S1 Nuclease Mapping Research Articles

Cloning of the Human Phospholipase C-γ1 Promoter and Identification of a DR6-type Vitamin D-responsive Element

The 5'-flanking region of the human phospholipase C-gamma1 gene was isolated from a human P1 genomic DNA library. The S1-nuclease mapping and primer extension analysis revealed that there is a single transcriptional start site located at 135 bases upstream from the translation start codon in the human phospholipase C-gamma1 gene. DNA sequence analysis showed that the sequence around the transcriptional start site is very GC-rich and has no TATA box. The fragment +135 to -877 in the 5'-flanking region of the human phospholipase C-gamma1 gene was subcloned into a luciferase reporter vector. The chimeric gene produced a high level of luciferase activity and responded to 1,25-(OH)2D3 in transiently transfected human keratinocytes. Deletion and mutation studies of the fragment +135 to -877 demonstrated a vitamin D-responsive element that contains a motif arranged as two direct repeats separated by 6 bases (DR6), AGGTCAgaccacTGGACA, located between -786 and -803 base pairs. Incubation of the oligonucleotide containing the DR6 with keratinocyte nuclear extracts produced a specific protein-DNA complex that shifted to a higher molecular weight form upon the addition of an antibody specific to the 1,25-(OH)2D3 receptor. Therefore, the 5'-flanking region of the human phospholipase C-gamma1 gene confers promoter activity and contains a DR6-type vitamin D-responsive element that mediates, at least in part, the enhanced expression of this gene in human keratinocytes by 1, 25-(OH)2D3.

Molecular cloning, upstream sequence and promoter studies of the human gene for the regulatory subunit RII α of cAMP-dependent protein kinase

The gene for the regulatory subunit RII α of cAMP-dependent protein kinase is highly regulated during spermatogenesis and a strong signal from a distinct short mRNA form is observed postmeiotically during spermatid elongation. This report presents the isolation and characterization of the 5′-flanking region (1.2 kb) and exon 1 of the human RII α gene. S1 nuclease mapping and primer extension experiments revealed the presence of a major transcriptional start site located 208 nucleotides upstream of start for translation. The 5′-flanking region of the RII α gene did not contain a TATA box and was highly G/C-rich. A basal promoter directing high levels of chloramphenicol acetyl transferase (CAT) activity was identified in the 5′-flanking sequence. Several potential binding sites for transcription factors were identified in this region, which may be responsible for the germ cell-specific regulation of this gene. We have previously reported that the human testis RII α cDNA contains a region (amino acids 45-75) with little or no homology to the corresponding rat skeletal muscle cDNA (Øyen, O., Myklebust, F., Scott, J.D., Cadd, G.G., McKnight, G.S., Hansson, V. and Jahnsen, T. (1990) Biol. Reprod. 43, 46–54). We examined whether this difference could arise due to organ-specific splice mechanisms or represented a species difference. We show that the low homology region of the human RII α cDNA resides entirely within exon 1, and does not originate from a tissue-specific alternate splicing of this distinct region.

Transcriptional analysis of type 1 capsule genes in Staphylococcus aureus.

Serotype 1 capsule of Staphylococcus aureus plays an important role in staphylococcal infections. A 14.6 kb chromosomal DNA fragment containing 13 cap1 genes responsible for the synthesis of type 1 capsule in S. aureus strain M has been previously cloned and sequenced in our laboratory. These genes are closely linked and are transcribed in one orientation. To study the transcription of these genes, we employed genetic complementation using a set of plasmids with various deletions in the 14.6 kb region to complement mutants mapped in each of the 13 genes. We found that there were six transcriptional units. By Northern hybridization using the entire gene cluster as a probe, we identified a large 14 kb band and several smaller bands ranging from 0.3 to 6 kb. The 14 kb band was also identified by various individual gene probes, suggesting that all 13 genes are transcribed into a large transcript. The 14 kb transcript and the smaller bands were not detected when a 533 bp fragment containing the potential promoter region upstream of the first gene (cap1A) was deleted from the chromosome, suggesting that the small transcripts are the degradation (processing) products of the 14 kb transcript. The activities of the promoters identified by genetic complementation tests were quantitatively measured by transcriptional fusions using xylE as the reporter gene. The result showed that the promoter preceding the first cap1 gene was 45-198-fold higher than the downstream promoters. The result from gene-fusion studies is in agreement with the Northern analysis in that only the transcript transcribed from the first promoter was detected. Taken together, these results suggest that the cap1 genes are organized as a large operon with at least five weak internal promoters, and the long polycistronic transcript is processed (degraded) into several smaller transcripts. The transcription initiation site of the primary transcript was mapped by S1 nuclease mapping and the major promoter was defined by deletion analysis of the promoter-xylE fusion. In addition, we showed that the internal promoters, despite their weak activities, were sufficiently effective at expressing their downstream genes required for producing capsule, compared to the wild-type strain.

