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PubMed2000

Antibiotic resistance in Gram-positive cocci.

Gram-positive cocci still predominate as a cause of nosocomial- and community-acquired infections. These organisms frequently reveal a high natural, intrinsic resistance to antimicrobials. Additionally, these bacteria are able to acquire resistance to frequently used drugs rapidly through selective pressure of the environment and via the genetic evolution of bacteria. The wide application of antimicrobials in medical and veterinary practice, usage of antibiotics in agriculture and common usage of antiseptics and disinfectants result in selective pressure. The use of antibiotics directly selects resistant variants to different antibiotics or disinfectants. The same genetic element (e.g. qac or smr) conferring resistance to some disinfectants are often present on the same plasmid conferring resistance to antibiotics. Selection of resistant variants occurs most frequently in the hospital environment. Staphylococcus aureus and enterococci are the most commonly isolated bacteria causing nosocomial infections. Among those giving therapeutic problems are methicillin-resistant staphylococci and vancomycin-resistant enterococci. Resistance to high levels of aminoglycosides or penicillins among hospital enterococcal strains can completely abolish synergism of the drugs. In these cases glycopeptides will be the drugs of choice in the treatment of serious infections. Recently S. aureus strains with decreased susceptibility to vancomycin has appeared. A mechanism for this elevated resistance, although intensively investigated, still remains unknown.

Aminoglycosides↗

PubMed1994

Urokinase overproduction results in increased skeletal metastasis by prostate cancer cells in vivo.

We previously reported that urokinase (uPA) is produced by the human prostate cancer cell line, PC-3, and could function as a growth factor for cells of the osteoblast phenotype. To examine the role of uPA in metastasis to the skeleton and to extraskeletal sites, we have developed a homologous model of uPA overexpression in a rat prostate cancer cell line. Full length cDNA encoding rat (r) uPA was isolated and subcloned as a 1.4-kilobase XbaI-BspHI fragment in the sense and antisense orientation into the Moloney murine leukemia retroviral vector pYN. The control (pYN) and experimental (pYN-ruPA, pYN-ruPA-AS) plasmids were transfected into Dunning R 3227, Mat LyLu rat prostate carcinoma cells. Experimental clones expressing at least 5-fold higher (pYN-ruPA) or 3-fold lower (pYN-ruPA-AS) than controls were selected, and control and experimental cells were inoculated into the left ventricles of inbred male Copenhagen rats. Animals were sacrificed at timed intervals to examine the evolution of metastatic lesions. Control animals developed metastases to the lumbar vertebrae resulting in spinal cord compression and hind limb paralysis at 20-21 days postinoculation. Animals inoculated with cells overexpressing uPA developed hind limb paralysis significantly earlier (by day 14-15 postinoculation). Additionally, more widespread skeletal (ribs, scapula, and femora) metastases were seen. Serum from experimental animals showed a progressive elevation in alkaline phosphatase levels, and histological examination of lumbar metastases revealed markedly increased osteoblastic activity over that observed in control animals. In contrast to this, animals inoculated with cells underexpressing uPA developed hind limb paralysis significantly later (days 25-29 postinoculation) and displayed decreased tumor metastasis. These studies support a role for the catalytic domain of uPA in enhancing both skeletal and nonskeletal prostate cancer invasiveness and are consistent with a role for the growth factor domain of uPA in mediating an osteoblastic skeletal response.

Animals↗

PubMedDate not supplied

Unusual intron in the immunoglobulin domain of the newly isolated murine CD4 (L3T4) gene.

The T-cell surface glycoprotein, CD4, is expressed predominantly on helper T cells and is thought to play a major role in cell-cell interactions. Monoclonal antibodies against CD4 have been shown to block numerous T-cell functions; moreover, recent results suggest that the CD4 molecule may be involved in transmembrane signal transduction. The human CD4 glycoprotein has also been shown to form at least part of the receptor for the AIDS virus, HIV-1. Elucidation of the functions of CD4 will be facilitated by the ability to manipulate the protein by genetic means. Because the mouse system is well suited for a variety of functional studies, we have isolated, sequenced and expressed cDNA clones encoding the murine CD4 (L3T4) glycoprotein. Comparison of the mouse and human CD4 sequences reveals striking evolutionary conservation of the cytoplasmic domain, suggesting that this region is essential for CD4 function. In addition, both the human and mouse CD4 gene contain a large intron in the coding region of the V-like domain. As no other members of the immunoglobulin gene superfamily have been shown to contain similarly placed introns, this finding may have important implications regarding the evolution of this gene family in particular and of introns in general.

