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.

What is a gene today?

Historians of genetics agree that multiple conceptions of the gene have coexisted at each stages in the history of genetics and that the resulting partial ambiguity has often contributed to the success of genetics, both because workers in different areas have needed to communicate and to draw on one another’s results despite wrestled with very different scientific challenges, and because empirical findings have often challenged the presuppositions of existing conceptions of the gene. Today, a number of different conceptions of the gene coexist in the biosciences. An ‘instrumental’ gene similar to that of classical genetics retains a critical role in the construction and interpretation of experiments in which the relationship between genotype and phenotype is explored via hybridization between organisms or directly between nucleic acid molecules. It also plays an important theoretical role in the foundations of disciplines such as quantitative genetics and population genetics. A ‘nominal’ gene, defined by the practice of genetic nomenclature, is a critical practical tool and allows communication between bioscientists in a wide range of fields to be grounded in welldefined sequences of nucleotides. This concept, however, does not embody major theoretical insights into genome structure or function. Instead, a ‘post-genomic’ conception of the gene embodies the continuing project of understanding how genome structure supports genome function, but with a deflationary picture of the gene as a structural unit. This final concept of the gene poses a significant challenge to earlier assumptions about the relationship between genome structure and function, and between genotype and phenotype.

A DHA14 drug efflux gene from Xanthomonas albilineans confers high-level albicidin antibiotic resistance in Escherichia coli

Aims: Identification of a gene for self-protection from the antibiotic-producing plant pathogen Xanthomonas albilineans, and functional testing by heterologous expression. Methods and Results: Albicidin antibiotics and phytotoxins are potent inhibitors of prokaryote DNA replication. A resistance gene (albF) isolated by shotgun cloning from the X. albilineans albicidin-biosynthesis region encodes a protein with typical features of DHA14 drug efflux pumps. Low-level expression of albF in Escherichia coli increased the MIC of albicidin 3000-fold, without affecting tsx-mediated albicidin uptake into the periplasm or resistance to other tested antibiotics. Bioinformatic analysis indicates more similarity to proteins involved in self-protection in polyketide-antibiotic-producing actinomycetes than to multi-drug resistance pumps in other Gram-negative bacteria. A complex promoter region may co-regulate albF with genes for hydrolases likely to be involved in albicidin activation or self-protection. Conclusions: AlbF is the first apparent single-component antibiotic-specific efflux pump from a Gram-negative antibiotic producer. It shows extraordinary efficiency as measured by resistance level conferred upon heterologous expression. Significance and Impact of the Study: Development of the clinical potential of albicidins as potent bactericidial antibiotics against diverse bacteria has been limited because of low yields in culture. Expression of albF with recently described albicidin-biosynthesis genes may enable large-scale production. Because albicidins are X. albilineans pathogenicity factors, interference with AlbF function is also an opportunity for control of the associated plant disease.

A diffusion approach to approximating preservation probabilities for gene duplicates

Consider a haploid population and, within its genome, a gene whose presence is vital for the survival of any individual. Each copy of this gene is subject to mutations which destroy its function. Suppose one member of the population somehow acquires a duplicate copy of the gene, where the duplicate is fully linked to the original gene's locus. Preservation is said to occur if eventually the entire population consists of individuals descended from this one which initially carried the duplicate. The system is modelled by a finite state-space Markov process which in turn is approximated by a diffusion process, whence an explicit expression for the probability of preservation is derived. The event of preservation can be compared to the fixation of a selectively neutral gene variant initially present in a single individual, the probability of which is the reciprocal of the population size. For very weak mutation, this and the probability of preservation are equal, while as mutation becomes stronger, the preservation probability tends to double this reciprocal. This is in excellent agreement with simulation studies.