DNA sequence and secondary structure of the mitochondrial small subunit ribosomal RNA coding region including a group-IC2 intron from the cultivated basidiomycete Agrocybe aegerita

Due to their structural complexity and their evolutionary dimension, rRNAs are the most investigated nucleic acids in prokaryotes, eukaryotes and organelles. However, no complete sequence of a mitochondrial small subunit (SSU) rRNA was available in the basidiomycotina subdivision. The mitochondrial gene encoding the SSU rRNA of the cultivated basidiomycete Agrocybe aegerita was cloned and its complete nucleotide sequence achieved; the 5′- and 3′-ends were localized by nuclease S1 mapping, leading to a size of 3277 nt. The secondary structure of the SSU rRNA (1906 nt in size) possessed all the helices and loops of the prokaryotic model; a unique modification was found in a conserved nucleotide predicted by the model: the nt 487 was A instead of C. The same modification, has been found in all the partial basidiomycete mitochondrial sequences available in databases. The Agrocybe aegerita SSU rRNA was characterized by large and unusual extensions leading to additional helices in the variable domains V4, V6 and V9, which were the longest of the known prokaryotic or mitochondrial SSU rRNAs. Nucleotide sequence analysis indicated a 1371-bp intron, belonging to subgroup-IC2, located in a conserved loop in the 3′-part of the SSU rRNA. This intron, which is the second example reported in a fungal mitochondrial SSU rDNA, encoded a putative protein (407 aa) sharing homologies with endonucleases involved in group-I intron mobility. This report constitutes the first complete mitochondrial SSU rRNA sequence and secondary structure of any member of the basidiomycotina subdivision.

Regulation of CO2 assimilation in Ralstonia eutropha: premature transcription termination within the cbb operon.

In the facultatively chemoautotrophic bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus), most genes required for CO2 assimilation via the Calvin cycle are organized within two highly homologous cbb operons located on the chromosome and on megaplasmid pHG1, respectively, of strain H16. These operons are subject to tight control exerted by a promoter upstream of the 5'-terminal cbbL gene that is regulated by the activator CbbR. The existence of subpromoters within the operons was now excluded, as determined with lacZ operon fusions to suitable cbb gene fragments in the promoter-probe vector pBK. Nevertheless, marked differential expression of the promoter-proximal ribulose-1,5-bisphosphate carboxylase-oxygenase genes cbbLS and the remaining distal genes occurs within the operons. Computer analysis revealed a potential stem-loop structure immediately downstream of cbbS that was suspected to be involved in the differential gene expression. Nuclease S1 mapping identified a major 3' end and a minor 3' end of the relatively stable cbbLS partial transcript just downstream of this structure. Moreover, operon fusions containing progressively deleted stem-loop structures showed that the structure primarily caused transcriptional termination downstream of cbbS rather than increased the segmental stability of the cbbLS transcript. Premature transcription termination thus represents an important mechanism leading to differential gene expression within the cbb operons.

Isolation and Characterization of the Human Cytochrome P450 CYP1B1 Gene

Previously, we identified a novel human cytochrome P450 cDNA that is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and represents the first member of a new subfamily designated cytochrome P4501B1 (CYP1B1; Sutter, T. R., Tang, Y. M., Hayes, C. L., Wo, Y. P., Jabs, E. W., Li, X., Yin, H., Cody, C. W., and Greenlee, W. F. (1994) J. Biol. Chem. 269, 13092-13099). Here, we report on the isolation and initial characterization of the CYP1B1 gene. The CYP1B1 gene maps to human chromosome 2 at 2p21-22 and contains three exons and two introns. The putative open reading frame starts in the second exon and is 1629 base pairs in length. Southern analysis using DNA probes directed to each of the three exons confirmed that CYP1B1 is a single copy gene. Human CYP1B1 differs from its two most closely related members of the cytochrome P450 superfamily, CYP1A1 and CYP1A2, in the number of exons (3 versus 7) and chromosome location (2 versus 15). A single transcription initiation site was identified by primer extension analysis and S1 nuclease mapping. Based on nucleotide sequence analysis, the CYP1B1 gene lacks a consensus TATA box in the promoter region and contains nine TCDD-responsive enhancer core binding motifs (5'-GCGTG-3') located within a 2.5-kilobase pair genomic fragment 5'-ward of the transcription initiation start site. Deletion analysis of chloramphenicol acetyltransferase reporter gene constructs containing 5' CYP1B1 genomic fragments indicates that a region from -1022 to -835 containing three of the nine core binding motifs contributes to the TCDD-inducible expression of CYP1B1.

