The compositional properties of human genes

Summary The present work represents the first attempt to study in greater detail previously proposed compositional correlations in genomes, based on a body of additional data relating to gene localizations as well as to extended flanking sequences extracted from gene banks. We have investigated the correlations that exist between (1) the GC levels of exons of human genes, and (2) the GC levels of either intergenic sequences or introns associated with the genes under consideration. In both cases, linear relationships with slopes close to unity were found. The similarity of the linear relationships indicates similar GC levels in intergenic sequences and introns located in the same isochores. Moreover, both intergenic sequences and introns showed GC levels 5–10% lower than the corresponding exons. The above findings considerably strengthen the previously drawn conclusion that coding and noncoding sequences (both inter- and intragenic) from the same isochores of the human genome are compositionally correlated. In addition, we find linear correlations between the GC levels of codon positions and of the intergenic sequences or introns associated with the corresponding genes, as well as among the GC levels of codon positions of genes.     

Silent DNA: speaking RNA language?

The sequence of silent DNA in the human genome (intergenic spacers, introns and synonymous codon positions of protein-coding genes) was found here to have the higher thermostability of corresponding RNA/RNA and RNA/DNA duplexes as compared with randomized sequence. This difference increased with elevation of GC content. The revealed effect was not due to correlation of RNA/RNA and RNA/DNA thermostabilities with thermostability of the DNA/DNA duplex, which, on the contrary, was lower than in the randomized sequence and lagged behind the elevation of GC content. The same picture was observed in the genomes of other warm-blooded vertebrates but not in the lower organisms. This finding suggests that RNA-RNA and RNA-DNA interactions could be involved in the putative function of silent DNA.     

Genome size and metabolic intensity in tetrapods: a tale of two lines

We show the negative link between genome size and metabolic intensity in tetrapods, using the heart index (relative heart mass) as a unified indicator of metabolic intensity in poikilothermal and homeothermal animals. We found two separate regression lines of heart index on genome size for reptiles-birds and amphibians-mammals (the slope of regression is steeper in reptiles-birds). We also show a negative correlation between GC content and nucleosome formation potential in vertebrate DNA, and, consistent with this relationship, a positive correlation between genome GC content and nuclear size (independent of genome size). It is known that there are two separate regression lines of genome GC content on genome size for reptiles-birds and amphibians-mammals: reptiles-birds have the relatively higher GC content (for their genome sizes) compared to amphibians-mammals. Our results suggest uniting all these data into one concept. The slope of negative regression between GC content and nucleosome formation potential is steeper in exons than in non-coding DNA (where nucleosome formation potential is generally higher), which indicates a special role of non-coding DNA for orderly chromatin organization. The chromatin condensation and nuclear size are supposed to be key parameters that accommodate the effects of both genome size and GC content and connect them with metabolic intensity. Our data suggest that the reptilian-birds clade evolved special relationships among these parameters, whereas mammals preserved the amphibian-like relationships. Surprisingly, mammals, although acquiring a more complex general organization, seem to retain certain genome-related properties that are similar to amphibians. At the same time, the slope of regression between nucleosome formation potential and GC content is steeper in poikilothermal than in homeothermal genomes, which suggests that mammals and birds acquired certain common features of genomic organization.     

真核生物DNA非编码区的组分分析

在全基因组水平上,用直方图、混沌表示灰度图、距离差异度和信息熵差异度四种方法,研究了拟南芥、线虫、果蝇的DNA内含子、基因间隔区DNA、外显子三种区域的核苷酸短序列组分及组分复杂度.结果表明：a.不同基因组之间,不管基因数目多少,用4种方法得到的外显子部分其组分复杂度都比较接近,而非编码区部分的组分复杂度却很大.这一点定量地说明了物种之间的复杂程度,主要不体现在编码区部分,而体现在非编码区部分.b.同一基因组中,内含子的核苷酸短序列组分复杂度都是相似的,外显子和intergenic DNA部分的组分复杂度也是相似的.c.内含子和intergenic DNA在转录、剪切、二级结构等方面有很大的不同,但它们在核苷酸短序列组分上的差异却很小,说明内含子和intergenic DNA在转录、剪切、二级结构上的不同并不通过核苷酸短序列组分来进行限制.     

