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At least 19 recordsLinked to original sources
PubMed2026

Long-range mRNA folding shapes expression and sequence of bacterial genes.

Bacterial gene expression is strongly influenced by local mRNA secondary structure, yet the impact of long-range folding remains poorly understood. Here, we show that sequences hundreds of nucleotides from the mRNA 5' end can act as potent repressors of gene expression through long-range base pairing to the ribosome binding site (RBS), subjecting anti-RBS sequences to negative selection. Using massively parallel reporter assays in Bacillus subtilis, we identify anti-RBS sequences as among the strongest determinants of reduced mRNA abundance across the transcript body. We demonstrate that distal anti-RBS elements engage in long-range folding with the Shine-Dalgarno sequence, blocking ribosome entry and promoting mRNA decay. Consistent with these repressive effects, anti-RBS-like sequences are depleted throughout diverse bacterial coding sequences but not from leaderless transcripts, and introducing distal anti-RBS to native genes reduces expression. Our findings establish that long-range mRNA folding is a conserved force shaping gene expression and constrains coding sequence evolution.

Bacillus subtilis↗
PubMed1989

A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae.

A homologous transformation system for Aspergillus oryzae is described. The system is based on an A. oryzae strain deficient in orotidine-5'-phosphate decarboxylase (pyrG) and the vector pAO4-2, which contains a functional A. oryzae pyrG gene as selection marker. Transformation of the A. oryzae pyrG mutant with circular pAO4-2 resulted in the appearance of Pyr+ transformants at a frequency of up to 20 per micrograms of DNA, whereas with linear pAO4-2 up to 200 transformants per micrograms DNA were obtained. In 75% of the Pyr+ transformants recombination events had occurred at the pyrG locus, most of which (90%) resulted in insertion of one or two copies of the vector and the others (10%) in a replacement of the mutant allele by the wild-type allele. Vector pAO4-2 is also capable of transforming a corresponding mutant of Aspergillus niger. This transformation system was used to introduce into A. oryzae the heterologous and non-selectable bacterial genes lacZ, encoding beta-galactosidase, and uidA, encoding beta-glucuronidase. Using the Aspergillus nidulans gpdA promoter to drive bacterial gene expression in A. oryzae, relatively high levels of activity, as well as protein per se, as judged by western blot analyses, were obtained.

Aspergillus↗
PubMed1985

Bacterial gene expression and biotechnology.

This brief review article has been intended to give a few up-to-date examples of the dramatic impact that our knowledge of gene expression (especially bacterial gene expression) has had in the area of biotechnology. This area is in a state of such rapid growth that it has only been possible to present a limited overview of the subject matter. We have tried to illustrate our points with examples from work in which we have had some direct involvement. It should be apparent that continued increase in our understanding of gene expression should provide additional opportunities for expanded application of the new methodology.

Bacteria↗
PubMed1986

Silent nucleotide substitutions and G + C content of some mitochondrial and bacterial genes.

The G + C content of DNA varies widely in different organisms, especially microorganisms. This variation is accompanied by changes in the nucleotide composition of silent positions in codons. (Silent positions are defined and explained in the text). These changes are mostly neutral or near neutral, and appear to result from mutation pressure in the direction of increasing either A + T (AT pressure) or G + C (GC pressure) content. Variations in G + C content are also accompanied by substitutions at replacement positions in codons. These substitutions produce changes in the amino acid content of homologous proteins. The examples studied were genes for 13 mitochondrial proteins in five species, and A and B genes for bacterial tryptophan synthase in four species. In microorganisms, varying AT and GC mutational pressures, presumably resulting from shifts in the DNA polymerase system, exert strong effects on molecular evolution by changing the G + C content of DNA. These effects may be greater than those of random drift. The effects of GC pressure on silent substitutions in the systems examined are several times as great as the effects on replacement substitutions. GC pressure is exerted on noncoding as well as coding regions in mitochondrial DNA. This is shown by the close correlation (correlation coefficient, 0.99) of the G + C content of the noncoding D loop of mitochondria with the G + C content of silent positions in the corresponding mitochondrial genes.

Amino Acids↗
PubMed1988

Rapid identification of bacterial genes that are lethal when cloned on multicopy plasmids.

A procedure to identify genes that are lethal when cloned on multicopy plasmids was developed. It depends on the ability of mini-Mu plasmid elements to be used for both in vivo cloning and generalized transduction of enterobacterial genes. The feasibility of this procedure was demonstrated by using the tetA gene of Tn10, which is lethal when in multiple copies in the presence of 25 micrograms of tetracycline per ml.

Cloning, Molecular↗
PubMed1989

A new procedure for the targeted inactivation of essential bacterial genes.

A new method is described for the exchange of a plasmid encoded mutant gene with its chromosomal counterpart. The method is based on positive selection and is applicable for the exchange of essential genes. The main features of the method are: (1) cloning of an antibiotic resistance marker (without its promoter) downstream of the cloned target gene, thus forming an artificial operon; (2) inactivating the target gene (and consequently also the antibiotic resistance gene) by inserting a strong transcriptional termination signal into it; and (3) selection for double recombinants by means of the antibiotic resistance gene.

Blotting, Southern↗
PubMed1985

Alterations upstream from the Shine-Dalgarno region and their effect on bacterial gene expression.

