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PubMed1993

Psoralen-modified oligonucleotide primers improve detection of mutations by denaturing gradient gel electrophoresis and provide an alternative to GC-clamping.

Denaturing gradient gel electrophoresis (DGGE), a mutation-scanning procedure separating DNA fragments differing by as little as a single base change, is widely used in studies of genomic nucleotide sequence variability. The efficiency of the technique is greatly enhanced by attaching, through polymerase chain reaction (PCR) incorporation, a long GC-tail to the test DNA sequence which, as a result, becomes analysable throughout. As synthesis of GC-rich specific PCR primers is costly and time-consuming, we attempted to clamp the DNA fragment using a psoralen derivative (ChemiClamp) that promotes photo-induced cross-linking at one end. We found that this procedure provides an attractive alternative to GC-clamp in DGGE (and temperature gradient gel electrophoresis) and should prove useful in both research and diagnostic laboratories.

Cross-Linking Reagents↗
PubMed2000

Investigation of the human serotonin 6 [5-HT6] receptor gene in bipolar affective disorder and schizophrenia.

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter that mediates a wide range of central nervous functions by activating multiple 5-HT receptor subtypes. A possible irregularity of serotonergic neurotransmission has been implicated in a variety of neuropsychiatric diseases. In the present study, we performed a systematic mutation scan of the complete coding region and splice junctions of the 5-HT(6) receptor gene to explore the contribution of this gene to the development of bipolar affective disorder and schizophrenia. Investigating 137 unrelated individuals (including 45 bipolar affective patients, 46 schizophrenic patients, and 46 unrelated controls), we identified six single base substitutions (126G/T, 267C/T, 873+30C/T, 873+128A/C, 1128G/C, 1376T/G). Comparing frequencies between patients and controls, we observed a significant overrepresentation of the 267C allele among bipolar patients (P=0. 023 not corrected for multiple testing). This finding was followed up in an independent sample of 105 bipolar family trios using a family-based association design. Fifty-one transmissions could be examined. In 30 cases allele 267C and in 21 cases allele 267T were transmitted to the affected offspring. Although this result was far from statistical significance (transmission disequilibrium test=1.59, P=0.208), the limited number of possible transmissions may have prevented detection of smaller effects. Our preliminary data suggest that bipolar affective disorder may be associated with variation in the 5-HT(6) gene. It will be important to extend the present analysis to larger samples. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:217-221, 2000.

Bipolar Disorder↗
PubMed2006

Brain derived neurotrophic factor (BDNF) gene variants and Alzheimer's disease, affective disorders, posttraumatic stress disorder, schizophrenia, and substance dependence.

Genetic variation at the locus encoding the brain derived neurotrophic factor (BDNF) has been implicated in some neuropsychiatric disorders such as Alzheimer's disease (AD), affective disorders (AFDs), schizophrenia, and substance dependence. We therefore performed a mutation scan of the BDNF gene to identify novel gene variants and examined the association between BDNF variants and several neuropsychiatric phenotypes in European American (EA) subjects and controls. Using denaturing high performance liquid chromatography (dHPLC), we identified a novel variant (G-712A) in the putative promoter region. This variant and two previously reported BDNF SNPs (C270T and Val66Met) were genotyped in 295 patients with AD, 108 with AFDs, 96 with posttraumatic stress disorder (PTSD), 84 with schizophrenia, 327 with alcohol and/or drug dependence, and 250 normal control subjects. No association was found between these three BDNF gene variants and AD, AFDs, PTSD, or schizophrenia. However, there was a nominally higher frequency of the G-712A G-allele and the G/G genotype in subjects with substance dependence than in controls (Allele: chi(2) = 4.080, df = 1, P = 0.043; Genotype: chi(2) = 7.225, df = 2, P = 0.027). Although after correction for multiple testing, the findings are not considered significant (threshold P-value was set at 0.020 by the program SNPSpD), logistic regression analyses confirmed the modest association between SNP G-712A and substance dependence, when the sex and age of subjects were taken into consideration. The negative results for AFDs, PTSD, and schizophrenia could be due to the low statistical power. Further study with larger samples is warranted.

Adult↗
PubMed1998

Adenosine A1 receptor and bipolar affective disorder: systematic screening of the gene and association studies.

