ABSTRACTcAMP regulates transcription of the gene encoding the a-subunit of human chorionic gonadotropin (hCG) in choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, we inserted fragments from the 5' flanking region of the a-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between positions -146 and -111. In the absence of cAMP, the a-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. We localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element.The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the a-subunit gene.Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone expressed in the placenta. Both the a-subunit and the P-subunit are required for biological activity (1). While a physiological regulator of hCG production has not been identified, the synthesis of both subunits can be stimulated by cAMP in placental explants and in human choriocarcinoma cells (2, 3). Recent reports from several laboratories have shown that cAMP regulates expression of the chorionic gonadotropin a-and ,-subunit genes, at least in part, at the level of transcription (refs. 4 and 5; A.M., R. Cox, and J.H.N., unpublished data).In the human a-subunit gene, the first 140 base pairs (bp) of 5' flanking sequence are sufficient to confer cAMP regulation to a heterologous gene after transfection and transient expression in choriocarcinoma cells (4). This suggests that a cAMP response element lies within this region. In the present study, we have constructed several expression vectors and have used a transient expression assay to localize this element to an 18-bp sequence that is repeated between positions -146 and -111 in the 5' flanking region of the a-subunit gene. A single copy ofthis cAMP response element is sufficient to confer the same degree of cAMP regulation as a 1500-bp fragment containing the a-subunit promoter. This response element functions independently of other promoter regulatory elements. PROCEDURES Construction of Vectors. Construction of the expression vector pHaCAT (Fig. 1A) was initiated by isolating a 1500-bp DNA fragment from the genomic clone of the human asubunit gene provided by J. Fiddes (6). ...
We demonstrate early proof of the principle that FDG-PET/CT-guided IMRT planning can selectively target and intensify treatment of head and neck disease while reducing critical normal tissue doses. Routine clinical use of such planning should not be engaged until the accuracy of FDG-PET/CT is fully validated. Future directions, including refinement of treatment to gross disease and radiologically uninvolved neck nodal levels, are discussed.
The activity of the M26 meiotic recombination hot spot of Schizosaccharomyces pombe depends on the presence of the heptamer 5-ATGACGT-3. Transplacement of DNA fragments containing the ade6-M26 gene to other chromosomal loci has previously demonstrated that the heptamer functions in some, but not all, transplacements, suggesting that hot spot activity depends on chromosomal context. In this study, hot spot activity was tested in the absence of gross DNA changes by using site-directed mutagenesis to create the heptamer sequence at novel locations in the genome. When created by mutagenesis of 1-4 bp in the ade6 and ura4 genes, the heptamer was active as a recombination hot spot, in an orientation-independent manner, at all locations tested. Thus, the heptamer sequence can create an active hot spot in other chromosomal contexts, provided that the gross chromosomal structure is not altered; this result is consistent with the hypothesis that a specific higher-order chromatin structure is required for M26 hot spot activity.Homologous recombination, the exchange of genetic information between homologous DNA duplexes, plays a vital role in the generation of genetic diversity, in the proper segregation of chromosomes during meiosis, and in the repair of DNA damage in somatic cells. Although homologous recombination occurs throughout the genome, some sites, termed hot spots, exhibit elevated frequencies of exchange. Recombination hot spots have been described in diverse organisms from bacteria to mammals (1, 2). Because hot spots appear to enhance a rate-limiting step of recombination, determination of the molecular mechanism by which hot spots act will increase our understanding of an important stage of homologous recombination.The M26 mutation of the fission yeast Schizosaccharomyces pombe creates a meiotic recombination hot spot in the ade6 gene. M26 is one of 394 mutations in ade6 described by Gutz (3) and results from a G 3 T transversion in the coding region of the ade6 gene (4, 5). Among these 394 mutations, M26 is unique; in heteroallelic crosses, it recombines with other ade6 mutations up to 15-fold more frequently than the adjacent M375 mutation does (3). M375 is an equivalent G 3 T transversion in the preceding codon and thus serves as an ideal genetic control for M26 (4). M26 undergoes gene conversion 10 times more frequently than M375 and demonstrates disparity of conversion, with preferential conversion of M26 to wild type, whereas M375 converts with near parity (3). M26 also increases the frequency of conversion of other ade6 mutations, with which it frequently coconverts; the frequency of coconversion decreases with increasing distance from M26 (6). These properties led Gutz (3) to propose that the M26 mutation creates a site that stimulates recombination near itself and, less frequently, at a distance.M26 is unique among eukaryotic hot spots in that hot spot activity is known to depend on a specific DNA sequence, the heptamer 5Ј-ATGACGT-3Ј, the first T in the heptamer sequence being the site of mut...
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