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PubMedDate not supplied

[Statistical characteristics of primary structures of the functional regions of the Escherichia coli genome. III. Computer recognition of coding regions].

We have presented the method for recognition of structural domains of DNA. This method uses statistical description of coding and non-coding regions in the form of stationary or nonstationary Marcov chain, which was introduced in our previous papers. Calculation of the probability that the given fragment of the DNA appears part of the coding region, is the main operation of this algorithm. The results, obtained for the number of E. coli DNA sequences showed the ability of the method to find the structural domains and correct reading frame, so as to give the estimation of the extent of protein expressivity. Provided necessary statistical data are available, the proposed method may be used for the analysis of DNA of other organisms.

Base Sequence↗

PubMed1986

Expression vectors based on the rac fusion promoter.

The -35 region of the rrnB P2 promoter and the -10 region of the lacZpo promoter-operator were fused to form the strong and regulatable rac promoter. Vectors were constructed that allow the attachment of protein-coding sequences to the beta-galactosidase alpha-peptide (LacZ alpha) in any reading frame. By introducing a high-copy-number mutation, the synthesis of a LacZ alpha-chloramphenicol acetyltransferase fusion protein reached more than 60% of total cell protein in Escherichia coli.

Acetyltransferases↗

PubMed1988

What constitutes the signal for the initiation of protein synthesis on Escherichia coli mRNAs?

Small DNA fragments (60 to 80 nucleotides), randomly obtained from a collection of 14 catabolic, biosynthetic or regulatory Escherichia coli genes, have been shot-gun cloned in place of the lacZ ribosome binding site. A total of 47 recombinants showing substantial beta-galactosidase synthesis (at least 1/30th of the wild-type) were isolated, and their newly acquired translational starts were characterized. Of these, 46 were found to carry a ribosome binding site from one of the original genes, and only one, a non-natural start. Moreover, 12 out of the 14 natural starts were found. The two that were not found are the only ones lacking a Shine-Dalgarno element. So, real starts are generally active in the lac mRNA, whereas the many sites (approx. 100 in this gene collection) that carry a Shine-Dalgarno element followed by AUG or GUG but are located in intra- or intergenic regions, or on non-transcribed strands, are inactive. I conclude that: (1) these "false" starts, being strongly discriminated against in the lac message, are presumably also inactive in their original mRNAs; (2) the discriminating information, being portable from one mRNA to another, must be contained within a small DNA region surrounding the starts. Indeed, I further show that it generally lies within a sequence of about 35 nucleotides bracketing real starts; and (3) this information must have a larger effect on initiation than the exact structure of the mRNA, because the discrimination persists despite a complete change of this structure. Previous statistical analysis has shown that real starts differ from false starts in having a non-random sequence composition from nucleotides -20 to +15 with respect to the start. To uncover whether these biases constitute the discriminating information or simply reflect coding constraints, translational starts were randomly searched in eukaryotic, largely non-coding, DNA. These "eukaryotic" starts all have an in-phase AUG or GUG, preceded by a typical Shine-Dalgarno sequence; outside these elements, the initiator region is strikingly rich in A, and poor in C. These biases match those found around real starts, demonstrating that they are indeed part of the initiation signal. Finally, I describe a simple procedure for introducing any DNA fragment in place of the lac operator site on the E. coli chromosome.

Base Sequence↗

PubMed1992

Regulated expression of a mammalian nonsense suppressor tRNA gene in vivo and in vitro using the lac operator/repressor system.

We have exploited the Escherichia coli lac operator/repressor system as a means to regulate the expression of a mammalian tRNA gene in vivo and in vitro. An oligonucleotide containing a lac operator (lacO) site was cloned immediately upstream of a human serine amber suppressor (Su+) tRNA gene. Insertion of a single lac repressor binding site at position -1 or -32 relative to the coding region had no effect on the amount of functional tRNA made in vivo, as measured by suppression of a nonsense mutation in the E. coli chloramphenicol acetyltransferase gene following cotransfection of mammalian cells. Inclusion of a plasmid expressing the lac repressor in the transfections resulted in 75 to 98% inhibition of suppression activity of lac operator-linked tRNA genes but had no effect on expression of the wild-type gene. Inhibition could be quantitatively relieved with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG). Similarly, transcription in vitro of lac operator-linked tRNA genes in HeLa cell extracts was repressed in the presence of lac repressor, and this inhibition was reversible with IPTG. These results demonstrate that the bacterial lac operator/repressor system can be used to reversibly control the expression of mammalian genes that are transcribed by RNA polymerase III.

