Perfect Palindromic Sequence Research Articles

Identification of AflR Binding Sites in the Genome of Aspergillus flavus by ChIP-Seq.

We report here the AflR binding motif of Aspergillus flavus for the first time with the aid of ChIP-seq analysis. Of the 540 peak sequences associated with AflR binding events, 66.8% were located within 2 kb upstream (promoter region) of translational start sites. The identified 18-bp binding motif was a perfect palindromic sequence, 5′-CSSGGGWTCGAWCCCSSG’3′ with S representing G or C and W representing A or T. On closer examination, we hypothesized that the 18-bp motif sequence identified contained two identical parts (here called motif A and motif B). Motif A was in positions 8–18 on the upper strand, while motif B was in positions 11-1 on the bottom strand. The inferred length and sequence of the putative motif identified in A. flavus were similar to previous findings in A. parasiticus and A. nidulans. Gene ontology analysis indicated that AflR bound to other genes outside the aflatoxin biosynthetic gene cluster.

Increasing Avermectin Production in Streptomyces avermitilis by Manipulating the Expression of a Novel TetR-Family Regulator and Its Target Gene Product.

Avermectins produced by Streptomyces avermitilis are commercially important anthelmintic agents. The detailed regulatory mechanisms of avermectin biosynthesis remain unclear. Here, we identified SAV3619, a TetR-family transcriptional regulator designated AveT, to be an activator for both avermectin production and morphological differentiation in S. avermitilis. AveT was shown to indirectly stimulate avermectin production by affecting transcription of the cluster-situated activator gene aveR. AveT directly repressed transcription of its own gene (aveT), adjacent gene pepD2 (sav_3620), sav_7490 (designated aveM), and sav_7491 by binding to an 18-bp perfect palindromic sequence (CGAAACGKTKYCGTTTCG, where K is T or G and Y is T or C and where the underlining indicates inverted repeats) within their promoter regions. aveM (which encodes a putative transmembrane efflux protein belonging to the major facilitator superfamily [MFS]), the important target gene of AveT, had a striking negative effect on avermectin production and morphological differentiation. Overexpression of aveT and deletion of aveM in wild-type and industrial strains of S. avermitilis led to clear increases in the levels of avermectin production. In vitro gel-shift assays suggested that C-5-O-B1, the late pathway precursor of avermectin B1, acts as an AveT ligand. Taken together, our findings indicate positive-feedback regulation of aveT expression and avermectin production by a late pathway intermediate and provide the basis for an efficient strategy to increase avermectin production in S. avermitilis by manipulation of AveT and its target gene product, AveM.

A single parS sequence from the cluster of four sites closest to oriC is necessary and sufficient for proper chromosome segregation in Pseudomonas aeruginosa.

Among the mechanisms that control chromosome segregation in bacteria are highly-conserved partitioning systems comprising three components: ParA protein (a deviant Walker-type ATPase), ParB protein (a DNA-binding element) and multiple cis-acting palindromic centromere-like sequences, designated parS. Ten putative parS sites have been identified in the P. aeruginosa PAO1 genome, four localized in close proximity of oriC and six, diverged by more than one nucleotide from a perfect palindromic sequence, dispersed along the chromosome. Here, we constructed and analyzed P. aeruginosa mutants deprived of each single parS sequence and their different combinations. The analysis included evaluation of a set of phenotypic features, chromosome segregation, and ParB localization in the cells. It was found that ParB binds specifically to all ten parS sites, although with different affinities. The P. aeruginosa parS mutant with all ten parS sites modified (parS null) is viable however it demonstrates the phenotype characteristic for parA null or parB null mutants: slightly slower growth rate, high frequency of anucleate cells, and defects in motility. The genomic position and sequence of parS determine its role in P. aeruginosa biology. It transpired that any one of the four parS sites proximal to oriC (parS1 to parS4), which are bound by ParB with the highest affinity, is necessary and sufficient for the parABS role in chromosome partitioning. When all these four sites are mutated simultaneously, the strain shows the parS null phenotype, which indicates that none of the remaining six parS sites can substitute for these four oriC-proximal sites in this function. A single ectopic parS2 (inserted opposite oriC in the parS null mutant) facilitates ParB organization into regularly spaced condensed foci and reverses some of the mutant phenotypes but is not sufficient for accurate chromosome segregation.

