Putative CAAT Boxes Research Articles

Cloning, characterization, and heterologous expression of a dextranase gene from Penicillium pinophilum SMCU3-14

A DNA fragment encoding dextranase was cloned fromPenicillium pinophilum SMCU3-14 using the genome walking approach. Sequence analysis of the gene (SMCU-DEX) revealed a putative CAAT box at 165 (CCAAT), a putative TATA box at 93 (TATAA) in the 5 0 -noncoding region, and a polyadenylation signal (AATAAG) in the 3 0 -noncoding region. A cDNA sequence analysis revealed no evidence of introns. The deduced open reading frame is 1824 bp in length and encodes a predicted protein of 608 amino acids (molecular weight (MW) of 66 kDa), with a putative N-terminal 20-amino acid signal peptide, giving a predicted mature protein of 588 amino acids (MW of 64 kDa) that belongs to glycosyl hydrolase family 49, as with other fungal dextranases. This is the first report of a dextranase gene sequence from P. pinophilum. The cDNA was cloned and expressed in Escherichia coli, and the transformants showed dextranase activity on a dextran-containing agar medium. Crude extracts from the transformants analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis containing blue dextran revealed a distinct specific band of dextranase activity at an MW of approximately 66 kDa. This recombinant dextranase is likely to have valuable and cost-effective applications in medicine and industry.

Cloning and characterization of ribonuclease T2 gene (RNHe30) from the basidiomycete, Hericium erinaceum

A gene encoding a ribonuclease T2 (RNase T2) family enzyme, RNHe30, was cloned from Hericium erinaceum by PCR. The deduced amino acid sequence from the complimentary DNA (cDNA) (1074 bp) encodes a 302-aa protein (RNase He30) that has the consensus amino acid sequences of RNase T2 family enzymes including the putative signal peptide. The presence of five introns in the genomic DNA was confirmed by comparison of the cDNA and genomic DNA sequences. The promoter region contains a putative CAAT box and a consensus TATA box. Genes coding homologous enzymes were also identified in various other basidiomycetes. A phylogenetic tree of RNase T2s from these fungi was constructed from a multiple alignment of the deduced amino acid sequences. The tree showed that the enzymes were divided into two main groups.

Transcriptional regulation of the rat apelin receptor gene: promoter cloning and identification of an Sp1 site necessary for promoter activity

The genomic structure and transcriptional regulation of the rat apelin receptor (APJR) were analysed by rapid amplification of 5' cDNA ends (5'-RACE), transient expression assays and DNA-protein interaction. Analysis of the 5'-flanking region of a rat genomic clone shows no TATA box, but a putative CAAT box and several putative binding sites for transcription factors are present. Two transcriptional start sites were identified by 5'-RACE, RNase protection and primer extension analyses. Promoter activity was exhibited in the APJR- expressing SH-SY5Y cell line as well as in COS-7 and Chinese hamster ovary (CHO-K1) cells. Consecutive 5'-deletion analysis revealed the highest promoter activity in a region between bp -966 and -165. DNaseI footprint analysis revealed seven protected regions and electrophoretic mobility shift, super-shift and competition assays identified individual DNA-protein complexes capable of binding Sp1, estrogen receptor (ER)alpha, glucocorticoid receptor and CCAAT enhancer binding protein (C/EBP)gamma transcription factors. Site-directed mutagenesis identified an individual Sp1 motif that plays a major role in activation of the APJR promoter and also demonstrated constitutive transcriptional regulation of the promoter by estrogen and glucocorticoid receptors. Promoter regulation by the cAMP-dependent signal cascade was also shown.

Characterization of a rice class II metallothionein gene: Tissue expression patterns and induction in response to abiotic factors

Data mining the complete rice genome sequences revealed a genomic fragment encoding a characteristic metallothionein (MT) protein, and its full-length cDNA was isolated from rice developing seeds by RT-PCR. This cDNA, designated OsMT-II-1a, contains an open reading frame of 264 bp encoding a protein of 87 amino acid residues. The predicted amino acid sequence was shown to have structural features characteristic of plant class II MT proteins. By sequence analysis of its 5'-flanking region, one putative TATA box, four putative CAAT boxes, and several short sequences homologous to previously reported regulatory cis-elements were identified. Northern blot analysis showed that accumulation of OsMT-II-1a mRNA is specifically abundant in developing seeds and 2-day glumes after pollination, and OsMT-II-1a transcription can markedly be induced by H2O2, paraquat, SNP, ethephon, ABA and SA, but barely by metal ions or other exogenous abiotic factors such as low temperature and PEG. These results coincide with the prediction of existing regulatory cis-elements in its 5'-flanking region. Taken together, the above results suggest that the processes of pollination and seed development might be mediated, at least in part, by expression of the OsMT-II-1a gene that is regulated by several abiotic factors.

