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Results for “plasmid evolution”

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PubMed1988

Different cis-acting DNA elements control expression of the human apolipoprotein AI gene in different cell types.

In mammals, the gene coding for apolipoprotein AI (apoAI), a protein of the plasma lipid transport system, is expressed only in the liver and the intestine. A series of plasmids containing various lengths of sequences flanking the 5' end of the human apoAI gene were constructed and assayed for transient expression after introduction into cultured human hepatoma (HepG2), colon carcinoma (Caco-2), and epithelial (HeLa) cells. The results showed that while most of these constructs are expressed in HepG2 and Caco-2 cells, none of them is expressed in HeLa cells. In addition, the results indicated that a DNA segment located between nucleotides -256 and -41 upstream from the transcription start site of this gene is necessary and sufficient for maximal levels of expression in HepG2 but not in Caco-2 cells, while a DNA segment located between nucleotides -2052 and -192 is required for maximal levels of expression in Caco-2 cells. Moreover, it was shown that the -256 to -41 DNA segment functions as a hepatoma cell-specific transcriptional enhancer with both homologous and heterologous promoters. These results indicate that different cis- and possibly trans-acting factors are involved in the establishment and subsequent regulation of expression of the apoAI gene in the mammalian liver and intestine.

Animals↗

PubMed2003

In vivo regulation of the chicken cardiac troponin T gene promoter in zebrafish embryos.

The chicken cardiac troponin T (cTnT) gene is representative of numerous cardiac and skeletal muscle-specific genes that contain muscle-CAT (MCAT) elements within their promoters. We examined the regulation of the chicken cTnT gene in vivo in zebrafish embryos, and in vitro in cardiomyocyte, myoblast, and fibroblast cultures. Defined regions of the cTnT promoter were linked to the green fluorescent protein (GFP) gene for in vivo analysis, and the luciferase gene for in vitro analysis. Injection of the cTnT promoter constructs into fertilized zebrafish eggs resulted in GFP expression in both heart and skeletal muscle cells reproducing the pattern of expression of the endogenous cTnT gene in the chicken embryo. Promoter deletion analysis revealed that the cis-regulatory regions responsible for cardiac and skeletal muscle-specific expression functioned in an equivalent manner in both in vitro and in vivo environments. In addition, we show that mutation of the poly-ADP ribose polymerase-I (PARP-I) binding site adjacent to the distal MCAT element in the chicken cTnT promoter produced a non-cell-specific promoter in vitro and in the zebrafish. Thus, the PARP-I transcriptional regulatory mechanism that governs muscle specificity of the chicken cTnT promoter is conserved across several chordate classes spanning at least 350 million years of evolution.

Animals↗

PubMed1990

Structural diversity and evolution of human receptor-like protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPases), together with protein tyrosine kinases, regulate the tyrosine phosphorylation that controls cell activities and proliferation. Previously, it has been recognized that both cytosolic PTPases and membrane associated, receptor-like PTPases exist. In order to examine the structural diversity of receptor-like PTPases, we isolated human cDNA clones that cross-hybridized to a Drosophila PTPase cDNA clone, DPTP12, under non-stringent hybridization conditions. The cDNA clones thus isolated included LCA and six other novel receptor-like PTPases, named HPTP alpha, beta, gamma, delta, epsilon, and zeta. The cytoplasmic regions of HPTP alpha and epsilon are highly homologous, and are composed of two tandemly duplicated PTPase-like domains. The extracellular regions of HPTP alpha and epsilon are, respectively, 123 amino acids and 27 amino acids, and do not have obvious similarity to any known protein. The cytoplasmic region of HPTP beta contains only one PTPase domain. The extracellular region of HPTP beta, which is 1599 amino acids, is composed of 16 fibronectin type-III repeats. HPTP delta is very similar to leukocyte common antigen related molecule (LAR), in both the extracellular and cytoplasmic regions. Partial sequences of HPTP gamma and zeta indicate that they are highly homologous and contain two PTPase-like domains. The PTPase-like domains of HPTP alpha, beta and delta expressed in Escherichia coli had tyrosine phosphatase activities.

Amino Acid Sequence↗

PubMed1992

Signal peptidase I of Bacillus subtilis: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases.

