Isochores Merit the Prefix 'Iso'

The isochore concept in human genome sequence was challenged in an analysis by the International Human Genome Sequencing Consortium (IHGSC). We argue here that a statement in IGHSC analysis concerning the existence of isochore is incorrect, because it had applied an inappropriate statistical test. To test the existence of isochores should be equivalent to a test of homogeneity of windowed GC%. The statistical test applied in the IHGSC's analysis, the binomial test, is however a test of a sequence being random on the base level. For testing the existence of isochore, or homogeneity in GC%, we propose to use another statistical test: the analysis of variance (ANOVA). It can be shown that DNA sequences that are rejected by binomial test may not be rejected by the ANOVA test.     

Isochores and the evolutionary genomics of vertebrates

The nuclear genomes of vertebrates are mosaics of isochores, very long stretches (>300kb) of DNA that are homogeneous in base composition and are compositionally correlated with the coding sequences that they embed. Isochores can be partitioned in a small number of families that cover a range of GC levels (GC is the molar ratio of guanine+cytosine in DNA), which is narrow in cold-blooded vertebrates, but broad in warm-blooded vertebrates. This difference is essentially due to the fact that the GC-richest 10-15% of the genomes of the ancestors of mammals and birds underwent two independent compositional transitions characterized by strong increases in GC levels. The similarity of isochore patterns across mammalian orders, on the one hand, and across avian orders, on the other, indicates that these higher GC levels were then maintained, at least since the appearance of ancestors of warm-blooded vertebrates. After a brief review of our current knowledge on the organization of the vertebrate genome, evidence will be presented here in favor of the idea that the generation and maintenance of the GC-richest isochores in the genomes of warm-blooded vertebrates were due to natural selection.     

Isochores and replication time zones: a perfect match

The mammalian genome is not a random sequence but shows a specific, evolutionarily conserved structure that becomes manifest in its isochore pattern. Isochores, i.e. stretches of DNA with a distinct sequence composition and thus a specific GC content, cause the chromosomal banding pattern. This fundamental level of genome organization is related to several functional features like the replication timing of a DNA sequence. GC richness of genomic regions generally corresponds to an early replication time during S phase. Recently, we demonstrated this interdependency on a molecular level for an abrupt transition from a GC-poor isochore to a GC-rich one in the NF1 gene region; this isochore boundary also separates late from early replicating chromatin. Now, we analyzed another genomic region containing four isochores separated by three sharp isochore transitions. Again, the GC-rich isochores were found to be replicating early, the GC-poor isochores late in S phase; one of the replication time zones was discovered to consist of one single replicon. At the boundaries between isochores, that all show no special sequence elements, the replication machinery stopped for several hours. Thus, our results emphasize the importance of isochores as functional genomic units, and of isochore transitions as genomic landmarks with a key function for chromosome organization and basic biological properties.     

Isochores,GC3 and mutation biases in the human genome

In this work we re-examined the hypothesis that the variation in GC content in the human genome is due to different regional mutational biases. For this purpose we inferred the mutational pattern by using mutation databases that are available for many genes associated with human genetic diseases. The assumption of this approach is that such mutations reflect the actual frequency distribution of mutations as they arise in the population. Four classes of genes, classified according to their GC₃ level, were included in this study: GC₃-poor genes (GC₃<45%), genes with intermediate GC₃ content (45%3<60%), GC₃-rich genes (60%3<75%) and very GC₃-rich genes (GC₃>75%). Our results show that most genes are under AT mutational biases, with very little variation compared to the expectations of neutral GC level. It is noteworthy that the mutational patterns in the GC₃-rich genes do not appear to account for their GC₃-richness. Instead, GC₃-rich and very GC₃-rich genes exhibit patterns of mutations that yield expectations of neutral GC₃ content that are much lower than their actual GC₃.     

