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The nucleotide sequence of a 1.4-kb region in the 3'-flanking region of the murine c-fos oncogene has been determined. This region contains an estrogen-response element which is located almost 5 kb downstream from the c-fos promoter.
BACKGROUND: Toxoplasma gondii (T. gondii) exhibits substantial genetic diversity, and different parasite lineages are associated with distinct epidemiological distributions and biological characteristics. Accurate molecular characterization of T. gondii strains is important for understanding parasite population structure and transmission patterns. However, existing genotyping approaches often require multiple loci, extensive experimental procedures, or complex data analysis. Therefore, simplified and reliable molecular markers for rapid lineage differentiation are still needed. METHODS: In this study, comparative genomic analysis was performed using representative T. gondii strains with well-defined genetic backgrounds and virulence phenotypes. The ROP18 genomic region, including partial genomic sequences, 5' flanking regions, coding sequence (CDS), and 3' flanking regions, was analyzed to identify informative polymorphic signatures. A short conserved sequence region containing lineage-associated polymorphic sites was identified within the ROP18 3' flanking region. Based on these sequence signatures, HRM-PCR and TaqMan MGB probe-based real-time PCR assays were developed and evaluated using plasmid standards and representative T. gondii genomic DNA samples. RESULTS: Phylogenetic analyses based on different ROP18 genomic regions demonstrated distinct clustering patterns among analyzed strains. Although the ROP18 3' flanking region was highly conserved, a short conserved sequence region containing informative polymorphic sites was identified, and the combination of these sites generated three distinct lineage-associated ROP18 patterns. Analysis of publicly available genomic datasets further demonstrated that individual strains contained one of these defined patterns rather than multiple patterns simultaneously. The developed HRM-PCR assay successfully discriminated the three ROP18-associated patterns based on distinct melting profiles with good reproducibility. Furthermore, the TaqMan MGB probe-based assay enabled specific identification of different ROP18-associated patterns through defined probe-recognition combinations and showed good analytical performance. CONCLUSION: This study identifies novel lineage-associated molecular signatures within the ROP18 3' flanking region and establishes complementary HRM-PCR and TaqMan MGB probe-based approaches for rapid molecular differentiation of T. gondii strains. These findings highlight the potential of conserved non-coding regions adjacent to functionally important genes as informative targets for parasite genotyping and provide a practical complementary tool for epidemiological surveillance and strain characterization.
The 3'-flanking region of the perfringolysin O (theta-toxin) gene (pfoA) of Clostridium perfringens was analyzed by chromosome walking. A total of 5,363 bp of the downstream region of the pfoA gene was sequenced and four open reading frames were found. ORF54 and ORF80 were found to be homologous to genes coding for membrane-bound transporter proteins of other bacteria and the beta-galactosidase gene (bgaB) of Bacillus stearothermophilus, respectively. ORF80 was named the pbg gene. Clones which showed beta-galactosidase activities were selected from a lambda FIXII genomic library of C. perfringens by blue plaque screening using X-Gal as a substrate. Four clones whose plaques showed blue appearances were obtained. Two of the four clones hybridized with the pbg probe but the others did not, indicating that there are two distinct beta-galactosidase genes in C. perfringens. The pbg gene was subcloned into pBR322 and was successfully expressed in Escherichia coli, suggesting that the pbg gene codes for a beta-galactosidase of C. perfringens.
Since several lines of evidence implicate the 3'-flanking region in regulating alpha1(I) collagen gene transcription, we analyzed 12. 4-kilobase pairs of 3'-flanking sequence of the murine alpha1(I) collagen gene for transcriptional elements. A region of the 3'-flanking region stimulated expression of the heterologous beta-globin gene promoter in an enhancer trap plasmid and of the alpha1(I) collagen gene promoter in a collagen-luciferase reporter gene construct when located 3' to the luciferase reporter gene. DNase I footprinting analysis demonstrated the presence of three regions where DNA binding proteins specifically interact within this 3'-stimulatory region. Inspection of the DNA sequence revealed a consensus E-box, a binding site for basic helix-loop-helix proteins, in one of the protein binding sites. Mobility shift assays demonstrated that upstream stimulatory factors (USF) USF-1 and USF-2 bind to this E-box. Mutating the E-box in the context of the 3'-flanking region confirmed that it contributes to the enhancement of transcriptional activity of the alpha1(I) collagen gene promoter. Mutations in all three protein binding sites abolished transcriptional activation by the 3'-flanking region, suggesting a complex interaction among the trans-acting factors in enhancing transcriptional activity. Thus, a region of the 3'-flanking region of the alpha1(I) collagen gene stimulates transcription of the alpha1(I) collagen gene promoter, and USF-1 and USF-2 contribute to this transcriptional stimulation.
