Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “regulatory sequence design”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources
PubMed1998

CPRF4a, a novel plant bZIP protein of the CPRF family: comparative analyses of light-dependent expression, post-transcriptional regulation, nuclear import and heterodimerisation.

Several DNA-binding proteins with conserved basic region/leucine zipper domains (bZIP) have been isolated from parsley. They all recognise defined ACGT-containing elements (ACEs), including ACE(PcCHSII) in the Light Regulatory Unit LRU1 of the CHS promoter which confers light responsiveness. A new member of this Common Plant Regulatory Factor (CPRF) family, designated CPRF4a, has been cloned, which displays sequence similarity to HBP-1a from wheat, as well as to other plant bZIP proteins. CPRF4a specifically binds as a homodimer to ACE(PcCHSII) and forms heterodimers with CPRF1 but not with CPRF2. In adult parsley plants, CPRF2 and CPRF4a mRNAs are found in all tissues and organs in which the chalcone synthase gene CHS is expressed. In protoplasts from suspension cultured cells, UV irradiation (290-350 nm) did not cause an increase in levels of CPRF1, CPRF2, or CPRF4a mRNA, whereas the corresponding CPRF proteins accumulated within 15 min of light treatment. Furthermore, the rapid light-mediated increase of CPRF proteins was insensitive to transcriptional inhibitors, suggesting that a post-transcriptional mechanism controls CPRF accumulation. CPRFs as well as Arabidopsis thaliana G-box binding factors (GBFs) are selectively transported from the cytosol into the nucleus, as shown in an in vitro nuclear transport system prepared from evacuolated parsley protoplasts, indicating that cytosolic compounds are involved in regulated nuclear targeting of plant bZIP factors.

Acyltransferases↗
PubMed1999

Recognizing the pleckstrin homology domain fold in mammalian phospholipase D using hidden Markov models.

Phospholipase D was first described in plant tissue but has recently been shown to occur in mammalian cells where it is activated by cell surface receptors. Its mode of activation by receptors in unclear. Biochemical studies suggest that it may occur downstream of other effector proteins and that small GTP-dependent regulatory proteins may be involved. The sequence in a non-designated region of mammalian phospholipase D1 and 2 shows similarity to a structural domain that is present in signalling proteins that are regulated by protein kinases or heterotrimeric G-proteins. Mammalian phospholipase D has structural similarities with other lipid signalling phospholipases and thus may be regulated by receptors in an analogous fashion.

Adaptor Proteins, Signal Transducing↗
PubMed1995

MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data.

The identification of potential regulatory motifs in new sequence data is increasingly important for experimental design. Those motifs are commonly located by matches to IUPAC strings derived from consensus sequences. Although this method is simple and widely used, a major drawback of IUPAC strings is that they necessarily remove much of the information originally present in the set of sequences. Nucleotide distribution matrices retain most of the information and are thus better suited to evaluate new potential sites. However, sufficiently large libraries of pre-compiled matrices are a prerequisite for practical application of any matrix-based approach and are just beginning to emerge. Here we present a set of tools for molecular biologists that allows generation of new matrices and detection of potential sequence matches by automatic searches with a library of pre-compiled matrices. We also supply a large library (> 200) of transcription factor binding site matrices that has been compiled on the basis of published matrices as well as entries from the TRANSFAC database, with emphasis on sequences with experimentally verified binding capacity. Our search method includes position weighting of the matrices based on the information content of individual positions and calculates a relative matrix similarity. We show several examples suggesting that this matrix similarity is useful in estimating the functional potential of matrix matches and thus provides a valuable basis for designing appropriate experiments.

Base Sequence↗
PubMed1989

Osteocalcin: characterization and regulated expression of the rat gene.

