Flanking DNA Fragments Research Articles

Ets‐1/ Elk‐1 is a critical mediator of dipeptidyl‐peptidase III transcription in human glioblastoma cells

Dipetidyl-peptidase III is a metallopeptidase involved in a number of physiological processes and its expression has been reported to increase with the histological aggressiveness of human ovarian primary carcinomas. Because no information regarding the regulation of its expression was available, experiments were designed to clone, define and characterize the promoter region of the human dipeptidyl-peptidase III (DPP-III) gene. In this study, we cloned a 1038 bp 5'-flanking DNA fragment of the human DPP-III gene for the first time and demonstrated strong promoter activity in this region. Deletion analysis revealed that as few as 45 nucleotides proximal to the transcription start site retained approximately 40% of the activity of the full-length promoter. This promoter lacked the TATA box but contained multiple GC boxes and a single CAAT box. Similarly, two Ets-1/Elk-1-binding motifs are present in the first 25 nucleotides from the transcription start site. Binding of Ets-1/Elk-1 proteins to these motifs was visualized by electrophoretic mobility shift and chromatin immunoprecipitation assays. Mutations of these binding sites abolished not only binding of the Ets protein, but also the intrinsic promoter activity. Increased DNA-binding activity of Ets-1/Elk-1 by v-Ha-ras also augmented the mRNA level and promoter activity of this gene. Similarly, co-transfection of DPP-III promoter-reporter constructs with Ets-1 expression vector led to a significant increase in promoter activity. From these results, we conclude that Ets-1/Elk-1 plays a critical role in transcription of the human DPP-III gene.

A high-throughput genome-walking method and its use for cloning unknown flanking sequences

We developed a PCR-based high-throughput genome-walking protocol. The novelty of this protocol is in the random introduction of unique walker primer binding sites into different regions of the genome efficiently by taking advantage of the rolling circle mode of DNA synthesis by Phi29 DNA polymerase after annealing the partially degenerate primers to the denatured genomic DNA. The inherent strand-displacement activity of the Phi29 DNA polymerase displaces the 5′ ends of downstream strands and DNA synthesis continues, resulting in a large number of overlapping fragments that cover the whole genome with the unique walker adapter attached to the 5′ end of all the genomic DNA fragments. The directional genome walking can be performed using a locus-specific primer and the walker primer and Phi29 DNA polymerase-amplified genomic DNA fragments as template. The locus-specific primer will determine the position and direction of the genome walk. Two rounds of successive PCR amplifications by locus-specific and walker primers and their corresponding nested primers effectively amplify the flanking DNA fragments. The desired PCR fragment can be either cloned or sequenced directly using another nested, locus-specific primer. We successfully used this protocol to isolate and sequence 5′ flanking regions/promoters of selected plant genes.

Evidence for a Link Between Pathogenicity and the Role of ImpBacterial Transport Effector Proteins in Soybean Infection by Xanthomonas axonopodis pv. glycines

Xanthomonas axonopodis pv. glycines (Xag) is the causal agent of bacterial pustule disease of soybeans. A nonpathogenic mutant of Xag (M715) was constructed employing transposon mutagenesis which showed similar epiphytic survival in planta to its wild type strain (YR32). The objective of this work was to identify and to analyze genes involved in pathogenicity in Xag YR32. Inverse Polymerase Chain Reaction (IPCR) was used to isolate the DNA flanking transposon insertion. A 1.3 kb flanking DNA fragment was sequenced and analyzed employing BLAST program to study homology, the position of transposon insertion and to predict the structure and function of the gene. One of the Open Reading Frames (ORFs) shared homology with inner membrane proteins (imps) of Xanthomonas axonopodis pv. citri (GenBank accession No. NC 003919). Northern blot analysis revealed that an imps gene was monocistronic and the size of imps mRNA in YR32 was slightly longer than in M715. Reverse Transcriptase- PCR analysis demonstrated that the imps transcript in M715 was much less abundant than in the wild type YR32. Transposon (mini-Tn5-Kmr-Tpr) was determined to be inserted close to the end of C-terminal region of imps gene and might be sufficient to destabilize the imps transcript in M715 and so influence effectors transportation from Xag to plant cell.

