Evolution of DNA or amino acid sequences with dependent sites

A framework is outlined to study the evolution of DNA or amino acid sequences, if sequence sites do not evolve independently. The units of evolution are nonoverlapping subsequences of length l. Each subsequence evolves independently of the others, but within a subsequence the sequences show a Markov order one dependency. We describe an algorithm to mimic the evolution of such sequences. The influence of dependencies between sites on distance estimates and the reliability of tree reconstruction methods is investigated. We show that an inappropriate model of sequence evolution in the tree reconstruction process will lead to a nonempty Felsenstein zone. Finally, we describe a method to infer l from sequence data. Examples from the evolution of DNA sequences as well as from amino acids are given.     

Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage

We have used two selection techniques to study sequence-specific DNA recognition by the zinc finger, a small, modular DNA-binding minidomain. We have chosen zinc fingers because they bind as independent modules and so can be linked together in a peptide designed to bind a predetermined DNA site. In this paper, we describe how a library of zinc fingers displayed on the surface of bacteriophage enables selection of fingers capable of binding to given DNA triplets. The amino acid sequences of selected fingers which bind the same triplet are compared to examine how sequence-specific DNA recognition occurs. Our results can be rationalized in terms of coded interactions between zinc fingers and DNA, involving base contacts from a few alpha-helical positions. In the paper following this one, we describe a complementary technique which confirms the identity of amino acids capable of DNA sequence discrimination from these positions.     

Dissociation, childhood trauma, and the response to fluoxetine in bulimic patients

Zuckerkandl and Pauling (1962, "Horizons in Biochemistry," pp. 189-225, Academic Press, New York) first noticed that the degree of sequence similarity between the proteins of different species could be used to estimate their phylogenetic relationship. Since then models have been developed to improve the accuracy of phylogenetic inferences based on amino acid or DNA sequences. Most of these models were designed to yield distance measures that are linear with time, on average. The reliability of phylogenetic reconstruction, however, depends on the variance of the distance measure in addition to its expectation. In this paper we show how the method of generalized least squares can be used to combine data types, each most informative at different points in time, into a single distance measure. This measure reconstructs phylogenies more accurately than existing non-likelihood distance measures. We illustrate the approach for a two-rate mutation model and demonstrate that its application provides more accurate phylogenetic reconstruction than do currently available analytical distance measures.     

Functional roles of the conserved aromatic amino acid residues at position 108 (motif IV) and position 196 (motif VIII) in base flipping and catalysis by the N6-adenine DNA methyltransferase from Thermus aquaticus

The DNA methyltransferase (Mtase) from Thermus aquaticus (M.TaqI) catalyzes the transfer of the activated methyl group of S-adenosyl-L-methionine to the N6 position of adenine within the double-stranded DNA sequence 5'-TCGA-3'. To achieve catalysis M.TaqI flips the target adenine out of the DNA helix. On the basis of the three-dimensional structure of M.TaqI in complex with the cofactor and its structural homology to the C5-cytosine DNA Mtase from Haemophilus haemolyticus, Tyr 108 and Phe 196 were suggested to interact with the extrahelical adenine. The functional roles of these two aromatic amino acid residues in M.TaqI were investigated by mutational analysis. The obtained mutant Mtases were analyzed in an improved kinetic assay, and their ability to flip the target base was studied in a fluorescence-based assay using a duplex oligodeoxynucleotide containing the fluorescent base analogue 2-aminopurine at the target position. While the mutant Mtases containing the aromatic amino acid Trp at position 108 or 196 (Y108W and F196W) showed almost wild-type catalytic activity, the mutant Mtases with the nonaromatic amino acid Ala (Y108A and F196A) had a strongly reduced catalytic constant. Y108A was still able to flip the target base, whereas F196A was strongly impaired in base flipping. These results indicate that Phe 196 is important for stabilizing the extrahelical target adenine and suggest that Tyr 108 is involved in placing the extrahelical target base in an optimal position for methyl group transfer. Since both aromatic amino acids belong to the conserved motifs IV and XIII found in N6-adenine and N4-cytosine DNA Mtases as well as in N6-adenine RNA Mtases, a similar function of aromatic amino acid residues within these motifs is expected for the different Mtases.     