In the present study we sought to identify genetic variation in the adenosine A1 receptor (A1AR) gene on chromosome 1q31-32.1, which through alteration of protein function or level of expression might contribute to the genetic predisposition to bipolar affective disorder. We performed a systematic mutation scan of the whole coding sequence as well as 5' and 3' untranslated regions by means of single-strand conformation analysis. The region upstream to the coding sequence we investigated contains two functional promoters. Screening 42 patients with bipolar affective disorder, we detected 11 DNA sequence variants (48T/A, 267 + 275C/T, 805T/G, 1777C/A, 1827C/T, 1904C/T, 2126G/T, 2294insT, 2776C/T, 2777del36, 2819T/G). Determining the frequency of these variants in 42 anonymous blood donors, we observed a non-significant (P < 0.06) trend towards an underrepresentation of the 2126T variant in patients when compared to controls. On the other hand, the 2777del36 and the 2819G variant were not found among the controls. These findings were followed up in a large independent replication sample. However, we were not able to confirm the initial findings in the second sample. Our data suggest that genetically determined variation of the A1AR and its two promoters do not play a major role in the development of bipolar affective disorder.

Base Sequence↗
PubMed2000

Regulation of the promoter for human immunoglobulin gamma3 germ-line transcription and its interaction with the 3'alpha enhancer.

The mechanism underlying the differential regulation of switching to human IgG subclasses is still largely unknown. We demonstrate that the region upstream of the initiation sites for gamma3 germ-line (GL) transcripts contains a functional promoter which is synergistically induced by IL-4, antibody to CD40 and phorbol dibutyrate in transient transfection assays in the human DG75 cell line. Linker-scanning mutations identified multiple elements in the 3' half of the evolutionarily conserved sequence that are required for inducibility. Electrophoretic mobility shift assays showed that Stat6 and NF-kappaB p50 / p65 are induced after stimulation, and bind to specific sequence motifs within the promoter. Overexpression of Stat6, NF-kappaB p50 / p65 and C / EBPgamma synergistically induced the GL gamma3 promoter. Insertion of DNA segments from the human 3' IgH regions, which may function as a locus control region for switch recombination, greatly activated the promoter in an orientation-independent manner. Duplication of the enhancer fragments resulted in a further increase of promoter activity. The greater enhancement of the HS1,2 fragment from the 3' alpha1 rather than the alpha2 locus may suggest a mechanistic explanation for the differential expression of various isotypes.

B-Lymphocytes↗
PubMed2002

Single-strand conformation polymorphism-based analysis of mitochondrial cytochrome c oxidase subunit 1 reveals significant substructuring in hookworm populations.

Sequence heterogeneity in a portion of the mitochondrial cytochrome c oxidase subunit 1 gene (pcox1) was measured for the hookworms, Ancylostoma caninum from Australia, A. duodenale from China, and Necator americanus from China and Togo using single-strand conformation polymorphism (SSCP) analysis combined with DNA sequencing. The pcox1 sequences were characterised for individual nematodes displaying genetic variation within each of the three species, and those were compared with pcox1 sequences of four other species of hookworm. While intraspecific variation in the pcox1 sequence ranged from 0.5 to 8.6% for A. caninum, 0.3 to 3.3% forA. duodenale, and 0.3 to 4.3% for N. americanus, interspecific differences varied from 4.8 to 12.9%. Sequence data also provided information on nucleotide compositions and substitution patterns. Genetically distinct groups were detected within A. caninum and A. duodenale, indicating significant population substructuring within these species. Also, N. americanus individuals from China all differed from those from Togo at four nucleotide positions, supporting a previous proposal (based on ribosomal DNA sequence data) that N. americanus may represent a species complex. The findings indicated the value of pcox1 sequence data and the mutation scanning approach for studying the genetic structures of hookworm populations, which should have important epidemiological relevance.

Ancylostomatoidea↗
PubMed2002

An update on conformation sensitive gel electrophoresis.

