetalis occurs.

Using DNA hybridization to only ‘count’ the a-genes the four forms of a-thalassaemia have been classically defined by the actual absence of a number of genes coding for z-globin. However, mapping of the a-locur has not always borne this out. EcoRI produces a fragment 22.5 kb long in normal individuals, which contains both a-genes per chromosome. Some thalassaemic genes have an EcoRI fragment of 20 kb, suggesting a deletion of 2-3 kb. It can be shown that this fragment contains only one r-globin gene, confirming the clarnical deletion model. O n the other hand, other thalassaemic patients show a ‘normal’ 22.5 kb EcoRI fragment which contains two a-globin genes, although it is genetically a thalassaemic locus. Thus the gene sequences are present but in some way are inactive. Similarly a smaller (2.6 kb) EcoRI fragment containing one non-functional a-globin has been described (Orkin ef al, 1979a).

f?- Thalassaemia Gene mapping has shown that 8“ or p+ thalassaemia patients with absent or greatly reduced

8-chain synthesis and normal HbA2 levels almost invariably have the globin gene present as normal (Mears et al, 1978a; Flavell et al, 1979). The disease then must be due, in the same way as for non-functional a-globin genes, to some defect in expression; either transcription, process- ing and transport of the primary transcript or translation of the mRNA.

Three patients are known, all of Indian ancestry, in which a partial deletion has been found in the B-globin gene (Flavell et al , 1979; Orkin et al , 1979b). This deletion removes 600 base pairs of DNA from the &locus, including the 3’ half of the gene plus probably a segment of both the major IVS and the DNA flanking the 3‘ end of the gene (Fig 2). Interestingly,

GY 4 Y S P Bg B E B E X B E P B E P X

HbF synthesis

L I Hb Kenya +++++

+ G y 4 y H P F H +++++

I J Spa Thalossaernio +++ - Hb Lepore +

U ’ Deletion-type‘ ? tholossoemio

FIG 2. Deletions around the non-x-globin locus which show a phenotypic effect. Details are in the text. The levels of HbF synthesis are given on an arbitrary scale by means of illustration.

Annotation 345

although all three patients clinically exhibit homozygous /3-thalassaemia, the deletion is present on only one chromowme, the other having a non-deletion pattern.

A gene with this deletion is non-functional as it cannot produce an active mRNA, although it could, conceivably, direct the synthesis of a shortened RNA. The reticulocytes of at least one of these patients contain no 1-globin mRNA (P. Tolstoshev quoted in Flavell et al, 1979). However, whether the absence of globin RNA is due to this deletion having some effect on normal expression pathways, or increasing the degradation of transcripts is not known. It is possible that the deletion appeared in a gene which was already non-functional (i.e. thalassae- mic) and in fact it may not be the caux of the absence of transcription products.

6Po T/ialassaemia 61’ thalassaemia is characterized by a lack of both HbA and HbA:. The apparent cause of the

defect is straight-forward, mapping studies showing a deletion of the whole of the 1 and part or all of the 6-globin genes (Mears et al, 107th; Ottolenghi et al, 1979; Fritsch et al, 1979). The deletion in one patient has been fully mapped (Bernards et at, 1979b) and is shown in Fig 2.

Fusion Huetrioglobins Protein sequencing data showed that Hb Lepore was made up of N-terminal 6-globin

peptides fused to C-terminal 1-globin peptides (Baglioni, 1962). Similarly Hb Kenya has a y-globin-like N terminus and a C terminus comprising 1-globin peptides (Huisman et al, 1972). Hb Lepore carriers exhibit thalassaemia, presumably because the gene is under 6-gene control. Hb Kenya shows a high level of HbF synthesis. As expected, the map around the Hb Lcpore locus shows a gene fusion of the 5’ half of the S-gene to the 3’ half of the 8-gene, with a deletion of the intergenic DNA (Flavell et al, 1978; Mears et a l , 1978b) (Fig 2). A larger deletion including the 6-gene and all the 6 / p and linking DNA would be expected for Hb Kenya but no mapping data is as yet available.

Hereditary Persistence $Fetal Haemoglobin Hereditary persistence of fetal haemoglobin (HPFH) is a complex, heterogenous condition.

