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PubMed1992

Current status of bacterial resistance to third-generation cephalosporins.

A review of the evolution of bacterial resistance to third-generation cephalosporins is presented, focusing mainly on the prototypical member of this group-cefotaxime. Third-generation cephalosporins generally remain highly active against most Enterobacteriaceae, staphylococci, streptococci, Haemophilus, and Neisseriaceae. Only enterobacteria with a high frequency of mutant derepressed strains that hyperproduce chromosomally mediated beta-lactamase, Pseudomonas spp., and some glucose nonfermenter Gram-negative bacilli have demonstrated increased levels of resistance. The significance of derepressed strains and of the recently described extended-spectrum, plasmid-mediated beta-lactamases to the usefulness of the third-generation cephalosporins is discussed.

Cefotaxime↗

PubMedDate not supplied

Resistance to beta-lactam antibiotics by re-modelling the active site of an E. coli penicillin-binding protein.

The beta-lactam antibiotics kill bacteria by inhibiting a set of penicillin-binding proteins (PBPs) that catalyse the final stages of peptidoglycan synthesis. In some bacteria the development of intrinsic resistance to beta-lactam antibiotics by the reduction in the affinity of PBPs causes serious clinical problems. The introduction of beta-lactam antibiotics that are resistant to hydrolysis by beta-lactamases may also result in the emergence of intrinsic resistance among the Enterobacteriaceae. The clinical problems that would arise from the emergence of resistant PBPs in enterobacteria have led us to examine the ease with which Escherichia coli can gain resistance to beta-lactams by the production of altered PBPs. The development of resistant PBPs also provides an interesting example of enzyme evolution, since it requires a subtle re-modeling of the enzyme active centre so that it retains affinity for its peptide substrate but excludes the structurally analogous beta-lactam antibiotics. We show here that only four amino-acid substitutions need to be introduced into PBP 3 of E. coli to produce a strain possessing substantial levels of resistance to a wide variety of cephalosporins. We also show that transfer of the gene encoding the resistant PBP 3 from the chromosome to a plasmid could result in the spread of intrinsic resistance not only to other strains of E. coli but also to other enterobacterial species.

Anti-Bacterial Agents↗

PubMed1995

Novel use of a selectable fusion gene as an "in-out" marker for studying genetic loss in mammalian cells.

Recent demonstrations of loss of heterozygosity in a wide variety of human cancers suggest that large multilocus genetic deletions (presumably including tumor suppressor genes) constitute a major class of genetic alteration in human carcinogenesis. Here we show that a bifunctional fusion gene (Hytk), suitable for both positive and negative selection, is an effective marker for studying genetic loss in mammalian cells with minimal interference from point-mutational changes. Studies with a transgenic V79 cell line in which a single functional copy of Hytk was stably inserted into the genome in a retroviral vector showed that loss of the marker (and presumably flanking cellular genetic material) could be induced efficiently by ionizing radiation (gamma-rays and fast neutrons) but only weakly by the powerful point-mutagen benzo[a]pyrene diol epoxide. In a first application of the system, we provide evidence that radiation-induced loss can occur through an indirect mechanism after a high-frequency event. Collectively, our results suggest that the Hytk marker should be a valuable tool for studying genome position effects on the tolerance of genetic loss in cultured human cells that represent different stages in clonal evolution and tumor progression.

3T3 Cells↗

PubMed1987

Complete cDNA sequence of human complement Cls and close physical linkage of the homologous genes Cls and Clr.

Overlapping molecular clones encoding the complement subcomponent Cls were isolated from a human liver cDNA library. The nucleotide sequence reconstructed from these clones spans about 85% of the length of the liver Cls messenger RNAs, which occur in three distinct size classes around 3 kilobases in length. Comparisons with the sequence of Clr, the other enzymatic subcomponent of Cl, reveal 40% amino acid identity and conservation of all the cysteine residues. Beside the serine protease domain, the following sequence motifs, previously described in Clr, were also found in Cls: (a) two repeats of the type found in the Ba fragment of complement factor B and in several other complement but also noncomplement proteins, (b) a cysteine-rich segment homologous to the repeats of epidermal growth factor precursor, and (c) a duplicated segment found only in Clr and Cls. Differences in each of these structural motifs provide significant clues for the interpretation of the functional divergence of these interacting serine protease zymogens. Hybridizations of Clr and Cls probes to restriction endonuclease fragments of genomic DNA demonstrate close physical linkage of the corresponding genes. The implications of this finding are discussed with respect to the evolution of Clr and Cls after their origin by tandem gene duplication and to the previously observed combined hereditary deficiencies of Clr and Cls.

