Evolutionary genomics of the Fox genes: origin of gene families and the ancestry of gene clusters

Over the past decade genomic approaches have begun to revolutionise the study of animal diversity. In particular, genome sequencing programmes have spread beyond the traditional model species to encompass an increasing diversity of animals from many different phyla, as well as unicellular eukaryotes that are closely related to the animals. Whole genome sequences allow researchers to establish, with reasonable confidence, the full complement of any particular family of genes in a genome. Comparison of gene complements from appropriate genomes can reveal the evolutionary history of gene families, indicating when both gene diversification and gene loss have occurred. More than that, however, assembled genomes allow the genomic environment in which individual genes are found to be analysed and compared between species. This can reveal how gene diversification occurred. Here, we focus on the Fox genes, drawing from multiple animal genomes to develop an evolutionary framework explaining the timing and mechanism of origin of the diversity of animal Fox genes. Ancient linkages between genes are a prominent feature of the Fox genes, depicting a history of gene clusters, some of which may be relevant to understanding Fox gene function.

Gene duplication and an accelerated evolutionary rate in 11S globulin genes are associated with higher protein synthesis in dicots as compared to monocots

Background: Seed storage proteins are a major source of dietary protein, and the content of such proteins determines both the quantity and quality of crop yield. Significantly, examination of the protein content in the seeds of crop plants shows a distinct difference between monocots and dicots. Thus, it is expected that there are different evolutionary patterns in the genes underlying protein synthesis in the seeds of these two groups of plants. Results: Gene duplication, evolutionary rate and positive selection of a major gene family of seed storage proteins (the 11S globulin genes), were compared in dicots and monocots. The results, obtained from five species in each group, show more gene duplications, a higher evolutionary rate and positive selections of this gene family in dicots, which are rich in 11S globulins, but not in the monocots. Conclusion: Our findings provide evidence to support the suggestion that gene duplication and an accelerated evolutionary rate may be associated with higher protein synthesis in dicots as compared to monocots.

Divergent evolutionary pattern of starch biosynthetic pathway genes in grasses and dicots

Starch is the most widespread and abundant storage carbohydrate in crops and its production is critical to both crop yield and quality. As regards the starch content in the seeds of crop plants, there are distinct difference between grasses (Poaceae) and dicots. However, few studies have described the evolutionary pattern of genes in the starch biosynthetic pathway in these two groups of plants. In this study, therefore, an attempt was made to compare the evolutionary rate, gene duplication and selective pattern of the key genes involved in this pathway between the two groups, using five grasses and five dicots as materials. The results showed (i) distinct differences in patterns of gene duplication and loss between grasses and dicots; duplication in grasses mainly occurred prior to the divergence of grasses, whereas duplication mostly occurred in individual species within the dicots; there is less gene loss in grasses than in dicots; (ii) a considerably higher evolutionary rate in grasses than in dicots in most gene families analyzed; (iii) evidence of a different selective pattern between grasses and dicots; positive selection may have occurred asymmetrically in grasses in some gene families, e.g. AGPase small subunit. Therefore, we deduced that gene duplication contributes to, and a higher evolutionary rate is associated with, the higher starch content in grasses. In addition, two novel aspects of the evolution of the starch biosynthetic pathway were observed.

Identifying antimicrobial resistance genes of human clinical relevance within Salmonella isolated from food animals in Great Britain

To investigate the occurrence of antimicrobial resistance genes of human clinical relevance in Salmonella isolated from livestock in Great Britain. Two hundred and twenty-five Salmonella enterica isolates were characterized using an antimicrobial resistance gene chip and disc diffusion assays. Plasmid profiling, conjugation experiments and identification of Salmonella genomic island 1 (SGI1) were performed for selected isolates. Approximately 43% of Salmonella harboured single or multiple antimicrobial resistance genes with pig isolates showing the highest numbers where 96% of Salmonella Typhimurium harboured one or more resistance genes. Isolates harbouring multiple resistances divided into three groups. Group 1 isolates harboured ampicillin/streptomycin/sulphonamide/tetracycline resistance and similar phenotypes. This group contained isolates from pigs, cattle and poultry that were from several serovars including Typhimurium, 4,[5],12:i:-, Derby, Ohio and Indiana. All Group 2 isolates were from pigs and were Salmonella Typhimurium. They contained a non-sul-type class 1 integron and up to 13 transferrable resistances. All Group 3 isolates harboured a class 1 integron and were isolated from all animal species included in the study. Most isolates were Salmonella Typhimurium and harboured SGI1. Salmonella isolated from livestock was shown to harbour antimicrobial resistance genes although no or little resistance to third-generation cephalosporins or ciprofloxacin, respectively, was detected. The preponderance in pigs of multidrug-resistant Salmonella Typhimurium makes it important to introduce control measures such as improved biosecurity to ensure that they do not pass through the food chain and limit human therapeutic options.