Bordetella bronchiseptica의 alcaligin siderophore 생합성 유전자인 alcA에 관한 연구

돼지 위축성 비염과 개의 kennel cough의 원인균인 B. bronchiseptica는 각 숙주의 상부 호흡기관의 점막에 집락을 형성하는 병원균으로서 철이 부족한 환경에서 hydroxamate type의 alcaligin이 라는 siderophore를 생산한다. Alcaligin의 생합성에 관련하는 구조유전자 중 alcA 유전자의 기능을 밝히고자 alcA 결손돌연변이주 구축을 통하여 확인하였다. alcA 유전자 결손 돌연변이를 위해 0.6 kb alcA 5' flanking DNA와 0.7 kb alcA 3' flanking DNA fragment들을 pCP1.11을 주형으로 하여 PCR법으로 증폭한 후, 5' flanking과 3' flanking DNA가 연결된 재조합 suicide vector pDMl을 구축하여 세포 접합을 통해 B. bronchiseptica로 도입시켰다. 도입된 pDM1으로부터 allelic exchange법에 의해 alcA 유전자가 결손된 돌연변이주 B. bronchiseptica H1을 얻을 수 있었다. B. bronchiseptica H1은 야생형인 B. bronchiseptica에 비하여 alcaligin siderophore를 거의 생성하지 못하였다. alc 오페론 중 promoter와 alcA 유전자만을 가지는 재조합 플라스미드를 B. bronchiseptica Hl에 도입하였을 때 alcaligin siderophore의 생산이 회복됨을 확인할 수 있었다. 이상의 결과로부터 alcA 유전자가 alcaligin 생합성에서 매우 중요한 역할을 수행하는 것을 알 수 있었다. Bordetella bronchiseptica, the agent of swine atrophic rhinitis and kennel cough in dogs, is a mucosal pathogen and produces the hydroxamate type alcaligin siderophore under iron-limited conditions. Genes involved in alcaligin siderophore biosynthesis are contained in an alcABCDE operon. In order to provide direct evidence for the role of AlcA in alcaligin biosynthesis, we needed a B. bronchiseptica mutant carrying alcA gene deletion. A 0.6 kb alcA 5'-flanking and 0.7kb 3'-flanking DNA fragments were PCR amplified with the use of pCP1.11 as a template DNA. The 5'-and 3'-flanking DNA fragments were joined in a suicide plasmid, resulting in a recombinant suicide plasmid pDM1. After introduction of pDM1 into B. bronchiseptica by conjugation, the allelic exchange technique was performed and a B. bronchiseptica alcA deletion mutant, named B. bronchiseptica H1, was obtained. The mutant strain produced reduced amount of siderophore as expected. When a plasmid containing complete alcA gene was transformed back into the mutant, the complemented mutant recovered ability of siderophore production. These results indicated that AlcA is one of essential components for the alcaligin siderophore biosynthesis. The mutant strains obtained in this study will be used in the further studies for the biochemical function of AlcA.

Kloning DNA Genom Pengapit Transposon dari Mutan Nonpatogenik Xanthomonas axonopodis pv. glycines M715

The objective of this work is to clone flanking DNA derived from Tn-5 mutagenesis of wild type strain Xanthomonas axonopodis pv. glycines as first step to clone and to identify the gene involved in pathogenicity mechanism. We have localized the flanking DNA fragment from a nonpathogenic mutant of Xag M715. Southern hybridization analysis using 2.8 kb EcoRI from pYR103 as a probe showed that the fragment is located within 2.0 kb PstI fragment. A 0.7 kb flanking DNA was amplified using inverse PCR technique, and inserted into pGEM-T Easy vector generating a 3.7 kb recombinant plasmid (pAA01). Southern hybridization analysis of the wild type (YR32) with pAA01 as a probe indicated a hybridization signal located at approximately 3.0 kb Pst I fragment. DNA sequence analysis revealed that the DNA fragment has a 64% identity to a vir gene of Bacillus anthracis