Effect of the anilinopyrimidine fungicide pyrimethanil on the cystathionine β-lyase of Botrytis cinerea

Previous studies, carried out in our laboratory, upon the mode of action of anilinopyrimidines (APs) suggested that these fungicides inhibit the biosynthesis of methionine and that the primary target could be the cystathionine β-lyase (CBL). More recent works carried on with strains of Botrytis cinerea highly resistant to APs (Ani R1) suggested that one major gene ( Ani 1) confers this phenotype and that this gene segreged independently of the gene of resistance to dicarboximides. This confirmed the fact that the target site of APs must be different from that of dicarboximides and suggested that the mechanism of resistance to APs could be due to a mutation in Ani 1 gene. In this report, we tried to find if the CBL of B. cinerea could be the target site of the APs, pyrimethanil and cyprodinil. To do this, first we searched for an inhibitory effect of APs on the enzyme activity of the B. cinerea CBL and second, we compared the nucleotide sequences of the B. cinerea CBL gene, we called metC, of 3 strains sensitive to APs (Ani S) and 10 strains Ani R1. We showed that aminoethoxyvinylglycine at 2.5 μM strongly inhibited, in a competitive manner, a crude extract of the CBL isolated from a strain Ani S or from a strain Ani R1. On the other hand, APs, at 0.1 mM, slightly inhibited the CBL isolated from B. cinerea. This inhibitory effect which was uncompetitive was not different between the strain Ani S and the strain Ani R1. We sequenced and analysed the metC gene (Accession No.: AF211176). Based on the cDNA sequence, we found that this gene spans 1377 bp and is interrupted by one intron. We found consensus sequences for the putative CAAT-box, TATA-box, transcription-site, polyadenylation, and splicing signals. The metC gene codes for a polypeptide chain of 459 amino-acids and has a predicted molecular weight of 49,087 Da. The analysis of the sequence polymorphism in 13 strains did not allow us to discriminate between the sensitive strains and the resistant ones. So, it is assumed that the CBL of B. cinerea does not seem to be the primary target site of APs and probably does not correspond to the Ani 1 gene.

Analysis of the Brugia malayi HSP70 promoter using a homologous transient transfection system

Biolistic transient transfection of Brugia malayi embryos with constructs driving the expression of a luciferase reporter gene was used to identify regions of the upstream sequence of the heat shock protein 70 (HSP70) gene of B. malayi necessary for transgene expression. Analysis of 1160 nucleotides upstream of the start codon of the HSP70 gene identified several potentially important elements, including putative CAAT and TATA boxes, a core promoter domain, a polypurine stretch, and a spliced leader addition site. Nested deletion analysis of the HSP70 upstream domain mapped the promoter of the HSP70 gene to the region 396 to 31 nucleotides upstream of the start codon. This encompassed the putative CAAT and TATA boxes, and putative core promoter. Deletion of the putative CAAT box did not result in any diminution of reporter activity, while constructs in which the TATA box or core promoter were deleted retained roughly half of the activity of the undeleted construct. Unlike the native gene, transcripts derived from constructs containing the HSP70 upstream sequences were not trans-spliced. However, incorporation of the 495 nucleotides downstream of the start codon (encompassing exon 1, intron 1 and part of exon 2) resulted in the production of transcripts that were correctly cis- and trans-spliced. Similarly, a construct containing the 495 downstream nucleotides in which most of exon 1 was deleted, was correctly cis- and trans-spliced. This finding suggests that downstream intron sequences in addition to the splice leader addition site are necessary for trans-splicing in B. malayi.

Purification and properties of an extracellular exoinulinase from Penicillium sp. strain TN-88 and sequence analysis of the encoding gene.

An exoinulinase, P-I, was purified from the culture filtrate of Penicillium sp. strain TN-88 grown on inulin. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis with an apparent Mr of 81 kDa. The purified enzyme had extremely high specific activity, 743 U/mg, toward inulin. Inulinase activity was optimal at pH 4.0 and 55 degrees C. A genomic DNA and cDNAs encoding this protein were cloned and sequenced. The exoinulinase gene (inuD) was present as a single copy in the genome. An open reading frame of 2,106 bp was interrupted by a single intron of 56 bp, and encoded a 25-amino acid signal peptide and a 677-amino acid mature protein. The mature protein contained two Cys residues and eight potential N-linked glycosylation sites. The 5'-noncoding region had a putative CAAT box at position -239. Four distinct transcription start points were observed at positions -98 (A), -91 (A), -80 (A), and -76 (A) from the start codon. The exoinulinase gene inuD was located 860-bp upstream of the previously reported endoinulinase gene inuC in the opposite direction of transcription.