High prevalence of multidrug-tolerant bacteria and associated antimicrobial resistance genes isolated from ornamental fish and their carriage water

Background: Antimicrobials are used to directly control bacterial infections in pet (ornamental) fish and are routinely added to the water these fish are shipped in to suppress the growth of potential pathogens during transport. Methodology/Principal Findings: To assess the potential effects of this sustained selection pressure, 127 Aeromonas spp. isolated from warm and cold water ornamental fish species were screened for tolerance to 34 antimicrobials. Representative isolates were also examined for the presence of 54 resistance genes by a combination of miniaturized microarray and conventional PCR. Forty-seven of 94 Aeromonas spp. isolates recovered from tropical ornamental fish and their carriage water were tolerant to >= 15 antibiotics, representing seven or more different classes of antimicrobial. The quinolone and fluoroquinolone resistance gene, qnrS2, was detected at high frequency (37% tested recent isolates were positive by PCR). Class 1 integrons, IncA/C broad host range plasmids and a range of other antibiotic resistance genes, including floR, blaTEM21, tet(A), tet(D), tet(E), qacE2, sul1, and a number of different dihydrofolate reductase and aminoglycoside transferase coding genes were also detected in carriage water samples and bacterial isolates. Conclusions: These data suggest that ornamental fish and their carriage water act as a reservoir for both multi-resistant bacteria and resistance genes.

Genetic diversity among Escherichia coli O157 : H7 isolates and identification of genes linked to human infections

An Escherichia coli oligonucleotide microarray based on three sequenced genomes was validated for comparative genomic microarray hybridization and used to study the diversity of E. coli O157 isolates from human infections and food and animal sources. Among 26 test strains, 24 (including both Shiga toxin [Stx]-positive and -negative strains) were found to be related to the two sequenced E. coli O157:117 strains, EDL933 and Sakai. However, these strains showed much greater genetic diversity than those reported previously, and most of them could not be categorized as either lineage I or H. Some genes were found more often in isolates from human than from nonhuman sources; e.g., ECs1202 and ECs2976, associated with stx2AB and stx1AB, were in all isolates from human sources but in only 40% of those from nonhuman sources. Some (but not all) lineage I-specific or -dominant genes were also more frequently associated with isolates from human. The results suggested that it might be more effective to concentrate our efforts on finding markers that are directly related to infection rather than those specific to certain lineages. In addition, two Stx-negative O157 cattle isolates (one confirmed to be 117) were significantly different from other Stx-positive and -negative E. coli O157:117 strains and were more similar to MG1655 in their gene content. This work demonstrates that not all E. coli O157:117 strains belong to the same clonal group, and those that were similar to E. coli K-12 might be less virulent.

Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria

We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended-spectrum beta-lactamases. Validation of the array with control strains demonstrated a 99% correlation between polymerase chain reaction and array results. There was also good correlation between phenotypic and genotypic results for a large panel of Escherichia coli and Salmonella isolates. Some differences were also seen in the number and type of resistance genes harboured by E. coli and Salmonella strains. The array provides an effective, fast and simple method for detection of resistance genes in clinical isolates suitable for use in diagnostic laboratories, which in future will help to understand the epidemiology of isolates and to detect gene linkage in bacterial populations. (C) 2008 Published by Elsevier B.V. and the International Society of Chemotherapy.

