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 {"file": "coffeebrk_NBK2345/A17.nxml", "text": "Only those changes in DNA sequence that have functional consequences are known as disease-causing mutations. One such frequently occurring mutation causes a premature stop codon to appear in the middle of a protein-coding sequence of messenger RNA (mRNA). Stop codons (a triplet of nucleotides: UAA, UAG, or UGA) normally signal the end of the stretch of mRNA that is translated into protein so that when one appears early, the result can be a truncated protein that could have nasty consequences for the host organism.\nHowever, a mechanism known as \"nonsense-mediated decay\" has evolved to detect these harmful RNAs, and sequence analysis suggests that it may have been conserved in eukaryotic organisms, including humans. In yeast, three proteins have been identified that are required to seek and destroy the partly translated RNAs: Upf1p, Upf2p, and Upf3p.\n\nNonsense-mediated decay (NMD) in yeast, as a model for NMD in humans\nRibonuclear proteins that bind to mRNAs in the nucleus remain associated with the mRNA as it becomes attached to the ribosome. When a premature stop codon is present, one of these proteins could be Upf3p. If Upf3p, or another as yet unidentified factor, is recognized by the surveillance complex (represented here by the eye), then the NMD mechanism is triggered. In yeast, this trigger may be assisted by the binding of Upf2p to Upf3p, after which the Upf1p helicase unwinds the mRNA, leaving it open for degradation by a decapping enzyme and exonuclease. Should the premature stop codon not be recognized, translation of the mRNA proceeds and results in the production of a truncated protein.\nUpf1p is an RNA unwinding enzyme, a helicase, that requires ATP for activity. Unfortunately, Upf1p will unwind pretty much anything, not just the problem mRNAs. So Upf2p and Upf3p are thought to be required to help Upf1p discriminate between nonsense and \"real\" mRNAs.\nHow do the core proteins work in synergy to trigger nonsense-mediated decay? One possibility is that Upf3p, along with several other ribonuclear proteins, may first bind to an mRNA as it is being exported from the nucleus en route to the ribosome, the site of protein synthesis. If the mRNA is fully translated into protein, Upf3p and the other protein factors are displaced. However, if there is a premature stop codon, Upf3p and cohorts may sit tight and mark the mutant mRNA as one that needs to be disposed of.\nExperiments have shown that Upf3p can bind Upf2p. Once bound, Upf2p could signal to the \"termination complex\", a mixed bag of termination factors that includes Upf1p. This results in the release of the incomplete polypeptide from the ribosome, mRNA unwinding by Upf1p and, exposure of the mRNA for total degradation by exonuclease.\nAlthough this model is attractive, more experiments are required to show that this actually happens in a living yeast cell.\nMany of the mutations that form a premature stop codon lead to human disease, for example, those in BRCA1 that lead to breast cancer, or those in NF1 that lead to neurofibromatosis type 1, to name just two. There are two ways by which nonsense-mediated decay can play a role in the disease process. The first occurs when the machinery is functioning correctly: if mutant mRNAs are removed, then there will be a reduction in the amount of mRNA and protein available in the cell. The second is when a mutation occurs in the nonsense-mediated decay process itself, such as a mutation in RENT1, a human homolog of Upf1p, resulting in a population of truncated proteins, which could be harmful when targeted to their site of function.\n\n\n\n\nUse BLAST to search for relatives of yeast Upf1p\nCreated: October 13, 1999\nClick on the link below to start an html tutorial.\nFind relatives of yeast Upf1p\nLive PubMed searches\n(1) Upf1p\n\n(2) Nonsense mediated decay\n\n(3) Premature stop codon AND disease\n\n(4) REVIEWS\n\nRelated resources\n\nGeneMap\n\n\nOMIM\n\n\nEntrez Gene\n", "pairs": [["litarch_figures/df/45/coffeebrk_NBK2345/A642.jpg", "\nNonsense-mediated decay (NMD) in yeast, as a model for NMD in humans\nRibonuclear proteins that bind to mRNAs in the nucleus remain associated with the mRNA as it becomes attached to the ribosome. When a premature stop codon is present, one of these proteins could be Upf3p. If Upf3p, or another as yet unidentified factor, is recognized by the surveillance complex (represented here by the eye), then the NMD mechanism is triggered. In yeast, this trigger may be assisted by the binding of Upf2p to Upf3p, after which the Upf1p helicase unwinds the mRNA, leaving it open for degradation by a decapping enzyme and exonuclease. Should the premature stop codon not be recognized, translation of the mRNA proceeds and results in the production of a truncated protein.\n", ""]], "interleaved": [["Only those changes in DNA sequence that have functional consequences are known as disease-causing mutations. One such frequently occurring mutation causes a premature stop codon to appear in the middle of a protein-coding sequence of messenger RNA (mRNA). Stop codons (a triplet of nucleotides: UAA, UAG, or UGA) normally signal the end of the stretch of mRNA that is translated into protein so that when one appears early, the result can be a truncated protein that could have nasty consequences for the host organism."], ["However, a mechanism known as \"nonsense-mediated decay\" has evolved to detect these harmful RNAs, and sequence analysis suggests that it may have been conserved in eukaryotic organisms, including humans. In yeast, three proteins have been identified that are required to seek and destroy the partly translated RNAs: Upf1p, Upf2p, and Upf3p."], ["litarch_figures/df/45/coffeebrk_NBK2345/A642.jpg", "\nNonsense-mediated decay (NMD) in yeast, as a model for NMD in humans\nRibonuclear proteins that bind to mRNAs in the nucleus remain associated with the mRNA as it becomes attached to the ribosome. When a premature stop codon is present, one of these proteins could be Upf3p. If Upf3p, or another as yet unidentified factor, is recognized by the surveillance complex (represented here by the eye), then the NMD mechanism is triggered. In yeast, this trigger may be assisted by the binding of Upf2p to Upf3p, after which the Upf1p helicase unwinds the mRNA, leaving it open for degradation by a decapping enzyme and exonuclease. Should the premature stop codon not be recognized, translation of the mRNA proceeds and results in the production of a truncated protein.\n", ""], ["Nonsense-mediated decay (NMD) in yeast, as a model for NMD in humans"], ["Ribonuclear proteins that bind to mRNAs in the nucleus remain associated with the mRNA as it becomes attached to the ribosome. When a premature stop codon is present, one of these proteins could be Upf3p. If Upf3p, or another as yet unidentified factor, is recognized by the surveillance complex (represented here by the eye), then the NMD mechanism is triggered. In yeast, this trigger may be assisted by the binding of Upf2p to Upf3p, after which the Upf1p helicase unwinds the mRNA, leaving it open for degradation by a decapping enzyme and exonuclease. Should the premature stop codon not be recognized, translation of the mRNA proceeds and results in the production of a truncated protein."], ["Upf1p is an RNA unwinding enzyme, a helicase, that requires ATP for activity. Unfortunately, Upf1p will unwind pretty much anything, not just the problem mRNAs. So Upf2p and Upf3p are thought to be required to help Upf1p discriminate between nonsense and \"real\" mRNAs."], ["How do the core proteins work in synergy to trigger nonsense-mediated decay? One possibility is that Upf3p, along with several other ribonuclear proteins, may first bind to an mRNA as it is being exported from the nucleus en route to the ribosome, the site of protein synthesis. If the mRNA is fully translated into protein, Upf3p and the other protein factors are displaced. However, if there is a premature stop codon, Upf3p and cohorts may sit tight and mark the mutant mRNA as one that needs to be disposed of."], ["Experiments have shown that Upf3p can bind Upf2p. Once bound, Upf2p could signal to the \"termination complex\", a mixed bag of termination factors that includes Upf1p. This results in the release of the incomplete polypeptide from the ribosome, mRNA unwinding by Upf1p and, exposure of the mRNA for total degradation by exonuclease."], ["Although this model is attractive, more experiments are required to show that this actually happens in a living yeast cell."], ["Many of the mutations that form a premature stop codon lead to human disease, for example, those in BRCA1 that lead to breast cancer, or those in NF1 that lead to neurofibromatosis type 1, to name just two. There are two ways by which nonsense-mediated decay can play a role in the disease process. The first occurs when the machinery is functioning correctly: if mutant mRNAs are removed, then there will be a reduction in the amount of mRNA and protein available in the cell. The second is when a mutation occurs in the nonsense-mediated decay process itself, such as a mutation in RENT1, a human homolog of Upf1p, resulting in a population of truncated proteins, which could be harmful when targeted to their site of function.\n"], ["\n\n"], ["Use BLAST to search for relatives of yeast Upf1p"], ["Created: October 13, 1999"], ["Click on the link below to start an html tutorial."], ["Find relatives of yeast Upf1p"], ["Live PubMed searches"], ["(1) Upf1p\n"], ["(2) Nonsense mediated decay\n"], ["(3) Premature stop codon AND disease\n"], ["(4) REVIEWS\n"], ["Related resources"], ["\nGeneMap\n"], ["\nOMIM\n"], ["\nEntrez Gene\n"]]}