Differential expression of two sporulation specific σ factors of Streptomyces aureofaciens correlates with the developmental stage

In previous experiments, the Streptomyces aureofaciens (Sa) rpoZ, and sigF genes have been shown to encode putative σ factors essential in differentiation. In an attempt to investigate expression of these genes during differentiation, we have performed S1 nuclease mapping using RNA prepared from Sa in various developmental stages. Two putative promoters were identified upstream of the rpoZ coding region. The promoters significantly differed in their strength, and were active in distinct developmental stages; the weaker, rpoZ-P1, was active only in substrate mycelium, and the stronger, rpoZ-P2, was induced at the beginning of aerial mycelium formation. Transcriptional analysis of sigF revealed two apparent transcription start points, both being detectable only in the late phase of aerial mycelium formation. No sigF transcription was detected in an rpoZ-disrupted Sa strain. Promoter-bearing DNA fragments from rpoZ and sigF were inserted into several promoter-probe vectors, to give expression patterns consistent with the results of direct RNA analysis. The results implicate temporally different expression of rpoZ and sigF during the differentiation of Sa, and direct or indirect dependence of sigF expression on the rpoZ-encoded putative σ factor, thus indicating a cascade of σ factors in Streptomyces development.

Differential tissue-specific expression of neurofibromin isoform mRNAs in rat.

We have cloned the full-length cDNA encoding the rat homolog of type I neurofibromin isoform, a protein product of a gene linked to neurofibromatosis type 1. Rat type I neurofibromin isoform is composed of 2,820 amino acid residues and shares about 98.5% amino acid identity with the human counterpart. By S1 nuclease mapping analysis of the alternatively spliced neurofibromin mRNAs in adult rat tissues, we showed that type I isoform mRNA was predominantly expressed in the brain, pituitary, and testis, while type II mRNA, carrying a 63-nucleotide insertion in the region coding for the domain related to GTPase-activating protein, was predominantly expressed in most other tissues, such as heart, kidney, and ovary. Furthermore, type II mRNA is predominant in the testis at age 1 week, while type I mRNA becomes predominant at 3 weeks and is subsequently expressed at higher levels, as seen in the adult testis. In contrast, type I mRNA is the predominant form in the brain throughout the postnatal development. Thus, the relative expression levels of type I and type II mRNAs may be specific to the tissues and to the developmental stage of certain tissues.

Structural Organization and Chromosomal Assignment of the Human 14-3-3 η Chain Gene (YWHAH)

14-3-3 protein, a brain-specific protein, is thought to be a multifunctional protein involved in the activation of tyrosine and tryptophan hydroxylases, the inhibition or activation of protein kinase C, and the activation of signal transduction. The human 14-3-3 η chain gene was isolated and its structure was determined. It is composed of two exons separated by one long intron (approximately 8 kb) and spans about 10 kb. A transcription initiation site was identified by a combination of S1 nuclease mapping, primer extension analysis, and RACE methods. In the 5′-flanking region, we found four GC box sequences, four anti-GC box sequences, a TATA box-like sequence, CAAT box-like sequences, a C/EBP element, two AP-2 sequences, an AP-3 sequence, an Oct-6-like sequence, six E boxes, and a CRE sequence. FISH with DNA probes of the human 14-3-3 η chain gene mapped the 14-3-3 η chain gene to chromosome 22q12.1–q13.1.

