Functional consequences of sequence alterations in the ATM gene

The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is a high molecular weight, protein (similar to350 kDa) containing a C-terminal protein kinase domain and a number of other putative domains not yet functionally defined. The majority of ATM gene mutations in A-T patients are truncating, resulting in prematurely terminated products that are highly unstable. Missense mutations within the kinase domain and elsewhere in the molecule alter the stability of the protein and lead to loss of protein kinase activity. Only rarely are patients observed with two missense mutations and this gives rise to a milder disease phenotype. Evidence for a dominant interfering effect on normal ATM kinase activity has been reported in cell lines transfected with missense mutant ATM and in cell lines from some A-T heterozygotes. The dominant negative effect of mutant ATM is manifested by an enhancement of cellular radiosensitivity and may be responsible for the cancer predisposition observed in carriers of ATM missense mutations. In this review, we explore the domain structure of the ATM molecule, sites of interaction with other proteins and the consequences of specific amino acid changes on function. (C) 2003 Elsevier B.V. All rights reserved.

Origin and diversity of the Sox transcription factor gene family: genome-wide analysis in Fugu rubripes

The SOX family of transcription factors are found throughout the animal kingdom and are important in a variety of developmental contexts. Genome analysis has identified 20 Sox genes in human and mouse, which can be subdivided into 8 groups, based on sequence comparison and intron-exon structure. Most of the SOX groups identified in mammals are represented by a single SOX sequence in invertebrate model organisms, suggesting a duplication and divergence mechanism has operated during vertebrate evolution. We have now analysed the Sox gene complement in the pufferfish, Fugu rubripes, in order to shed further light on the diversity and origins of the Sox gene family. Major differences were found between the Sox family in Fugu and those in humans and mice. In particular, Fugu does not have orthologues of Sry, Sox,15 and Sox30, which appear to be specific to mammals, while Sox19, found in Fugu and zebrafish but absent in mammals, seems to be specific to fishes. Six mammalian Sox genes are represented by two copies each in Fugu, indicating a large-scale gene duplication in the fish lineage. These findings point to recent Sox gene loss, duplication and divergence occurring during the evolution of tetrapod and teleost lineages, and provide further evidence for large-scale segmental or a whole-genome duplication occurring early in the radiation of teleosts. (C) 2004 Elsevier B.V. All rights reserved.

The Caenorhabditis briggsae genome contains active CbmaT1 and Tcb1 transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.

Multiple genotypes of Chlamydia pneumoniae identified in human carotid plaque

Chlamydia pneumoniae is an obligate intracellular respiratory pathogen that causes 10% of community-acquired pneumonia and has been associated with cardiovascular disease. Both whole-genome sequencing and specific gene typing suggest that there is relatively little genetic variation in human isolates of C. pneumoniae. To date, there has been little genomic analysis of strains from human cardiovascular sites. The genotypes of C. pneumoniae present in human atherosclerotic carotid plaque were analysed and several polymorphisms in the variable domain 4 (VD4) region of the outer-membrane protein-A (ompA) gene and the intergenic region between the ygeD and uridine kinase (ygeD-urk) genes were found. While one genotype was identified that was the same as one reported previously in humans (respiratory and cardiovascular), another genotype was found that was identical to a genotype from non-human sources (frog/koala).

Detection and discrimination of herpes simplex viruses, Haemophilus ducreyi, Treponema pallidum, and Calymmatobacterium (Klebsiella) granulomatis from genital ulcers

Background. Genital ulcer disease (GUD) is commonly caused by pathogens for which suitable therapies exist, but clinical and laboratory diagnoses may be problematic. This collaborative project was undertaken to address the need for a rapid, economical, and sensitive approach to the detection and diagnosis of GUD using noninvasive techniques to sample genital ulcers. Methods. The genital ulcer disease multiplex polymerase chain reaction (GUMP) was developed as an inhouse nucleic acid amplification technique targeting serious causes of GUD, namely, herpes simplex viruses (HSVs), Haemophilus ducreyi, Treponema pallidum, and Klebsiella species. In addition, the GUMP assay included an endogenous internal control. Amplification products from GUMP were detected by enzyme linked amplicon hybridization assay (ELAHA). Results. GUMP-ELAHA was sensitive and specific in detecting a target microbe in 34.3% of specimens, including 1 detection of HSV-1, three detections of HSV-2, and 18 detections of T. pallidum. No H. ducreyi has been detected in Australia since 1998, and none was detected here. No Calymmatobacterium ( Klebsiella) granulomatis was detected in the study, but there were 3 detections during ongoing diagnostic use of GUMP-ELAHA in 2004 and 2005. The presence of C. granulomatis was confirmed by restriction enzyme digestion and nucleotide sequencing of the 16S rRNA gene for phylogenetic analysis. Conclusions. GUMP-ELAHA permitted comprehensive detection of common and rare causes of GUD and incorporated noninvasive sampling techniques. Data obtained by using GUMP-ELAHA will aid specific treatment of GUD and better define the prevalence of each microbe among at-risk populations with a view to the eradication of chancroid and donovanosis in Australia.