Conformation-sensitive gel electrophoresis (CSGE) was developed as a method of heteroduplex analysis to screen large multi-exon genes for sequence variation. The novelty of the method was in the use of a non-proprietary acrylamide gel matrix that used 1,4-bis (acrolyl) piperazine (BAP) as a cross linker with ethylene glycol and formamide as mildly denaturing solvents. The denaturing environment enhances the conformation polymorphism present in DNA heteroduplexes containing variations as small as single nucleotide polymorphisms (SNPs). CSGE has also been adapted for use on a fluorescent platform (F-CSGE) that resulted in higher throughput and sensitivity. Variation in sensitivity of CSGE has been studied extensively. The results demonstrate that the nature of the mismatched base in a defined sequence context has the most profound effect on the conformation of the heteroduplex. Additionally, the size of the PCR product, as well as the location of the mismatch within the PCR product, are two important parameters that determine the resolution of the mismatch-containing heteroduplexes during CSGE. Like any other mutation scanning technique, CSGE can have limited resolution of two closely linked sequence variations. For specific genes, like BRCA1 and BRCA2 where multiple SNPs are present in the coding sequence, each CSGE shift has to be sequenced to define the exact nature of the sequence change. In conclusion, CSGE scanning provides a powerful, cost-efficient way to scan genes with high sensitivity and specificity.

Base Sequence↗
PubMed1985

The adenovirus-2 early EIIa transcription unit possesses two overlapping promoters with different sequence requirements for EIa-dependent stimulation.

The EIa-inducible, EIIa transcription unit of adenovirus-2 is transcribed early in infection from two start sites (+1 or EIIaE1 and -26 or EIIaE2), neither of which is preceded by canonical TATA box elements. Analysis of promoter deletion and linker scanning mutations for in vivo transcriptional activity after transfection into HeLa cells has indicated the existence of two overlapping promoters in the EIIaE gene. Two regions, each approximately 30 nucleotides upstream from start sites EIIaE1 and EIIaE2, function as TATA box substitutes. A sequence centered at position -42 (with respect to the major start site at position +1) is essential for transcription from both sites, while an element further upstream, localized between nucleotides -91 and -62, is also required for efficient EIIaE transcription, with the 3' border being dispensable for EIIaE2 transcription. Analysis of the entire series of EIIaE mutants, co-transfected with an EIa-containing plasmid, revealed that no unique sequence elements in the EIIaE1 promoter region between -97 and +1 were responsible for the stimulation of EIIaE1 transcription by EIa. In contrast, the EIa-mediated augmentation of EIIaE2 template activity was mainly dependent upon a sequence, the 5'-TTAAATTT-3' putative TATA box substitute, located around position -59.

Adenoviridae↗
PubMed1992

Regulation of transcription of immunoglobulin germ-line gamma 1 RNA: analysis of the promoter/enhancer.

Antibody class switching is achieved by recombinations between switch (S) regions which consist of tandemly repeated sequences located 5' to Ig heavy chain constant (CH) region genes. RNA transcripts from specific unrearranged or germ-line Ig CH genes are induced in IgM+ B cells prior to their undergoing class switch recombination to the same CH genes. Thus, the antibody class switch appears to be directed by induction of accessibility, as assayed by transcription of germ line CH genes. For example, IL-4 induces transcripts from the mouse germ-line C gamma 1 and C epsilon genes to which it also directs switch recombination. We report here that the 150 bp region upstream of the first initiation site of RNA transcribed from the murine germ-line C gamma 1 gene, contains promoter and enhancer elements responsible for basal level transcription and inducibility by anti-Ig phorbol myristate acetate (PMA) and for synergy of these inducers with IL-4 in a surface IgM+ B cell line, L10A6.2 and a surface IgG2a+ B cell line, A20.3. Linker-scanning mutations demonstrated that multiple interdependent elements are required for inducibility by PMA and also for synergy with IL-4. Within the 150 bp region are several consensus sequences that bind known or putative transcription factors, including a C/EBP binding site--IL-4 responsive element, four CACCC boxes, a PU box, a TGF beta inhibitory element (TIE), an alpha beta-interferon response element (alpha beta-IRE) and an AP-3 site. The relationship between transcription regulated by these elements and the regulation of endogenous germ-line gamma 1 transcripts and switching to IgG1 is discussed.

Animals↗
PubMed1993

Analysis of gene expression in mouse preimplantation embryos demonstrates that the primary role of enhancers is to relieve repression of promoters.

