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Modeling and simulating the evolution of resistance against antibiotics.

An epidemiological model is proposed for the spread of resistance against antibiotics in populations of bacterial pathogens. The host population, which is assumed to be constant, is divided into three compartments, viz. susceptible, hosts infected by an antibiotic-sensitive strain and hosts infected by a resistant strain. It is further assumed that susceptibles can be infected by either strain and that there is a possibility for cross-infection between hosts infected by the two strains. The rate of cross-infection can be enhanced by mutations or the transfer of plasmids conferring resistance. Equilibrium analysis was performed in order to determine which of the strain 'wins' the competition by the host. It is assumed that the eventual shift in the competition between the two strains is due to treatment by antibiotic (selective pressure).

Amikacin↗

Restoration of blood flow by using continuous perimuscular infiltration of plasmid DNA encoding subterranean mole rat Spalax ehrenbergi VEGF.

The optimal vector, regulatory sequences, and method of delivery of angiogenic gene therapy are of considerable interest. The Spalax ehrenbergi superspecies live in subterranean burrows at low oxygen tensions and its tissues are highly vascularized. We tested whether continuous perimuscular administration of Spalax vascular endothelial growth factor (VEGF) DNA could increase tissue perfusion in a murine hindlimb ischemia model. Placebo or VEGF +/- internal ribosome entry site (IRES) was continuously administrated perimuscularly in the ischemic zone by using an infusion pump. None of the mice in the VEGF-treated group (>50 microg) developed visible necrosis vs. 33% of the placebo group. Microscopic necrosis was observed only in the placebo group. Spalax VEGF muscular infiltration resulted in a faster and more complete restoration of blood flow. The restoration of blood flow by VEGF was dose-dependent and more robust and rapid when using the VEGF-IRES elements. The flow restoration using continuous perimuscular infiltration was faster than single i.m. injections. Vessel density was higher in the VEGF and VEGF-IRES (-) groups compared with the placebo. Continuous perimuscular administration of angiogenic gene therapy offers a new approach to restore blood flow to an ischemic limb. Incorporation of an IRES element may assist in the expression of transgenes delivered to ischemic tissues. Further studies are needed to determine whether VEGF from the subterranean mole rat Spalax VEGF is superior to VEGF from other species. If so, 40 million years of Spalax evolution underground, including adaptive hypoxia tolerance, may prove important to human angiogenic gene therapy.

Animals↗

The type 1 human immunodeficiency virus Tat binding protein is a transcriptional activator belonging to an additional family of evolutionarily conserved genes.

The type 1 human immunodeficiency virus Tat protein is a powerful transcriptional activator when bound to an RNA structure (TAR) present at the extreme 5' terminus of viral mRNA. Since transcriptional activation requires binding of Tat to RNA, it has been suggested that Tat enhances initiation or elongation through a direct interaction with cellular transcription factors. Here we show through protein fusion experiments that the previously identified cellular Tat binding protein, TBP-1, although unable to bind DNA, is a strong transcriptional activator when brought into proximity of several promoter elements. Transcriptional activity depends upon the integrity of at least two highly conserved domains: one resembling a nucleotide-binding motif and the other motif common to proteins with helicase activity. Our studies further reveal that TBP-1 represents one member of a large, highly conserved gene family that encodes proteins demonstrating strong amino acid conservation across species. Finally, we identified a second family member that, although 77% similar to TBP-1, does not activate transcription from the promoters examined. This finding, together with the observation that TBP-1 does not activate each promoter examined, suggests that this gene family may encode promoter-specific transcriptional activators.

ATPases Associated with Diverse Cellular Activitie↗

Increasing PCR fragment stability and protein yields in a cell-free system with genetically modified Escherichia coli extracts.

