[show abstract][hide abstract] ABSTRACT: Pterin-4alpha-carbinolamine dehydratase (PCD) is a key enzyme in the regeneration pathway of tetrahydrobiopterin. Previously, we isolated and reported the Drosophila melanogaster gene encoding PCD. In the present study, we isolated and characterized the Drosophila virilis gene encoding PCD. The Drosophila virilis PCD gene has two introns and an open reading frame to encode a protein of 101 amino acids. The amino acid sequence of Drosophila virilis PCD shows a 83% homology to that of the Drosophila melanogaster PCD protein. From the alignment of the nucleotide sequence in the 5'-flanking region of the Drosophila melanogaster and Drosophila virilis PCD genes, we found four conserved sequences. Using a transient transfection assay, we showed that one of the conserved sequences (-127 to approximately -115) is critical for expression, also the minimal promoter region between -127 and +51 is necessary for the efficient expression of Drosophila melanogaster PCD.
Molecules and Cells 11/2001; 12(2):233-8. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: The promoter region of the Drosophila melanogaster TFIIS gene was characterized by transient expression assay. Serial deletion analysis of the promoter region showed that the promoter region between -112 and +113 is required for the efficient expression of the D. melanogaster TFIIS gene. The results also suggest that the DNA fragments between -112 and -54 and between +94 and +113 contain the vital elements for the expression. The importance of these fragments was further substantiated by the findings that the sequences in these fragments of the D. melanogaster TFIIS gene are conserved in the 5'-flanking regions of the Drosophila virilis TFIIS gene. The comparison of the nucleotide sequences in the 5'-flanking region of the D. melanogaster and D. virilis TFIIS genes revealed that the three regions, -85--59, +76-+126, and the vicinity of the transcription initiation site of the D. melanogaster TFIIS gene, are conserved. It is very interesting that the long downstream DNA between +76 and +126 is highly conserved with 90% identities between the two species. The downstream promoter region between +94 and +113 of the D. melanogaster TFIIS gene was further analyzed by transient expression and band mobility shift assays. The results obtained suggest that the region between +94 and +113 is probably recognized by nuclear factors and that the sequence (+98)AGTAAACAACAT(+109) seems to make a great contribution to promoter activity of the D. melanogaster TFIIS gene.
Biochimica et Biophysica Acta 05/2001; 1518(3):276-81. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: The TATA box binding protein (TBP) is a general transcription factor required for initiation by all three eukaryotic RNA polymerases. Previously, we found that the promoter region of the Drosophila melanogaster TBP gene contains three sequences similar to the DNA replication-related element (DRE) (5'-TATCGATA). In the present study, we found that the DRE-like sequences are also present in the promoter of the Drosophila virilis TBP gene, suggesting a role for these sequences in TBP expression. Band mobility shift assays revealed that oligonucleotides containing sequences similar to the DRE of D. melanogaster TBP gene promoter form specific complexes with a factor in a Kc cell nuclear extract and with recombinant DRE-binding factor (DREF). Furthermore, these complexes were either supershifted or diminished by monoclonal antibodies to DREF. Transient luciferase assays demonstrated that induction of mutations in two DRE-related sequences at positions -223 and -63 resulted in an extensive reduction of promoter activity. Thus, the DRE-DREF system appears to be involved in the expression of the D. melanogaster TBP gene.
[show abstract][hide abstract] ABSTRACT: We have isolated and characterized a Drosophila melanogaster gene encoding the sepiapterin reductase (SR). The gene does not have introns. The 5'- and 3'-RACE analysis, which determined the transcription start point (tsp) and polyadenylation site, respectively, showed that the gene produces single mRNA species. The potential promoter region lacks distinct TATAAA or CCAAT box consensus sequences. RNA blot analysis revealed that the gene encodes a 1.4kb transcript that could be detected throughout development and in both heads and bodies of adults. The Drosophila SR gene maps to 15A on the X chromosome.
[show abstract][hide abstract] ABSTRACT: The Drosophila trp gene encodes a light-activated Ca(2+) channel subunit, which is a prototypical member of a novel class of channel proteins. Previously identified trp mutants are all recessive, loss-of-function mutants characterized by a transient receptor potential and the total or near-total loss of functional TRP protein. Although retinal degeneration does occur in these mutants, it is relatively mild and slow in onset. We report herein a new mutant, Trp(P365), that does not display the transient receptor potential phenotype and is characterized by a substantial level of the TRP protein and rapid, semi-dominant degeneration of photoreceptors. We show that, in spite of its unusual phenotypes, Trp(P365) is a trp allele because a Trp(P365) transgene induces the mutant phenotype in a wild-type background, and a wild-type trp transgene in a Trp(P365) background suppresses the mutant phenotype. Moreover, amino acid alterations that could cause the Trp(P365) phenotype are found in the transmembrane segment region of the mutant channel protein. Whole-cell recordings clarified the mechanism underlying the retinal degeneration by showing that the TRP channels of Trp(P365) are constitutively active. Although several genes, when mutated, have been shown to cause retinal degeneration in Drosophila, the underlying mechanism has not been identified for any of them. The present studies provide evidence for a specific mechanism for massive degeneration of photoreceptors in Drosophila. Insofar as some human homologs of TRP are highly expressed in the brain, a similar mechanism could be a major contributor to degenerative disorders of the brain.