Telomeres in Warm-Blooded Vertebrates Are Composed of GC-Rich Isochores

We have hybridized the vertebrate telomeric sequence (TTAGGG)n on DNA compositional fractions from 13 mammalian species and 3 avian species, representing 9 and 3 orders, respectively. Our results indicate that the 50- to 100-kb fragments derived from telomeric regions are composed of GC-rich and GC-richest isochores. Previous works from our laboratory demonstrated that single-copy sequences from the human H3 isochore family (the GC-richest and gene-richest isochore in the human genome) share homology with compositionally correlated compartments of warm-blooded vertebrates. This correlation suggested that the GC-richest isochores are, as in the human genome, the gene-richest regions of warm-blooded vertebrates' genome. Moreover, this evidence suggests that telomeric regions are the most gene-dense region of all warm-blooded vertebrates. The implications of these findings are discussed.     

The genetical control and tissue-specificity of esterase isozymes in hexaploid wheat

A comparative genetic analysis of esterase (E.C.3.1.1.1) isozymes of wheat cultivar Chinese Spring in endosperm, embryo, coleoptile, leaf and root tissues revealed eight sets of isozymes characterised by different tissue specificities, pI ranges and the chromosomal locations of their controlling genes. This data was considered together with previously published work, resulting in a proposed rationalization of nine sets of wheat esterase isozymes. Although this classification included two sets of isozymes controlled by genes on the short arms of homoeologous group 3 chromosomes and three sets on the long arms of the same chromosomes, for which no recombination evidence of genetic distinctness has been obtained among either group, it is argued that the different characteristics of the various sets warrant retention of separate set nomenclatures. Previously unreported esterase genes includeEst-9, a low pI, monomeric, embryo-specific group with controlling genes on chromosomes 3BS and 3DS and two further members ofEs-1,Est-H1 inHordeum vulgare andEst-S ^l1 inAegilops longissima.     

Host- and tissue-specificity of moss-associated Galerina and Mycena determined from amplicon pyrosequencing data

The genetic diversity of two agaricoid saprotroph genera, Galerina and Mycena, and their distribution across bryophyte host species, and within bryophytes' photosynthetic and senescent tissues, was assessed using data from two pyrosequencing biodiversity inventories of bryophilous fungi. A total of 9 498 Galerina and 5 731 Mycena reads were mapped to branches broadly distributed throughout backbone trees, reflecting no obvious evolutionary specialization of particular fungal lineages to moss hosts/substrata. Although a few OTUs occurred with equal frequency across the hosts, most exhibited some degree of specialization to one or more bryophytes, indicating that the quality of different mosses as substratum varies between species. With one exception, all Galerina and Mycena OTUs were more frequent and abundant in senescent than photosynthetic tissues, likely reflecting saprotrophic nutritional modes in the fungi. A single Mycena OTU showed considerable colonization of both tissues, which may reflect an opportunistic parasitic or endophytic lifestyle.     

Dose-dependence, sex- and tissue-specificity, and persistence of radiation-induced genomic DNA methylation changes

Radiation is a well-known genotoxic agent and human carcinogen that gives rise to a variety of long-term effects. Its detrimental influence on cellular function is actively studied nowadays. One of the most analyzed, yet least understood long-term effects of ionizing radiation is transgenerational genomic instability. The inheritance of genomic instability suggests the possible involvement of epigenetic mechanisms, such as changes of the methylation of cytosine residues located within CpG dinucleotides. In the current study we evaluated the dose-dependence of the radiation-induced global genome DNA methylation changes. We also analyzed the effects of acute and chronic high dose (5Gy) exposure on DNA methylation in liver, spleen, and lung tissues of male and female mice and evaluated the possible persistence of the radiation-induced DNA methylation changes. Here we report that radiation-induced DNA methylation changes were sex- and tissue-specific, dose-dependent, and persistent. In parallel we have studied the levels of DNA damage in the exposed tissues. Based on the correlation between the levels of DNA methylation and DNA damage we propose that radiation-induced global genome DNA hypomethylation is DNA repair-related.     

