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PubMed2004

Cellular engineering in a minimal microbe: structure and assembly of the terminal organelle of Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a minimal microbe with respect to cell envelope composition, biosynthetic and regulatory capabilities and genome size, yet it possesses a remarkably complex, multifunctional terminal organelle. This membrane-bound extension of the mycoplasma cell is defined by the presence of an electron-dense core that appears as paired, parallel bars oriented longitudinally and enlarging at the distal end to form a terminal button. Most non-cytadhering mutants of M. pneumoniae isolated to date exhibit defects in the architecture of the terminal organelle. Detailed characterization of those mutants has revealed the identities of many component proteins of the terminal organelle as well as the likely order in which some of those components are required. Additional questions regarding the composition of the electron-dense core, the means by which the terminal organelle is duplicated during cell division and the manner in which this process is regulated remain to be answered. Thus, it seems that there is much to be learned about cellular engineering and spatial regulation in these 'simple' cell wall-less bacteria.

Adhesins, Bacterial↗
PubMed2006

Strand bias in complementary single-nucleotide polymorphisms of transcribed human sequences: evidence for functional effects of synonymous polymorphisms.

BACKGROUND: Complementary single-nucleotide polymorphisms (SNPs) may not be distributed equally between two DNA strands if the strands are functionally distinct, such as in transcribed genes. In introns, an excess of A<-->G over the complementary C<-->T substitutions had previously been found and attributed to transcription-coupled repair (TCR), demonstrating the valuable functional clues that can be obtained by studying such asymmetry. Here we studied asymmetry of human synonymous SNPs (sSNPs) in the fourfold degenerate (FFD) sites as compared to intronic SNPs (iSNPs). RESULTS: The identities of the ancestral bases and the direction of mutations were inferred from human-chimpanzee genomic alignment. After correction for background nucleotide composition, excess of A-->G over the complementary T-->C polymorphisms, which was observed previously and can be explained by TCR, was confirmed in FFD SNPs and iSNPs. However, when SNPs were separately examined according to whether they mapped to a CpG dinucleotide or not, an excess of C-->T over G-->A polymorphisms was found in non-CpG site FFD SNPs but was absent from iSNPs and CpG site FFD SNPs. CONCLUSION: The genome-wide discrepancy of human FFD SNPs provides novel evidence for widespread selective pressure due to functional effects of sSNPs. The similar asymmetry pattern of FFD SNPs and iSNPs that map to a CpG can be explained by transcription-coupled mechanisms, including TCR and transcription-coupled mutation. Because of the hypermutability of CpG sites, more CpG site FFD SNPs are relatively younger and have confronted less selection effect than non-CpG FFD SNPs, which can explain the asymmetric discrepancy of CpG site FFD SNPs vs. non-CpG site FFD SNPs.

Algorithms↗
PubMed2004

Distribution of HIV-1 in the genomes of AIDS patients.

The localization of HIV-1 proviruses in compositional DNA fractions from 27 AIDS patients during the chronic phase of the disease with depletion of CD4+ and different levels of viremia showed the following. (1) At low viremia, proviruses are predominantly localized in the GC-richest isochores, which are characterized by an open chromatin structure; this result mimics findings on HIV-1 integration in early infected cells in culture. (2) At higher viremia, an increased distribution of proviruses in GC-poor isochores (which match the GC poorness of HIV-1) was found; this suggests a selection of cells in which the 'isopycnic' localization leads to a higher expression of proviruses and, in turn, to higher viremia. (3) At the highest viremia, integrations in GC-rich isochores are often predominant again, but generally not at the same level as in (1); this may be the consequence of new integrations from the extremely abundant RNA copies.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Genetic variation in trophectoderm function in parthenogenetic mouse embryos.