Escherichia coli isolates from extraintestinal organs of livestock animals harbour diverse virulence genes and belong to multiple genetic lineages

Escherichia coli, the most common cause of bacteraemia in humans in the UK, can also cause serious diseases in animals. However the population structure, virulence and antimicrobial resistance genes of those from extraintestinal organs of livestock animals are poorly characterised. The aims of this study were to investigate the diversity of these isolates from livestock animals and to understand if there was any correlation between the virulence and antimicrobial resistance genes and the genetic backbone of the bacteria and if these isolates were similar to those isolated from humans. Here 39 E. coli isolates from liver (n=31), spleen (n=5) and blood (n=3) of cattle (n=34), sheep (n=3), chicken (n=1) and pig (n=1) were assigned to 19 serogroups with O8 being the most common (n=7), followed by O101, O20 (both n=3) and O153 (n=2). They belong to 29 multi-locus sequence types, 20 clonal complexes with ST23 (n=7), ST10 (n=6), ST117 and ST155 (both n=3) being most common and were distributed among phylogenetic group A (n=16), B1 (n=12), B2 (n=2) and D (n=9). The pattern of a subset of putative virulence genes was different in almost all isolates. No correlation between serogroups, animal hosts, MLST types, virulence and antimicrobial resistance genes was identified. The distributions of clonal complexes and virulence genes were similar to other extraintestinal or commensal E. coli from humans and other animals, suggesting a zoonotic potential. The diverse and various combinations of virulence genes implied that the infections were caused by different mechanisms and infection control will be challenging.

Low relative humidity triggers RNA-directed de novo DNA methylation and suppression of genes controlling stomatal development

Environmental cues influence the development of stomata on the leaf epidermis, and allow plants to exert plasticity in leaf stomatal abundance in response to the prevailing growing conditions. It is reported that Arabidopsis thaliana ‘Landsberg erecta’ plants grown under low relative humidity have a reduced stomatal index and that two genes in the stomatal development pathway, SPEECHLESS and FAMA, become de novo cytosine methylated and transcriptionally repressed. These environmentally-induced epigenetic responses were abolished in mutants lacking the capacity for de novo DNA methylation, for the maintenance of CG methylation, and in mutants for the production of short-interfering non-coding RNAs (siRNAs) in the RNA-directed DNA methylation pathway. Induction of methylation was quantitatively related to the induction of local siRNAs under low relative humidity. Our results indicate the involvement of both transcriptional and post-transcriptional gene suppression at these loci in response to environmental stress. Thus, in a physiologically important pathway, a targeted epigenetic response to a specific environmental stress is reported and several of its molecular, mechanistic components are described, providing a tractable platform for future epigenetics experiments. Our findings suggest epigenetic regulation of stomatal development that allows for anatomical and phenotypic plasticity, and may help to explain at least some of the plant’s resilience to fluctuating relative humidity.

Genistein protects prostate cells against hydrogen peroxide-induced DNA damage and induces expression of genes involved in the defence against oxidative stress

Prostate cancer is one of the most frequent cancer types in Western societies and predominately occurs in the elderly male. The strong age-related increase of prostate cancer is associated with a progressive accumulation of oxidative DNA damage which is presumably supported by a decline of the cellular antioxidative defence during ageing. Risk of developing prostate cancer is much lower in many Asian countries where soy food is an integral part of diet. Therefore, isoflavones from soy were suggested to have chemopreventive activities in prostate cells. Here, we have investigated the hypothesis that the soy-isoflavone genistein could protect DNA of LAPC-4 prostate cells from oxidative stress-related damage by enhancing the expression of antioxidative genes and proteins. A 24 h preincubation with genistein (1-30 microM) protected cells from hydrogen peroxide-induced DNA damage, as determined by the comet assay. Analysis of two cDNA macroarrays, each containing 96 genes of biotransformation and stress response, revealed a modulated expression of 3 genes at 1 microM and of 19 genes at 10 microM genistein. Real-time PCR confirmed the induction of three genes encoding products with antioxidant activities, namely glutathione reductase (2.7-fold), microsomal glutathione S-transferase 1 (1.9-fold) and metallothionein 1X (6.3-fold), at 1-30 microM genistein. 17Beta-estradiol, in contrast, decreased the expression of metallothionein 1X at 0.3 microM (2.0-fold), possibly pointing to an estrogen receptor-mediated regulation of this gene. Immunocytochemical staining revealed an induction of metallothionein proteins at 30 microM genistein, while their intracellular localization was unaffected. Metallothioneins were previously found to protect cells from hydrogen peroxide-induced DNA damage. Hence, our findings indicate that genistein protects prostate cells from oxidative stress-related DNA damage presumably by inducing the expression of antioxidative products, such as metallothioneins. Genistein, therefore, might counteract the age-related decline of important antioxidative defence systems which in turn maintain DNA integrity.