Distributions of exons and introns in the human genome

The human genome is revisited using exon and intron distribution profiles. The 26,564 annotated genes in the human genome (build October, 2003) contain 233,785 exons and 207,344 introns. On average, there are 8.8 exons and 7.8 introns per gene. About 80% of the exons on each chromosome are < 200 bp in length. < 0.01% of the introns are < 20 bp in length and < 10% of introns are more than 11,000 bp in length. These results suggest constraints on the splicing machinery to splice out very long or very short introns and provide insight to optimal intron length selection. Interestingly, the total length in introns and intergenic DNA on each chromosome is significantly correlated to the determined chromosome size with a coefficient of correlation r = 0.95 and r = 0.97, respectively. These results suggest their implication in genome design.     

Excision sequences in the mitochondrial genome of yeast

We report an analysis of the sequences used in the excision of the mitochondrial genomes of 22 spontaneous and ten ethidium bromide (EtBr)-induced Saccharomyces cerevisiae petite mutants. In all cases, excision sequences were found to be perfect direct repeats, often flanked on one or both sides by regions of patchy homology. Sequences used in the excision of the genomes of spontaneous petites were always located in the AT spacers and GC clusters of intergenic regions of the genome; the GC clusters corresponded to ori and ori^s sequences, namely to canonical and surrogate origins of DNA replication, respectively. In the case of the ethidium bromide-induced petites, excision sequences were found not only in intergenic sequences, but also in the introns and exons of mitochondrial genes.     

Pheromone 4 gene of Euplotes octocarinatus.

We have cloned and sequenced a 1.7 kb macronuclear chromosome encoding the pheromone 4 gene of Euplotes octocarinatus. The sequence of the secreted pheromone is preceded by a 42 amino acid leader peptide, which ends with a lysine residue. The sequence coding for the leader peptide contains information for a putative signal peptide and is interrupted by a 772 bp intron as shown by comparison with a cDNA clone. A 64 bp intron and a 145 bp intron interrupt the sequence coding for the secreted pheromone. The three introns contain typical 5' and 3' splice junctions and a putative branch point site. The small introns have a low GC content. The large intron has a GC content similar to that of the pheromone 4 gene exons. The amino acid sequence of pheromone 4, deduced from both the genomic DNA and the cDNA of pheromone 4, shows that the secreted pheromone consists of 85 amino acids. One of its amino acids is encoded by a UGA codon. Since it has been shown for pheromone 3 of E. octocarinatus that UGA is translated as cysteine, it is assumed that the UGA codon encodes cysteine in pheromone 4 as well. The 164 bp noncoding region upstream of the leader peptide is AT-rich and contains an inverted repeat capable of forming a stem-loop structure with a stem of 11 bp. The 151 bp noncoding region at the 3' end of the chromosome contains a putative polyadenylation sequence and an inverted repeat. The macronuclear molecule is flanked by telomeres and carries the pentanucleotide motif TTGAA, located at a distance of 17 nucleotides from the telomeres. This motif has been suggested to be involved in the formation of macronuclear chromosomes.     

The exon-intron organization of the human erythrocyte alpha-spectrin gene.

The human erythrocyte alpha-spectrin gene which spans 80 kbp has been cloned from human genomic DNA as overlapping lambda recombinants. The exon-intron junctions were identified and the exons mapped. The gene is encoded by 52 exons whose sizes range from 684 bp to the smallest of 18 bp. The donor and acceptor splice site sequences match the splice site consensus sequences, with the exception of one splice site where a donor sequence begins with -GC. The size and location of exons do not correlate with the 106-amino-acid repeat, except in three locations where the surrounding codons are conserved as well. The lack of correspondence between exons and 106-amino-acid repeat is interpreted to reflect the appearance of a spectrin-like gene from a minigene early in the evolution of eukaryotes. Since current evidence indicates that introns were present in genes before the divergence of prokaryotes and eukaryotes, it is possible that the original distribution of introns within the minigene has been lost by the random deletion of introns from the spectrin gene.     