 {"file": "coffeebrk_NBK2345/A16.nxml", "text": "The Pax6 group of genes belongs to a larger class of homeobox-containing genes, found in organisms from yeast to humans. They code for transcription factors and are distinguished by the presence of a specific DNA-binding motif (a homeodomain) that serves to regulate gene expression. The \"helix-turn-helix\" 3D structure of the homeodomain is the same structure that is seen in bacterial gene regulatory proteins, suggesting that this is an ancient conformation that has been conserved throughout billions of years of evolution.\nIn mice, sea squirts, and squid, Pax6 has been shown to activate the program that leads to eye formation during the development of the organism. In mouse, where the Pax6 gene is expressed in the developing eye and brain, a mutation called Small eye (Sey) results from defects in Pax6. This makes it a good model for studying aniridia, a condition caused by a mutation in human Pax6 in which an incomplete iris can lead to poor vision, light sensitivity, and a tendency to develop progressive glaucoma.\n\nThe structure of the paired domain found in human Pax6\nTwo distinct domains are found in Pax6 \u2014 a homeodomain and a paired domain. Recent interest has focused on the paired domain, mutations in which cause several human disorders, including aniridia. This figure depicts the structure as ribbons drawn through the main carbon backbone of the protein (red) and through the phosphate atoms of the DNA backbone (blue). Mutations in the paired domain interfere with the DNA-binding properties of Pax6, altering its function, and causing a detrimental effect on the health of an individual.\n(Reproduced from Xu, H.E., Rould, M.A., Xu W., Epstein, J.A., Maas, R.L. and Pabo, C.O. (1999) 'Crystal structure of the human Pax6 paired domain-DNA complex reveals specific roles for the linker region and carboxy-terminal subdomain in DNA binding' Genes Dev. 13, 1263-1275, with permission.)\n\nAlthough the eyes of vertebrates have a single lens, the compound eye of Drosophila consists of about 750 units, each unit containing a lens, retina, and photoreceptor cells. Even so, parallels exist between these two types of eye. In flies, the eye precursor cells differentiate into these distinct units at a distinct step of development, when ey, a Pax6 homolog, can be detected.\nRecently, a second Drosophila \nPax6 gene was reported and named twin of eyeless (toy). Perhaps surprisingly, toy in some ways is more similar to the evolutionarily distant vertebrate Pax6 proteins than to Ey. In particular, fly Toy and mouse Pax6 have a similar DNA-binding pattern; they have a much higher affinity for DNA than Ey. This can be attributed to the mutation of a single residue (Asn?Gly) in a highly conserved part of Ey, known as the paired domain. \nThe existence of two Pax6 genes in flies, but not in vertebrates, suggests that a gene duplication event occurred sometime during fly evolution. That Toy is more closely related to vertebrate Pax6 suggests that Toy is the more ancient form, which gave rise to Ey. The key point in the evolution in these two genes was probably when the Asn?Gly mutation occurred, radically altering the DNA-binding function of Ey. At this point Ey could have become dependent on Toy for its activation, triggering a divergence in function of the two proteins. Today, genetic evidence suggests that Toy is found upstream of Ey in the regulatory pathway of eye development, and they each regulate distinct developmental events.\n\n\n\nSearch PubMed for Pax6 mutations\nCreated: September 29, 1999\nClick on the link below to start an html tutorial.\nFind information on Pax6 mutations\n\n\n\nSearch GeneMap99 for Pax6\nCreated: September 29, 1999\nClick on the link below to start an html tutorial.\nWhere is Pax6 found in the human genome?\n\n\n\nUse BLAST to search for proteins similar to Pax6\nCreated: September 29, 1999\nClick on the link below to start an html tutorial.\nSee how Drosophila Ey compares to other Pax6 proteins", "pairs": [["litarch_figures/df/45/coffeebrk_NBK2345/A543.jpg", "\nThe structure of the paired domain found in human Pax6\nTwo distinct domains are found in Pax6 \u2014 a homeodomain and a paired domain. Recent interest has focused on the paired domain, mutations in which cause several human disorders, including aniridia. This figure depicts the structure as ribbons drawn through the main carbon backbone of the protein (red) and through the phosphate atoms of the DNA backbone (blue). Mutations in the paired domain interfere with the DNA-binding properties of Pax6, altering its function, and causing a detrimental effect on the health of an individual.\n(Reproduced from Xu, H.E., Rould, M.A., Xu W., Epstein, J.A., Maas, R.L. and Pabo, C.O. (1999) 'Crystal structure of the human Pax6 paired domain-DNA complex reveals specific roles for the linker region and carboxy-terminal subdomain in DNA binding' Genes Dev. 13, 1263-1275, with permission.)\n\n", ""]], "interleaved": [["The Pax6 group of genes belongs to a larger class of homeobox-containing genes, found in organisms from yeast to humans. They code for transcription factors and are distinguished by the presence of a specific DNA-binding motif (a homeodomain) that serves to regulate gene expression. The \"helix-turn-helix\" 3D structure of the homeodomain is the same structure that is seen in bacterial gene regulatory proteins, suggesting that this is an ancient conformation that has been conserved throughout billions of years of evolution."], ["In mice, sea squirts, and squid, Pax6 has been shown to activate the program that leads to eye formation during the development of the organism. In mouse, where the Pax6 gene is expressed in the developing eye and brain, a mutation called Small eye (Sey) results from defects in Pax6. This makes it a good model for studying aniridia, a condition caused by a mutation in human Pax6 in which an incomplete iris can lead to poor vision, light sensitivity, and a tendency to develop progressive glaucoma."], ["litarch_figures/df/45/coffeebrk_NBK2345/A543.jpg", "\nThe structure of the paired domain found in human Pax6\nTwo distinct domains are found in Pax6 \u2014 a homeodomain and a paired domain. Recent interest has focused on the paired domain, mutations in which cause several human disorders, including aniridia. This figure depicts the structure as ribbons drawn through the main carbon backbone of the protein (red) and through the phosphate atoms of the DNA backbone (blue). Mutations in the paired domain interfere with the DNA-binding properties of Pax6, altering its function, and causing a detrimental effect on the health of an individual.\n(Reproduced from Xu, H.E., Rould, M.A., Xu W., Epstein, J.A., Maas, R.L. and Pabo, C.O. (1999) 'Crystal structure of the human Pax6 paired domain-DNA complex reveals specific roles for the linker region and carboxy-terminal subdomain in DNA binding' Genes Dev. 13, 1263-1275, with permission.)\n\n", ""], ["The structure of the paired domain found in human Pax6"], ["Two distinct domains are found in Pax6 \u2014 a homeodomain and a paired domain. Recent interest has focused on the paired domain, mutations in which cause several human disorders, including aniridia. This figure depicts the structure as ribbons drawn through the main carbon backbone of the protein (red) and through the phosphate atoms of the DNA backbone (blue). Mutations in the paired domain interfere with the DNA-binding properties of Pax6, altering its function, and causing a detrimental effect on the health of an individual."], ["(Reproduced from Xu, H.E., Rould, M.A., Xu W., Epstein, J.A., Maas, R.L. and Pabo, C.O. (1999) 'Crystal structure of the human Pax6 paired domain-DNA complex reveals specific roles for the linker region and carboxy-terminal subdomain in DNA binding' Genes Dev. 13, 1263-1275, with permission.)\n"], ["Although the eyes of vertebrates have a single lens, the compound eye of Drosophila consists of about 750 units, each unit containing a lens, retina, and photoreceptor cells. Even so, parallels exist between these two types of eye. In flies, the eye precursor cells differentiate into these distinct units at a distinct step of development, when ey, a Pax6 homolog, can be detected."], ["Recently, a second Drosophila \nPax6 gene was reported and named twin of eyeless (toy). Perhaps surprisingly, toy in some ways is more similar to the evolutionarily distant vertebrate Pax6 proteins than to Ey. In particular, fly Toy and mouse Pax6 have a similar DNA-binding pattern; they have a much higher affinity for DNA than Ey. This can be attributed to the mutation of a single residue (Asn?Gly) in a highly conserved part of Ey, known as the paired domain. "], ["The existence of two Pax6 genes in flies, but not in vertebrates, suggests that a gene duplication event occurred sometime during fly evolution. That Toy is more closely related to vertebrate Pax6 suggests that Toy is the more ancient form, which gave rise to Ey. The key point in the evolution in these two genes was probably when the Asn?Gly mutation occurred, radically altering the DNA-binding function of Ey. At this point Ey could have become dependent on Toy for its activation, triggering a divergence in function of the two proteins. Today, genetic evidence suggests that Toy is found upstream of Ey in the regulatory pathway of eye development, and they each regulate distinct developmental events."], ["\n\n"], ["Search PubMed for Pax6 mutations"], ["Created: September 29, 1999"], ["Click on the link below to start an html tutorial."], ["Find information on Pax6 mutations"], ["\n\n"], ["Search GeneMap99 for Pax6"], ["Created: September 29, 1999"], ["Click on the link below to start an html tutorial."], ["Where is Pax6 found in the human genome?"], ["\n\n"], ["Use BLAST to search for proteins similar to Pax6"], ["Created: September 29, 1999"], ["Click on the link below to start an html tutorial."], ["See how Drosophila Ey compares to other Pax6 proteins"]]}