Amino Acid Sequence↗

PubMed2002

A DNA methyltransferase can protect the genome from postdisturbance attack by a restriction-modification gene complex.

In prokaryotic genomes, some DNA methyltransferases form a restriction-modification gene complex, but some others are present by themselves. Dcm gene product, one of these orphan methyltransferases found in Escherichia coli and related bacteria, methylates DNA to generate 5'-C(m)CWGG just as some of its eukaryotic homologues do. Vsr mismatch repair function of an adjacent gene prevents C-to-T mutagenesis enhanced by this methylation but promotes other types of mutation and likely has affected genome evolution. The reason for the existence of the dcm-vsr gene pair has been unclear. Earlier we found that several restriction-modification gene complexes behave selfishly in that their loss from a cell leads to cell killing through restriction attack on the genome. There is also increasing evidence for their potential mobility. EcoRII restriction-modification gene complex recognizes the same sequence as Dcm, and its methyltransferase is phylogenetically related to Dcm. In the present work, we found that stabilization of maintenance of a plasmid by linkage of EcoRII gene complex, likely through postsegregational cell killing, is diminished by dcm function. Disturbance of EcoRII restriction-modification gene complex led to extensive chromosome degradation and severe loss of cell viability. This cell killing was partially suppressed by chromosomal dcm and completely abolished by dcm expressed from a plasmid. Dcm, therefore, can play the role of a "molecular vaccine" by defending the genome against parasitism by a restriction-modification gene complex.

Animals↗

PubMed2005

Comparative functional genomics revealed conservation and diversification of three enhancers of the isl1 gene for motor and sensory neuron-specific expression.

Islet-1 (Isl1) is a member of the Isl1 family of LIM-homeodomain transcription factors (LIM-HD) that is expressed in a defined subset of motor and sensory neurons during vertebrate embryogenesis. To investigate how this specific expression of isl1 is regulated, we searched for enhancers of the isl1 gene that are conserved in vertebrate evolution. Initially, two enhancer elements, CREST1 and CREST2, were identified downstream of the isl1 locus in the genomes of fugu, chick, mouse, and human by BLAST searching for highly similar elements to those originally identified as motor and sensory neuron-specific enhancers in the zebrafish genome. The combined action of these elements is sufficient for completely recapitulating the subtype-specific expression of the isl1 gene in motor neurons of the mouse spinal cord. Furthermore, by direct comparison of the upstream flanking regions of the zebrafish and human isl1 genes, we identified another highly conserved noncoding element, CREST3, and subsequently C3R, a similar element to CREST3 with two CDP CR1 recognition motifs, in the upstream regions of all other isl1 family members. In mouse and human, CRESTs are located as far as more than 300 kb away from the isl1 locus, while they are much closer to the isl1 locus in zebrafish. Although all of zebrafish CREST2, CREST3, and C3R activate gene expression in the sensory neurons of zebrafish, CREST2 of mouse and human does not have the sequence necessary for sensory neuron-specific expression. Our results revealed both a remarkable conservation of the regulatory elements regulating subtype-specific gene expression in motor and sensory neurons and the dynamic process of reorganization of these elements whereby each element increases the level of cell-type specificity by losing redundant functions with the other elements during vertebrate evolution.

Animals↗

PubMed1999

Enhanced hydrogen production by a mutant of Rhodobacter sphaeroides having an altered light-harvesting system.

A stable mutant of the photosynthetic bacterium Rhodobacter sphaeroides with an altered light-harvesting (LH) system (P3 mutant) was obtained by UV irradiation and characterized. The mutant exhibited a 2.7-fold decrease in the core antennal (LH1) content and 1.6-fold increase in peripheral antennal (LH2) content compared to the wild-type strain. The H2 evolution rates in the P3 mutant under 800- and 850-nm light, corresponding to the absorption maxima of LH2, were 1.5 times higher than in the wild-type strain. The wild-type absorption spectrum was restored in the P3 mutant when a 1.1-kb PCR-amplified fragment containing the puf promoter and pufQBA genes was ligated into a pRK-415 derivative and introduced into it. The transformant showed lower H2 production rates at 800 and 850 nm than the P3 strain carrying the control plasmid, indicating that the accelerated H2 production in the P3 mutant was a result of alterations in the LH system.