The developing oocyte constitutes the source of a unique and essential molecular legacy that supports embryo metabolism for a substantial period after fertilization and that also directs important epigenetic events that prepare the embryonic genome for transcription and faithful execution of the developmental program. Parthenogenetically activated embryos provide a useful tool with which to examine how maternally derived factors contribute to early development. They also provide a means for evaluating genetic effects on the maternal genomic imprinting process. We report here that the genetic background of the oocyte affects trophectoderm function at the blastocyst stage. Parthenogenetic embryos obtained from activated (B6D2)F1 oocytes hatch efficiently in culture, whereas parthenogenones from C57BL/6 oocytes hatch less efficiently. Fertilized embryos of both strains hatch efficiently. The (B6D2)F1 parthenogenones also undergo blastocoel re-expansion after treatment with cytoskeletal inhibitors more rapidly than do C57BL/6 parthenogenones and exhibit a moderately greater abundance of the Na+, K(+)-ATPase alpha 1 subunit mRNA. Surprisingly, parthenogenones of both strains undergo blastocoel re-expansion more rapidly than do their normal fertilized counterparts. Parthenogenones of both types are able to attach efficiently in culture after removal of the zona pellucida. These observations indicate that significant genetic effects of maternal genotype on trophectoderm function are revealed in the absence of a paternal genetic contribution and that trophectoderm function also differs between parthenogenetic embryos and fertilized embryos. The differences observed between parthenogenetic and fertilized embryos indicate a likely role for one or more imprinted genes in the development of hatching and blastocoel expansion ability. The effect of maternal genotype on parthenogenetic embryo phenotype is consistent with possible differences in maternal genome imprinting or differences in ooplasm composition that have long-term effects on development. The specific differences in hatching and blastocoel re-expansion between parthenogenones of the two strains may be the result of differences in the activity or expression of a hatching enzyme or other molecules that affect fluid accumulation within the blastocyst, such as components of junctional complexes or proteins that regulate Na+, K(+)-ATPase activity.

Animals↗
PubMed2005

Microsatellite megatracts in the maize (Zea mays L.) genome.

Long tracts (megatracts) of (CAG)n, (TAG)n, and (GAA)n microsatellite sequences capable of forming composite DNA segments were found in the maize (Zea mays L.) genome. Some of the (CAG)n and (TAG)n megatracts were organized in clusters of up to 1 Mb on several chromosomes, as detected by fluorescence in situ hybridization (FISH), as well as on extended DNA fibers. Extensive polymorphism was found among different maize inbred lines with respect to the number and size of microsatellite megatract clusters on the A chromosomes. Polymorphism was also common among B chromosomes of different nuclei in the inbred line Zapalote Chico. Different retrotransposable elements were often inserted into the microsatellite tracts. Size variation in some (TAG)n and (GAA)n megatracts was observed in consecutive generations among siblings of the inbred lines, indicating that these loci are highly unstable and predisposed to dynamic mutations similar to those described in mammalian systems.

Blotting, Southern↗
PubMed1993

Similarity of the 5' and 3'-TAR secondary structures in HIV-1.

The structure of the TAR RNA element transcribed at the 5' end of the R region of the human immunodeficiency virus is compared to the structure of its duplicate sequence in the 3' R region of the viral genome. Based on the 5'-TAR secondary structure already described, we assessed by RNase T1 primer extension assay the degree of similarity between the 5'-TAR and the 3'-TAR RNA secondary structures. We also analysed the influence of modifications in the flanking sequences. We show that the secondary structures of the 5'-TAR and the 3'-TAR are very similar and are not influenced by the flanking sequences.

Base Composition↗
PubMed1998

Dipodascus capitatus, Dipodascus spicifer and Geotrichum clavatum: genomic characterization.

The G + C contents of 25 strains of Dipodascus capitatus, Dipodascus spicifer and Geotrichum clavatum were found to be heterogeneous on basis of derivative graphs of the melting profiles. Strains showing similar derivative graphs of the melting curve exhibited high levels of DNA homology (80-100%); strains showing dissimilar derivative graphs exhibited low levels of DNA homology (5 to 45%). Being considered separate taxa on basis of these parameters, D. capitatus, D. spicifer and G. clavatum could be identified by a combination of the key characteristics growth on xylose, cellobiose, salicin and arbutin.

