Elevated GC Content Research Articles

Characterization of single-cell cis-regulatory elements informs implications for cell differentiation.

Cis-regulatory elements (CREs) govern the specific patterns and dynamics of gene expression in cells during development, which are the fundamental mechanisms behind cell differentiation. However, the genomic characteristics of single-cell CREs (scCREs) closely linked to cell differentiation during development remain unclear. To explore this, we systematically analyzed ∼250,000 putative scCREs obtained from snATAC-seq analysis of the developing mouse cerebellum. We found that over 80% of these scCREs show pleiotropic effects, being active in two or more cell types. The pleiotropic degrees of proximal and distal scCREs are positively correlated with the density and diversity of transcription factor (TF) binding motifs and GC content. There is a negative correlation between the pleiotropic degrees of scCREs and their distances to the nearest TSSs, and proximal scCREs display higher relevance strengths than distal ones. Furthermore, both proximal and distal scCREs related to cell differentiation exhibit enhanced sequence-level evolutionary conservation, increased density and diversity of TF binding motifs, elevated GC content, and greater distances from their nearest genes. Together, our findings reveal the general genomic characteristics of putative scCREs and provide insights into the genomic and evolutionary mechanisms by which scCREs regulate cell differentiation during development.

Plasma cell-free DNA promise monitoring and tissue injury assessment of COVID-19

Coronavirus 2019 (COVID-19) is a complex disease that affects billions of people worldwide. Currently, effective etiological treatment of COVID-19 is still lacking; COVID-19 also causes damages to various organs that affects therapeutics and mortality of the patients. Surveillance of the treatment responses and organ injury assessment of COVID-19 patients are of high clinical value. In this study, we investigated the characteristic fragmentation patterns and explored the potential in tissue injury assessment of plasma cell-free DNA in COVID-19 patients. Through recruitment of 37 COVID-19 patients, 32 controls and analysis of 208 blood samples upon diagnosis and during treatment, we report gross abnormalities in cfDNA of COVID-19 patients, including elevated GC content, altered molecule size and end motif patterns. More importantly, such cfDNA fragmentation characteristics reflect patient-specific physiological changes during treatment. Further analysis on cfDNA tissue-of-origin tracing reveals frequent tissue injuries in COVID-19 patients, which is supported by clinical diagnoses. Hence, our work demonstrates and extends the translational merit of cfDNA fragmentation pattern as valuable analyte for effective treatment monitoring, as well as tissue injury assessment in COVID-19.

Shared properties of gene transfer agent and core genes revealed by comparative genomics of Alphaproteobacteria.

Gene transfer agents (GTAs) are phage-like particles that transfer pieces of cellular genomic DNA to other cells. Homologues of the Rhodobacter capsulatus GTA (RcGTA) structural genes are widely distributed in the Alphaproteobacteria and particularly well conserved in the order Rhodobacterales. Possible reasons for their widespread conservation are still being discussed. It has been suggested that these alphaproteobacterial elements originate from a prophage that was present in an ancestral bacterium and subsequently evolved into a GTA that is now widely maintained in extant descendant lineages. Here, we analysed genomic properties that might relate to the conservation of these alphaproteobacterial GTAs. This revealed that the chromosomal locations of the GTA gene clusters are biased. They primarily occur on the leading strand of DNA replication, at large distances from long repetitive elements, and thus are in regions of lower plasticity, and in areas of extreme GC skew, which also accumulate core genes. These extreme GC skew regions arise from the preferential use of codons with an excess of G over C, a distinct phenomenon from the elevated GC content that has previously been found to be associated with GTA genes. The observed properties, along with their high level of conservation, show that GTA genes share multiple features with core genes in the examined lineages of the Alphaproteobacteria.

Comparative analysis reveals within-population genome size variation in a rotifer is driven by large genomic elements with highly abundant satellite DNA repeat elements