An osteocalcin gene was isolated from a rat genomic DNA library, and sequence analysis indicated that the mRNA is represented in 953 nucleotide segment of DNA consisting of 4 exons and 3 introns. Although the introns in the rat gene are larger, its overall organization is similar to the human gene. Analysis of the 5' flanking sequences of the rat gene shows a modular organization of the promotor as reflected by the the presence of at least 3 classes of regulatory elements. These include (1) typical sequences associated with most genes transcribed by RNA polymerase II (e.g. TATA, CAAT, AP1, AP2), (2) a series of consensus sequences for cyclic nucleotide responsive elements and several hormone receptor binding-sites (estrogen, thyroid and clusters of AG-rich putative Vitamin D responsive elements); and (3) a 24 nucleotide highly conserved sequence between the rat and human gene having a CAAT motif as a central element, designated as an "osteocalcin box." Two regulatory factors of osteocalcin gene expression have been identified. First, contained within the 600 nucleotides immediately upstream from the transcription initiation site are sequences which support Vitamin D dependent transcription of the rat osteocalcin gene. 1,25(OH)2D3 increases osteocalcin mRNA by 6-20 fold increases. In contrast, up to a 200 fold increase in osteocalcin gene expression occurs with mineralization of the extracellular matrix produced by osteoblasts. We propose osteocalcin is a bone-specific marker protein of the mature osteoblast in a mineralizing matrix.

Animals↗
PubMed2001

A silencer inhibitor confers specific expression of intestinal trefoil factor in gobletlike cell lines.

Intestinal trefoil factor (ITF) is selectively expressed in intestinal goblet cells. Previous studies identified cis-regulatory elements in the proximal promoter of ITF, but these were insufficient to recapitulate the exquisite tissue- and cell-specific expression of native ITF in vivo. Preliminary studies suggested that goblet cell-specific expression of murine ITF requires elements far upstream that include a silencer element that effectively prevents ITF expression in non-goblet cells. Transient transfection studies using native or mutant ITF 5'-flanking sequences identified a region that restores expression in goblet cells. This element, designated goblet cell silencer inhibitor (GCSI) element, enables human and murine goblet cell-like cell lines to override the silencing effect of more proximal elements. The GCSI has no intrinsic enhancer activity and regulates expression only when the silencer element is present. Ligation of GCSI and silencer elements to sucrase-isomaltase conferred goblet cell-specific expression. Goblet cells but not non-goblet cells possess a nuclear protein that binds to the GCSI regulatory element (GCSI binding protein; GCSI-BP). Both transient transfection and gel mobility shift assay studies localize the GCSI and GCSI-BP to -2216 to -2204. We conclude that goblet cell-specific transcription of ITF in vivo depends on a regulatory element designated GCSI.

Animals↗
PubMed1994

Characterisation of cDNA and genomic clones encoding homologues of the 65 kDa regulatory subunit of protein phosphatase 2A in Arabidopsis thaliana.

Two cDNA species encoding sequences homologous to the 65 kDa regulatory subunit (PR 65) of protein phosphatase 2A (PP2A) have been isolated from an Arabidopsis thaliana cDNA library. These were designated pDF1 and pDF2. pDF1 is 1795 bp long and by comparison with the human and porcine PP2A regulatory subunit sequences represents a full-length clone. It encodes a predicted polypeptide of 587 amino acid residues. pDF2 is truncated at the 5' end by 237 bp. The complete nucleotide sequences have been determined for both cDNA species. Comparison of the nucleotide and the deduced amino acid sequences showed that the two sequences were homologous but not identical and therefore must be derived from two different genes. Northern blot analysis was performed on total RNA and poly(A)+ RNA isolated from seed at various stages of development and from young leaf material of Brassica napus L. (oilseed rape). Both cDNA probes hybridised to a single major mRNA species of ca. 2.2 kb. The highest level of expression was observed in the total RNA from developing rape seed at about 33 days after flowering, and the transcript level in the poly(A)+ RNA of the seed was higher than in young leaf of oilseed rape. Southern blot analysis was performed on two varieties of A. thaliana and B. napus genomic DNA; this identified a small family of genes in A. thaliana consisting of at least 2 or 3 members and a larger multigene family in B. napus of at least 5 or 6 members. Two independent genomic clones were isolated from an A. thaliana genomic library. Sequencing of a fragment common to both revealed that the sequence was identical in both clones and, therefore, they were assumed to contain the same genomic sequence. The genomic sequence selected, designated regA, is 3639 bp long and the coding sequence contains eleven introns. The gene encodes a predicted polypeptide of 590 amino acid residues. The sequence comparison with both cDNA sequences showed that it is homologous but not identical to the two, confirming that at least three different genes exist in A. thaliana which encode PR65 of PP2A.

