Comparative genome analysis--a novel strategy to study the genetics of host-parasite interaction.
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Animals↗
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Genomic analysis reveals chromosomal variation in natural populations of the uncultured psychrophilic archaeon Cenarchaeum symbiosum.
Molecular phylogenetic surveys have recently revealed an ecologically widespread crenarchaeal group that inhabits cold and temperate terrestrial and marine environments. To date these organisms have resisted isolation in pure culture, and so their phenotypic and genotypic characteristics remain largely unknown. To characterize these archaea, and to extend methodological approaches for characterizing uncultivated microorganisms, we initiated genomic analyses of the nonthermophilic crenarchaeote Cenarchaeum symbiosum found living in association with a marine sponge, Axinella mexicana. Complex DNA libraries derived from the host-symbiont population yielded several large clones containing the ribosomal operon from C. symbiosum. Unexpectedly, cloning and sequence analysis revealed the presence of two closely related variants that were consistently found in the majority of host individuals analyzed. Homologous regions from the two variants were sequenced and compared in detail. The variants exhibit >99.2% sequence identity in both small- and large-subunit rRNA genes and they contain homologous protein-encoding genes in identical order and orientation over a 28-kbp overlapping region. Our study not only indicates the potential for characterizing uncultivated prokaryotes by genome sequencing but also identifies the primary complication inherent in the approach: the widespread genomic microheterogeneity in naturally occurring prokaryotic populations.
Animals↗
Genomic analysis and growth-phase-dependent regulation of the SEF14 fimbriae of Salmonella enterica serovar Enteritidis.
Salmonella enterica serovar Enteritidis is a leading cause of food poisoning in the USA and Europe. Although Salmonella serovars share many fimbrial operons, a few fimbriae are limited to specific Samonella serovars. SEF14 fimbriae are restricted to group D Salmonella and the genes encoding this virulence factor were acquired relatively recently. Genomic, genetic and gene expression studies have been integrated to investigate the ancestry, regulation and expression of the sef genes. Genomic comparisons of the Salmonella serovars sequenced revealed that the sef operon is inserted in leuX in Salmonella Enteritidis, Salmonella Paratyphi and Salmonella Typhi, and revealed the presence of a previously unidentified 25 kb pathogenicity island in Salmonella Typhimurium at this location. Salmonella Enteritidis contains a region of homology between the Salmonella virulence plasmid and the chromosome downstream of the sef operon. The sef operon itself consists of four co-transcribed genes, sefABCD, and adjacent to sefD there is an AraC-like transcriptional activator that is required for expression of the sef genes. Expression of the sef genes was optimal during growth in late exponential phase and was repressed during stationary phase. The regulation was coordinated by the RpoS sigma factor.
Amino Acid Sequence↗
Genomic analysis of mouse retinal development.
The vertebrate retina is comprised of seven major cell types that are generated in overlapping but well-defined intervals. To identify genes that might regulate retinal development, gene expression in the developing retina was profiled at multiple time points using serial analysis of gene expression (SAGE). The expression patterns of 1,051 genes that showed developmentally dynamic expression by SAGE were investigated using in situ hybridization. A molecular atlas of gene expression in the developing and mature retina was thereby constructed, along with a taxonomic classification of developmental gene expression patterns. Genes were identified that label both temporal and spatial subsets of mitotic progenitor cells. For each developing and mature major retinal cell type, genes selectively expressed in that cell type were identified. The gene expression profiles of retinal Müller glia and mitotic progenitor cells were found to be highly similar, suggesting that Müller glia might serve to produce multiple retinal cell types under the right conditions. In addition, multiple transcripts that were evolutionarily conserved that did not appear to encode open reading frames of more than 100 amino acids in length ("noncoding RNAs") were found to be dynamically and specifically expressed in developing and mature retinal cell types. Finally, many photoreceptor-enriched genes that mapped to chromosomal intervals containing retinal disease genes were identified. These data serve as a starting point for functional investigations of the roles of these genes in retinal development and physiology.
Animals↗
Insights into extreme thermoacidophily based on genome analysis of Picrophilus torridus and other thermoacidophilic archaea.