Amino Acid Sequence↗

PubMed1989

Suppressors of yeast actin mutations.

Suppressors of a temperature-sensitive mutation (act1-1) in the single actin gene of Saccharomyces cerevisiae were selected that had simultaneously acquired a cold-sensitive growth phenotype. Five genes, called SAC (suppressor of actin) were defined by complementation tests; both suppression and cold-sensitive phenotypes were recessive. Three of the genes (SAC1, SAC2 and SAC3) were subjected to extensive genetic and phenotypic analysis, including molecular cloning. Suppression was found to be allele-specific with respect to actin alleles. The sac mutants, even in ACT1+ genetic backgrounds, displayed phenotypes similar to those of actin mutants, notably aberrant organization of intracellular actin and deposition of chitin at the cell surface. These results are interpreted as being consistent with the idea that the SAC genes encode proteins that interact with actin, presumably as components or controllers of the assembly or stability of the yeast actin cytoskeleton. Two unexpected properties of the SAC1 gene were noted. Disruptions of the gene indicated that its function is essential only at temperatures below about 17 degrees and all sac1 alleles are inviable when combined with act1-2. These properties are interpreted in the context of the evolution of the actin cytoskeleton of yeast.

Actins↗

PubMed1985

The nucleotide sequence of the ilvBN operon of Escherichia coli: sequence homologies of the acetohydroxy acid synthase isozymes.

Three acetohydroxy acid synthase isozymes, AHAS I (ilvBN), AHAS II (ilvGM) and AHAS III (ilvIH) catalyze the first step of the parallel isoleucine-valine biosynthetic pathway in Escherichia coli. Previous DNA sequence and protein purification data have shown that AHAS II and AHAS III are composed of large and small subunits encoded in the ilvGMEDA and ilvIH operons, respectively. Recent protein purification and characterization data have demonstrated that the AHAS I isozyme is also composed of large and small subunits (L. Eoyang, L. and P. M. Silverman [1984] J. Bacteriol. 157:184-189). Now the complete DNA sequence of the operon encoding the AHAS I isozyme has been determined. These data show that both AHAS I subunits (Mr 60,400 and Mr 11,100) are encoded in this operon. The coordinant regulation of both genes of the ilvBN operon has also been demonstrated. Comparisons of the DNA sequences of the genes encoding all three AHAS isozymes have been performed. Conserved homologies were observed between both the large and small subunits of all three isozymes. The closest homology was seen between the AHAS I and AHAS II isozymes. On the basis of these comparisons a rationale for the evolution of the AHAS isozymes in E. coli has been proposed.

Acetolactate Synthase↗

PubMed1997

Cloning and characterization of a cDNA encoding a bacteriophage-type RNA polymerase from the higher plant Chenopodium album.

We have cloned a full-length cDNA from the higher plant Chenopodium album coding for a single subunit bacteriophage-type RNA polymerase. The cDNA isolated from an actively growing cell suspension culture recognized a 3.8 kb transcript on Northern blots. The open reading frame comprises 987 amino acids with a predicted molecular mass of 112 kDa. A comparison of the protein sequence with those of the two known fungal mitochondrial RNA polymerases, from Saccharomyces cerevisiae and Neurospora crassa , reveals extensive homology between the three enzymes. with complete conservation of all catalytically essential amino acids. The putative mitochondrial RNA polymerase from C.album , as well as homologous sequences from rice and barley, which have been partially cloned, lack two catalytically non-essential regions of up to 176 amino acids near the C-terminus present in the two fungal mitochondrial RNA polymerases. The extreme N-terminus of the cloned C.album RNA polymerase displays features of a potential mitochondrial transit sequence. In phylogenetic trees constructed to compare the evolutionary relationships between the different single subunit RNA polymerases the C.album sequence forms a subgroup together with the S.cerevisiae and the N.crassa mitochondrial RNA polymerases, well separating from both bacteriophage enzymes and plasmid-encoded RNA polymerases found in mitochondria of many fungi and some higher plants.