Amino Acid Sequence↗
PubMed1991

Genomic structure of murine macrophage inflammatory protein-1 alpha and conservation of potential regulatory sequences with a human homolog, LD78.

The gene for a murine macrophage inflammatory cytokine, MIP-1 alpha, belongs to a newly recognized superfamily encoding small, inducible peptides shown to be up-regulated in association with cellular activation or transformation (tentatively designated the scy, or small cytokine, gene family). Secreted scy family peptides as a group, and MIP-1 alpha in particular, have inflammatory and mitogenic activities, and the family has been divided into CXC and CC subfamilies according to the spacing of conserved cysteine residues in the primary amino acid sequences. We have isolated and characterized a genomic clone encoding the CC subfamily member MIP-1 alpha. The organization of the murine MIP-1 alpha gene into three exons interrupted by two introns is identical to that found for other members of the CC subfamily (e.g., huLD78, muJE, huJE/MCP-1, muTCA3, and hul-309), which has been taken as evidence of evolution from a common ancestral gene. With the exception of the ratPF4 gene, which shares the two-intron/three-exon pattern typical of the CC subfamily, sequenced genes encoding CXC subfamily peptides (e.g., hulL-8 and hulP-10) include an additional intervening sequence that creates a fourth exon. Genomic nucleotide sequences 5' of the MIP-1 alpha cap site are highly homologous to corresponding regions of the human gene encoding a CC peptide variously designated as LD78/GOS19/pAT464, including consensus regulatory motifs in common, reinforcing the contention that MIP-1 alpha and LD78 may be interspecies homologs.

Amino Acid Sequence↗
PubMed2006

[Functional analyses of TcrA-a TPR-containing regulatory protein in Streptomyces coelicolor A3(2)].

In Streptomyces coelicolor A3(2), SCO5433 encodes a TPR domain containing protein designated TcrA (TPR containing regulator A). TcrA is similar in amino acid sequence to AfsR, a well-characterized global regulatory protein in S. coelicolor A3(2). Disruption of tcrA enhanced the production of spore pigment on MM containing glucose or mannitol and also resulted in more diffusible pigment production on MM containing mannitol or MS agar medium, but no significant effects on morphological differentiation were observed. Complementation of tcrA mutation restored the phenotype of tcrA mutant to that of the wild-type strain. These results suggest that tcrA is involved in the regulation of secondary metabolism under defined conditions. In S. coelicolor A3(2), the AfsK/AfsR system positively regulates the production of secondary metabolites while the results of our work suggest that there might be a TcrA-dependent pathway that negatively regulates secondary metabolism.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequencing and characterization of Pseudomonas putida catR: a positive regulator of the catBC operon is a member of the LysR family.

Pseudomonas putida utilizes the catBC operon for growth on benzoate as a sole carbon source. This operon is positively regulated by the CatR protein, which is encoded from a gene divergently oriented from the catBC operon. The catR gene encodes a 32.2-kilodalton polypeptide that binds to the catBC promoter region in the presence or absence of the inducer cis-cis-muconate, as shown by gel retardation studies. However, the inducer is required for transcriptional activation of the catBC operon. The catR promoter has been localized to a 385-base-pair fragment by using the broad-host-range promoter-probe vector pKT240. This fragment also contains the catBC promoter whose -35 site is separated by only 36 nucleotides from the predicted CatR translational start. Dot blot analysis suggests that CatR binding to this dual promoter-control region, in addition to inducing the catBC operon, may also regulate its own expression. Data from a computer homology search using the predicted amino acid sequence of CatR, deduced from the DNA sequence, showed CatR to be a member of a large class of procaryotic regulatory proteins designated the LysR family. Striking homology was seen between CatR and a putative regulatory protein, TfdS.