Cloning and sequence characteristics of the genomic gene of a rice metallothionein

Northern blot analysis showed that a metallothionein gene,ricMT, is expressed strongly in the stem of rice with an expression level that could be more than 100-fold stronger than in leaf blades. The results suggest that the 5′ upstream region flanking the coding sequence of thericMT may contain a fairly strong promoter. To elucidate its regulation and promoter structure, the genomic clones ofricMT were screened out from a rice genomic library and a fragment of about 4 084 bp was sequenced. The fragment included a 5′ upstream region of ca. 2 970 bp, a transcription region of ca. 690 bp and a 3′ downstream region of ca. 420 bp. Computer analysis of the sequence homology showed that the 5′ upstream region included a putative TATA box, a putative CAAT box, and a typical metal-responsive element TGCGCGCG. The results will promote further understanding of the mechanisms of gene regulation and metal response of plant metallothionein proteins.

Molecular cloning and nucleotide sequence of a gene encoding a cotton palmitoyl-acyl carrier protein thioesterase

A cotton genomic clone containing a 17.4-kb DNA segment was found to encompass a palmitoyl-acyl carrier protein (ACP) thioesterase ( Fat B1) gene. The gene spans 3.6 kb with six exons and five introns, and is apparently the first plant FatB acyl-ACP thioesterase gene to be completely sequenced. The six exons are identical in nucleotide sequence to the open reading frame of the corresponding cDNA, and would encode a preprotein of 413 amino acids. The preprotein can clearly be identified as a FatB acyl-ACP thioesterase from its similarity to the deduced amino acid sequences of other FatB thioesterase preproteins. A 5′-flanking region of 914 bp was sequenced, with the potential TATA basal promoter 324 bp upstream from the ATG initiation codon. The 5′-flanking sequence also has a putative CAAT box and two presumptive basic region helix-loop-helix (bHLH) elements with the consensus motif CANNTG (termed an E box), implicated as being a positive regulatory element in seed-specific gene expression.

Structure of the Human Type II 3β-Hydroxysteroid Dehydrogenase/Δ5-Δ4Isomerase (3β-HSD) Gene: Adrenal and Gonadal Specificity

While classical 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase deficiency (3 beta-HSD) is a known cause of adrenal hyperplasia resulting in ambiguous genitalia and adrenal insufficiency at birth, nonclassical or late-onset 3 beta-HSD deficiency is found in an important proportion of women with androgen excess. We have previously isolated and sequenced the cDNA and gene for the human type I 3 beta-HSD, which represents the main species expressed in the placenta and skin. Recently, we isolated, sequenced, and expressed the functional cDNA encoding type II 3 beta-HSD, which is the predominant 3 beta-HSD expressed in human adrenals and gonads. The present study describes the isolation and complete sequence of the corresponding type II 3 beta-HSD gene, which is the form most likely responsible for human 3 beta-HSD deficiency. The structural gene contains four exons of 57, 231, 165, and 1,214 bp, respectively, separated by introns of 128, 3,383, and 2,162 bp. DNA sequence analysis of the 5'-flanking region reveals the existence of two putative TATA boxes situated 28 and 140 nucleotides upstream from the transcription start site whereas two putative CAAT boxes are located 57 and 38 nucleotides upstream from the TATA boxes, respectively. A restriction fragment length pattern specific for each gene has been characterized. The present findings should provide the tools required for detailed analysis of the molecular basis of 3 beta-HSD deficiency as well as of normal sex steroid biosynthesis.

Genomic structure of human lysosomal glycosylasparaginase

The gene structure of the human lysosomal enzyme glycosylasparaginase was determined. The gene spans 13 kb and consists of 9 exons. Both 5′ and 3′ untranslated regions of the gene are uninterrupted by introns. A number of transcriptional elements were identified in the 5′ upstream sequence that includes two putative CAAT boxes followed by TATA-like sequences together with two AP-2 binding sites and one for Sp1. A 100 bp CpG island and several ETF binding sites were also found. Additional AP-2 and Sp1 binding sites are present in the first intron. Two polyadenylation sites are present and appear to be functional. The major known glycosylasparaginase gene defect G488→C, which causes the lysosomal storage disease aspartylglycosaminuria (AGU) in Finland, is located in exon 4. Exon 5 encodes the post-translational cleavage site for the formation of the mature α/β subunits of the enzyme as well as a recently proposed active site threonine, Thr206.

Multiple mRNAs of rat brain alpha-crystallin B chain result from alternative transcriptional initiation.