Escherichia coli cell-free protein synthesis is a highly productive system that can be applied to high throughput expression from polymerase chain reaction (PCR) products in 96-well plates for proteomic studies as well as protein evolution. However, linear DNA instability appears to be a major limitation of the system. We modified the genome of the E. coli strain A19 by removing the endA gene encoding the endonuclease I and replacing the recCBD operon (in which recD encodes the exonuclease V) by the lambda phage recombination system. Using the cell extract from this new strain increased the stability of PCR products amplified from a plasmid containing the cat gene. This resulted in CAT (chloramphenicol acetyltransferase) production from PCR products comparable to that from plasmids (500-600 microg/ml) in a batch reaction. We show that cell-free protein synthesis reactions using PCR products amplified from genomic DNA and extended with the T7 promoter and the T7 terminator give the same high yields of proteins (550 microg/ml) in 96-well plates. With this system, it was possible to rapidly express a range of cytoplasmic and periplasmic proteins.

Bacterial Proteins↗

Induction by cAMP of the mRNA encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken. Identification and characterization of a cDNA clone for the enzyme.

Previous work from our laboratory (Hod, Y., Utter, M. F., and Hanson, R. W. (1982) J. Biol. Chem. 257, 13787-13794) has demonstrated that chicken kidney contains both mitochondrial and cytosolic forms of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) and that the two forms are distinct proteins. Using poly(A+) RNA from chicken kidney, a double-stranded cDNA library was constructed. DNA clones containing sequences complementary to the mRNA for the cytosolic form of phosphoenolpyruvate carboxykinase were initially identified by colony hybridization with 32P-labeled cDNA transcribed from an RNA fraction enriched for the enzyme mRNA. The identity of plasmids containing phosphoenolpyruvate carboxykinase cDNA was confirmed by hybrid-selected translation. Mature mRNA for cytosolic phosphoenolpyruvate carboxykinase of the chicken is 2.8 kilobases in length, similar to that previously noted for mRNA coding for the same enzyme in the rat. The cDNA for the chicken enzyme hybridizes with several restriction fragments of the corresponding cDNA for the rat cytosolic phosphoenolpyruvate carboxykinase, indicating conservation of nucleotide sequences during evolution. Wide spread conservation of sequence homology is also demonstrated by the hybridization of the cDNA for the rat phosphoenolpyruvate carboxykinase with a 2.8-kilobase RNA from the livers of a variety of vertebrates including amphibian, avian, and primate species. Specific mRNA coding for the cytosolic form of phosphoenolpyruvate carboxykinase was present in chicken kidney but absent from the liver, even in animals starved for 48 h. However, the administration of cAMP to normal fed chickens caused a rapid induction of phosphoenolpyruvate carboxykinase mRNA. These findings suggest that the gene for the cytosolic enzyme in chicken liver can be expressed if the proper hormonal stimuli are present.

Animals↗

Expression of hepatitis C virus hypervariable region 1 and its clinical significance.

AIM: To explore the properties of hypervariable region 1 (HVR1) in the envelope 2 gene of hepatitis C virus by analyzing the reactivity of HVR1 fusion proteins from different Chinese HCV strains with sera of patients with chronic hepatitis C and by comparing their reactivity between interferon therapy responders and non-responders. METHODS: Gene fragments of HVR1 of four HCV strains (three genotype 1b and one genotype 2a) were amplified from pGEMT-E2 plasmids and sub-cloned into pQE40 vectors respectively to construct recombinant expression plasmids which expressed HVR1 fused downstream to DHFR in Escherichia coli strain TG1. The purified DHFR- HVR1 proteins were then used to detect the anti-HVR1 antibodies in 70 serum samples of patients with chronic hepatitis C. RESULTS: Four DHFR- HVR1 fusion proteins were successfully expressed in E.coli (320-800 ug fusion proteins per 100 ml culture). Each fusion protein (SH1b, BJ1b, SD1b and SD2a) reacted with 72.8 % (51/70), 60 % (42/70), 48.6 % (34/70), and 58.6 % (41/30) of the anti-HCV positive patients' sera respectively by ELISA. 57 % (4/7) of non responders reacted with all four HVR1 fusion proteins, while only 15.3 % (2/13) of responders reacted with all of them. The O.D. values of sera from IFN therapy responders were significantly higher than those of non responders (P<0.05). CONCLUSION: The selected HVR1 fusion proteins expressed in E. coli can broadly react with HCV-infected patients' sera. The intensity and/or quality of the immune response against HCV may be a critical factor determining the response to interferon treatment. With the evolution of virus strains, anti-HVR1 antibodies can not neutralize all the quasispecies. A polyvalent and high immunogenic vaccine comprising a mixture of several HVR1 sequences that cover the reactivity of most HCV isolates may be useful.