Enhancers are generally viewed simply as extensions of promoters, lacking a function of their own. However, previous studies of mouse preimplantation embryos revealed that 1-cell embryos can utilize enhancer-responsive promoters efficiently without an enhancer, whereas 2-cell embryos require an enhancer to achieve the same levels of expression. This suggested that enhancers relieved a repression in 2-cell embryos that is absent in 1-cell embryos. Results presented here demonstrate first that the ability of 1-cell embryos to dispense with enhancers does not result from the absence of specific activation proteins. Under conditions where GAL4-VP16 activated a GAL4-dependent promoter in both embryos, GAL4-VP16 activated a GAL4-dependent enhancer only in 2-cell embryos. Moreover, the role of an enhancer is not to compensate for either changes in promoter requirements, or for reduced levels of promoter-specific transcription factors. Linker-scanning mutations in a natural promoter revealed that both embryos utilized the same promoter elements, and comparison of different promoters revealed that these embryos have equivalent transcriptional capacities. In addition, titration experiments revealed less Sp1 activity in 1-cell embryos where enhancers are dispensable than in 2-cell embryos where enhancers are required. Therefore, we propose that the primary function of enhancers, first evident with formation of a mouse 2-cell embryo, is to prevent repression of weak promoters, probably by altering chromatin structure. Consistent with this hypothesis is the fact that butyrate, an agent that alters chromatin structure, stimulated promoters in 2-cell embryos, but not in 1-cell embryos.

Animals↗
PubMed2001

Relation between the flexibility of the WPD loop and the activity of the catalytic domain of protein tyrosine phosphatase SHP-1.

The conserved WPD loop of protein tyrosine phosphatases play an important role in the catalytic activity and the invariant aspartate residue acts as a general acid/base catalyst in the dephosphorylation reaction. In our previous report, we have demonstrated that the catalytic activities of the PTPs are influenced by the flexibility and stability of the WPD loop in its active "open" conformation [Yang et al., 1998]. Phosphatases with a more flexible WPD loop generally have higher specific activity. In this report, we modify the WPD loop of SHP-1 by alanine-scan mutation of the residues flanking the loop and measure their effects on the catalytic activity of the phosphatase. We show that the S418A, V424A, S426A, E427A, and P428A mutants increase the phosphatase activity, possibly due to the increased flexibility of the WPD loop, whereas the L417A, L417G and P425A mutants decrease its phosphatase activity. In addition, we propose that the two-proline residues in the WPD loop (Pro(420) and Pro(425) in SHP-1) work as pivotal points through a conserved hydrophobic network and allows residues between the pivotal points to have maximum flexibility in enhancing the phosphatase activity. Furthermore, our data suggest that the hydrolysis of the phosphoryl-cysteine intermediate, not its formation, is the rate-limiting step with p-nitrophenyl phosphate as the substrate while both the steps are rate-limiting with phosphotyrosine as the substrate.

Alanine↗
PubMed1995

Human adenosine A1 receptor gene: systematic screening for DNA sequence variation and linkage mapping on chromosome 1q31-32.1 using a silent polymorphism in the coding region.

Adenosine is a major inhibitory neuromodulator in the central nervous system. One of the receptors mediating the central effects of adenosine is the adenosine A1 receptor. We performed a systematic mutation scan of the coding region of the adenosine A1 receptor gene to explore its variability in the general population. Investigating 40 unrelated healthy subjects by single-strand conformation analysis no sequence changes of likely functional relevance were observed. We detected, however, a frequent T to G substitution at nucleotide position 716 which constitutes the first variant described in an adenosine receptor gene. It was used for fine scale linkage mapping of the A1 gene. Employing a polymerase-chain-reaction-based restriction assay, we genotyped 7 CEPH families (Centre d'Etude du Polymorphisme Humaine) and mapped the receptor in a gene cluster around the renin gene on chromosome 1q31-32.1. In addition, we utilized the 716T/G polymorphism to demonstrate biallelic expression of the adenosine A1 receptor gene in adult human brain.

Adult↗
PubMed1997

Human 5-HT5A receptor gene: systematic screening for DNA sequence variation and linkage mapping on chromosome 7q34-q36 using a polymorphism in the 5' untranslated region.