Thermoacidophiles are prokaryotic microorganisms with the stunning capability to survive and multiply at extremely low pH and simultaneously at high temperatures. The mechanisms by which these organisms, exclusively members of the Archaea, cope with their harsh surroundings are poorly understood. The genome sequences of several representatives of the thermoacidophilic genera Picrophilus, Thermoplasma and Sulfolobus have recently become available. Genome-wide comparison has revealed a number of features as possible facets of the overall acidophilic survival strategy of the most thermoacidophilic organisms known, such as a high ratio of secondary over primary transport systems, the composition of the respiratory chain, and the frequent genetic input via lateral gene transfer (LGT) during evolution.
Acclimatization↗
Genome analysis of lactic acid bacteria in food fermentations and biotechnological applications.
Lactic acid bacteria are an important group of microorganisms, several of which are used in fermented food processes. Lactococcus lactis is a non-pathogenic, non-invasive and non-colonising gram-positive lactic acid bacterium, the genome sequence of which has been established. A great deal is known about the genetics, vectors, gene expression systems and protein secretion apparatus of this bacterium. Recently, recombinant strains of L. lactis have been developed that might provide in vivo delivery of cytokines and specific antigens across mucosal surfaces to the immune system of animals.
Biotechnology↗
Integrative genomic analysis of protein kinase C (PKC) family identifies PKCiota as a biomarker and potential oncogene in ovarian carcinoma.
The protein kinase C (PKC) family plays a key regulatory role in a wide range of cellular functions as well as in various cancer-associated signal transduction pathways. Here, we investigated the genomic alteration and gene expression of most known PKC family members in human ovarian cancer. The DNA copy number of PKC family genes was screened by a high-resolution array-based comparative genomic hybridization in 89 human ovarian cancer specimens. Five PKC genes exhibited significant DNA copy number gains, including PKCiota (43.8%), PKCbeta1 (37.1%), PKCgamma (27.6%), PKCzeta (22.5%), and PKCtheta (21.3%). None of the PKC genes exhibited copy number loss. The mRNA expression level of PKC genes was analyzed by microarray retrieval approach. Two of the amplified PKC genes, PKCiota and PKCtheta, were significantly up-regulated in ovarian cancer compared with normal ovary. Increased PKCiota expression correlated with tumor stage or grade, and PKCiota overexpression was seen mostly in ovarian carcinoma but not in other solid tumors. The above results were further validated by real-time reverse transcription-PCR with 54 ovarian cancer specimens and 24 cell lines; overexpression of PKCiota protein was also confirmed by tissue array and Western blot. Interestingly, overexpressed PKCiota did not affect ovarian cancer cell proliferation or apoptosis in vitro. However, decreased PKCiota expression significantly reduced anchorage-independent growth of ovarian cancer cells, whereas overexpression of PKCiota contributed to murine ovarian surface epithelium transformation in cooperation with mutant Ras. We propose that PKCiota may serve as an oncogene and a biomarker of aggressive disease in human ovarian cancer.
Biomarkers, Tumor↗
Genomic analysis of murine pulmonary tissue following carbonyl chloride inhalation.
Carbonyl chloride (phosgene) is a toxic industrial compound widely used in industry for the production of synthetic products, such as polyfoam rubber, plastics, and dyes. Exposure to phosgene results in a latent (1-24 h), potentially life-threatening pulmonary edema and irreversible acute lung injury. A genomic approach was utilized to investigate the molecular mechanism of phosgene-induced lung injury. CD-1 male mice were exposed whole body to either air or a concentration x time amount of 32 mg/m3 (8 ppm) phosgene for 20 min (640 mg x min/m3). Lung tissue was collected from air- or phosgene-exposed mice at 0.5, 1, 4, 8, 12, 24, 48, and 72 h postexposure. RNA was extracted from the lung and used as starting material for the probing of oligonucleotide microarrays to determine changes in gene expression following phosgene exposure. The data were analyzed using principal component analysis to determine the greatest sources of data variability. A three-way analysis of variance based on exposure, time, and sample was performed to identify the genes most significantly changed as a result of phosgene exposure. These genes were rank ordered by p values and categorized based on molecular function and biological process. Some of the most significant changes in gene expression reflect changes in glutathione synthesis and redox regulation of the cell, including upregulation of glutathione S-transferase alpha-2, glutathione peroxidase 2, and glutamate-cysteine ligase, catalytic subunit (also known as gamma-glutamyl cysteine synthetase). This is in agreement with previous observations describing changes in redox enzyme activity after phosgene exposure. We are also investigating other pathways that are responsive to phosgene exposure to identify mechanisms of toxicity and potential therapeutic targets.