Journal Article↗

PubMed1998

Beta-amyrin synthase--cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants.

Beta-amyrin, a typical pentacyclic triterpene having an oleanane skeleton, is one of the most commonly occuring triterpenes in nature and is biosynthesized from (3S)-2,3-oxidosqualene. The enzyme, beta-amyrin synthase, catalyzing the cyclization of oxidosqualene into beta-amyrin, generates five rings and eight asymmetric centers in a single transformation. A homology-based PCR method was attempted to obtain the cDNA of this enzyme from the hairy root of Panax ginseng which produces oleanane saponins together with dammarane-type saponins. Two sets of degenerate oligonucleotide primers were designed at the regions which are highly conserved among known oxidosqualene cyclases (OSCs). Nested PCRs using these primers successfully amplified the core fragment which revealed the presence of two OSC clones PNX and PNY. Specific amplification of each clone by 3'-RACE and 5'-RACE was carried out to obtain the whole sequences. The two clones exhibited 60% amino acid identity to each other. A full-length clone of PNY was ligated into the yeast expression vector pYES2 under the GAL1 promoter to give pOSC(PNY). Beta-amyrin production was observed with the mutant yeast lacking lanosterol synthase, transformed by this plasmid. The sequence of pOSC(PNY) contains an open reading frame of 2289 nucleotides which codes for 763 amino acids with a predicted molecular mass of 88 kDa. Sequence comparison with other OSCs showed a high level of similarity with lanosterol, cycloartenol and lupeol synthases. The other clone, pOSC(PNX), was shown to be cycloartenol synthase by similar expression in yeast. The present studies have revealed that distinct OSC exists for triterpene formation in higher plants, and the high level of similarity with cycloartenol synthase indicates close evolutional relationship between sterol and triterpene biosynthesis.

Amino Acid Sequence↗

PubMed2004

Purifying selection masks the mutational flexibility of HIV-1 reverse transcriptase.

DNA and RNA polymerases share a core architecture composed of three structurally conserved motifs: A, B, and C. Although the amino acid sequences of these motifs are highly conserved between closely related organisms, variation across broader evolutionary distances suggests that only a few residues in each motif are indispensable for polymerase function. To test this, we constructed libraries of human immunodeficiency virus type-1 (HIV-1) containing random single amino acid replacements in motif B of reverse transcriptase (RT), and we used selection in culture to assess RT function. Despite the nearly absolute constancy of motif B in vivo, virus replicating in culture tolerated a range of conservative and nonconservative substitutions at 10 of the 11 amino acid positions examined. These included residues that are invariant across all retroviral subfamilies and highly conversed in diverse retroelements. Several mutants retained wild type infectivity, and serial passage experiments revealed replacements that were neutral or even beneficial to viral fitness. In addition, a number of the selected variants exhibited altered susceptibility to the nucleoside analog inhibitors AZT and 3TC. Taken together, these data indicate that HIV-1 tolerates a range of substitutions at conserved RT residues and that selection against slightly deleterious mutations (purifying selection) in vivo masks a large repertoire of viable phenotypic variants. This mutational flexibility likely contributes to HIV-1 evolution in response to changing selection pressures in infected individuals.

Amino Acid Motifs↗

PubMed2002

Herpesvirus genetics has come of age.

The genetic analysis of the large and complex herpesviruses has been a constant challenge to herpesvirologists. Elegant methods have been developed to produce mutants in infected cells that rely on the cellular recombination machinery. Bacterial artificial chromosomes (BACs), single copy F-factor-based plasmid vectors of intermediate insert capacity, have now enabled the cloning of complete herpesvirus genomes. Infectious virus genomes can be shuttled between Escherichia coli and eukaryotic cells. Herpesvirus BAC DNA engineering in E. coli by homologous recombination requires neither restriction sites nor cloning steps and allows the introduction of a wide variety of DNA modifications. Such E. coli-based technology has provided a safe, fast and effective approach to the systematic mining of the information stored in herpesvirus genomes as a result of their intimate co-evolution with their specific hosts for millions of years. Use of this technique could lead to new developments in clinical virology and basic virology research, and increase the usage of viral genomes as investigative tools and vectors.