The first contiguous estrogen receptor gene from a fish, Oreochromis aureus: evidence for multiple transcripts

The O. aureus estrogen receptor (OaER) gene of 40.4 kb containing ten exons is the first complete piscine gene to be cloned. There are two extra introns: intron I that divides the 5′ UTR into two exons, and intron V that intersperses D and E 1 exons. Except for I and V, other introns have identical positions to those of human ER gene. All the donor and acceptor splice sites exhibit consensus sequences. The promoter lacks consensus TATA and CAAT boxes. This region exhibits several putative regulatory elements. A functional imperfect ERE deviating at two bases is located in the leader exon, thus suggesting that this gene is autoregulated. The OaER gene lacks an A region whereas its C and E domains are highly conserved. Within the ER subfamily, OaER exhibits the longest F domain of 77 amino acids. OaER has a long 3′ UTR constituting > 1 2 of its transcript. Using RT-PCR and S1 nuclease mapping, we report for the first time the usage of both alternative transcriptional start sites and polyadenylation signals during estrogen-induced OaER expression. Thus, O. aureus may have four species of ER transcripts differing structurally in their transcriptional start sites and lengths of their 3′ UTR.

Regulation of the operon responsible for broad-spectrum mercury resistance in Streptomyces lividans 1326.

The broad-spectrum mercury resistance of Streptomyces lividans 1326 is mediated by six open reading frames (orf). These are arranged in two divergently transcribed operons. The orfs mer A (mercuric reductase) and mer B (organolyase) form one of the two operons. These genes and their regulation were further studied by deletion analysis and transcriptional fusion to the reporter gene xylE in the plasmid pXE4. An increase in XylE activity in response to the presence of mercuric ions was observed. The function of ORF2 (MerT) and ORF3 (MerP) as mercury-specific transport proteins, previously postulated based on the structural features of the predicted proteins, was confirmed. Transcription of the mer genes starts within the intercistronic region and two divergent promoters were identified by S1 nuclease mapping. Expression of the genes was negatively regulated by the product of orf1, now called merR. The repressor function was confirmed by gel retardation assays. MerR, produced in Escherichia coli, bound to two sites (operators) in the fragment containing the promoter region between merA and merR. Addition of mercuric ions and phenylmercuric acetate prevented the binding of MerR.

Sequence of the Polypyrimidine Tract of the 3'-Terminal 3' Splicing Signal Can Affect Intron-Dependent Pre-mRNA Processing In Vivo

Most pre-mRNAs require an intron for efficient processing in higher eukaryotes. However, not all introns can provide this function. For example, transcripts synthesized from a variant of the human beta-globin gene lacking its second intervening sequence (IVS2), yet retaining its first intervening sequence (IVS1), exhibit multiple defects in mRNA biogenesis. To investigate why, we transfected into monkey cells plasmids containing the human beta-globin gene and variants of it altered in (i) IVS1, (ii) the 3'-terminal exon, and (iii) the polyadenylation signal. The beta-globin RNAs accumulated in these cells were analyzed by quantitative S1 nuclease mapping for nuclear accumulation, intron excision, polyadenylation and cytoplasmic accumulation. We found that the 3' splicing signal of IVS1, with multiple purines interrupting its polypyrimidine tract, could efficiently function as an internal 3' splicing signal; however, it could not efficiently function as the 3'-terminal 3' splicing signal for any of these steps in intron-dependent mRNA biogenesis unless (i) its polypyrimidine tract was made uninterrupted in pyrimidines, or (ii) specific sequences were deleted from the 3'-terminal exon. We conclude that whether an intron can provide the function necessary for efficient processing of intron-dependent pre-mRNA is dependent upon the ability of its 3' splicing signal to define the 3'-terminal exon. On the practical side, this finding means one needs to consider both the sequence and location of the intron to be included in an intron-dependent gene to obtain efficient expression in vivo.

An S1 Nuclease Mapping Method for Detection of Low Abundance Transcripts

As the extracellular nuclease of Aspergillus, S1 nuclease can split single and double-stranded DNA into oligo- or mononucleotides, while preferentially digests single-stranded nucleic acids. Furthermore, the existence of S1 can be the standard to identify Aspergillus and used to evaluate the severity of Aspergillosis. Herein, a simple and sensitive fluorescent sensing platform for S1 assay was developed based on the S1-induced DNA strand scission and the difference in affinity of graphene oxide (GO) for single-stranded DNA containing different bases. This platform was applied to monitor S1 activity and study the kinetics in real time. Results indicated that the detection limit is 0.5 U/mL. The Km and kcat at 45 °C, are 1.4 ± 0.12 μM and 0.6 min− 1, respectively. Moreover, by monitoring the effect of chemical drugs on S1 activity, we found that 2 mM of erythromycin, sodium penicillin, carbenicillin disodium and ampicillin can inhibit S1 activity about 8%, 60%, 61% and 66%, respectively, while gentamycin sulfate is a stimulator. Overall, the assay platform based on graphene oxide quenched fluorescence probe is successfully constructed to study the enzymatic activity of S1 and used for screening antibiotics.