Amino Acid Sequence↗

PubMed1989

Responsiveness of the Epstein-Barr virus NotI repeat promoter to the Z transactivator is mediated in a cell-type-specific manner by two independent signal regions.

Cells latently infected with Epstein-Barr virus (EBV) can be activated to express lytic-cycle polypeptides by the introduction of the EBV-encoded Z transactivator, indicating that this protein has a pivotal role in virus reactivation. We examined the target specificity of the Z transactivator in short-term contransfection assays and found that the most responsive target to Z transactivation was the divergent NotI repeat promoter, located within the EBV BamHI H fragment. In contrast, target plasmids containing the cat gene linked to heterologous viral promoters were not activated by cotransfection with the Z gene. S1 nuclease analysis of RNA from chemically induced B95-8 cells and from Vero cells cotransfected with NotI repeat promoter-CAT and Z showed that Z transactivation increased the level of correctly initiated, stable RNA transcripts. The NotI repeat gene (ntr) gives rise to a highly abundant mRNA species after chemical induction of lytic virus replication, but no protein product had been previously identified. Using monospecific antiserum raised against a synthetic peptide from the BHLF1 open reading frame, we demonstrated that the ntr gene encodes a protein product that is found in nuclear patches colocalizing with nucleoli. A series of deletions introduced into the upstream sequences of the NotI-repeat-promoter revealed two separate Z-response regions. The minimal promoter region between -7 and -155 of the leftward RNA cap site and an upstream region between -644 and -902 were both independently capable of conferring Z responsiveness. However, the minimal region, which was activated by Z cotransfection in Vero cells, was poorly responsive in lymphocytes, whereas the response of the far-upstream region to Z cotransfection was lymphocyte specific. In its human host, EBV infects both epithelial and lymphocyte populations. This dual lifestyle may have led to the evolution of multiple Z-response signals that enable the Z transactivator to interact with both cell-specific promoter and enhancer factors.

Animals↗

PubMed2000

Effect of the irradiated microenvironment on the expression and retrotransposition of intracisternal type A particles in hematopoietic cells.

We have previously demonstrated that the frequency of transformation of the factor-dependent hematopoietic cell line FDCP-1JL26 was dramatically increased when cells were cocultured with the irradiated bone marrow cell line D2XRII. In many of our factor-independent subclonal cell lines that we examined, transformation to factor independence appeared to be due to the retrotransposition of intracisternal type A particles (IAP) into the growth factor genes that are normally required for survival and growth of FDCP-1JL26 cells. To determine the role of the irradiated microenvironment in the evolution of factor-independent cells, we have examined the expression and retrotransposition of IAPs after exposure to the irradiated bone marrow stromal cell line D2XRII. Differential display and Northern blot analysis demonstrated that IAPs were overexpressed in a nonautocrine factor-independent subclonal cell line, FI7CL2. The frequency of retrotransposition was determined by the introduction of the IAP-neo(RT) plasmid into FDCP-1JL26 cells. The IAP-neo(RT) contains a neomycin resistance gene (neo) that only becomes active after retrotransposition, and thus the frequency of retrotransposition in FDCP-1JL26 cells was quantified by determining the frequency of neo-resistant cells.No significant increases in the expression of IAPs were observed after the cells were exposed to the irradiated stromal cells. This observation is in agreement with the observation that no increase in the frequency of retrotransposition could be detected. These results suggest that the irradiated bone marrow may have a passive role in the selection of factor-independent cells. During cocultivation, bone marrow stromal cells may provide a factor(s) to hematopoietic cells that allow it to survive in medium lacking IL-3. At random, a retrotransposition may occur that provides a selective advantage to the hematopoietic cells. In the absence of the irradiated stromal cells, the hematopoietic cells are perhaps more likely to die and therefore are not available for a random retrotransposition event to occur. This model is to be distinguished from an active role in which the irradiated microenvironment would synthesize or activate a factor(s) that promotes retrotransposition.