Adult↗

Genetic dissection of histone function.

Mutational analysis is an essential tool for understanding the functions of genes within a living organism. The budding yeast Saccharomyces cerevisiae provides an excellent model system for dissecting the genetics of histone function at the molecular and cellular levels. A simple gene organization, plus a wide variety of genetic strategies, makes it possible to directly manipulate a specific histone gene in vitro and then examine the expression of mutant alleles in vivo. Recent methods for manipulating the yeast histone genes have been designed to facilitate both side-directed analysis of structure/function relationships and unbiased screens targeted at specific functional pathways. The conservation of histone and nucleosome structure throughout evolution means that the principles discovered through genetic studies in yeast will be broadly applicable to the chromatin of more complex eukaryotes.

Cell Cycle↗

An iteron-related domain is associated to Motif 1 in the replication proteins of geminiviruses: identification of potential interacting amino acid-base pairs by a comparative approach.

Geminiviruses encode a replication initiator protein, Rep, which binds in a sequence-specific fashion to iterated DNA motifs (iterons) functioning as essential elements for virus-specific replication. By using the iterons of more than one hundred geminiviruses as heuristic devices, we have identified a Rep subdomain 8 to 10 residues in length, whose primary structure varies among viruses harboring different iterons, but which is similar among viruses with identical iterons, regardless of their differences in host range, insect vector, geographical origin or genome structure. Close analysis of this iteron-related domain (IRD) revealed consistent correlations between specific Rep residues and defined nucleotides of its cognate iteron, thus providing important insights about the molecular code which dictates the Rep preference for specific DNA sequences. A model of potential Rep-iteron contacts is proposed. The identified IRD is adjacent to a conserved motif characteristic of a superfamily of rolling-circle (RC) replication proteins, and secondary structure predictions suggest that those Rep subdomains form together the core of a novel DNA-binding domain possessing a beta-sheet as recognition subdomain, which is apparently conserved in the replication proteins of nanoviruses, circoviruses, microviruses, and a variety of ssDNA plasmids of eubacteria, archaebacteria and red algae. The evolutionary implications of these findings are discussed.

Amino Acid Motifs↗

A novel nuclear localization signal in the human single-minded proteins SIM1 and SIM2.

Human Single-minded 1 (SIM1) and SIM2 genes were found as homologs of Drosophila sim gene which plays a key role in the midline cell lineage of the central nervous system. SIM proteins belong to a family of transcription factors, called bHLH/PAS. Here we examined the intracellular localization of SIM proteins using the expression constructs of whole SIM2 or SIM1 protein fused with enhanced green fluorescent protein (EGFP). The transient expression analysis revealed the nuclear localization of SIM proteins in the cultured cells. To identify the nuclear localization signal, we made expression constructs of EGFP-fusion protein consisting of various portions of SIM proteins. Transfection assay showed the presence of NLS activity in the small region of 23 and 21 amino acid residues at the central part of SIM2 and SIM1 proteins, respectively. Further analysis with amino acid substitution of this small region of SIM2 protein revealed the critical role of five amino acid residues (Arg367, Lys373, Pro385, Tyr386, and Gln389) in NLS activity. The consensus sequence of RKxxKx[K/R]xxxxKxKxRxxPY was estimated as a presumptive NLS in SIM proteins from various species. Thus, the NLS consisting of a cluster of basic amino acids with Pro and Tyr at the C-terminal end is novel and well conserved in the SIM proteins during evolution.