Endogenous endothelin-1 mediates cardiac hypertrophy and switching of myosin heavy chain gene expression in rat ventricular myocardium

We investigated the role of endogenous endothelin-1 in the development of cardiac hypertrophy in vivo under pressure overload conditions. Endothelin-1, a potent vasoconstrictor peptide, has recently been shown to act as a growth factor of myocardial cells in culture. We examined the effect of an endothelin-A receptor antagonist (FR139317) on the development of right ventricular hypertrophy in rats with monocrotaline-induced pulmonary hypertension. Three groups of rats were studied: those given monocrotaline alone or monocrotaline plus FR139317 and those given vehicle alone (control group). The ratio of right ventricular systolic pressure to aortic systolic pressure was similarly elevated in rats treated with monocrotaline and monocrotaline plus FR139317. The right ventricular/left ventricular weight ratio was increased in monocrotaline-treated rats but lower in rats treated with monocrotaline plus FR139317 than in those treated with monocrotaline alone (p < 0.01). As a biochemical marker of hypertrophy, the isoform ratio of beta-myosin heavy chain protein was determined for the right ventricular tissue samples. This ratio was increased in all monocrotaline-treated rats but was lower (p < 0.01) in rats given monocrotaline plus FR139317 than in those given monocrotaline alone. The isoform ratio of beta-myosin heavy chain messenger ribonucleic acid quantitated by S1 nuclease mapping also was lower (p < 0.025) in rats receiving monocrotaline plus FR139317 than in those receiving monocrotaline alone. These data suggest that blocking the action of endothelin-1 with a receptor antagonist ameliorates cardiac hypertrophy in this model system, and that this action is not mediated by ameliorating hemodynamic changes.

Isolation, identification, and transcriptional specificity of the heat shock sigma factor sigma32 from Caulobacter crescentus

We report the identification of the Caulobacter crescentus heat shock factor sigma32 as a 34-kDa protein that copurifies with the RNA polymerase holoenzyme. The N-terminal amino acid sequence of this protein was determined and used to design a degenerate oligonucleotide as a probe to identify the corresponding gene, rpoH, which encodes a predicted protein with a molecular mass of 33,659 Da. The amino acid sequence of this protein is similar to those of known bacterial heat shock sigma factors of Escherichia coli (41% identity), Pseudomonas aeruginosa (40% identity), and Citrobacter freundii (38% identity). The isolated C. crescentus gene complements the growth defect of an E. coli rpoH deletion strain at 37 degrees C, and Western blot (immunoblot) analysis confirmed that the gene product is related to the E. coli sigma32 protein. The purified RpoH protein in the presence of RNA polymerase core enzyme specifically recognizes the heat shock-regulated promoter P1 of the C. crescentus dnaK gene, and base pair substitutions in either the -10 or -35 region of this promoter abolish transcription. S1 nuclease mapping indicates that rpoH transcripts originate from two promoters, P1 and P2, under the normal growth conditions. The P2 promoter is similar to the sigma32 promoter consensus, and the P2-specific transcript increases dramatically during heat shock, while the P1-specific transcript remains relatively constant. These results suggest that although the structure and function of C. crescentus sigma32 appear to be very similar to those of its E. coli counterpart, the C. crescentus rpoH gene contains a novel promoter structure and may be positively autoregulated in response to environmental stress.

Identification of a Melanocyte-Type Promoter of the Microphthalmia-Associated Transcription Factor Gene

Microphthalmia-associated transcription factor (MITF), the human homolog of the mouse microphthalmia gene product, regulates melanocyte-specific transcription of the tyrosinase gene that codes for an essential enzyme in melanin biosynthesis. In this study, we have cloned and characterized the human genomic DNA segment containing a melanocyte-type exon and its 5′-flanking region of the MITF gene. A major transcriptional initiation site was assigned by primer extension and S1 nuclease mapping analyses using melanoma RNA. Subsequently, the fusion genes, containing the identified 5′-flanking region upstream from the firefly luciferase gene, were constructed and were introduced into pigmented melanoma cells or HeLa cells which do not express MITF mRNA. Transient expression assays show that the 5′-flanking region of 2.3 kb is able to confer preferential expression of a luciferase gene in pigment cells. These results establish that the MITF gene contains a melanocyte-specific promoter.

Gene expression and transcriptional regulation of thrombopoietin.

