Antisense RNA complementary to c-fos mRNA was produced in mouse 3T3 cells by gene transfer techniques. Transcriptional units were constructed consisting of a steroid-inducible mouse mammary tumor virus (MMTV) promoter, mouse or human 5' c-fos gene fragments in either the sense (5' to 3') or antisense (3' to 5') orientation, and splice and poly(A) signals from the human J3-globin gene. A gene that confers neomycin resistance was included in the vectors to allow isolation of stable transformants. Dexamethasone caused a marked induction of hybrid MMTV-fos-globin RNA. Induction of the hybrid transcript containing antisense c-fos RNA decreased colony formation following DNA transfer and inhibited the proliferation of cells into which the antisense transcriptional unit had been integrated. In contrast, colony formation and cell proliferation were not inhibited by induction of hybrid RNA containing c-fos RNA sequences in the sense orientation. These results indicate that the strategy of generating antisense RNA to inhibit gene expression may be useful in delineating the function of protooncogenes. The c-fos gene product appears to have a required role in normal cell division.

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Recombinant adeno-associated viruses (rAAV) containing only the inverted terminal repeats (ITR) from the wild-type virus are capable of stable integration into the host cell genome, and expression of inserted genes in cultured cells. We have now defined the ability of rAAV to introduce genes into primary hematopoietic progenitors. A vector was constructed containing the coding sequences for beta- galactosidase (beta-gal), including a nuclear localization signal, under the control of a strong viral promotor. Infectious vector particles were prepared by cotransfection of the vector plasmid with a second plasmid that contained the coding sequences for AAV proteins into adenovirus-infected human embryonic kidney cells. These vector preparations transferred and expressed the beta-gal gene in human K562 erythroleukemia and Detroit 6 cells. Positive immunoselection yielded a population of enriched CD34+ cells that were transduced with the rAAV beta-gal vector. Nuclear localized enzyme expression was documented in 60% to 70% of infected cells. Progenitor-derived colonies that developed after 2 weeks in clonogenic cultures were shown to have viral- associated DNA at an estimated copy number of 1 to 2 per cell using a semiquantitative polymerase chain reaction (PCR) method. Integration of AAV into hematopoietic progenitors was documented using wild-type virus, as its genome may integrate at a preferred site on chromosome 19. Our data suggest that rAAV will transfer and express genes in primitive hematopoietic progenitors with high frequency, and support the development of this vector system for therapeutic gene transfer.

Three groups ofrecombinant bacteriophage containing coding sequences for dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP' oxidoreductase, EC 1.5.1.3) were isolated from two human DNA clone libraries. One recombinant (AhDHFR-1) contains three exons that encode the COOH-terminal portion of human DHFR.The other two human DHFR genes (hDHFR-k, and hDHRF-fr2) lack introns. hDHFR-.f2 contains several in-phase termination codons and is only 93% homologous to the normal human DHFR coding sequences, whereas hDHFR-01 has an open reading frame and is virtually identical to the coding sequence of the normal DHFR gene. The region ofDNA sequence homology between each intronless gene and the normal DHFR gene extends 2.9 kilobases beyond the end of the coding sequences. At the 3' end of this homologous sequence, each intronless gene has an A-rich tract. The lack of introns and the presence of the 3' A-rich tract suggest that hDHFR-tpl and hDHFR-qi2 were derived from processed RNA molecules. A short DNA sequence, 60 nucleotides 5' to the ATG start codon in AhDHFR-af2, is directly repeated immediately after the 3' A-rich tract; such terminal direct repeats also flank integrated proretroviruses and transposable DNA elements and are thought to be the hallmark of inserted DNA sequences.Several recent observations suggest that certain nonfunctional genes (pseudogenes) and repetitive DNA sequences may be derived from RNA molecules that have been converted into DNA and inserted into the genome. Pseudogenes for small nuclear RNA have 5' ends that coincide with the 5' end of small nuclear RNA (1). Furthermore, these small nuclear RNA pseudogenes are flanked by short directly repeated DNA sequences, a hallmark of DNA insertion also found flanking integrated proretroviral sequences and transposable DNA elements (2, 3). Interspersed repetitive DNA sequences ofthe Alu family sometimes terminate in A-rich tracts and often are flanked by directly repeated DNA sequences (4, 5), features which suggest that many members ofthe Alu family may be derived from RNA molecules that have been converted into DNA. Several pseudogenes have been described that are related to functional structural genes by virtue of sequence homology but appear to have lost their introns precisely according to the rules of RNA splicing (6-9). Intronless pseudogenes for human immunoglobulin A light chain (8) and ,B-tubulin (9) terminate in a 3' A-rich tract, another characteristic feature of RNA processing. An intronless mouse a-globin pseudogene (6, 7) and the human ,Btubulin intronless pseudogene (9) are flanked by short directly repeated DNA sequences, suggesting DNA insertion. Fur-

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