Journal of Neuroscience 02/2000; 20(2):649-59. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Previously we showed that the 5'-flanking regions between -261 and -207 of the Drosophila melanogaster TBP (TATA box binding protein) gene is important for its expression. We further made serial deletion mutants in this region and analyzed their promoter activities using the transient transfection assay. We found that the 16 bp deletion from -261 to -245 greatly reduces the promoter activity of the Drosophila TBP gene. The 16 bp DNA element contains half of a 11 bp long palindromic sequence, CTTTT-GAAAAG. Disruption of the palindromic sequence by site-directed mutagenesis severely affected promoter activity. In addition, the electrophoretic mobility shift assay showed that the oligonucleotide containing the palindromic sequence can make specific DNA/protein complexes when it was mixed with the Drosophila nuclear extract, suggesting that it interacts with nuclear protein(s). Our data suggest that the palindromic sequence has a critical role in the expression of the Drosophila TBP gene.
Molecules and Cells 01/2000; 9(6):673-7. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have isolated and characterized a novel Drosophila melanogaster gene (noe) that is specifically and abundantly expressed in the central nervous system (CNS). The gene, which maps to 74B on the left arm of third chromosome, encodes a protein of 74 amino acids with no significant similarity to known protein sequences. The deduced amino acid sequence of the gene product is rich in basic amino acids, especially the lysine, and contains five potential phosphorylation sites. The noe gene lacks introns and seems to produce two transcripts by alternative polyadenylations. The promoter region deduced from 5'-RACE analysis contains a sequence similar to the TATA-box consensus sequence. RNA blot analysis detected 1.0 kb noe transcripts that are expressed from the third-instar larval stage to the adult stage and which are predominantly found in the adult heads. In situ hybridizations to tissue sections showed that the gene is abundantly expressed in neuronal cell bodies as well as in the neurophiles of adult and larval CNS (brain, optic lobe, and thoracic ganglia of adults and larval brain).
Molecules and Cells 01/1999; 8(6):750-7. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have analyzed two tandem promoters, separated by only about 400 bp, of the purple (pr) gene of Drosophila melanogaster, by fusing them to the firefly luciferase reporter gene and employing a transient expression assay with Drosophila S2 cells. Both the distal promoter and the proximal promoter were found to function in S2 cells and an about 700 bp long region (-270 to +421), containing both promoters, was sufficient to effect maximal promoter activity. When the two promoters were analyzed separately, the distal promoter was found to be much stronger in its function than the proximal promoter. At least three different kinds of cis elements near the transcription start site appear to play crucial roles in driving constitutive expression from the distal promoter. On the other hand, only a single cis element, which may play a role in tissue-specific expression, appears to be important for the activity of the proximal promoter in S2 cells. We propose that the clustering of important cis elements near the transcription start sites may be responsible for the selective regulation of the two tandem promoters.
Molecules and Cells 01/1999; 8(6):685-90. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have analyzed the 5'-flanking regions of the Drosophila genes encoding the TFIIB and TATA box-binding proteins (TBP) and mapped the regulatory regions required for their efficient expression. We found that the 500 bp long region (-439 to +60) and the 401 bp long region (-261 to +138) are required for the efficient expression of TFIIB and TBP genes, respectively. In the TFIIB promoter region, the upstream DNA between -439 and -280 and the downstream DNA between +8 and +60 are necessary for the stimulation of promoter activity. The upstream DNA between -439 and -280 stimulates transcription in an orientation dependent manner. In the TBP promoter region, the upstream DNA between -261 and -207, and the downstream DNA between +15 and +138 are necessary for the stimulation of promoter activity. The upstream DNA (-261 to -207) required for TBP promoter activity contains a 11 bp long palindromic sequence and a DNA replication-related element sequence. Particularly, we could find that the downstream promoter regions of TFIIB and TBP genes contain the conserved nucleotide sequences, suggesting the presence of a common regulatory mechanism for the expression of these two genes.
