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PubMed1980

Intragenomic DNA sequence homologies in the chicken and other members of the class Aves: DNA re-association under reduced stringency conditions.

We have investigated the intragenomic DNA sequence homologies of twelve species of birds representing five orders, and emphasizing Galliformes. This study differs in two important ways from the classical approaches taken in constructing and evaluating phylogenies based on DNA sequence similarities. Comparisons are made on the basis of sequence homologies within genomes of related birds, rather than between genomes. DNA is reassociated at 50 degrees C in 0.5M phosphate buffer; these conditions allow formation and detection of duplexes containing more mismatch than would normally be permitted using more stringent conditions, affording an opportunity to observe more ancient sequence homologies. Thermal stability profiles of DNA duplexes formed under these conditions are the basis of comparison; three general patterns were observed. This approach emphasizes differences in sequence composition between genomes while the more traditional method of intergenomic tracer DNA hybridization at higher stringency emphasizes sequence similarities. No correlation was found between taxonomic position and intragenomic sequence composition, either within or between lineages. The thermal stability profiles of DNA duplexes formed within avian genomes did not reflect the biological similarities inferred from morphology, karyotype, and studies of interspecific hybridization. While all of the differences observed could have occurred over geological time, it was surprising that the genomes of the domestic chicken and the Red Jungle Fowl (Gallus gallus) differ in their sequence compositions. It appears that amolification/reduction events and/or positional changes occur rather often during evolution of a lineage.

Animals↗
PubMed2005

"Word" preference in the genomic text and genome evolution: different modes of n-tuplet usage in coding and noncoding sequences.

Extensive work on n-tuplet occurrence in genomic sequences has revealed the correlation of their usage with sequence origin. Parallel to that, there exist different restrictions in the nucleotide composition of coding and noncoding sequences that may result in distinct modes of usage of n-tuplets. The relatively simple approaches described herein focus on such differences. They are based on simple summation measures of n-tuplet frequencies, computed after filtering the background nucleotide composition. Among the main targets of this work is to draw some conclusions on the qualitative differences in the composition of genomic sequences depending on their functionality. Moreover, an evolutionary model is formulated, including simple forms of ubiquitous events of genome dynamics: genomic fusions, genome shuffling due to transpositions, replication slippage, and point mutations. This model is shown to be able to reproduce all the statistical features of genomic sequences discussed herein.

Base Sequence↗
PubMed2005

Comparative analysis of the base composition and codon usages in fourteen mycobacteriophage genomes.

To study the possible codon usage and base composition variation in the bacteriophages, fourteen mycobacteriophages were used as a model system here and both the parameters in all these phages and their plating bacteria, M. smegmatis had been determined and compared. As all the organisms are GC-rich, the GC contents at third codon positions were found in fact higher than the second codon positions as well as the first + second codon positions in all the organisms indicating that directional mutational pressure is strongly operative at the synonymous third codon positions. Nc plot indicates that codon usage variation in all these organisms are governed by the forces other than compositional constraints. Correspondence analysis suggests that: (i) there are codon usage variation among the genes and genomes of the fourteen mycobacteriophages and M. smegmatis, i.e., codon usage patterns in the mycobacteriophages is phage-specific but not the M. smegmatis-specific; (ii) synonymous codon usage patterns of Barnyard, Che8, Che9d, and Omega are more similar than the rest mycobacteriophages and M. smegmatis; (iii) codon usage bias in the mycobacteriophages are mainly determined by mutational pressure; and (iv) the genes of comparatively GC rich genomes are more biased than the GC poor genomes. Translational selection in determining the codon usage variation in highly expressed genes can be invoked from the predominant occurrences of C ending codons in the highly expressed genes. Cluster analysis based on codon usage data also shows that there are two distinct branches for the fourteen mycobacteriophages and there is codon usage variation even among the phages of each branch.

Bacteriophages↗
PubMed2001

Nuclear DNA content and base composition in 28 taxa of Musa.