Amino Acid Sequence↗
PubMed2001

Protein phosphatase 2A: identification in Oryza sativa of the gene encoding the regulatory A subunit.

A 2225 bp cDNA, designated RPA1, was isolated from an Oryza sativa cDNA library. Analysis revealed a 1761 bp coding sequence with 15 non-identical repeat units. The ORF encoded the A regulatory subunit of protein phosphatase 2A (PP2A-A) as ascertained by complementation of the yeast tpd3 mutant defective in this gene. The corresponding genomic DNA from a rice genome BAC library revealed that the gene contains eleven introns. The rice genome contains only a single copy of this gene as judged by Southern blot analysis. The PP2A protein is highly conserved in nature; the rice protein shows 88% amino acid identity with its counterparts in Arabidopsis or Nicotiana tabacum.

Amino Acid Sequence↗
PubMed1987

Sequence-specific interactions of a pea nuclear factor with light-responsive elements upstream of the rbcS-3A gene.

Pea nuclear extracts were used in gel retardation assays and DNase I footprinting experiments to identify a protein factor that specifically interacts with regulatory DNA sequences upstream of the pea rbcS-3A-gene. This factor, designated GT-1, binds to two short sequences (boxes II and III) in the -150 region that are known to function as light-responsive elements (LREs) in transgenic tobacco. Binding of GT-1 to homologous sequences further upstream (boxes II and III in the -220 region) indicates that these boxes comprise the redundant LRE that functions in vivo when boxes II and III are deleted. In both box II and box II, methylation interference experiments demonstrate that two adjacent G residues are critical for GT-binding. Single Gs present in boxes III and III are also important. Since GT-1 is present in nuclear extracts from leaves of light-grown and dark-adapted pea plants, its regulatory role does not depend on de novo synthesis. Thus if GT-1 binds differentially in vivo it must be postranslationally modified or sterically blocked from binding by another factor in response to light.

Base Sequence↗
PubMed1997

Specific interactions between the K domains of AG and AGLs, members of the MADS domain family of DNA binding proteins.

MADS domain (for MCM1, AG, DEFA and SRF) proteins are regulatory proteins found in all major eukaryotic kingdoms. Plant MADS domain regulatory proteins have a region of moderate sequence similarity that has been designated as the K domain, and its predicted coiled-coil structure suggests a role in establishing a protein-protein interaction. In vivo studies with the Arabidopsis AGAMOUS (AG) protein have indicated that the K domain is important for AG function. Using a bait fusion protein containing the K domain and the C-terminal region of AG in a yeast two-hybrid selection, 156 clones that encode potential AG-interacting proteins were identified. These clones each encode one of four highly related MADS domain proteins: AGL2, AGL4, AGL6 and AGL9. Additional analysis showed that the K domain of AG alone was able to bind the K domains of these AGLs. This binding was further confirmed by immunoprecipitation experiments using in vitro synthesized AG and AGL K domains. These results strongly suggest that AG interacts with AGL2, AGL4, AGL6 and AGL9 in vivo. Based on these results and previous observations, it is proposed that the AG function requires interaction with at least one of these AGL proteins, and such interactions contribute to the functional specificity of the AG protein.

AGAMOUS Protein, Arabidopsis↗
PubMed1998

A transcriptional regulator of the LuxR-UhpA family, FlcA, controls flocculation and wheat root surface colonization by Azospirillum brasilense Sp7.

Genetic complementation of a spontaneous mutant, impaired in flocculation, Congo red binding, and colonization of root surface, led to the identification of a new regulatory gene in Azospirillum brasilense Sp7, designated flcA. The deduced amino acid sequence of flcA shared high similarity with a family of transcriptional activators of the LuxR-UphA family. The most significant match was with the AgmR protein, an activator for glycerol metabolism in Pseudomonas aeruginosa. Derivatives of Sp7 resulting from site-directed Tn5 mutagenesis in the flcA coding sequence were constructed by marker exchange. Characterization of the resulting mutant strains showed that flcA controls the production of capsular polysaccharides, the flocculation process in culture, and the colonization of the root surface of wheat. This study provides new information on the genetic control of the mechanism of plant root colonization by Azospirillum.

