On the complexity measures of genetic sequences

MOTIVATION: It is well known that the regulatory regions of genomes are highly repetitive. They are rich in direct, symmetric and complemented repeats, and there is no doubt about the functional significance of these repeats. Among known measures of complexity, the Ziv-Lempel complexity measure reflects most adequately repeats occurring in the text. But this measure does not take into account isomorphic repeats. By isomorphic repeats we mean fragments that are identical (or symmetric) modulo some permutation of the alphabet letters. RESULTS: In this paper, two complexity measures of symbolic sequences are proposed that generalize the Ziv-Lempel complexity measure by taking into account any isomorphic repeats in the text (rather than just direct repeats as in Ziv-Lempel). The first of them, the complexity vector, is designed for small alphabets such as the alphabet of nucleotides. The second is based on a search for the longest isomorphic fragment in the history of sequence synthesis and can be used for alphabets of arbitrary cardinality. These measures have been used for recognition of structural regularities in DNA sequences. Some interesting structures related to the regulatory region of the human growth hormone are reported.     

Gene conversion: mechanisms, evolution and human disease

Gene conversion, one of the two mechanisms of homologous recombination, involves the unidirectional transfer of genetic material from a 'donor' sequence to a highly homologous 'acceptor'. Considerable progress has been made in understanding the molecular mechanisms that underlie gene conversion, its formative role in human genome evolution and its implications for human inherited disease. Here we assess current thinking about how gene conversion occurs, explore the key part it has played in fashioning extant human genes, and carry out a meta-analysis of gene-conversion events that are known to have caused human genetic disease.     

An absence of cutaneous neurofibromas associated with a 3-bp inframe deletion in exon 17 of the NF1 gene (c.2970-2972 delAAT): evidence of a clinically significant NF1 genotype-phenotype correlation

Neurofibromatosis type 1 (NF1) is characterized by cafe-au-lait spots, skinfold freckling, and cutaneous neurofibromas. No obvious relationships between small mutations (<20 bp) of the NF1 gene and a specific phenotype have previously been demonstrated, which suggests that interaction with either unlinked modifying genes and/or the normal NF1 allele may be involved in the development of the particular clinical features associated with NF1. We identified 21 unrelated probands with NF1 (14 familial and 7 sporadic cases) who were all found to have the same c.2970-2972 delAAT (p.990delM) mutation but no cutaneous neurofibromas or clinically obvious plexiform neurofibromas. Molecular analysis identified the same 3-bp inframe deletion (c.2970-2972 delAAT) in exon 17 of the NF1 gene in all affected subjects. The Delta AAT mutation is predicted to result in the loss of one of two adjacent methionines (codon 991 or 992) ( Delta Met991), in conjunction with silent ACA-->ACG change of codon 990. These two methionine residues are located in a highly conserved region of neurofibromin and are expected, therefore, to have a functional role in the protein. Our data represent results from the first study to correlate a specific small mutation of the NF1 gene to the expression of a particular clinical phenotype. The biological mechanism that relates this specific mutation to the suppression of cutaneous neurofibroma development is unknown.     

New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras

A new computer program, called Mallard, is presented for screening entire 16S rRNA gene libraries of up to 1,000 sequences for chimeras and other artifacts. Written in the Java computer language and capable of running on all major operating systems, the program provides a novel graphical approach for visualizing phylogenetic relationships among 16S rRNA gene sequences. To illustrate its use, we analyzed most of the large libraries of cloned bacterial 16S rRNA gene sequences submitted to the public repository during 2005. Defining a large library as one containing 100 or more sequences of 1,200 bases or greater, we screened 25 of the 28 libraries and found that all but three contained substantial anomalies. Overall, 543 anomalous sequences were found. The average anomaly content per clone library was 9.0%, 4% higher than that previously estimated for the public repository overall. In addition, 90.8% of anomalies had characteristic chimeric patterns, a rise of 25.4% over that found previously. One library alone was found to contain 54 chimeras, representing 45.8% of its content. These figures far exceed previous estimates of artifacts within public repositories and further highlight the urgent need for all researchers to adequately screen their libraries prior to submission. Mallard is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/.     