The nuclear DNA content of 28 taxa of Musa was assessed by flow cytometry, using line PxPC6 of Petunia hybrida as an internal standard. The 2C DNA value of Musa balbisiana (BB genome) was 1.16 pg, whereas Musa acuminata (AA genome) had an average 2C DNA value of 1.27 pg, with a difference of 11% between its subspecies. The two haploid (IC) genomes, A and B, comprising most of the edible bananas, are therefore of similar size, 0.63 pg (610 million bp) and 0.58 pg (560 million bp), respectively. The genome of diploid Musa is thus threefold that of Arabidopsis thaliana. The genome sizes in a set of triploid Musa cultivars or clones were quite different, with 2C DNA values ranging from 1.61 to 2.23 pg. Likewise, the genome sizes of tetraploid cultivars ranged from 1.94 to 2.37 pg (2C). Apparently, tetraploids (for instance, accession I.C.2) can have a genome size that falls within the range of triploid genome sizes, and vice versa (as in the case of accession Simili Radjah). The 2C values estimated for organs such as leaf, leaf sheath, rhizome, and flower were consistent, whereas root material gave atypical results, owing to browning. The genomic base composition of these Musa taxa had a median value of 40.8% GC (SD = 0.43%).

Algorithms↗
PubMed2002

Global features of the Pseudomonas putida KT2440 genome sequence.

The compositional bias of the G+C, di- and tetranucleotide contents in the 6 181 862 bp Pseudomonas putida KT2440 genome was analysed in sliding windows of 4000 bp in steps of 1000 bp. The genome has a low GC skew (mean 0.066) between the leading and lagging strand. The values of GC contents (mean 61.6%) and of dinucleotide relative abundance exhibit skewed Gaussian distributions. The variance of tetranucleotide frequencies, which increases linearly with increasing GC content, shows two overlapping Gaussian distributions of genome sections with low (minor fraction) or high variance (major fraction). Eighty per cent of the chromosome shares similar GC contents and oligonucleotide bias, but 105 islands of 4000 bp or more show atypical GC contents and/or oligonucleotide signature. Almost all islands provide added value to the metabolic proficiency of P. putida as a saprophytic omnivore. Major features are the uptake and degradation of organic chemicals, ion transport and the synthesis and secretion of secondary metabolites. Other islands endow P. putida with determinants of resistance and defenceor with constituents and appendages of the cell wall. A total of 29 islands carry the signature of mobile elements such as phage, transposons, insertion sequence (IS) elements and group II introns, indicating recent acquisition by horizontal gene transfer. The largest gene carries the most unusual sequence that encodes a multirepeat threonine-rich surface adhesion protein. Among the housekeeping genes, only genes of the translational apparatus were located in segments with an atypical signature, suggesting that the synthesis of ribosomal proteins is uncoupled from the rapidly changing translational demands of the cell by the separate utilization of tRNA pools.

Base Composition↗
PubMed2003

Bacteriophage ST64B, a genetic mosaic of genes from diverse sources isolated from Salmonella enterica serovar typhimurium DT 64.

The complete sequence of the double-stranded DNA (dsDNA) genome of the Salmonella enterica serovar Typhimurium ST64B bacteriophage was determined. The 40,149-bp genomic sequence of ST64B has an overall G+C content of 51.3% and is distinct from that of P22. The genome architecture is similar to that of the lambdoid phages, particularly that of coliphage lambda. Most of the putative tail genes showed sequence similarity to tail genes of Mu, a nonlambdoid phage. In addition, it is likely that these tail genes are not expressed due to insertions of fragments of genes related to virulence within some of the open reading frames. This, together with the inability of ST64B to produce plaques on a wide range of isolates, suggests that ST64B is a defective phage. In contrast to the tail genes, most of the head genes showed similarity to those of the lambdoid phages HK97 and HK022, but these head genes also have significant sequence similarities to those of several other dsDNA phages infecting diverse bacterial hosts, including Escherichia, Pseudomonas, Agrobacterium, Caulobacter, Mesorhizobium, and Streptomyces: This suggests that ST64B is a genetic mosaic that has acquired significant portions of its genome from sources outside the genus Salmonella.

Bacteriophages↗
PubMedDate not supplied

Compositional constraints in the extremely GC-poor genome of Plasmodium falciparum.