BackgroundEukaryotic genomes are known to display an enormous variation in size, but the evolutionary causes of this phenomenon are still poorly understood. To obtain mechanistic insights into such variation, previous studies have often employed comparative genomics approaches involving closely related species or geographically isolated populations within a species. Genome comparisons among individuals of the same population remained so far understudied—despite their great potential in providing a microevolutionary perspective to genome size evolution. The rotifer Brachionus asplanchnoidis represents one of the most extreme cases of within-population genome size variation among eukaryotes, displaying almost twofold variation within a geographic population.ResultsHere, we used a whole-genome sequencing approach to identify the underlying DNA sequence differences by assembling a high-quality reference genome draft for one individual of the population and aligning short reads of 15 individuals from the same geographic population including the reference individual. We identified several large, contiguous copy number variable regions (CNVs), up to megabases in size, which exhibited striking coverage differences among individuals, and whose coverage overall scaled with genome size. CNVs were of remarkably low complexity, being mainly composed of tandemly repeated satellite DNA with only a few interspersed genes or other sequences, and were characterized by a significantly elevated GC-content. CNV patterns in offspring of two parents with divergent genome size and CNV patterns in several individuals from an inbred line differing in genome size demonstrated inheritance and accumulation of CNVs across generations.ConclusionsBy identifying the exact genomic elements that cause within-population genome size variation, our study paves the way for studying genome size evolution in contemporary populations rather than inferring patterns and processes a posteriori from species comparisons.

GC-biased gene conversion in X-chromosome palindromes conserved in human, chimpanzee, and rhesus macaque.

Gene conversion is GC-biased across a wide range of taxa. Large palindromes on mammalian sex chromosomes undergo frequent gene conversion that maintains arm-to-arm sequence identity greater than 99%, which may increase their susceptibility to the effects of GC-biased gene conversion. Here, we demonstrate a striking history of GC-biased gene conversion in 12 palindromes conserved on the X chromosomes of human, chimpanzee, and rhesus macaque. Primate X-chromosome palindrome arms have significantly higher GC content than flanking single-copy sequences. Nucleotide replacements that occurred in human and chimpanzee palindrome arms over the past 7 million years are one-and-a-half times as GC-rich as the ancestral bases they replaced. Using simulations, we show that our observed pattern of nucleotide replacements is consistent with GC-biased gene conversion with a magnitude of 70%, similar to previously reported values based on analyses of human meioses. However, GC-biased gene conversion since the divergence of human and rhesus macaque explains only a fraction of the observed difference in GC content between palindrome arms and flanking sequence, suggesting that palindromes are older than 29 million years and/or had elevated GC content at the time of their formation. This work supports a greater than 2:1 preference for GC bases over AT bases during gene conversion and demonstrates that the evolution and composition of mammalian sex chromosome palindromes is strongly influenced by GC-biased gene conversion.

A database of sequenced genomes of different Streptomyces albus J1074 strains and uses thereof

Streptomyces albus J1074 has recently emerged as a powerful chassis strain for natural product research and discovery, as well as model to investigate various aspects of actinobacterial biology. A number of genetic tools have been developed to facilitate the use of this strain for the aforementioned purposes. One of the promising approaches is to introduce into J1074 genome mutations that would improve the antibiotic-producing capacity of J1074. Particularly, we reported a collection of spontaneous and genomically engineered J1074 mutants carrying mutation in genes for ribosomal protein S12, RNA polymerase beta subunit etc. We took advantage of this collection to build an in-house database which would host (both current and future) the primary and curated sequencing data for these mutants. The database is available at https://biotools.online/media/. The main benefits of the database lie in the known pedigree of the strains, which allows deep interpretation of the data. For example, there is lively – and unresolved – debate on the origins and consequences of the GC composition in actinobacteria. A better understanding of this issue should improve our knowledge of genome evolution in bacteria and will have a number of biotechnological ramifications. We used our Streptomyces albus J1074 dataset as an experimental model to reveal genome-wide spectrum of mutation, which appears to be biased towards elevated GC content. We also included the high-quality genomes of the other streptomycetes into our databse for comparative purposes. The genomic GC content in streptomycetes varies from 75 % to 66.5 %, with median value being 72 %. The GC content of secondary metabolic genes of S. coelicolor is less variable than that of primary metabolic genes, an indicative of different selection pressure on these gene groups. Along with selective constraints, the peculiarities of Streptomyces DNA mismatch repair might contribute to the skewed GC content of their genomes. Further uses of the database may include the development of a more precise knowledge of the mutation rate as well as population genetic processes within this species and genus.

Comparative Chloroplast Genomics of Corydalis Species (Papaveraceae): Evolutionary Perspectives on Their Unusual Large Scale Rearrangements.