Cloning of Genomic DNA Flanking Transposon in the Nonpathogenic Mutant of Xanthomonas axonopodis pv. glycines M715

The objective of this work is to clone flanking DNA derived from Tn-5 mutagenesis of wild type strain Xanthomonas axonopodis pv. glycines as first step to clone and to identify the gene involved in pathogenicity mechanism. We have localized the flanking DNA fragment from a nonpathogenic mutant of Xag M715. Southern hybridization analysis using 2.8 kb EcoRI from pYR103 as a probe showed that the fragment is located within 2.0 kb PstI fragment. A 0.7 kb flanking DNA was amplified using inverse PCR technique, and inserted into pGEM-T Easy vector generating a 3.7 kb recombinant plasmid (pAA01). Southern hybridization analysis of the wild type (YR32) with pAA01 as a probe indicated a hybridization signal located at approximately 3.0 kb PstI fragment. DNA sequence analysis revealed that the DNA fragment has a 64% identity to a vir gene of Bacillus anthracis.

Strong expression of the rice catalase gene CatB promoter in protoplasts and roots of both a monocot and dicots

The rice ( Oryza sativa L.) catalase (EC 1.11.1.6) gene CatB is expressed in roots and cultured cells. We examined the promoter activity of its 5′-flanking region in a monocot and in two dicots. Transient expression assays in rice Oc and tobacco BY-2 suspension cell protoplasts showed that CatB’s 5′-flanking DNA fragments (nucleotides –1066 to +298) had about 20 and 3–4 times as much promoter activity, respectively, as the CaMV 35S promoter. Serial deletion analyses of the CatB promoter region revealed that the shortest fragment (–56 to +298) still had about 10 times as much promoter activity as the CaMV 35S promoter in rice protoplasts. In tobacco protoplasts, the activity of the fragment (–56 to +298) was about half of the CaMV 35S promoter. Transgenic rice and Arabidopsis plants carrying GUS genes driven by the 5′-truncated CatB promoters were generated and their GUS activity was examined. The region ranging from –329 to +298 showed preferential expression in the roots of rice and Arabidopsis, and in the shoot apical meristems of Arabidopsis. In situ hybridization revealed that CatB was highly expressed in branch root primordia and root apices of rice. Fusion of the GUS gene to the region (–329 to +298) conferred strong expression in these same areas, indicating that the presence of this region was sufficient to express CatB specifically in the roots. There may be new regulatory element(s) in this region, because it contained no previously known cis-regulatory elements specific for gene expression in roots.

Dynamic Regulation of Cyclooxygenase-2 Promoter Activity by Isoforms of CCAAT/Enhancer-binding Proteins

To elucidate the mechanism by which isoforms of CCAAT/enhancer-binding proteins regulate cyclooxygenase-2 expression, we determined by a novel technique binding of six isoforms of this transactivator to two sequence-specific CCAAT/enhancer-binding protein (-132/-125) and cyclic AMP (-59/-53) regulatory elements in human foreskin fibroblasts treated with phorbol 12-myristate 13-acetate for 4 h. The delta isoform bound to these two elements at basal state, which was displaced by full-length as well as two truncated beta isoforms, a 41-kDa liver-enriched activating protein and a 16-kDa liver-enriched inhibitory protein, after phorbol ester stimulation. Kinetic analysis shows time-dependent changes in beta and delta binding that were concordant with time-dependent increase in cyclooxygenase-2 induction. Overexpression of the 16-kDa beta isoform blocked the promoter activity and protein level induced by phorbol ester. Paradoxically, it increased binding of beta isoforms to the sequence-specific promoter DNA but suppressed cyclooxygenase-2 promoter activation by p300 cotransfection. These findings provide new insight into the regulation of cyclooxygenase-2 promoter by an interplay between two opposite beta isoforms and p300 co-activator.

Inverse PCR. Genomic DNA cloning.

