The cysteine-rich angiogenic protein 61 (Cyr61) is an extracellular matrix-associated, heparin-binding protein that mediates cell adhesion, stimulates cell migration, and enhances growth factor-induced cell proliferation. Cyr61 also promotes chondrogenic differentiation and induces neovascularization. In this study, we show that a 2-kb fragment of the Cyr61 promoter, which confers growth factor-inducible expression in cultured fibroblasts, is able to drive accurate expression of the reporter gene lacZ in transgenic mice. Thus, transgene expression was observed in the developing placenta and embryonic cardiovascular, skeletal, and central and peripheral nervous systems. The sites of transgene expression are consistent with those observed of the endogenous Cyr61 gene as determined by in situ hybridization and immunohistochemistry. The transgene expression in the cardiovascular system does not require the serum response element, a promoter sequence essential for transcriptional activation of Cyr61 by serum growth factors in cultured fibroblasts. Because the serum response element contains the CArG box, a sequence element implicated in cardiovascular-specific gene expression, the nonessential nature of this sequence for cardiovascular expression of Cyr61 is unexpected. Furthermore, the Cyr61 promoter-driven lacZ expression is inducible in granulation tissue during wound healing, as is synthesis of the endogenous Cyr61 protein, suggesting a role for Cyr61 in wound healing. Consistent with this finding, purified Cyr61 protein promotes the healing of a wounded fibroblast monolayer in culture. In addition, we mapped the mouse Cyr61 gene to the distal region of chromosome 3. Together, these results define the functional Cyr61 promoter in vivo, and suggest a role of Cyr61 in wound healing through its demonstrated angiogenic activities upon endothelial cells and its chemotactic and growth promoting activities upon fibroblasts.

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC--AT transitions or AT-*TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT-*GC transitions and for one cell line with a GC-*CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT--GC transitions had mutated back to the wild-type gpt sequence via GC-*AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC->CG transversion had mutated via GC--AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG--GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.Analysis of the molecular mechanisms of mutagenesis in mammalian cells has been hindered by the inability to efficiently recover chromosomal genes from the cells. To overcome this problem, our laboratory recently has developed a system (3) that combines mutagenesis of a gene that is chromosomally integrated in mammalian cells, clonal purification of the mutant cells, excision of the gene, and transfer of the gene into bacteria for DNA sequencing. The key feature of this system is the use of a retroviral shuttle vector as the element in which the gene is located. The target gene for mutagenesis is the Escherichia coli gpt gene, which codes for the enzyme xanthine-guanine phosphoribosyltransferase (GPT). The gene was incorporated into the retroviral vector pZipNeoSV(X)1 (5) and introduced into murine A9 cells, which lack activity of the enzyme hypoxanthine-guanine phosphoribosyltransferase (10), a mammalian enzyme that is similar in function to GPIT. In hypoxanthineguanine phosphoribosyltransferase-deficient cells, transformants that produce functional GPIT can be selected in HAT medium, and those that have lost the ability to produce functional GPIT can be selected with 6-thioguanine. The retroviral vector has long terminal repeats, which allow it to integrate into mam...