Animals↗

PubMed1981

Distribution of the insertion sequence IS1 in gram-negative bacteria.

Translocation of DNA segments is a recombinational event seen in both eukaryotic and prokaryotic chromosomes, and it is thought to be involved in controlling gene expression and in the evolution of chromosomes. In bacteria, insertion (IS) and transposable (Tn) elements not only translocate their own DNA, but also promote the rearrangement of both bacterial chromosomes and the plasmic genomes carrying them. The insertion element IS1 is one such element which is 768 base pairs long. IS1 is involved in the generation of deletion mutations and in the fusion of two different plasmid genomes. It can also promote the translocation of DNA segments flanked by two copies of IS1 to give rise to transposable elements responsible for antibiotic resistance and enterotoxin production. We report here the distribution of the IS1 sequence in various bacterial DNAs, particularly in the family Enterobacteriaceae. Comparison of the results with the phylogenetic relationship of these bacteria suggests that IS1 was transferred from one bacterium to another after their divergence and in some bacteria the copy number of IS1 increased by translocation. The increase in the number of copies of IS1 in bacteria may increase the probability of the genetic rearrangement responsible for the generation of resistance and enterotoxin plasmids, the existence of which is a serious problem in medical microbiology.

Acinetobacter↗

PubMed2003

Potent anti-tumor effects of an active site mutant of human manganese-superoxide dismutase. Evolutionary conservation of product inhibition.

Mn-SOD serves as the primary cellular defense against oxidative damage by converting superoxide radicals (O(2)(-)) to O(2) and H(2)O(2). A unique characteristic of this mitochondrial anti-oxidant enzyme is the conservation from bacteria to man of a rapidly formed product inhibited state. Using site-directed mutagenesis, we have generated an active site mutant (H30N) of human Mn-SOD, which exhibits significantly reduced product inhibition and increased enzymatic efficiency. Overexpression of the H30N enzyme causes anti-proliferative effects in vitro and anti-tumor effects in vivo. Our results provide a teleological basis for the phylogenetically invariant nature of position His-30 and the evolutionary conservation of product inhibition. These data also provide more direct intracellular evidence for the signaling role associated with H(2)O(2).

Animals↗

PubMed1996

Functional analysis in Saccharomyces cerevisiae of naturally occurring amino acid substitutions in human dihydrolipoamide dehydrogenase.

Dihydrolipoamide dehydrogenase is a common component of mammalian multienzyme complexes that decarboxylate alpha-ketoacids and catabolize glycine. The common function is to reoxidize a reduced lipoate component of each complex, thereby preparing that lipoate for another round of catalysis. Regions within dihydrolipoamide dehydrogenase involved in association with other proteins of the complexes are poorly defined, and despite high amino acid sequence conservation through evolution, it is unknown if dihydrolipoamide dehydrogenases are functionally equivalent across species. To address this issue, we asked whether the human enzyme could restore function to the alpha-ketoacid dehydrogenase complexes in a yeast strain deficient in endogenous dihydrolipoamide dehydrogenase. This dihydrolipoamide dehydrogenase null mutant will not grow on non-fermentable carbon sources. The human enzyme expressed from a CEN plasmid complemented the growth phenotype and restored full activity to the pyruvate and alpha-ketoglutarate dehydrogenase complexes. Human dihydrolipoamide dehydrogenases with selected amino acid substitutions were then tested in the null strain for their ability to restore function. Substitutions tested represented naturally occurring candidate mutations identified in an individual with inactive dihydrolipoamide dehydrogenase. A K37E change had full function while a P453L change resulted in reduced dihydrolipoamide dehydrogenase abundance in the mitochondria and no detectable catalytic activity.

Amino Acids↗

PubMed2000

Role of Horizontal Gene Transfer in the Evolution of Fungi.