 {"file": "coffeebrk_NBK2345/A33.nxml", "text": "The world of small RNAs just got bigger. In Caenorhabditis elegans, two small temporal RNAs produced by the ribonuclease Dicer have previously been shown to be involved in regulating developmental timing. These RNAs \u2014 lin-4 and let-7, 22 and 21 nucleotides in length, respectively \u2014 act as antisense repressors of messenger RNA translation and, until recently, they were the only known RNAs of this class. But three papers published in Science now show that lin-4 and let-7 probably belong to a large family of single-stranded RNAs, 20 - 24 nucleotides in length, called microRNAs (miRNAs). These results indicate that post-transcriptional regulation by small RNAs is more common than previously believed.\nLagos-Quintana and colleagues used complementary DNA libraries constructed from a size-fractionated RNA population to identify 14 new miRNAs in Drosophila melanogaster and 19 new miRNAs in humans. Lau et al. created a cDNA library enriched for Dicer products, distinguished from other oligonucleotides by their small size, 5'-monophosphate group and 3'-hydroxyl group, to identify 54 novel miRNAs in C. elegans. Finally, using size-selected cDNA cloning together with computational methods, Lee and Ambros identified 15 miRNAs in C. elegans, 11 of which matched those identified by Lau and co-workers. In all cases, they showed the miRNAs were not simply the breakdown products of mRNAs or structural RNAs.\nThese papers identified 91 different miRNAs in total, about 12% of which have been conserved through evolution. Moreover, Lau and colleagues found that \u223c85% of the miRNAs identified in C. elegans had homologues in the 90%-complete C. briggsae genome sequence.\nmiRNAs are produced through processing, probably by Dicer, of a \u223c70-nucleotide precursor stem-loop structure. Either the 5' or the 3' arm of the precursor can be released to form the miRNA, with one exception. miR-56, identified by Lau et al., exists in two forms, resulting from processing of both the 5' and 3' arms of the precursor stem. How miRNA excision occurs has yet to be defined.\nThe mir genes often cluster together in the genome; for example, Lagos-Quintana and colleagues showed that mir-3, -4, -5, and -6 form a gene cluster in the Drosophila genome. The mir gene clusters investigated so far are co-expressed, and Lau and co-workers predicted that, in C. elegans, the gene cluster mir-35 to -41 is transcribed to form a single RNA precursor, which is processed to produce miR-35 to -41. Some mir genes have multiple genomic copies, and some miRNAs are highly homologous.\nAll three groups investigated the expression of miRNAs and found that, in some cases, it was both stage- and tissue-specific. For example, Lee and Ambros found that mir-1 is expressed stage-specifically in mouse embryogenesis and tissue-specifically in the human heart. These regulated expression patterns indicate an involvement in developmental control.\nmiRNAs have been proposed to function as \"riboregulators\", regulating gene expression by binding sequence-specifically to mRNAs, thereby blocking translation. The challenge now is to define the potential targets of miRNAs and their exact functions. There are probably many miRNAs yet to be identified and, if they are found to be as numerous and diverse as the miRNAs identified in these papers, they could have a range of regulatory functions. These authors seem to have discovered a small fortune, and the world of small RNAs could turn out to be very big indeed.\nStory by Rachel Smallridge, Nature Reviews Molecular Cell Biology\n\n\n\n\nSearch the Bookshelf\nCreated: June 5, 2002\nClick on the link below to start an html tutorial.\n\ncan additional resources be researched?\n\nLive PubMed searches\n\n(1) lin-4\n\n\n(2) Dicer ribonuclease protein\n\nAdditional resources\n\nC. elegans genome\n\n\nDrosophila genome\n", "pairs": [], "interleaved": []}
-{"file": "coffeebrk_NBK2345/A622.nxml", "text": "An impressive number of bacteria\u2014about 30,000 species\u2014are represented in GenBank. However, our view of the microbial world is both scant and skewed. A recent estimate suggests that the sea may support as many as 2 million different bacteria, and a ton of soil might contain 4 million (1). Less than half of the bacteria represented in GenBank\u2014about 13,000\u2014have been formally described, and almost all of these (90%) lie within 4 of the 40 bacterial divisions (2). Similar or greater paucity of knowledge also exists for archaea and viruses (3). \nSampling \"wild\" microorganisms leads to the discovery of new species and novel metabolisms, which may be important from both a basic science and a practical perspective (for example, see Refs 4,5 [search PubMed]). For example, if we characterized the community in the human gut, it would be easier to spot non-native organisms in food poisoning outbreaks. Pathogens that may underlie neurological syndromes that present with features of infection would stand out against the background flora (1). Engineered communities of microorganisms might also be able to assist clean up of environmental disasters or create sustainable energy sources. \nExploring bacterial diversity is typically done by amplifying rRNA genes, in particular 16S rRNA genes, from DNA samples isolated from a habitat. The sequences are then compared to each other and to the 16S rRNA sequences from known species. If no close match to an existing 16S rRNA gene sequence is found, then the test sequence is thought to represent a new bacterium and is listed in GenBank as \"uncultured bacterium\". Even in well-studied, discrete places like the human mouth, new groups of uncultured bacteria continue to be discovered all the time. A newly identified organism has to be isolated and cultured in the lab to be described further; but many bugs are just not amenable to monoculture\u2014they have adapted to living in a specific environment and may need to be part of a complex community to survive (1-3).\n16S rRNA genes are considered standard because they are thought to be conserved across vast taxonomic distance (they are critical for protein translation), yet show some sequence variation between closely related species. However, one problem with using rRNA genes is that they are often present in multiple copy numbers; therefore, other representative genes may be used for sampling specific populations. \nWhole Genome Shotgun Sequencing of Environmental Samples\nNew approaches to environmental sampling are emerging (6\u20139). One of these used a microarray to discover and assist in the isolation of new viruses (6); another used a shotgun clone and sequencing method to explore marine viral communities (9). Two others have used whole genome shotgun (WGS) sequencing on a population of bacteria, obviating the need to isolate each organism before sequencing can begin (7,8). These methods, used in combination with existing methods, may provide shortcuts to the discovery of new genes and give a holistic persective to microbial populations. \nOne recent study used a WGS approach to explore a sample from an acid mine drainage biofilm (7; AADL00000000). These investigators report that near-complete genomes for Leptospirillum Group II and Ferroplasma Type II were assembled, along with more fragmentory assemblies for Leptospirillum Group III, Thermoplasmatales archaeon gpl, and Ferroplasma acidarmanus Type I. Analysis of the results provided some insight into how such organisms survive in an extreme environment.\nIn another test case of the WGS method, Venter et al. (8) sampled water from the Sargasso Sea\u2014one of the most well-characterized regions of ocean in the world. The major set of samples produced 1.66 million short sequences, some of which could be grouped together into larger genomic pieces. There remained about 400,000 paired-end reads and singleton reads. \nFinding the Data\nUsing a WGS method to sequence an undefined population as opposed to a single organism adds significant complexity to the assembly process and to the identification of genes. About 25% of the assembled data from the Sargasso Sea had 3X coverage or greater; these well-sampled portions were used to cluster the sequence by \u201corganism\u201d. \nThe assembled sequences have been deposited in the WGS division of GenBank, with the project Accession number AACY01000000; thus, there are 811,372 WGS contigs in GenBank with the Accession numbers AACY01000001\u2013AACY01811372. 