We have identified a folate receptor gene upstream of the chicken β-globin locus and separated from it by a 16 kbp region of silent chromatin. We find that this receptor is expressed only at a stage of erythroid differentiation (CFU-E) preceding the activation of β-globin genes, consistent with the role of folate receptors in proliferation. This discovery raises the question of how these two loci are regulated during erythropoiesis. Our data suggest that the folate receptor gene and the β-globin locus are regulated independently. We show that a 3.3 kbp DNA region upstream of the folate receptor gene is sufficient to induce strong expression of a transgene in CFU-E stage cells. We also find that the region between the β-globin locus and the folate receptor gene is fully methylated and condensed at this stage of differentiation. Its 3Ј boundary coincides with the 5Ј β-globin insulator. We speculate that the 5Ј β-globin boundary element might be important for the proper regulation of two adjacent domains activated at two different stages during differentiation.

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disorder characterized by the presence of multiple gastrointestinal polyps and an increased risk for various types of cancers. Inactivating germline mutations of the LKB1 gene, which encodes a serine/threonine kinase, are responsible for the majority of PJS cases. Here, we show that the heteromeric complex containing the molecular chaperones Hsp90 and Cdc37/p50 interacts with the kinase domain of LKB1. Treatment of cells with either geldanamycin or novobiocin, two pharmacological inhibitors of Hsp90 causes the destabilization of LKB1. Furthermore, geldanamycin treatment leads to the ubiquitination and the rapid degradation of LKB1 by the proteasome-dependent pathway. In addition, we found that a LKB1 point mutation identified in a sporadic testicular cancer, weakens the interaction of LKB1 with both Hsp90 and Cdc37/p50 and enhances its sensitivity to the destabilizing effect of geldanamycin. Collectively, our results demonstrate that the Hsp90/Cdc37 complex is a major regulator of the stability of the LKB1 tumor suppressor. Furthermore, these data draw attention to the possible adverse consequences of antitumor drugs that target Hsp90, such as antibiotics related to geldanamycin, which could disrupt LKB1 function and promote the development of polyps and carcinomatous lesions. Oncogene (2003) 22, 9165-9175.

This study identifies a region of the adeno-associated virus type 2 (AAV-2) rep gene (nucleotides 190 to 540 of wild-type AAV-2) as a cis-acting Rep-dependent element able to promote the replication of transiently transfected plasmids. This viral element is also shown to be involved in the amplification of integrated sequences in the presence of adenovirus and Rep proteins.It was previously reported that efficient recombinant adenoassociated virus (AAV) production using stable rep-cap cell lines correlated with a 100-fold amplification of the AAV-2 genes upon adenovirus infection (3,9). This phenomenon, which occurred despite the absence of inverted terminal repeats (ITRs) generated extrachromosomal double-stranded DNA molecules harboring the rep-cap genes and required the activity of the adenovirus DNA binding protein, cellular polymerases, and Rep proteins (9). A question that remained unanswered was whether the rep-cap amplification was dependent on the activity of an as-yet-unidentified viral origin of replication present within the viral genome.To answer this question, we investigated if a rep-cap-containing plasmid was able to replicate following transient transfection into adenovirus-infected cells. 293 cells were transfected with plasmid pRCtag containing the rep-cap genome with the ITRs deleted, ligated to a 3Ј tag sequence, and then mock or adenovirally infected. After DNA extraction, replication was assessed by digestion with DpnI or MboI followed by Southern blot analysis using a tag probe (Fig. 1). Cleavage by DpnI indicates that both strands are methylated in the absence of replication of the transfected DNA; cleavage by MboI occurs only if both strands are unmethylated as a result of two rounds of replication. In the absence of adenovirus, the pRCtag plasmid did not replicate (Fig. 1B, lanes 5 and 6). In contrast, upon adenoviral infection, a fraction of the plasmid DNA was susceptible to MboI digestion (Fig. 1B, lane 9), indicating that some input rep-cap molecules had replicated. After DpnI digestion, high-molecular-weight resistant bands were detected as weak signals, suggesting that pRCtag replication generated products which are heterogenous in size (Fig. 1B, lane 8).To identify the cis element(s) involved in pRCtag replica- , and analyzed on a Southern blot by using a tag probe. As a control (lanes 1, 2, and 3), untransfected pRCtag plasmid DNA mixed with 10 g of total DNA from 293 cells was digested with DpnI or MboI and similarly analyzed using the tag probe. The expected 1,430-bp DpnI-MboI fragment hybridizing to the tag probe is indicated. 9991on May 12, 2018 by guest

