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Results for “Terminator Regions, Genetic”

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PubMed1987

A 12-base-pair sequence is an essential element of the ribosomal gene terminator in Xenopus laevis.

rRNA transcription in Xenopus laevis terminates near a 7-base-pair (bp) conserved sequence (T3 box) located 200 bp upstream of the site of transcription initiation for the adjacent gene promoter. We present evidence here that a 12-bp element containing the T3 box is an essential part of the terminator. Using an oocyte injection assay, we found that the 12-bp element (but not the T3 box alone) severely reduced the amount of RNA detectable at sites downstream from itself and that the T3 box within the 12-bp element was required to specify the formation of correct 3' ends. This requirement for the 12-bp element was also seen in pulse-label experiments by using a homogenate of oocyte nuclei, but the present data did not allow us to determine the exact mechanism by which the 12-bp element acts. Removal of the T3 region from its normal location allowed a significant amount of readthrough transcripts to accumulate, indicating that additional sequences may be required for complete terminator function.

Animals↗

PubMed1991

3' processing in Dictyostelium: unusual sequence requirements and interaction with a downstream promoter.

We have determined the sequence requirements for 3'-processing in Dictyostelium and find that a single AATAAA site, embedded in an A/T-rich environment is sufficient. A synthetic oligonucleotide containing the additional GT/T-rich element, which is necessary for 3'-processing in higher eukaryotes, is not used in Dictyostelium. On the basis of reports suggesting termination signals (upstream terminators) in Dictyostelium promoters, we investigated possible interactions between processing signals and regulatory elements. Our data suggest that upstream termination enhances transcription from a downstream promoter.

Base Sequence↗

PubMed1975

In vitro synthesis of enzymes of the tryptophan operon of Escherichia coli.

In vitro synthesis of enzymes of the tryptophan (trp) operon of E. coli was studied in an extract prepared from E. coli, which is programmed with purified DNA from trp transducing phages with mutations that effect the expression of the trp genes in various ways. Our results show that control of transcription, i.e. initiation and termination, is similar in vivo and in vitro.

Anthranilate Synthase↗

PubMed1986

Isolation and sequence analysis of the gene (cpdB) encoding periplasmic 2',3'-cyclic phosphodiesterase.

The cpdB gene encodes a periplasmic 2',3'-cyclic phosphodiesterase (3'-nucleotidase). This enzyme has been purified previously and the gene is located at 96 min on the Escherichia coli chromosome. In this study the cpdB gene was cloned from ClaI-cleaved DNA, and the gene product was identified. DNA blotting experiments showed that the recombinant plasmid contains a deletion with respect to the expected genomic fragment of approximately 4 kilobases, which extends into the vector. Furthermore, the gene was absent from three other recombinant libraries. Together, these findings suggest the presence in the genome of an adjacent gene whose product is lethal when it is present on a multicopy plasmid. The nucleotide sequence of the cpdB gene was also determined. The 5' and 3' untranslated sequences contain characteristic sequences that are involved in the initiation and termination of transcription, including two possible promoters, one of which may contain two overlapping -10 sequences. A strong Shine-Dalgarno sequence is followed by an open reading frame which corresponds to a protein having a molecular weight of 70,954. The first 19 amino acid residues have the characteristics of a signal peptide. The 3' untranslated sequence contains two putative rho-independent transcription terminators having low thermodynamic stability.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed2005

A versatile vector system for multiple gene expression in plants.

Today, cloning vectors that have been specifically designed to facilitate the fusion, overexpression or down-regulation of a variety of genes in plant cells are available from various sources. In most cases, their basic design allows the cloning of a single target gene, typically under a specific promoter, in parallel with the expression of selection and/or marker genes from the same vector. However, new and versatile systems now exist that expand the user's choice to a large number of promoters and terminators, and various autofluorescent tags confer the ability to express multiple genes from a single transformation vector.

Cloning, Molecular↗

PubMed1985

Transcription signals in a region essential for replication of plasmid R6K.

A DNA fragment encoding for the enzyme chloramphenicol acetyltransferase (CAT) but lacking the CAT promoter sequence was used to probe transcription signals in a DNA fragment (640 base pairs) from plasmid R6K that plays a central role in R6K DNA replication. The R6K DNA fragment analyzed contains the seven 22-bp direct repeats which express R6K incompatibility and are required in cis for activity of all three of the R6K origins of replication. In addition to the previously identified promoter sequences for the R6K initiation protein pi (P pi), a relatively weak transcriptional promoter upstream to P pi was detected. On the opposite strand an efficient (94%) transcriptional terminator was identified. This terminator is located upstream to nucleotide sequences active in the repression of R6K DNA replication.