Promoter specificity determinants of T7 RNA polymerase

The high specificity of T7 RNA polymerase (RNAP) for its promoter sequence is mediated, in part, by a specificity loop (residues 742-773) that projects into the DNA binding cleft (1). Previous work demonstrated a role for the amino acid residue at position 748 (N748) in this loop in discrimination of the base pairs (bp) at positions -10 and -11 (2). A comparison of the sequences of other phage RNAPs and their promoters suggested additional contacts that might be important in promoter recognition. We have found that changing the amino acid residue at position 758 in T7 RNAP results in an enzyme with altered specificity for the bp at position -8. The identification of two amino acid:base pair contacts (i.e., N748 with the bp at -10 and -11, and Q758 with the bp at -8) provides information concerning the disposition of the specificity loop relative to the upstream region of the promoter. The results suggest that substantial rearrangements of the loop (and/or the DNA) are likely to be required to allow these amino acids to interact with their cognate base pairs during promoter recognition.     

Methods for the detection of non-random base substitution in virus genes: models of synonymous nucleotide substitution in picornavirus genes

A substantial fraction of phylogenetic divergence between closely related RNA virus genes is generally accounted for by synonymous (non-amino acid changing) point mutation. Viral evolution may be a complicated phenomena, governed by many different processes. However in this study we ask whether there are any properties in the patterns of synonymous nucleotide substitutions in three different Picornavirus genes that permit the process of accumulation of synonymous point mutation in these genes to be distinguished from some of the simplest most basic evolutionary models. We conclude that while the observed patterns in the occurrence of synonymous point substitution are consistent with those predicted by a model in which base mutation is equi-probable along a gene, and the probability of synonymous substitution determined only by local codon usage, some patterns in the actual nucleotides exchanged remain to be explained.     

Establishment of the block against sperm penetration in parthenogenetically activated bovine oocytes matured in vitro

It was demonstrated previously that replication of plasmids derived from bacteriophage lambda (so-called lambda plasmids) is inhibited in wild-type Escherichia coli cells starved for isoleucine and arginine whereas it proceeds under the same conditions in relA mutants. Since replication of other replicons during the stringent or relaxed response depends on the nature of the deprived amino acid, we investigated replication of lambda plasmids in E. coli relA+ and relA- strains starved for different amino acids. We found that replication of lambda plasmids is generally inhibited during the stringent, but not relaxed, response. Differences between cells starved for different amino acids, although reproducible, were not dramatic. Amino acid starvation was previously proposed as a method for amplification of lambda plasmid DNA in vivo. We found that during amino acid limitation lambda plasmids replicate more extensively in the relA mutants than during amino acid starvation. The efficiency of plasmid DNA amplification was found to be dependent on the kind of limited amino acid; in relA- bacteria limited for leucine we observed about 10-fold plasmid amplification. Some lambda plasmid replication was also found under these conditions in the relA+ host. The mechanism of the stringent control of lambda plasmid DNA replication has already been proposed. Here the possible mechanism of the regulation of lambda plasmid replication during amino acid limitation is presented.     

Codon usage and base composition in Rickettsia prowazekii

Codon usage and base composition in sequences from the A + T-rich genome of Rickettsia prowazekii, a member of the alpha Proteobacteria, have been investigated. Synonymous codon usage patterns are roughly similar among genes, even though the data set includes genes expected to be expressed at very different levels, indicating that translational selection has been ineffective in this species. However, multivariate statistical analysis differentiates genes according to their G + C contents at the first two codon positions. To study this variation, we have compared the amino acid composition patterns of 21 R. prowazekii proteins with that of a homologous set of proteins from Escherichia coli. The analysis shows that individual genes have been affected by biased mutation rates to very different extents: genes encoding proteins highly conserved among other species being the least affected. Overall, protein coding and intergenic spacer regions have G + C content values of 32.5% and 21.4%, respectively. Extrapolation from these values suggests that R. prowazekii has around 800 genes and that 60-70% of the genome may be coding.     