Base Sequence↗

PubMed1983

Synthesis of two polypeptide subunits of an aminoacyl tRNA synthetase as a single polypeptide chain.

E. coli aminoacyl tRNA synthetases are typically comprised of a single type of polypeptide chain. Glycine tRNA synthetase is an exception, and is comprised of two different subunits. Previous work showed that glyS encodes both subunits in a tandem arrangement of coding regions which are in the same reading frame. Nine nucleotides separate the TAA stop of the first coding segment (alpha-subunit) from the ATG start of the second one (beta-subunit). A plasmid containing glyS was put into four different ochre suppressor strains. In three of them, significant quantities of an alpha-beta fusion protein were synthesized in maxicells, in genetic backgrounds which retained cellular proteases. This shows that the fusion protein is stable in vivo and suggests that Gly-tRNA synthetase is operationally a single polypeptide which is the ancestor of the two subunits.

Amino Acid Sequence↗

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↗

PubMed1988

A mammalian viral enhancer confers transcriptional regulation in yeast.

Transcriptional enhancers are DNA sequences that regulate RNA transcription from linked promoters by binding to cellular proteins (trans-activators). In the mammalian virus SV40, initiation of transcription is controlled, in part, by a strong 72-base pair enhancer. We show that yeast cells contain a factor that binds specifically to a key DNA motif in the SV40 enhancer, the P element that is essential for viral transcription in mammalian cells. The P element shows sequence similarity to a yeast DNA transcriptional regulatory element, GCN4, that controls transcription of genes that code for amino acid biosynthetic enzymes. Insertion of the SV40 enhancer or single or multiple copies of the P element itself upstream from the cytochrome Cyc-1 promoter places the hybrid viral-yeast transcription unit under metabolic control in yeast cells. These studies suggest that the SV40 P element and its complementary trans-activator represent a conserved transcriptional control mechanism that operates on widely divergent functions in evolution.

Base Sequence↗

PubMed1983

Repression of glycoprotein synthesis and release of surface coat during transformation of Trypanosoma brucei.

The biosynthesis of the variant surface glycoprotein (VSG) and its release from the surface of Trypanosoma brucei 427 variant clone MITat 1.4 (117) during in vitro transformation of bloodstream trypomastigotes to procyclic trypomastigotes was investigated. After transfer to the transformation medium at 27 degrees C, VSG synthesis is repressed with a half-time, t1/2 = 30 min. Concomitantly VSG-specific mRNA is lost suggesting that repression operates at the transcriptional level. The expression-linked extra gene copy, which codes for VSG, is retained during and after completion of transformation. After repression of VSG synthesis, surface VSG is shed from the cells into the culture medium. During release part of VSG (apparent mol. wt. 61 000) is proteolytically cleaved to a product (apparent mol. wt. 51 000) which represents the N-terminal domain of the protein as judged by the absence of the carbohydrate moiety normally linked to the C terminus.

Animals↗

PubMed1989

Investigation into the nature of a Bacillus promoter cloned into a promoter-probe plasmid.

The alpha-amylase-coding gene (amy) of Bacillus amyloliquefaciens NCP1 was cloned into the Bacillus subtilis promoter probe vector pPL603b.1, using a BglII digest of chromosomal DNA. The resulting plasmid, pVC102, was shown to have a BglII site within the insert. It was determined that this was the result of the fortuitous co-cloning of 2.88-kb and 0.92-kb BglII fragments separated in NCP1 DNA by approx. 3 kb. Unexpectedly, this co-cloning was readily repeated. Subcloning showed that while the 2.88-kb amy-bearing fragment was sufficient for amylase production, it might not have been capable of promoting sufficient levels of chloramphenicol resistance under the conditions used in the cloning experiments. The promoter on the 0.92-kb BglII fragment was more efficient, although its sequence differed from the canonical promoter sequence recognised by B. subtilis RNA polymerase E.sigma 43. As other promoter-bearing fragments from NCP1 DNA operated equally efficiently when cloned into pPL603b.1, the reason for the repeated co-cloning of the 2.88-kb and 0.92-kb NCPI BglII fragments may well be due to structural parameters, whereby certain nucleotide sequences are more readily cloned than others.

Bacillus↗

PubMed1990

A family of expression vectors based on the rrnB P2 promoter of Escherichia coli.