Acetyltransferases↗

PubMed2006

Involvement of DNA curvature in intergenic regions of prokaryotes.

It is known that DNA curvature plays a certain role in gene regulation. The distribution of curved DNA in promoter regions is evolutionarily preserved, and it is mainly determined by temperature of habitat. However, very little is known on the distribution of DNA curvature in termination sites. Our main objective was to comprehensively analyze distribution of curved sequences upstream and downstream to the coding genes in prokaryotic genomes. We applied CURVATURE software to 170 complete prokaryotic genomes in a search for possible typical distribution of DNA curvature around starts and ends of genes. Performing cluster analyses and other statistical tests, we obtained novel results regarding various factors influencing curvature distribution in intergenic regions, such as growth temperature, A+T composition and genome size. We also analyzed intergenic regions between converging genes in 15 selected genomes. The results show that six genomes presented peaks of curvature excess larger than 3 SDs. Insufficient statistics did not allow us to draw further conclusion. Our hypothesis is that DNA curvature could affect transcription termination in many prokaryotes either directly, through contacts with RNA polymerase, or indirectly, via contacts with some regulatory proteins.

Cluster Analysis↗

PubMed1990

An undecamer DNA sequence directs termination of human ribosomal gene transcription.

Previously we have shown that a repetitive 18 bp sequence motif, the Sal box (AGGTCGACCAGA/TT/ANTCCG), present in the 3' terminal spacer of mouse rDNA constitutes a termination signal for RNA polymerase I (pol I). Similar sequence elements which are functionally analogous to the murine terminator are present in the spacer of human rDNA. However, the human termination signal is shorter encompassing only 11 bp (GGGTCGACCAG) which correspond to the proximal part of the mouse sequence. Two out of the five human Sal box elements are functionally inactive due to natural point mutations which damage factor binding. A similar sequence motif with a 10 of 11 base identity with the downstream terminators is located upstream of the human transcription initiation site. The upstream element interacts with the same factor(s) as the downstream terminators and is also capable to stop elongating human RNA polymerase I. Despite the human and mouse factors exert different electrophoretic mobilities in gel retardation assays, UV-crosslinking and proteolytic clipping experiments indicate that both the sizes and the tertiary structure of the Sal box binding proteins of both species are very similar. When bound to DNA, both the human and the mouse factor terminate transcription of pol I from the heterologous species. The results implicate that changes in signal sequences necessary for termination have been accompanied by compensatory changes in the DNA binding domain of the protein(s) interacting with the termination signal. In contrast, the protein-protein interactions between the termination factor and the transcribing RNA polymerase I appear to have been conserved during evolution.

Animals↗

PubMed1987

Functional elements of DNA upstream from the integrase operon that are conserved in bacteriophages 434 and lambda.

A 1488-bp restriction fragment of bacteriophage 434 DNA contains the integrase promoter and an adjacent nucleotide sequence (t'I) resembling a Rho-independent terminator. To identify and quantitate transcription termination, DNA segments were cloned into a plasmid between the galactose promoter and assayable galactokinase gene and tested for termination. Whereas the entire fragment effectively terminated transcription, a 331-bp restriction fragment containing the t'I terminator had only weak terminator activity. Random sequential deletions of the 434 DNA segment defined a strong terminator 650-bp upstream from t'I. This proposed Rho-independent terminator called tL4 consists of a 7-bp stem and 6-nt loop followed by a uridine-rich region in the RNA. Phage lambda contains an even stronger tL4 terminator that differs in 4 nt from 434 tL4. Thus, despite some sequence divergence, terminator activity has been conserved in these phages. The 434 DNA segment was also tested for promoter activity. Rightward promoter activity (opposite to pL in the phage) was located about 200 bp to the right of tL4 and was followed by an open reading frame (ORF) capable of encoding a 91 amino acid protein. Promoter activity in the same approximate location was also found in phage lambda. Thus the rightward promoter, the tL4 and t'I terminators, and ORF-55 all are elements in this segment of the genome that are conserved for function despite sequence divergence.

Amino Acid Sequence↗

PubMed1987

Transcripts within the replication origin, oriC, of Escherichia coli.