A vector containing the leftward promoter (pL) as transcription initiation signal and a synthetic, easily adaptable translation initiation region have been constructed. We have used the expression system to assess the relevance of sequences upstream from the Shine-Dalgarno (SD) region in the translational-initiation process. To this end, a series of structural variants of the prototype ribosome-binding site were used to direct the synthesis of both mature human fibroblast interferon and beta-galactosidase (beta-gal). It was found that alterations 5' to the SD element can considerably affect the rate of mRNA translation. The observation that the relative efficiency of the various 5'-untranslated regions depends on the downstream coding information implies that secondary (and/or tertiary) structure formation is of major importance in the initiation process. But an mRNA folding, in which the SD and ATG determinant are set free in single-stranded regions, does not unconditionally guarantee an efficient initiation of translation.

Base Sequence↗
PubMed1989

Expression of a bacterial gene in a trypanosomatid protozoan.

A simple and reproducible assay for DNA-mediated transfection in the trypanosomatid protozoan Leptomonas seymouri has been developed. The assay is based on expression of the Escherichia coli chloramphenicol acetyl transferase (CAT) gene flanked by Leptomonas DNA fragments that are likely to contain necessary elements for gene expression in trypanosomes. After electroporation of cells in the presence of plasmid DNA, CAT activity was detected in crude cell lysates. No activity was detected when the orientation of the L. seymouri mini-exon sequence (placed upstream of the CAT gene) was reversed, or in additional control experiments. This system provides a method for defining transcriptional control elements in trypanosomes.

Animals↗
PubMed1990

Stable incorporation of a bacterial gene into adult rat skeletal muscle in vivo.

We have developed a novel technique to incorporate and stably express foreign genes in adult rat skeletal muscle in vivo. Endogeneous satellite cells in skeletal muscle regenerating from bupivacaine damage were infected with an injected retrovirus containing the Escherichia coli beta-galactosidase gene under the promoter control of the Moloney murine leukemia virus long-terminal repeat. Constitutive and stable expression of beta-galactosidase activity was observed in muscle fibers after 6 days and 1 mo of muscle regeneration. Two patterns of expression were observed, diffuse expression within fibers and focal expression associated with the sarcolemma. This technique will allow future experiments with muscle-specific genes and promoters to study the physiological regulation of skeletal muscle gene expression in the intact adult mammal. Furthermore, the technique of stimulating stem cell proliferation to allow retroviral-mediated gene transfer may be generally applicable to other tissues.

Animals↗
PubMedDate not supplied

[Binding of RNA-polymerase from Escherichia coli with oligodeoxyribonucleotides homologous to transcribed and non-transcribed DNA stands in the "-10"-promoter region of bacterial genes].

By using synthetic oligodeoxynucleotides related to the "-10" regions of spc and lacUV5 E. coli promoters we have shown, that the efficient binding occurs with oligodeoxynucleotides non-transcribed DNA strands. The duplexes containing oligodeoxynucleotides from the non-transcribed and transcribed DNA strands are also bound effectively by RNA polymerase. Oligodeoxynucleotides related to the non-transcribed DNA strands can effectively inhibit the RNA synthesis catalyzed by E. coli RNA polymerase. The data obtained allowed to consider the possible mechanism of selective binding of oligodeoxynucleotides by E. coli RNA polymerase and inhibition of the transcription.

Base Sequence↗
PubMed1990

Regulation of bacterial gene expression by metal-protein complexes.

Metal ions are essential cofactors in several transacting bacterial gene regulators. Upon binding of the metal, the receptor proteins act either as repressors of gene expression or, in other systems, as transcriptional activators. Other metal-dependent regulatory proteins may function, directly or indirectly, as sensors of the cellular oxygen status, and may even be mediators in light-responsive gene regulation.

Bacterial Proteins↗
PubMed1988

Transformation of Phycomyces with a bacterial gene for kanamycin resistance.

Phycomyces protoplasts transformed with a plasmid containing the bacterial gene for kanamycin resistance grow in the presence of G418, a kanamycin analogue. The plasmid also contains a Phycomyces DNA sequence that supports autonomous replication in yeast. We obtained about 250 transformants per microgram DNA or one per 5000 viable protoplasts. The transformant phenotype is retained under selective conditions and lost in the majority of the vegetative spores. Recovered plasmids and Southern analysis indicate that the plasmid probably replicates autonomously in Phycomyces.

DNA Restriction Enzymes↗
PubMed1989

Transfection with bacterial genes as a marker for cells seeded on vascular flow surfaces.

Autologous seeding of vascular grafts has been in use since 1972; however, the fate of seeded cells has never been determined. While short-term retention has been determined by radioactively labeling cells, long-term studies of seeded cells have not been possible due to the lack of an appropriate marker system. We have developed a long-term marker system for endothelial cells by transfecting the cells with bacterial genes that can be detected by fluorescentally-labeled antibodies to these markers. Two bacterial genes, neo and cat both carried by a pSV2 plasmid construct were used to co-transfect cells. Transfects were selected by growth in the presence of G418. Transfected clones were expanded into monolayers that stained positive for cat by fluorescence, and retained the normal cobblestone morphology and factor VIII staining of endothelial cells. By stably transfecting cells with bacterial genes these cells can now be used to seed vascular grafts and follow the long-term fate of seeded cells.

Animals↗
PubMed1985

[Expression in Chinese hamster transformant TK+-cells of the bacterial gene for beta-lactamase].

Using monospecific immune antiserum the expression of beta-lactamase bacterial gene (from the plasmid pBR322) in transformed TK+ Chinese hamster cells was studied. It is shown that in some TK+-clones the beta-lactamase gene is expressed and this expression is stimulated by an inhibitor of DNA methylation-5-azacytidine. Two physically linked genes (the Herpes simplex thymidine kinase gene, and the beta-lactamase gene) introduced into the Chinese hamster cells on the same plasmid are found to be expressed independently.

Animals↗