Base Composition↗
PubMed2006

Prediction of whole-genome DNA-DNA similarity, determination of G+C content and phylogenetic analysis within the family Pasteurellaceae by multilocus sequence analysis (MLSA).

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies, including DNA-DNA similarity, G+C content and representative phylogeny of bacteria. At present, DNA-DNA hybridizations are still considered the gold standard in species descriptions. However, this method is time-consuming and troublesome, and datasets can vary significantly between experiments as well as between laboratories. For the same reasons, full matrix hybridizations are rarely performed, weakening the significance of the results obtained. The authors established a universal sequencing approach for the three genes recN, rpoA and thdF for the Pasteurellaceae, and determined if the sequences could be used for predicting DNA-DNA relatedness within the family. The sequence-based similarity values calculated using a previously published formula proved most useful for species and genus separation, indicating that this method provides better resolution and no experimental variation compared to hybridization. By this method, cross-comparisons within the family over species and genus borders easily become possible. The three genes also serve as an indicator of the genome G+C content of a species. A mean divergence of around 1 % was observed from the classical method, which in itself has poor reproducibility. Finally, the three genes can be used alone or in combination with already-established 16S rRNA, rpoB and infB gene-sequencing strategies in a multisequence-based phylogeny for the family Pasteurellaceae. It is proposed to use the three sequences as a taxonomic tool, replacing DNA-DNA hybridization.

Base Composition↗
PubMed1997

Amplification and fusion of long fragments of hepatitis C virus genome.

The 'long PCR' was used for amplification of hepatitis C virus (HCV) subgenomic fragments from liver. After testing several commercially available systems, it was found that Tth as the major enzyme is superior to using Taq. Employing a mixture of Tth and Vent polymerase (rTth polymerase, XL, Perkin Elmer) it was possible to amplify 4.6-kb and 9-kb fragments from biological samples containing as little as 10(2) and 10(4) viral copies, respectively. It was also demonstrated that 'long PCR' is useful for joining together large size amplification products.

Artificial Gene Fusion↗
PubMed2006

Strict host-symbiont cospeciation and reductive genome evolution in insect gut bacteria.

Host-symbiont cospeciation and reductive genome evolution have been identified in obligate endocellular insect symbionts, but no such example has been identified from extracellular ones. Here we first report such a case in stinkbugs of the family Plataspidae, wherein a specific gut bacterium is vertically transmitted via "symbiont capsule." In all of the plataspid species, females produced symbiont capsules upon oviposition and their gut exhibited specialized traits for capsule production. Phylogenetic analysis showed that the plataspid symbionts constituted a distinct group in the gamma-Proteobacteria, whose sister group was the aphid obligate endocellular symbionts Buchnera. Removal of the symbionts resulted in retarded growth, mortality, and sterility of the insects. The host phylogeny perfectly agreed with the symbiont phylogeny, indicating strict host-symbiont cospeciation despite the extracellular association. The symbionts exhibited AT-biased nucleotide composition, accelerated molecular evolution, and reduced genome size, as has been observed in obligate endocellular insect symbionts. These findings suggest that not the endocellular conditions themselves but the population genetic attributes of the vertically transmitted symbionts are probably responsible for the peculiar genetic traits of these insect symbionts. We proposed the designation "Candidatus Ishikawaella capsulata" for the plataspid symbionts. The plataspid stinkbugs, wherein the host-symbiont associations can be easily manipulated, provide a novel system that enables experimental approaches to previously untouched aspects of the insect-microbe mutualism. Furthermore, comparative analyses of the sister groups, the endocellular Buchnera and the extracellular Ishikawaella, would lead to insights into how the different symbiotic lifestyles have affected their genomic evolution.

Animals↗
PubMed1999

Biased amino acid composition in repeat regions of Plasmodium antigens.