Signal peptidases (SPases) remove signal peptides from secretory proteins. The sipS (signal peptidase of subtilis) gene, which encodes an SPase of Bacillus subtilis, was cloned in Escherichia coli and was also found to be active in E.coli. Its overproduction in B.subtilis resulted in increased rates of processing of a hybrid beta-lactamase precursor. The SipS protein consisted of 184 amino acids (mol. wt 21 kDa). The protein showed sequence similarity with the leader peptidases of E.coli and Salmonella typhimurium, and the mitochondrial inner membrane protease I of Saccharomyces cerevisiae. Patterns of conserved amino acids present in these four proteins were also detected in the Sec11 subunit of the SPase complex of S.cerevisiae and the 18 and 21 kDa subunits of the canine SPase complex. Knowledge of the sequence of SipS was essential for the detection of these similarities between prokaryotic and eukaryotic SPases. The data suggest that these proteins, which have analogous functions, belong to one class of enzymes, the type I SPases.

Amino Acid Sequence↗

PubMed2001

Analysis of the NF-kappaB p50 dimer interface by diversity screening.

An in vivo screen has been devised for NF-kappaB p50 activity in Escherichia coli exploiting the ability of the mammalian transcription factor to emulate a prokaryotic repressor. Active intracellular p50 was shown to repress the expression of a green fluorescent protein reporter gene allowing for visual screening of colonies expressing active p50 on agar plates. A library of mutants was constructed in which the residues Y267, L269, A308 and V310 of the dimer interface were simultaneously randomised and twenty-five novel functional interfaces were selected which repressed the reporter gene to similar levels as the wild-type protein. The leucine-269 alanine-308 core was repeatedly, but not exclusively, selected from the library whilst a diversity of predominantly non-polar residues were selected at positions 267 and 310. These results indicate that L269 and A308 may form a hot spot of interaction and allow an insight into the processes of dimer selectivity and evolution within this family of transcription factors.

Amino Acid Sequence↗

PubMed2004

Evolution of an arbitrary sequence in solubility.

We have investigated the evolvability of an insoluble random polypeptide, RP3-34, to a soluble form through iterative mutation and selection with the aid of the green fluorescent protein (GFP) folding reporter. To assess the solubility of the polypeptides in the selected clones of each generation, the polypeptide genes were detached from the GFP fusions and expressed with a His(6) tag. The solubility of the variant random polypeptides increased in each generation within the scope of the evolutionary process, and the polypeptides assumed a soluble form from the fourth generation. Analysis of the synonymous and nonsynonymous mutations found in the deduced amino acid sequence of the selected polypeptides revealed that selection had accelerated the evolutionary rate. The solubility and hydrophobicity of the polypeptides and the 25 arbitrarily chosen random polypeptides found in a previously prepared library were determined, analyzed, and interpreted from the landscape on the protein sequence space. This study showed the evolvability of an insoluble arbitrary sequence toward a soluble one, hence, it provides a new perspective on the field of artificial evolution.

Amino Acid Sequence↗

PubMed1984

Isolation of an evolutionarily conserved epidermal growth factor receptor cDNA from human A431 carcinoma cells.

Complementary DNA corresponding to total poly(A)+-RNA from the human A431 epidermoid carcinoma cell line was cloned in the phage expression vector lambda gt 11. An epidermal growth factor (EGF) receptor cDNA clone was obtained by screening of the expression library with a rabbit polyclonal antibody (IgG), raised to the purified A431 EGF receptor, in combination with [125I]protein A of S. aureus. The cloned cDNA was able to select, by hybridization, messenger RNA which was translated in Xenopus oocytes and yielded an immunoprecipitable EGF receptor protein of Mr = 160,000. The insert of this cDNA (phEGFR-1), is approximately 880 base pairs in length and encodes the carboxyterminal portion of the EGF receptor protein. Its sequence is evolutionarily conserved among vertebrates as shown by hybridization to unique chromosomal DNA sequences from human, baboon, dog, rat, mouse and frog.

Amino Acid Sequence↗

PubMed1988

A yeast and a human CCAAT-binding protein have heterologous subunits that are functionally interchangeable.

The S. cerevisiae HAP2 and HAP3 gene products have been shown to recognize CCAAT-containing transcription elements as a HAP2/HAP3 multisubunit heterologous complex, or heteromer. Recently, we have demonstrated that the human sequence-specific DNA-binding protein, CP1, also recognizes CCAAT-containing transcription elements as a heteromer. Mammalian cells contain at least three distinct CCAAT-binding proteins, all of which appear to be multisubunit complexes composed of heterologous subunits. One of these proteins, CP1, has DNA-binding properties that are virtually identical to the yeast HAP2/HAP3 complex. These two proteins bind to the same target sequences, make the same DNA contacts, and are affected in a similar manner by mutations in the CCAAT element. Most surprisingly, the subunits of CP1 and HAP2/HAP3 are functionally interchangeable. That is, the yeast/human hybrid complexes that are formed retain the ability to specifically recognize CCAAT elements.