Administration, Inhalation↗
FASSM: enhanced function association in whole genome analysis using sequence and structural motifs.
We present an algorithm to detect remote homology, which arises through circular permutation and discontinuous domains. It is also helpful in detecting small domain proteins that are characterized by few conserved residues. The input to the algorithm is a set of multiply aligned protein sequence profiles. This method, coded as FASSM, examines the sequence conservation and positions of protein family signatures or motifs for the annotation of protein sequences and to facilitate the analysis of their domains. The overall coverage of FASSM is 93% in comparison to other validation tools like HMM and IMPALA. The method is especially useful for difficult relationships such as discontinuous domains during whole-genome surveys and is demonstrated to perform accurate family associations at sequence identities as low as 15%.
Algorithms↗
Comparative genome analysis identifies the vitamin D receptor gene as a direct target of p53-mediated transcriptional activation.
p53 is the most frequently mutated tumor suppressor gene in human neoplasia and encodes a transcriptional coactivator. Identification of p53 target genes is therefore key to understanding the role of p53 in tumorigenesis. To identify novel p53 target genes, we first used a comparative genomics approach to identify p53 binding sequences conserved in the human and mouse genome. We hypothesized that potential p53 binding sequences that are conserved are more likely to be functional. Using stringent filtering procedures, 32 genes were newly identified as putative p53 targets, and their responsiveness to p53 in human cancer cells was confirmed by reverse transcription-PCR and real-time PCR. Among them, we focused on the vitamin D receptor (VDR) gene because vitamin D3 has recently been used for chemoprevention of human tumors. VDR is induced by p53 as well as several other p53 family members, and analysis of chromatin immunoprecipitation showed that p53 protein binds to conserved intronic sequences of the VDR gene in vivo. Introduction of VDR into cells resulted in induction of several genes known to be p53 targets and suppression of colorectal cancer cell growth. In addition, p53 induced VDR target genes in a vitamin D3-dependent manner. Our in silico approach is a powerful method for identification of functional p53 binding sites and p53 target genes that are conserved among humans and other organisms and for further understanding the function of p53 in tumorigenesis.
Animals↗
Comparative whole-genome analysis of virulent and avirulent strains of Porphyromonas gingivalis.
We used Porphyromonas gingivalis gene microarrays to compare the total gene contents of the virulent strain W83 and the avirulent type strain, ATCC 33277. Signal ratios and scatter plots indicated that the chromosomes were very similar, with approximately 93% of the predicted genes in common, while at least 7% of them showed very low or no signals in ATCC 33277. Verification of the array results by PCR indicated that several of the disparate genes were either absent from or variant in ATCC 33277. Divergent features included already reported insertion sequences and ragB, as well as additional hypothetical and functionally assigned genes. Several of the latter were organized in a putative operon in W83 and encoded enzymes involved in capsular polysaccharide synthesis. Another cluster was associated with two paralogous regions of the chromosome with a low G+C content, at 41%, compared to that of the whole genome, at 48%. These regions also contained conserved and species-specific hypothetical genes, transposons, insertion sequences, and integrases and were located adjacent to tRNA genes; thus, they had several characteristics of pathogenicity islands. While this global comparative analysis showed the close relationship between W83 and ATCC 33277, the clustering of genes that are present in W83 but divergent in or absent from ATCC 33277 is suggestive of chromosomal islands that may have been acquired by lateral gene transfer.
Bacterial Capsules↗
Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli.