Fragile DNA Motifs Trigger Mutagenesis at Distant Chromosomal Loci in Saccharomyces cerevisiae

DNA sequences capable of adopting non-canonical secondary structures have been associated with gross-chromosomal rearrangements in humans and model organisms. Previously, we have shown that long inverted repeats that form hairpin and cruciform structures and triplex-forming GAA/TTC repeats induce the formation of double-strand breaks which trigger genome instability in yeast. In this study, we demonstrate that breakage at both inverted repeats and GAA/TTC repeats is augmented by defects in DNA replication. Increased fragility is associated with increased mutation levels in the reporter genes located as far as 8 kb from both sides of the repeats. The increase in mutations was dependent on the presence of inverted or GAA/TTC repeats and activity of the translesion polymerase Polζ. Mutagenesis induced by inverted repeats also required Sae2 which opens hairpin-capped breaks and initiates end resection. The amount of breakage at the repeats is an important determinant of mutations as a perfect palindromic sequence with inherently increased fragility was also found to elevate mutation rates even in replication-proficient strains. We hypothesize that the underlying mechanism for mutagenesis induced by fragile motifs involves the formation of long single-stranded regions in the broken chromosome, invasion of the undamaged sister chromatid for repair, and faulty DNA synthesis employing Polζ. These data demonstrate that repeat-mediated breaks pose a dual threat to eukaryotic genome integrity by inducing chromosomal aberrations as well as mutations in flanking genes.

Genetic Control of Amadori Product Degradation in Bacillus subtilis via Regulation of frlBONMD Expression by FrlR

Bacillus subtilis is capable of degrading fructosamines. The phosphorylation and the cleavage of the resulting fructosamine 6-phosphates is catalyzed by the frlD and frlB gene products, respectively. This study addresses the physiological importance of the frlBONMD genes (formerly yurPONML), revealing the necessity of their expression for growth on fructosamines and focusing on the complex regulation of the corresponding transcription unit. In addition to the known regulation by the global transcriptional regulator CodY, the frl genes are repressed by the convergently transcribed FrlR (formerly YurK). The latter causes repression during growth on substrates other than fructosamines. Additionally, we identified in the first intergenic region of the operon an FrlR binding site which is centrally located within a 38-bp perfect palindromic sequence. There is genetic evidence that this sequence, in combination with FrlR, contributes to the remarkable decrease in the transcription downstream of the first gene of the frl operon.

Regulation of the Bacillus subtilis Divergent yetL and yetM Genes by a Transcriptional Repressor, YetL, in Response to Flavonoids

DNA microarray analysis revealed that transcription of the Bacillus subtilis yetM gene encoding a putative flavin adenine dinucleotide-dependent monooxygenase was triggered by certain flavonoids during culture and was derepressed by disruption of the yetL gene in the opposite orientation situated immediately upstream of yetM, which encodes a putative MarR family transcriptional regulator. In vitro analyses, including DNase I footprinting and gel retardation analysis, indicated that YetL binds specifically to corresponding single sites in the divergent yetL and yetM promoter regions, with higher affinity to the yetM region; the former region overlaps the Shine-Dalgarno sequence of yetL, and the latter region contains a perfect 18-bp palindromic sequence (TAGTTAGGCGCCTAACTA). In vitro gel retardation and in vivo lacZ fusion analyses indicated that some flavonoids (kaempferol, apigenin, and luteolin) effectively inhibit YetL binding to the yetM cis sequence, but quercetin, galangin, and chrysin do not inhibit this binding, implying that the 4-hydroxyl group on the B-ring of the flavone structure is indispensable for this inhibition and that the coexistence of the 3-hydroxyl groups on the B- and C-rings does not allow antagonism of YetL.