GPNN: Power studies and applications of a neural network method for detecting gene-gene interactions in studies of human disease

Background: The identification and characterization of genes that influence the risk of common, complex multifactorial disease primarily through interactions with other genes and environmental factors remains a statistical and computational challenge in genetic epidemiology. We have previously introduced a genetic programming optimized neural network (GPNN) as a method for optimizing the architecture of a neural network to improve the identification of gene combinations associated with disease risk. The goal of this study was to evaluate the power of GPNN for identifying high-order gene-gene interactions. We were also interested in applying GPNN to a real data analysis in Parkinson's disease. Results: We show that GPNN has high power to detect even relatively small genetic effects (2-3% heritability) in simulated data models involving two and three locus interactions. The limits of detection were reached under conditions with very small heritability (

Molecular cloning and characterization of the mouse Na+ sulfate cotransporter gene (Slc13a4): structure and expression

Sulfate is an essential ion required for numerous functions in mammalian physiology. Due to its hydrophilic nature, cells require sulfate transporters on their plasma membranes to allow entry of sulfate into cells. In this study, we identified a new mouse Na+-sulfate cotransporter (mNaS2), characterized its tissue distribution and determined its cDNA and gene (Slc13a4) structures. mNaS2 mRNA was expressed in placenta, brain, lung, eye, heart, testis, thymus and liver. The mouse NaS2 cDNA spans 3384 nucleotides and its open frame encodes a protein of 624 amino acids. Slc13a4 maps to mouse chromosome 6131 and contains 16 exons, spanning over 40 kb in length. Its 5'-flanking region contains CART- and GC-box motifs and a number of putative transcription factor binding sites, including GATA-1, MTF-1, STAT6 and HNF4 consensus sequences. This is the first study to define the tissue distribution of mNaS2 and resolve its cDNA and gene structures, which will allow us to investigate mNaS2 gene expression in vivo and determine its role in mammalian physiology.

Silencing of the ACC synthase gene ACACS2 causes delayed flowering in pineapple [Ananas comosus (L.) Merr.]

Flowering is a crucial developmental stage in the plant life cycle. A number of different factors, from environmental to chemical, can trigger flowering. In pineapple, and other bromeliads, it has been proposed that flowering is triggered by a small burst of ethylene production in the meristem in response to environmental cues. A 1-amino-cyclopropane-1-carboxylate synthase (ACC synthase) gene has been cloned from pineapple (ACACS2), which is induced in the meristem under the same environmental conditions that induce flowering. Two transgenic pineapple lines have been produced containing co-suppression constructs designed to down-regulate the expression of the ACACS2 gene. Northern analysis revealed that the ACACS2 gene was silenced in a number of transgenic plants in both lines. Southern hybridization revealed clear differences in the methylation status of silenced versus non-silenced plants by the inability of a methylation-sensitive enzyme to digest within the ACACS2 DNA extracted from silenced plants, indicating that methylation is the cause of the observed co-suppression of the ACACS2 gene. Flowering characteristics of the transgenic plants were studied under field conditions in South East Queensland, Australia. Flowering dynamics studies revealed significant differences in flowering behaviour, with transgenic plants exhibiting silencing showing a marked delay in flowering when compared with non-silenced transgenic plants and control non-transformed plants. It is argued that the ACACS2 gene is one of the key contributors towards triggering 'natural flowering' in mature pineapples under commercial field conditions.

The effect of larval age on morphology and gene expression during ascidian metamorphosis

Metamorphosis is both an ecological and a developmental genetic transition that an organism undergoes as a normal part of ontogeny. Many organisms have the ability to delay metamorphosis when conditions are unsuitable. This strategy carries obvious benefits, but may also result in severe consequences for older larvae that run low on energy. In the marine environment, some lecithotrophic larvae that have prolonged periods in the plankton may begin forming postlarval and juvenile structures that normally do not appear until after settlement and the initiation of metamorphosis. This precocious activation of the postlarval developmental program may reflect an adaptation to increase the survival of older, energy-depleted larvae by allowing them to metamorphose more quickly. In the present study, we investigate morphological and genetic consequences of delay of metamorphosis in larvae of Herdmania momus (a solitary stolidobranch ascidian). We observe significant morphological and genetic changes during prolonged larval life, with older larvae displaying significant changes in RNA levels, precocious migration of mesenchyme cells, and changes in larval shape including shortening of the tail. While these observations suggest that the older H. momus larvae are functionally different from younger larvae and possibly becoming more predisposed to undergo metamorphosis, we did not find any significant differences in gene expression levels between postlarvae arising from larvae that metamorphosed as soon as they were competent and postlarvae developing from larvae that postponed metamorphosis. This recalibration, or convergence, of transcript levels in the early postlarva suggests that changes that occur during prolonged larval life of H. momus are not necessarily associated with early activation of adult organ differentiation. Instead, it suggests that an autonomous developmental program is activated in H. momus upon the induction of metamorphosis regardless of the history of the larva.