The recognition and phagocytosis of microbes by macrophages is a principal aspect of innate immunity that is conserved from insects to humans. Drosophila melanogaster has circulating macrophages that phagocytose microbes similarly to mammalian macrophages, suggesting that insect macrophages can be used as a model to study cell-mediated innate immunity. We devised a double-stranded RNA interference-based screen in macrophage-like Drosophila S2 cells, and have defined 34 gene products involved in phagocytosis. These include proteins that participate in haemocyte development, vesicle transport, actin cytoskeleton regulation and a cell surface receptor. This receptor, Peptidoglycan recognition protein LC (PGRP-LC), is involved in phagocytosis of Gram-negative but not Gram-positive bacteria. Drosophila humoral immunity also distinguishes between Gram-negative and Gram-positive bacteria through the Imd and Toll pathways, respectively; however, a receptor for the Imd pathway has not been identified. Here we show that PGRP-LC is important for antibacterial peptide synthesis induced by Escherichia coli both in vitro and in vivo. Furthermore, totem mutants, which fail to express PGRP-LC, are susceptible to Gram-negative (E. coli), but not Gram-positive, bacterial infection. Our results demonstrate that PGRP-LC is an essential component for recognition and signalling of Gram-negative bacteria. Furthermore, this functional genomic approach is likely to have applications beyond phagocytosis.
Actins↗
Spatiotemporal and genomic analysis of carbapenem resistance elements in Enterobacterales from hospital inpatients and natural water ecosystems of an Irish city.
Carbapenemase-producing Enterobacterales (CPE) is a diverse group of often multidrug-resistant organisms. Surveillance and control of infections are complicated due to the inter-species spread of carbapenemase-encoding genes (CEGs) on mobile genetic elements (MGEs), including plasmids and transposons. Due to wastewater discharges, urban water ecosystems represent a known reservoir of CPE. However, the dynamics of carbapenemase-bearing MGE dissemination between Enterobacterales in humans and environmental waters are poorly understood. We carried out whole-genome sequencing, combining short- and long-sequencing reads to enable complete characterization of CPE isolated from patients, wastewaters, and natural waters between 2018 and 2020 in Galway, Ireland. Isolates were selected based on their carriage of Class A blaKPC-2 (n = 6), Class B blaNDM-5 (n = 12), and Class D blaOXA-48 (n = 21) CEGs. CEGs were plasmid-borne in all but two isolates. OXA-48 dissemination was associated with a 64 kb IncL plasmid (62%), in a broad range of Enterobacterales isolates from both niches. Conversely, blaKPC-2 and blaNDM-5 genes were usually carried on larger and more variable multireplicon IncF plasmids in Klebsiella pneumoniae and Escherichia coli, respectively. In every isolate, each CEG was surrounded by a gene-specific common genetic environment which constituted part, or all, of a transposable element that was present in both plasmids and the bacterial chromosome. Transposons Tn1999 and Tn4401 were associated with blaOXA-48 and blaKPC-2, respectively, while blaNDM-5 was associated with variable IS26 bound composite transposons, usually containing a class 1 integron.IMPORTANCESince 2018, the Irish National Carbapenemase-Producing Enterobacterales (CPE) Reference Laboratory Service at University Hospital Galway has performed whole-genome sequencing on suspected and confirmed CPE from clinical specimens as well as patient and environmental screening isolates. Understanding the dynamics of CPE and carbapenemase-encoding gene encoding mobile genetic element (MGE) flux between human and environmental reservoirs is important for One Health surveillance of these priority organisms. We employed hybrid assembly approaches for improved resolution of CPE genomic surveillance, typing, and plasmid characterization. We analyzed a diverse collection of human (n = 17) and environmental isolates (n = 22) and found common MGE across multiple species and in different ecological niches. The conjugation ability and frequency of a subset of these plasmids were demonstrated to be affected by the presence or absence of necessary conjugation genes and by plasmid size. We characterize several MGE at play in the local dissemination of carbapenemase genes. This may facilitate their future detection in the clinical laboratory.
Humans↗
Optimized conditions of PacI and SwaI for genomic analysis of X. axonopodis pv. vesicatoria by PFGE.
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DNA Restriction Enzymes↗
Comparative genomic analysis of equilibrative nucleoside transporters suggests conserved protein structure despite limited sequence identity.