Deleterious mutations at the mitochondrial ND3 gene in South American marsh rats (Holochilus)

Statistical analyses of DNA sequences have revealed patterns of nonneutral evolution in mitochondrial DNA of mice, humans, and Drosophila. Here we report patterns of mitochondrial sequence evolution in South American marsh rats (genus Holochilus). We sequenced the complete mitochondrial ND3 gene in 82 Holochilus brasiliensis and 21 H. vulpinus to test the neutral prediction that the ratio of nonsynonymous to synonymous nucleotide changes is the same within and between species. Within H. brasiliensis we observed a greater number of amino acid polymorphisms than expected based on interspecific comparisons. This contingency table analysis suggests that many amino acid polymorphisms are mildly deleterious. Several tests of the frequency distribution also revealed departures from a neutral, equilibrium model, and these departures were observed for both nonsynonymous and synonymous sites. In general, an excess of rare sites was observed, consistent with either a recent selective sweep or with populations not at mutation-drift equilibrium.     

Protein identification using mass spectrometric information

In an effort to gain an understanding of the value of the information in different mass spectrometric measurements for protein identification, the genome of Saccharomyces cerevisiae was studied in silico. We calculate how constraining the knowledge of the mass of a proteolytic peptide is as a function of mass and mass accuracy. We also assess the value for protein identification of additional information concerning a proteolytic peptide, including the presence or absence of a given amino acid, the number of exchangeable hydrogens, the N-terminal sequence, and the masses of mass spectrometrically produced fragment ions. Knowledge of the relative value of these different constraints is useful in the design of efficient protein identification experiments. Finally, we describe a software tool, PepFrag, for searching protein and DNA sequence databases that can use different types of mass spectrometric information to restrict the search.     

Representation of amino acid sequences as two-dimensional point patterns

An algorithm for the representation of amino acid sequences as two-dimensional point patterns (2-D plot) is described. The algorithm is based on chaos game representation (CGR) for DNA sequences and was extended for amino acid sequences. The 2-D plot depicts the sequentiality of amino acids and the amino acid composition of a protein. Changes in a protein sequence as insertion, deletion and repeats of amino acids are characterized by specific geometrical properties and changes in the 2-D plots. The 2-D plot may be considered as a two-dimensional "fingerprint" of a protein. The properties of the algorithm are explained by user-defined amino acid sequences. As an example the 2-D plots of two selected heart proteins are generated. The sequences of these proteins are obtained from the protein sequence database SWISS-PROT.     

Formation of the amino acid-DNA complexes by hexavalent and trivalent chromium in vitro: importance of trivalent chromium and the phosphate group

We have recently shown that a substantial fraction of all Cr-DNA adducts in chromate-exposed cells are represented by ternary complexes involving amino acids or glutathione bridged by Cr-(III) to DNA. The tridentate amino acids such as cysteine, glutamic acid, and histidine were predominantly found cross-linked to DNA. The mechanism by which Cr can cross-link these amino acids to DNA has been modeled by reacting DNA and trivalent and hexavalent chromium with cysteine and histidine. The formation of a Cr(III)-amino acid binary complex was required before Cr(III) reacted with DNA to yield a ternary complex. Cr(III)-pretreated DNA did not bind cysteine or histidine even after prolonged incubations. Reduction of Cr(VI) in the presence of DNA gave rise to an extensive cross-linking of cysteine and histidine. Addition of DNA to Cr(VI) mixtures at the start of reduction or after the reduction was complete had little effect on the level of ternary complexes indicating that Cr(III)-amino acid binary complexes were DNA-attacking species. In order to identify DNA groups involved in the ternary complex formation, pre-formed Cr(III)-histidine complexes were reacted with nucleosides and nucleotide monophosphates followed by separation and analysis of the products. The incubation of the Cr(III)-histidine complexes with nucleotide monophosphates but not with nucleosides gave rise to ternary complexes that contained both histidine and Cr, showing the primary importance of the phosphate group in this reaction. All four DNA nucleotides were capable of the ternary complex formation with Cr(III) and histidine. No apparent base preference in the amino acid cross-linking was also found in the reaction of Cr(III)/cysteine and Cr(VI)/cysteine mixtures with oligonucleotides of base-specific composition.     