Evolutionary conservation and mutational spectrum of BMPR2 gene

A variety of mutations in the bone morphogenetic protein receptor type 2 ( BMPR2) have been identified in patients with pulmonary arterial hypertension. In this study, using our BMPR2 mutation database and BMPR-II protein sequences from eight distantly related species, we defined the relationship among evolutionary conservation, mutation frequency and mutation distribution. As a whole, BMPR2 is evolving slower than the average for mammalian protein-encoding genes. As expected, the kinase domain is evolving more slowly than the extracellular ligand-binding and C-terminal domains. A detailed map of evolutionary conservation shows that there are repeating peaks and valleys within the C-terminal domain, representing higher and lower evolutionary conservation. We observed a strong correlation between evolutionary conservation and the distribution of mutations along the gene. All except two, of the nineteen missense mutations occur in absolutely conserved amino acids among the vertebrate homologs. In addition, we identified six mutational hotspots ( P < 0.05) by comparing the observed distribution of mutations to the pattern expected from a random multinomial distribution. Furthermore, analysis of the sequence environment surrounding the mutations revealed a specific pattern of mutagenesis. Over 22% of all single base-paired substitutions and 30% of all deletions and insertions are situated within tandem or non-tandem direct repeats of at least 5-bp and may be explained by slipped-mispairing model of mutagenesis. Also, over 59% of single base-paired substitutions versus 20% of deletions and insertions are located in perfect palindromic sequences that could produce “hairpin-loop” secondary structures with relatively high thermodynamic stability under physiological conditions. In addition, 3.7% of single base-paired substitutions versus 30% of deletions and insertions are located either within or in close proximity to the Krawczak and Cooper consensus sequence (TG A/G A/G G/T A/C). Further study of the mechanism of mutagenesis in BMPR2 may help identify other potentially mutable sites and differentiate between deleterious mutations and harmless polymorphic variants.

Molecular determinants of the hpa regulatory system of Escherichia coli: the HpaR repressor.

The HpaR-mediated regulation of the hpa-meta operon (Pg promoter) of the 4-hydroxyphenylacetic acid catabolic pathway of Escherichia coli has been studied. The HpaR regulator was purified to homogeneity showing that it is able to bind selectively to 4-hydroxyphenylacetic, 3-hydroxyphenylacetic and 3,4-dihydroxyphenylacetic acids, which act as inducers of the system. The role of HpaR as a repressor and the requirement for cAMP receptor protein for maximal activity have been confirmed by in vitro transcription analyses. Two DNA operators, OPR1 and OPR2, have been identified in the intergenic region located between the hpa-meta operon and the hpaR gene. The OPR1 operator contains a perfect palindromic sequence overlapping the transcriptional +1 start site of the Pg promoter. The OPR2 operator shows a similar but imperfect palindromic sequence and is located far downstream of the +1 start site of the Pr promoter. The binding of HpaR to OPR2 displays a clear cooperativity with OPR1 binding. Based on the above observations and the results of permanganate footprinting experiments, a repression mechanism for HpaR is postulated. A 3-dimensional model of HpaR, generated by comparison with the crystal structures of the homologous regulators, MarR and MexR, suggests that HpaR is a dimer that contains a typical winged-helix DNA binding motif in each subunit.

Contribution of downstream promoter elements to transcriptional regulation of the rice tungro bacilliform virus promoter.

Downstream sequences influence activity of the rice tungro bacilliform virus (RTBV) promoter in protoplasts derived from cultured rice cells. We previously identified a DNA element located between positions +50 and +90 relative to the transcription start site to which rice nuclear proteins bind. In this study, using DNA UV crosslinking assays, we show that two rice nuclear proteins bind specifically to this DNA element. We demonstrate that the DNA element enhances RTBV promoter activity in a copy number-dependent manner when transferred to a position upstream of the promoter. In addition, using electrophoretic mobility shift assays, we show that at least two novel nuclear proteins from rice cell suspension cultures bind to a subregion (from +50 to +59) of the DNA element and that a protein from rice root, but not shoot, nuclear extracts interacts with a perfect palindromic sequence motif located within the sequence +45 to +59. Furthermore, a position-dependent GAGA motif, present in three copies within downstream promoter sequences from +1 to +50, is involved in the regulation of RTBV promoter activity.

Transcriptional regulation of the Bacillus subtilis bscR-CYP102A3 operon by the BscR repressor and differential induction of cytochrome CYP102A3 expression by oleic acid and palmitate.

The adjacent yrhI and yrhJ genes were identified by the Bacillus subtilis genome sequencing project. We now report that yrhJ (renamed CYP102A3) encodes a cytochrome P450 and that yrhI (renamed bscR) encodes a repressor that negatively regulates the transcription of the bscR-CYP102A3 operon. The transcriptional initiation site of bscR has been mapped by primer extension analysis. An 18-bp perfect palindromic sequence centered 65.5 bp downstream from the transcriptional initiation site of bscR has been identified as the binding site for BscR by gel mobility shift assays. Base substitutions in the 18-bp inverted repeat resulted in derepression of the bscR-xylE transcriptional fusion in vivo. bscR-xylE fusion studies and Northern blot analysis revealed that oleic acid and palmitate could induce the expression of the bscR-CYP102A3 operon to a considerable extent. However, only oleic acid was capable of preventing the binding of BscR to its operator DNA in vitro, suggesting that the induction of CYP102A3 expression by oleic acid and palmitate in B. subtilis might be mediated through different mechanisms.