Amino Acid Sequence↗
PubMed2001

Identification of polymorphisms in the 5'-untranslated region of the TAFI gene: relationship with plasma TAFI levels and risk of venous thrombosis.

BACKGROUND AND OBJECTIVES: Thrombin activatable fibrinolysis inhibitor (TAFI) plays an important role in hemostasis, functioning as a potent fibrinolysis inhibitor. TAFI gene variations may contribute to plasma TAFI levels and thrombotic risk. DESIGN AND METHODS: We sequenced a 2083-bp region of the 5'-regulatory region of the TAFI gene in 127 healthy subjects searching for variations, and correlated identified polymorphisms with plasma TAFI levels. TAFI polymorphisms were examined as risk factors for venous thrombosis by determining their prevalence in 388 patients with deep venous thrombosis (DVT) and in 388 controls. RESULTS: Seven novel polymorphisms were identified: -152 A/G, -438 A/G, -530 C/T, -1053 T/C, -1102 T/G, -1690 G/A, and -1925 T/C. -152 A/G, -530 C/T and -1925 T/C were found to be in strong linkage disequilibrium, as were the -438 A/G, -1053 T/C, -1102 T/G and -1690 G/A. Plasma TAFI levels were higher in -438GG/-1053CC/-1102GG/-1690AA homozygotes than in -438AG/-1053TC/-1102TG/-1690GA heterozygotes, and -438AA/-1053TT/-1102TT/-1690GG homozygotes had the lowest TAFI levels (p=0.0003). TAFI concentrations in -152AA/-530CC/-1925TT homozygotes were somewhat higher but not significantly different from levels observed for -152AG/-530CT/-1925TC heterozygotes. Taken in combination, -438AG/-1053TC/-1102TG/-1690GA and -438AA/-1053TT/-1102TT/-1690GG yielded an OR for DVT of 0.8 (95%CI: 0.6-1). In subjects aged <35 years the OR was 0.7 (95%CI: 0.5-1.1). The OR for -152AG/-530CT/-1925TC was 1 (95%CI: 0.5-2.2) in the whole group of patients and controls, whereas in subjects aged <35 years the OR was 0.1 (95%CI: 0.02-0.9). INTERPRETATION AND CONCLUSIONS: Polymorphisms in the TAFI promoter determine plasma antigen levels and may influence the risk of venous thrombophilia.

5' Untranslated Regions↗
PubMed2001

Characterisation of the pncA gene in Mycobacterium tuberculosis isolates from Gauteng, South Africa.

SETTING: The use of pyrazinamide (PZA) is important for the treatment of Mycobacterium tuberculosis as it is bactericidal to semi-dormant mycobacteria that are not affected by other drugs. The incidence of resistance to PZA and other drugs used in the treatment of M. tuberculosis is increasing in South Africa. OBJECTIVE: To characterise the pncA gene of M. tuberculosis isolates from Gauteng, South Africa, and to develop a rapid diagnostic method. DESIGN: The pncA gene and the putative regulatory gene were characterised by sequence analysis in a total of six PZA susceptible and 15 resistant isolates. The association with classical PZA susceptibility testing and PZase activity was determined. RESULTS: All PZA-resistant isolates were PZase negative as well as resistant to at least one other anti-tuberculosis drugs. Mutations were identified throughout the length of the pncA gene in 10/15 PZA-resistant isolates. Five lacked PZase activity, but the wild type pncA sequence was present. In all six PZase-positive strains, a PZA-susceptible pattern was obtained on BACTEC and the wild type pncA sequence was present. CONCLUSION: Sequencing is an effective means to identify mutations in the pncA gene in M. tuberculosis and therefore resistance to PZA. The fact that some PZA-resistant M. tuberculosis isolates lack mutations in the pncA gene suggests that alternative mechanisms for drug resistance exist. In PZase negative strains with no genetic changes which are resistant to 100 microg/ml and susceptible to 300 microg/ml, 300 microg/ml may be a more reliable breakpoint.

