Retroviral vectors are used for the efficient transfer of foreign genes into mammalian cells. We report here the construction of murine retrovirus-based vectors carrying the full-length cDNA for human hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) and from which the enhancer sequences, the "CAAT box," and the "TATA box" in the long terminal repeats (LTRs) have been deleted.

The organization of the X-linked gene for human hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8.) has been determined by a combination of restriction endonuclease mapping, heteroduplex analysis and DNA sequence analysis of overlapping genomic clones. The entire gene is 42 kilobases in length and split into 9 exons. The sizes of the 7 internal exons and the exon-intron boundaries are identical to those of mouse HPRT gene. The 5' end of the gene lacks the prototypical 5' transcriptional regulatory sequence elements but contains extremely GC-rich sequences and five GC hexanucleotide motifs (5'-GGCGGG-3'). These structural features are very similar to those found in the mouse HPRT gene and to some of the regulatory signals common to a class of constitutively expressed "housekeeping" genes. Several transcriptional start sites have been identified by nuclease protection studies. Extensive sequence homology between the mouse and human genes is found in the 3' non-coding portion of the gene.

Differentiated primary rat hepatocyte cultures have been infected with retroviral vectors expressing human hypoxanthine/guanine phosphoribosyltransferase or the transposon TnS neomycin-resistance gene. Expression of the markers was detected only after infection of the cells during a short period of cell replication and transient dedifferentiation from days 1 to 5 of culture. Provirus integrated during that period remains fully expressed during the entire subsequent stationary period of culture up to at least 25 days. Selection with the neomycin analogue G418 of cells infected with the neomycin vector led to the appearance of cells with hepatocyte morphology in which newly synthesized albumin was detectable by immunoprecipitation, indicating successful infection of hepatocytes.Gene therapy models have recently been developed based on the concept of the repopulation of a major organ, such as mammalian bone marrow, with cells genetically modified in vitro by infection with retroviral vectors (1-4). In addition to bone marrow, another important potential target organ for therapeutically useful gene transfer is the mammalian liver. Unfortunately, the murine leukemia viruses (MuLV) used to construct most retroviral vectors do not infect newborn, adult, or regenerating liver cells in vivo (5-8), although fetal liver can be infected and continues to express retrovirally transduced genes postnatally (7,8). While the relationship between the in vivo and in vitro systems is not entirely clear, we have chosen to examine as a model system the response of primary liver cultures to retroviral vectors expressing easily detectable reporter genes.Primary hepatocyte cultures exhibit many adult liverspecific functions-including gluconeogenesis and the expression of albumin, alcohol dehydrogenase, and type I pyruvate kinase-during their early-lag (days 0-2) and latestationary (after day 11) phases of culture (9-16). During the intervening phase of logarithmic growth, these mature liverspecific functions are transiently but markedly decreased, and fetal hepatocyte functions such as a1-fetoprotein and type III pyruvate kinase are expressed (12). We now show that these adult primary rat hepatocyte cultures (9, 10) are in fact susceptible to infection with MuLV-based retroviral vectors during a period of transient dedifferentiation in vitro. MATERIALS AND METHODSRetroviral Vectors. The construction and preparation of retroviral vectors expressing the human hypoxanthine/guanine phosphoribosyltransferase (HPRT) cDNA and the transposon TnS neomycin-resistance gene have been described (2,17,18,39) (Fig. 1). Amphotropic virus was made from the PA12 helper line grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum as described by Miller et al. (19). In some instances, virus preparations were concentrated from DMEM containing 10% fetal calf serum or from minimal essential medium containing 10% fetal calf serum by filtration through an Amicon YM100 filter (20) to titers of approximately 2-5 x 106 per ml as determi...

A gene bank of DNA from plant growth-promoting Pseudomonas sp. strain B10 was constructed using the broad host-range conjugative cosmid pLAFR1. The recombinant cosmids contained insert DNA averaging 21.5 kilobase pairs in length. Nonfluorescent mutants of Pseudomonas sp. strain B10 were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, or UV light and were defective in the biosynthesis of its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin. No yellow-green, fluorescent mutants defective in the production of pseudobactin were identified. Nonfluorescent mutants were individually complemented by mating the gene bank en masse and identifying fluorescent transconjugants. Eight recombinant cosmids were sufficient to complement 154 nonfluorescent mutants. The pattern of complementation suggests that a minimum of 12 genes arranged in four gene clusters is required for the biosynthesis of pseudobactin. This minimum number of genes seems reasonable considering the structural complexity of pseudobactin.

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