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At least 991 records · Page 55Linked to original sources
PubMed1992

Characterization of a stable eukaryotic cell line expressing the Rous sarcoma virus integrase.

The Rous sarcoma virus integration protein (IN) is required for efficient integration of viral DNA into the host genome. IN was expressed in mouse C127 cells using a bovine papillomavirus vector. This system utilizes the mouse metallothionein promoter and the SV40 late polyadenylation signal for efficient expression of IN. A stable cell line derived from a single hygromycin-resistant colony was characterized. The expression of IN increased significantly upon Zn2+ induction of the metallothionein promoter, but did not respond to "superinduction" protocols. Full-length nonphosphorylated IN was the major product of expression. A minor product resulting from initiation of translation at an internal Met codon was also produced. The expressed IN did not exhibit the polypeptide heterogeneity at its COOH-terminus nor phosphorylation as is seen when IN is immunoprecipitated from virions. Using subcellular fractionation and indirect immunofluorescence, IN was primarily localized to nuclei and in some cells appeared to concentrate at discrete loci within the nuclei.

Animals↗
PubMed1982

Probing cII and himA action at the integrase promoter pi of bacteriophage lambda.

Plasmids were constructed to supply cII-coded protein for activation of the phage promoter pI. Using a fusion which expresses lacZ from pI. We can accurately follow activation of pI without having to assay int activity in vivo. A large excess of cII protein compared to a normal lytic infection stimulates lacZ expression about 10-fold over the basal level. The int-c226 constitutive allele of pI is not further activated by cII even though its level of lacZ expression is less than the maximal cII-activated expression from wild type pI. A himA-deleted strain also shows activation, demonstrating that there is no absolute requirement for the himA gene product for cII-stimulated transcription at pI.

Bacteriophage lambda↗
PubMed2006

A series of 5-aminosubstituted 4-fluorobenzyl-8-hydroxy-[1,6]naphthyridine-7-carboxamide HIV-1 integrase inhibitors.

A series of 5-amino derivatives of 8-hydroxy[1,6]-naphthyridine-7-carboxamide exhibiting sub-micromolar potency against replication of HIV-1 in cell culture was identified. One of these analogs, compound 12, displayed excellent pharmacokinetic properties when dosed orally in rats and in monkeys. This compound was demonstrated to be efficacious against replication of simian-human immunodeficiency virus (SHIV) 89.6P in infected rhesus macaques.

Amination↗
PubMed2006

A novel diketo phosphonic acid that exhibits specific, strand-transfer inhibition of HIV integrase and anti-HIV activity.

We have synthesized novel phosphonic acid analogues of beta-diketo acids. Interestingly, the phosphonic acid isostere, 2, of our anti-HIV compound, 1, was an inhibitor of only the strand transfer step, in stark contrast to 1. Compound 2 had lower anti-HIV activity than 1, but was more active and less toxic than the phosphonic acid analogue of L-708906. These isosteric compounds represent the first examples of beta-diketo phosphonic acids of structural, synthetic, and antiviral interest.

Acetoacetates↗
PubMed2006

Sensitive and specific LC-ESI-MS/MS method for the determination of a styrylquinoline, BA011FZ041, a potent HIV anti-integrase agent, in rat plasma.

A LC-MS/MS method was validated for the determination of BA011FZ041, a styrylquinoline derivative. After addition of BA011FZ055 as internal standard (IS), the method involved solid phase extraction (SPE), LC separation with an ether-phenyl column and quantification by MS/MS after positive ESI. The calibration curve, ranging from 1 to 500 ng/mL was fitted to a 1/x-weighted quadratic regression model. Lower limit of quantification (LLOQ) was 1 ng/mL using 100 microL of plasma. Intra- and inter-assay precision and accuracy values were within the regulatory limits. The method was successfully applied to the determination of BA011FZ041 in rat plasma and PBMCs after i.v. dosing.

Animals↗
PubMed2005

Non-equivalent interactions between amino-terminal domains of neighboring lambda integrase protomers direct Holliday junction resolution.

The bacteriophage lambda site-specific recombinase (Int), in contrast to other family members such as Cre and Flp, has an amino-terminal domain that binds "arm-type" DNA sequences different and distant from those involved in strand exchange. This defining feature of the heterobivalent recombinases confers a directionality and regulation that is unique among all recombination pathways. We show that the amino-terminal domain is not a simple "accessory" element, as originally thought, but rather is incorporated into the core of the recombination mechanism, where it is well positioned to exert its profound effects. The results reveal an unexpected pattern of intermolecular interactions between the amino-terminal domain of one protomer and the linker region of its neighbor within the tetrameric Int complex and provide insights into those features distinguishing an "active" from an "inactive" pair of Ints during Holliday junction resolution.

Alleles↗
PubMed2005

Phage phiC31 integrase: a new tool in plastid genome engineering.