We have analyzed the compositional properties of coding (protein encoding) and non-coding sequences of Plasmodium falciparum, a unicellular parasite characterized by an extremely AT-rich genome. GC% levels, base and dinucleotide frequencies were studied. We found that among the various factors that contribute to the properties of the sequences analyzed, the most relevant are the compositional constraints which operate on the whole genome.

Animals↗
PubMed2003

Metagenomic profiling: microarray analysis of an environmental genomic library.

Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms. However, conventional library-screening techniques permit characterization of relatively few environmental clones. Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities. A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing approximately 1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls. COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species. The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe. Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes. Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture. Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition. Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms.

Base Sequence↗
PubMed2004

Correlations between genomic GC levels and optimal growth temperatures are not 'robust'.

Musto et al. [FEBS Lett. 573 (2004) 73] studied the correlations between GC levels and optimal growth temperatures in 20 prokaryotic families. They reported that positive correlations are generally observed, and many of these are significant. Here, we have shown that these correlations are not "robust," i.e., correlation coefficients and/or significance of correlations can be considerably influenced by exclusion of very few (even as small as one) species from each dataset. The sensitivity of correlations is assumed as a result of high levels of bias in the family datasets. We concluded that solely based on these data, one cannot establish that GC contents of prokaryotic genomes increase as a result of growth temperature increments.

Adaptation, Physiological↗
PubMed1997

A survey of polypeptide deformylase function throughout the eubacterial lineage.

N-terminal formylation of ribosome-synthesized polypeptides is assumed to be among the most conserved features that distinguish the eubacterial line of descent from other living phyla. In order to assess the ancientness of this trait, def genes encoding polypeptide deformylase were characterized from four eubacterial species, Lactococcus lactis, Bacillus subtilis, Calothrix PCC7601 and Thermotoga maritima, taking advantage of the conditional viability of the def mutants of Escherichia coli. Altogether, eight sequences of polypeptide deformylase have been obtained from all the eubacterial sources which were investigated, either through systematic genome sequence analysis or through genetic screening, yielding a highly homologous family. A gene putatively encoding Met-tRNAi formyltransferase, fmt, was found downstream of the deformylase gene except in L. lactis, Mycoplasma genitalium, Calothrix PCC7601 and T. maritima. These results argue strongly for the ancestral character of N-terminal formylation in eubacteria. Most of the wide deviations of amino acid usage observed in def- and fmt-encoded proteins among species is best accounted for by the nucleotide composition of genomes. Furthermore, the species of origin of each protein appears to be more recognizable than its function, considering only its amino acid composition.

Amidohydrolases↗
PubMed2006

Cloning of environmental genomic fragments as physical markers for monitoring microbial populations in coking wastewater treatment system.

The association between community functional shift and dynamics of genomic DNA composition can be used to identify functionally relevant populations as indicator organisms for systems monitoring. In this work, fingerprinting-based community DNA hybridization was used to monitor community structural dynamics and identify genomic fragments whose abundance shifts were concomitant to changes in COD removal capacity in a reactor. A laboratory-scale anaerobic-anoxic-oxic fixed biofilm system treating coking wastewater was operated with (LR mode) or without effluent recirculation (LNR mode). The contribution to total chemical oxygen demand (COD) removal by the anoxic reactor increased from 4% in LNR mode to 26% in LR mode. Long primer RAPD (randomly amplified polymorphic DNA) community fingerprints of the anoxic reactor also changed most significantly from the one similar to the anaerobic reactor to one similar to the oxic reactor. DNA hybridization revealed one signature band of 2.1 kb shared by the anoxic and oxic reactors in LR, but not LNR mode. Clone library profiling of this band resulted in one predominant 2.1-kb genomic fragment (B3) with no homologous sequences in GenBank. Real-time polymerase chain reaction indicated that copy numbers of B3 in the anoxic reactor under LR mode were 69 times higher than that under LNR mode, concomitant to a significant increase in COD removal capacity in this reactor. The different patterns of distribution of B3 in the laboratory system and a comparable malfunctioning industrial system demonstrated the potential of this genomic fragment as physical markers in systems monitoring. In addition, this genomic fragment may allow sequence-guided isolation of the host microbe.

Bacteria↗
PubMedDate not supplied

[Localization of DNA sequences tightly associated with synaptonemal complex in compositional fractions of golden hamster].