Equilibrative nucleoside transporters (ENTs) are a recently characterized and poorly understood group of membrane proteins that are important in the uptake of endogenous nucleosides required for nucleic acid and nucleoside triphosphate synthesis. Despite their central importance in cellular metabolism and nucleoside analog chemotherapy, no human ENT gene has been described and nothing is known about gene structure and function. To gain insight into the ENT gene family, we used experimental and in silico comparative genomic approaches to identify ENT genes in three evolutionarily diverse organisms with completely (or almost completely) sequenced genomes, Homo sapiens, Caenorhabditis elegans and Drosophila melanogaster. We describe the chromosomal location, the predicted ENT gene structure and putative structural topologies of predicted ENT proteins derived from the open reading frames. Despite variations in genomic layout and limited ortholog protein sequence identity (< or =27.45%), predicted topologies of ENT proteins are strikingly similar, suggesting an evolutionary conservation of a prototypic structure. In addition, a similar distribution of protein domains on exons is apparent in all three taxa. These data demonstrate that comparative sequence analyses should be combined with other approaches (such as genomic and proteomic analyses) to fully understand structure, function and evolution of protein families.
Alternative Splicing↗
Two-dimensional bacterial genome display: a method for the genomic analysis of mycobacteria.
Annually, Mycobacterium tuberculosis is the cause of approximately three million deaths worldwide. It would appear that currently available therapies for this disease are inadequate. The identification of genes involved in mycobacterial virulence will facilitate the design of new prophylactic and therapeutic interventions. A method for high-resolution comparison of bacterial genomes has been developed to facilitate the identification of genes possibly involved in the virulence of clinically relevant mycobacteria. This 'two-dimensional bacterial genome display' (2DBGD) method utilizes two-dimensional DNA electrophoresis to separate, on the basis of size and G+C content, genomic fragments generated with different restriction endonucleases. The use of this method to identify genomic differences between species, strains and, most importantly, isogenic mutants of mycobacteria is reported. That 2DBGD can be used to identify differences resulting from either insertional mutagenesis using a gentamicin-resistance gene or from a frameshift mutation is demonstrated.
Bacterial Proteins↗
Complete hepatitis B virus genome analysis in HBsAg positive mothers and their infants with fulminant hepatitis B.
BACKGROUND: After perinatal transmission of hepatitis B virus, infants of anti-HBe positive HBsAg carrier mothers may develop fulminant hepatitis B. Previously it has been suggested, that fulminant hepatitis B in adults was associated with specific mutations in the HBV-genome. The aim of this study was to investigate, whether specific viral variants are associated with fulminant hepatitis B in young infants. METHODS: The complete HBV-genomes of five mothers and their infants with fulminant hepatitis were isolated from the sera, amplified and directly sequenced. RESULTS: Between 6 and 43 base pair exchanges between the HBV genomes of the infants and their mothers were identified. The mutations spread over the entire virus genome. Nucleotide exchanges in the basic core promotor and precore region were identified in all cases. A heterogeneous virus population was detected in four mothers. CONCLUSIONS: Many new mutations were proved to emerge during fulminant hepatitis B in infants, who had been perinatally infected. HBeAg negative variants were the predominant population in all children, whereas these mutants could only be detected as subpopulations in four mothers. The data suggest that the selection of a specific HBeAg negative viral strain may be associated with the development of fulminant hepatitis B in children.
Adult↗
Whole-genome analysis of animal A- and B-type cyclins.
BACKGROUND: Multiple A- and B-type cyclins have been identified in animals, but their study is complicated by varying degrees of functional redundancy. A non-essential phenotype may reflect redundancy with a known or as yet unknown gene. Complete sequencing of several animal genomes has allowed us to determine the size of the mitotic cyclin gene family and therefore to start to address this issue. RESULTS: We analyzed the Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens genomes to identify known and novel A- and B-type cyclin genes and distinguish them from related pseudogenes. We find only a single functional A-type cyclin gene in invertebrates but two in vertebrates. In addition to the single functional cyclin A gene, the C. elegans genome contains numerous cyclin A pseudogenes. In contrast, the number and relationship of B-type cyclins varies considerably between organisms but all contain at least one cyclin B1-like gene and a cyclin B3 gene. CONCLUSIONS: There are three conserved families of mitotic cyclins in animals: A-, B3- and B-type. The precise number of genes within the A- and B-type families varies in different organisms, possibly as an adaptation to their distinct developmental strategies.
Amino Acid Sequence↗