Amino Acid Motifs↗

A search for hyperglycosylation signals in yeast glycoproteins.

N-oligosaccharides of Saccharomyces cerevisiae glycoproteins are classified as core and mannan types. The former contain 13-14 mannoses whereas mannan-type structures consist of an inner core extended with an outer chain of up to 200-300 mannoses, a process known as hyperglycosylation. The selection of substrates for hyperglycosylation poses a theoretical and practical question. To identify hyperglycosylation determinants, we have analyzed the influence of the second amino acid (Xaa) of the sequon in this process using the major exoglucanase as a model. Our results indicate that negatively charged amino acids inhibit hyperglycosylation, whereas positively charged counterparts promote it. On the basis of the tridimensional structure of Exg1, we propose that Xaa influences the orientation of the inner core making it accessible to mannan polymerase I in the appropriate position for the addition of alpha-1,6-mannoses. The presence of Glu in the Xaa of the second sequon of the native exoglucanase suggests that negative selection may drive evolution of these sites. However, a comparison of invertases secreted by S. cerevisiae and Pichia anomala suggests that hyperglycosylation signals are also subjected to positive selection.

Amino Acid Sequence↗

Genetic organization of the duplicated vap region of the Dichelobacter nodosus genome.

The recombinant plasmid pJIR318 contains a fragment of the Dichelobacter nodosus genome which is associated with virulence. Sequence analysis of the pJIR318 insert has shown that it contains four vap (virulence-associated protein) genes which are homologous to open reading frames found on the Escherichia coli F plasmid and the Neisseria gonorrhoeae cryptic plasmid (M. E. Katz, R. A. Strugnell, and J. I. Rood, Infect. and Immun. 60:4586-4592, 1992). The plasmid pJIR318 hybridizes to three regions of the D. nodosus genome, each of which has now been isolated. Regions 1 and 3 were found to be adjacent in the genome of D. nodosus A198, and the order of the vap genes in vap regions 1 and 2 were shown to be identical. Partial sequence analysis and Southern blot analysis of the vap regions showed that the three regions probably arose by a duplication event(s) followed by insertions and/or deletions. A recombinant plasmid, pJIR749, was isolated from a library of a benign D. nodosus strain, 305. This plasmid contained sequences from both ends of vap region 2. Analysis of pJIR749 showed that the sequences on either side of vap region 2 were separated by 324 bp in the genome of benign strain 305 and that the orientations of the sequences were different. It is clear that a simple insertion or deletion event did not generate the benign and virulent strains studied. A model which describes the evolution of the duplicated vap regions in D. nodosus A198 is presented.

Bacteria, Anaerobic↗

The solitary long terminal repeats of ERV-9 endogenous retrovirus are conserved during primate evolution and possess enhancer activities in embryonic and hematopoietic cells.

The solitary long terminal repeats (LTRs) of ERV-9 endogenous retrovirus contain the U3, R, and U5 regions but no internal viral genes. They are middle repetitive DNAs present at 2,000 to 4,000 copies in primate genomes. Sequence analyses of the 5" boundary area of the erythroid beta-globin locus control region (beta-LCR) and the intron of the embryonic axin gene show that a solitary ERV-9 LTR has been stably integrated in the respective loci for at least 15 million years in the higher primates from orangutan to human. Functional studies utilizing the green fluorescent protein (GFP) gene as the reporter in transfection experiments show that the U3 region of the LTRs possesses strong enhancer activity in embryonic cells of widely different tissue origins and in adult cells of blood lineages. In both the genomic LTRs of embryonic placental cells and erythroid K562 cells and transfected LTRs of recombinant GFP plasmids in K562 cells, the U3 enhancer activates synthesis of RNAs that are initiated from a specific site 25 bases downstream of the AATAAA (TATA) motif in the U3 promoter. A second AATAAA motif in the R region does not serve as the TATA box or as the polyadenylation signal. The LTR-initiated RNAs extend through the R and U5 regions into the downstream genomic DNA. The results suggest that the ERV-9 LTR-initiated transcription process may modulate transcription of the associated gene loci in embryonic and hematopoietic cells.

