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At least 73 records · Page 4Linked to original sources
PubMed2005

Cyclic peptides, a chemical genetics tool for biologists.

The genomics era has presented scientists with an overwhelming amount of sequence information that encodes for a continuously expanding array of metabolic pathways and regulatory networks. In order to unravel this elaborate maze, effective approaches need to be pioneered, and cyclic peptides represent an exciting new tool that can aid this challenging task. There are considerable benefits to cyclic peptides including enhanced protein binding affinity and metabolic stability, but perhaps most important is the ability to biologically synthesize them using intein chemistry. This capacity for genetic encoding allows biologists access to diverse, intracellular small-molecule libraries that can be tailored for numerous chemical genetics approaches. An increasing number of molecular strategies are being developed to aid in the implementation of cyclic peptides in forward and reverse genetics experiments, which will provide researchers with a powerful toolbox to complement existing genomic methodologies.

Animals↗
PubMed2001

Colicin A hybrids: a genetic tool for selection of type II secretion-proficient Pseudomonas strains.

The gram-negative bacterium Pseudomonas aeruginosa secretes the majority of its extracellular proteins by the type II secretion mechanism, a two-step process initiated by translocation of signal peptide-bearing exoproteins across the inner membrane. The periplasmic forms are transferred across the outer membrane by a machinery consisting of 12 xcp gene products. Although the type II secretion machinery is conserved among gram-negative bacteria, interactions between the secreted proteins and the machinery are specific. The lack of a selectable phenotype has hampered the development of genetic strategies for studying type II secretion. We report a novel strategy to identify rare events, such as those that allow heterologous secretion or identification of extragenic suppressors correcting xcp defects. This is based on creating a host-vector system where the non-secretory phenotype is lethal. The original tool we designed is a hybrid protein containing elastase and the pore-forming domain of colicin A.

Bacterial Proteins↗
PubMed2004

Interneuron Diversity series: Molecular and genetic tools to study GABAergic interneuron diversity and function.

Structural and functional diversity of GABAergic interneurons has become increasingly central in our understanding of the elemental steps of information processing in the brain. The use of different molecular, electrophysiological and anatomical techniques has provided a wealth of new information regarding GABAergic interneurons over the past decade but it has also led to confusion regarding the number of subtypes of GABAergic interneurons. Combinatorial approaches that also consider multiple parameters seem now to offer renewed hope for finally clarifying the structural diversity of GABAergic interneurons. New molecular techniques have become a powerful tool for exposing the functional diversity of GABAergic neurons at the cellular, microcircuit and systems levels. This article reviews literature regarding molecular tools that have been used, or that appear promising for future attempts, to classify GABAergic interneurons. Some important limitations will also be indicated.

Animals↗
PubMed1987

Transfer of plasmid-borne tuf mutations to the chromosome as a genetic tool for studying the functioning of EF-TuA and EF-TuB in the E. coli cell.

The elongation factor EF-Tu of E. coli is a multifunctional protein that lends itself extremely well to studies concerning structure-function relationships. It is encoded by two genes: tufA and tufB. Mutant species of EF-Tu have been obtained by various genetic manipulations, including site- and segment-directed mutagenesis of tuf genes on a vector. The presence of multiple tuf genes in the cell, both chromosomal and plasmid-borne, hampers the characterization of the mutant EF-Tu. We describe a procedure for transferring plasmid-borne tuf gene mutations to the chromosome. Any mutation engineered by genetic manipulation of tuf genes on a vector can be transferred both to the tufA and the tufB position on the chromosome. The procedure facilitated the functional characterization of some of our recently obtained tuf mutations. Of particular relevance is, that it enabled us for the first time to obtain a mutant tufB on the chromosome, encoding an EF-TuB resistant to kirromycin. It thus became possible to study the consequences for growth of tufA inactivation by insertion of bacteriophage Mu. The preliminary evidence obtained suggests that an EF-TuA, active in polypeptide synthesis, is essential for growth whereas such an EF-TuB is dispensable.

