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 HPRTprotein..Transfection of HPRTdeficient mouse IA9 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.Current methods of molecular biology, including the techniques of recombinant -DNA construction and cloning, rapid nucleotide sequence analysis, the design and construction of transducing vectors, and techniques oftransfection ofeukaryotic cells with foreign genes, have made it possible to clone and characterize a large number of eukaryotic genes. One gene of particular interest, not only for basic studies of eukaryotic gene regulation but also for understanding of several important human genetic diseases, is the gene encoding the enzyme hypoxanthine phosphoribosyltransferase (HPRT) (1). This enzyme catalyzes vital steps in the reutilization pathway for purine biosynthesis, and its deficiency leads to forms ofgouty arthritis and to the devastating Lesch-Nyhan disease (2,3). The HPRT locus is known to be X linked in the human and other mammalian genomes, and the availability of a cloned HPRT gene would facilitate studies ofthe organization ofa particularly interesting region of the human X chromosome and of the mechanisms of inactivation of specific and well-mapped regions of the X chromosome. Recently, we succeeded in isolating a human genomic clone containing a portion ofthe HPRT (4) by a combination of gene transfection into enzyme-deficient mouse cells and localization of human sequences by hybridization with probes of middle repetitive human (Alu) sequence, as described (4). A 15-kilobase (kb) EcoRI/BamilI fragment derived from p6B2aE2 and free ofrepetitive human sequences ,has been subcloned into the BamHI and EcoRI-sites ofpBR322 and shown to hybridize to a discrete human cytoplasmic poly(A)+RNA approximately 1.5 kb long, presumably. representing the mRNA for HPRT (4). DNA from this subcloned plasmid, called pBR1.5, was prepared by detergent lysis (5) of chloramphenicol-treated (6) transformed SF8 Escherichia coli (7) followed. by cesium chloride/ethidium bromide ultracentrifugation (8). This DNA was cleaved with EcoRI/BamrHI and the cloned insert was isolated by trough electroelution onto a dialysis membrane (9) The colonies on the replica plates were transferred to Whatman 541 paper, amplified on chloramphenicol plates overnight at 37QC by the method ofGergen et at (13), denatured and fixed to the filters, and screened for HPRT cDNA sequences by hybridization with the gel-purified insert from pBR1.5 containing the HPRT fragment free ofrepeat sequences. The filter replicas of the...

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.

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.

No abstract