Isochores exhibit evidence of genes interacting with the large-scale genomic environment

The genomes of mammals and birds can be partitioned into megabase-long regions, termed isochores, with consistently high, or low, average C + G content. Isochores with high CG contain a mixture of CG-rich and AT-rich genes, while high-AT isochores contain predominantly AT-rich genes. The two gene populations in the high-CG isochores are functionally distinguishable by statistical analysis of their gene ontology categories. However, the aggregate of the two populations in CG isochores is not statistically distinct from AT-rich genes in AT isochores. Genes tend to be located at local extrema of composition within the isochores, indicating that the CG-enriching mechanism acted differently when near to genes. On the other hand, maximum-likelihood reconstruction of molecular phylogenetic trees shows that branch lengths (evolutionary distances) for third codon positions in CG-rich genes are not substantially larger than those for AT-rich genes. In the context of neutral mutation theory this argues against any strong positive selection. Disparate features of isochores might be explained by a model in which about half of all genes functionally require AT richness, while, in warm-blooded organisms, about half the genome (in large coherent blocks) acquired a strong bias for mutations to CG. Using mutations in CG-rich genes as convenient indicators, we show that approximately 20% of amino acids in proteins are broadly substitutable, without regard to chemical similarity.     

The Biased Distribution of Alus in Human Isochores Might Be Driven by Recombination

Alu retrotransposons do not show a homogeneous distribution over the human genome but have a higher density in GC-rich (H) than in AT-rich (L) isochores. However, since they preferentially insert into the L isochores, the question arises: What is the evolutionary mechanism that shifts the Alu density maximum from L to H isochores? To disclose the role played by each of the potential mechanisms involved in such biased distribution, we carried out a genome-wide analysis of the density of the Alus as a function of their evolutionary age, isochore membership, and intron vs. intergene location. Since Alus depend on the retrotransposase encoded by the LINE1 elements, we also studied the distribution of LINE1 to provide a complete evolutionary scenario. We consecutively check, and discard, the contributions of the Alu/LINE1 competition for retrotransposase, compositional matching pressure, and Alu overrepresentation in introns. In analyzing the role played by unequal recombination, we scan the genome for Alu trimers, a direct product of Alu–Alu recombination. Through computer simulations, we show that such trimers are much more frequent than expected, the observed/expected ratio being higher in L than in H isochores. This result, together with the known higher selective disadvantage of recombination products in H isochores, points to Alu–Alu recombination as the main agent provoking the density shift of Alus toward the GC-rich parts of the genome. Two independent pieces of evidence—the lower evolutionary divergence shown by recently inserted Alu subfamilies and the higher frequency of old stand-alone Alus in L isochores—support such a conclusion. Other evolutionary factors, such as population bottlenecks during primate speciation, may have accelerated the fast accumulation of Alus in GC-rich isochores.     

Lack of tissue-specificity of calcium-activated neutral proteases from skeletal muscle and lung of rabbit

Calcium-activated neutral proteases (CANPs) with high sensitivity (microCANP) and low sensitivity (mCANP) to calcium ions were purified individually from rabbit skeletal muscle and rabbit lung and compared as to their electrophoretic properties, calcium requirements and peptide mapping of fragments produced by S. aureus V8 protease digestion of separated subunits. All of the results suggested that there is no difference between the microCANPs as well as between the mCANPs obtained from the two tissues, with respect to the chemical and enzymatic properties. However, the contents of CANPs in these tissues were different.     

The tissue-specificity associated region and motif of an emx2 downstream enhancer CNE2.04 in zebrafish

BackgroundExpression level of EMX2 plays an important role in the development of nervous system and cancers. CNE2.04, a conserved enhancer downstream of emx2, drives fluorescent protein expression in the similar pattern of emx2.MethodsCNE2.04 truncated or motif-mutated transgenic reporter plasmids were constructed and injected into the zebrafish fertilized egg with Tol2 mRNA at the unicellular stage of zebrafish eggs. The green fluorescence expression patterns were observed at 24, 48, and 72 hpf, and the fluorescence rates of different tissues were counted at 48 hpf.ResultsCompared to CNE2.04, CNE2.04-R400 had comparable enhancer activity, while the tissue specificity of CNE2.04-L400 was obviously changed. Motif CCCCTC mutation obviously changed the enhancer activity, while motif CCGCTC mutations also changed it.ConclusionDue to their correlation with tissue specificity, CNE2.04-R400 is associated with the tissue-specificity of CNE2.04, and motif CCCCTC plays an important role in the enhancer activity of CNE2.04.     