Nucleotide sequence of the gene for human prothrombin

A human genomic DNA library was screened for the gene coding for human prothrombin with a cDNA coding for the human protein. Eighty-one positive lambda phage were identified, and three were chosen for further characterization. These three phage hybridized with 5' and/or 3' probes prepared from the prothrombin cDNA. The complete DNA sequence of 21 kilobases of the human prothrombin gene was determined and included a 4.9-kilobase region that was previously sequenced. The gene for human prothrombin contains 14 exons separated by 13 intervening sequences. The exons range in size from 25 to 315 base pairs, while the introns range from 84 to 9447 base pairs. Ninety percent of the gene is composed of intervening sequence. All the intron splice junctions are consistent with sequences found in other eukaryotic genes, except for the presence of GC rather than GT on the 5' end of intervening sequence L. Thirty copies of Alu repetitive DNA and two copies of partial KpnI repeats were identified in clusters within several of the intervening sequences, and these repeats represent 40% of the DNA sequence of the gene. The size, distribution, and sequence homology of the introns within the gene were then compared to those of the genes for the other vitamin K dependent proteins and several other serine proteases.     

Genomic structure of murine mitochondrial DNA polymerase-gamma

We have sequenced a genomic clone of the gene encoding the mouse mitochondrial DNA polymerase. The gene consists of 23 exons, which span approximately 13.2 kb, with exons ranging in size from 53 to 768 bp. All intron-exon boundaries conform to the GT-AG rule. By comparison with the human genomic sequence, we found remarkable conservation of the gene structure; the intron-exon borders are in almost identical locations for the 22 introns. The 5' upstream region contains approximately 300 bp of homology between the mouse and human sequences that presumably contain the promoter element. This region lacks any obvious TATA domain and is relatively GC rich, consistent with the housekeeping function of the mitochondrial DNA polymerase. Finally, within the 5' flanking region, both mouse and human genes have a region of 73 bp with high homology to the tRNA-Arg gene.     

DNA sequence alterations affect nucleosome array formation of the chicken ovalbumin gene

The role of the large amount (more than half of the genome) of noncoding DNA in higher organisms is not well understood. DNA evolved to function in the context of chromatin, and the possibility exists that some of the noncoding DNA serves to influence chromatin structure and function. In this age of genomics and bioinformatics, genomic DNA sequences are being searched for informational content beyond the known genetic code. The discovery that period-10 non-T, A/T, G (VWG) triplets are among the most abundant motifs in human genomic DNA suggests that they may serve some function in higher organisms. In this paper, we provide direct evidence that the regular oscillation of period-10 VWG that occurs in the chicken ovalbumin gene sequence with a dinucleosome-like period facilitates nucleosome array formation. Using a linker histone-dependent in vitro chromatin assembly system that spontaneously aligns nucleosomes into a physiological array, we show that nucleosomes tend to avoid DNA regions with low period-10 VWG counts. This avoidance leads to the formation of an array with a nucleosome repeat equal to half the period value of the oscillation in period-10 VWG, as determined by Fourier analysis. Two different half-period deletions in the wild-type DNA sequence altered the nucleosome array, as predicted computationally. In contrast, a full-period deletion had an insignificant effect on the nucleosome array formed, also consistent with the prediction. An inversion mutation, with no DNA sequences deleted, again altered the nucleosome array formed, as predicted computationally. Hence, a VWG dinucleosome signal is plausible.     

Differential GC content between exons and introns establishes distinct strategies of splice-site recognition

During evolution segments of homeothermic genomes underwent a GC content increase. Our analyses reveal that two exon-intron architectures have evolved from an ancestral state of low GC content exons flanked by short introns with a lower GC content. One group underwent a GC content elevation that abolished the differential exon-intron GC content, with introns remaining short. The other group retained the overall low GC content as well as the differential exon-intron GC content, and is associated with longer introns. We show that differential exon-intron GC content regulates exon inclusion level in this group, in which disease-associated mutations often lead to exon skipping. This group's exons also display higher nucleosome occupancy compared to flanking introns and exons of the other group, thus "marking" them for spliceosomal recognition. Collectively, our results reveal that differential exon-intron GC content is a previously unidentified determinant of exon selection and argue that the two GC content architectures reflect the two mechanisms by which splicing signals are recognized: exon definition and intron definition.