Globin gene switching is a well-described model of eucaryotic developmental control. In the case of the human alpha-globin gene cluster, migration of erythropoietic activity from the embryonic yolk sac to the fetal liver is parallaled by the zeta-globin gene silencing and enhanced expression of the alpha-globin genes. To map critical cis determinants of this switch, the human zeta-globin gene, the alpha-globin gene, and chimeric recombinants were introduced into the mouse genome. Consistent with previous studies, expression of the individual alpha- and zeta-globin transgenes was found to be developmentally appropriate. Contrary to current models, however, the alpha- and zeta-globin gene promoters were not sufficient to establish this control. Instead, full silencing of the zeta-globin gene required the combined activities of this promoter, transcribed region, and 3'-flanking sequences. Individually, the silencing activities of the zeta-globin gene promoter and 3'-flanking region were minimal but increased markedly when both regions were present. The zeta-globin transcribed region appeared to contribute to gene silencing by a mechanism specifically activated in definitive erythroblasts in the fetal liver. These data demonstrate that a complex set of controls, requiring at least three determinants and involving at least two independent mechanisms, is necessary for full developmental silencing of the human zeta-globin gene.
Previous reports have provided inconsistent data as to the cis-regulatory elements that are essential for correct expression of the gene for the pro alpha 1 (I) chain of type I procollagen (COL1A1) in the many tissues in which the protein is synthesized. Here, two internally deleted minigene versions of the human COL1A1 gene were used to prepare transgenic mice. The constructs made it possible to test regulatory sequences in the normal context of the gene. Also, in contrast to the reporter genes used in previous experiments, the constructs made it possible to assay quantitatively expression of the exogenous genes relative to expression of the endogenous COL1A1 gene, both as mRNA and as protein. The average level of expression of the minigenes varied among three transgenic lines, but the ratio of expression of the minigenes to expression of the endogenous gene was the same in all transgenic mice of a given line. Within the same line, the ratio of expression was essentially the same in nine or more tissues in which expression of the endogenous gene varied widely. Also, the ratio of expression within a given line was the same in 15-day-old embryos and in mice ranging in age from 4 days to 4 months. In addition, the ratio remained constant during repair of a surgical wound. The results demonstrated, therefore, that the minigene constructs with about 2.3 kb of the promoter region and about 2 kb of the 3'-flanking region contained all of the sequences necessary for correct expression of the genes in a tissue-specific and development-specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)
The 3' flanking region of the interleukin 6 gene is polymorphic due to insertions of different size. Within this region lies a sequence of approximately 500 base pairs that is AT rich. Based on flanking sequence information we have constructed oligonucleotides which prime the polymerase chain reaction (PCR) and amplify this AT rich region. The amplification products visualized by agarose gel electrophoresis gave fragment sizes for both homozygous and heterozygous individuals that were concordant with those observed by conventional genomic blotting techniques. Alleles that could not be typed by Southern analysis were resolved with this approach. These results illustrate the value of PCR for the rapid detection of length polymorphisms such as those due to variable numbers of tandem repeats. In contrast to RFLP analysis this procedure takes less than a day to perform, is cheaper, avoids the use of radioactivity and requires far less substrate DNA. Three different human alleles were sequenced, and differences were detected that were due to both large duplications and loss of one or two bases, suggesting that AT rich regions identify highly polymorphic loci. The same primers also amplified non-human primate DNA, allowing a comparison of the human sequence with that of the common chimpanzee and baboon.
The 5'- and 3'-flanking regions of HPRA, a cucumber gene that encodes hydroxypyruvate reductase, were evaluated for regulatory activity with respect to light responsiveness and organ specificity. To define the functional regions of the 5'-flanking region of HPRA, a series of deletions was generated and the remaining portions fused to the beta-glucuronidase (GUS) reporter gene (uidA) containing a minimal 35S promoter truncated at -90. The region from -66 to +39 was found to be necessary for light-regulated expression of the uidA reporter gene, while the region from -382 to -67 was found to be necessary for its leaf-specific expression. Further deletion of the HPRA 5' flanking region to -590 resulted in high levels of root expression, suggesting the presence of a negative regulatory element responsible for silencing root expression of the HPRA gene between -590 and -383. The 3'-flanking region of the HPRA gene downstream of the polyadenylation site contains several sequence motifs resembling regulatory elements present in the promoters of several light-responsive genes. An 823 bp portion of the HPRA 3'-flanking region containing these putative regulatory elements enhanced GUS expression in leaves when placed downstream of the uidA reporter gene in the forward orientation, but not in the reverse orientation. When placed 5' of the -90 35S promoter, the 823 bp fragment enhanced slightly, independently of orientation, the root tip-specific expression pattern intrinsic to the -90 35S promoter, indicating that in some cases this region can act as a transcriptional enhancer.