Two major classes of mRNAs for the alpha-crystallin B chain (or alpha(B)crystallin), about 0.9 and 1.2 kilobases in length, are expressed in rat brain. To examine the structures of these mRNAs, we isolated cDNA clones from rat brain and genomic DNA from rat liver. Characterization of these clones as well as Northern blot analysis indicated that the various mRNAs differed in the lengths of their 5' leader sequences. RNase protection assays revealed that the gene for alpha-crystallin B chain contains multiple start sites. The transcriptional start sites of the longer mRNAs are preceded by a putative CAAT box and that of the shorter mRNA by a putative TATA box. The shorter mRNA encodes the alpha-crystallin B chain protein, whereas the longer mRNA contained three extra small open reading frames upstream of the AUG start codon for the protein. The shorter mRNA is abundant in lens, heart, muscle, and kidney, while the longer mRNAs are constitutively expressed at low levels in a wide variety of tissues. The shorter mRNA was increased by treatment with phorbol 12-myristate 13-acetate in rat C6 glioma cells. Since there is only a single copy of the alpha-crystallin B chain gene, our results indicate that the two classes of mRNAs are generated by alternative transcriptional initiation from different promoters and their expressions are regulated differentially.

Cloning and functional analysis of the rat ornithine decarboxylase-encoding gene

We have isolated a functional gene ( ODC) encoding rat ornithine decarboxylase (ODC; EC 4.1.1.17) from a partial rat liver genomic DNA bank. The entire gene is located on a 7776-bp BamHI fragment and was shown to comprise twelve exons, of which ten encode the ODC protein (exons III–XII). Introduction of the BamHI fragment into an ODC-deficient hamster cell line restores ODC activity, indicating that the gene is functional. Comparison of the structure and nucleotide (nt) sequence of the rat ODC gene with recently reported mouse ODC genes, reveals that the gene is highly conserved. Primer extension analysis and RNA sequencing demonstrates that the transcription start point of rat ODC mRNA is located 303 nt upstream from the A residue in the start codon. Compared with our previously published sequence of the rat ODC cDNA, this indicates that a short sequence at the extreme 5′ end of our cDNA clone represents a cloning artefact. The correct 5′ leader of ODC mRNA, which is very G + C rich (62%), can be folded into a highly stable secondary structure, which may play a role in the translational control of ODC activity. Like in mouse, the promoter region of rat ODC is also extremely rich in G + C, and contains a TATA box and several putative SP1-binding sites. Possible binding sites for other transcription factors, like AP-1, AP-2 and CREB, can also be observed in the promoter region and, moreover, in the first intro. With the help of constructs containing different portions of the promoter region fused to the bacterial chloramphenicol acetyltransferase-encoding gene ( cat), the function and boundaries of the rat ODC promoter were analyzed. For transient expression in Rat-1 cells, sequences between 78 and 105 bp upstream of the cap site were found to be essential. In this region, only a putative CAAT box (CCGAT) is observed. Deletion of 4 nt within this sequence motif clearly diminished the promoter activity, thereby lending further support to its functional significance.

Structure and Expression of the Rice Glutelin Multigene Family

A near full-length cDNA and three genomic clones for rice (Oryza sativa L.) glutelin were isolated and studied. Based on nucleic acid sequence and Southern blot analyses, the three isolated glutelin genomic clones were representative members of three gene subfamilies each containing five to eight copies. A comparison of DNA sequences displayed by relevant regions of these genomic clones showed that two subfamilies, represented by clones Gt1 and Gt2, were closely related and evolved by more recent gene duplication events. The 5'-flanking and coding sequences of Gt1 and Gt2 displayed at least 87% homology. In contrast, Gt3 showed little or no homology in the 5'-flanking sequences upstream of the putative CAAT boxes and exhibited significant divergence in all other portions of the gene. Conserved sequences in the 5'-flanking regions of these genes were identified and discussed in light of their potential regulatory role. The derived primary sequences of all three glutelin genomic clones showed significant homology to the legume 11 S storage proteins indicating a common gene origin. A comparison of the derived glutelin primary sequences showed that mutations were clustered in three peptide regions. One peptide region corresponded to the highly mutable hypervariable region of legume 11 S storage proteins, a potential target area for protein modification. Expression studies indicated that glutelin mRNA transcripts are differentially accumulated during endosperm development. Promoters of Gt2 and Gt3 were functional as they direct transient expression of chloramphenicol acetyltransferase in cultured plant cells.

Plant expression signals of the Agrobacterium T-cyt gene.

Within the 5' and 3' non-coding regions of the T-cyt gene from the octopine T-DNA of Agrobacterium tumefaciens sequences required for expression of this gene in plant cells were identified by deletion mutagenesis. The results show that 184 bp of the 5' non-coding region and 270 bp of the 3' non-coding region are sufficient for wild-type expression. Within the 5' non-coding region two essential expression signals were identified: (1.) an activator element located between -185 and -129 with respect to the ATG start codon and (2.) one out of two TATA boxes. Deletions of the activator element or the two TATA boxes resulted in nonfunctional genes. Deletion of the upstream TATA box and both putative CAAT boxes did not significantly affect expression. Within the 3' non-coding region, the polyadenylation box most distal to the stop codon was not essential for expression, but sequences more upstream, including a second polyadenylation box were found to be required for wild-type expression.