The chloroplast genome (plastome) of angiosperms (particularly photosynthetic members) is generally highly conserved, although structural rearrangements have been reported in a few lineages. In this study, we revealed Corydalis to be another unusual lineage with extensive large-scale plastome rearrangements. In the four newly sequenced Corydalis plastomes that represent all the three subgenera of Corydalis, we detected (1) two independent relocations of the same five genes (trnV-UAC-rbcL) from the typically posterior part of the large single-copy (LSC) region to the front, downstream of either the atpH gene in Corydalis saxicola or the trnK-UUU gene in both Corydalis davidii and Corydalis hsiaowutaishanensis; (2) relocation of the rps16 gene from the LSC region to the inverted repeat (IR) region in Corydalis adunca; (3) uniform inversion of an 11–14 kb segment (ndhB-trnR-ACG) in the IR region of all the four Corydalis species (the same below); (4) expansions (>10 kb) of IR into the small single-copy (SSC) region and corresponding contractions of SSC region; and (5) extensive pseudogenizations or losses of 13 genes (accD, clpP, and 11 ndh genes). In addition, we also found that the four Corydalis plastomes exhibited elevated GC content in both gene and intergenic regions and high number of dispersed repeats. Phylogenomic analyses generated a well-supported topology that was consistent with the result of previous studies based on a few DNA markers but contradicted with the morphological character-based taxonomy to some extent. This study provided insights into the evolution of plastomes throughout the three Corydalis subgenera and will be of value for further study on taxonomy, phylogeny, and evolution of Corydalis.

Genetic innovations: Transposable element recruitment and de novo formation lead to the birth of orphan genes in the rice genome

AbstractOrphan genes are genetic innovations that lack homologs in other lineages. Orphan genes can rapidly originate and become substantially functional, yet the mechanisms underlying their origins are still largely unknown in plants. Here, we investigated the origin of orphan genes in the Oryza sativa ssp. japonica “Nipponbare” genome using genome‐wide comparisons with 10 closely related Oryza species. We identified a total of 37 orphan genes in the Nipponbare genome that show short sequence lengths, elevated GC content, and absence of introns. Interestingly, half of the identified orphan genes originated by way of a distinctive mechanism that involved the generation of new coding sequences through independent and rapid divergence within the inserted transposable element. Our results provide valuable insight into genetic innovations in the model rice genome that formed on a very short timescale.

Codon selection reduces GC content bias in nucleic acids encoding for intrinsically disordered proteins.

Protein-coding nucleic acids exhibit composition and codon biases between sequences coding for intrinsically disordered regions (IDRs) and those coding for structured regions. IDRs are regions of proteins that are folding self-insufficient and which function without the prerequisite of folded structure. Several authors have investigated composition bias or codon selection in regions encoding for IDRs, primarily in Eukaryota, and concluded that elevated GC content is the result of the biased amino acid composition of IDRs. We substantively extend previous work by examining GC content in regions encoding IDRs, from 44 species in Eukaryota, Archaea, and Bacteria, spanning a wide range of GC content. We confirm that regions coding for IDRs show a significantly elevated GC content, even across all domains of life. Although this is largely attributable to the amino acid composition bias of IDRs, we show that this bias is independent of the overall GC content and, most importantly, we are the first to observe that GC content bias in IDRs is significantly different than expected from IDR amino acid composition alone. We empirically find compensatory codon selection that reduces the observed GC content bias in IDRs. This selection is dependent on the overall GC content of the organism. The codon selection bias manifests as use of infrequent, AT-rich codons in encoding IDRs. Further, we find these relationships to be independent of the intrinsic disorder prediction method used, and independent of estimated translation efficiency. These observations are consistent with the previous work, and we speculate on whether the observed biases are causal or symptomatic of other driving forces.

De Novo Assembly of Two Swedish Genomes Reveals Missing Segments from the Human GRCh38 Reference and Improves Variant Calling of Population-Scale Sequencing Data

The current human reference sequence (GRCh38) is a foundation for large-scale sequencing projects. However, recent studies have suggested that GRCh38 may be incomplete and give a suboptimal representation of specific population groups. Here, we performed a de novo assembly of two Swedish genomes that revealed over 10 Mb of sequences absent from the human GRCh38 reference in each individual. Around 6 Mb of these novel sequences (NS) are shared with a Chinese personal genome. The NS are highly repetitive, have an elevated GC-content, and are primarily located in centromeric or telomeric regions. Up to 1 Mb of NS can be assigned to chromosome Y, and large segments are also missing from GRCh38 at chromosomes 14, 17, and 21. Inclusion of NS into the GRCh38 reference radically improves the alignment and variant calling from short-read whole-genome sequencing data at several genomic loci. A re-analysis of a Swedish population-scale sequencing project yields > 75,000 putative novel single nucleotide variants (SNVs) and removes > 10,000 false positive SNV calls per individual, some of which are located in protein coding regions. Our results highlight that the GRCh38 reference is not yet complete and demonstrate that personal genome assemblies from local populations can improve the analysis of short-read whole-genome sequencing data.