Biological Evolution↗

PubMed1989

The phosphofructokinase genes of yeast evolved from two duplication events.

Yeast phosphofructokinase (PFK) is an octameric enzyme composed of four alpha-subunits and four beta-subunits, encoded by the genes PFK1 and PFK2, respectively. PFK1 was mapped 23 cM distal to ADE3 on chromosome VII, and PFK2 30 cM proximal to RNA1 on chromosome XIII. The entire nucleotide sequences for the two genes were obtained by sequencing both DNA strands. Only one major open reading frame was found for each gene. They encode 987 aa for PFK1 (Mr 107,984) and 959 aa for PFK2 (Mr 104,589). Both genes show a biased codon usage. The deduced amino acid sequences showed: (i) 20% homology between the N- and the C-terminal halves of each subunit, (ii) 55% homology between the two subunits, and (iii) significant homologies to the PFK sequences from human and rabbit muscle (42%), Escherichia coli (34%), and Bacillus (36%). These data support the view that two gene duplication events occurred in the evolution of the yeast PFK genes. The first duplication event took place soon after the separation of prokaryotic and eukaryotic lineage and the second in Saccharomyces later in the phylogeny. Functional domains in the yeast subunits were deduced by comparison to the rabbit muscle enzyme.

Amino Acid Sequence↗

PubMed2003

How does an enzyme evolved in vitro compare to naturally occurring homologs possessing the targeted function? Tyrosine aminotransferase from aspartate aminotransferase.

Aspartate aminotransferase (AATase) and tyrosine aminotransferase (TATase) are Escherichia coli paralogs that share 43% sequence identity. A plausible model posits that TATase arose from a duplication of an ancestral AATase-like enzyme. Directed evolution of AATase to an enzyme having TATase activity was undertaken in order to compare the evolved AATase variants with homologous TATases. Eight rounds of DNA shuffling and in vivo selection followed by a backcross with WT AATase produced enzymes that exhibited 100-270-fold increases in k(cat)/K(m)(Phe) and had as much as 11% of the tyrosine aminotransferase activity of WT E.coli TATase. Amino acid substitutions in 11 clones from rounds 7 and 8 were compared with conserved residues in AATases and TATases. The findings are conveniently and compactly illustrated by the use of Venn diagrams and set theory notation. A statistically significant (0.001 or=75% identical) in AATases and variable (<75% identical) in TATases. Very few mutations occur in the intersection (set AAT intersection TAT) of amino acid residues that are conserved in both enzyme types. Seven mutations from set AAT-TAT were combined by site-directed mutagenesis to give a construct that is 60% as active as the best round 8 enzyme, which has 13 amino acid replacements. The Venn diagrams may provide a generally useful tool to highlight the most important specificity determinants for rational redesign. Amino acid replacements were mapped onto the crystal structure of a hydrocinnamate complex of a designed TATase. Five of the seven positions most frequently substituted in the evolved clones are within 15 A of the phenyl side-chain, but only six of the 48 positions that were mutated once or twice are within that radius. Context dependence, neutral mutations, different selective pressures, and stochastic components provide explanations for the observation that many of the substitutions found in the directly evolved enzymes differ from the corresponding amino acids found in the modern natural TATases.

Amino Acid Sequence↗

PubMed2001

Fidelity of replication of repetitive DNA in mutS and repair proficient Escherichia coli.

Replication fidelity is not constant among strains within a species or at all genetic loci within a genome. Altered fidelity of replication may affect patterns of pathogenesis and the evolution of these strains. We have been studying replication fidelity in Escherichia coli, both in laboratory attenuated strains and in food-borne pathogens. To understand the altered patterns of mutagenesis at the molecular level, we used a shuttle vector plasmid with a tRNA mutational marker gene which had been altered to include homopolymeric runs of five, seven and nine [G:C] pairs, as well as non-repetitive DNA. Replication of the plasmid in mutS strains resulted in a 20-fold increase in mutant progeny plasmids. The mutations were almost all (>90%) frameshift mutations, while base substitution mutations were rare. Most mutations were insertions or deletions of one or two [G:C] pairs in the longest homopolymeric runs. Larger deletions (5 to >70bp), also targeted to the repetitive sequence, were likewise common. Mutations increased exponentially with the length of the homopolymeric run. These patterns of mutation, including unexpectedly high levels in repair proficient strains, led to an examination of the E. coli K-12 genome for homopolymeric DNA. This sequence motif was found to be rare, particularly in genes and open reading frames. Amino acid homotrimers were found to avoid usage of homopolymeric codons, even when they are preferred among synonymous codons in E. coli. There appears to be active selection against tandem direct nucleotide repeats in the E. coli genome, correlated with the inability of the organism to accurately replicate such sequence.