AbstractInverse PCR (IPCR) was designed for amplifying anonymous flanking genomic DNA regions (1 2). The technique involves the digestion of source DNA, circulation of restriction fragments, and amplification using oligonucleotides that prime the DNA synthesis directed away from the core region of a known sequence, i.e., opposite of the direction of primers used in normal or standard PCR Fig. 1). Prior to the invention of the polymerase chain reaction (PCR), the acquisition of a specific DNA fragment usually entailed the construction and screening of DNA libraries, and the traditional “walking” into flanking DNA fragments involved the successive probing of libraries with clones obtained in the prior screening. These time-consuming procedures could be replaced by IPCR. Because IPCR can be used to efficiently and rapidly amplify regions of unknown sequence flanking any identified segment of cDNA or genomic DNA, researchers do not need to construct and screen DNA libraries to obtain additional unidentified DNA sequence information using this technique. Some recombinant phage or plasmids may be unstable in bacteria and amplified libraries tend to lose them. IPCR eliminates this problem. Diagram of IPCR for genomic DNA cloning. The procedure consists of four steps: genomic DNA isolation, circularization of double-stranded DNA, reopening of the circular DNA, and amplification of reverse DNA fragment. The black and open bars represent the known and unknown sequence regions of double-stranded cDNA, respectively. RE: restriction enzymesite; 65p: gene-specific primer. KeywordsStandard Polymerase Chain ReactionRecombinant PhageInverse Polymerase Chain ReactionPolymerase Chain Reaction MutagenesisIntramolecular LigationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Cell-specific and developmental regulation of rabbit surfactant protein B promoter in transgenic mice.

Surfactant protein B (SP-B) is expressed tissue specifically in the lung and is developmentally regulated. To identify genomic regions that control SP-B expression, we analyzed SP-B promoter activity in transgenic mice containing rabbit SP-B 5'-flanking DNA fragments linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Results showed that whereas the -2,176/+39-bp fragment failed to express CAT, shorter fragments of -730/+39 and -236/+39 bp expressed CAT tissue specifically in the lung. Further deletion of 5'-flanking DNA to -136 bp resulted in no expression of CAT. Immunostaining demonstrated that both -730/+39- and -236/+39-bp regions expressed CAT specifically in alveolar type II and Clara cells. The -236/+39-bp region expressed CAT at a significantly lower level than the -730/+39-bp region. CAT expression in mice containing the -730/+39-bp region was detected in embryonic day 14 lung and attained maximum levels in day 18 lung, indicating that the developmental expression of CAT was similar to that of SP-B. These data show that the DNA elements necessary for cell type-specific expression are located within -236/+39 bp of the SP-B gene. Additionally, these data suggest that the -2,176/-730- and -730/-236-bp regions contain the DNA elements that repress and enhance SP-B gene transcription, respectively.

The transposon A(R)4-24P[white, rosy] in Drosophila melanogaster is subject to position-effect variegation at a non-centromeric insertion site.

The white gene within the transposon A(R)4-24P[white,rosy] inserted at cytological location 24D1-2 in the euchromatic portion of the Drosophila melanogaster genome exhibits a mosaic pattern of expression which is modified by temperature and Y-chromosome number, as in cases of classical position-effect variegation (PEV). The eye colour of the flies in this variegated stock remains mosaic in the presence of the PEV modifier Su(var)3-6, slightly less so with Su(var)3-9 and Su(var)2-5, and full suppression of variegation occurs in the presence of Su(var)3-7. We have induced further transposition of A(R)4-24 and isolated two mosaic stocks with this transgene at new cytological locations. In these stocks, the A(R)4-24 transposon was flanked by the same genomic DNA fragments as in the original location. Spontaneous loss of these fragments leads to reversion of the variegated eye colour to wild-type. We suggest that the flanking DNA fragments from 24D1-2 are capable of inducing position-effect variegation without any association with centromeric heterochromatin. In situ hybridisation and Southern analysis demonstrate that the 5' flanking genomic fragment contains repeated sequences which are abundantly present in heterochromatin.