Genes Translocated into the Plastid Inverted Repeat Show Decelerated Substitution Rates and Elevated GC Content.

Plant chloroplast genomes (plastomes) are characterized by an inverted repeat (IR) region and two larger single copy (SC) regions. Patterns of molecular evolution in the IR and SC regions differ, most notably by a reduced rate of nucleotide substitution in the IR compared to the SC region. In addition, the organization and structure of plastomes is fluid, and rearrangements through time have repeatedly shuffled genes into and out of the IR, providing recurrent natural experiments on how chloroplast genome structure can impact rates and patterns of molecular evolution. Here we examine four loci (psbA, ycf2, rps7, and rps12 exon 2–3) that were translocated from the SC into the IR during fern evolution. We use a model-based method, within a phylogenetic context, to test for substitution rate shifts. All four loci show a significant, 2- to 3-fold deceleration in their substitution rate following translocation into the IR, a phenomenon not observed in any other, nontranslocated plastid genes. Also, we show that after translocation, the GC content of the third codon position and of the noncoding regions is significantly increased, implying that gene conversion within the IR is GC-biased. Taken together, our results suggest that the IR region not only reduces substitution rates, but also impacts nucleotide composition. This finding highlights a potential vulnerability of correlating substitution rate heterogeneity with organismal life history traits without knowledge of the underlying genome structure.

Side Effects: Substantial Non-Neutral Evolution Flanking Regulatory Sites

Side Effects: Substantial Non-Neutral Evolution Flanking Regulatory Sites

Sequence-Specific Targeting of Dosage Compensation in Drosophila Favors an Active Chromatin Context

The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at “entry sites” that contain a consensus sequence motif (“MSL recognition element” or MRE). However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome.

The mitochondrial genome of the arbuscular mycorrhizal fungus Gigaspora margarita reveals two unsuspected trans-splicing events of group I introns.

• Arbuscular mycorrhizal fungi (AMF) are ubiquitous organisms that benefit ecosystems through the establishment of an association with the roots of most plants: the mycorrhizal symbiosis. Despite their ecological importance, however, these fungi have been poorly studied at the genome level. • In this study, total DNA from the AMF Gigaspora margarita was subjected to a combination of 454 and Illumina sequencing, and the resulting reads were used to assemble its mitochondrial genome de novo. This genome was annotated and compared with those of other relatives to better comprehend the evolution of the AMF lineage. • The mitochondrial genome of G. margarita is unique in many ways, exhibiting a large size (97 kbp) and elevated GC content (45%). This genome also harbors molecular events that were previously unknown to occur in fungal mitochondrial genomes, including trans-splicing of group I introns from two different genes coding for the first subunit of the cytochrome oxidase and for the small subunit of the rRNA. • This study reports the second published genome from an AMF organelle, resulting in relevant DNA sequence information from this poorly studied fungal group, and providing new insights into the frequency, origin and evolution of trans-spliced group I introns found across the mitochondrial genomes of distantly related organisms.

In Silico Prediction of Evolutionarily Conserved GC-Rich Elements Associated with Antigenic Proteins of Plasmodium falciparum

The Plasmodium falciparum genome being AT-rich, the presence of GC-rich regions suggests functional significance. Evolution imposes selection pressure to retain functionally important coding and regulatory elements. Hence searching for evolutionarily conserved GC-rich, intergenic regions in an AT-rich genome will help in discovering new coding regions and regulatory elements. We have used elevated GC content in intergenic regions coupled with sequence conservation against P. reichenowi, which is evolutionarily closely related to P. falciparum to identify potential sequences of functional importance. Interestingly, ~30% of the GC-rich, conserved sequences were associated with antigenic proteins encoded by var and rifin genes. The majority of sequences identified in the 5′ UTR of var genes are represented by short expressed sequence tags (ESTs) in cDNA libraries signifying that they are transcribed in the parasite. Additionally, 19 sequences were located in the 3′ UTR of rifins and 4 also have overlapping ESTs. Further analysis showed that several sequences associated with var genes have the capacity to encode small peptides. A previous report has shown that upstream peptides can regulate the expression of var genes hence we propose that these conserved GC-rich sequences may play roles in regulation of gene expression.