Many malarial antigens contain extensive arrays of tandemly repeated short amino acid sequences, and much of the antibody response induced by malaria infections is directed against these repeats. Indeed, it has been hypothesized that these repeats function to elicit a relatively ineffective T-cell-independent antibody response by the host. In order to test this hypothesis, tandem repeats of Plasmodium species were examined for a bias in composition favoring amino acids likely to form epitopes for the antibody. The genome of Plasmodium is very A+T-rich, and nucleotide compositional bias will, in itself, lead to a high proportion of hydrophilic amino acids. When this bias was controlled for, Plasmodium antigens did not show a higher proportion of hydrophilic amino acids than expected, but there was a significant reduction in the proportion of hydrophobic amino acids in the repeats of the antigens. The amino acid composition of the repeats was thus strikingly different from those seen both in the remainder of the antigens and in a sample of Plasmodium falciparum housekeeping genes.

Amino Acid Sequence↗
PubMed1998

Genome size and GC-percent in vertebrates as determined by flow cytometry: the triangular relationship.

Genome size and GC-percent were determined by means of a special method of DNA flow cytometry in 154 vertebrate species. For the total dataset, a highly significant positive correlation was found between both parameters. The overall distribution of points is not linear but triangular: a wide range of GC-percent values is observed at the lower end of genome size range, whereas with an increase in genome size the lower limit for GC-percent is elevated, gradually approaching the upper limit (about 46%). In teleost fishes, which occupy the lower part of genome size range, the negative relationship between both parameters was observed. Two positive linear relationships were found between mean genome size and GC-percent of the main vertebrate groups (one includes fishes, amphibians, and mammals, the other consists of reptiles and birds, which show the higher GC-percent for their genome sizes). Distribution of variance between taxonomic levels indicates that GC-percent is more evolutionarily conservative than genome size in anamniotes. Anuran amphibians show the greatest part of genome size variability at the lower taxonomic levels as compared to other vertebrates (with no additional variance already above the genus level). The data obtained with different methods are compared. It is shown that the proposed method can provide useful data for studies on genome evolution and biodiversity.

Animals↗
PubMed2003

An isochore map of the human genome based on the Z curve method.

The distribution of the G+C content in the human genome has been studied by using a windowless technique derived from the Z curve method. The most important findings presented in this paper are twofold. First, abrupt variations of the G+C content along human chromosome sequences are the main variation patterns of G+C content. It is found that at some sites, the G+C content undergoes abrupt changes from a G+C-rich region to a G+C-poor region alternatively and vice versa. Second, it is shown that long domains with relatively homogeneous G+C content along each chromosome do exist. These domains are thought to be isochores, which usually have sharp boundaries. Consequently, 56 isochores longer than 3 Mb have been identified in chromosomes 1-22, X and Y. Boundaries, size and G+C content of each isochore identified are listed in detail. As an example to demonstrate the power of the method, the boundary between the Classes III and II isochores of the MHC sequence has been determined and found to be at 2,477,936, which is in good agreement with the experimental evidence. A homogeneity index is introduced to measure the homogeneity of G+C content in isochores. We emphasize that the homogeneity of G+C content is relative. The isochores in which the G+C content keeps absolutely constant do not exist. Isochore structures appear to be a basic organization of the human genome. Due to the relevance to many important biological functions, the clarification of isochore structures will provide much insight into the understanding of the human genome.

Algorithms↗
PubMed2003

A survey of small RNA-encoding genes in Escherichia coli.

Small RNA (sRNA) molecules have gained much interest lately, as recent genome-wide studies have shown that they are widespread in a variety of organisms. The relatively small family of 10 known sRNA-encoding genes in Escherichia coli has been significantly expanded during the past two years with the discovery of 45 novel genes. Most of these genes are still uncharacterized and their cellular roles are unknown. In this survey we examined the sequence and genomic features of the 55 currently known sRNA-encoding genes in E.coli, attempting to identify their common characteristics. Such characterization is important for both expanding our understanding of this unique gene family and for improving the methods to predict and identify sRNA-encoding genes based on genomic information.

Base Composition↗
PubMed2006

The genome sequence of Methanosphaera stadtmanae reveals why this human intestinal archaeon is restricted to methanol and H2 for methane formation and ATP synthesis.