It is clinically silent. Although the patients have a reduced level or absence of adult haemo- globin, it is compensated by a raised level of fetal haemoglobin. A recent review of these conditions has been published (Wood et al, 1979).

Mapping has been performed on the y-locus of *f’y-HPFH from two Negro patients. These studies demonstrate a deletion of DNA which includes both the /3- and 6-globin genes and part of the DNA between the 6 and y-globin genes (Fig 2) (Fritsch et al, 1979). Accurate mapping of these genes required the use of DNA probes prepared not from mRNA copies but from a cloned recombinant molecule containing the DNA between the 6 and y genes of a normal individual.

HbF Synthesis At about the time of birth the synthesis of fetal haemoglobin (HbF), which contains

y-globin, is turned off and the /3-chains present in adult haemoglobin (HbA) begin to be

346 Annotation

synthesized. The nature of this ‘switch’ is of great interest as it represents a developmental change which is both simple in terms of product and relatively amenable to study.

The ‘switch’ is not absolute; normal individuals express low levels of HbF, representing - 1% non a-globin synthesis as y-globin. Patients with some of the haemoglobinopathies described above express higher levels, suggesting that their y-globin genes are less well repressed. Individuals homozygous for the Negro form of HPFH have no HbA or HbA2 synthesis but have apparently fully derepressed y-globin genes, as they are clinically normal apart from slight microcytosis. The level of HbF in hcterozygotes for HPFH is usually less than 30%. Hb Kenya patients appear to have similar levels of HbF. No homozygotes for Hb Kenya are known, but heterozygotes’ red cells contain 7-23% Hb Kenya (representing A? synthesis) plus 4 9 % “7-globin. Thus total HbF plus Hb Kenya levels from these individuals are about the same as heterozygous HPFH individuals.

These two conditions can be considered as the extremes of derepressed y-globin synthesis. Homozygotes with 6@” thalassaemia, like HPFH homozygotes, have no HbA or HbA2, but, unlike HPFH patients, have a thalassaemic condition. Thus their y-globin genes are not sufficiently derepressed to compensate for the absence of 6- and @-globin synthesis-although the condition is milder than @“-thalassaemia. Parents of homozygous 8”-thalassaemics have HbF in the region of 5-2O%. Heterozygotes for Hb Lepore have a significantly higher level of HbF than @” thalassaemia suggesting a very slight derepression of the y-locus (Marinucci et al, 1979).

When the maps around the globin genes of persons with these conditions are analysed a pattern emerges (Fig 2). All the conditions involve deletions, unlike the 8-thalassaemias where, in general, there is no deletion and there is no enhanced y-globin synthesis. Although no map is available for the Hb Kenya gene, Huisman et al(1972) proposed from the protein sequence that the fiision eliminated the DNA between the A*/ and @-genes. They suggested that the removal of DNA from this region caused the derepression of the y-locus, and that the control region (the ‘7-@ switch’) was located in this region. The mapping of the Negro-type HPFH genes supports this hypothesis, the deletion extends from the 3’ side of the @-gene to 5 kb to the 5’ side of the &gene, suggesting that the control element is on this DNA. The deletions which cause S@’ thalassaemia and Hb Lepore might narrow down the DNA segment to that between 6 and @ genes. However, it must be noted that there is a gradation of derepression, and that Hb Lepore and 6@” thalassaemia do not show fully derepressed y-globin.

There may be several explanations. There may be several switches, the deletion of only some of which cause only partial derepression. The deletion of the 8-@ intergenic DNA might cause derepression of the y-locus, but secondary mutations elsewhere moderate their activity. Alternatively, the increasing degree of malfunction of the switch may be due to the increasing size of deletion of DNA per se, rather than specific sequences. The gross morphology of the DNA or the chromatin could be disrupted such that the normal control mechanisms are increasingly faulty.

Antenatal Diagnosis In certain parts of the world the thalassaemias and sickle-celldisease constitute a major public

health problem. Even where rare, as in Britain, the suffering, both physical and social, by the individual patient is considerable.