The 3' flanking region of the Escherichia coli rnpB gene-encoding M1 RNA, the RNA component of RNase P, contains a 113 bp repeated sequence. This sequence, successively reiterating 3.5 times, includes the region for intrinsic termination. In vivo termination of transcription occurs mostly at the first terminator (T1). Analysis of deletions at the 3' flanking region revealed that the second terminator (T2) and third (T3) are functional in vivo and that the sequences preceding the region coding for an RNA-terminator hairpin and U-rich 3' tail are essential for efficient termination. Transcripts terminating at T2 and T3 were also processed at the 3' end in a manner similar to those terminating at T1.
We report that the expression of the Bacillus megaterium bmlP1 gene is subject to negative regulation by the bmlP1 3' flanking region. This repression occurred both in B. megaterium and in Escherichia coli. When the bmlP1 promoter was replaced with a heterologous promoter or when the orientation of the bmlP1 3' flanking region was reversed, the inhibitory effect was still observed. However, the bmlP1 3' flanking region was unable to exert repression on a heterologous gene when fused downstream in either orientation, and it was incapable of acting in trans. Dot blot and Northern blot analyses revealed that the repression occurred at the RNA level. Deletion analysis showed that the regulatory site responsible for the repression is located within a 116-bp region immediately following the bmlP1 gene. Possible mechanisms for this repression are discussed.
We have determined the identity of about 700 nucleotides in the 3' flanking region of the gene for M1 RNA, the RNA component of RNase P (EC 3.1.26.5). This region begins with a 113-base-pair segment of DNA, which is repeated approximately 3.5 times. The first repeating unit originates within the gene sequence for the 3' end of mature M1 RNA. The repeating units are highly conserved but diverge considerably after the partial fourth repeat. Segments of sequence homologous to the repeats have been identified both upstream in the M1 RNA transcription unit and downstream from the repeating units. Several overlapping open reading frames, with the potential to encode small basic proteins, have been identified in the repeated sequences. The structure of the M1 RNA gene 3' flanking region is very similar to the corresponding region at the Tyr T locus. In vitro and in vivo, transcription of the M1 RNA gene appears to terminate about 40 nucleotides downstream from the 3' terminus of mature M1 RNA. Therefore, an RNA processing event is involved in the biosynthesis of M1 RNA.
The 3'-flanking region of the beta-galactosidase gene (pbg), which is located downstream of the perfringolysin O gene (pfoA), and the 5'-flanking region of the collagenase gene (colA) of Clostridium perfringens strains NCTC8237 and 13, respectively, were analyzed. Southern analysis revealed that the colA gene is located 6.5 kb downstream of the pbg gene in the chromosome of C. perfringens. Sequence analysis showed that between the pbg and colA genes were the arcABDC and ahrC genes, whose putative products were quite similar to enzymes of the arginine deiminase pathway of Pseudomonas aeruginosa and the arginine repressor/activator of Bacillus subtilis, respectively. It is concluded that the genomic structure of the pfoA-colA region consists of pfoR-pfoA-ORF54-pbg-arcABDC-ahrC-colA.
The cysteine proteinase I gene of Dictyostelium discoideum is a developmentally regulated single copy gene. Specific sites in the 5' and the 3' flanking regions of the gene were cleaved by an endogenous nuclease when the gene was being transcribed. The majority of these sites were not cut when the gene was inactive. A dramatic change in the pattern of micrococcal nuclease and DNase I hypersensitive sites occurred in the 5' flanking region when transcription commenced at the 8 h stage of development. The major sites, doublets at -220/-300 bp and -670/-770 bp upstream of the transcription start site, corresponded to those cut by the endogenous nuclease. When transcription subsequently ceased the hypersensitive sites did not significantly change, indicating the gene remained in an activated state. The micrococcal nuclease hypersensitive sites in the 3' flanking region did not change significantly during development.
In rat liver, the two major phenobarbital (PB)-inducible cytochrome P450s, P450b (P450IIB1), and P450e (P450IIB2), are encoded by approximately 2.1-kb mRNAs showing more than 97% nucleotide sequence identity. Almost half of the sequence differences are concentrated in two short divergent segments in exon 7. An additional 4.8-kb P450b/P450e RNA, inducible by Aroclor and by PB, hybridizes with a classical P450b probe and with the 3' extension of the PB23 insert, a cloned P450b-like cDNA (Affolter et al., DNA 5, 209-218, 1986). The 4.8-kb form has now been detected in several rats under a variety of induction conditions. DNA sequencing of the 5' portion of the PB23 insert showed it is derived from a P450b gene. The 4.8-kb RNA hybridized with an oligonucleotide probe that recognizes P450b mRNA, but did not hybridize detectably with one that recognizes P450e mRNA. The 4.8-kb RNA and the PB23 insert doubtless represent P450b RNAs polyadenylated at a downstream site. DNA sequence analysis of the PB23 3' extension (which represents the 3' end of the P450b gene) and of the P450e gene 3'-flanking region demonstrated that the near identity of the P450b and P450e genes extends for at least 920 bp beyond the major polyadenylation site. This region was doubtless part of the original duplication that gave rise to the P450b and P450e genes. A short divergent segment is present in the 3'-flanking region of near sequence identity; the segment is embedded in simple sequence DNA that contains a mixed alternating pyrimidine/purine tract.