Relationships Among Stop Codon Usage Bias, Its Context, Isochores, and Gene Expression Level in Various Eukaryotes

It is well known that stop codons play a critical role in the process of protein synthesis. However, little effort has been made to investigate whether stop codon usage exhibits biases, such as widely seen for synonymous codon usage. Here we systematically investigate stop codon usage bias in various eukaryotes as well as its relationships with its context, GC3 content, gene expression level, and secondary structure. The results show that there is a strong bias for stop codon usage in different eukaryotes, i.e., UAA is overrepresented in the lower eukaryotes, UGA is overrepresented in the higher eukaryotes, and UAG is least used in all eukaryotes. Different conserved patterns for each stop codon in different eukaryotic classes are found based on information content and logo analysis. GC3 contents increase with increasing complexity of organisms. Secondary structure prediction revealed that UAA is generally associated with loop structures, whereas UGA is more uniformly present in loop and stem structures, i.e., UGA is less biased toward having a particular structure. The stop codon usage bias, however, shows no significant relationship with GC3 content and gene expression level in individual eukaryotes. The results indicate that genomic complexity and GC3 content might contribute to stop codon usage bias in different eukaryotes. Our results indicate that stop codons, like synonymous codons, exhibit biases in usage. Additional work will be needed to understand the causes of these biases and their relationship to the mechanism of protein termination. [Reviewing Editor: Dr. Manyuan Long]     

Expected Relationship Between the Silent Substitution Rate and the GC Content: Implications for the Evolution of Isochores

The relationship between the silent substitution rate (K _s) and the GC content along the genome is a focal point of the debate about the origin of the isochore structure in vertebrates. Recent estimation of the silent substitution rate showed a positive correlation between K _s and GC content, in contradiction with the predictions of both the regional mutation bias model and the selection or biased gene conversion model. The aim of this paper is to help resolve this contradiction between theoretical studies and data. We analyzed the relationship between K _s and GC content under (1) uniform mutation bias, (2) a regional mutation bias, and (3) mutation bias and selection. We report that an increase in K _s with GC content is expected under mutation bias because of either nonequilibrium of the isochore structure or an increasing mutation rate from AT toward GC nucleotides in GC-richer isochores. We show by simulations that CpG deamination tends to increase the mutation rate with GC content in a regional mutation bias model. We also demonstrate that the relationship between K _s and GC under the selectionist or biased gene conversion model is positive under weak selection if the mutation selection equilibrium GC frequency is less than 0.5. Received: 28 March 2001 / Accepted: 16 May 2001     

Relationship between the tissue-specificity of mouse gene expression and the evolutionary origin and function of the proteins

Background  

The combination of complete genome sequence information with expression data enables us to characterize the relationship between a protein's evolutionary origin or functional category and its expression pattern. In this study, mouse proteins were assigned into functional and phyletic groups and the gene expression patterns of the different protein groupings were examined by microarray analysis in various mouse tissues.     

The Decline of Isochores in Mammals: An Assessment of the GC ContentVariation Along the Mammalian Phylogeny

Whether isochores, the large-scale variation of the GC content in mammalian genomes, are being maintained has recently been questioned. It has been suggested that GC-rich isochores originated in the ancestral amniote genome but that whatever force gave rise to them is no longer effective and that isochores are now disappearing from mammalian genomes. Here we investigated the evolution of the GC content of 41 coding genes in 6 to 66 species of mammals by estimating the ancestral GC content using a method which allows for different rates of substitution between sites. We found a highly significant decrease in the GC content during early mammalian evolution, as well as a weaker but still significant decrease in the GC content of GC-rich genes later in at least three groups of mammals: primates, rodents, and carnivores. These results are of interest because they confirm the recently suggested disappearance of GC-rich isochores in some mammalian genomes, and more importantly, they suggest that this disappearance started very early in mammalian evolution.This article contains online supplementary material.