Annotation 347

One solution is to identify marriages at risk and to perform antenatal diagnosis at sufficiently early times to be able to offer an abortion, if an affected fetus is detected. Antenatal diagnosis of haemoglobinopathies is a highly skilled operation, usually requiring sampling of fetal blood. O n the other hand, gene mapping, or identification of key globin-bearing restriction frag- ments, requires only a sample of fetal DNA, easily isolated from the fetal fibroblasts obtained from an amniocentesis sample. The preceding sections show that homozygotes of several haemoglobinopathies can readily be identified using Southern blotting. This has already been achieved for the rare condition of S/3' thalassaemia (Orkin et a1,1978) and is obviously possible for Hb Lepore and for those thalassaemias characterized by a deletion around the a or P genes.

Important diagnostic potential has conic from the work of Kan & Dozy (1978a), who showed that 87% of P-globin genes with the sickle niutation (HbS) were carried on a 13 kb HpaI fragment, and the normal P-globin gene is rarely found on a HpaI fragment of this size, a iiormal HpaI fragment being 7.6 kb. This fortuitous linkage is presumed to have arisen because an individual in whom the HbS gene first arose carried this HpaI polymorphism, and the close physical linkage between the site and the fi-gene is only rarely disrupted by genetic recombina- tion. In any case, this observation has proved useful for carrying out antenatal diagnosis of sickle-cell disease.

After a parental study to indicate that the HbS gene is indeed carried on a 13 kb fragment, it is possible to test if a fetus is normal, heterozygous or homozygous for HbS. Kan & Dozy (1978b) have performed such an antenatal diagnosis in conjuqtion with fetal blood sampling, and showed it to be accurate. It may also be possible to diagnose a large number of non-deletion /3-thalassaemia homozygotes using a similar method, identifying the linkage of affected genes to distinctive restriction fragments by family studies.

Should these methods prove practical in the seriously affected regions, a contribution to solving this major health probleni would have been made.

Perspectives Gene mapping provides an analytical tool, easily applied to small amounts of DNA. The

knowledge acquired of the gross morphology of normal and defective globin genes is now being improved in definition by the cloning of these genes and the determination of the DNA sequences in and around them. However, these are still descriptive methods. Investigation of the mechanism of expression of the globin genes and the nature of the developmental switches involved will require the use of in vitro systems in which an isolated gene can be expressed. Gene mapping, meanwhile, will continue to be important as a means of readily screening large numbers of patients and providing a description of their gene structure. The use of these techniques in antenatal diagnosis will also increase and develop to become a safer and more reliable alternative to fetal blood sampling.

Note added in prooj Antenatal diagnosis of non-deletion type /3-thalassaemia has recently been performed using amniotic fluid fibroblast DNA, by showing the linkage of the affected Bglobin genes to polymorphisms in the y-globin gene locus (Little et al , Nature, in press).

Biochemistry Department, St Mary's Hospital Medical School, London

IAN J. JACKSON ROBERT WILLIAMSON

348 Annotation

REFERENCES

BAGLIONI, C. (1%2) The fusion oftwo peptide chains in Hemoglobin Lepore and its interpretation as a genetic deletion. Proceedings ofthe National Academy of Sciences of the United States of America, 48, 1880-1886.

BERNARDS, R., LnTLE, P.F.R., ANNISON, G., WII.I.IAM- SON, R. & FLAVELL, R.A. (1979a) The structure of the human Gy "y 6 fl globin gene locus. Proceedingsof the National Academy ofSciences ofthe United States of America, 76, 4827-4831.

BERNARDS, R., KOWER, J.M. & FLAVELL, R.A. (1979b) Physical mapping of the globin gene deletion in 6fl"-thalassaemia. Gene, 6,265-280.

DEISSEROTH, A., NIENHUIS, A., TURNER, P., VELEY, R., ANDERSON, W.F., RUDDLE, F., LAWRENCE, J., CREACEN, R. & KUcmR LAYATI, R. (1977) Locaha- tion ofthe human a-globin gene to chromosome 16 in cell hybrids by molecular hybridisation. Cell, 12, 205-2 18.