Analysis of sequences controlling tissue-specific and hyperproliferation-related keratin 6 gene expression in transgenic mice.

Keratin 6 (K6) is an intermediate filament protein found in hair follicles and in several internal stratified epithelia. This keratin has been the focus of special attention because it is also strongly induced in epidermal interfollicular keratinocytes in hyperproliferative situations and in certain conditions leading to abnormal differentiation. To localize and identify the sequences controlling this complex expression pattern, and because of their potential use in transgenic mouse models and gene therapy strategies for epidermal hyperproliferative disorders, we have thoroughly analyzed a 9 kbp region of this gene previously shown to direct proper tissue-specific and inducible expression in transgenic mice. To reproduce the K6 constitutive expression pattern, cooperation is necessary between elements located in at least two different regions, one distal between -9 and -4 kbp and one proximal between -830 and -125 bp with respect to the CAP site. The ability to induce expression under hyperproliferative conditions resides in the 2.4 kbp fragment preceding the transcription start site. When this DNA fragment was analyzed in more detail, we found that all subfragments tested contained regulatory elements necessary for inducible expression. Thus, a complex organization of K6 regulatory elements emerges, as both the constitutive and the inducible expressions of this gene are under the control of multiple elements dispersed throughout relatively large 5' flanking DNA fragments. These findings will allow the expression of cloned genes in transgenic mouse skin in response to pathological or applied hyperproliferative stimuli, avoiding the effects of their constitutive expression in other epithelia.

Developmental regulatory elements in the 5' flanking DNA of the Drosophila choline acetyltransferase gene.

Choline acetyltransferase (ChAT, EC 2.3.1.6) catalyzes the production of the neurotransmitter acetylcholine, and is an essential factor for neurons to be cholinergic. We have analyzed regulation of the Drosophila ChAT gene during development by examining the β-galactosidase expression pattern in transformed lines carrying different lengths of 5' flanking DNA fused to a lacZ reporter gene. The largest fragment tested, 7.4 kb, resulted in the most extensive expression pattern in embryonic and larval nervous system and likely reflects all the cis-regulatory elements necessary for ChAT expression. We also found that 5' flanking DNA located between 3.3 kb and 1.2 kb is essential for the reporter gene expression in most of the segmentally arranged embryonic sensory neurons as well as other distinct cells in the CNS. The existence of negative regulatory elements was suggested by the observation that differentiating photoreceptor cells in eye imaginal discs showed the reporter gene expression in several 1.2 kb and 3.3 kb transformants but not in 7.4 kb transformants. Furthermore, we have fused the 5' flanking DNA fragments to a wild type ChAT cDNA and used these constructs to transform Drosophila with a Cha mutant background. Surprisingly, even though different amounts of 5' flanking DNA resulted in different spatial expression patterns, all of the positively expressing cDNA transformed lines were rescued from lethality. Our results suggest that developmental expression of the ChAT gene is regulated both positively and negatively by the combined action of several elements located in the 7.4 kb upstream region, and that the more distal 5' flanking DNA is not necessary for embryonic survival and development to adult flies.

Stress responses and metabolic regulation of glyceraldehyde-3-phosphate dehydrogenase genes in Arabidopsis.

We report here effects of three environmental conditions, heat shock, anaerobic treatment, and carbon source supply, on expression of nuclear genes encoding chloroplast (GapA and GapB) and cytosolic (GapC) glyceraldehyde-3-phosphate dehydrogenase from Arabidopsis thaliana. The steady-state mRNA level of the GapC increased when Arabidopsis plants were transferred from normal growth condition to heat-shock, anaerobiosis, or increased sucrose supply conditions. In contrast, the steady-state mRNA levels for GapA and GapB genes were unaffected or decreased transiently under the same treatments. To identify the cis-acting regulatory elements, transgenic tobacco plants containing a 820-bp GapC 5'-flanking DNA fragment and beta-glucuronidase (Gus) fusion were constructed. Analyses of these transgenic plants indicate that this 820-bp DNA fragment is sufficient to confer both heat-shock and anaerobic responses. These results suggest that transcriptional level control is involved in regulation of GapC expression under these stress conditions. Histochemical analysis of Gus activity indicates that expression of the GapC is cell-type specific and is probably linked to the metabolic activity of the cells.