Adenosine Triphosphatases↗

PubMed2003

Origin and evolutionary process of the CNS elucidated by comparative genomics analysis of planarian ESTs.

Among the bilateral animals, a centralized nervous system is found in both the deuterostome and protostome. To address the question of whether the CNS was derived from a common ancestor of deuterostomes and protostomes, it is essential to know kinds of genes existed in the CNS of the putative common ancestor and to trace the evolutionary divergence of genes expressed in the CNS. To answer these questions, we took a comparative approach using different species, particularly focusing on one of the lower bilateral animals, the planarian (Platyhelminthes, Tricladida), which is known to possess a CNS. We determined the nucleotide sequence of ESTs from the head portion of planarians, obtaining 3,101 nonredundant EST clones. As a result of homology searches, we found that 116 clones had significant similarity to known genes related to the nervous system. Here, we compared these 116 planarian EST clones with all ORFs of the complete genome sequences of the human, fruit fly, and nematode, and showed that >95% of these 116 nervous system-related genes, including genes involved in brain or neural morphogenesis, were commonly shared among these organisms, thus providing evidence at the molecular level for the existence of a common ancestral CNS. Interestingly, we found that approximately 30% of planarian nervous system-related genes had homologous sequences in Arabidopsis and yeast, which do not possess a nervous system. This implies that the origin of nervous system-related genes greatly predated the emergence of the nervous system, and that these genes might have been recruited toward the nervous system.

Animals↗

PubMed2006

Effector-repressor interactions, binding of a single effector molecule to the operator-bound TtgR homodimer mediates derepression.

The RND family transporter TtgABC and its cognate repressor TtgR from Pseudomonas putida DOT-T1E were both shown to possess multidrug recognition properties. Structurally unrelated molecules such as chloramphenicol, butyl paraben, 1,3-dihydroxynaphthalene, and several flavonoids are substrates of TtgABC and activate pump expression by binding to the TtgR-operator complex. Isothermal titration calorimetry was employed to determine the thermodynamic parameters for the binding of these molecules to TtgR. Dissociation constants were in the range from 1 to 150 microm, the binding stoichiometry was one effector molecule per dimer of TtgR, and the process was driven by favorable enthalpy changes. Although TtgR exhibits a large multidrug binding profile, the plant-derived compounds phloretin and quercetin were shown to bind with the highest affinity (K(D) of around 1 microm), in contrast to other effectors (chloramphenicol and aromatic solvents) for which exhibited a more reduced affinity. Structure-function studies of effectors indicate that the presence of aromatic rings as well as hydroxyl groups are determinants for TtgR binding. The binding of TtgR to its operator DNA does not alter the protein effector profile nor the effector binding stoichiometry. Moreover, we demonstrate here for the first time that the binding of a single effector molecule to the DNA-bound TtgR homodimer induces the dissociation of the repressor-operator complex. This provides important insight into the molecular mechanism of effector-mediated derepression.

Anti-Infective Agents↗

PubMed1988

Evolution and mutagenesis of the mammalian excision repair gene ERCC-1.

The human DNA excision repair protein ERCC-1 exhibits homology to the yeast RAD10 repair protein and its longer C-terminus displays similarity to parts of the E. coli repair proteins uvrA and uvrC. To study the evolution of this 'mosaic' ERCC-1 gene we have isolated the mouse homologue. Mouse ERCC-1 harbors the same pattern of homology with RAD10 and has a comparable C-terminal extension as its human equivalent. Mutation studies show that the strongly conserved C-terminus is essential in contrast to the less conserved N-terminus which is even dispensible. The mouse ERCC-1 amino acid sequence is compatible with a previously postulated nuclear location signal and DNA-binding domain. The ERCC-1 promoter harbors a region which is highly conserved in mouse and man. Since the ERCC-1 promoter is devoid of all classical promoter elements this region may be responsible for the low constitutive level of expression in all mouse tissues and stages of embryogenesis examined.