Animals↗

DNA microarray analysis of genome dynamics in Yersinia pestis: insights into bacterial genome microevolution and niche adaptation.

Genomics research provides an unprecedented opportunity for us to probe into the pathogenicity and evolution of the world's most deadly pathogenic bacterium, Yersinia pestis, in minute detail. In our present work, extensive microarray analysis in conjunction with PCR validation revealed that there are considerable genome dynamics, due to gene acquisition and loss, in natural populations of Y. pestis. We established a genomotyping system to group homologous isolates of Y. pestis, based on profiling or gene acquisition and loss in their genomes, and then drew an outline of parallel microevolution of the Y. pestis genome. The acquisition of a number of genomic islands and plasmids most likely induced Y. pestis to evolve rapidly from Yersinia pseudotuberculosis to a new, deadly pathogen. Horizontal gene acquisition also plays a key role in the dramatic evolutionary segregation of Y. pestis lineages (biovars and genomovars). In contrast to selective genome expansion by gene acquisition, genome reduction occurs in Y. pestis through the loss of DNA regions. We also theorized about the links between niche adaptation and genome microevolution. The transmission, colonization, and expansion of Y. pestis in the natural foci of endemic plague are parallel and directional and involve gradual adaptation to the complex of interactions between the environment, the hosts, and the pathogen itself. These adaptations are based on the natural selections against the accumulation of genetic changes within genome. Our data strongly support that the modern plague originated from Yunnan Province in China, due to the arising of biovar orientalis from biovar antiqua rather than mediaevalis.

Adaptation, Physiological↗

An intracellular actin motor in bacteria?

Actin performs structural as well as motor-like functions in eukaryotic cells. Orthologues of actin have also been identified in bacteria, where they perform an essential function during cell growth. Bacterial actins are implicated in the maintenance of rod-shaped cell morphology, and appear to form a cytoskeletal structure, localising as helical filaments underneath the cell membrane. Recently, a plasmid-borne actin orthologue has been shown to perform a mitotic-like function during segregation of a plasmid, and chromosomally encoded actin proteins were found to play an important role in chromosome segregation. Based on the findings that actin filaments are dynamic structures in two bacterial species, we propose that actins perform motor functions rather than a purely structural role in bacteria. We suggest that an intracellular motor exists in bacteria that could be derived from an ancestral actin motor that was present in cells early in evolution.

Actins↗

Cytogenetic heterogeneity of genetically marked and metastatically competent "dominant" tumor cell clones.

Examination of tumors usually shows them to consist of phenotypically and clonally heterogeneous cell subpopulations. On the other hand, previous studies from our laboratory have provided compelling evidence for the rapid evolution, or overgrowth, of single "dominant" clones during the course of primary tumor growth. Thus in one such study, syngeneic CBA mice were injected with a mixture of 50-100 different genetically tagged clones of a mouse mammary carcinoma called SP1. The vast majority of these clones were non-metastatic. The different clones were tagged by random integrations of foreign DNA using calcium phosphate-mediated transfection of the plasmid pSV2neo, the resultant primary tumors were found to consist of a single dominant clone, called B5, which was also shown to be metastatically competent. The detection of single dominant clones such as B5 in primary tumors can be reconciled with the concept of tumor cell heterogeneity if it could be shown that the dominant clone was in fact heterogeneous for other genetic or phenotypic markers, i.e., was homogeneous only for the plasmid-based genetic marker used for its detection. To study this question, we examined the karyotypes of several sublines of B5, two derived from a primary advanced tumor and one from a spontaneous lung metastasis. We indeed found evidence to support the existence of marked cellular heterogeneity within and between the three sublines examined. Thus, while all three retained common cytogenetic markers, each also expressed unique markers. Moreover, karyotypic heterogeneity within a given subline was observed. Thus the concept of clonal dominance of primary tumors by metastatically competent cell subpopulations is not incompatible with the concept of the cellular heterogeneity of tumors. The implications of the results are discussed.