The archetypal TATA-box deficient G؉C-rich promoter of the murine adenosine deaminase gene (Ada) requires a 48-bp minimal self-sufficient promoter element (MSPE) for function. This MSPE was used to isolate a novel full-length cDNA clone that encodes a 66-kDa murine G؉C-rich promoter binding protein (mGPBP). The mGPBP mRNAs are ubiquitously expressed as either 3.0-or 3.5-kb forms differing in 3 polyadenylation site usage. Purified recombinant mGPBP, in the absence of any other mammalian cofactors, binds specifically to both the murine Ada gene promoter's MSPE and the nonhomologous human Topo II␣ gene's G؉C-rich promoter. In situ binding assays, immunoprecipitation, and Western blot analyses demonstrated that mGPBP is a nuclear factor that can form complexes with TATA-binding protein, TFIIB, TFIIF, RNA polymerase II, and P300/CBP both in vitro and in intact cells. In cotransfection assays, increased mGPBP expression transactivated the murine Ada gene's promoter. Sequestering of GPBP present in HeLa cell nuclear extract by immunoabsorption completely and reversibly suppressed extract-dependent in vitro transcription from the murine Ada gene's G؉C-rich promoter. However, transcription from the human Topo II␣ gene's TATA box-containing G؉C-rich promoter was only partially suppressed and the adenovirus major late gene's classical TATA box-dependent promoter is totally unaffected under identical assay conditions. These results implicate GPBP as a requisite G؉C-rich promoter-specific transcription factor and provide a mechanistic basis for distinguishing transcription initiated at a TATA box-deficient G؉C-rich promoter from that initiated at a TATA box-dependent promoter.Promoters that govern the transcription of mammalian genes by RNA polymerase II fall broadly into three types: the classical TATA box-dependent promoters, the initiator element (Inr)-dependent promoters, and the GϩC-rich promoters, which also have been referred to as CpG islands. Transcription initiation at the TATA box-dependent promoters is dictated by the direct interaction of the TATA box with the TATA-binding protein (TBP) as a first and rate-limiting step (reviewed in reference 8). Likewise, transcription initiation at Inr-dependent promoters occurs when the Inr element interacts with sequence-specific Inr-binding proteins (35,36). The mechanism by which transcription initiation sites within GϩC-rich promoters are recognized by the RNA polymerase II transcription machinery is currently unelucidated.

The molecular mechanism of reversion induced by 5-bromodeoxyuridine (BrdU) replication-dependent mutagenesis in mammalian cells was studied. Murine cells with single mutant copies of the E. coli gpt gene integrated chromosomally as part of a shuttle vector were mutagenized with BrdU, and GPT+ revertants were selected. Thirteen mutant cell lines (each of which had a gpt gene that varied from the wild-type gene by a different GC----AT base transition in the coding region) were mutagenized, and only four were found to be effectively reverted. All revertant gpt genes that were analyzed had reverted via AT----GC base transition at the original site of mutation, thus demonstrating that replication-dependent mutagenesis by BrdU causes AT----GC transitions. The nine cell lines that were nonrevertible by BrdU replication-dependent mutagenesis could be mutated by this protocol to ouabain resistance as effectively as the four revertible lines, indicating that the nonrevertible lines were susceptible to such mutagenesis. Thus, differences among the cell lines in frequencies of HATr revertants generated by BrdU replication-dependent mutagenesis could not be attributed to differences in general susceptibility of the lines to the mutagenic protocol. The revertible and nonrevertible lines could not be separated according to the position of the original GC----AT transition in the gpt coding region. However, there was evidence that the DNA base sequence flanking the site of mutation affected the susceptibility of that site to BrdU replication-dependent mutagenesis. For example, six of the cell lines tested had gpt genes in which the mutant T residue was immediately adjacent on its 3' side to an A residue, and all six were found to be nonrevertible by BrdU replication-dependent mutagenesis. Furthermore, a target AT base pair flanked by GC base pairs in opposite orientation and either immediately adjacent to or one base removed from the target site on both the 5' and 3' sides appeared to have an increased susceptibility to BrdU replication-dependent mutagenesis.

Polyethylene glycol-mediated fusion of Chinese hamster ovary (CHO) cells with mouse C11D cells produced interspecific somatic cell hybrids which slowly segregated CHO chromosomes. Cytogenetic and isozyme analysis of HAT-and bromodeoxyuridine-selected hybrid subclones and of members of a hybrid clone panel retaining different combinations of CHO chromosomes enabled provisional assignments of the following enzyme loci to CHO chromosomes: TK, GALK, and ACPJ to chromosome 7; TK and GALK to chromosome Z13; ACPI, ADA, and ITPA to chromosome Z8; and ADA and ITPA to chromosome Z9. These genetic markers reflect the origin of each of these Z group chromosomes and indicate the functional activity of alleles located on rearranged chromosomes. Identification of diploid electrophoretic shift mutations for ADA and ITPA was consistent with those observations. Assignment of the functional TK locus in TK+'-CHO-AT3-2