|
|
|
|
|
|
Kinetic Analysis of tRNA-Directed Transcription Antitermination of the Bacillus subtilis glyQS Gene In Vitro. Frank J. Grundy, 2004.Binding of uncharged tRNA to the nascent transcript promotes readthrough of a leader region transcription termination signal in genes regulated by the T box transcription antitermination mechanism . Each gene in the T box family responds independently to its cognate tRNA, with specificity determined by base pairing of the tRNA to the leader at the anticodon and acceptor ends of the tRNA . tRNA binding stabilizes an antiterminator element in the transcript that sequesters sequences that participate in formation of the terminator helix . tRNAGly-dependent antitermination of the Bacillus subtilis glyQS leader was previously demonstrated in a purified in vitro assay system . This assay system was used to investigate the kinetics of transcription through the glyQS leader and the effect of tRNA and transcription elongation factors NusA and NusG on transcriptional pausing and antitermination . Several pause sites, including a major site in the loop of stem III of the leader, were identified, and the effect of modulation of pausing on antitermination efficiency was analyzed . We found that addition of tRNAGly can promote antitermination as long as the tRNA is added before the majority of the transcription complexes reach the termination site, and variations in pausing affect the requirements for timing of tRNA addition . Inhibition of Human Immunodeficiency Virus Type 1 Tat-trans-Activation-Responsive Region Interaction by an Antiviral Quinolone Derivative. Sara Richter, 2004.WM5, a 6-aminoquinolone derivative, binds with high affinity to the bulge of the trans-activation-responsive region (TAR), whereas it displays low binding affinity for the loop and stem regions of TAR and for random RNA and DNA sequences . Furthermore, WM5 disrupts the natural protein-nucleic acid complex with a 50% inhibitory concentration in the low micromolar range in both in vitro and in vivo assays . Characterization of Humanized Antibodies Secreted by Aspergillus niger. Michael Ward, 2004.Two different humanized immunoglobulin G1(  ) antibodies and an Fab' fragment were produced by Aspergillus niger . The antibodies were secreted into the culture supernatant . Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase . After antibody assembly, cleavage by A . niger KexB protease allowed the release of free antibody . Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached . Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains . The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex6GlcNAc2 to Hex15GlcNAc2 . An aglycosyl mutant form of antibody was also produced . No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function .
Transposition of Cyanobacterium Insertion Element ISY100 in Escherichia coli. Akihiro Urasaki, 2002.The genome of the cyanobacterium Synechocystis sp . strain PCC6803 has nine kinds of insertion sequence (IS) elements, of which ISY100 in 22 copies is the most abundant . A typical ISY100 member is 947 bp long and has imperfect terminal inverted repeat sequences . It has an open reading frame encoding a 282-amino-acid protein that appears to have partial homology with the transposase encoded by a bacterial IS, IS630, indicating that ISY100 belongs to the IS630 family . To determine whether ISY100 has transposition ability, we constructed a plasmid carrying the IPTG (isopropyl-ß-D-thiogalactopyranoside)-inducible transposase gene at one site and mini-ISY100 with the chloramphenicol resistance gene, substituted for the transposase gene of ISY100, at another site and introduced the plasmid into an Escherichia coli strain already harboring a target plasmid . Mini-ISY100 transposed to the target plasmid in the presence of IPTG at a very high frequency . Mini-ISY100 was inserted into the TA sequence and duplicated it upon transposition, as do IS630 family elements . Moreover, the mini-ISY100-carrying plasmid produced linear molecules of mini-ISY100 with the exact 3' ends of ISY100 and 5' ends lacking two nucleotides of the ISY100 sequence . No bacterial insertion elements have been shown to generate such molecules, whereas the eukaryotic Tc1/mariner family elements, Tc1 and Tc3, which transpose to the TA sequence, have . These findings suggest that ISY100 transposes to a new site through the formation of linear molecules, such as Tc1 and Tc3, by excision . Some Tc1/mariner family elements leave a footprint with an extra sequence at the site of excision . No footprints, however, were detected in the case of ISY100, suggesting that eukaryotes have a system that repairs a double strand break at the site of excision by an end-joining reaction, in which the gap is filled with a sequence of several base pairs, whereas prokaryotes do not have such a system . ISY100 transposes in E . coli, indicating that it transposes without any host factor other than the transposase encoded by itself . Therefore, it may be able to transpose in other biological systems . Molecular Cloning of Endo-ß-D-1,4-Glucanase Genes, rce1, rce2, and rce3, from Rhizopus oryzae. Tatsuki Moriya, 2003.Three endoglucanase genes, designated the rce1, rce2, and rce3 genes, were isolated from Rhizopus oryzae as the first cellulase genes from the subdivision Zygomycota . All the amino acid sequences deduced from the rce1, rce2, and rce3 genes consisted of three distinct domains: cellulose binding domains, linker domains, and catalytic domains belonging to glycosyl hydrolase family 45 . The rce3 gene had two tandem repeated sequences of cellulose binding domains, while rce1 and rce2 had only one . rce1, rce2, and rce3 had various lengths of linker sequences . Quantum Dots as Strain- and Metabolism-Specific Microbiological Labels. J. A. Kloepfer, 2003.Biologically conjugated quantum dots (QDs) have shown great promise as multiwavelength fluorescent labels for on-chip bioassays and eukaryotic cells . However, use of these photoluminescent nanocrystals in bacteria has not previously been reported, and their large size (3 to 10 nm) makes it unclear whether they inhibit bacterial recognition of attached molecules and whether they are able to pass through bacterial cell walls . Here we describe the use of conjugated CdSe QDs for strain- and metabolism-specific microbial labeling in a wide variety of bacteria and fungi, and our analysis was geared toward using receptors for a conjugated biomolecule that are present and active on the organism's surface . While cell surface molecules, such as glycoproteins, make excellent targets for conjugated QDs, internal labeling is inconsistent and leads to large spectral shifts compared with the original fluorescence, suggesting that there is breakup or dissolution of the QDs . Transmission electron microscopy of whole mounts and thin sections confirmed that bacteria are able to extract Cd and Se from QDs in a fashion dependent upon the QD surface conjugate .
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