Methanosphaera stadtmanae has the most restricted energy metabolism of all methanogenic archaea. This human intestinal inhabitant can generate methane only by reduction of methanol with H2 and is dependent on acetate as a carbon source. We report here the genome sequence of M. stadtmanae, which was found to be composed of 1,767,403 bp with an average G+C content of 28% and to harbor only 1,534 protein-encoding sequences (CDS). The genome lacks 37 CDS present in the genomes of all other methanogens. Among these are the CDS for synthesis of molybdopterin and for synthesis of the carbon monoxide dehydrogenase/acetyl-coenzyme A synthase complex, which explains why M. stadtmanae cannot reduce CO2 to methane or oxidize methanol to CO2 and why this archaeon is dependent on acetate for biosynthesis of cell components. Four sets of mtaABC genes coding for methanol:coenzyme M methyltransferases were found in the genome of M. stadtmanae. These genes exhibit homology to mta genes previously identified in Methanosarcina species. The M. stadtmanae genome also contains at least 323 CDS not present in the genomes of all other archaea. Seventy-three of these CDS exhibit high levels of homology to CDS in genomes of bacteria and eukaryotes. These 73 CDS include 12 CDS which are unusually long (>2,400 bp) with conspicuous repetitive sequence elements, 13 CDS which exhibit sequence similarity on the protein level to CDS encoding enzymes involved in the biosynthesis of cell surface antigens in bacteria, and 5 CDS which exhibit sequence similarity to the subunits of bacterial type I and III restriction-modification systems.

Adenosine Triphosphate↗
PubMed2003

Transcription-coupled TA and GC strand asymmetries in the human genome.

Analysis of the whole set of human genes reveals that most of them present TA and GC skews, that these biases are correlated to each other and are specific to gene sequences, exhibiting sharp transitions between transcribed and non-transcribed regions. The GC asymmetries cannot be explained solely by a model previously proposed for (G+T) skew based on transitions measured in a small set of human genes. We propose that the GC skew results from additional transcription-coupled mutation process that would include transversions. During evolution, both processes acting on a large majority of genes in germline cells would have produced these transcription-coupled strand asymmetries.

Base Composition↗
PubMed1999

Strand asymmetry and codon usage bias in the chloroplast genome of Euglena gracilis.

It is shown that the two strands of the chloroplast genome from Euglena gracilis are asymmetric with regards to nucleotide composition. This asymmetry switches at both the origin of replication and a location that is halfway around the circular genome from the origin. In both halves of the genome the leading strand is G+T-rich, having a bias toward G over C and T over A, and the lagging strand is A+C-rich. This asymmetry is probably the result of a difference in mutation dynamics between the leading and lagging strands. In addition to composition asymmetry, the two strands differ with regards to coding content. In both halves of the genome the vast majority of genes are coded by the leading strand. These two aspects of strand asymmetry are then applied to a statistical test for selection on codon usage. The results indicate that selection on codon usage is limited to genes on the leading strand; no gene on the A+C-rich lagging strand shows evidence for selection, suggesting that highly expressed genes are coded predominantly on the strand of DNA that is the leading strand during replication. On the basis of these observations it is proposed that the coding strand bias is generated by selection to code highly expressed genes on the leading strand to coordinate the direction of replication and transcription, thereby increasing the potential rate of both reactions.

Animals↗
PubMed2006

First exons and introns--a survey of GC content and gene structure in the human genome.

Most transcriptional regulatory elements are located in non-coding DNA. In particular, some first introns play a vital role in transcriptional control and splicing. The length and GC-content of first exons and introns in complex organisms suggests that these structural units are likely to be important functional elements in large genomes. Hence, in this paper we perform a systematic comparison of exon-intron structure and GC content on all known genes in the human genome. Our in-silico analysis found that the GC content of introns and exons varies significantly depending on their length. On average, the first intron of a gene is significantly longer than other introns in the same gene. Our results also show that first introns and exons are more GC rich than last and internal. This study provides insight into the structure of eukaryotic genes. These results confirm and expand the previously identified regulatory potential of first exons and introns.

Base Composition↗