Experimental validation of the regulated expression of large numbers of non-coding RNAs from the mouse genome

Recent large-scale analyses of mainly full-length cDNA libraries generated from a variety of mouse tissues indicated that almost half of all representative cloned sequences did flat contain ail apparent protein-coding sequence, and were putatively derived from non-protein-coding RNA (ncRNA) genes. However, many of these clones were singletons and the majority were unspliced, raising the possibility that they may be derived from genomic DNA or unprocessed pre-rnRNA contamination during library construction, or alternatively represent nonspecific transcriptional noise. Here we Show, using reverse transcriptase-dependent PCR, microarray, and Northern blot analyses, that many of these clones were derived from genuine transcripts Of unknown function whose expression appears to be regulated. The ncRNA transcripts have larger exons and fewer introns than protein-coding transcripts. Analysis of the genomic landscape around these sequences indicates that some cDNA clones were produced not from terminal poly(A) tracts but internal priming sites within longer transcripts, only a minority of which is encompassed by known genes. A significant proportion of these transcripts exhibit tissue-specific expression patterns, as well as dynamic changes in their expression in macrophages following lipopolysaccharide Stimulation. Taken together, the data provide strong support for the conclusion that ncRNAs are an important, regulated component of the mammalian transcriptome.

Exploring synonymous codon usage preferences of disulfide-bonded and non-disulfide bonded cysteines in the E-coli genome

High-quality data about protein structures and their gene sequences are essential to the understanding of the relationship between protein folding and protein coding sequences. Firstly we constructed the EcoPDB database, which is a high-quality database of Escherichia coli genes and their corresponding PDB structures. Based on EcoPDB, we presented a novel approach based on information theory to investigate the correlation between cysteine synonymous codon usages and local amino acids flanking cysteines, the correlation between cysteine synonymous codon usages and synonymous codon usages of local amino acids flanking cysteines, as well as the correlation between cysteine synonymous codon usages and the disulfide bonding states of cysteines in the E. coli genome. The results indicate that the nearest neighboring residues and their synonymous codons of the C-terminus have the greatest influence on the usages of the synonymous codons of cysteines and the usage of the synonymous codons has a specific correlation with the disulfide bond formation of cysteines in proteins. The correlations may result from the regulation mechanism of protein structures at gene sequence level and reflect the biological function restriction that cysteines pair to form disulfide bonds. The results may also be helpful in identifying residues that are important for synonymous codon selection of cysteines to introduce disulfide bridges in protein engineering and molecular biology. The approach presented in this paper can also be utilized as a complementary computational method and be applicable to analyse the synonymous codon usages in other model organisms. (c) 2005 Elsevier Ltd. All rights reserved.

Genome-wide analysis of mammalian promoter architecture and evolution

Mammalian promoters can be separated into two classes, conserved TATA box-enriched promoters, which initiate at a welldefined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3' UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis-acting elements associated with them.

Translation of the flavivirus Kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging

Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.

Dynamics of gene expression in an artificial genome

Complex systems techniques provide a powerful tool to study the emergent properties of networks of interacting genes. In this study we extract models of genetic regulatory networks from an artificial genome, represented by a sequence of nucleotides, and analyse how variations in the connectivity and degree of inhibition of the extracted networks affects the resulting classes of behaviours. For low connectivity systems were found to be very stable. Only with higher connectivity was a significant occurrence of chaos found. Most interestingly, the peak in occurrence of chaos occurs perched on the edge of a phase transition in the occurrence of attractors.