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter that mediates a wide range of sensory, motor, and cortical functions by activating multiple 5-HT receptor subtypes. In the present study we performed a systematic mutation scan of the complete coding region of the 5-HT5A receptor to explore its variability in the general population. Investigating 46 unrelated healthy subjects by single-strand conformation analysis no sequence changes of likely functional relevance were observed. The detection of a frequent G-->C substitution at position -19 was used for fine scale linkage mapping of the 5-HT5A gene. Employing a polymerase-chain-reaction based assay we genotyped 7 CEPH families (Centre d'Etude du Polymorphisme Humaine) and mapped the receptor to genetic markers on chromosome 7q34-q36.

Chromosome Mapping↗
PubMed1997

Genome amplification and long-distance movement functions associated with the central domain of tobacco etch potyvirus helper component-proteinase.

The tobacco etch potyvirus (TEV) helper component-proteinase (HC-Pro, 460 amino acid residues) is a multifunctional protein involved in aphid-mediated transmission, genome amplification, polyprotein processing, and long-distance movement. To investigate the interrelationships between three of these functions, 25 alanine-scanning mutations affecting clusters of charged residues were introduced into the HC-Pro coding sequence. The resulting mutants were analyzed with respect to HC-Pro proteolytic activity in vitro, genome amplification in protoplasts, and long-distance movement in tobacco plants. Three classes of mutants were identified. Class I mutants (total of 17) were capable of genome amplification, long-distance movement, and HC-Pro proteolysis with efficiencies similar to parental virus. The class III mutant (total of 1) encoded a proteolytically debilitated HC-Pro and was replication-defective. Class II mutants (total of 7) encoded proteolytically active HC-Pro, but each exhibited a suppressed amplification phenotype that was characterized by a progressive shutoff during the course of infection in protoplasts. The class II mutants also exhibited defects in long-distance movement, accumulating to relative levels of 0 to 7.5% in noninoculated tissue. Wild-type HC-Pro supplied in trans was able to partially rescue the class II mutant amplification defects in protoplasts and long-distance movement defects in plants, although the extent of complementation of movement function varied for each mutant. Six of the seven class II mutations affected the central region of HC-Pro between residues 126 and 300, whereas only one affected the C-terminal proteolytic domain. These results indicate that the central region of HC-Pro is necessary for efficient genome amplification and long-distance movement, and that the one or more HC-Pro functions involved in these processes is at least partially trans-active. Additionally, the long-distance movement properties of a previously characterized HC-Pro-defective mutant (TEV-GUS/CCCE) were characterized further using grafted nontransgenic and HC-Pro-expressing transgenic plants. The results indicated that HC-Pro is required in both inoculated and noninoculated tissues to complement the TEV-GUS/CCCE movement defects.

Alanine↗
PubMed2006

Robustness of single-base extension against mismatches at the site of primer attachment in a clinical assay.

DNA genotyping is important for epidemiological and clinical studies and diagnosis for individuals. Genotyping error can strongly influence the outcome of such investigations. One possible reason for genotyping error is additional DNA sequence variation, which can lead to allelic dropout. Based on a published study where allelic dropout occurred in genotyping the cholesteryl ester transfer protein TaqIB polymorphism by a TaqMan-based method, we investigated the susceptibility of the single-base extension (SBE)-based GenoSNIP method to additional sequence variation at the primer attachment site. SBE genotyping was applied to 147 patient samples with known alleles and to synthetic SBE templates. Variables were positions of nucleotide mismatches, yield of SBE reactions, primer design, and ratio of alleles in the template. No allelic dropout occurred when genotyping the TaqIB polymorphism regardless of the reported nucleotide mismatch. Yields of SBE assays critical for allelic dropout were decreased in the presence of the reported nucleotide mismatch depending on SBE assay design. In a systematic mutation scan, only the position immediately adjacent to the polymorphism caused allelic dropout under standard conditions. Depending on SBE assay design, changes in allelic ratio due to a nucleotide mismatch were similar in appearance to changes due to sample mixture or copy number variation. In conclusion, we found the SBE genotyping assays to be relatively robust against interfering DNA variations. The importance of appropriate design and validation of assays, especially in regard to critical yields and potentially interfering nucleotide mismatches, should be emphasized particularly in clinical settings. Care should be taken when interpreting observed changes in the allelic ratio, which could be caused by nucleotide mismatches, sample mixtures, or copy number variation.

Alleles↗
PubMed2002

Molecular simulation reveals structural determinants of the hanatoxin binding in Kv2.1 channels.