Adenocarcinoma↗

Mef2c is activated directly by Ets transcription factors through an evolutionarily conserved endothelial cell-specific enhancer.

Members of the Myocyte Enhancer Factor 2 (MEF2) family of transcription factors play key roles in the development and differentiation of numerous cell types during mammalian development, including the vascular endothelium. Mef2c is expressed very early in the development of the endothelium, and genetic studies in mice have demonstrated that mef2c is required for vascular development. However, the transcriptional pathways involving MEF2C during endothelial cell development have not been defined. As a first step towards identifying the transcriptional factors upstream of MEF2C in the vascular endothelium, we screened for transcriptional enhancers from the mouse mef2c gene that regulate vascular expression in vivo. In this study, we identified a transcriptional enhancer from the mouse mef2c gene sufficient to direct expression to the vascular endothelium in transgenic embryos. This enhancer is active in endothelial cells within the developing vascular system from very early stages in vasculogenesis, and the enhancer remains robustly active in the vascular endothelium during embryogenesis and in adulthood. This mef2c endothelial cell enhancer contains four perfect consensus Ets transcription factor binding sites that are efficiently bound by Ets-1 protein in vitro and are required for enhancer function in transgenic embryos. Thus, these studies identify mef2c as a direct transcriptional target of Ets factors via an evolutionarily conserved transcriptional enhancer and establish a direct link between these two early regulators of vascular gene expression during endothelial cell development in vivo.

Animals↗

Protein microarrays guide tolerizing DNA vaccine treatment of autoimmune encephalomyelitis.

The diversity of autoimmune responses poses a formidable challenge to the development of antigen-specific tolerizing therapy. We developed 'myelin proteome' microarrays to profile the evolution of autoantibody responses in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS). Increased diversity of autoantibody responses in acute EAE predicted a more severe clinical course. Chronic EAE was associated with previously undescribed extensive intra- and intermolecular epitope spreading of autoreactive B-cell responses. Array analysis of autoantigens targeted in acute EAE was used to guide the choice of autoantigen cDNAs to be incorporated into expression plasmids so as to generate tolerizing vaccines. Tolerizing DNA vaccines encoding a greater number of array-determined myelin targets proved superior in treating established EAE and reduced epitope spreading of autoreactive B-cell responses. Proteomic monitoring of autoantibody responses provides a useful approach to monitor autoimmune disease and to develop and tailor disease- and patient-specific tolerizing DNA vaccines.

Animals↗

E2F-4 and E2F-5, two members of the E2F family, are expressed in the early phases of the cell cycle.

The E2F transcription factors play a role in regulating the expression of genes required for cell proliferation. Their activity appears to be regulated by association with the retinoblastoma protein (pRb) and the pRb-related proteins p107 and p130. In vivo, pRb is found in complex with a subset of E2F components--namely, E2F-1, E2F-2, and E2F-3. Here we describe the characterization of cDNAs encoding two unusual E2Fs, E2F-4 and E2F-5, each identified by the ability of their gene product to interact with p130 in a yeast two-hybrid system. E2F-4 and -5 share common sequences with E2F-1, E2F-2, and E2F-3 and, like these other E2Fs, the ability to heterodimerize with DP-1, thereby acquiring the ability to bind an E2F DNA recognition sequence with high affinity. However, in contrast to E2F-1, E2F-4 and E2F-5 fail to bind pRb in a two-hybrid assay. Moreover, they show a unique pattern of expression in synchronized human keratinocytes: E2F-4 and E2F-5 mRNA expression is maximal in mid-G1 phase before E2F-1 expression is detectable. These findings suggest that E2F-4 and E2F-5 may contribute to the regulation of early G1 events including the G0/G1 transition.

Amino Acid Sequence↗