Increased DNA Methylation at the AXIN1 Gene in a Monozygotic Twin from a Pair Discordant for a Caudal Duplication Anomaly

The AXIN1 gene has been implicated in caudal duplication anomalies. Its coding region was sequenced in both members of a monozygotic ( MZ) twin pair discordant for a caudal duplication anomaly, but no mutation was found. Using bisulfite sequencing, we examined methylation at the promoter region of the AXIN1 gene in these twins and in twin and age-matched singleton controls. Methylation of the promoter region in peripheral blood mononucleated cells was variable among individuals, including MZ pairs. In the MZ pair discordant for the caudal duplication, this region of the affected twin was significantly more methylated than that of the unaffected twin (), which was significantly more P < .0001 methylated than those of the controls (). We have confirmed that this CpG island does function as a promoter P = .02 in vitro and that its activity is inversely proportional to the extent of methylation. This finding raises the possibility that hypermethylation of the AXIN1 promoter, by mechanisms as yet undetermined, is associated with the malformation. This case may be paradigmatic for some cases of MZ discordance.

Rapid screening of 4000 individuals for germ-line variations in the BRAF gene

Background: The BRAF gene is frequently somatically altered in malignant melanoma. A majority of variations are at the valine 600 residue leading to a V600E substitution that constitutively activates the kinase. We screened 4000 patient and control DNAs for germ-line variations at the valine 600 residue. Methods: We developed a novel assay by adapting single-base variation assays and software for MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry to screen for all 5 reported variants at codon 600 of the BRAF gene. We screened a case-control collection comprising samples from 1082 melanoma patients and 154 of their unaffected relatives from 1278 families and from 2744 individuals from 659 unselected twin families with no history of melanoma. A panel of 66 melanoma cell lines was used for variation-positive controls. Results: All melanoma cell lines that we had found previously to carry a codon 600 variation were verified in this study. Three of the 4 possible variants (V600E n = 47, V600K n = 2, V600R n = 1) were detected, but no case of V600D was available. No germ-line variants were found in the samples from the 3980 melanoma patients or from the control individuals. Conclusions: This new assay is a high-throughput, automated alternative to standard sequencing and can be used as a rapid initial screen for somatic variants associated with melanoma. Germ-line variants at valine 600 are unlikely to exist and do not contribute to the reported role of the BRAF gene in melanoma predisposition. (c) 2006 American Association for Clinical Chemistry.

Replication of association of IL1 gene complex members with ankylosing spondylitis in Taiwanese Chinese

Objective: To test the association of interleukin 1 ( IL1) gene family members with ankylosing spondylitis ( AS), previously reported in Europid subjects, in an ethnically remote population. Methods: 200 Taiwanese Chinese AS patients and 200 ethnically matched healthy controls were genotyped for five single nucleotide polymorphisms ( SNPs) and the IL1RN. VNTR, markers previously associated with AS. Allele, genotype, and haplotype frequencies were compared between cases and controls. Results: Association of alleles and genotypes of the markers IL1F10.3, IL1RN. 4, and IL1RN. VNTR was observed with AS ( p < 0.05). Haplotypes of pairs of these markers and of the markers IL1RN.6/1 and IL1RN.6/2 were also significantly associated with AS. The strongest associations observed were with the marker IL1RN. 4, and with the two-marker haplotype IL1RN.4-IL1RN.VNTR ( both p = 0.004). Strong linkage disequilibrium was observed between all marker pairs except those involving IL1B-511 ( D' 0.4 to 0.9, p < 0.01). Conclusions: The IL1 gene cluster is associated with AS in Taiwanese Chinese. This finding provides strong statistical support that the previously observed association of this gene cluster with AS is a true positive finding.