The Taq1 polymorphism in the 3' flanking region of the PI gene has been reported to be associated with chronic obstructive pulmonary disease (COPD). We have studied the frequency of the Taq1 polymorphism in 117 Kuwaiti Arabs and 110 Russians using PCR/RFLP. The frequency of this polymorphism was found to be 0.235 in the Arabs and 0.027 in the Russians. Such a striking difference in allele frequencies could be due to a 'founder effect' in the Kuwaiti population. However: it may also be that this mutation provides a selective advantage, thus accounting for its fixation at a rather high frequency in some populations. Our results suggest that ethnic composition is a very important factor which should be taken into consideration when studying the association of the Taq1 polymorphism with COPD.
Alu family sequences are middle repetitive short interspersed elements (SINEs) dispersed throughout vertebrate genomes that can modulate gene transcription. The human (h) GH locus contains 44 complete and four partial Alu elements. An Sx Alu repeat lies in close proximity to the hGH-1 and hGH-2 genes in the 3'-flanking region. Deletion of the Sx Alu repeat in reporter constructs containing hGH-1 3'-flanking sequences increased reporter activity in transfected pituitary GC cells, suggesting this region contained a repressor element. Analysis of multiple deletion fragments from the 3'-flanking region of the hGH-1 gene revealed a strong orientation- and position-independent silencing activity mapping between nucleotides 2158 and 2572 encompassing the Sx Alu repeat. Refined mapping revealed that the silencer was a complex element comprising four discrete entities, including a core repressor domain (CRD), an antisilencer domain (ASE) that contains elements mediating the orientation-independent silencer activity, and two domains flanking the CRD/ASE that modulate silencer activity in a CRD-dependent manner. The upstream modulator domain is also required for orientation-independent silencer function. EMSA with DNA fragments representing all of the silencer domains yielded a complex pattern of DNA-protein interactions indicating that numerous GC cell nuclear proteins bind specifically to the CRD, ASE, and modulator domains. The silencer is GH promoter dependent and, in turn, its presence decreases the rate of promoter-associated histone acetylation resulting in a significant decrease of RNA polymerase II recruitment to the promoter. The silencer may provide for complex regulatory control of hGH gene expression in pituitary cells.
V511 and V513 are Chinese hamster cell lines with acquired resistance to topoisomerase II (topo II) directed agents. These cell lines were obtained by mutagenizing Chinese hamster V79 cells with N-methyl-N'-nitro-N-nitrosoguanidine and subsequently selecting in etoposide (VP-16). We have previously shown that this resistance is not associated with alterations in drug uptake. To elucidate whether any alterations in the functionally important domains of topo II alpha were associated with VP-16 resistance, we used reverse transcriptase-polymerase chain reaction, single-strand conformational polymorphism analysis, and subsequent sequencing of topo II alpha from V79, V511, and V513 to search for mutations in five major functional domains including the regions of the consensus ATP binding sequences (Motif A and Motif B/dinucleotide binding site), the DNA binding domain, and the 5' and 3' flanking regions of the DNA binding position. The V511 cells showed no mutational changes in these regions. However, the topo II alpha gene from V513 showed a point mutation at nucleotide 2552 that resulted in a glycine-to-aspartate mutation at amino acid position 851 in the 3' flanking region of the DNA binding site. This mutation at amino acid position 851 in V513 cells is associated with reduced VP-16-induced cleavable complex formation demonstrated by potassium-sodium dodecyl sulfate assay and band-depletion analysis. Our results suggest that the mutation at amino acid position 851 may play a role in drug resistance, presumably by interfering with enzyme-DNA binding.
The Dexras1 gene responds to glucocorticoids with a rapid and profound induction. A glucocorticoid response element (GRE) was identified in the 3'-flanking region (2.3 kb downstream of poly(A) signal) of the human Dexras1 gene. This element conferred rapid glucocorticoid responsiveness when inserted into a homologous promoter-driven luciferase reporter. A point mutation within the 15-bp GRE abolished this glucocorticoid responsiveness.