Thrombopoietin (TPO) is predominantly expressed in the liver among various tissues that express TPO transcripts. To investigate the transcriptional regulation of the human TPO gene in the liver, we determined the major transcription initiation site by means of 5'-RACE and Northern blotting. From these analyses, we concluded that TPO gene transcription started at various points, and the transcription initiation sites of the human TPO gene were localized downstream, close to a point we determined by S1 nuclease mapping. The human TPO promoter region contains consensus sequences of GATA, Evi-1, and Ets binding sites. We used the hepatocellular carcinoma cell line, HepG2, that expresses TPO mRNA to analyze its promoter activity by transfecting various reporter plasmids containing a sequentially 5'-deleted human TPO promoter. Although GATA binding factors increased the promoter activity, their effect was independent of the GATA binding consensus sequence. On the other hand, Evi-1 did not affect transcription. Moreover, we defined the core promoter region, in which an Ets binding consensus sequence was located. The deletion or mutation of the Ets binding site resulted in a loss of the promoter activity. These results suggested that TPO is regulated by the Ets family of transcription factors.

Characterization of the Promoter Region of the Human Transforming Growth Factor-β Type II Receptor Gene

Diminished cellular responsiveness to transforming growth factor-beta (TGF-beta) is frequently correlated with decreased transcription of the type II receptor for TGF-beta (TGF-beta RII). We have cloned and characterized the human TGF-beta RII promoter and, using S1 nuclease mapping and 5' rapid amplification of cDNA ends polymerase chain reaction, have identified five alternative transcription start sites within the region -33 to +57. DNA transfection experiments and electrophoretic mobility shift assays have revealed the existence of five distinct regulatory regions including two positive regulatory elements and two negative regulatory elements in addition to the core promoter region. The first positive regulatory element (-219 to -172) interacts with two distinct nuclear protein complexes, at least one of which appears to be a previously unidentified transcription factor. The second positive regulatory element (+1 to +35) also interacts with two separate protein complexes, both of which appear to be novel transcription factors. Deletion of either positive regulatory element markedly decreased expression of the target gene, suggesting that both positive regulatory elements are necessary for basal expression levels of TGF-beta RII.

The bovine DNA polymerase β promoter: cloning, characterization and comparison with the human core promoter

The core promoter of the human DNA polymerase β (βPol)-encoding gene (POLβ) is regulated through cis-elements for the ATF/CREB protein(s), and GC box-binding and initiation-site-binding proteins. The mechanism of promoter regulation has been studied using a nuclear extract transcription system from HeLa cells [Narayan et al., J. Biol. Chem. 269 (1994) 12755–12763]. To study the homologous promoter (ppolβ) in a bovine system, we cloned and characterized the 5′-flanking region of the bovine gene (polβ). A 15.3-kb fragment of bovine genomic DNA containing the first two exons and 11 kb of 5′-flanking region was isolated from a testis library in bacteriophage λEMBL3. S1 nuclease mapping and primer extension analysis of the 5′-end of the polβ mRNA identified the major transcription start point (tsp), which is located 142-bp 5′ of the translational start codon. In transient expression assays using a bovine cell line, analysis of various 5′-deletion mutants demonstrated that a fragment of only 91-bp 5′ of the tsp had promoter activity similar to that of a 1.37-kb fragment, so that cis-elements for basal transcription are located within this approx. 100-bp core promoter, as in the human promoter (pPOLβ). Comparison of the core promoters from the bovine and human genes revealed striking similarity, including an almost precise match of the tsp, the ATF/CREB-binding and Spl-binding sites, and the spacing separating them.

Complete sequences and organization of the rrnA operon from Campylobacter jejuni TGH9011 (ATCC43431)

The rrnA ribosomal RNA (rRNA) operon of Campylobacter jejuni (Cj) TGH9011 (ATCC43431) was cloned and sequenced to completion, rRNAs were then characterized by primer extension and S1 nuclease mapping analysis. The secondary structure models of Cj 16S and 23S rRNAs were constructed, and the models were compared to the corresponding models from other eubacterial rRNA. The analysis presented a typical 5'-promoter-16S-tRNAs-23S-5S-terminator-3′ prokaryotic rRNA operon structure. However, an unusual organization of the intercistronic tRNAs was observed where the two tRNAs, tRNA Ala and tRNA Ile, were present in the order 5′-16S-tRNA Ala-tRNA Ile-23S-3′, which is opposite of the typical 5′-16S-tRNA Ile-tRNA Ala-23S-3′ structure observed in other bacteria.