Plastid transformation offers the unique advantages of high-level transgene expression and increased transgene containment compared with conventional transgenic technologies. The process relies on the homologous recombination machinery of the plastid incorporating foreign DNA into the plastome, which restricts the method to species where this type of incorporation works well. However, Pal Maliga and colleagues have recently reported a novel approach for integrating foreign DNA into the plastid genome that works independently of homologous recombination.

Bacteriophages↗
PubMed2006

Structure-based mutagenesis of the integrase-LEDGF/p75 interface uncouples a strict correlation between in vitro protein binding and HIV-1 fitness.

LEDGF/p75 binding-defective IN mutant viruses were previously characterized as replication-defective, yet RNAi did not reveal an essential role for the host factor in HIV-1 replication. Correlative analyses of protein binding and viral fitness were expanded here by targeting 12 residues at the IN-LEDGF/p75 binding interface. Whereas many of the resultant viruses were defective, the majority of the INs displayed wild-type in vitro integration activities. Though an overall trend of parallel loss of LEDGF/p75 binding and HIV-1 infectivity was observed, a strict correlation was not. His-tagged IN(A128Q), derived from a phenotypically wild-type virus, failed to pull-down LEDGF/p75, but IN(A128Q) was effectively recovered in a reciprocal GST pull-down assay. Under these conditions, IN(H171A), also derived from a phenotypically wild-type virus, interacted less efficiently than a previously described interaction-defective mutant, IN(Q168A). Thus, the relative affinity of the in vitro IN-LEDGF/p75 interaction is not a universal predictor of IN mutant viral fitness.

Cell Line↗
PubMed2002

Differential affinity and cooperativity functions of the amino-terminal 70 residues of lambda integrase.

The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein that binds two different classes of DNA-binding sites within its recombination target sites. The several functions of Int are apportioned between a large carboxy-terminal domain that cleaves and ligates DNA at each of its four "core-type" DNA-binding sites and a small amino-terminal domain, whose primary function is binding to each of its five "arm-type" DNA sites, which are distant from the core region. Int bridges between the two classes of binding sites are facilitated by accessory DNA-bending proteins that along with Int comprise higher-order recombinogenic complexes. We show here that although the 64 amino-terminal residues of Int bind efficiently to a single arm site, this protein cannot form doubly bound complexes on adjacent arm sites. However, 1-70 Int does show the same cooperative binding to adjacent arm sites as the full length protein. We also found that 1-70 Int specifies cooperative interactions with the accessory protein Xis when the two are bound to their adjacent cognate sites P2 and X1, respectively. To complement the finding that these two amino-terminal domain functions (along with arm DNA binding) are all specified by residues 1-70, we determined that Thr75 is the first residue of the minimal carboxy-terminal domain, thereby identifying a specific interdomain linker region. We have measured the affinity constants for Int binding to each of the five arm sites and the cooperativity factors for Int binding to the two pairs of adjacent arm sites, and we have identified several DNA structural features that contribute to the observed patterns of Int binding to arm sites. Taken together, the results highlight several interesting features of arm DNA binding that invite speculation about additional levels of complexity in the regulation of lambda site-specific recombination.

Amino Acid Sequence↗
PubMed2002

Specificity in DNA recognition by phage integrases.

The lambda-related (lambdoid) coliphages are related to one another by frequent natural recombination and maintain a high level of functional polymorphism for several activities of the phages. Arguments are presented that the polymorphism of the integration module results from selection (presumably frequency-dependent) for new (not improved) specificities of site recognition. Analysis of phages lambda and HK022 by Weisberg and collaborators previously showed that changes in five noncontiguous amino acids could switch site recognition specificity. Phage 21 and defective element e14, which integrate at the same site, differ in recognition specificity for both core and arm sites. In vitro assays of e14 and 21 insertion and excision confirm this conclusion. Inhibition by ds arm site oligonucleotides defines the sequence specificity more precisely.

Bacteriophage lambda↗
PubMedDate not supplied

Resolution of synthetic att-site Holliday structures by the integrase protein of bacteriophage lambda.

Site-specific recombination of the bacteriophage lambda genome into and out of the host bacterial genome is postulated to involve the formation of Holliday structure intermediates by reciprocal single-strand exchanges. Synthetic analogues of the predicted recombination intermediates are resolved in vitro by the protein product of the lambda int gene. Some of the structural features and reaction conditions for this genetic recombination can now be defined.

Bacteriophage lambda↗
PubMed2005

Enantioselective total synthesis of a novel polyketide natural product (+)-integrasone, an HIV-1 integrase inhibitor.

Enantioselective total synthesis of the recently isolated, novel polyketide natural product (+)-integrasone has been accomplished from the readily available Diels-Alder adduct of cyclopentadiene and p-benzoquinone. An enzymatically desymmetrized epoxyquinone building block has been elaborated through a series of regio- , chemo- and stereocontrolled steps to the final bicyclic framework of the natural product.

Bridged Bicyclo Compounds, Heterocyclic↗