The carboxyl terminus of the S3 segment (S3C) in voltage-gated potassium channels was suggested to be the binding site of gating modifier toxins like hanatoxin. It has also been proposed to have a helical secondary structural arrangement. The currently available structures in high resolution for such channel molecules are restricted to regions illustrating the pore function. Therefore no further direct experimental data to elucidate the detailed mechanism for such toxin binding can be derived. In order to examine the putative three-dimensional structure of S3C and to analyze the residues required for hanatoxin binding, molecular simulation and docking were performed, based on the solution structure of hanatoxin and the structural information from mutational scanning data for the S3C fragment in Kv2.1. Our results indicate that hydrophobic and electrostatic interactions are both utilized to stabilize the toxin binding. Precise docking residues and the appropriate orientation for binding regarding amphipathic environments are also described. Compared with the functional data proposed by previous studies, the helical structural arrangement for the C-terminus of the S3 segment in voltage-gated potassium channels can therefore be further emphasized and analyzed. The possible location/orientation for toxin binding with respect to membrane distribution around the S3C segment is also discussed in this paper.

Binding Sites↗
PubMed1991

Analysis of sequence elements important for the synthesis and control of ribosomal RNA in E coli.

The regulation of the synthesis of ribosomal RNA is a key problem for the understanding of bacterial growth. Many different regulatory mechanisms involving cis and trans acting components participate in a concerted way to achieve the very efficient, flexible and coordinated production of this class of molecules. We have studied three different sequence regions within a ribosomal RNA transcription unit which are believed to control different stages of ribosomal RNA expression. In the first part of the study the function of AT-rich sequences upstream of the -35 hexamer of rRNA promoter P1 in the activation of rRNA transcription was analyzed. We confirm that a sequence dependent bend upstream of P1 is responsible for the high promoter activity. Experiments employing linker scanning mutations demonstrated that the distance as well as the angular orientation of the bent DNA is crucial for the degree of activation. In addition, the effect of the trans activating protein Fis on the transcription initiation of promoter P1 was investigated. We can show, using the abortive initiation assay, that the predominant effect of Fis is due to an increase in the affinity of RNA polymerase for the promoter (binding constant KB) while the isomerisation rate (kf) from a closed to an open RNA polymerase promoter complex is not altered significantly. We also describe the characterization of sequence determinants important for stringent regulation and growth rate control. Evidence is provided that the discriminator motif GCGC is a necessary but not sufficient element for both types of control. Furthermore we show that not simply a particular DNA primary structure but the higher order conformation of the complete promoter region is recognized and triggers the two regulatory mechanisms, both of which are apparently mediated by the effector molecule guanosine tetraphosphate (ppGpp). Finally, we have carried out a systematic mutational analysis of the rrnB leader region preceding the structural gene for 16S RNA. We could demonstrate that highly conserved sequence elements within the rrnB leader, which were believed to be involved in transcription antitermination have post-transcriptional functions. We present evidence that these sequence elements direct the biogenesis of active ribosomal particles.

Base Sequence↗
PubMed2004

Identification of the functional elements in the promoter region of human DNA topoisomerase IIIbeta gene.

In this study, we have isolated and characterized the promoter region of the human DNA topoisomerase IIIbeta (hTOP3beta) gene. The 5' RACE assay showed a short exon 1 encoding only the 35-bp untranslated region and suggested the presence of multiple transcription initiation sites. The hTOP3beta gene promoter lacks a canonical TATA box or initiation element and is moderately high in GC content. Transient expression of a luciferase reporter gene under the control of serially deleted 5'-flanking sequence identified an activator element between -141 and -119 upstream of the transcription initiation site and a second regulatory element between -91 and -71. On the basis of scanning mutations of triple nucleotides, we demonstrated that a 5'GGAACC3' element between -117 and -112 plays a critical role in the up-regulation of the basal transcription activity. Changing the 5'GGAACC3' sequence leads to markedly reduced promoter activity. Gel mobility shift assays revealed that the 5'GGAACC3' element is required for DNA binding by the transcription factor complex. These observations lead to the conclusion that the positive regulatory region including the 5'GGAACC3' core element is essential for efficient expression of the hTOP3beta gene as well as for the binding of as yet unidentified regulatory factor(s).

5' Flanking Region↗