Molecules and Cells 01/1999; 8(6):770-6. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have isolated and characterized the cDNA encoding Drosophila melanogaster sepiapterin reductase (SR). The amino acid sequence deduced from the cDNA sequence was 29% identical to those of mammalian SRs. The active site residues proposed from the three-dimensional structure of mouse SR are well conserved in Drosophila SR. The protein-coding region of the cDNA was expressed in Escherichia coli as a histidine fusion protein, and the resulting recombinant protein proved to have SR activity. The SR activity of the recombinant protein was inhibited by two indoleamines, N-acetyl serotonin and melatonin. Southern analysis suggests that the Drosophila SR gene is encoded by a single copy gene. RNA blot analysis revealed that the gene expresses 1.5 kb mRNA in both adult heads and bodies.
Biochimica et Biophysica Acta 12/1998; 1443(1-2):239-44. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have isolated and characterized the cDNA and the genomic DNA encoding Drosophila melanogaster pterin 4alpha-carbinolamine dehydratase (PCD). The amino acid sequence deduced from the cDNA sequence was very similar to those of PCDs previously reported in other species (19-57% identity). The protein coding region of the cDNA was expressed in E. coli as a histidine fusion protein, and the expressed protein proved to have PCD activity. The characterization of the Drosophila genomic clone revealed that the Drosophila PCD gene is interrupted by two introns. The potential promoter region, deduced from the determination of the transcription start point (tsp), lacks the distinct TATAAA box consensus sequence.
Biochimica et Biophysica Acta 11/1998; 1388(1):273-8. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have characterized the bovine and the human cDNAs encoding the NAP-22 (22 kDa neuronal tissue-enriched acidic protein) homologs. Both bovine and human cDNAs encode proteins of 227 amino acids. The deduced amino acid sequences of the bovine and the human proteins are 63% and 65% identical, respectively, to that of rat NAP-22 protein, strongly suggesting that both the cDNAs characterized encode NAP-22 proteins. They also share 45% and 41% amino acid sequence identities with chicken CAP-23 (23 kDa cytoskeleton associated protein). Several important protein motifs, including myristoylation and phosphorylation sites, are well conserved in sequences and positions in all three mammalian NAP-22 proteins and chicken CAP-23 proteins. The bovine cDNA was characterized further. Southern analysis of the bovine genomic DNA suggests that the bovine NAP-22 protein is encoded by a single-copy gene. RNA blot analysis revealed that the bovine gene for NAP-22 protein encodes a 1.7 kb transcript that is present only in the brain. Our data suggest that the four proteins, bovine and human NAP-22 homologs, rat NAP-22, and chicken CAP-23, have homologous functions in different organisms.
Molecules and Cells 09/1998; 8(4):471-7. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have sequenced the 5'-flanking region of the Drosophila melanogaster norpA gene and characterized its promoter. The potential promoter region, which was deduced from the determination of the transcription start point (tsp), lacks a distinct TATA box sequence. Deletion analysis of the promoter region suggests that the minimal promoter necessary for efficient expression of the gene is located between -138 (PstI) and +278 relative to the tsp. Within this minimal promoter region, at least two downstream regulatory elements responsible for the stimulation of gene expression seem to exist in the DNA fragments between +44 and +121 and between +214 and +278. Among these, the DNA fragment between +44 and +121 affects promoter activity more dramatically (about 6-7 fold). This DNA fragment contains the consensus promoter element previously reported to be important for photoreceptor cell-specific expression, and this promoter element seems to be working in the norpA gene expression.
Molecules and Cells 01/1998; 7(6):795-9. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have isolated and characterized a genomic clone encoding the Drosophila melanogaster transcription factor IIB (TFIIB). The coding region of the TFIIB gene is interrupted by three short introns. The 5'-flanking region of the gene lacks the typical TATA box sequence like those of other known genes encoding the general transcription factors. In addition, the 5'-flanking region of the gene contains several common DNA sequences present in Drosophila TBP and TFIIS genes, suggesting the common regulation mechanism of gene expression. RNA blot analysis revealed that the gene expresses 1.6 kb, 1.3 kb and 1.2 kb mRNAs throughout development and in adults. Deletion analysis of the promoter region shows that the minimal promoter necessary for efficient expression is located between -698 (PstI) and +60 relative to the transcription start point. Within this minimal promoter region, the upstream regulatory element responsible for the stimulation of gene expression may exist in the DNA fragment between -698 (PsfI) and -351 (StuI).
Molecules and Cells 07/1997; 7(3):374-9. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have characterized a genomic clone encoding the Drosophila melanogaster transcriptional elongation factor, TFIIS. The coding region of the TFIIS gene is interrupted by a short intron. The potential promoter region, deduced from the determination of the transcription start point (tsp), lacks distinct TATAAA or CCAAT box consensus sequences. Southern analysis and the in situ hybridization to chromosomes suggests that it is single-copy gene which is localized to the 35B region on the left arm of the second chromosome.
Biochimica et Biophysica Acta 06/1995; 1262(1):99-103. · 4.66 Impact Factor