Primer-template misalignments during leading strand DNA synthesis account for the most frequent spontaneous mutations in a quasipalindromic region in Escherichia coli

Spontaneous mutant sequences which differ from the starting DNA sequence by the specific correction of quasipalindromic to perfect palindromic sequence are hallmarks of mutagenesis mediated by misalignments directed by palindromic complementarity. The mutant sequences are specifically predicted by templated, but ectopic, DNA polymerization on a misaligned DNA substrate. In a previous study, we characterized a spontaneous frameshift hotspot near a 17 bp quasipalindromic DNA sequence within the mutant chloramphenicol acetyl transferase (CAT) gene of plasmid pJT7. A one base-pair insertion hotspot, ectopically templated by misalignment mediated by palindromic complementarity, was shown to occur more frequently during synthesis of the leading than the lagging DNA strand. Here we analyze the misalignment mechanisms that can account for the DNA sequences of 123 additional spontaneous frameshift mutations (22 distinct genotypes) occurring in the same quasipalindromic DNA region in plasmids pJT7 and p7TJ (a pJT7 derivative with the CAT gene in the inverse orientation). Approximately 80% of the small frameshift mutants in each plasmid are predicted by palindromic misalignments of the leading strand. Smaller numbers of mutations are consistent with other DNA misalignments, including those predicted by simple slippage of the nascent DNA strand on its template. The results show that remarkable changes in the mutation spectra of a reporter gene may not be revealed by measurements of mutation frequency.

Genomic organization and promoter activity of the maize starch branching enzyme I gene.

Starch branching enzymes (SBE) which catalyse the formation of alpha-1,6-glucan linkages are of crucial importance for the quantity and quality of starch synthesized in plants. In maize (Zea mays L.), three SBE isoforms (SBEI, IIa and IIb) have been identified and shown to exhibit differential expression patterns. As a first step toward understanding the regulatory mechanisms controlling their expression, we isolated and sequenced a maize genomic DNA (-2190 to +5929) which contains the entire coding region of SBEI (Sbe1) as well as 5'-and 3'-flanking sequences. Using this clone, we established a complete genomic organization of the maize Sbe1 gene. The transcribed region consists of 14 exons and 13 introns, distributed over 5.7kb. A consensus TATA-box and a G-box containing a perfect palindromic sequence, CCACGTGG, were found in the 5'-flanking region. Genomic Southern blot analysis indicated that two Sbe1 genes with divergent 5'-flanking sequences exist in the maize genome, suggesting the possibility that they are differentially regulated. A chimeric construct containing the 5'-flanking region of Sbe1 (-2190 to +27) fused to the beta-glucuronidase gene (pKG101) showed promoter activity after it was introduced into maize endosperm suspension cells by particle bombardment. 1998 Elsevier Science B.V.

Autoregulation of the Plasmid Addiction Operon of Bacteriophage P1

The P1 plasmid addiction operon increases the apparent stability of a plasmid that carries it by killing plasmid-free (cured) segregants. The operon consists of a gene encoding an endotoxin responsible for death on curing (doc), preceded by a gene encoding a relatively unstable antidote that can prevent host death (phd). When the copy number of the operon was increased, expression of a lacZ reporter fused to the promoter of the operon decreased, indicating that expression of the operon was stabilized by an autoregulatory circuit. Transcription of the lacZ reporter was repressed about 10-fold when phd, without doc, was expressed from an exogenous promoter. DNase I footprinting showed that Phd binds a perfect 10-base pair palindromic DNA sequence and, at higher concentrations, an adjacent, imperfect palindrome. The palindromic sites are located between the -10 region of the putative promoter and the start codon of phd. Electrophoretic mobility of DNA containing the promoter region was retarded in the presence of Phd and further retarded in the presence of Phd and Doc. When doc was co-expressed with phd, repression of the lacZ fusion was enhanced more than 100-fold. Thus, both products of the addiction operon participate in its autoregulation.

Transcription termination at intrinsic terminators: the role of the RNA hairpin.