We describe here the construction of a family of expression vectors, based on the P2 promoter of the Escherichia coli rrnB gene by removing regulatory sequences downstream of the Pribnow-box and replacing them with the lac operator. These vectors allow cloning of foreign genes in such a way that their products are synthesized either in the form of fusion proteins of different length, or without fusion partners, with or without the original translational initiation signals. One of the vectors contains a synthetic oligothreonine-coding sequence that helps to stabilize the product of the cloned gene. These vectors allow high-level regulated expression of foreign genes, even if their products are relatively short peptides.

Amino Acid Sequence↗

PubMed1981

The sequence of human serum albumin cDNA and its expression in E. coli.

A recombinant plasmid has been constructed which contains the mature protein coding region of the human serum albumin (HSA) gene. Bacteria containing this plasmid synthesize HSA protein under control of the E. coli trp promoter-operator. The DNA sequence and predicted protein sequence of HSA were determined from the cDNA plasmid and are compared to existing data obtained from direct protein sequencing. The DNA sequence predicts a mature protein of 585 amino acids preceded by a 24 amino acid "prepro" peptide.

Amino Acid Sequence↗

PubMed1990

Translational regulation of the expression of ribosomal protein genes in Xenopus laevis.

The mRNAs coding for ribosomal proteins (rp-mRNA) are subjected to translational control during Xenopus oogenesis and embryogenesis, and also during nutritional changes in Xenopus cultured cells. This regulation, which appears to respond to the cellular need for new ribosomes, operates by changing the fraction of rp-mRNA engaged on polysomes, each translated rp-mRNA molecule always remaining fully loaded with ribosomes. All rp-mRNAs analyzed up to now show this translational behavior, and also share some structural features in their untranslated portions. In particular they all have rather short 5' untranslated regions, similar to each other, and always start at the very 5' end with a stretch of several pyrimidines. Fusion to a reporter-coding sequence of the 5' untranslated region of r-protein S19 has shown that this is involved in the translational regulation.

Animals↗

PubMed1991

Point mutations upstream of the yeast ADH2 poly(A) site significantly reduce the efficiency of 3'-end formation.

The sequences directing formation of mRNA 3' ends in Saccharomyces cerevisiae are not well defined. This is in contrast to the situation in higher eukaryotes in which the sequence AAUAAA is known to be crucial to proper 3'-end formation. The AAUAAA hexanucleotide is found upstream of the poly(A) site in some but not all yeast genes. One of these is the gene coding for alcohol dehydrogenase, ADH2. Deletion or a double point mutation of the AAUAAA has only a small effect on the efficiency of the reaction, and in contrast to the mammalian system, it is most likely not operating as a major processing signal in the yeast cell. However, we isolated point mutations which reveal that a region located approximately 80 nucleotides upstream of the poly(A) site plays a critical role in either transcription termination, polyadenylation, or both. These mutations represent the first point mutations in yeasts which significantly reduce the efficiency of 3'-end formation.

Alcohol Dehydrogenase↗

PubMed1990

Codon recognition mechanisms in plant chloroplasts.

In chloroplasts, all 61 sense codons are found in chloroplast (cp) DNA sequences coding for proteins. However among the sequenced cp tRNAs or tRNA genes, tRNAs with anticodons complementary to codons CUU/C (Leu), CCU/C (Pro), GCU/C (Ala) and CGC/A/G (Arg) [or CGC/A (Arg) in Marchantia] have not been found. In this paper we show that cp tRNA(Ala)(U*GC) cp tRNA(Pro)(U*GG) and cp tRNA(Arg)(ICG) are able to decode the corresponding four-codon family. In the case of leucine codons CUU/C, we show that 'U:U and U:C wobble' mechanisms can operate to allow the reading of these codons by cp tRNA(Leu)(UAm7G).

Amino Acid Sequence↗

PubMed2026

Functional chimeric mRNAs encode proteins in mammalian immunity.

Individual mammalian mRNAs and proteins are typically believed to originate from single genomic loci, with isoform diversity arising through cis-splicing of pre-mRNA. Whether mRNA from distant genes can undergo trans-splicing to generate functionally relevant chimeric transcripts has remained unclear. Here we develop a pipeline combining long-read direct RNA sequencing with non-targeted and targeted validation to identify chimeric transcripts in macrophages. Chromatin conformation capture studies reveal that inflammation induces interchromosomal DNA interactions, positioning parent genes proximally to facilitate the formation of chimeric mRNA. Notably, we identify a protein-coding chimeric mRNA representing a fusion between the pore-forming protein gasdermin D (GSDMD)1,2 and a C-terminal domain translated out of frame from Tmem106a (Gsdmd-Tmem106a) in mice. We show that inflammasome priming upregulates Gsdmd-Tmem106a, with the protein localizing to the plasma membrane. After activation of the inflammasome, GSDMD-TMEM106A directly interacts with canonical GSDMD N termini to accelerate and enhance pore formation and IL-1β release. Finally, we show that GSDMD-TMEM106A balances host defence and immunopathology in vivo: its loss protects against lethal sepsis but compromises antibacterial defence, whereas overexpression enhances host protection while increasing sepsis lethality. We establish that protein-coding chimeric mRNAs formed by regulated transcript fusion events are operative during inflammation and immunity.