498,641 of the WGS contigs are assembled into 232,442 scaffolds, the rest remain \u201csingleton\u201d WGS contigs; all but 10,685 of the scaffolds are made up of two contigs only. For the organism genomes listed in1, 301 of the total scaffolds plus 36 singleton WGS contigs were used; the remainder have not been associated with any particular organism. \nAll of the short sequence reads, including those that were not included in the assembly, can be found in the Trace Archive. \nThe assemblies were then further clustered into 30 tentative organism \u201cbins\u201d based on depth of coverage, oligonucleotide frequencies and similarities to previously sequenced genomes. Of these, 12 are of sufficient size to be considered a genome assembly, while the remaining 16 are relatively small single scaffolds (1). All organism bins have been assigned a taxonomy ID, and have been placed in the taxonomic tree. shows the graphical representation of the cf. Shewanella SAR-1 \u201cgenome\u201d sequence.\n\n\n(a)\nGenome view of cf. Shewanella SAR-1, constructed from the whole genome shotgun sequence derived from Sargasso Sea environmental samples (8). Genes have been classified according to the COG functional categories of the protein products, and color-coded accordingly. note that the actual order of the scaffolds is unknown, so in this representation they have been ordered by size. Clicking on the image reveals the gene sequences and approximate location. (b) Selecting one of the genes (in this case, the blue gene around position 2619000) shows the results of an automated BLAST search (BLink). This gene is similar to L-sorbosone dehydrogenase from a variety of bacteria, archaea, and fungi. L-sorbosone dehydrogenase is an enzyme required for the biosynthesis of L-ascorbic acid, a product widely used in the food industry as a vitamin and antioxidant.\n\nEach of the 28 organism \u201cgenomes\u201d can be viewed in a similar mannner (see1).\nThe organism bins assembled from the Sargasso Sea WGS environmental sample dataset (8)\n|    | Organism Bin                  | Description                                                                                                       | Data              | Further Reading   |\n|---:|:------------------------------|:------------------------------------------------------------------------------------------------------------------|:------------------|:------------------|\n|  0 | Genome Assemblies             | Genome Assemblies                                                                                                 | Genome Assemblies | Genome Assemblies |\n|  1 | cf. Alphaproteobacteria SAR-1 | Oligotrophic Typical of marine bacterioplankton                                                                   | Genome GenBank    | PubMed Books      |\n|  2 | cf. Archaea SAR-1             | One of the three major domains of lifeOften inhabit extreme environments                                          | Genome GenBank    | PubMed Books      |\n|  3 | cf. Bacteria SAR-1            | One of the three major domains of life                                                                            | Genome GenBank    | PubMed Books      |\n|  4 | cf. Burkholderia SAR-1        | Gram-negative bacilliAerobicFound in a variety of aquatic environments                                            | Genome GenBank    | PubMed Books      |\n|  5 | cf. Gammaproteobacteria SAR-1 | Purple bacteria Some plant pathogens                                                                              | Genome GenBank    | PubMed Books      |\n|  6 | cf. Microbulbifer SAR-1       | Marine bacteria that degrade and recycle complex carbohydrates                                                    | Genome GenBank    | PubMed Books      |\n|  7 | cf. Prochlorococcus SAR-1     | Smallest known photosynthetic organism The most abundant in the ocean                                             | Genome GenBank    | PubMed Books      |\n|  8 | cf. Proteobacteria SAR-1      | Phylum includes nitrogen-fixing bacteria and enteric bacteria                                                     | Genome GenBank    | PubMed Books      |\n|  9 | cf. Pseudomonadaceae SAR-1    | Gram-negative rods Often motile Includes many plant and a few animal pathogens                                    | Genome GenBank    | PubMed Books      |\n| 10 | cf. Shewanella SAR-1          | Versatile metabolism Potential biotech applications such as heavy metal or chlorinated solvent reduction          | Genome GenBank    | PubMed Books      |\n| 11 | cf. Shewanella SAR-2*         | Versatile metabolism Potential biotech applications such as heavy metal or chlorinated solvent reduction          | Genome GenBank    | PubMed Books      |\n| 12 | cf. Streptomyces SAR-1        | Superficially similar to fungi(filaments and spores) Common in many habitats                                      | Genome GenBank    | PubMed Books      |\n| 13 | Single Scaffolds              | Single Scaffolds                                                                                                  | Single Scaffolds  | Single Scaffolds  |\n| 14 | cf. Actinobacteria SAR-1      | High G+C group of Gram-positive bacteria Most found in soil Some pathogens                                        | GenBank           | PubMed Books      |\n| 15 | cf. Bordetella SAR-1          | Gram-negative coccobacilliStrict aerobes                                                                          | GenBank           | PubMed Books      |\n| 16 | cf. Burkholderiaceae SAR-1    | Occupy diverse ecological nichesMay have potential for biotech applications but also involved in human infections | GenBank           | PubMed Books      |\n| 17 | cf. Caulobacter SAR-1         | Found in oligotrophic environments Prosthecate (having appendages)                                                | GenBank           | PubMed Books      |\n| 18 | cf. Crenarchaeota SAR-1       | ArchaealMost species are motileTolerant of extreme acidity and temperature                                        | GenBank           | PubMed Books      |\n| 19 | cf. Cyanobacteria SAR-1       | Aquatic and photosyntheticOften called \u201dblue-green algae\u201d                                                         | GenBank           | PubMed Books      |\n| 20 | cf. Enterobacteriaceae SAR-1  | Large Gram-negative rodsFacultative anaerobes                                                                     | GenBank           | PubMed Books      |\n| 21 | cf. Haemophilus SAR-1         | Gram-negative rodsLike to grow on blood agarSome pathogens                                                        | GenBank           | PubMed Books      |\n| 22 | cf. Magnetococcus SAR-1       | Gram-negative coccus Magnetic bacteriaUsually located at sediment-water interface                                 | GenBank           | PubMed Books      |\n| 23 | cf. Magnetospirillum SAR-1    | Magnetic bacteria                                                                                                 | GenBank           | PubMed Books      |\n| 24 | cf. Ralstonia SAR-1           | Includes medically and economically important plant and animal pathogens                                          | GenBank           | PubMed Books      |\n| 25 | cf. Rhizobiales SAR-1         | Involved in nitrogen fixation, often in symbiotic relationships with plants                                       | GenBank           | PubMed Books      |\n| 26 | cf. Sinorhizobium SAR-1       | Symbiotic nitrogen fixation in plant root nodules                                                                 | GenBank           | PubMed Books      |\n| 27 | cf. Spirochaetales SAR-1      | Spiral rodsSome pathogens (e.g. Borrelia burgdorferi - Lyme disease)                                              | GenBank           | PubMed Books      |\n| 28 | cf. Streptomycetaceae SAR-1   | Typically aerobic and found in soil Some parasitic forms                                                          | GenBank           | PubMed Books      |\n| 29 | cf. Vibrionaceae SAR-1        | Gram-negative, non-sporing rods Generally motileMany strains of Vibrio genus cause infection                      | GenBank           | PubMed Books      |\ncf. is used to designate an unidentified species of the genus. Therefore, \u201ccf. Burkholderia\u201d means \u201csomething that is like the genus Burkholderia\u201c (in this case, by sequence similarity).\nAs each organism bin could actually represent several different unidentified species, a strain name cannot be assigned, so instead, the suffix \"SAR-#\" identifies each bin as a \u201cSargasso Sea cyber-species\u201d.\n* cf. Shewanella SAR-2: two distinct Shewanella genomes were constructed from the dataset.\n A variety of approaches suggested that there are at least 1000 species represented in the Sargasso Sea samples (8). Burkholderia species were represented in a high proportion (a genus that includes human and plant pathogens and some environmentally important bacteria), as were two distinct species closely related to Shewanella oneidensis. Both of these genera require a more nutrient-rich environment than the open ocean can offer, suggesting that they originated from microhabitats such as marine snow. The cyanobacterium Prochlorococcus was also relatively abundant in some samples.\nAlthough the primary focus of this study was on bacterial populations, WGS environmental sampling may be an equally valid approach for exploring plasmids (2), phage, viruses, and eukaryotic microbes.