Changes in restricted human cellular DNA fragments containing globin gene sequences in thalassemias and related disorders

702

178

Restriction endonuclease mapping of the human globin genes revealed a genetic variation in a Hpa I recognition site about 5000 nucleotides from the 3' end of the P-glo in structural gene. Instead of a normal 7.6-kilobase (kb) fragment which contains the globin structural gene, 7.0-kb and 13.0-kb variants were detecte. Both variants were found in eople of African origin and were not detected in Asians or Caucasians. The 13.0-kb variant is frequently associated with the sickle hemoglobin mutation and may be useful for the prediction of the sickle cell gene in prenatal diagnosis. Polymorphism in a restriction enzyme site could be considered as a new class of genetic marker and may offer a new approach to linkage analysis and anthropological studies. Polymorphism in structural genes in the human is a well-known phenomenon which has been utilized for many types of genetic analyses. Usually, the normal and variant genes or gene products are identified by structural studies, functional assays, or immunological methods. Recently, Southern (1) introduced a new method for analysis of DNA consisting of restriction endonuclease digestion of the genomic DNA, electrophoretic separation of the DNA fragments, and identification of the structural genes in these fragments by hybridization analysis. This method has been used to study the organization of many eukaryotic genomes, including the human globin genes (2, 3).During study of the human globin genes by the Southern method, we detected genetic variations of a restriction endonuclease site close to the human fl-globin structural gene. The variations were found in people of African origin, and one variant was commonly associated with the fl-globin structural mutation, hemoglobin S. This type of polymorphism may be useful for linkage analysis, prenatal diagnosis, or anthropological studies.MATERIALS AND METHODS Preparation of Cellular DNA. DNA was prepared from leukocytes, placentas, and cultured fibroblasts according to described methods (4,5), except that 100,ug of proteinase K per ml was used to digest the cells at 550C, and the phenol-extracted DNA was extensively dialyzed against 1 mM Tris-HCI, pH 7. Electrophoresis and Identification of Globin Genes.Samples (10 ;g) of digested DNAs were applied to 6-mm-thick horizontal 0.8% agarose (SeaKem) gels in a buffer (pH 8.05) containing 0.04 M Tris-acetate, 0.02 M Na acetate, 0.018 M NaCl, and 0.02 M EDTA and were electrophoresed at 50 V for 14 hr. The gels were stained for 30 min in ethidium bromide (10 Mg/ml in H20) and photographed under ultraviolet light.The DNAs were transferred to nitrocellulose filters with 0.90 M NaCl/0.09 M Na citrate for 40 hr (1). The filters were dried in a vacuum oven at 80°C for 2 hr and presoaked in 2-4 ml of 50% (vol/vol) formamide/0.45 M NaCI/0.045 M Na citrate containing 200 Mg of yeast tRNA and 200 ,ug of denatured salmon sperm DNA per ml and 1% (vol/vol) Denhardt's solution (9). The filters were wrapped in adhesive polyethylene (Saran-Wrap) and incubated at 41°C for 16 hr.[32P]cDN...

“…Thus, we could order the 5.5-, 3.7-, 2.1-, and 1.6-kb EcoRI fragments as 5' (3, 3' (3, 5' 6, and 3' 6 fragments, respectively. This order is in agreement with that of Mears et al (2).…”

Section: Resultssupporting

confidence: 83%

“…Four fragments, 5.5, 3.7, 2.1, and 1.6 kb in length, were present in normal and hydrops DNA and absent in HPFH DNA, in which the #(-and 6-globin structural genes are deleted. These four fragments were derived from the (3-and 6-globin structural genes, both of which contain an EcoRI site in the coding sequences (2,12).…”

Section: Resultsmentioning

confidence: 99%

Polymorphism of DNA sequence adjacent to human beta-globin structural gene: relationship to sickle mutation.

Kan¹,

Dozy²

1978

702

178

“…The hybridization signal was amplified as described by Woo et al (23). Positive plaques were rescreened and small-scale preparations of DNA were made (24 (26); this gives the normal DNA band pattern, whereas the aj30 allele is associated with the (at least) partial deletion of fl and a genes (27), producing the extra 3.4-kb EcoRI band (28). Previous studies on genomic DNA (5, 6) and cloned human globin genes (3) allowed us to assign each of the bands shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

Molecular cloning of human epsilon-globin gene.