Transcription start and termination sites were mapped in the E. coli replication origin, oriC. Outward transcription from within oriC (promoters Pori-r and Pori-l) was found to start in vivo at position 178 for Pori-l and at positions 294 and 304 for Pori-r, respectively. These transcripts were terminated after 100-150 bases, at terminators designated Tori-l and Tori-r. Transcription from the 16 kd promoter, which lies clockwise adjacent to oriC and promotes transcription toward oriC, started at position 757 and gave transcripts with 3' ends at several positions within and to the left of the minimal replication origin. However, the majority of transcripts traversed the whole oriC region, and were not terminated within the DNA segment tested. Transcription of the chromosomal 16 kd gene was negatively regulated by DnaA protein and positively affected by dam methylation. The possible function of these transcripts is discussed.

DNA Replication↗

PubMed1984

Structural and functional organization of the gpt gene region of Escherichia coli.

The nucleotide sequence was determined for the Escherichia coli region containing the gpt gene, which encodes the enzyme xanthine-guanine phosphoribosyl transferase (XGPRT; EC 2.4.2.22). Restriction enzyme and sequence analyses have allowed us to locate precisely the gpt gene (16.9-kDal XGPRT) with respect to other genes in this region, notably phoE. Genes gpt and phoE are pointing towards each other and are separated by about 1840 bp. Available sequence data and protein analyses [Overbeeke et al., J. Mol. Biol. 163 (1983) 513-522, and this paper] indicate the presence, between gpt and phoE, of two additional genes. These genes are oriented the same way as gpt and code for proteins of 49 and 15.7 kDal, respectively. By in vitro transcription with E. coli RNA polymerase and nuclease S1 analysis, we have identified a promoter upstream of gpt. The short intercistronic region between gpt and the 49-kDal protein gene contains a rho-independent termination signal that closely precedes and partially overlaps another promoter. It appears from these data that gpt transcription is essentially monocistronic, giving rise to RNA of approx. 555 nucleotides, whereas the 49-kDal and 15.7-kDal protein genes are transcribed from their own promoter.

Amino Acid Sequence↗

PubMed1999

Linear expression elements: a rapid, in vivo, method to screen for gene functions.

The increasing accumulation of genomic sequence information has accentuated the need for new methods to efficiently assess gene function and to prepare reagents to study these functions. Toward solving this general problem in functional genomics, we report a method by which any PCR-amplified open-reading frame (ORF) can be noncovalently linked to a eukaryotic promoter and terminator, and directly injected into animals to produce local gene expression. We also demonstrate that ORFs can be delivered into mice to produce antibodies specific for the encoded foreign protein by simply attaching mammalian promoter and terminator sequences. This technology makes it possible to screen large numbers of genes rapidly for their functions in vivo or to produce immune responses without the necessity of cloning, bacterial propagation, or protein purification.

Animals↗

PubMed1992

Characterization of transcription initiation and termination signals of the proteinase genes of Lactococcus lactis Wg2 and enhancement of proteolysis in L. lactis.

The transcription initiation signals of the prtP and prtM genes specifying the proteolytic activity of Lactococcus lactis subsp. cremoris Wg2 were mapped by primer extension. The strength of these promoters was analyzed with promoter-screening vector pGKV410, and they appeared to be weaker than previously isolated promoters of strain Wg2. In addition, a putative transcription terminator downstream of the prtP gene was characterized by using the terminator-screening vector pGKV259. The putative terminator decreased the transcription activity of lactococcal promoter P59 by approximately 70% in both Bacillus subtilis and L. lactis. Deletion of a part of the stem-loop structure of the terminator decreased the negative effect on transcription, indicating that the structure could indeed function as a terminator of transcription. The proteolytic activity of the lactococcal host was enhanced by placing the originally oppositely oriented prt genes in tandem and replacing the relatively weak promoters upstream of the prt genes with the stronger promoter, P32, from the chromosome of L. lactis Wg2.

Bacterial Proteins↗

PubMed1991

Construction and characterization of promoter-probe vectors for Corynebacteria using the kanamycin-resistance reporter gene.

Several multicopy promoter-probe plasmid vectors have been constructed that replicate in Brevibacterium lactofermentum and related coryneform amino acid-producing bacteria. Transcriptional activity is detected by the expression of a promoter-less aminoglycoside phosphotransferase gene (kan) derived from transposon Tn5; expression of this gene confers kanamycin resistance in B. lactofermentum. An efficient transcriptional terminator from the B. lactofermentum trp operon has been inserted upstream of the kan coding region to prevent significant transcriptional readthrough from vector promoters. The cat gene from Streptomyces acrimycini or the hygromycin-resistance gene from S. hygroscopicus are used as primary selection markers in the promoter-probe plasmid vectors. Using the promoter-probe vectors described in this paper, we have cloned several transcriptionally active fragments from the endogenous plasmid pBL1 of B. lactofermentum into Escherichia coli and/or B. lactofermentum.