\nThe plasmid bins assembled from the Sargasso Sea WGS environmental sample dataset (8)\n|    | Plasmid Bin    | Data    |\n|---:|:---------------|:--------|\n|  0 | Plasmid pSAR-1 | GenBank |\n|  1 | Plasmid pSAR-2 | GenBank |\n|  2 | Plasmid pSAR-3 | GenBank |\n|  3 | Plasmid pSAR-4 | GenBank |\n|  4 | Plasmid pSAR-5 | GenBank |\n|  5 | Plasmid pSAR-6 | GenBank |\n|  6 | Plasmid pSAR-7 | GenBank |\n|  7 | Plasmid pSAR-8 | GenBank |\n|  8 | Plasmid pSAR-9 | GenBank |\nReferences\nGenomes\nBrowse \u201cgenomes\u201d by organism bin\n\nBLAST\n\nSearch environmental samples data by sequence similarity\n\nTaxonomy\n\nView taxonomic tree of all organism bins\n\nGenBank\n\nGenome assemblies in GenBank\n\nTrace Archive\n\nAll Sargasso Sea traces\n", "pairs": [["litarch_figures/df/45/coffeebrk_NBK2345/A636.jpg", "\n\n(a)\nGenome view of cf. Shewanella SAR-1, constructed from the whole genome shotgun sequence derived from Sargasso Sea environmental samples (8). Genes have been classified according to the COG functional categories of the protein products, and color-coded accordingly. note that the actual order of the scaffolds is unknown, so in this representation they have been ordered by size. Clicking on the image reveals the gene sequences and approximate location. (b) Selecting one of the genes (in this case, the blue gene around position 2619000) shows the results of an automated BLAST search (BLink). This gene is similar to L-sorbosone dehydrogenase from a variety of bacteria, archaea, and fungi. L-sorbosone dehydrogenase is an enzyme required for the biosynthesis of L-ascorbic acid, a product widely used in the food industry as a vitamin and antioxidant.\n\nEach of the 28 organism \u201cgenomes\u201d can be viewed in a similar mannner (see Table 1).\n", ""]], "interleaved": [["An impressive number of bacteria\u2014about 30,000 species\u2014are represented in GenBank. However, our view of the microbial world is both scant and skewed. A recent estimate suggests that the sea may support as many as 2 million different bacteria, and a ton of soil might contain 4 million (1). Less than half of the bacteria represented in GenBank\u2014about 13,000\u2014have been formally described, and almost all of these (90%) lie within 4 of the 40 bacterial divisions (2). Similar or greater paucity of knowledge also exists for archaea and viruses (3). "], ["Sampling \"wild\" microorganisms leads to the discovery of new species and novel metabolisms, which may be important from both a basic science and a practical perspective (for example, see Refs 4,5 [search PubMed]). For example, if we characterized the community in the human gut, it would be easier to spot non-native organisms in food poisoning outbreaks. Pathogens that may underlie neurological syndromes that present with features of infection would stand out against the background flora (1). Engineered communities of microorganisms might also be able to assist clean up of environmental disasters or create sustainable energy sources. "], ["Exploring bacterial diversity is typically done by amplifying rRNA genes, in particular 16S rRNA genes, from DNA samples isolated from a habitat. The sequences are then compared to each other and to the 16S rRNA sequences from known species. If no close match to an existing 16S rRNA gene sequence is found, then the test sequence is thought to represent a new bacterium and is listed in GenBank as \"uncultured bacterium\". Even in well-studied, discrete places like the human mouth, new groups of uncultured bacteria continue to be discovered all the time. A newly identified organism has to be isolated and cultured in the lab to be described further; but many bugs are just not amenable to monoculture\u2014they have adapted to living in a specific environment and may need to be part of a complex community to survive (1-3)."], ["16S rRNA genes are considered standard because they are thought to be conserved across vast taxonomic distance (they are critical for protein translation), yet show some sequence variation between closely related species. However, one problem with using rRNA genes is that they are often present in multiple copy numbers; therefore, other representative genes may be used for sampling specific populations. "], ["Whole Genome Shotgun Sequencing of Environmental Samples"], ["New approaches to environmental sampling are emerging (6\u20139). One of these used a microarray to discover and assist in the isolation of new viruses (6); another used a shotgun clone and sequencing method to explore marine viral communities (9). Two others have used whole genome shotgun (WGS) sequencing on a population of bacteria, obviating the need to isolate each organism before sequencing can begin (7,8). These methods, used in combination with existing methods, may provide shortcuts to the discovery of new genes and give a holistic persective to microbial populations. "], ["One recent study used a WGS approach to explore a sample from an acid mine drainage biofilm (7; AADL00000000). These investigators report that near-complete genomes for Leptospirillum Group II and Ferroplasma Type II were assembled, along with more fragmentory assemblies for Leptospirillum Group III, Thermoplasmatales archaeon gpl, and Ferroplasma acidarmanus Type I. Analysis of the results provided some insight into how such organisms survive in an extreme environment."], ["In another test case of the WGS method, Venter et al. (8) sampled water from the Sargasso Sea\u2014one of the most well-characterized regions of ocean in the world. The major set of samples produced 1.66 million short sequences, some of which could be grouped together into larger genomic pieces. There remained about 400,000 paired-end reads and singleton reads. "], ["Finding the Data"], ["Using a WGS method to sequence an undefined population as opposed to a single organism adds significant complexity to the assembly process and to the identification of genes. About 25% of the assembled data from the Sargasso Sea had 3X coverage or greater; these well-sampled portions were used to cluster the sequence by \u201corganism\u201d. "], ["The assembled sequences have been deposited in the WGS division of GenBank, with the project Accession number AACY01000000; thus, there are 811,372 WGS contigs in GenBank with the Accession numbers AACY01000001\u2013AACY01811372. 498,641 of the WGS contigs are assembled into 232,442 scaffolds, the rest remain \u201csingleton\u201d WGS contigs; all but 10,685 of the scaffolds are made up of two contigs only. For the organism genomes listed in1, 301 of the total scaffolds plus 36 singleton WGS contigs were used; the remainder have not been associated with any particular organism. "], ["All of the short sequence reads, including those that were not included in the assembly, can be found in the Trace Archive. "], ["The assemblies were then further clustered into 30 tentative organism \u201cbins\u201d based on depth of coverage, oligonucleotide frequencies and similarities to previously sequenced genomes. Of these, 12 are of sufficient size to be considered a genome assembly, while the remaining 16 are relatively small single scaffolds (1). All organism bins have been assigned a taxonomy ID, and have been placed in the taxonomic tree. shows the graphical representation of the cf. Shewanella SAR-1 \u201cgenome\u201d sequence."], ["litarch_figures/df/45/coffeebrk_NBK2345/A636.jpg", "\n\n(a)\nGenome view of cf. Shewanella SAR-1, constructed from the whole genome shotgun sequence derived from Sargasso Sea environmental samples (8). Genes have been classified according to the COG functional categories of the protein products, and color-coded accordingly. note that the actual order of the scaffolds is unknown, so in this representation they have been ordered by size. Clicking on the image reveals the gene sequences and approximate location. (b) Selecting one of the genes (in this case, the blue gene around position 2619000) shows the results of an automated BLAST search (BLink). This gene is similar to L-sorbosone dehydrogenase from a variety of bacteria, archaea, and fungi. L-sorbosone dehydrogenase is an enzyme required for the biosynthesis of L-ascorbic acid, a product widely used in the food industry as a vitamin and antioxidant.\n\nEach of the 28 organism \u201cgenomes\u201d can be viewed in a similar mannner (see Table 1).\n", ""], ["\n(a)\nGenome view of cf. Shewanella SAR-1, constructed from the whole genome shotgun sequence derived from Sargasso Sea environmental samples (8). Genes have been classified according to the COG functional categories of the protein products, and color-coded accordingly. note that the actual order of the scaffolds is unknown, so in this representation they have been ordered by size. Clicking on the image reveals the gene sequences and approximate location. (b) Selecting one of the genes (in this case, the blue gene around position 2619000) shows the results of an automated BLAST search (BLink). This gene is similar to L-sorbosone dehydrogenase from a variety of bacteria, archaea, and fungi. L-sorbosone dehydrogenase is an enzyme required for the biosynthesis of L-ascorbic acid, a product widely used in the food industry as a vitamin and antioxidant."], ["\nEach of the 28 organism \u201cgenomes\u201d can be viewed in a similar mannner (see1)."], ["The organism bins assembled from the Sargasso Sea WGS environmental sample dataset (8)"], ["|    | Organism Bin                  | Description                                                                                                       | Data              | Further Reading   |\n|---:|:------------------------------|:------------------------------------------------------------------------------------------------------------------|:------------------|:------------------|\n|  0 | Genome Assemblies             | Genome