Proudfoot¹,

Baralle²

1979

Human a-like globin genes were investigated by use of rabbit P-globin cDNA plasmid as a cross-species hybridization probe. Normal and 2°/SB thalassemic DNA were compared by filter hybridization procedures. It roved possible to demonstrate that the rabbit probe detected ad A7, 6, a, a0, and 6PB human globin genes as well as an additional unidentified a-like globin gene. By use of an agarose gel elution procedure, fractions of HindIII-digested DNA enriched for a-like globin genes were purified. One of these fractions, 8.0 kilobases in size, was cloned into X788, and EK2 X HindIII vector. A positive clone was obtained and characterized by restriction mapping and sequence analysis. The sequence data obtained predicted an amino acid sequence that exactly matches a part of human &globin. The human non--globin locus is now nearly complete. 6, A, and y human globin genes have already been cloned and analyzed. We describe here the cloning of the remaining non-x-globin gene, e.The globin gene system of humans provides an excellent model system for the study of gene expression in eukaryotes, both during and after development. In the yolk sac of early embryos two genes, E and A, produce embryonic hemoglobin ({2e2). Later in the fetal liver there is a switch to the expression of y and a genes, producing fetal hemoglobin (a2Y'2). Finally, at birth there is a second switch to the bone marrow synthesis of adult hemoglobins a2#2 and a262 (ratio 40:1) by the (3, 6, and a genes (1). The genetic defects that cause thalassemia are associated with this gene system and could prove very useful in establishing how these genes are regulated during development and in adult life (2).Great advances have been made in the structural analysis of this gene system. The 13, 3, and a part of the y genes (3, 4) have been cloned from genomic DNA. Their linkage and restriction site maps have been established by analysis of these clones and also by direct analysis of genomic DNA (5, 6) by use of human globin cDNA plasmids as probes (7,8). Similarly, the two linked a-globin genes have been mapped by genomic DNA analysis (9). However, no information has been obtained on the genes coding for the embryonic hemoglobin.We describe in this paper the molecular cloning of an 8-kilobase (kb) human DNA fragment containing the -globin gene.This clone was isolated by use of a rabbit /3-globin cDNA plasmid (10) as a crosshybridization probe. MATERIALS AND METHODSAnalysis of Genomic and Cloned DNAs by Restriction, Filter Hybridization, and Sequence Determination. Genomic (both normal and 10/3130 thalassemic) and cloned DNAs were digested with various restriction enzymes-EcoRI, HindIII, BamHI, and Pst I (Boehringer, Mannheim, W. Germany); Xba I and Bgl II (BioLabs)-under standard conditions. Digests were fractionated on 0.8-1.5% agarose gels, with a HindIII digest of wild-type X DNA (11) and a Hae III digest of PBR322 (12) as size markers. Gels for filter hybridization were denatured in alkali (0.2 M NaOH/0.6 M NaCl, 1 hr, 200C), neutralized (1.0 M Tris-HCI, ...

“…2), the extent of hybridization to DNA-paper is usually 2-3 times greater than to DNA-nitrocellulose after the stringent washes (0.1 concentrated NaCI/Cit/0.1% sodium dodecyl sulfate at 500C for [1][2] (7) observed that anionic dextran polymers accelerate reannealing of DNA in solution. Although such compounds should also accelerate hybridization of probes to immobilized DNA, the net effect of using such polymers in two-phase hybridizations was not easy to predict because self-annealing of the probe and hybridization to the DNA-paper could have been accelerated to different extents and because the backgrounds might have increased.…”

Section: Kb (Lanes B-d)mentioning

confidence: 99%

“…Southern's procedure (1) for transferring restriction fragments from agarose gels to nitrocellulose has been an essential part of recent advances in analyzing and purifying many DNA fragments and has also been used more recently to identify mutant globin genes in individuals with thalessemia (2) or sickle cell trait (3). However, several aspects of this method have not yet been optimized: (i) Large restriction fragments are not transferred from agarose gels efficiently, and fragments smaller than 0.3-0.5 kilobase (kb) do not bind well to nitrocellulose.…”

mentioning

confidence: 99%

Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate.

Wahl¹,

Stern²,

Stark³

1979

2,897

865

We describe a technique for transferring electrophoretically separated bands of double-stranded DNA from agarose gels to diazobenzyloxymethyl-paper. Controlled cleavage of the DNA in situ by sequential treatment with dilute acid, which causes partial depurination, and dilute alkali, which causes cleavage and separation of the strands, allows the DNA to leave the gel rapidly and completely, with an efficiency independent of its size. Covalent attachment of DNA to paper prevents losses during subsequent hybridization and washing steps and allows a single paper to be reused many times. Ten percent dextran sulfate, originally found to accelerate DNA hybridization in solution by about 10-fold [. G. Wetmur (1975) Biopolymers 14, [2517][2518][2519][2520][2521][2522][2523][2524], accelerates the rate of hybridization of randomly cleaved double-stranded DNA probes to immobilized nucleic acids by as much as 100-fold, without increasing the background significantly.Southern's procedure (1) for transferring restriction fragments from agarose gels to nitrocellulose has been an essential part of recent advances in analyzing and purifying many DNA fragments and has also been used more recently to identify mutant globin genes in individuals with thalessemia (2) or sickle cell trait (3). However, several aspects of this method have not yet been optimized: (i) Large restriction fragments are not transferred from agarose gels efficiently, and fragments smaller than 0.3-0.5 kilobase (kb) do not bind well to nitrocellulose. (if) Some of the DNA bound noncovalently to nitrocellulose is removed by stringent posthybridization washes, reducing the intensity of the signal and limiting repeated uses of the transfer. (iii) The technique is relatively slow; up to 2 weeks can be required to detect fragments derived from unique genes of higher eukaryotes.One modification of Southern's technique follows from our previous work, which showed that RNA and single-stranded DNA could be linked covalently to diazobenzyloxymethyl (DBM)-cellulose and used in hybridization reactions (4), that RNA could be transferred from agarose gels to DBM-paper (5), and that small fragments of DNA could be transferred from composite agarose-acrylamide gels to DBM-paper (6). Transfer of DNA fragments of any size from agarose gels to paper has now been optimized. An important improvement follows from Wetmur's finding (7) that 10% dextran sulfate greatly accelerates hybridization reactions in solution. This anionic polymer gives an even larger acceleration in a two-phase system with double-stranded, randomly cleaved, denatured DNA as probe.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3683MATERIALS AND METHODS General Methods. Conditions for cleaving the DNA samples with restriction enzymes, for separating the fragments ,according to size by electrophoresis in agarose gels (8), and for preparing n...