Animals↗

PubMed1992

Fission yeast cdc21+ belongs to a family of proteins involved in an early step of chromosome replication.

The cdc21+ gene of Schizosaccharomyces pombe was originally identified in a screen for cdc mutants affecting S phase and nuclear division. Here we show that the cdc21+ gene product belongs to a family of proteins implicated in DNA replication. These include the Saccharomyces cerevisiae MCM2 and MCM3 proteins, which are needed for the efficient function of certain replication origins, and S.cerevisiae CDC46, which is required for the initiation of chromosome replication. The cdc21 mutant is defective in the mitotic maintenance of some plasmids, like mcm2 and mcm3. The mutant arrests with a single nucleus containing two genome equivalents of DNA, and maintains a cytoplasmic microtubular configuration. Activation of most, but not all, replication origins in the mutant may result in failure to replicate a small proportion of the genome, and this could explain the arrest phenotypes. Using the polymerase chain reaction technique, we have identified new cdc21(+)-related genes in S.cerevisiae, S.pombe and Xenopus laevis. Our results suggest that individual members of the cdc21(+)-related family are highly conserved in evolution.

Amino Acid Sequence↗

PubMed2004

pdf1, a palmitoyl protein thioesterase 1 Ortholog in Schizosaccharomyces pombe: a yeast model of infantile Batten disease.

Infantile Batten disease is a severe neurodegenerative storage disorder caused by mutations in the human PPT1 (palmitoyl protein thioesterase 1) gene, which encodes a lysosomal hydrolase that removes fatty acids from lipid-modified proteins. PPT1 has orthologs in many species, including lower organisms and plants, but not in Saccharomyces cerevisiae. The fission yeast Schizosaccharomyces pombe contains a previously uncharacterized open reading frame (SPBC530.12c) that encodes the S. pombe Ppt1p ortholog fused in frame to a second enzyme that is highly similar to a previously cloned mouse dolichol pyrophosphatase (Dolpp1p). In the present study, we characterized this interesting gene (designated here as pdf1, for palmitoyl protein thioesterase-dolichol pyrophosphate phosphatase fusion 1) through deletion of the open reading frame and complementation by plasmids bearing mutations in various regions of the pdf1 sequence. Strains bearing a deletion of the entire pdf1 open reading frame are nonviable and are rescued by a pdf1 expression plasmid. Inactivating mutations in the Dolpp1p domain do not rescue the lethality, whereas mutations in the Ppt1p domain result in cells that are viable but abnormally sensitive to sodium orthovanadate and elevated extracellular pH. The latter phenotypes have been previously associated with class C and class D vacuolar protein sorting (vps) mutants and vacuolar membrane H(+)-ATPase (vma) mutants in S. cerevisiae. Importantly, the Ppt1p-deficient phenotype is complemented by the human PPT1 gene. These results indicate that the function of PPT1 has been widely conserved throughout evolution and that S. pombe may serve as a genetically tractable model for the study of human infantile Batten disease.

Amino Acid Sequence↗

PubMed1997

The lytic enzyme of the pneumococcal phage Dp-1: a chimeric lysin of intergeneric origin.

We have localized, cloned and characterized the genes coding for the lytic system of the pneumococcal phage Dp-1. The lytic enzyme of this phage (Pal), previously identified as an N-acetyl-muramoyl-L-alanine amidase, shows a modular organization similar to that described for the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. The construction of chimeric enzymes between pneumococcus and bacteria (or phages) that belong to different Gram-positive families has shown that the interchange of functional domains switches enzyme specificity. Interestingly, Pal appears to be a natural chimeric enzyme of intergeneric origin, that is the N-terminal domain was highly similar to that of the murein hydrolase coded by a gene found in the phage BK5-T that infects Lactococcus lactis, whereas the C-terminal domain was homologous to those found in the lytic enzymes of the pneumococcal system that is responsible for the binding to the choline residues present in the cell wall substrate. Biochemical analysis of Pal revealed that this enzyme shares important properties with those of the major LytA101 autolysin found in an atypical, clinical pneumococcal isolate. These peculiar characteristics have been ascribed to a modified C-terminal domain. The natural chimeric enzyme described here provides further support for the theory of modular evolution of proteins and its characteristics also furnish interesting clues on the molecular mechanisms involved in the more invasive types of atypical pneumococci.