The synaptonemal complex isolated from the spermatocyte nuclei by exhaustive hydrolysis of the latter by DNase II contains tightly associated DNA sequences (SCAR DNA). Here we studied the compositional properties of a cloned family of SCAR DNA of golden hamster, namely we performed the localization of 27 SCAR DNA clones on compositionally fractionated genomic DNA from golden hamster. We observed that sequences of the SCAR DNA family are mainly localized in the GC-poor isochore families L1 and L2, that showed 63% hybridization signals. This means that 37% of signals is referred to the GC-rich isochores, indicating the presence of SCAR DNA overall the genome, even if each isochore family presents differences in density and sequence type. Moreover, the SCAR DNA sequences containing regions of homology with LINE/SINE repeats were observed in all the isochore families. The compositional localization of SCAR DNA is in agreement with the hypothesis that SC and SCAR DNA participate in the chromatin organization during the meiosis prophase I, which should result in the attachment of chromatin loops to lateral elements of SC along the whole length of the latter.

Animals↗
PubMed2006

Differential genome contents of nontypeable Haemophilus influenzae strains from adults with chronic obstructive pulmonary disease.

Haemophilus influenzae is an important cause of otitis media in children and lower respiratory infection in adults with chronic obstructive pulmonary disease (COPD). Patients with COPD experience periodic exacerbations that are associated with acquisition of new bacterial strains. However, not every strain acquisition is associated with exacerbation. To test the hypothesis that genetic differences among strains account for differences in pathogenic potential, a microarray consisting of 4,992 random 1.5- to 3-kb genomic fragments of an exacerbation strain was constructed. Competitive hybridization was performed using six strains associated with exacerbation as well as five strains associated with asymptomatic colonization. Seven sequences that were absent in all five colonization strains and present in at least two exacerbation strains were identified. One such sequence was a previously unreported gene with high homology to the meningococcal immunoglobulin A (IgA) protease gene, which is distinct from the previously described H. influenzae IgA protease. To assess the distribution of the seven sequences among well-characterized strains of H. influenzae, 59 exacerbation strains and 73 asymptomatic colonization strains were screened by PCR for the presence of these sequences. The presence or absence of any single sequence was not significantly associated with exacerbations of COPD. However, logistic regression and subgroup analysis identified combinations of the presence and absence of genes that are associated with exacerbations. These results indicate that patterns of genes are associated with the ability of strains of H. influenzae to cause exacerbations of COPD, supporting the concept that differences in pathogenic potential are based in part on genomic differences among infecting strains, not merely host factors.

Adult↗
PubMed2004

Driving change: the evolution of alternative genetic codes.

Pioneering studies in the 1960s that elucidated the genetic code suggested that all extant forms of life use the same genetic code. This early presumption has subsequently been challenged by the discovery of deviations of the universal genetic code in prokaryotes, eukaryotic nuclear genomes and mitochondrial genomes. These studies have revealed that the genetic code is still evolving despite strong negative forces working against the fixation of mutations that result in codon reassignment. Recent data from in vitro, in vivo and in silico comparative genomics studies are revealing significant, previously overlooked links between modified nucleosides in tRNAs, genetic code ambiguity, genome base composition, codon usage and codon reassignment.

Animals↗
PubMed2001

M13 cloning of mung bean nuclease digested PCR fragments as a means of gap closure within A/T-rich, genome sequencing projects.

Obtaining the complete DNA sequence of a genome is often not straightforward. After standard shotgun sequencing strategies have been employed there are often gaps remaining and these can be the most intractable regions, frequently containing repeat sequences, "uncloneable" sequences and/or regions of potential secondary structure or differential base composition. In genomes with a high A/T content, such as Plasmodium falciparum and Dictyostelium discoideum, solving these gaps is a particularly difficult problem as the sequences concerned are "fragile" and easily denatured, commonly uncloneable and have a paucity of good oligonucleotide priming sites. Reported here is a simple, yet reliable method for determining the sequence of A/T-rich gap-spanning PCR products. This method relies on the slippage of the specificity of mung bean nuclease so that it digests A/T-rich double-stranded DNA into a set of deletion fragments that can then be cloned into M13, sequenced and the original sequence assembled therefrom.