Colorectal cancer and the relationship between genes and the environment

Colorectal cancer (CRC) is a significant cause of morbidity and mortality in developed countries, with both genetic and environmental factors contributing to the etiology and progression of the disease. Several risk factors have been identified, including positive family history, red meat intake, smoking, and alcohol intake. Protective factors include vegetables, calcium, hormone replacement therapy, folate, nonsteroidal anti-inflammatory drugs, and physical activity. The interaction between these environmental factors, in particular diet and genes, is an area of growing interest. Currently, oncogenes, tumor suppressor genes, and mismatch repair genes are believed to play an essential role in colorectal carcinogenesis. When considering the genetics of CRC, only 10% of cases are inherited and only 2-6% can be ascribed to the highly penetrant genes, such as APC, hMLH and hMSH2. Lower penetrance genes combined with a Western-style diet contribute to the majority of sporadic CRCs. The purpose of this article is to give a brief overview of the epidemiologic studies that have been conducted and present the major findings. Here, we examine the molecular events in CRC, with particular focus on the interaction between genes and environment, and review the most current research in this area.

Adaptive evolution of four microcephaly genes and the evolution of brain size in anthropoid primates

The anatomical basis and adaptive function of the expansion in primate brain size have long been studied; however, we are only beginning to understand the genetic basis of these evolutionary changes. Genes linked to human primary microcephaly have received much attention as they have accelerated evolutionary rates along lineages leading to humans. However, these studies focus narrowly on apes, and the link between microcephaly gene evolution and brain evolution is disputed. We analyzed the molecular evolution of four genes associated with microcephaly (ASPM, CDK5RAP2, CENPJ, MCPH1) across 21 species representing all major clades of anthropoid primates. Contrary to prevailing assumptions, positive selection was not limited to or intensified along the lineage leading to humans. In fact we show that all four loci were subject to positive selection across the anthropoid primate phylogeny. We developed clearly defined hypotheses to explicitly test if selection on these loci was associated with the evolution of brain size. We found positive relationships between both CDK5RAP2 and ASPM and neonatal brain mass and somewhat weaker relationships between these genes and adult brain size. In contrast, there is no evidence linking CENPJ and MCPH1 to brain size evolution. The stronger association of ASPM and CDK5RAP2 evolution with neonatal brain size than with adult brain size is consistent with these loci having a direct effect on prenatal neuronal proliferation. These results suggest that primate brain size may have at least a partially conserved genetic basis. Our results contradict a previous study that linked adaptive evolution of ASPM to changes in relative cortex size; however, our analysis indicates that this conclusion is not robust. Our finding that the coding regions of two widely expressed loci has experienced pervasive positive selection in relation to a complex, quantitative developmental phenotype provides a notable counterexample to the commonly asserted hypothesis that cisregulatory regions play a dominant role in phenotypic evolution. Key words: ASPM, MCPH1, CDK5RAP2, CENPJ, brain, neurogenesis, primates.

Variation in clonality and antibiotic-resistance genes among multiresistant Salmonella enterica serotype typhimurium phage-type U302 (MR U302) from humans, animals, and foods

Since 1990 multiresistant (MR) Salmonella enterica serotype Typhimurium definitive phage-type (DT) 104 (MR DT104) and closely related phage types have emerged as a worldwide health problem in humans and food animals. In this study the presence of the bla(CARB-2) (ampicillin), cmlA (chloramphenicol), aadA2 (streptomycin/spectinomycin), sul1 (sulphonamide), and tetG (tetracycline) resistance genes in isolates of one such phage type, U302, have been determined. In addition bla(TEM) I primers have been used for the detection of TEM-type beta-lactamases. Isolates have also been characterized by plasmid profile and pulsed field gel electrophoresis (PFGE). Thirty-three of 39 isolates were positive for blaCARB-2, cmlA, aadA2, sul1 and tetG, four for bla(TEM), aadA2 and sul1, one for aadA2 and sul1, and one for blaTEM only. bla(TEM)-mediated ampicillin resistance was transferred to Escherichia coli K12 from three isolates along with other resistance markers, including resistance to chloramphenicol, streptomycin, spectinomycin, sulphonamides, and tetracyclines. Strains carried up to 6 plasmids and 34 plasmid profiles were identified. Although the majority of strains (33/39) produced a PFGE profile identical to that predominant in MR DT104, six different patterns were generated demonstrating the presence of various clones within MR U302. The results show that the majority of the MR U302 strains studied possessed the same antibiotic resistance genes as MR DT104. However, isolates with distinctive PFGE patterns can have different mechanisms of resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracyclines. Such resistance genes may be borne on transmissible plasmids.