Partial phenotypic correction of human Lesch-Nyhan (hypoxanthine-guanine phosphoribosyltransferase-deficient) lymphoblasts with a transmissible retroviral vector.

A human Lesch-Nyhan (hereditary, severe hypoxanthine-guanine phosphoribosyltransferase (HPR transferase) deficiency) B-lymphoblast line was infected with an amphotropic retroviral vector containing human HPR transferase cDNA under transcriptional control of viral long terminal repeat sequences. Of 17 clones isolated, 12 integration groups were defined by analysis of restriction enzyme digests of their genomic DNA with HPR transferase and viral long terminal repeat probes. These groups had HPR transferase activity restored to levels of 4 to 23% of normal values. Aberrant metabolic parameters associated with severe deficiency of HPR transferase activity, i.e. elevated rates of purine excretion, increased accumulation of hypoxanthine, elevated 5-phosphoribosyl-1-pyrophosphate contents, altered nucleoside triphosphate pools, resistance to toxic effects of 6-thioguanine, were partially to nearly completely corrected; the degree of correction generally corresponded to the degree of restoration of HPR transferase activity. The integration of the HPR transferase gene was found to be variably stable during 9 months of culture of the virally transformed lymphoblasts under nonselective conditions. The HPR transferase gene-infected lines reverted to resistance to 20 microM 6-thioguanine, i.e. severe HPR transferase deficiency, at frequencies of 10(-6) to in excess of 10(-5) per generation. The reversions were accompanied by either a loss or rearrangement of the integrated HPR transferase sequences or by retention of the sequences in an unaltered form.

Elements in the long terminal repeat of murine retroviruses enhance stable transformation by thymidine kinase gene.

We have investigated the effects of long terminal repeats (LTRs) of murine retroviruses on the frequency of obtaining stable transfectants by the herpes virus thymidine kinase (TK) gene. The results indicate that addition of LTRs enhances the number of TK+ transformants by 10-20 fold. A 5-12 fold enhancement was also observed when chromosomal DNA from either human or hamster cells was mixed with a plasmid containing LTR sequences and transfected onto LTR- cells. The LTR sequences involved in the enhancement were localized in the region which contains tandem repeats. All other regions of the LTR did not show any enhancement of stable TK+ transfectants. The location or the orientation of the enhancer sequences with respect to the TK gene did not exert any influence on the frequency of transformation. The enhancement effect does not appear to be linked to either increased numbers of chromosomal integrations or elevated levels of transcription of the TK gene.

Isolation and characterization of a full-length expressible cDNA for human hypoxanthine phosphoribosyl transferase.

We have cloned a full-length 1.6-kilobase cDNA of a human mRNA coding for hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) into a simian virus 40-based expression vector and have determined its full nucleotide sequence. The inferred amino acid sequence agrees with a partial amino acid sequence determined for authentic human HPRT protein. Transfection of HPRT-deficient mouse LA9 cells with the purified plasmid leads to the expression of human HPRT enzyme activity in cells stably transfected and selected for enzyme activity in hypoxanthine/aminopterin/thymidine medium.

Isolation of a genomic clone partially encoding human hypoxanthine phosphoribosyltransferase.

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 Escherichia 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, pBR1.5, 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 gene fragments. This has been confirmed by using pBR1.5 as a probe to isolate an authentic and expressible human HPRT cDNA clone from a library prepared by H. Okayama and P. Berg.