AT Rich Sequence↗
PubMed1987

Mono- through hexanucleotide composition of the Escherichia coli genome: a Markov chain analysis.

Several statistical methods were tested for accuracy in predicting observed frequencies of di- through hexanucleotides in 74,444 bp of E. coli DNA. A Markov chain was most accurate overall, whereas other methods, including a random model based on mononucleotide frequencies, were very inaccurate. When ranked highest to lowest abundance, the observed frequencies of oligonucleotides up to six bases in length in E. coli DNA were highly asymmetric. All ordered abundance plots had a wide linear range containing the majority of the oligomers which deviated sharply at the high and low ends of the curves. In general, values predicted by a Markov chain closely followed the overall shape of the ordered abundance curves. A simple equation was derived by which the frequency of any nucleotide longer than four bases in the E. coli genome (or any genome) can be relatively accurately estimated from the nested set of component tri- and tetranucleotides by serial application of a 3rd order Markov chain. The equation yielded a mean ratio of 1.03 +/- 0.94 for the observed-to-expected frequencies of the 4,096 hexanucleotides. Hence, the method is a relatively accurate but not perfect predictor of the length in nucleotides between hexanucleotide sites. Higher accuracy can be achieved using a 4th order Markov chain and larger data sets. The high asymmetry in oligonucleotide abundance means that in the E. coli genome of 4.2 X 10(6) bp many relatively short sequences of 7-9 bp are very rare or absent.

Base Composition↗
PubMed2006

Involvement of DNA curvature in intergenic regions of prokaryotes.

It is known that DNA curvature plays a certain role in gene regulation. The distribution of curved DNA in promoter regions is evolutionarily preserved, and it is mainly determined by temperature of habitat. However, very little is known on the distribution of DNA curvature in termination sites. Our main objective was to comprehensively analyze distribution of curved sequences upstream and downstream to the coding genes in prokaryotic genomes. We applied CURVATURE software to 170 complete prokaryotic genomes in a search for possible typical distribution of DNA curvature around starts and ends of genes. Performing cluster analyses and other statistical tests, we obtained novel results regarding various factors influencing curvature distribution in intergenic regions, such as growth temperature, A+T composition and genome size. We also analyzed intergenic regions between converging genes in 15 selected genomes. The results show that six genomes presented peaks of curvature excess larger than 3 SDs. Insufficient statistics did not allow us to draw further conclusion. Our hypothesis is that DNA curvature could affect transcription termination in many prokaryotes either directly, through contacts with RNA polymerase, or indirectly, via contacts with some regulatory proteins.

Cluster Analysis↗
PubMed1992

Modeling of heteroduplex formation during PCR from mixtures of DNA templates.

We have investigated the capability of PCR to amplify a specific locus from each template in a mixture of allelic DNAs. Modeling experiments employed a 300-bp HOX2B segment as target and utilized, as distinct template "alleles," genomic DNAs from 1 human and from 2 chimpanzees known to differ in sequence at the target. Two modes were used: (1) mixtures of PCR products that had been previously amplified individually from a single template and (2) PCR amplification en masse from composite human/chimpanzee genomic DNA templates. Products generated by either mode were separated by denaturing gradient gel electrophoresis (DGGE). Detection of a "trace" allele mixed with a "dominant" one was possible by simple ethidium bromide staining of the gel up to a sensitivity of 1 part in 20. A balanced mixture was represented by 5:5 and 4:3:3 mixtures of allelic PCR products or genomic templates; an uneven mixture of dominant and trace alleles, by 9:1 and 8:1:1 mixtures. For 5:5, two homoduplex bands and two heteroduplex bands of equal intensity were generated. For 9:1, the trace homoduplex disappears whereas the two heteroduplexes are easily visible. For 8:1:1, four heteroduplexes and one homoduplex were observed; homoduplexes and heteroduplexes formed from the trace alleles were not visible. These experiments demonstrate that PCR can amplify mixed allelic templates in direct proportion to the stoichiometric fraction of each template. Trace species are captured as heteroduplexes with the most abundant species and are clearly displaced on denaturing gradient gels from the dominant homoduplex species. Our analytical studies can be applied to analysis of sequence variants in DNA collected from cancerous or infected tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