DEISSEROTH, A., NEINHUIS, A., LAWRENCE, J., GILES, R., TURNER, P. & RUDDLE, F. (1978) Chromosomal location of the human fl-globin gene on human chromosome 11 in somatic cell hybrids. Proceedings ofthe National Academy ofsciences ofthe United States of America, 75,14561460.

FLAVELL, R.A., BERNARDS, R., KOOTER, J.M., DE BOER, E., L ~ L E , P.F.R., ANNISON, G. & WILLIAMSON, R. (1979) The structure of the fl-globin gene in fl-tha- lassaemia. Nucleic Acids Research, 6,2749-2760.

FLAVAELL, R.A., KOOTER, J.M., DE BOER, E., LIITLE, P.F.R. & WILLIAMSON, R. (1978) Analysis of the 6-fl-globin gene loci in normal and Hb-Lepore DNA; direct demonstration of gene linkage and intergene distance. Cell, 15,2541.

FRIBCH, E.F., LAWN, R.M. & MANIATIS, T. (1979) Characterisation of deletions which affect the expression of fetal globin genes in man. Nature, 279, 598-603.

HUISMAN, T.H.J., WRIGIITSTONE, R.N., WILSON,J.B.. SCHROEDER, W.A. & KENDALL, A.G. (1972) Hemoglobin Kenya, the product of a fusion of y and fl polypeptide chains. Archives of Biochemistry and Biophysics, 153, 85M53.

JEFFREYS, A.J. (1979) DNA sequence variants in the Gy-Ay-6-b globin genes in man. Cell, 18, 1-10.

KAN, Y.W. &DOZY, A.M. (1978a) Polymorphism of DNA sequences adjacent to the human 8-globin structural gene: relationship to sickle mutation. Pro- ceedings of the National Academy of Sciences of the United States of America, 75,5631-5635.

KAN, Y.W. & KOZY, A.M. (1978b) Antenatal diag- nosis of sickle-eel1 anaemia by DNA analysis of amniotic fluid cells. Lancet, ii, 91G912.

LAWN, R.M., FRITSCH, E.F., PARKER, R.C., BLAKE,G.

& MANIATIS, T. (1978) Isolation and characterisa- tion of linked j and 6 globin genes from a cloned library of human DNA. Cell, 15, 1157-1174.

NORMAN, B., SULLIVAN M. & LEDER, P. (1978) Comparison of cloned mouse a and fl globin genes: conservation of intervening sequence location and extragenic homology. Proceedings of the National Academy of Sciences of the United States of America, 75, 618745191.

L~ITLE, P.F.R., CURTIS, P., COU~ELLE, Crt., VAN DEN BERG, J., DALGLEISH, R. W.M., MALCOLM, S.,

SON, R. (1978) Isolation and partial sequence of recombinant plasmids containing a-, fl- and y-glo- bin cDNA fragments. Nuture, 273,64@643.

LIITLE, P.F.R., FLAVELL, R.A., KOOTER, J.M., ANNI- SON, G. 8; WILLIAMSON, R. (1979) Structure ofthe human foetal globin gene locus. Nature, 278,

MARRINUCCI, M., MAVILIO, F., MASSA, A,, G.413- RIANELLI, M., FONTANAROSA, P.P., SAMOGGIA, P. & TEMORI, L. (1979) Haenioglobin Lepore trait: haematological and structural studies on the Italian popuiation. British Journal of Haematology, 42,

MEARS, J.G., RAMIREZ, F., LEIBOWITZ, D., NAKAMURA, F., BLOOM, A,, KONOTEY-AHULU, F. & BANK, A. (1978a) Changes in restricted human cellular DNA fragments containing globin gene sequences in tha- lassemia and related disorders. Proceedings of the National Academy of Sciences of the United States of America, 75, 1222-1226.

MEARS, J.G., RAMIREZ, F., LEIBOWITZ, D. & BANK, A. (1978b) Organisation of human fl and 6 globin genes in cellular DNA and the presence of intra- genic inserts. Cell, 15, 15-23.

ORKIN, S.H. (1978) The duplicated human a-globin genes lie close together in cellular DNA. Proceedings of the National Academy ofSciences ofthe United States of America, 75,5950-5954.