Non-B DNA-forming sequences and WRN deficiency independently increase the frequency of base substitution in human cells

Although alternative DNA secondary structures (non-B DNA) can induce genomic rearrangements, their associated mutational spectra remain largely unknown. The helicase activity of WRN, which is absent in the human progeroid Werner syndrome, is thought to counteract this genomic instability. We determined non-B DNA-induced mutation frequencies and spectra in human U2OS osteosarcoma cells and assessed the role of WRN in isogenic knockdown (WRN-KD) cells using a supF gene mutation reporter system flanked by triplex- or Z-DNA-forming sequences. Although both non-B DNA and WRN-KD served to increase the mutation frequency, the increase afforded by WRN-KD was independent of DNA structure despite the fact that purified WRN helicase was found to resolve these structures in vitro. In U2OS cells, ～70% of mutations comprised single-base substitutions, mostly at G·C base-pairs, with the remaining ～30% being microdeletions. The number of mutations at G·C base-pairs in the context of NGNN/NNCN sequences correlated well with predicted free energies of base stacking and ionization potentials, suggesting a possible origin via oxidation reactions involving electron loss and subsequent electron transfer (hole migration) between neighboring bases. A set of ～40,000 somatic mutations at G·C base pairs identified in a lung cancer genome exhibited similar correlations, implying that hole migration may also be involved. We conclude that alternative DNA conformations, WRN deficiency and lung tumorigenesis may all serve to increase the mutation rate by promoting, through diverse pathways, oxidation reactions that perturb the electron orbitals of neighboring bases. It follows that such "hole migration" is likely to play a much more widespread role in mutagenesis than previously anticipated.     

Rapid Method for Identification of Interconnections between Functionally and/or Evolutionarily Related Biological Sequences

It is now common practice to retrieve, by key words, highly specialized selections of sequences from general-purpose databases such as EMBL, GenBank, etc. The sequences included in a selection are often interconnected, which means that there are duplications, embeddings, intersections, homology, common structural elements. Knowledge of these interconnections is necessary for further processing of the sequences. We propose a rapid (single scan) method for identification of such interconnections by means of complexity analysis that generalizes the Lempel–Ziv approach. Analysis of a selection of 5"-flanking regions of vertebrate growth hormone genes from EMBL is presented as an example.     

Promoter shuffling has occurred during the evolution of the vertebrate growth hormone gene

Comparative studies of vertebrate gene promoter regions seldom detect gross rearrangements ('promoter shuffling') since such analyses usually employ relatively similar DNA sequences. Conversely, attempts to compare evolutionarily more divergent promoter sequences have been largely unsuccessful owing to the inability of conventional alignment procedures to deal with gross rearrangements. These limitations have been circumvented in the present study by using the novel technique of complexity analysis to identify modular components ('blocks') in the growth hormone (GH) gene promoter sequences of some 22 vertebrate species, from salmon to human. Significant rearrangement of blocks was found to have occurred, indicating that they have evolved as independent units. Some blocks appear to be ubiquitous, whereas others are restricted to a specific taxon. Considerable variation between orthologous GH gene promoters was apparent in terms of block length, copy number and relative location. It may be inferred that a wide variety of different mutational mechanisms have operated upon the GH gene promoter over evolutionary time. These include gross changes such as deletion, duplication, amplification, elongation, contraction, transposition, inversion and fusion, as well as the slow, steady accumulation of single base-pair substitutions. Thus the patchwork structure of the modular GH promoter region, and those of its paralogous GH2 and prolactin (PRL) counterparts, have continually been shuffled into new combinations through the rearrangement of pre-existing blocks. Although some of these changes may have had no influence on promoter function, others could have served to alter either the level of gene expression or the responsiveness of the promoter to external stimuli.     

Changes in primary DNA sequence complexity influence the phenotypic consequences of mutations in human gene regulatory regions

No general rules have been proposed to account for the functional consequences of gene regulatory mutations. In a first attempt to establish the nature of such rules, an analysis was performed of the DNA sequence context of 153 different single base-pair substitutions in the regulatory regions of 65 different human genes underlying inherited disease. Use of a recently proposed measure of DNA sequence complexity (taking into account the level of structural repetitiveness of a DNA sequence, rather than simply the oligonucleotide composition) has served to demonstrate that the concomitant change in local DNA sequence complexity surrounding a substituted nucleotide is related to the likelihood of a regulatory mutation coming to clinical attention. Mutations that led to an increase in complexity exhibited higher odds ratios in favour of pathological consequences than mutations that led to a decrease or left complexity unchanged. This relationship, however, was discernible only for pyrimidine-to-purine transversions. Odds ratios for other types of substitution were not found to be significantly associated with local changes in sequence complexity, even though a trend similar to that observed for Y-->R transversions was also apparent for transitions. These findings suggest that the maintenance of a defined level of DNA sequence complexity, or at least the avoidance of an increase in sequence complexity, is a critical prerequisite for the function of gene regulatory regions.