Africa, Southern↗
PubMed1999

BTEB2, a Krüppel-like transcription factor, regulates expression of the SMemb/Nonmuscle myosin heavy chain B (SMemb/NMHC-B) gene.

We have recently characterized the promoter region of the rabbit embryonic smooth muscle myosin heavy chain (SMemb/NMHC-B) gene and identified the 15-bp sequence, designated SE1, located at -105 from the transcriptional start site as an important regulatory element for its transcriptional activity in a smooth muscle cell (SMC) line. In this study, we attempted to isolate cDNA clones encoding for the transcription factors that control the expression of the SMemb gene through binding to this cis-regulatory element. We screened a lambdagt11 cDNA library prepared from C2/2 cells, a rabbit-derived SMC line, by using a radiolabeled concatenated oligonucleotide containing SE1 as a probe. Sequence analysis revealed that one of the cDNA clones corresponds to the rabbit homologue of basic transcriptional element binding protein-2 (BTEB2), which has previously been identified as one of the Krüppel-like transcription factor. Gel mobility shift assays and antibody supershift analyses with nuclear extracts from C2/2 cells indicate that BTEB2 is a major component of nuclear factor:SE1 complexes. Furthermore, a glutathione S-transferase-BTEB2 fusion protein binds to the SE1 in a sequence-specific manner. In support of the functionality of BTEB2 binding, basal promoter activity and BTEB2-induced transcriptional activation were markedly attenuated by the disruption of the SE1. In adult rabbit tissues, BTEB2 mRNA was most highly expressed in intestine, urinary bladder, and uterus. BTEB2 mRNA levels were downregulated in rabbit aorta during normal development. Moreover, immunohistochemical analysis indicated a marked induction of BTEB2 protein in the neointimal SMC after balloon injury in rat aorta. These results suggest that BTEB2 mediates the transcriptional regulation of the SMemb/NMHC-B gene and possibly plays a role in regulating gene expression during phenotypic modulation of vascular SMC.

Age Factors↗
PubMed2003

DNA vaccines: improving expression of antigens.

DNA vaccination is a relatively recent development in vaccine methodology. It is now possible to undertake a rational step-by-step approach to DNA vaccine design. Strategies may include the incorporation of immunostimulatory sequences in the backbone of the plasmid, co-expression of stimulatory molecules, utilisation of localisation/secretory signals, and utilisation of the appropriate delivery system, for example. However, another important consideration is the utilisation of methods designed to optimise transgene expression. In this review we discuss the importance of regulatory elements, kozak sequences and codon optimisation in transgene expression.

Journal Article↗
PubMed1994

High-copy suppression of glucose transport defects by HXT4 and regulatory elements in the promoters of the HXT genes in Saccharomyces cerevisiae.

HXT4, a new member of the hexose transporter (HXT) family in Saccharomyces cerevisiae was identified by its ability to suppress the snf3 mutation in multicopy. Multicopy HXT4 increases both high and low affinity glucose transport in snf3 strains and increases low and high transport in wild-type strains. Characterization of HXT4 led to the discovery of a new class of multicopy suppressors of glucose transport defects: regulatory elements in the promoters of the HXT genes. We have designated these sequences DDSEs (DNA sequence dependent suppressing element). Multicopy HXT4 and DDSEs in the HXT1, HXT2, HXT3 and HXT4 promoters were found to restore growth to snf3 and grr1 strains on low glucose media. The DDSE in the HXT4 promoter was refined to a 340-bp sequence 450 bp upstream of the HXT4 translational start. This region was found to contain an 183-amino acid open reading frame. Extensive analysis indicates that the DNA sequence itself and not the encoded protein is responsible for suppression. The promoters of SNF3 and of other glycolytic genes examined did not suppress snf3 in multicopy. Suppression of snf3 by DDSE is dependent on the presence of either HXT2 or HXT3.

Amino Acid Sequence↗