MITOCHONDRIAL DNA IN THE OOGAMOCHLAMYS CLADE (CHLOROPHYCEAE): HIGH GC CONTENT AND UNIQUE GENOME ARCHITECTURE FOR GREEN ALGAE(1).

Most mitochondrial genomes in the green algal phylum Chlorophyta are AT-rich, circular-mapping DNA molecules. However, mitochondrial genomes from the Reinhardtii clade of the Chlorophyceae lineage are linear and sometimes fragmented into subgenomic forms. Moreover, Polytomella capuana, from the Reinhardtii clade, has an elevated GC content (57.2%). In the present study, we examined mitochondrial genome conformation and GC bias in the Oogamochlamys clade of the Chlorophyceae, which phylogenetic data suggest is closely related to the Reinhardtii clade. Total DNA from selected Oogamochlamys taxa, including four Lobochlamys culleus (H. Ettl) Pröschold, B. Marin, U. G. Schlöss. et Melkonian strains, Lobochlamys segnis (H. Ettl) Pröschold, B. Marin, U. G. Schlöss. et Melkonian, and Oogamochlamys gigantea (O. Dill) Pröschold, B. Marin, U. G. Schlöss. et Melkonian, was subjected to Southern blot analyses with cob and cox1 probes, and the results suggest that the mitochondrial genome of these taxa is represented by multiple-sized linear DNA fragments with overlapping homologies. On the basis of these data, we propose that linear mitochondrial DNA with a propensity to become fragmented arose in an ancestor common to the Reinhardtii and Oogamochlamys clades or even earlier in the evolutionary history of the Chlorophyceae. Analyses of partial cob and cox1 sequences from these Oogamochlamys taxa revealed an unusually high GC content (49.9%-65.1%) and provided evidence for the accumulation of cob and cox1 pseudogenes and truncated sequences in the mitochondrial genome of all L. culleus strains examined.

Gene Expression and Isoform Variation Analysis using Affymetrix Exon Arrays

BackgroundAlternative splicing and isoform level expression profiling is an emerging field of interest within genomics. Splicing sensitive microarrays, with probes targeted to individual exons or exon-junctions, are becoming increasingly popular as a tool capable of both expression profiling and finer scale isoform detection. Despite their intuitive appeal, relatively little is known about the performance of such tools, particularly in comparison with more traditional 3' targeted microarrays. Here, we use the well studied Microarray Quality Control (MAQC) dataset to benchmark the Affymetrix Exon Array, and compare it to two other popular platforms: Illumina, and Affymetrix U133.ResultsWe show that at the gene expression level, the Exon Array performs comparably with the two 3' targeted platforms. However, the interplatform correlation of the results is slightly lower than between the two 3' arrays. We show that some of the discrepancies stem from the RNA amplification protocols, e.g. the Exon Array is able to detect expression of non-polyadenylated transcripts. More importantly, we show that many other differences result from the ability of the Exon Array to monitor more detailed isoform-level changes; several examples illustrate that changes detected by the 3' platforms are actually isoform variations, and that the nature of these variations can be resolved using Exon Array data. Finally, we show how the Exon Array can be used to detect alternative isoform differences, such as alternative splicing, transcript termination, and alternative promoter usage. We discuss the possible pitfalls and false positives resulting from isoform-level analysis.ConclusionThe Exon Array is a valuable tool that can be used to profile gene expression while providing important additional information regarding the types of gene isoforms that are expressed and variable. However, analysis of alternative splicing requires much more hands on effort and visualization of results in order to correctly interpret the data, and generally results in considerably higher false positive rates than expression analysis. One of the main sources of error in the MAQC dataset is variation in amplification efficiency across transcripts, most likely caused by joint effects of elevated GC content in the 5' ends of genes and reduced likelihood of random-primed first strand synthesis in the 3' ends of genes. These effects are currently not adequately corrected using existing statistical methods. We outline approaches to reduce such errors by filtering out potentially problematic data.