Assemblies                                                                                                 | Genome Assemblies | Genome Assemblies |\n|  1 | cf. Alphaproteobacteria SAR-1 | Oligotrophic Typical of marine bacterioplankton                                                                   | Genome GenBank    | PubMed Books      |\n|  2 | cf. Archaea SAR-1             | One of the three major domains of lifeOften inhabit extreme environments                                          | Genome GenBank    | PubMed Books      |\n|  3 | cf. Bacteria SAR-1            | One of the three major domains of life                                                                            | Genome GenBank    | PubMed Books      |\n|  4 | cf. Burkholderia SAR-1        | Gram-negative bacilliAerobicFound in a variety of aquatic environments                                            | Genome GenBank    | PubMed Books      |\n|  5 | cf. Gammaproteobacteria SAR-1 | Purple bacteria Some plant pathogens                                                                              | Genome GenBank    | PubMed Books      |\n|  6 | cf. Microbulbifer SAR-1       | Marine bacteria that degrade and recycle complex carbohydrates                                                    | Genome GenBank    | PubMed Books      |\n|  7 | cf. Prochlorococcus SAR-1     | Smallest known photosynthetic organism The most abundant in the ocean                                             | Genome GenBank    | PubMed Books      |\n|  8 | cf. Proteobacteria SAR-1      | Phylum includes nitrogen-fixing bacteria and enteric bacteria                                                     | Genome GenBank    | PubMed Books      |\n|  9 | cf. Pseudomonadaceae SAR-1    | Gram-negative rods Often motile Includes many plant and a few animal pathogens                                    | Genome GenBank    | PubMed Books      |\n| 10 | cf. Shewanella SAR-1          | Versatile metabolism Potential biotech applications such as heavy metal or chlorinated solvent reduction          | Genome GenBank    | PubMed Books      |\n| 11 | cf. Shewanella SAR-2*         | Versatile metabolism Potential biotech applications such as heavy metal or chlorinated solvent reduction          | Genome GenBank    | PubMed Books      |\n| 12 | cf. Streptomyces SAR-1        | Superficially similar to fungi(filaments and spores) Common in many habitats                                      | Genome GenBank    | PubMed Books      |\n| 13 | Single Scaffolds              | Single Scaffolds                                                                                                  | Single Scaffolds  | Single Scaffolds  |\n| 14 | cf. Actinobacteria SAR-1      | High G+C group of Gram-positive bacteria Most found in soil Some pathogens                                        | GenBank           | PubMed Books      |\n| 15 | cf. Bordetella SAR-1          | Gram-negative coccobacilliStrict aerobes                                                                          | GenBank           | PubMed Books      |\n| 16 | cf. Burkholderiaceae SAR-1    | Occupy diverse ecological nichesMay have potential for biotech applications but also involved in human infections | GenBank           | PubMed Books      |\n| 17 | cf. Caulobacter SAR-1         | Found in oligotrophic environments Prosthecate (having appendages)                                                | GenBank           | PubMed Books      |\n| 18 | cf. Crenarchaeota SAR-1       | ArchaealMost species are motileTolerant of extreme acidity and temperature                                        | GenBank           | PubMed Books      |\n| 19 | cf. Cyanobacteria SAR-1       | Aquatic and photosyntheticOften called \u201dblue-green algae\u201d                                                         | GenBank           | PubMed Books      |\n| 20 | cf. Enterobacteriaceae SAR-1  | Large Gram-negative rodsFacultative anaerobes                                                                     | GenBank           | PubMed Books      |\n| 21 | cf. Haemophilus SAR-1         | Gram-negative rodsLike to grow on blood agarSome pathogens                                                        | GenBank           | PubMed Books      |\n| 22 | cf. Magnetococcus SAR-1       | Gram-negative coccus Magnetic bacteriaUsually located at sediment-water interface                                 | GenBank           | PubMed Books      |\n| 23 | cf. Magnetospirillum SAR-1    | Magnetic bacteria                                                                                                 | GenBank           | PubMed Books      |\n| 24 | cf. Ralstonia SAR-1           | Includes medically and economically important plant and animal pathogens                                          | GenBank           | PubMed Books      |\n| 25 | cf. Rhizobiales SAR-1         | Involved in nitrogen fixation, often in symbiotic relationships with plants                                       | GenBank           | PubMed Books      |\n| 26 | cf. Sinorhizobium SAR-1       | Symbiotic nitrogen fixation in plant root nodules                                                                 | GenBank           | PubMed Books      |\n| 27 | cf. Spirochaetales SAR-1      | Spiral rodsSome pathogens (e.g. Borrelia burgdorferi - Lyme disease)                                              | GenBank           | PubMed Books      |\n| 28 | cf. Streptomycetaceae SAR-1   | Typically aerobic and found in soil Some parasitic forms                                                          | GenBank           | PubMed Books      |\n| 29 | cf. Vibrionaceae SAR-1        | Gram-negative, non-sporing rods Generally motileMany strains of Vibrio genus cause infection                      | GenBank           | PubMed Books      |"], ["cf. is used to designate an unidentified species of the genus. Therefore, \u201ccf. Burkholderia\u201d means \u201csomething that is like the genus Burkholderia\u201c (in this case, by sequence similarity)."], ["As each organism bin could actually represent several different unidentified species, a strain name cannot be assigned, so instead, the suffix \"SAR-#\" identifies each bin as a \u201cSargasso Sea cyber-species\u201d."], ["* cf. Shewanella SAR-2: two distinct Shewanella genomes were constructed from the dataset."], [" A variety of approaches suggested that there are at least 1000 species represented in the Sargasso Sea samples (8). Burkholderia species were represented in a high proportion (a genus that includes human and plant pathogens and some environmentally important bacteria), as were two distinct species closely related to Shewanella oneidensis. Both of these genera require a more nutrient-rich environment than the open ocean can offer, suggesting that they originated from microhabitats such as marine snow. The cyanobacterium Prochlorococcus was also relatively abundant in some samples."], ["Although the primary focus of this study was on bacterial populations, WGS environmental sampling may be an equally valid approach for exploring plasmids (2), phage, viruses, and eukaryotic microbes."], ["The plasmid bins assembled from the Sargasso Sea WGS environmental sample dataset (8)"], ["|    | Plasmid Bin    | Data    |\n|---:|:---------------|:--------|\n|  0 | Plasmid pSAR-1 | GenBank |\n|  1 | Plasmid pSAR-2 | GenBank |\n|  2 | Plasmid pSAR-3 | GenBank |\n|  3 | Plasmid pSAR-4 | GenBank |\n|  4 | Plasmid pSAR-5 | GenBank |\n|  5 | Plasmid pSAR-6 | GenBank |\n|  6 | Plasmid pSAR-7 | GenBank |\n|  7 | Plasmid pSAR-8 | GenBank |\n|  8 | Plasmid pSAR-9 | GenBank |"], ["References"], ["Genomes"], ["Browse \u201cgenomes\u201d by organism bin\n"], ["BLAST"], ["\nSearch environmental samples data by sequence similarity\n"], ["Taxonomy"], ["\nView taxonomic tree of all organism bins\n"], ["GenBank"], ["\nGenome assemblies in GenBank\n"], ["Trace Archive"], ["\nAll Sargasso Sea traces\n"]]}
 {"file": "coffeebrk_NBK2345/A31.nxml", "text": "It's been a long time coming, but now two papers report a clear cut identification by linkage mapping of a gene involved in a common human disorder \u2014 Crohn's disease (CD). Importantly, they also indicate how the innate immune system might be involved in the aetiology of CD, because the identified gene \u2014 NOD2 \u2014 encodes an intracellular receptor for bacterial lipopolysaccharides (LPS) that activates NF\u03baB, a target of the innate immune signalling pathway and a transcriptional regulator of inflammatory genes.\nCD is a chronic inflammatory gut disorder, thought to be caused by an abnormal inflammatory response to enteric microbes. In 1996, a CD susceptibility locus, IBD1, was identified on chromosome 16. Little progress has been made since, but it is this locus that the two research teams \u2014 one European, the other US-based \u2014 tackled in their studies, using positional-cloning and candidate-gene strategies, respectively.