Characteristic distribution of bases and codons around the initiation and termination codons in whole reading frames in bacteria and yeast genomes

Recently the complete nucleotide sequence of the entire genome was determined for yeast and a few kinds of bacterium. To see characteristic features of base sequence in the cistron (actually the open reading frame, ORF) and in the regions around a cistron (ORF), the biases of appearance frequency of bases from the base ratio were studied statistically. In the regions before the base biases were observed. The characteristic base distribution patterns were similar to all the cases of bacteria, but different from yeast. The base biases are reflected on the appearance frequency of amino acids near the N-termini and C-termini of proteins. The characteristic biases found in the amino acid sequence of the N-terminal part of bacteria proteins are different from that in yeast proteins.     

Selective recognition of tandemly repeated DNA sequences by homo- and heterodimers of short peptides

The non-covalent and specific protein-protein interaction is critical to the specificity and the cooperativity of the DNA-binding by protein dimers. We have designed and synthesized three sets of peptide dimers with covalent- or noncovalent artificial dimerization modules to elucidate the structural and thermodynamic aspects for the sequence-specific DNA-binding by protein dimers. We have investigated the DNA binding of covalent dimer, noncovalent homodimer and noncovalent heterodimer with specific or nonspecific DNA sequences by gel mobility shift assay. Although the amino acid sequence of DNA-binding region of these peptide dimers are the same, the selectivity and the cooperativity of DNA binding by these peptide dimers were found to be different.     

Chaos game representation of proteins

The present report proposes a new method for the chaos game representation (CGR) of different families of proteins. Using concatenated amino acid sequences of proteins belonging to a particular family and a 12-sided regular polygon, each vertex of which represents a group of amino acid residues leading to conservative substitutions, the method can generate the CGR of the family and allows pictorial representation of the pattern characterizing the family. An estimation of the percentages of points plotted in different segments of the CGR (grid points) allows quantification of the nonrandomness of the CGR patterns generated. The CGRs of different protein families exhibited distinct visually identifiable patterns. This implies that different functional classes of proteins follow specific statistical biases in the distribution of different mono-, di-, tri-, or higher order peptides along their primary sequences. The potential of grid counts as the discriminative and diagnostic signature of a family of proteins is discussed.     

ComEA is a DNA receptor for transformation of competent Bacillus subtilis

Competent cells of Bacillus subtilis efficiently bind and internalize DNA. ComEA and the seven proteins encoded by the comG operon are required in vivo for the binding step. We show here that ComEA, a bitopic membrane protein, is itself capable of high-affinity DNA binding. A domain necessary for DNA binding is located at the C-terminus of ComEA. Proteins with similar 60-80 amino acid residue domains are widespread among bacteria and higher organisms. ComEA shows a marked preference for double-stranded DNA and can bind to oligomers as small as 22 bp in length. DNA binding by ComEA exhibits no apparent base sequence specificity. Using a membrane vesicle DNA-binding assay system we show that in the absence of cell wall, ComEA is still required for DNA binding, whereas the requirement for the ComG proteins is bypassed. We conclude that the ComG proteins are needed in vivo to provide access of the binding domain of ComEA to exogenous DNA. Possible specific roles for the ComG proteins are discussed.     

The genome sequence of Rickettsia prowazekii and the origin of mitochondria

We describe here the complete genome sequence (1,111,523 base pairs) of the obligate intracellular parasite Rickettsia prowazekii, the causative agent of epidemic typhus. This genome contains 834 protein-coding genes. The functional profiles of these genes show similarities to those of mitochondrial genes: no genes required for anaerobic glycolysis are found in either R. prowazekii or mitochondrial genomes, but a complete set of genes encoding components of the tricarboxylic acid cycle and the respiratory-chain complex is found in R. prowazekii. In effect, ATP production in Rickettsia is the same as that in mitochondria. Many genes involved in the biosynthesis and regulation of biosynthesis of amino acids and nucleosides in free-living bacteria are absent from R. prowazekii and mitochondria. Such genes seem to have been replaced by homologues in the nuclear (host) genome. The R. prowazekii genome contains the highest proportion of non-coding DNA (24%) detected so far in a microbial genome. Such non-coding sequences may be degraded remnants of 'neutralized' genes that await elimination from the genome. Phylogenetic analyses indicate that R. prowazekii is more closely related to mitochondria than is any other microbe studied so far.