Characterization of the rat pulmonary surfactant protein A promoter.

The expression of the pulmonary surfactant protein A (SP-A) is developmentally regulated and controlled by several hormones. In an attempt to characterize cis-acting elements involved in the regulation of SP-A expression, we have cloned the 5' flanking sequence of the rat SP-A gene. The promoter region contains a TATA box but no CAAT box. The transcription start site has been identified by anchored polymerase chain reaction and S1 nuclease mapping of the mature and precursor transcripts. S1 mapping of precursor transcripts has confirmed the stimulating effect of glucocorticoids on SP-A rat gene transcription in vivo. This hormonal effect may be mediated by a putative glucocorticoid responsive element located 140 bp upstream from the initiation site and protected against DNase 1 digestion in footprinting experiments. In vitro transcription of a G-free reporter cassette linked to the 212-bp 5' flanking DNA fragment has established that this putative promoter region is functional. Efficient transcription of the G-free reporter cassette was obtained with cell-free fetal lung extracts, whereas no transcript was detectable with cell-free liver extracts. Comparative analysis of the human and rat 5' flanking sequences shows the presence of strongly conserved motifs, unrelated to previously known consensus sequences. Some of these motifs, specifically protected in DNase 1 footprinting studies, could therefore be involved in the regulation of SP-A gene expression.

Pituitary pro-opiomelanocortin gene expression requires synergistic interactions of several regulatory elements.

The pro-opiomelanocortin (POMC) gene is expressed very early during pituitary development, before expression of the other pituitary hormone genes, growth hormone and prolactin, and before expression of the Pit-1/GHF-1 transcription factor which activates those genes. Thus, analysis of the POMC promoter should provide markers of the early stages of pituitary development at the time when cells are being committed to expression of one or the other pituitary hormone. We have previously localized the rat POMC promoter to a 543-bp 5'-flanking DNA fragment of the gene using transfection and transgenic mice experiments. We have now used mutagenesis and in vitro protein-DNA binding studies to define three domains of the promoter which have distinct and complementary activities. Within these domains which require each other for full activity, at least nine regulatory elements were defined by in vitro footprinting and replacement mutagenesis. Each element appeared equally important for promoter activity, as mutagenesis of any element had similar effect on promoter activity. Most of the elements bound different AtT-20 nuclear proteins in gel mobility shift experiments. Whereas only two elements appeared to be binding sites for the known transcription factors AP-1 and chicken ovalbumin upstream promoter, the seven other elements appeared to bind nuclear proteins with novel properties. Thus, in contrast to the predominant role of Pit-1/GHF-1 in transcription of the growth hormone and prolactin genes, the control of an early pituitary gene, POMC, appears to depend on the synergistic interaction of several regulatory elements which bind different nuclear proteins.

Pituitary Pro-Opiomelanocortin Gene Expression Requires Synergistic Interactions of Several Regulatory Elements

The pro-opiomelanocortin (POMC) gene is expressed very early during pituitary development, before expression of the other pituitary hormone genes, growth hormone and prolactin, and before expression of the Pit-1/GHF-1 transcription factor which activates those genes. Thus, analysis of the POMC promoter should provide markers of the early stages of pituitary development at the time when cells are being committed to expression of one or the other pituitary hormone. We have previously localized the rat POMC promoter to a 543-bp 5'-flanking DNA fragment of the gene using transfection and transgenic mice experiments. We have now used mutagenesis and in vitro protein-DNA binding studies to define three domains of the promoter which have distinct and complementary activities. Within these domains which require each other for full activity, at least nine regulatory elements were defined by in vitro footprinting and replacement mutagenesis. Each element appeared equally important for promoter activity, as mutagenesis of any element had similar effect on promoter activity. Most of the elements bound different AtT-20 nuclear proteins in gel mobility shift experiments. Whereas only two elements appeared to be binding sites for the known transcription factors AP-1 and chicken ovalbumin upstream promoter, the seven other elements appeared to bind nuclear proteins with novel properties. Thus, in contrast to the predominant role of Pit-1/GHF-1 in transcription of the growth hormone and prolactin genes, the control of an early pituitary gene, POMC, appears to depend on the synergistic interaction of several regulatory elements which bind different nuclear proteins.