Sequence analysis and distribution in Salmonella enterica serovars of IS3-like elements

The genome of Salmonella enterica serovar Enteritidis was shown to possess three IS3-like insertion elements, designated IS1230A, B and C, and each was cloned and their respective deoxynucleotide sequences determined. Mutations in elements IS1230A and B resulted in frameshifts in the open reading frames that encoded a putative transposase to be inactive. IS1230C was truncated at nucleotide 774 relative to IS1230B and therefore did not possess the 3' terminal inverted repeat. The three IS1230 derivatives were closely related to each other based on nucleotide sequence similarity. IS1230A was located adjacent to the sef operon encoding SEF14 fimbriae located at minute 97 of the genome of S. Enteritidis. IS1230B was located adjacent to the umuDC operon at minute 42.5 on the genome, itself located near to one terminus of an 815-kb genome inversion of S. Enteritidis relative to S. Typhimurium. IS1230C was located next to attB, the bacteriophage P22 attachment site, and proB, encoding gamma-glutamyl phosphate reductase. A truncated 3' remnant of IS1230, designated IS1230T, was identified in a clinical isolate of S. Typhimurium DT193 strain 2391. This element was located next to attB adjacent to which were bacteriophage P22-like sequences. Southern hybridisation of total genomic DNA from eighteen phage types of S. Enteritidis and eighteen definitive types of S. Typhimurium showed similar, if not identical, restriction fragment profiles in the respective serovars when probed with IS1230A.

Sequence-related structural DNA anomalies affect restriction enzyme activity and DNA polymerase I binding

DNA serves as a target molecule for several types of enzymes and may assume a wide variety of structural motifs depending upon the local sequence. The BssHII restriction site (GC)3 resides in a 9bp region of alternating pyrimidine and purine residues within the &phis;X174 genome. Such sequences are known to demonstrate non-canonical helical behavior under the appropriate conditions. The kinetics of BssHII cleavage was investigated in supercoiled and linear plasmid DNA, and in a 323bp DNA fragment obtained via amplification of &phis;X174. The rate of enzyme cleavage was enhanced in the supercoiled form and in the presence of 50μM cobalt hexamine. Similarly, cobalt hexamine was also found to enhance TaqI activity directly adjacent to the (GC)3 region. ^ Initial DNA polymerase I binding studies (including a gel mobility shift assay and a protection assay) indicated a notable interaction between DNA polymerase I and the BssHII site. An in-depth study revealed that equilibrium binding of DNA polymerase I to the T7 RNA polymerase promoter was comparable to that of the (GC)3 site, however the strongest interaction was observed with a cruciform containing region. Increasing the ionic strength of the solution environment, including the addition of DNA polymerase I reaction buffer significantly decreased the equilibrium dissociation constant values. ^ It is suggested that the region within or around the BssHII site experiences a conformational change generating a novel structure under the influence of supercoiled tension or 50μM cobalt hexamine. It is proposed that this transition may enhance enzyme activity and binding by providing an initial enzyme-docking site—the rate-limiting step in restriction enzyme kinetics. The high binding potential of DNA polymerase I for each of the motifs described, is hypothesized to be due to recognition of the structural DNA anomalies by the 3′–5′ exonuclease domain. ^

Targeted sequencing for high-resolution evolutionary analyses following genome duplication in salmonid fish : Proof of concept for key components of the insulin-like growth factor axis

Acknowledgements This study was funded by a Natural Environment Research Council grant (NERC, project code: NBAF704). FML is funded by a NERC Doctoral Training Grant (Project Reference: NE/L50175X/1). RLS was an undergraduate student at the University of Aberdeen and benefitted from financial support from the School of Biological Sciences. DJM is indebted to Dr. Steven Weiss (University of Graz, Austria), Dr. Takashi Yada (National Research Institute of Fisheries Science, Japan), Dr. Robert Devlin (Fisheries and Oceans Canada, Canada), Prof. Samuel Martin (University of Aberdeen, UK), Mr. Neil Lincoln (Environment Agency, UK) and Prof. Colin Adams/Mr. Stuart Wilson (University of Glasgow, UK) for providing salmonid material or assisting with its sampling. We are grateful to staff at the Centre for Genomics Research (University of Liverpool, UK) (i.e. NERC Biomolecular Analysis Facility – Liverpool; NBAF-Liverpool) for performing sequence capture/Illumina sequencing and providing us with details on associated methods that were incorporated into the manuscript. Finally, we are grateful to the organizers of the Society of Experimental Biology Satellite meeting 'Genome-powered perspectives in integrative physiology and evolutionary biology' (held in Prague, July 2015) for inviting us to contribute to this special edition of Marine Genomics and hosting a really stimulating meeting.