The development of retroviral vectors that target specific cell types could have important implications for the design of gene therapy strategies. A chimeric protein containing the polypeptide hormone erythropoietin and part of the env protein of ecotropic Moloney murine leukemia virus was engineered into the virus. This murine virus became several times more infectious for murine cells bearing the erythropoietin receptor, and it also became infectious for human cells bearing the erythropoietin receptor. This type of tissue-specific targeting by means of ligand-receptor interactions may have broad applications to a variety of gene delivery systems.

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...

We have identified 12 individuals who are heterozygous for a chromosome with three a-globin genes. We determined the presence of the third a-globin locus by restriction endonuclease digestion and hybridization with a-globin cDNA probes. The three a-globin loci resided in an elongated fragment on digestion with &oRI, BamHI, and Xba I, and the third locus was present in-an additional 3.7-kilobase fragment on digestion with Hpa I, Sac I, and Bgl I. The locations of the restriction sites are compatible with a nonhomologous crossover mechanism producing the triple a loci. The frequency of the triple a loci was 0.0036 in American blacks and <0.004 in Sardinians, but was higher in Greek Cypriots (0.05). Individuals with five a-globin genes (aa/aaa) apparently showed no clinical or hematologic abnormalities. The human a-globin structural genes are duplicated (1, 2), and the two a-globin loci are located 3.7 kilobases (kb) apart (3, 4) on chromosome 16 (5). One or both of these loci may be deleted in the a thalassemia syndromes, givlig rise to the genotypes of a thalassemia-2 (-a) (6) or a thala semia-l (--), respectively (7,8). Two molecular mechanisms that produce the a thalassemia-2 genotype have been defined. A single 3' a-globin locus may remain as a result of deletion of a region of DNA about 4.0 kb long containing the entire 5' a-globin locus (4). Alternatively, the remaining a-globin locus may result from the nonhomologous crossover of the two a-globin loci (3, 9). Our studies of people who originated from Africa, Asia, and the Mediterranean area, where the a thalassemia-2 genotype occurs frequently, indicated that the nonhomologous crossover mechanism predominates (9).Unequal crossing-over of a pair of structural loci produces, in addition to one chromosome with a single locus, an opposite chromosome which bears three structural loci. Thus, hemoglobin Lepore represents the A1hgle 6-( fusion gene produced by the nonhomologous crossing-over of the 3-and f3-globin genes (10), whereas hemoglobins P Congo (11, 12) and Miyada (13) are examples of a 3l-6 fusion gene residing on a chromosome together with the 3-and f3-globin genes. Nonhomologous crossover of the two a-globin genes should therefore produce, in addition to the single a locus of the a thalassemia-2 genotype, a chromosome with three a-globin structural loci (14). In this study, we describe several individuals who carry a chromosome with triple a-globin gene loci (triple a loci). [a-32P]dTTP (360 Ci/mmol) (4). These cDNAs were prepared from two different chimeric plasmids which contained a-6lobin cDNA sequences linked to pBR322 by HindIII sites: (i) plasmid pRP 7 with a-globin cDNA sequences corresponding to the region from the second amino acid to the HindIII site (amino acids 90 and 91), and (ii) plasmid pRP 10 with sequences corresponding to the region from the HindIII site to the 3' noncoding region (i7). The cDNA inserts were excised by digestion with HindIII and separated by electrophoresis on 5% polyacrylamide gel. The nick-translated plasmi...

A B S T R A C T The a-thalassemia-2 (a-thal-2) genotype or mild a-thalassemia gene consists of a single structural a-globin gene on the chromosome that normally bears two a-globin genes. We used blot hybridization to investigate variation in the molecular organization of this genotype and to determine the distributions of these variations in the world population. Two different patterns of gene organization responsible for the a-thal-2 genotype were found: the first was the result of a 4.2-kilobase pair deletion involving the normal 5' a-globin gene (leftward deletion a-thal-2 genotype), and the second probably the result of a crossover deletion of a DNA fragment bridging the two normal a-globin genes (rightward deletion a-thal-2-genotype). The rightward deletion was found in all 9 Black subjects, all 8 Mediterranean subjects, and 4 of 13 Chinese subjects. The leftward deletion was found in four and the nondeletion a-thalassemia lesion was found in five of the nine remaining Chinese subjects. It is likely that these deletions are related to specific DNA sequences that determine DNA recombinational events.

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A human tRNALys gene was converted to an amber suppressor by site-specific mutagenesis of the anticodon. The mutated tRNALys gene directed synthesis of a tRNA that suppressed the UAG amber nonsense mutation in beta O thalassemia mRNA. Such genes may be used to detect other nonsense mutations in mammalian cells and may provide an approach to gene therapy for beta O thalassaemia due to nonsense mutations.

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