We previously reported that a 350-bp region of the adeno-associated virus (AAV) type 2 rep gene contains a cis-acting element responsible for the Rep-dependent replication of a transiently transfected rep-cap plasmid. In this study, we further report that replicated rep-cap sequences can be packaged into AAV capsids in the absence of the inverted terminal repeats.The usual procedure for recombinant adeno-associated virus (rAAV) assembly involves transfection of the vector and the rep-cap plasmid into cells which are either infected with adenovirus or cotransfected with an adenoviral helper plasmid (4,11,15). Despite the lack of homologous sequences between the rep-cap and vector sequences, the precise characterization of rAAV preparations indicated that they were contaminated to various extents with particles containing rep-cap AAV sequences. We designated these contaminating particles rep positive (rep ϩ ), because they were able to transfer a Rep function, as detected by a replication center assay (RCA) (10). Previous studies have indicated that most of these particles were replication competent and that they arose from nonhomologous recombination events between the rep-cap plasmid and the inverted terminal repeats (ITRs) in the rAAV vector (1, 14). Deletion of critical ITR sequences involved in the nonhomologous recombination events prevented the formation of such replication-competent particles. However, rAAV preparations remained contaminated by replication-defective AAV particles containing rep-cap genomes, further suggesting that these sequences had been packaged in the absence of the ITRs (14).Altogether, these observations suggested that some additional cis-acting elements were present in the rep-cap sequences, allowing their replication and encapsidation. We and others have previously reported the presence of a cis-acting replication element (CARE) located in the 5Ј portion of the rep gene (9, 12). The CARE was localized in a 350-bp region that included the p5 promoter and the 5Ј portion of the rep coding sequence (nucleotides 190 to 540 of wild-type AAV), and it was demonstrated that this element behaved in vitro and in vivo as a Rep-dependent origin of replication in the absence of both ITRs (9).In the present study, we investigated whether the presence of CARE could also lead to the packaging of rep-cap sequences into AAV capsids in the absence of the viral ITRs. A critical element in this study was the need to distinguish between DNA truly packaged inside the particles and that simply contaminating the preparations. The conventional procedure to extract viral DNA from a cell lysate or a purified rAAV stock relies on the use of DNase I to digest contaminating DNA before extraction of packaged sequences. The activity of DNase I was checked by mixing purified rAAVLZ particles (rAAV encoding the nucleus-localized ␤-galactosidase) with up to 400 ng of X174 DNA (either double or single stranded). After digestion for 1 h at 37°C with 50 U of DNase I (Roche) in 500 l of Dulbecco modified Eagle medium, pack...

The gene encoding the silk protein P25 in Bombyx mori is expressed in the posterior silk gland (PSG) cells and repressed in the middle silk gland (MSG) cells. To identify the factors involved in this transcriptiondependent spatial restriction, we examined the P25 chromatin in PSG and MSG nuclei by DNase I-aided ligation-mediated PCR and analyzed the expression of various P25-lacZ constructs in biolistically treated silk glands. P25 promoter activation depends on two cis-acting elements. One coincides with the target sequence of SGFB, a silk gland-specific factor present in all silk gland nuclei, but bound to its target DNA sequence in only PSG cells. The interaction of the other element with a factor that we named PSGF is also exclusive to PSG cells. Placed ahead of a non-P25-related basal promoter, the SGFB and PSGF elements are sufficient to drive posterior-cell transcription. Collectively, our data support the hypothesis that the spatial restriction of P25 expression is driven by the stabilization of SGFB onto its target sequence by the action of PSGF.Organogenesis proceeds by selective gene activation and repression, resulting in the appropriate complement of transregulatory factors that define the different cell types. Identifying these factors that govern each category of cell is one step toward understanding gene selection during embryogenesis. Silk gland organogenesis in Bombyx mori is completed in the embryo as evidenced by the spatial organization of fibroin-and sericin-secreting cells in, respectively, posterior and middle positions (2). As a consequence, the five single-copy genes encoding the various silk proteins of Bombyx are expressed in the embryo in distinct groups of the ϳ750 cells that constitute the silk gland epithelium.One such gene encodes the protein P25, a silk polypeptide that binds to the heavy and light subunits of fibroin. This gene is highly expressed in the posterior silk gland (PSG) cells and repressed in the middle silk gland (MSG) cells (3,4,30).To understand the regulation of P25 expression, an in vivo analysis was performed with transgenic Drosophila melanogaster. These studies demonstrated that the Bombyx promoter is regulated by Drosophila trans-acting factors in the salivary gland, the silk gland homolog in the fruit fly (1). As in the Bombyx silk gland, the activity of the P25 promoter in the salivary gland is positionally determined and is controlled by upstream sequences located within the 441 bp of proximal DNA.To identify putative regulatory elements, in vitro DNA-protein interactions between silk gland nuclear factors and synthetic DNA probes were explored, and two possible regulators with different properties were discovered (5). The first, BMFA, is a ubiquitous protein thought to be involved in the repression of silk gland-expressed genes at molting, including those encoding silk proteins. The second, SGFB, is a silk gland-specific regulatory protein expressed in both PSG and MSG cells and thus unable, by itself, to specify PSG expression. Three other proteins (TRI...