\n\nDNA sequence electropherograms of the NOD2 gene\n A portion of NOD2 exon 11 DNA sequence from control and three CD-affected individuals. The control sequence codes for full-length NOD2 protein. The patients from families 1 and 6 are heterozygous for a cytosine insertion at position 3020 in the NOD2 gene. The wild-type sequence in these panels is in the upper position and is read GCC-CTT-GAA. The sequence containing the cytosine insert is in the lower position and is read GCC-CCT-TGA. The extra cytosine base (marked by the arrows) causes a framshift mutation to occur, and the TGA sequence immediately downstream is recognized as a stop codon, causing the NOD2 protein to be truncated. The patient from family 7 is homozygous for the same cytosine insertion. \nHugot et al. took a decisive step when they identified association of CD to an allele of a chromosome-16 microsatellite marker. Despite the borderline significance of this association, the authors went on to identify putative transcripts in the region of this marker, and identified over 30 single nucleotide polymorphisms (SNPs) by sequencing the region from affected and unaffected individuals. Several turned out to be non-synonymous variants in a chromosome \u2014 16 gene, NOD2. Three of these SNPs \u2014 each independently associated with disease susceptibility \u2014 altered the leucine-rich repeat (LRR) region of NOD2, which is required for LPS recognition.\nHaving previously identified NOD2, Ogura et al. considered it a candidate for CD because of its chromosome-16 location. On sequencing the gene from CD individuals, they identified an insertion that caused two frameshift mutations in the LRR region and the premature truncation of NOD2. In in vitro assays, this mutant NOD2 produced considerably diminished levels of NF\u03baB activation in response to bacterial LPS compared to wild-type NOD2.\nSo how could NOD2 contribute to susceptibility to CD? The innate immune system regulates the immediate immune response to bacterial pathogens, components of which are recognized in host immune cells by specific receptors, such as NOD2. A defect in this recognition might lead to an exaggerated inflammatory reaction being mediated by the adaptive immune system. Alternatively, NOD2 might act to trigger cytokines that dampen inflammatory responses. Although NOD2 does not account for all susceptibility to CD, it does provide a first glimpse into the aetiology of the disease and should speed the discovery of other CD loci and future therapies, and improve its diagnosis. These papers are hopefully the first of many such successes in grappling with the genetic basis of multifactorial, common disease.\nStory by Jane Alfred, Nature Reviews Genetics\n\n\n\n\nSearch the genome for the NOD2 gene polymorphisms\nCreated: August 6, 2001\nClick on the link below to start an html tutorial.\nAre there additional polymorphisms in the NOD2 gene?\n\nLive PubMed searches\n\n(1) NOD2\n\n\n(2) IBD1\n\n\n(3) REVIEWS\n\nAdditional NCBI resources\n\nNOD2 in Entrez Gene\n\n\nGenes and Disease\n\n\nMedline Plus\n", "pairs": [["litarch_figures/df/45/coffeebrk_NBK2345/A560.jpg", "\nDNA sequence electropherograms of the NOD2 gene\n A portion of NOD2 exon 11 DNA sequence from control and three CD-affected individuals. The control sequence codes for full-length NOD2 protein. The patients from families 1 and 6 are heterozygous for a cytosine insertion at position 3020 in the NOD2 gene. The wild-type sequence in these panels is in the upper position and is read GCC-CTT-GAA. The sequence containing the cytosine insert is in the lower position and is read GCC-CCT-TGA. The extra cytosine base (marked by the arrows) causes a framshift mutation to occur, and the TGA sequence immediately downstream is recognized as a stop codon, causing the NOD2 protein to be truncated. The patient from family 7 is homozygous for the same cytosine insertion. \n", ""]], "interleaved": [["It's been a long time coming, but now two papers report a clear cut identification by linkage mapping of a gene involved in a common human disorder \u2014 Crohn's disease (CD). Importantly, they also indicate how the innate immune system might be involved in the aetiology of CD, because the identified gene \u2014 NOD2 \u2014 encodes an intracellular receptor for bacterial lipopolysaccharides (LPS) that activates NF\u03baB, a target of the innate immune signalling pathway and a transcriptional regulator of inflammatory genes."], ["CD is a chronic inflammatory gut disorder, thought to be caused by an abnormal inflammatory response to enteric microbes. In 1996, a CD susceptibility locus, IBD1, was identified on chromosome 16. Little progress has been made since, but it is this locus that the two research teams \u2014 one European, the other US-based \u2014 tackled in their studies, using positional-cloning and candidate-gene strategies, respectively."], ["litarch_figures/df/45/coffeebrk_NBK2345/A560.jpg", "\nDNA sequence electropherograms of the NOD2 gene\n A portion of NOD2 exon 11 DNA sequence from control and three CD-affected individuals. The control sequence codes for full-length NOD2 protein. The patients from families 1 and 6 are heterozygous for a cytosine insertion at position 3020 in the NOD2 gene. The wild-type sequence in these panels is in the upper position and is read GCC-CTT-GAA. The sequence containing the cytosine insert is in the lower position and is read GCC-CCT-TGA. The extra cytosine base (marked by the arrows) causes a framshift mutation to occur, and the TGA sequence immediately downstream is recognized as a stop codon, causing the NOD2 protein to be truncated. The patient from family 7 is homozygous for the same cytosine insertion. \n", ""], ["DNA sequence electropherograms of the NOD2 gene"], [" A portion of NOD2 exon 11 DNA sequence from control and three CD-affected individuals. The control sequence codes for full-length NOD2 protein. The patients from families 1 and 6 are heterozygous for a cytosine insertion at position 3020 in the NOD2 gene. The wild-type sequence in these panels is in the upper position and is read GCC-CTT-GAA. The sequence containing the cytosine insert is in the lower position and is read GCC-CCT-TGA. The extra cytosine base (marked by the arrows) causes a framshift mutation to occur, and the TGA sequence immediately downstream is recognized as a stop codon, causing the NOD2 protein to be truncated. The patient from family 7 is homozygous for the same cytosine insertion. "], ["Hugot et al. took a decisive step when they identified association of CD to an allele of a chromosome-16 microsatellite marker. Despite the borderline significance of this association, the authors went on to identify putative transcripts in the region of this marker, and identified over 30 single nucleotide polymorphisms (SNPs) by sequencing the region from affected and unaffected individuals. Several turned out to be non-synonymous variants in a chromosome \u2014 16 gene, NOD2. Three of these SNPs \u2014 each independently associated with disease susceptibility \u2014 altered the leucine-rich repeat (LRR) region of NOD2, which is required for LPS recognition."], ["Having previously identified NOD2, Ogura et al. considered it a candidate for CD because of its chromosome-16 location. On sequencing the gene from CD individuals, they identified an insertion that caused two frameshift mutations in the LRR region and the premature truncation of NOD2. In in vitro assays, this mutant NOD2 produced considerably diminished levels of NF\u03baB activation in response to bacterial LPS compared to wild-type NOD2."], ["So how could NOD2 contribute to susceptibility to CD? The innate immune system regulates the immediate immune response to bacterial pathogens, components of which are recognized in host immune cells by specific receptors, such as NOD2. A defect in this recognition might lead to an exaggerated inflammatory reaction being mediated by the adaptive immune system. Alternatively, NOD2 might act to trigger cytokines that dampen inflammatory responses. Although NOD2 does not account for all susceptibility to CD, it does provide a first glimpse into the aetiology of the disease and should speed the discovery of other CD loci and future therapies, and improve its diagnosis. These papers are hopefully the first of many such successes in grappling with the genetic basis of multifactorial, common disease."], ["Story by Jane Alfred, Nature Reviews Genetics\n"], ["\n\n"], ["Search the genome for the NOD2 gene polymorphisms"], ["Created: August 6, 2001"], ["Click on the link below to start an html tutorial."], ["Are there additional polymorphisms in the NOD2 gene?\n"], ["Live PubMed searches"], ["\n(1) NOD2\n"], ["\n(2) IBD1\n"], ["\n(3) REVIEWS\n"], ["Additional NCBI resources"], ["\nNOD2 in Entrez Gene\n"], ["\nGenes and Disease\n"], ["\nMedline Plus\n"]]}