Bacteriophage mu↗
PubMed1992

Human congenital anomalies: application of new genetic tools and concepts.

The discovery of nontraditional inheritance and of developmental gene families common to all metazoans has revolutionized research on human congenital anomalies. The most gratifying results of these new concepts are summarized in Table 5: several human syndromes have already been related to their causative genes. Classic embryonic mechanisms (ie, gastrulation, segmentation, adhesion) also have been correlated with specific molecules. Perhaps most optimistic are the facts that many of the relevant molecules can be viewed as growth factors and that many anomalies are examples of extended or arrested development known as heterochrony. The implication is that genetic screening and preconceptional diagnosis will allow treatment of congenital malformations by supplementation/suppression of the maternofetal unit. This future specialty of "gestational endocrinology" is already being used for embryonic therapy of 21-hydroxylase deficiency.

Alleles↗
PubMedDate not supplied

Species identification using genetic tools: the value of nuclear and mitochondrial gene sequences in whale conservation.

DNA sequence analysis is a powerful tool for identifying the source of samples thought to be derived from threatened or endangered species. Analysis of mitochondrial DNA (mtDNA) from retail whale meat markets has shown consistently that the expected baleen whale in these markets, the minke whale, makes up only about half the products analyzed. The other products are either unregulated small toothed whales like dolphins or are protected baleen whales such as humpback, Bryde's, fin, or blue whales. Independent verification of such mtDNA identifications requires analysis of nuclear genetic loci, but this is technically more difficult than standard mtDNA sequencing. In addition, evolution of species-specific sequences (i.e., fixation of sequence differences to produce reciprocally monophyletic gene trees) is slower in nuclear than in mitochondrial genes primarily because genetic drift is slower at nuclear loci. When will use of nuclear sequences allow forensic DNA identification? Comparison of neutral theories of coalescence of mitochondrial and nuclear loci suggests a simple rule of thumb. The "three-times rule" suggests that phylogenetic sorting at nuclear loci is likely to produce species-specific sequences when mitochondrial alleles are reciprocally monophyletic and the branches leading to the mtDNA sequences of a species are three times longer than the average difference observed within species. A preliminary test of the three-times rule, which depends on many assumptions about the species and genes involved, suggests that blue and fin whales should have species-specific sequences at most neutral nuclear loci, whereas humpback and fin whales should show species-specific sequences at fewer nuclear loci. Partial sequences of actin introns from these species confirm the predictions of the three-times rule and show that blue and fin whales are reciprocally monophyletic at this locus. These intron sequences are thus good tools for the identification of these species and will afford a chance to identify putative hybrid blue/fin whales thought to have entered the retail market after 1989.

Actins↗
PubMed1990

Retroviruses as genetic tools to isolate transcriptionally active chromosomal regions.

By exploiting the ability of retroviruses to move genes into random sites of mammalian genomes and by exploiting some features of their replication, retrovirus vectors have been developed that select for instances in which the virus integrates into expressed genes. Since integrated proviruses tag transcriptionally active sites, the vectors provide a means to identify and isolate promoters active in different cell types. Furthermore, the viruses may be useful as insertional mutagens, since they select for instances in which integration occurs into expressed sites. This reduces the number of integrants needed to screen for loss of gene function and may enable genes controlling phenotypes in mammalian cells to be isolated.

Chromosomes↗
PubMed1994

Characterization of the auxin-inducible SAUR-AC1 gene for use as a molecular genetic tool in Arabidopsis.