Brevibacterium↗

PubMed1991

Expression and cellular localisation of porcine transmissible gastroenteritis virus N and M proteins by recombinant vaccinia viruses.

Porcine transmissible gastroenteritis virus (TGEV) nucleoprotein and integral membrane protein genes were cloned into the vaccinia virus insertion vector, pGS20, in the correct orientation for expression under the control of the vaccinia P7.5K promoter. Recombinant vaccinia viruses were generated by in vivo homologous recombination of the insertion vector with the WR strain of vaccinia virus. Nucleoprotein (N) expressed by both recombinant vaccinia virus and TGEV had a relative molecular mass (Mr) of 47,000 and was susceptible to degradation at the C-terminus yielding discrete breakdown products. The integral membrane protein (M) expressed by a recombinant vaccinia virus and TGEV was sensitive to endoglycosidase H reducing the mature polypeptide of Mr 29,000 to a species of Mr 27,000. Expression of M by recombinant vaccinia virus was inhibited during early infection due to a cryptic vaccinia virus transcriptional termination signal within the TGEV coding sequence. Indirect immunofluorescence showed that both N and M were only localised in the cell cytoplasm of either TGEV or recombinant vaccinia virus immunoprecipitated specific TGEV antigens from lysates of TGEV infected cells but had little significant TGEV neutralising activity in vitro.

Acetylglucosaminidase↗

PubMed1985

Thermosensitivity of a DNA recognition site: activity of a truncated nutL antiterminator of coliphage lambda.

Antitermination is an important transcriptional control. In bacteriophage lambda, the presence of the nut antiterminators between the promoters and terminators results in relatively unhindered transcription when the lambda N gene product and necessary host factors are supplied. This antitermination system has been rendered thermosensitivity by modification of the nut site. A fragment of lambda DNA [74 base pairs (bp) in length]that contained the 17-bp nutL core sequence, but lacked the 8-bp boxA sequence, was cloned in a pp-N-tL1-galK plasmid between the pp promoter and gene N. This fragment mediated antitermination of transcription at 30 degrees C, as measured by assaying galK gene expression in Escherichia coli. At 42 degrees C, however, antitermination at the lambda tL1 terminator was abolished. Antitermination at 42 degrees C was restored by replacing the 74-bp nutL fragment with longer sequences containing both nutL and boxA or by cloning a synthetic boxA sequence ahead of the 74-bp nutL fragment. Thus, efficient antitermination required both boxA and the 17-bp nutL core, with the latter becoming conditionally defective when the boxA sequence was deleted.

Bacteriophage lambda↗

PubMed1990

Expression of a ciliate gene in Escherichia coli using a suppressor tRNA to read the UAA and UAG glutamine codons.

Most ciliates use a particular genetic code where the standard stop codons UAA and UAG encode glutamine. Ciliate genes cannot therefore be expressed in heterologous systems such as Escherichia coli. To overcome this problem, we worked out a system of inducible suppression to permit efficient readthrough of UAAs and UAGs: a strong UAA tRNA suppressor that inserts glutamic acid was cloned downstream from a tac promoter whose efficiency was reduced by a transcription terminator. This system proved to be operational (1) to suppress UAG mutations by wobble pairing in an E. coli lacI-lacZ gene fusion and (2) to read through at least eight UAA glutamine codons in a Paramecium alpha-tubulin gene, as detected by Western blotting and colony hybridization. This work opens the way for cloning Ciliate genes from expression libraries and for expressing particular sequences without extended in vitro mutagenesis. A similar approach can be envisaged for expression of genes from Mycoplasma, mitochondria or other genomes that use non-standard genetic codes.

Animals↗

PubMed2003

Assembly of linear functional expression elements with DNA fragments digested with asymmetric restriction endonucleases.

A three-step method is described to assemble linear functional expression elements for rapid gene expression. In step one, a eukaryotic promoter, an open reading frame and a transcription terminator are PCR-amplified individually with primers containing recognition sites of asymmetric restriction endonucleases. In step two, the three DNA fragments, digested with corresponding restriction endonucleases, are directionally joined by T4 DNA ligase to assemble linear functional expression elements. In step three, the resultant linear functional expression elements are amplified with element-specific primers by a second round PCR, followed by transfection into Chinese hamster ovary cells for gene expression.

Animals↗