 {"file": "coffeebrk_NBK2345/A28.nxml", "text": "There are many ways to monitor the onset of gene expression, but so far it has been impossible to detect its down-regulation. This problem might have been solved now, as Terskikh and colleagues report in Science a simple method to follow promoter activity.\nLast year, a red fluorescent protein (drFP583) was identified in tropical corals, further increasing the wide spectrum of possibilities to light up cells in different colors. Not satisfied with just one color, Terskikh and colleagues introduced random mutations into drFP583, and found one mutant (called E5) that changes its fluorescence from green to red in a time-dependent manner. As E5 switches from green to red fluorescence over time, it can be used as a timer for gene expression. During the first hours of activity of a promoter, green fluorescence is predominant, whereas sustained activity of the promoter leads to a mixture of green and red fluorescence. A few hours after the promoter is turned off, only red fluorescence remains.\n\nThe change in fluorescence of E5 over time in C. elegans.\n The E5 mutant was placed under the control of a heat shock promoter and injected into C. elegans embryos. Green fluoresence was detected 2 hours into the recovery phase following a standard heat shock treatment (1 hour incubation at 33\u00b0). The embryos were documented under bright field (DIC), with a FITC filter, with a PE filter, and with an overlay at 3.5, 7.5, and 50 hours following heat shock. Yellow fluorescence, as seen in the overlay column at 7.5 hours, indicates a combination of green and red fluorescence. \nTerskikh and colleagues verified these predictions in three experimental systems. First they monitored up- and down-regulation of E5 expression in Tet-on and Tet-off mammalian expression systems. Then they followed the activity of a heat-shock promoter during heat-induced stress ofCaenorhabditis elegans. Last, they traced the expression of a homeobox gene involved in the patterning of anterior structures in Xenopus laevis. In all cases, green fluorescence correctly indicated the onset of gene expression and was replaced with red fluorescence when expression ceased.\nSo after decades of blue-stained embryos, we'll now have to get used to seeing gene expression in green and red.\nStory contributed by Raluca Gagescu, Nature Reviews Molecular Cell Biology\n\n\n\n\nVAST search for structures similar to E5\nCreated: January 22, 2001\nClick on the link below to start an html tutorial.\n\nSearch for structures similar to E5\nLive PubMed searches\n\n(1) Coral proteins\n\n\n(2) Heat-shock promoters\n\n\n(3) REVIEWS\n\nAdditional NCBI resources\n\nNCBI Structure Group\n\n\nTaxonomic tree of jelly fish & red coral\n", "pairs": [["litarch_figures/df/45/coffeebrk_NBK2345/A557.jpg", "\nThe change in fluorescence of E5 over time in C. elegans.\n The E5 mutant was placed under the control of a heat shock promoter and injected into C. elegans embryos. Green fluoresence was detected 2 hours into the recovery phase following a standard heat shock treatment (1 hour incubation at 33\u00b0). The embryos were documented under bright field (DIC), with a FITC filter, with a PE filter, and with an overlay at 3.5, 7.5, and 50 hours following heat shock. Yellow fluorescence, as seen in the overlay column at 7.5 hours, indicates a combination of green and red fluorescence. \n", ""]], "interleaved": [["There are many ways to monitor the onset of gene expression, but so far it has been impossible to detect its down-regulation. This problem might have been solved now, as Terskikh and colleagues report in Science a simple method to follow promoter activity."], ["Last year, a red fluorescent protein (drFP583) was identified in tropical corals, further increasing the wide spectrum of possibilities to light up cells in different colors. Not satisfied with just one color, Terskikh and colleagues introduced random mutations into drFP583, and found one mutant (called E5) that changes its fluorescence from green to red in a time-dependent manner. As E5 switches from green to red fluorescence over time, it can be used as a timer for gene expression. During the first hours of activity of a promoter, green fluorescence is predominant, whereas sustained activity of the promoter leads to a mixture of green and red fluorescence. A few hours after the promoter is turned off, only red fluorescence remains."], ["litarch_figures/df/45/coffeebrk_NBK2345/A557.jpg", "\nThe change in fluorescence of E5 over time in C. elegans.\n The E5 mutant was placed under the control of a heat shock promoter and injected into C. elegans embryos. Green fluoresence was detected 2 hours into the recovery phase following a standard heat shock treatment (1 hour incubation at 33\u00b0). The embryos were documented under bright field (DIC), with a FITC filter, with a PE filter, and with an overlay at 3.5, 7.5, and 50 hours following heat shock. Yellow fluorescence, as seen in the overlay column at 7.5 hours, indicates a combination of green and red fluorescence. \n", ""], ["The change in fluorescence of E5 over time in C. elegans."], [" The E5 mutant was placed under the control of a heat shock promoter and injected into C. elegans embryos. Green fluoresence was detected 2 hours into the recovery phase following a standard heat shock treatment (1 hour incubation at 33\u00b0). The embryos were documented under bright field (DIC), with a FITC filter, with a PE filter, and with an overlay at 3.5, 7.5, and 50 hours following heat shock. Yellow fluorescence, as seen in the overlay column at 7.5 hours, indicates a combination of green and red fluorescence. "], ["Terskikh and colleagues verified these predictions in three experimental systems. First they monitored up- and down-regulation of E5 expression in Tet-on and Tet-off mammalian expression systems. Then they followed the activity of a heat-shock promoter during heat-induced stress ofCaenorhabditis elegans. Last, they traced the expression of a homeobox gene involved in the patterning of anterior structures in Xenopus laevis. In all cases, green fluorescence correctly indicated the onset of gene expression and was replaced with red fluorescence when expression ceased."], ["So after decades of blue-stained embryos, we'll now have to get used to seeing gene expression in green and red."], ["Story contributed by Raluca Gagescu, Nature Reviews Molecular Cell Biology\n"], ["\n\n"], ["VAST search for structures similar to E5"], ["Created: January 22, 2001"], ["Click on the link below to start an html tutorial."], ["\nSearch for structures similar to E5"], ["Live PubMed searches"], ["\n(1) Coral proteins\n"], ["\n(2) Heat-shock promoters\n"], ["\n(3) REVIEWS\n"], ["Additional NCBI resources"], ["\nNCBI Structure Group\n"], ["\nTaxonomic tree of jelly fish & red coral\n"]]}
 {"file": "coffeebrk_NBK2345/A643.nxml", "text": "Are you surprised that the tiny chihuahua belongs to the same species as the imposing great dane? The domestic dog species (Canis familiaris) includes more than 400 breeds that differ, for example in their appearance (size, coat length, and color) and behavior, (guarding, herding, and hunting). \nMore than 150 breeds are officially recognized by the American Kennel Club, which assigns each breed to one of seven groups or a miscellaneous class, based on the uses for which the breeds were originally developed. \nIn a recent study, Parker et al. studied the genetic relationships among a diverse range of dog breeds. They found that most breeds of dog fall into four groups\u2014three \"modern\" categories and one \"ancient\" group that may date back to antiquity. \nThe modern categories include breeds that have been around for fewer than 400 years:\nWorking dogs/guard dogs, e.g., mastiff, bulldog, boxer\nHerding dogs, e.g., Belgian sheepdog, collie, shetland sheepdog\nHunting dogs, e.g., scent hounds, terriers, spaniels, pointers, retrievers \nThis genetic classification of breeds grouped dogs together in a way that matched similarities in morphology and geographical origin. However, there were some surprises. For example, the oldest of all dog breeds are commonly believed to be the Pharaoh Hound and Ibizan Hound, which resemble the ancient Egyptian dogs drawn on tomb walls more than 5000 years ago. However, this study failed to detect their ancient lineage. This may be because they are modern recreations of old breeds or because current tools are unable to detect their ancient genes.\nIn contrast, a diverse group of dog breeds appears to be most related to the dog's ancient ancestor, the grey wolf. These breeds include dogs whose appearances resemble the wolf (e.g., the Siberian Husky) and dogs that do not (e.g., the cuddlely Sharpei). Breeds that belong to this ancient grouping are diverse and originate from different continents, e.g., the Afghan from the Middle East, the Basenji from Africa, the Tibetan Terrier from Tibet, the Pekingese from China, and the Alaskan Malamute from the Arctic. \nParker et al. looked at microsatellites to find the genetic differences between breeds of domestic dogs. Microsatellites are short segments of DNA that contain repeats of DNA sequence. The repeats usually occur in a noncoding part of the gene, and their number is highly variable. Analysis of the microsatellites of 414 dogs representing 85 different breeds revealed that the degree of genetic differentiation between dog breeds is much higher than that found between human populations on different continents!\nGiven that most modern dog breeds have existed for fewer than 400 years, it is surprising that dog breeds are genetically distinct. But a dog can be matched to its breed by its individual genotype. Of 414 dogs tested, only 4 dogs were assigned to the wrong breed.\nThis apparent genetic isolation of dog breeds through selective breeding was reinforced by the formation of breed clubs in the mid-19th century. Rules such as the \"Breed Barrier Rule\" states that \"no dog may become a registered member of a breed unless its dam and sire are registered members\". Such selective breeding generates not only genetically diverse breeds of dog but also leads to the accumulation of mutations and inherited diseases. By using a genetic classification of dog breeds, scientists will be able to select breeds of dogs that share the same ancient mutations and genetic predisposition to diseases that some humans have. Analysis of this DNA is more likely to yield information about the diseases and the mutations responsible for them. \nReferences\nTaxonomy\n\nTaxonomy information for Canis familiaris\n\nLive PubMed Searches\n\nEvolution of the domestic dog\n\n\nMicrosatellites and dogs\n\n\nDog breeds and genetic diseases\n\nResources\nDog Genome ProjectFred Hutchinson Cancer Research Center\n\nDog Genome ProjectUniversity of California, Berkeley", "pairs": [], "interleaved": []}