Multiplex Asymmetric PCR-Based Oligonucleotide Microarray for Detection of Drug Resistance Genes Containing Single Mutations in Enterobacteriaceae

ABSTRACT A multiplex asymmetric PCR (MAPCR)-based microarray method was developed for the detection of 10 known extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamase genes in gram-negative bacteria and for the typing of six important point mutations (amino acid positions 35, 43, 130, 179, 238, and 240) in the bla SHV gene. The MAPCR is based on a two-round reaction to promote the accumulation of the single-stranded amplicons amenable for microarray hybridization by employing multiple universal unrelated sequence-tagged primers and elevating the annealing temperature at the second round of amplification. A strategy to improve the discrimination efficiency of the microarray was constituted by introducing an artificial mismatch into some of the allele-specific oligonucleotide probes. The microarray assay correctly identified the resistance genes in both the reference strains and some 111 clinical isolates, and these results were also confirmed for some isolates by direct DNA sequence analysis. The resistance genotypes determined by the microarray correlated closely with phenotypic MIC susceptibility testing. This fast MAPCR-based microarray method should prove useful for undertaking important epidemiological studies concerning ESBLs and plasmid-mediated AmpC enzymes and could also prove invaluable as a preliminary screen to supplement phenotypic testing for clinical diagnostics.

Emergence of Mammalian Cell-Adapted Vesicular Stomatitis Virus from Persistent Infections of Insect Vector Cells

ABSTRACT Arboviruses (arthropod-borne viruses) represent quintessential generalists, with the ability to infect and perform well in multiple hosts. However, antagonistic pleiotropy imposed a cost during the adaptation to persistent replication of vesicular stomatitis virus in sand fly cells and resulted in strains that initially replicated poorly in hamster cells, even when the virus was allowed to replicate periodically in the latter. Once a debilitated strain started replicating continuously in mammalian cells, fitness increased significantly. Fitness recovery did not entail back mutations or compensatory mutations, but instead, we observed the replacement of persistence-adapted genomes by mammalian cell-adapted strains with a full set of new, unrelated sequence changes. These mammalian cell-adapted genomes were present at low frequencies in the populations with a history of persistence for up to a year and quickly became dominant during mammalian infection, but coexistence was not stable in the long term. Periodic acute replication in mammalian cells likely contributed to extending the survival of minority genomes, but these genomes were also found in strictly persistent populations.

Essentiality of the Early Transcript in the Replication Origin of the Lactococcal Prolate Phage c2

ABSTRACT The genome of the prolate-headed lytic lactococcal bacteriophage c2 is organized into two divergently oriented blocks consisting of the early genes and the late genes. These blocks are separated by the noncoding origin of DNA replication. We examined the functional role of transcription of the origin in a plasmid model system. Deletion of the early promoter PE1 abolished origin function. Introduction of mutations into PE1 which did not eliminate promoter activity or replacement of PE1 with an unrelated but functional promoter did not abolish replication. The A-T-rich region upstream of PE1, which is conserved in prolate phages, was not required for plasmid replication. Replacement of the PE1 transcript template sequence with an unrelated sequence with a similar G+C content abolished replication, showing that the sequence encoding the transcript is essential for origin function. Truncated transcript and internal deletion constructs did not support replication except when the deletion was at the very 3′ end of the DNA sequence coding for the transcript. The PE1 transcript could be detected for all replication-proficient constructs. Recloning in a plasmid vector allowed detection of PE1 transcripts from some fragments that did not support replication, indicating that stability of the transcript alone was not sufficient for replication. The data suggest that production of a transcript of a specific length and with a specific sequence or structure is essential for the function of the phage c2 origin in this model system.

Modular Structure of the Human Lamin B2 Replicator

ABSTRACT The cis-acting elements necessary for the activity of DNA replication origins in metazoan cells are still poorly understood. Here we report a thorough characterization of the DNA sequence requirements of the origin associated with the human lamin B2 gene. A 1.2-kb DNA segment, comprising the start site of DNA replication and located within a large protein-bound region, as well as a CpG island, displays origin activity when moved to different ectopic positions. Genomic footprinting analysis of both the endogenous and the ectopic origins indicates that the large protein complex is assembled in both cases around the replication start site. Replacement of this footprinted region with an unrelated sequence, maintaining the CpG island intact, abolishes origin activity and the interaction with hORC2, a subunit of the origin recognition complex. Conversely, the replacement of 17 bp within the protected region reduces the extension of the protection without affecting the interaction with hORC2. This substitution does not abolish the origin activity but makes it more sensitive to the integration site. Finally, the nearby CpG island positively affects the efficiency of initiation. This analysis reveals the modular structure of the lamin B2 origin and supports the idea that sequence elements close to the replication start site play an important role in origin activation.