Although evidence for horizontal gene transfer (HGT) in eukaryotes remains largely anecdotal, literature on HGT in fungi suggests that it may have been more important in the evolution of fungi than in other eukaryotes. Still, HGT in fungi has not been widely accepted because the mechanisms by which it may occur are unknown, because it is usually not directly observed but rather implied as an outcome, and because there are often equally plausible alternative explanations. Despite these reservations, HGT has been justifiably invoked for a variety of sequences including plasmids, introns, transposons, genes, gene clusters, and even whole chromosomes. In some instances HGT has also been confirmed under experimental conditions. It is this ability to address the phenomenon in an experimental setting that makes fungi well suited as model systems in which to study the mechanisms and consequences of HGT in eukaryotic organisms.

gene cluster↗

PubMed2006

Investigation of de novo totally random biosequences, Part II: On the folding frequency in a totally random library of de novo proteins obtained by phage display.

We present an investigation on theoretically possible protein structures which have not been selected by evolution and are, therefore, not present on our Earth ('Never Born Proteins' (NBP)). In particular, we attempt to assess whether and to what extent such polypeptides might be folded, thus acquiring a globular protein status. A library (ca. 10(9) clones) of totally random polypeptides, with a length of 50 amino acids, has been produced by phage display. The only structural bias in these sequences is a tripeptide substrate for thrombin: PRG, chosen according to the criteria described in the preceding Part I of this series. The presence of this substrate in an otherwise totally random sequence forms the basis for a qualitative experimental criterion which distinguishes unfolded from folded proteins, as folded proteins are more protected from protease digestion than unfolded ones. The investigation of 79 sequences, randomly selected from the initially large library, shows that over 20% of this population is thrombin-resistant, likely due to folding. Analysis of the amino acid sequences of these clones shows no significant homology to extant proteins, which indicates that they are indeed totally de novo. A few of these sequences have been expressed, and here we describe the structural properties of two thrombin-resistant randomly selected ones. These two de novo proteins have been characterized by spectroscopic methods and, in particular, by circular dichroism. The data show a stable three-dimensional folding, which is temperature-resistant and can be reversibly denatured by urea. The consequences of this finding within a library of 'Never Born Proteins' are discussed in terms of molecular evolution.

Circular Dichroism↗

PubMed1983

Sequence analysis of Gardner-Arnstein feline leukaemia virus envelope gene reveals common structural properties of mammalian retroviral envelope genes.

We have sequenced the envelope (env) gene and most of the adjacent 3' long terminal repeat (LTR) of Gardner-Arnstein feline leukaemia virus subtype B. The LTR of this virus contains, at corresponding positions, all signal sequence elements known from other retroviral LTRs. The deduced amino acid sequence of the longest open reading frame was compared with env polypeptide sequences of several murine leukaemia viruses. This allowed us to predict the positions of both p12/15env and gp70 polypeptides as well as a hydrophobic leader polypeptide. The env polypeptides of the different viruses show long stretches of homology and similar hydrophilicity profiles in the p12env region and in the carboxy-terminal half of gp70 (constant region). The most extensive variations are confined to certain parts of the amino-terminal half of gp70 (differential region). In this region, however, feline leukaemia virus and murine mink cell focus forming viruses are still closely related. A correspondingly spaced pattern of identical, short amino acid sequences appears in three different parts of the env polyprotein, suggesting its evolution from a primordial env-related precursor by tandem duplications.

Amino Acid Sequence↗

PubMed1993

Efficient splicing of an AU-rich antisense intron sequence.

For successful splicing in dicot plants the only recognised intron requirements are 5' and 3' splice sites and AU-rich sequences. We have investigated further the importance of AU-rich elements by analyzing the splicing of an AU-rich antisense intron sequence. Activation of cryptic splice sites on either side of the AU-rich sequence permitted the efficient removal of this essentially non-intron sequence by splicing. This splicing event not only confirms the importance of AU-rich sequences but also has implications for the evolution of interrupted genes and the expression of heterologous genes in transgenic plants.

Adenine↗

PubMed1997

HupUV proteins of Rhodobacter capsulatus can bind H2: evidence from the H-D exchange reaction.