The small auxin up RNA (SAUR) genes were originally characterized in soybean, where they encode a set of unstable transcripts that are rapidly induced by auxin. In this report, the isolation of a SAUR gene, designated SAUR-AC1, from Arabidopsis thaliana (L.) Heynh. ecotype Columbia is described. The promoter of the SAUR-AC1 gene contains putative regulatory motifs conserved among soybean SAUR promoters, as well as sequences implicated in the regulation of other genes in response to auxin. The transcribed region is approximately 500 bp in length and contains no introns. Highly conserved sequences located within the SAUR-AC1 transcript include the central portion of the coding region and a putative mRNA instability sequence (DST) located in the 3' untranslated region. Accumulation of SAUR-AC1 mRNA is readily induced by natural and synthetic auxins and by the translational inhibitor cycloheximide. Moreover, several auxin- and gravity-response mutants of Arabidopsis exhibit decreased accumulation of the SAUR-AC1 mRNA in elongating etiolated seedlings. In particular, in the axr2-1 mutant the SAUR-AC1 transcript accumulates to less than 5% of wild-type levels. These studies indicate that SAUR-AC1 will be a useful probe of auxin-induced gene expression in Arabidopsis and will facilitate the functional analysis of both transcriptional and posttranscriptional regulatory elements.

Amino Acid Sequence↗
PubMedDate not supplied

First isolation of bacteriophages for a spirochaete: potential genetic tools for Leptospira.

Three bacteriophages of the saprophytic aquicole bacterium Leptospira biflexa were isolated from sewage waters from the outskirts of Paris, France. These phages do not infect representative strains of the pathogenic species Leptospira interrogans, and their host range is restricted to serovar patoc of the saprophytic species. The phages were found to be lytic and no lysogenic state could be demonstrated. Electron micrographs showed that the phages were morphologically identical and had polyhedral heads and contractile tails. Their genomes were sensitive to restriction enzymes and consisted of double-stranded DNA. Pulsed-field agarose gel electrophoresis indicated that their genomes were linear: 60 kb for LE1 and 50 kb for LE3 and LE4.

Bacteriophages↗
PubMed2000

The "chip" as a specific genetic tool.

DNA microarrays are powerful tools for the analysis of the organization and regulation of the brain, in both illness and health. Such messenger RNA expression methods are outgrowths of a marriage between the several genome sequencing projects and a wide variety of physical, chemical, optical, and electronic systems. The advantages of microarray analyses include the ability to study the regulation of several genes or even the entire genome in a single experiment. However, there are substantive issues associated with the use of these tools that need to be considered before drawing conclusions about the genomic regulation of the brain. These issues include the loss of most anatomic (i.e., cellular and circuit) specificity, only fair sensitivity, lack of absolute quantitative data, poor comparability between studies, and high variability in sample values, to mention the most obvious. In this review we point to some of the solutions proposed for these problems and novel techniques and approaches for newer methods. Among these are methods for making arrays more sensitive, including nonarray messenger RNA expression systems. The future of this field and its links to deeper protein and cell biology are both emphasized.

Clinical Laboratory Techniques↗
PubMed1990

Bacteriophage Mu as a genetic tool to study Erwinia amylovora pathogenicity and hypersensitive reaction on tobacco.

Erwinia amylovora 1430 was shown to be sensitive to Mu G(-) particles. Infection resulted either in lytic development or in lysogenic derivatives with insertion of the Mu genome at many sites in the bacterial chromosome. We used the Mu d1Bx::Tn9 (lac Apr Cmr) derivative, called Mu dX, to identify mutants affected in pathogenicity and in their ability to induce a hypersensitive reaction (HR) on tobacco plants. Inoculation of 1,400 lysogenic derivatives on apple root calli led to the identification of 12 mutants in three classes: (i) class 1 mutants were nonpathogenic and unable to induce an HR on tobacco plants; (ii) class 2 mutants were nonpathogenic but retained the ability to induce an HR; and (iii) class 3 mutants showed attenuated virulence. Of the 12 mutants, 8 had a single insertion of the Mu dX prophage. For class 1 and 2 mutants, reversion to pathogenicity was concomitant with the loss of the Mu dX prophage. Furthermore, revertants from the class 1 mutants also recovered the ability to induce an HR on tobacco plants. Five of the six class 3 mutants were impaired in exopolysaccharide production. No changes of the envelope structure (lipopolysaccharide and outer membrane proteins) were correlated with differences in pathogenicity. One class 3 mutant did not produce any functional siderophore, suggesting that iron uptake could be involved in pathogenicity.

Bacteriophage mu↗