Amino Acid Sequence↗

PubMed1991

Evolved neomycin phosphotransferase from an isolate of Klebsiella pneumoniae.

A new aminoglycoside resistance gene (aphA1-IAB) confers high-level resistance to neomycin. The sequence of aphA1-IAB is closely related to aphA1 found in the transposons Tn4352, Tn903 and Tn602. For example, aphA1-IAB differs from aphA1-903 at five nucleotides that result in four amino acid replacements. The enzyme encoded by aphA1-IAB has a significantly higher turnover number with neomycin, kanamycin and G418 as substrates than does the aphA1-903 enzyme. A parsimonious phylogenetic tree suggests that aphA1-IAB evolved from an ancestral form that is closely related or identical to the aphA1 found in Tn903. The excess of replacement substitutions over silent substitutions in aphA1-IAB, as well as its convergence toward aphA3 from Staphylococcus aureus, is indicative of selective evolution. Our hypothesis to explain these results is that aphA1-IAB evolved under the selective pressure of neomycin use in relatively recent times.

Amino Acid Sequence↗

PubMed1992

Cloning and sequencing of the gene for the cytoplasmic inorganic pyrophosphatase from the thermoacidophilic archaebacterium Thermoplasma acidophilum.

The gene (ppa) from the thermoacidophilic archaebacterium Thermoplasma acidophilum, encoding the cytoplasmic pyrophosphatase, has been cloned. Two degenerate oligonucleotide probes, synthesized according to the N-terminal amino acid sequence of the isolated protein, were used to screen subgenomic libraries. The DNA-derived amino acid sequence of the archaebacterial enzyme allows, for the first time, comparative studies of cytoplasmic pyrophosphatases to be extended to all three urkingdoms. The archaebacterial pyrophosphatase more closely resembles the eubacterial enzymes on the basis of sequence similarity and subunit size. The majority of amino acid residues considered to be essential for hydrolysis of pyrophosphate seem to have been conserved throughout evolution, as inferred from the results of an alignment of sequences from all three urkingdoms.

Amino Acid Sequence↗

PubMed1989

Evolutionary perspectives on multiresistance beta-lactamase transposons.

A series of intragenic DNA probes, encoding the major part of the transposase resolvase and inverted repeats of transposons Tn3, Tn21, and Tn2501, were used in hybridization assays for homologous DNA sequences in 18 transposons studied. The tnpA and tnpR probes detected extensive homology with Tn3-like and Tn21-like elements for 11 transposons. This high degree of homology was confirmed with the 38- and 48-base-pair inverted-repeat oligonucleotide probes of Tn3, Tn21, and Tn2501. The Southern-type gel hybridization experiments localized the tnpA-homologous sequences on the physical DNA maps constructed. The genetic and physical maps of the transposons were compared, as were their nucleic acid sequence homologies. These comparisons suggested a subfamily of mobile elements distinct from but related to the Tn21 group. Based on these results, an evolutionary model is proposed and a pedigree is presented for the genesis of multiresistance beta-lactamase transposons.

Base Sequence↗

PubMed2005

Insights into the role of histone H3 and histone H4 core modifiable residues in Saccharomyces cerevisiae.

The biological significance of recently described modifiable residues in the globular core of the bovine nucleosome remains elusive. We have mapped these modification sites onto the Saccharomyces cerevisiae histones and used a genetic approach to probe their potential roles both in heterochromatic regions of the genome and in the DNA repair response. By mutating these residues to mimic their modified and unmodified states, we have generated a total of 39 alleles affecting 14 residues in histones H3 and H4. Remarkably, despite the apparent evolutionary pressure to conserve these near-invariant histone amino acid sequences, the vast majority of mutant alleles are viable. However, a subset of these variant proteins elicit an effect on transcriptional silencing both at the ribosomal DNA locus and at telomeres, suggesting that posttranslational modification(s) at these sites regulates formation and/or maintenance of heterochromatin. Furthermore, we provide direct mass spectrometry evidence for the existence of histone H3 K56 acetylation in yeast. We also show that substitutions at histone H4 K91, K59, S47, and R92 and histone H3 K56 and K115 lead to hypersensitivity to DNA-damaging agents, linking the significance of the chemical identity of these modifiable residues to DNA metabolism. Finally, we allude to the possible molecular mechanisms underlying the effects of these modifications.

Alleles↗