Intrinsic termination of transcription in Escherichia coli involves the formation of an RNA hairpin in the nascent RNA. This hairpin plays a central role in the release of the transcript and polymerase at intrinsic termination sites on the DNA template. We have created variants of the lambda tR2 terminator hairpin and examined the relationship between the structure and stability of this hairpin and the template positions and efficiencies of termination. The results were used to test the simple nucleic acid destabilization model of Yager and von Hippel and showed that this model must be modified to provide a distinct role for the rU-rich sequence in the nascent RNA, since a perfect palindromic sequence that is sufficiently long to form an RNA hairpin that could destabilize the entire putative 12-bp RNA-DNA hybrid does not trigger termination at the expected positions. Rather, our results show that both a stable terminator hairpin and the run of 6-8 rU residues that immediately follows are required for effective intrinsic termination and that termination occurs at specific and invariant template positions relative to these two components. Possible structural or kinetic modifications of the simple model are proposed in the light of these findings and of recent results implicating "inchworming" and possible conformational heterogeneity of transcription complexes in intrinsic termination. Thus, these findings argue that the structure and dimensions of the hairpin are important determinants of the termination-elongation decision and suggest that a complete mechanism is likely to involve specific interactions of the polymerase, the RNA terminator hairpin, and, perhaps, the dT-rich template sequence that codes for the run of rU residues at the 3' end of the nascent transcript.

DNA‐binding proteins for transcription enhancing region of HLA class I gene

Class I regulatory complex (CRC) located in the 5'-upstream region of MHC class I gene contains transcriptional enhancing sequences, called Enh A. This Enh A region contains tandem-arranged kappa B-like sites, one of which has a well-conserved perfect palindromic sequence. The second kappa B-like site, juxtaposed to the perfect palindrome, contains an imperfect palindromic sequence. In B-cell nuclear extracts, we have identified at least four sequence-specific protein complexes; three shared the repeated kappa B enhancer as their binding motifs. The perfect palindromic sequence facilities the binding of a complex termed BI, while the imperfect palindrome provides the binding sites for two other complexes, BII and BIII. The BII and BIII complexes exhibited binding crossreactivity with other kappa B-related motifs and recognized both the perfect and imperfect palindromic sequences, whereas the BI complex was specific for the perfect palindromic sequence which is unique to the class I promoters. A DNA segment outside the repeated kappa B enhancers probably binds the fourth complex, BIV. These complexes, except for the perfect palindrome-binding complex, differ from those described for the murine class I promoter. The binding characteristics of these factors suggest that the mechanism controlling the class I transcription may be quite complex.

DNA‐binding proteins for transcription enhancing region of HLA class I gene

Abstract: Class I regulatory complex (CRC) located in the 5′‐upstream region of MHC class I gene contains transcriptional enhancing sequences, called Enh A. This Enh A region contains tandem‐arranged κB‐like sites, one of which has a well‐conserved perfect palindromic sequence. The second κB‐like site, juxtaposed to the perfect palindrome, contains an imperfect palindromic sequence. In B‐cell nuclear extracts, we have identified at least four sequence‐specific protein complexes; three shared the repeated κB enhancer as their binding motifs. The perfect palindromic sequence facilitates the binding of a complex termed BI, while the imperfect palindrome provides the binding sites for two other complexes, BII and BIII. The BII and BIII complexes exhibited binding crossreactivity with other κB‐related motifs and recognized both the perfect and imperfect palindromic sequences, whereas the BI complex was specific for the perfect palindromic sequence which is unique to the class I promoters. A DNA segment outside the repeated κB enhancers probably binds the fourth complex, BIV. These complexes, except for the perfect palindrome‐binding complex, differ from those described for the murine class I promoter. The binding characteristics of these factors suggest that the mechanism controlling the class I transcription may be quite complex.

Characterization of the interaction of the human mineralocorticosteroid receptor with hormone response elements.

Although the mineralocorticosteroid receptor (MR) belongs to the superfamily of hormone-dependent transcription factors, the molecular mechanism by which it regulates gene expression is poorly understood. Binding of the MR to target gene promoters has never been characterized, and specific mineralocorticosteroid response elements (MREs) remain to be identified. The human MR (hMR) was overexpressed in Sf21 insect cells using the baculovirus system. The high degree of similarity between the glucocorticosteroid receptor (GR) and the MR prompted us to examine the DNA-binding properties of the recombinant MR with glucocorticosteroid-regulated genes. Gel shift mobility assays demonstrated that the recombinant receptor interacted with oligonucleotides containing perfect and imperfect palindromic sequences of GRE. A monoclonal anti-hMR antibody (FD4) induced a supershift of protein-DNA complexes and identified the MR in Western blot analysis. In vitro DNAse I protection assays with the hormone-regulated murine mammary tumour virus promoter showed that recombinant hMR generated four footprints whose limits encompassed the GRE motifs. By means of these two complementary approaches, no difference between the interaction of free, agonist- or antagonist-bound MR and DNA was detected. We provide evidence that hMR functions as a sequence-specific DNA-binding protein.

