Isolation of a genomic clone partially encoding human hypoxanthine phosphoribosyltransferase.

Abstract

Mouse cells deficient in the enzyme hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) have been transfected with total human DNA, and cells producing human enzyme were isolated by growth in selective medium. DNA from several such cell lines has been used to generate secondary transfectants that make human HPRT. Blots of the DNA of these secondary cells have been hybridized with total human DNA probes or with cloned human Alu sequences, and one of several common bands has been cloned in pBR322. Colonies of transformed Eacherichia coli containing human sequences were detected by their homology with human DNA, and subclones of resulting recombinant plasmids were prepared. Two subclones free of Alu sequences were found to contain human sequences that hybridized to human X chromosome DNA. One of these, pBRI.5, also hybridized to a single RNA band on gel blots of human and secondary transfectant cytoplasmic poly(A)+RNA but not to RNA from the parent mouse cell line. These results indicate that these clones represent human HPRT HPRT Assays. Cell lines were assayed for the presence of mouse or human HPRT on isoelectric focusing gels as described (9). DNA Preparation. Human placental DNA was prepared by the method of Graham (10). DNA from tissue culture cells was prepared as described by Pellicer et aL (11). Plasmid DNA was prepared from Escherichia coli by conventional methods (12).RNA Preparation. Cytoplasmic RNA was prepared according to Levis and Penman (13), and poly(A)+RNA was selected by oligo(dT)-cellulose chromatography (14).Blots. DNA gels were blotted according to Southern (15); acid pretreatment (16) was used when transfers included high molecular weight (25 kb) bands. RNA was electrophoresed in agarose gels and blotted to nitrocellulose filters according to Thomas (17). High-stringency hybridization conditions were: 0.5 M NaCl, 20 mM Hepes at pH 7.4, 0.1 mM EDTA, 30% (vol/vol) 5038The publication costs ofthis 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.

Ionizing radiation-induced mutagenesis

“…However, the cloning of the genomic and cDNA sequences for specific genes such as adenine phosphoribosyl transferase (aprt) (Lowy et al, 1980), hypoxanthine phosphorybosyl transferase (hprt) (Jolly et al, 1982;Konecki et al, 1982), dihydrofolate reductase (dhfr) (Chang et al, 1978) and thymidine kinase (tk) (Bradshaw & Deininger, 1984) has made it possible to study directly the effects of mutagens on endogenous and exogenous genes.…”

Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases.

“…A 2-,u amount of the various samples was spotted onto nitrocellulose sheets that had been soaked in 5 x SSC, pH 7.0 (1x SSC = 0.15 M NaCl-0.015 M sodium citrate) and dried. The filters containing DNA were baked in a vacuum oven at 80°C for 4 h. The blots were prehybridized for 8 h as described by Wahl et al (30) and then hybridized for 16 h at 42°C with 3 x 107 cpm of 32P-labeled total human DNA (108 to 2 x 108 cpm/4ig of DNA) which had been prepared via nick translation (20). After hybridization, the blots were washed four times for 15 min at room temperature with 2x SSC-0.1% sodium dodecyl sulfate (SDS), once in 1 x SSC-0.1% SDS for 30 min at 68°C, twice in 0.3x SSC-0.1% SDS for 30 min at 68°C, and once in 0.2x SSC for 30 min at 68°C.…”

“…The technique of DNA-mediated transfer of genes into mammalian cells has proven to be an effective means to enrich for and clone specific genes (16,18,19,35,36). Variations of the basic gene transfer procedure, including plasmid rescue, have increased the general usefulness of this technique considerably (18,19).…”

An appraisal of the application of recombinant DNA techniques to chromosome defects

“…If the gene is expressed at high levels in a particular cell, such as globin gene in erythropoietic cells, the correspondAbbreviations used: RFLP, restriction fragment length polymorphism; cML, chronic myelocytic leukaemia; BL, Burkitt's lymphoma; DMD, Duchenne muscular dystrophy; CF, cystic fibrosis. ing cDNA can be cloned by purification of the mRNA (Wilson et al, 1978). Alternatively, if a mutant cell line exists, the gene may be cloned by integrating foreign DNA into the cell and investigating the sequences that complement the mutation (Jolly et al, 1982). Whenever the protein sequence is known, a short corresponding DNA sequence can be predicted and synthesized (Woods et al, 1982).…”

“…Thus the mutant phenylalanine hydroxylase gene must be associated with the 7kb allele in the father which he has also transmltted to an unaffected sibling homozygous for the 7 kb band. Since this sibling is also homozygous for the 4kb HindIll band, the (Old & Higgs, 1983) Collagen Sykes, 1983;Prockop & Kivirikko, 1984) Human growth hormone (Moore et al, 1982) al-Antitrypsin (Kidd et al, 1983) Hypoxanthine phosphoribosyltransferase (Jolly et al, 1982;Brennand et al., 1982) Phenylalanine hydroxylase Factor IX (Choo et al, 1982) Factor VIII Glucose-6-phosphate dehydrogenase (Persico et al, 1981) Ornithine transcarbamoylase (Old et al, 1985) Antithrombin-3 (Bock et al, 1983) Low density lipoprotein receptor (Russell et al, 1983); 3-hydroxy-3-methylglutaryl-CoA reductase (Chin et al, 1982); apolipoproteins (Tolleshaug et al, 1983) Oncogenes (Hamlyn & Sikora, 1983) Immunoglobins Arnold et al, 1983) T-cell receptor (Toyonaga et al, 1984) . This is important for future genetic counselling and will help in the understanding of the factors involved in the non-disjunction resulting in the chromosomal trisomy.…”