ORKIN, S.H., ALTER, B.P., ALTAY, C., MAHONEY, M.J., LAZARUS, H., HOBBINS, J.C. & NATHAN, D.G. (1978) Application of restriction endonuclease mapping to the analysis and prenatal diagnosis of thalassemia caused by globin gene deletions. N e w England Joural ofMedicine, 299, 1 6 1 7 2 .

ORKIN, S.H., OLD, J., LAZARUS, H., ALTAY, C., GUR- GEY, A., WEATHERALL, D.J. & NATHAN, D.G. (1979a) The molecular basis of a-thalassaemia: fre- quent occurrence of dysfunctional a-loci among non-Asians with HbH disease. Cell, 17,33-42.

ORKIN, S.H., OLD, J., WEATHERALL, D.J. & NATHAN, D.G. (1979b) Partial deletion of b-globin gene

LEDER, A., MILLER, H.I., HAMER, lI.H., SEIDMAN,J.G.,

COURTNEY, M.G., WESTAWAY, D.A. & WILLIAM-

277-23 1.

557-566.

Annotation 349

DNA in certain patients with fl-thalassemia. Pro- ceedings of the National Academy of Sciences of the United States of America, 76, 240-2404.

A.M., POI LI, E., A ~ Q U A Y E , C.T.A., OLDHAM, J.H. & MASERA, G. (1979) Globin gene deletion in HPFH, G/3” thalassaemia and Hb Lepore disease. Nature, 278, 654-657.

POON, R., KAN, Y.W. & BOYER, H.W. (1978) Sequence of the 3’ noncoding and adjacent coding regions of y-globin mRNA. Nucleic Acids Research,

OlTOLENGIII, s., GIGI.ION1, B., COMI, P., GIANNI,

5,4625-4630. S M ~ H I E S , O.M., BLECIIL, A.E., DENNISTON-TIIOMP-

SON, K., NEWELL, N., RICHARDS, J.E., SLIGHTON, J.L., TUCKER, P.W. & BLATTNER, F.R. (1978) Clon- ing human fetal y-globin and mousc 1-type globin DNA; characterisation and partial sequence. Science, 202, 12861289.

SOUTHERN, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal qf Molecular Biology, 98, 503-5 17.

E. (1978) Restriction endonuclease analysis of human globin genes in cellular DNA. Biochemical and Biophysical Research Communications, 83,

TUAN, D., BIRO, P.A., DE RIEL, J.K., LAZARUS, H. &

SURREY, s., CHAMBERS, J.S., MUNI, 1). & SCHWAWZ,

1125-1 131.

FORG~T, R.G. (1979) Restriction endonuclcase map- ping of the human y-globin gene loci. Nurleic Acids Research, 6, 251 9-2544.

TILGHMAN, S.M., TIEMIER, D.C., SEIDMAN, J.G.,

LEDER, P. (1978) Intervening sequences of DNA identified in the structural portion of the mouse fl-globin gene. Proceedings ofthe National Academy of Sciences of the United States of America, 75, 725- 729.

VAN DEN BERG, J., V A N OOYEN, A., MANTEI, N., SCHAMBOCK, A., GROSVELI), G., FLAVELI., R.A. & WEISSMAN, C. (1978) Comparison of cloned rabbit and mouse p-globin genes, showing strong evolu- tionary divergence of two homologous pairs of introns. Nature, 276, 37-44.

WILLIAMSON, R. (1976) Direct measurement of the number of globin genes. British Medical Bulletin, 32, 246-250.

WILSON, J.T., WILSON, L.B., DE RIEL, J.K., VILLA- KOMAROW, L., EFWRADIADIS, A,, FORGET, B.G. & WEISSMANN, S.M. (1978) Insertion of synthetic copies of human globin genes into bacterial plas- mids. Nucleic Acids Research, 5,563-581.

WOOD, W.G., CLEGG, J.B. & WEATHERALL, D.J. (1979) Hereditary persistence of fetal haemoglobin (HPFH) and Gfl-thalassaemia. British Journal of Haematology, 43, 509-520.

PITERIN, B.M., SULLIVAN, M., MAIZEL, J.V. &