Evolution of an Autotransporter: Domain Shuffling and Lateral Transfer from Pathogenic Haemophilus toNeisseria

ABSTRACT The genomes of pathogenic Haemophilus influenzaestrains are larger than that of Rd KW20 (Rd), the nonpathogenic laboratory strain whose genome has been sequenced. To identify potential virulence genes, we examined genes possessed by Int1, an invasive nonencapsulated isolate from a meningitis patient, but absent from Rd. Int1 was found to have a novel gene termed lav, predicted to encode a member of the AIDA-I/VirG/PerT family of virulence-associated autotransporters (ATs). Associated withlav are multiple repeats of the tetranucleotide GCAA, implicated in translational phase variation of surface molecules. Laterally acquired by H. influenzae, lav is restricted in distribution to a few pathogenic strains, including H. influenzae biotype aegyptius and Brazilian purpuric fever isolates. The DNA sequence of lav is surprisingly similar to that of a gene previously described for Neisseria meningitidis. Sequence comparisons suggest that lavwas transferred relatively recently from Haemophilus toNeisseria, shortly before the divergence of N. meningitidis and Neisseria gonorrhoeae. Segments oflav predicted to encode passenger and β-domains differ sharply in G+C base content, supporting the idea that AT genes have evolved by fusing domains which originated in different genomes. Homology and base sequence comparisons suggest that a novel biotype aegyptius AT arose by swapping an unrelated sequence for the passenger domain of lav. The unusually mobile lav locus joins a growing list of genes transferred from H. influenzae to Neisseria. Frequent gene exchange suggests a common pool of hypervariable contingency genes and may help to explain the origin of invasiveness in certain respiratory pathogens.

Identification of an extracellular segment of the oxytocin receptor providing agonist-specific binding epitopes

The effects of the peptide hormone oxytocin are mediated by oxytocin receptors (OTRs) expressed by the target tissue. The OTR is a member of the large family of G-protein-coupled receptors. Defining differences between the interaction of agonists and antagonists with the OTR at the molecular level is of fundamental importance, and is addressed in this study. Using truncated and chimaeric receptor constructs, we establish that a small 12-residue segment in the distal portion of the N-terminus of the human OTR provides important epitopes which are required for agonist binding. In contrast, this segment does not contribute to the binding site for antagonists, whether peptide or non-peptide. It does, however, have a role in agonist-induced OTR signalling. Oxytocin is also an agonist at the vasopressin V1a receptor (V1aR). A chimaeric receptor (V1aRN-OTR) was engineered in which the N-terminus of the OTR was substituted by the corresponding, but unrelated, sequence from the N-terminus of the V1aR. We show that the V1aR N-terminus present in V1aRN-OTR fully restored both agonist binding and intracellular signalling to a dysfunctional truncated OTR construct. The N-terminal segment does not, however, contribute to receptor-selective agonism between the OTR and the V1aR. Our data establish a key role for the distal N-terminus of the OTR in providing agonist-specific binding epitopes.

Human Cytomegalovirus Mutant with Sequence-Dependent Resistance to the Phosphorothioate Oligonucleotide Fomivirsen (ISIS 2922)

ABSTRACT A human cytomegalovirus mutant that was isolated for resistance (10-fold) to the antisense oligonucleotide fomivirsen (ISIS 2922) exhibited cross-resistance to a modified derivative of fomivirsen with an identical base sequence but little or no resistance to an oligonucleotide with an unrelated sequence. No changes in the mutant’s DNA corresponding to the fomivirsen target sequence were found.

Abortive gap repair: underlying mechanism for Ds element formation.

The mechanism by which the maize autonomous Ac transposable element gives rise to nonautonomous Ds elements is largely unknown. Sequence analysis of native maize Ds elements indicates a complex chimeric structure formed through deletions of Ac sequences with or without insertions of Ac-unrelated sequence blocks. These blocks are often flanked by short stretches of reshuffled and duplicated Ac sequences. To better understand the mechanism leading to Ds formation, we designed an assay for detecting alterations in Ac using transgenic tobacco plants carrying a single copy of Ac. We found frequent de novo alterations in Ac which were excision rather than sequence dependent, occurring within Ac but not within an almost identical Ds element and not within a stable transposase-producing gene. The de novo DNA rearrangements consisted of internal deletions with breakpoints usually occurring at short repeats and, in some cases, of duplication of Ac sequences or insertion of Ac-unrelated fragments. The ancient maize Ds elements and the young Ds elements in transgenic tobacco showed similar rearrangements, suggesting that Ac-Ds elements evolve rapidly, more so than stable genes, through deletions, duplications, and reshuffling of their own sequences and through capturing of unrelated sequences. The data presented here suggest that abortive Ac-induced gap repair, through the synthesis-dependent strand-annealing pathway, is the underlying mechanism for Ds element formation.

trans-Golgi retention of a plasma membrane protein: mutations in the cytoplasmic domain of the asialoglycoprotein receptor subunit H1 result in trans-Golgi retention.

Unlike the wild-type asialoglycoprotein receptor subunit H1 which is transported to the cell surface, endocytosed and recycled, a mutant lacking residues 4-33 of the 40-amino acid cytoplasmic domain was found to be retained intracellularly upon expression in different cell lines. The mutant protein accumulated in the trans-Golgi, as judged from the acquisition of trans-Golgi-specific modifications of the protein and from the immunofluorescence staining pattern. It was localized to juxtanuclear, tubular structures that were also stained by antibodies against galactosyltransferase and gamma-adaptin. The results of further mutagenesis in the cytoplasmic domain indicated that the size rather than the specific sequence of the cytoplasmic domain determines whether H1 is retained in the trans-Golgi or transported to the cell surface. Truncation to less than 17 residues resulted in retention, and extension of a truncated tail by an unrelated sequence restored surface transport. The transmembrane segment of H1 was not sufficient for retention of a reporter molecule and it could be replaced by an artificial apolar sequence without affecting Golgi localization. The cytoplasmic domain thus appears to inhibit interaction(s) of the exoplasmic portion of H1 with trans-Golgi component(s) for example by steric hindrance or by changing the positioning of the protein in the membrane. This mechanism may also be functional in other proteins.