Amino Acid Sequence↗

PubMed1991

An Escherichia coli tyrosine transfer RNA is a leucine-specific transfer RNA in the yeast Saccharomyces cerevisiae.

While the Escherichia coli Su-3 (tyrT) tyrosine tRNA suppressor inserts only tyrosine at amber codons in E. coli, we show here that in Saccharomyces cerevisiae this tRNA inserts leucine and no significant amounts of any other amino acid. Thus, the E. coli tyrosine tRNA is functionally a leucine tRNA in yeast cytoplasm. This functional identity may correlate with a structural relationship between the E. coli tyrosine and yeast leucine tRNAs, which are both members of the uncommon type II class of tRNA structures. The results raise the possibility that in evolution a tRNA may be more closely related to a tRNA of different acceptor specificity, but of the same type class, than to one with the same amino acid specificity, but of a different type class.

Alleles↗

PubMed1996

Differential expression of the ToxR regulon in classical and E1 Tor biotypes of Vibrio cholerae is due to biotype-specific control over toxT expression.

The two major disease-causing biotypes of Vibrio cholerae, classical and El Tor, exhibit differences in their epidemic nature. Their behavior in the laboratory also differs in that El Tor strains produce two major virulence factors, cholera toxin (CT) and the toxin coregulated pilus (TCP), only under very restricted growth conditions, whereas classical strains do so in standard laboratory medium. Expression of toxin and TCP is controlled by two activator proteins, ToxR and ToxT, that operate in cascade fashion with ToxR controlling the synthesis of ToxT. Both biotypes express equivalent levels of ToxR, but only classical strains appear to express ToxT when grown in standard medium. In this report we show that restrictive expression of CT and TCP can be overcome in El Tor strains by expressing ToxT independently of ToxR. An El Tor strain lacking functional ToxT does not express CT or TCP, ruling out existence of a cryptic pathway for virulence regulation in this biotype. These results may have implications for understanding the evolution of El Tor strains toward reduced virulence with respect to classical strains.

Amino Acid Sequence↗

PubMed2004

Dimorphecolic acid is synthesized by the coordinate activities of two divergent Delta12-oleic acid desaturases.

Dimorphecolic acid (9-OH-18:2Delta(10)(trans)(,12)(trans)) is the major fatty acid of seeds of Dimorphotheca species. This fatty acid contains structural features that are not typically found in plant fatty acids, including a C-9 hydroxyl group, Delta(10),Delta(12)-conjugated double bonds, and trans-Delta(12) unsaturation. Expressed sequence tag analysis was conducted to determine the biosynthetic origin of dimorphecolic acid. cDNAs for two divergent forms of Delta(12)-oleic acid desaturase, designated DsFAD2-1 and Ds-FAD2-2, were identified among expressed sequence tags generated from developing Dimorphotheca sinuata seeds. Expression of DsFAD2-1 in Saccharomyces cerevisiae and soybean somatic embryos resulted in the accumulation of the trans-Delta(12) isomer of linoleic acid (18: 2Delta(9)(cis)(,12)(trans)) rather than the more typical cis-Delta(12) isomer. When co-expressed with DsFAD2-1 in soybean embryos or yeast, DsFAD2-2 converted 18:2Delta(9)(cis)(,12)(trans) into dimorphecolic acid. When DsFAD2-2 was expressed alone in soybean embryos or together with a typical cis-Delta(12)-oleic acid desaturase in yeast, trace amounts of the cis-Delta(12) isomer of dimorphecolic acid (9-OH-18:2Delta(10)(trans,)(12)(cis)) were formed from DsFAD2-2 activity with cis-Delta(12)-linoleic acid [corrected]. These results indicate that DsFAD2-2 catalyzes the conversion of the Delta(9) double bond of linoleic acid into a C-9 hydroxyl group and Delta(10)(trans) double bond and displays a substrate preference for the trans-Delta(12), rather than the cis-Delta(12), isomer of linoleic acid. Overall these data are consistent with a biosynthetic pathway of dimorphecolic acid involving the concerted activities of DsFAD2-1 and DsFAD2-2. The evolution of two divergent Delta(12)-oleic acid desaturases for the biosynthesis of an unusual fatty acid is unprecedented in plants.