Journal Article↗

PubMed1991

Fibronectin and collagen gene expression in healing experimental colonic anastomoses.

The temporal and spatial expression of fibronectin and type I and III collagen genes were studied 1-14 days after surgery in the healing rat colonic anastomosis using recombinant deoxyribonucleic acid techniques. Messenger ribonucleic acids (mRNAs) coding for fibronectin and type III collagen synthesis increased from the first day after operation and type I collagen synthesis increased from the second day after operation, as demonstrated by Northern hybridizations. Maximal mRNA production for fibronectin and collagens was seen at 2 and 7 days, respectively, after anastomosis. Activation of type I and III collagen genes in the anastomotic area was confined to tissues developing in the anastomotic line, the serosal surface and the submucosal layer. Strong fibronectin expression was observed in the same areas. The results suggest that genetic events leading to collagen synthesis in the anastomotic area start immediately after surgery. Maximal gene expression is not reached until 1 week after surgery.

Anastomosis, Surgical↗

PubMed1991

Poly(A) site choice in retroelements: deja vu all over again?

The primary transcripts encoded by retroelements contain two polyadenylation [poly(A)] sites, positioned 5' and 3' of the protein-coding sequences. To ensure efficient gene expression in these systems, a mechanism must operate that suppresses use of the 5' site, enhances use of the 3' site, or both. These possibilities have been examined in a recent series of experiments that have revealed two main regulatory themes. First, maximum 3' end processing can require sequences other than core poly(A) signals, which are transcribed only before the 3' site. Second, processing is partly inhibited when the site is positioned close to the promoter. These results should serve as the basis for a more detailed understanding of how the choice of the poly(A) site is regulated in these elements.

Base Sequence↗

PubMed2024

Serum lncRNA ITGB2-AS1 and ICAM-1 as novel biomarkers for rheumatoid arthritis and osteoarthritis diagnosis.

BACKGROUND: The complete circulating long non-coding RNAs (lncRNAs) signature of rheumatoid arthritis (RA) and osteoarthritis (OA) is still uncovered. The lncRNA integrin subunit beta 2 (ITGB2)-anti-sense RNA 1 (ITGB2-AS1) affects ITGB2 expression; however, there is a gap in knowledge regarding its expression and clinical usefulness in RA and OA. This study investigated the potential of serum ITGB2-AS1 as a novel diagnostic biomarker and its correlation with ITGB2 expression and its ligand intercellular adhesion molecule-1 (ICAM-1), disease activity, and severity in RA and primary knee OA patients. SUBJECTS: Forty-three RA patients, 35 knee OA patients, and 22 healthy volunteers were included. RESULTS: Compared with healthy controls, serum ITGB2-AS1 expression was upregulated in RA patients but wasn't significantly altered in knee OA patients, whereas serum ICAM-1 protein levels were elevated in both diseases. ITGB2-AS1 showed discriminative potential for RA versus controls (AUC = 0.772), while ICAM-1 displayed diagnostic potential for both RA and knee OA versus controls (AUC = 0.804, 0.914, respectively) in receiver-operating characteristic analysis. In the multivariate analysis, serum ITGB2-AS1 and ICAM-1 were associated with the risk of developing RA, while only ICAM-1 was associated with the risk of developing knee OA. A panel combining ITGB2-AS1 and ICAM-1 showed profound diagnostic power for RA (AUC = 0.9, sensitivity = 86.05%, and specificity = 91.67%). Interestingly, serum ITGB2-AS1 positively correlated with disease activity (DAS28) in RA patients and with ITGB2 mRNA expression in both diseases, while ICAM-1 positively correlated with ITGB2 expression in knee OA patients. CONCLUSION: Our study portrays serum ITGB2-AS1 as a novel potential diagnostic biomarker of RA that correlates with disease activity. A predictive panel combining ITGB2-AS1 and ICAM-1 could have clinical utility in RA diagnosis. We also spotlight the association of ICAM-1 with knee OA diagnosis. The correlation of serum ITGB2-AS1 with ITGB2 expression in both diseases may be insightful for further mechanistic studies.

Humans↗