The H-D exchange reaction has been measured with the D2-H2O system, for Rhodobacter capsulatus JP91, which lacks the hupSL-encoded hydrogenase, and R. capsulatus BSE16, which lacks the HupUV proteins. The hupUV gene products, expressed from plasmid pAC206, are shown to catalyze an H-D exchange reaction distinguishable from the H-D exchange due to the membrane-bound, hupSL-encoded hydrogenase. In the presence of O2, the uptake hydrogenase of BSE16 cells catalyzed a rapid uptake and oxidation of H2, D2, and HD present in the system, and its activity (H-D exchange, H2 evolution in presence of reduced methyl viologen [MV+]) depended on the external pH, while the H-D exchange due to HupUV remained insensitive to external pH and O2. These data suggest that the HupSL dimer is periplasmically oriented, while the HupUV proteins are in the cytoplasmic compartment.

Bacterial Proteins↗

PubMed1999

SHV-type beta-lactamases.

The group of plasmid-mediated SHV b-lactamases includes SHV-1 and at least twenty-three variants, most of which possess extended-spectrum (ES) activity against the newer broad-spectrum cephalosporins. Their likely ancestor is a chromosomal penicillinase of Klebsiella pneumoniae. SHV enzymes belong to the molecular class A of serine b-lactamases and share extensive functional and structural similarity with TEM b-lactamases. The three-dimensional structure of the SHV-1 b-lactamase possesses an active site wider than that of TEM-1 b-lactamase by 0.7 to 1.2 A. This results in subtle, yet important, differences in the positioning of critical active-site residues. SHV-1 b-lactamase behaves as a typical penicillinase hydrolyzing penicillins and early generation cephalosporins. SHV-1 b-lactamase has spread, via plasmids, to virtually all enterobacterial species but is encountered mostly in K. pneumoniae. ES SHV b-lactamases are found with increasing frequency in K. pneumoniae and other enterobacterial isolates and are now considered the most prevalent ES b-lactamases. These ES SHV b-lactamases confer a wide spectrum of resistance to b-lactams, including the new generation cephalosporins and monobactams, and are usually encoded by self-transmissible multi-resistant plasmids that are highly mobile. Extension of the hydrolytic spectrum of ES SHV enzymes to include oximino-b-lactams is seen as a result of substitutions of critical amino acid residues that alter the properties of the active site. These mutational changes, however, result in diminished hydrolytic activity against penicillins and an increased susceptibility to mechanism-based inhibitors. Understanding the substrate evolution, properties and modes of spread of these clinically important b-lactamases can help in formulating effective antibiotic policies and developing new antimicrobial agents.

Amino Acid Sequence↗

PubMed1987

Import and processing of human ornithine transcarbamoylase precursor by mitochondria from Saccharomyces cerevisiae.

Expression of the subunit precursor of the human mitochondrial matrix enzyme ornithine transcarbamoylase (OTCase; EC 2.1.3.3) was programmed in Saccharomyces cerevisiae from a 2-micron plasmid by using an inducible galactose operon promoter. In the presence of the inducing sugar (galactose), two polypeptides were specifically precipitable with anti-OTCase antiserum: the human OTCase precursor (40 kDa); and the mature OTCase subunit (36 kDa). When yeast cells containing these species were lysed and fractionated, the OTCase precursor was found to be associated with mitochondrial membranes, while the mature subunit was found partly with mitochondrial membranes and partly in the soluble mitochondrial matrix-containing fraction. When OTCase enzymatic activity was assayed in fractions similarly derived from an S. cerevisiae strain devoid of yeast OTCase activity (an arg3 mutant) but expressing human OTCase, activity was detected specifically in the mitochondrial matrix fraction. A mutant human OTCase precursor containing an artificial mutation in the NH2-terminal leader peptide (arginine-23 to glycine) was similarly examined. As was previously observed with mammalian mitochondria, this precursor failed both to reach the matrix compartment and to be proteolytically processed; it also failed to exhibit OTCase enzymatic activity. Presence of OTCase enzymatic activity in an arg3 strain expressing wild-type precursor was utilized to obtain selective growth in a medium devoid of arginine but supplemented with the OTCase substrate ornithine. We conclude that, during evolution, the pathway of mitochondrial import utilized by the human OTCase precursor is conserved between yeast and humans, and that, by using selective growth conditions, it may be possible to examine genetically this pathway in S. cerevisiae.

Enzyme Precursors↗