Calendula↗

PubMed2004

Evolution of naturally occurring 5' non-translated region variants of hepatitis C virus genotype 1b in selectable replicons.

Quasispecies shifts are essential for the development of persistent hepatitis C virus (HCV) infection. Naturally occurring sequence variations in the 5' non-translated region (NTR) of the virus could lead to changes in protein expression levels, reflecting selective forces on the virus. The extreme 5' end of the virus' genome, containing signals essential for replication, is followed by an internal ribosomal entry site (IRES) essential for protein translation as well as replication. The 5' NTR is highly conserved and has a complex RNA secondary structure consisting of several stem-loops. This report analyses the quasispecies distribution of the 5' NTR of an HCV genotype 1b clinical isolate and found a number of sequences differing from the consensus sequence. The consensus sequence, as well as a major variant located in stem-loop IIIa of the IRES, was investigated using self-replicating HCV RNA molecules in human hepatoma cells. The stem-loop IIIa mutation, which is predicted to disrupt the stem structure, showed slightly lower translation efficiency but was severely impaired in the colony formation of selectable HCV replicons. Interestingly, during selection of colonies supporting autonomous replication, mutations emerged that restored the base pairing in the stem-loop. Recloning of these altered IRESs confirmed that these second site revertants were more efficient in colony formation. In conclusion, naturally occurring variants in the HCV 5' NTR can lead to changes in their replication ability. Furthermore, IRES quasispecies evolution was observed in vitro under the selective pressure of the replicon system.

5' Untranslated Regions↗

PubMed1996

Molecular basis of the variable mitochondrial and peroxisomal localisation of alanine-glyoxylate aminotransferase.

The molecular basis of the variable species-specific peroxisomal and/or mitochondrial targeting of the enzyme alanine-glyoxylate aminotransferase 1 (AGT) has been studied in human fibroblasts by confocal immunofluorescence microscopy after intranuclear microinjection of various human, rabbit, marmoset, and feline AGT cDNA constructs. The expression of full-length human and rabbit AGT cDNA led to an exclusively peroxisomal distribution of AGT. However, the distribution of feline and marmoset AGT depended on the cDNA construct injected. In both species, injection of the short cDNAs (from transcripts that occur naturally in marmoset liver but not in feline liver) led to an exclusively peroxisomal distribution. However, injection of the long cDNAs (from transcripts that occur naturally in both species) led to most of the AGT being targeted to the mitochondria and only a small, yet significant, fraction to the peroxisomes. Reintroduction of the 'ancestral' first potential translation initiation site into human AGT cDNA led to an 'ancestral' distribution of AGT (i.e. both mitochondrial and peroxisomal). Deletion of the second potential translation start site from the long feline cDNA led to a distribution that was almost entirely mitochondrial, which suggests that most peroxisomal AGT encoded by the long cDNA results from internal translation initiation from this site with the consequent loss of the N-terminal mitochondrial targeting sequence. Expression of rabbit cDNA and the short marmoset and feline cDNAs in cells selectively deficient in the import of peroxisomal matrix proteins showed that peroxisomal AGT in all these species is imported via the peroxisomal targeting sequence type 1 (PTS1) import pathway. The almost complete functional dominance of the N-terminal mitochondrial targeting sequence over the C-terminal PTS. which was not due to any direct interference of the former with peroxisomal import, was maintained even when the unusual PTS1 of AGT (KKL in human) was replaced by the prototypical PTS1 SKL. The results demonstrate that the major determinant of alanine-glyoxylate aminotransferase subcellular distribution in mammals is the presence or absence of the mitochondrial targeting sequence rather than the peroxisomal targeting sequence. Various strategies have arisen during the evolution of mammals to enable the exclusion of the mitochondrial targeting sequence from the newly synthesised polypeptide, all of which involve the use of alternative transcription and/or translation initiation sites.

Alanine Transaminase↗