In this report, we describe an approach to detect the presence of abnormal alleles in those genetic diseases in which frequency of occurrence of the same mutation is high (e.g., cystic fibrosis and sickle cell disease), and in others in which multiple mutations cause the disease and the sequence variation in an affected member of a given family is known (e.g., hemophilia B). Initially, from each subject, the DNA fragment containing the putative mutation site is amplified by the polymerase chain reaction. For each fragment two reaction mixtures are then prepared. Each contains the amplified fragment, a primer (18-mer or longer) whose sequence is identical to the coding sequence of the normal gene immediately flanking the 5' end of the mutation site, and either an a-32P-labeled nucleotide corresponding to the normal coding sequence at the mutation site or an a-32P-labeled nucleotide corresponding to the mutant sequence. Single nucleotide primer extensions are then carried out and analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. As predicted by the Watson-Crick base-pair rule, in the wild type only the normal base, in an affected member only the mutant base, and in carriers both the normal and the mutant base are incorporated into the primer. Thus, an essential feature of the present methodology is that the base immediately 3' to the template-bound primer is one of those altered in the mutant, since in this way an extension of the primer by a single base will give an extended molecule characteristic of either the mutant or the wild type. The method is rapid and should be useful in carrier detection and prenatal diagnosis of every genetic disease with a known sequence variation.One goal of molecular biology is to identify the mutations that cause genetic diseases and to develop strategies and related technologies to diagnose them. Toward this end, in the past decade or so many methodological advances have been made to detect the human genetic abnormalities at the DNA level. These include indirect methods such as linkage analysis by the Southern blotting technique (1) in which the inheritance of a disorder is associated with the presence of a restriction fragment length polymorphism (RFLP)-e.g., Duchenne muscular dystrophy (2). Other indirect methods include RNase A cleavage at mismatches in probe RNA-sample DNA duplexes or denaturing gradient gel electrophoresis for mismatches in probe DNA-sample DNA duplexes-e.g., 3-thalassemia (3, 4). The direct methods include detection with the restriction enzymes or with the allele-specific oligonucleotide (ASO) probes-e.g., the sickle cell mutation (5, 6).A majority of the above approaches have now been combined with the polymerase chain reaction (PCR) for diagnosis of the sequence variations (7,8). Initially, the target DNA is amplified by PCR followed by analysis of the sequence variation by ASO hybridization (e.g., the sickle cell mutation; ref. 9), restriction enzyme analysis (e.g., the sickle cell mutation and a hemophilia B mutation;...

We report here the cDNA sequence for hu-

We investigated the feasibility of using retroviruses as vectors for transferring DNA sequences into animal cells. The thymidine kinase (tk) gene of herpes simplex virus was chosen as a convenient model. The internal BamHI fragments of a DNA clone of Moloney leukemia virus (MLV) were replaced with a purified BamHI DNA segment containing the tk gene. Chimeric genomes were created carrying the tk insert in both orientations relative to the MLV sequence. Each was transfected into TK-cells along with MLV helper virus, and TK+ colonies were obtained by selection in the presence of hypoxanthine, aminopterin, and thymidine (HAT). Virus collected from TK+-transformed, MLV producer cells passed the TK+ phenotype to TK-cells. Nonproducer cells were isolated, and TK+ transducing virus was subsequently rescued from them. The chimeric virus showed single-hit kinetics in infections. Virion and cellular RNA and cellular DNA from infected cells were all shown to contain sequences which hybridized to both MLV-and tk-specific probes. The sizes of these sequences were consistent with those predicted for the chimeric virus. In all respects studied, the chimeric MLV-tk virus behaved like known replication-defective retroviruses. These experiments suggest great general applicability of retroviruses as eucaryotic vectors.Techniques which allow the transfer of DNA into higher cells are the key to many studies of the function and regulation of eucaryotic genes. A number of methods have been employed to deliver DNA into cells, such as the uptake of calcium phosphate precipitates of naked DNA (13), the transfer of metaphase chromosomes (17), the injection of DNA directly into cells (18), and the fusing of DNA-carrying liposomes with cells (4). These techniques suffer from several weaknesses, including the lack of retrievability of transferred DNAs, low efficiencies of transfer, and an uncertainty concerning the physical state of the DNA which has entered the cells.An alternate approach which overcomes many of these problems has been the use of animal viruses as vectors to mediate the transfer of DNA sequences into cells (6, 7). A segment of DNA is inserted into a viral genome in the place of certain viral sequences. As a consequence, certain virus-encoded functions are lost, and the chimeric virus is rendered replication defective. Such chimeric genomes can be rescued from cells by coinfection of the cells with a competent t Present address:

The endogenous, vertically transmitted proviral DNAs of the ecotropic murine leukemia virus in AKR embryo fibroblasts were found to be hypermethylated relative to exogenous AKR murine leukemia virus proviral DNAs acquired by infection of the same cells. The hypermethylated state of the endogenous AKR murine leukemia virus proviruses in these cells correlated with the failure to express AKR murine leukemia virus and the lack of infectivity of cellular DNA. Induction of the endogenous AKR murine leukemia virus proviruses with the methylation antagonist 5-azacytidine suggested a causal connection between DNA methylation and provirus expression. Also found to be relatively hypermethylated and noninfectious were three of six Moloney murine leukemia virus proviral DNAs in an unusual clone of infected rat cells. Recombinant DNA clones which derived from a methylated, noninfectious Moloney provirus of this cell line were found to be highly active upon transfection, suggesting that a potentially active proviral genome can be rendered inactive by cellular DNA methylation. In contrast, in vitro methylation with the bacterial methylases MHpaII and MHhaI only slightly reduced the infectivity of the biologically active cloned proviral DNA. Recombinant DNA clones which derived from a second Moloney provirus of this cell line were noninfectious. An in vitro recombination method was utilized in mapping studies to show that this lack of infectivity was governed by mechanisms other than methylation.

We recently described a murine model for mucopolysaccharidosis VII in mice that have an inherited deficiency of 13-glucuronidase (P-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31). Affected mice, of genotype gus""s/gus"'s, present clinical manifestations similar to those of humans with mucopolysaccharidosis VII (Sly syndrome) and are shown here to have secondary elevations of other lysosomal enzymes. The mucopolysaccharidosis VII phenotype in both species includes dwarfism, skeletal deformities, and premature death. Lysosome storage is visualized within enlarged vesicles and correlates biochemically with accumulation of undegraded and partially degraded glycosaminoglycans. In this report we describe the consequences of introducing the human (3-glucuronidase gene, GUSB, into gushPs/gus 'PS mice that produce virtually no murine P-glucuronidase. Transgenic mice homozygous for the mucopolysaccharidosis VII mutation expressed high levels of human P-glucuronidase activity in all tissues examined and were phenotypically normal. (1,4,6,7). The MPSVII mice show excessive storage of undegraded glycosaminoglycans within lysosomes, and their leukocytes exhibit the characteristic inclusions described in humans with MPSVII (4).We recently isolated the human gene for P-glucuronidase from a genomic cosmid library (8); this provided us with the opportunity to study the expression of the human gene in transgenic mice homozygous for the gusmPs mutation. METHODSConstruction of Transgenic AMice. The human 3-glucuronidase cosmid clone, pHGUS, was digested with EcoRV to generate a 28-kilobase (kb) fragment containing the entire structural gene with 1.6 kb of 5'-flanking sequence andc3.8 kb of 3'-flanking sequence. This EcoRV fragment was purified by sedimentation in a 5-20% (wt/wt) sucrose gradient. The 38-ml gradient was centrifuged at 26,000 rpm in a Beckman SW 28 rotor for 20 hr at 40C. After precipitation in ethanol, the DNA fragment was resuspended in 10 mM Tris-HCl, pH 7.5/0.1 mM EDTA at a final concentration of 20 jig/ml. Male pronuclei of both F1 and F2 zygotes were microinjected as described (9). The F1 zygotes were derived from C57BL/6J

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The Clara cell is believed to be the progenitor of the peripheral airway epithelium, and it produces the surfactant proteins SP-A and SP-B, in addition to the 10-kDa Clara cell secretory protein (CCSP or CC10). To date, attempts to develop Clara cell lines have been unsuccessful. Most such attempts have involved the in vitro insertion of a transforming viral oncogene. We have reported previously the characterization of a differentiated conditionally immortalized murine lung Type II epithelial cell line, T7, from the H-2Kb-tsA58 transgenic mouse. We have also used this mouse model to derive Clara cell lines. In this model, the need for in vitro gene insertion is circumvented by the creation of a transgene, in which the large tumor antigen of a temperature-sensitive strain (tsA58) of the simian virus 40 (SV40) is fused with the major histocompatibility complex promoter H-2Kb. The promoter is active in a wide range of tissues and is induced by interferons (IFN). From the lungs of animals harboring the hybrid construct, we isolated and characterized Clara cells. The cells contain dense secretory granules and mitochondria typical of Clara cells, and express SP-A, SP-B, SP-D, and the Clara cell secretory protein, CC10. Withdrawal of the IFN and elevation of the incubation temperature permit normal cell differentiation similar to that of Clara cells in vivo. This cell line should be very useful for the investigation of normal Clara cell function and gene expression.

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