Substantial biases in ultra-short read data sets from high-throughput DNA sequencing

Novel sequencing technologies permit the rapid production of large sequence data sets. These technologies are likely to revolutionize genetics and biomedical research, but a thorough characterization of the ultra-short read output is necessary. We generated and analyzed two Illumina 1G ultra-short read data sets, i.e. 2.8 million 27mer reads from a Beta vulgaris genomic clone and 12.3 million 36mers from the Helicobacter acinonychis genome. We found that error rates range from 0.3% at the beginning of reads to 3.8% at the end of reads. Wrong base calls are frequently preceded by base G. Base substitution error frequencies vary by 10- to 11-fold, with A > C transversion being among the most frequent and C > G transversions among the least frequent substitution errors. Insertions and deletions of single bases occur at very low rates. When simulating re-sequencing we found a 20-fold sequencing coverage to be sufficient to compensate errors by correct reads. The read coverage of the sequenced regions is biased; the highest read density was found in intervals with elevated GC content. High Solexa quality scores are over-optimistic and low scores underestimate the data quality. Our results show different types of biases and ways to detect them. Such biases have implications on the use and interpretation of Solexa data, for de novo sequencing, re-sequencing, the identification of single nucleotide polymorphisms and DNA methylation sites, as well as for transcriptome analysis.

Turtle isochore structure is intermediate between amphibians and other amniotes

Vertebrate genomes are comprised of isochores that are relatively long (>100 kb) regions with a relatively homogenous (either GC-rich or AT-rich) base composition and with rather sharp boundaries with neighboring isochores. Mammals and living archosaurs (birds and crocodilians) have heterogeneous genomes that include very GC-rich isochores. In sharp contrast, the genomes of amphibians and fishes are more homogeneous and they have a lower overall GC content. Because DNA with higher GC content is more thermostable, the elevated GC content of mammalian and archosaurian DNA has been hypothesized to be an adaptation to higher body temperatures. This hypothesis can be tested by examining structure of isochores across the reptilian clade, which includes the archosaurs, testudines (turtles), and lepidosaurs (lizards and snakes), because reptiles exhibit diverse body sizes, metabolic rates, and patterns of thermoregulation. This study focuses on a comparative analysis of a new set of expressed genes of the red-eared slider turtle and orthologs of the turtle genes in mammalian (human, mouse, dog, and opossum), archosaurian (chicken and alligator), and amphibian (western clawed frog) genomes. EST (expressed sequence tag) data from a turtle cDNA library enriched for genes that have specialized functions (developmental genes) revealed using the GC content of the third-codon-position to examine isochore structure requires careful consideration of the types of genes examined. The more highly expressed genes (e.g., housekeeping genes) are more likely to be GC-rich than are genes with specialized functions. However, the set of highly expressed turtle genes demonstrated that the turtle genome has a GC content that is intermediate between the GC-poor amphibians and the GC-rich mammals and archosaurs. There was a strong correlation between the GC content of all turtle genes and the GC content of other vertebrate genes, with the slope of the line describing this relationship also indicating that the isochore structure of turtles is intermediate between that of amphibians and other amniotes. These data are consistent with some thermal hypotheses of isochore evolution, but we believe that the credible set of models for isochore evolution still includes a variety of models. These data expand the amount of genomic data available from reptiles upon which future studies of reptilian genomics can build.

Identification and characterization of lineage-specific genes within the Poaceae.

Using the rice (Oryza sativa) sp. japonica genome annotation, along with genomic sequence and clustered transcript assemblies from 184 species in the plant kingdom, we have identified a set of 861 rice genes that are evolutionarily conserved among six diverse species within the Poaceae yet lack significant sequence similarity with plant species outside the Poaceae. This set of evolutionarily conserved and lineage-specific rice genes is termed conserved Poaceae-specific genes (CPSGs) to reflect the presence of significant sequence similarity across three separate Poaceae subfamilies. The vast majority of rice CPSGs (86.6%) encode proteins with no putative function or functionally characterized protein domain. For the remaining CPSGs, 8.8% encode an F-box domain-containing protein and 4.5% encode a protein with a putative function. On average, the CPSGs have fewer exons, shorter total gene length, and elevated GC content when compared with genes annotated as either transposable elements (TEs) or those genes having significant sequence similarity in a species outside the Poaceae. Multiple sequence alignments of the CPSGs with sequences from other Poaceae species show conservation across a putative domain, a novel domain, or the entire coding length of the protein. At the genome level, syntenic alignments between sorghum (Sorghum bicolor) and 103 of the 861 rice CPSGs (12.0%) could be made, demonstrating an additional level of conservation for this set of genes within the Poaceae. The extensive sequence similarity in evolutionarily distinct species within the Poaceae family and an additional screen for TE-related structural characteristics and sequence discounts these CPSGs as being misannotated TEs. Collectively, these data confirm that we have identified a specific set of genes that are highly conserved within, as well as specific to, the Poaceae.