Nucleotide sequence of the ilvH-fruR gene region of Escherichia coli K12 and Salmonella typhimurium LT2

We have sequenced the fruR gene and flanking DNA fragments from Escherichia coli K12 and Salmonella typhimurium LT2. The fruR gene codes for a protein that represses the fru operon and activates the pps gene for PEP synthase. The corresponding open reading frame (ORF) FruR consists of 334 amino acid residues. The ORF contains an amino-terminal helix-turn-helix motif, characteristic of DNA-binding proteins and has similarity to known repressor proteins. The sequence is identical to that of the E. coli shl gene (mnemonic for suppressor-H-linked phenotype). It is flanked upstream by the ilvIH genes and downstream by the pbpB gene in both organisms and by orfB, a gene possibly involved in the regulation of cell division.

Promoter function and structure of the growth factorinducible immediate early genecyr61

cyr61 is an immediate early gene that is transcriptionally activated in 3T3 fibroblasts by serum, platelet-derived growth factor, fibroblast growth factor, and the tumor promoter TPA with kinetics similar to the induction of c-fos. cyr61 encodes a secreted protein that is associated with the cell surface and the extracellular matrix, and may play a role in cell-cell communication. We report here the complete nucleotide sequence of the mouse cyr61 gene, which contains four short introns. The transcription start site was mapped by S1 nuclease and primer extension analyses. A 2 kb 5' flanking DNA fragment functions as a serum-inducible promoter. This DNA fragment contains a poly(CA) sequence that can adopt the Z DNA form. In addition, it contains a sequence that resembles the serum response element (SRE) originally identified in the c-fos promoter. We show that deletion of the cry61 SRE-like sequence abrogates serum inducibility. Furthermore, this SRE-like sequence is sufficient to confer serum and growth factor inducibility when linked to a basal promoter, and binds the 67 kD serum response factor in vitro. We conclude that the cyr61 SRE functions as a serum response element and may account for the coordinate activation of cyr61 and c-fos.

Transgenic regulation of moth chorion gene promoters inDrosophila: Tissue, temporal, and quantitative control of four bidirectional promoters

Bidirectional chorion gene promoter regions from three silkmoth species, Bombyx mori, Antheraea pernyi, or Antheraea polyphemus (members of two different moth families), were tested for their ability to transcriptionally activate a bacterial marker gene (chloramphenicol acetyltransferase) in transformant Drosophila. Relatively short 5' flanking DNA fragments (272-367 bp) of chorion gene pairs are sufficient to confer a high degree of tissue and choriogenic stage specificity of expression to the marker gene. Thus, significant conservation of molecular interactions controlling transcription during choriogenesis is observed between the distantly related orders, Lepidoptera and Diptera. However, quantitative and fine temporal regulation in the Drosophila host does not fully parallel the in situ regulation in moths, indicating that some regulatory protein-DNA interactions have diversified in the approximately 250 million years since the last common ancestor of these insect groups. Limited in vitro mutagenesis of a B. mori promoter DNA has shown that a central 189-bp region includes elements sufficient for the qualitative specificity of chorion-specific expression. The same experiments have shown that a previously identified essential element, centered on the TCACGT hexamer, is not sufficient for chorion-specific expression: an additional essential element or elements are found farther upstream, within a 112-bp DNA region. Comparisons of silkmoth and Drosophila chorion gene promoter sequences have identified some candidates for cis-acting elements involved in the developmental regulation of chorion gene expression.