Human JC virus perfect palindromic nuclear factor 1-binding sequences important for glial cell-specific expression in differentiating embryonal carcinoma cells.

The brain cell specificity of the human papovavirus JC virus was examined by site-directed mutagenesis of the nuclear factor 1 (NF1) motifs within the viral regulatory region. The NF1 motif sites, located within the 98-bp tandem repeats that contain 6-bp perfect inverted palindromic sequences, were important for glial cell-specific expression of JC virus in differentiated embryonal carcinoma cells in vivo. The NF1 site on the late side of the repeats was not important, a fact confirmed by in vitro transcription studies. These observations were correlated with in vitro DNase I footprinting and mobility shift assays, which demonstrated specific interactions of factors in glial cell nuclear extracts with NF1 sites.

Two G-box-related sequences confer different expression patterns in transgenic tobacco.

We have analyzed the expression patterns conferred by two G-box-related motifs, a perfect palindromic sequence (PA, 5'-GCCACGTGGC-3') and motif I (Iwt, 5'-GTACGTGGCG-3'), in transgenic tobacco plants. A mutant version of motif I, Imu, was used as a negative control. PA is present in the promoters of several different genes, whereas Iwt is a conserved sequence found in abscisic acid-inducible promoters. Previously we have demonstrated that PA and Iwt, but not Imu, can bind to the tobacco transcription activator TAF-1 in vitro, with the PA sequence showing a 70-fold higher affinity as compared to Iwt. We found that tetramers of PA and Iwt, which differ by only 2 bp per 10-bp repeat, confer very different tissue-specific and expression patterns in transgenic tobacco plants. PA confers preferential expression in root tissues with a low level of activity in leaves, whereas Iwt directs developmentally regulated expression in seeds beginning 15 days after petals have fully expanded until seed maturation. Imu appears to be inactive because it gives the same expression pattern as the -90 cauliflower mosaic virus 35S promoter control. RNA gel blot analysis showed that the expression pattern of TAF-1 mRNA is similar to that directed by PA, suggesting that TAF-1 may be involved in the transcriptional regulation of PA.

Expression of luteinising hormone-β subunit chloramphenicol acetyltransferase (LH-β-CAT) fusion gene in rat pituitary cells: Induction by cyclic 3′-adenosine monophosphate (cAMP)

In this study we determined the activity of the rat luteinising hormone-β gene promoter in a heterologous rat pituitary cell line (GH 3 cells). 1.7 kb of LH-β 5′ flanking sequence and the first 5 bp of the 5′ untranslated region were ligated to the chloramphenicol acetyltransferase (CAT) receptor gene (LH-β-CAT) and transiently transfected by calcium phosphate precipitation into subconfluent cultures of GH 3 cells. Basal low-level CAT activity was only detected in GH 3 cells, being absent in two non-pituitary cell lines (BeWo and HeLa) RNase analysis revealed that mRNA from transfected GH 3 cells protected a fragment of labelled antisense probe of correct size for transcription initiation from the LH-β CAP site, confirming that promoter activity reflected correctly initiated LH-β-CAT fusion gene transcripts. CAT activity was consistently induced by an average of 3–5-fold from the full-length 1.7 kb promoter, in a dose- and time-dependent manner, by forskolin, dibutyryl cAMP, and 8-bromo cAMP implying presence of a cAMP-responsive cis-acting domain in the LH-β promoter region. Transfection of deletion mutants Δ-615-CAT, Δ-385-CAT and Δ-250-CAT each reduced forskolin inducibility to 1.7-fold but did not abolish induction completely suggesting a domain between −1.7 and −0.6 kb contained a cAMP-responsive element(s) (CRE). Further deletion of LH-β 5′ flanking sequences to Δ-85-CAT restored forskolin induction to wild-type levels (3–5-fold), suggesting the presence of a weak inhibitory element between −600 and −85 kb, and a cAMP-responsive domain in the proximal promoter region. The LH-β promoter does not contain perfect tandem repeat palindromic CRE DNA sequences, though there are several octanucleotide sequences differing by only 1 bp from AP-2 binding sites, the consensus CRE, and the vasointestinal peptide gene CRE. Although these data suggest that the LH-β gene is cAMP responsive this is likely mediated by several and complex protein interactions with multiple DNA sequences in the proximal and distal LH-β promoter enhancer.