Showing papers by "Cold Spring Harbor Laboratory published in 2001"
TL;DR: The results of an international collaboration to produce and make freely available a draft sequence of the human genome are reported and an initial analysis is presented, describing some of the insights that can be gleaned from the sequence.
Abstract: The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.
22,269 citations
TL;DR: Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals, and has a distinctive structure, which includes a helicase domain and dualRNase III motifs.
Abstract: RNA interference (RNAi) is the mechanism through which double-stranded RNAs silence cognate genes. In plants, this can occur at both the transcriptional and the post-transcriptional levels; however, in animals, only post-transcriptional RNAi has been reported to date. In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease, RISC, to destroy specific messenger RNAs. Here we identify an enzyme, Dicer, which can produce putative guide RNAs. Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi.
5,229 citations
TL;DR: In vivo evidence is provided that lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the Clr4 protein at heterochromatin-associated regions in fission yeast, defining a conserved pathway wherein sequential histone modifications establish a “histone code” essential for the epigenetic inheritance of heterochROMatin assembly.
Abstract: The assembly of higher order chromatin structures has been linked to the covalent modifications of histone tails. We provide in vivo evidence that lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the Clr4 protein at heterochromatin-associated regions in fission yeast. Both the conserved chromo- and SET domains of Clr4 are required for H3 Lys9 methylation in vivo. Localization of Swi6, a homolog of Drosophila HP1, to heterochomatic regions is dependent on H3 Lys9 methylation. Moreover, an H3-specific deacetylase Clr3 and a beta-propeller domain protein Rik1 are required for H3 Lys9 methylation by Clr4 and Swi6 localization. These data define a conserved pathway wherein sequential histone modifications establish a "histone code" essential for the epigenetic inheritance of heterochromatin assembly.
1,708 citations
TL;DR: Biochemical purification of the RNAi effector nuclease from cultured Drosophila cells reveals that one constituent of this complex is a member of the Argonaute family of proteins, which are essential for gene silencing in Caenorhabditis elegans, Neurospora, and Arabidopsis.
Abstract: Double-stranded RNA induces potent and specific gene silencing through a process referred to as RNA interference (RNAi) or posttranscriptional gene silencing (PTGS). RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (∼22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity are unknown. Here, we report the biochemical purification of the RNAi effector nuclease from cultured Drosophila cells. The active fraction contains a ribonucleoprotein complex of ∼500 kilodaltons. Protein microsequencing reveals that one constituent of this complex is a member of the Argonaute family of proteins, which are essential for gene silencing in Caenorhabditis elegans , Neurospora , and Arabidopsis . This observation begins the process of forging links between genetic analysis of RNAi from diverse organisms and the biochemical model of RNAi that is emerging from Drosophila in vitro systems.
1,602 citations
TL;DR: The combination of regulated addition and continuous replacement of synaptic receptors can stabilize long-term changes in synaptic efficacy and may serve as a general model for how surface receptor number is established and maintained.
1,106 citations
TL;DR: The power of RNA interference, the process by which double-stranded RNA induces the silencing of homologous endogenous genes, is slowly becoming clear, and might help to develop an effortless tool to probe gene function in cells and animals.
Abstract: Imagine being able to knock out your favourite gene with only a day's work. Not just in one model system, but in virtually any organism: plants, flies, mice or cultured cells. This sort of experimental dream might one day become reality as we learn to harness the power of RNA interference, the process by which double-stranded RNA induces the silencing of homologous endogenous genes. How this phenomenon works is slowly becoming clear, and might help us to develop an effortless tool to probe gene function in cells and animals.
1,051 citations
TL;DR: It is concluded that Apaf-1 is inactivated in metastatic melanomas, which leads to defects in the execution of apoptotic cell death, which may contribute to the low frequency of p53 mutations observed in this highly chemoresistant tumour type.
Abstract: Metastatic melanoma is a deadly cancer that fails to respond to conventional chemotherapy and is poorly understood at the molecular level. p53 mutations often occur in aggressive and chemoresistant cancers but are rarely observed in melanoma. Here we show that metastatic melanomas often lose Apaf-1, a cell-death effector that acts with cytochrome c and caspase-9 to mediate p53-dependent apoptosis. Loss of Apaf-1 expression is accompanied by allelic loss in metastatic melanomas, but can be recovered in melanoma cell lines by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine (5aza2dC). Apaf-1-negative melanomas are invariably chemoresistant and are unable to execute a typical apoptotic programme in response to p53 activation. Restoring physiological levels of Apaf-1 through gene transfer or 5aza2dC treatment markedly enhances chemosensitivity and rescues the apoptotic defects associated with Apaf-1 loss. We conclude that Apaf-1 is inactivated in metastatic melanomas, which leads to defects in the execution of apoptotic cell death. Apaf-1 loss may contribute to the low frequency of p53 mutations observed in this highly chemoresistant tumour type.
990 citations
TL;DR: It is shown that distinct site-specific histone H3 methylation patterns define euchromatic and heterochromatic chromosomal domains within a 47-kilobase region of the mating-type locus in fission yeast.
Abstract: Eukaryotic genomes are organized into discrete structural and functional chromatin domains. Here, we show that distinct site-specific histone H3 methylation patterns define euchromatic and heterochromatic chromosomal domains within a 47-kilobase region of the mating-type locus in fission yeast. H3 methylated at lysine 9 (H3 Lys9), and its interacting Swi6 protein, are strictly localized to a 20-kilobase silent heterochromatic interval. In contrast, H3 methylated at lysine 4 (H3 Lys4) is specific to the surrounding euchromatic regions. Two inverted repeats flanking the silent interval serve as boundary elements to mark the borders between heterochromatin and euchromatin. Deletions of these boundary elements lead to spreading of H3 Lys9 methylation and Swi6 into neighboring sequences. Furthermore, the H3 Lys9 methylation and corresponding heterochromatin-associated complexes prevent H3 Lys4 methylation in the silent domain.
760 citations
TL;DR: A comparative analysis of the structural relationships among vertebrate PTP domains is presented and a comprehensive resource for sequence analysis of phosphotyrosine-specific PTPs is provided.
Abstract: With the current access to the whole genomes of various organisms and the completion of the first draft of the human genome, there is a strong need for a structure-function classification of protein families as an initial step in moving from DNA databases to a comprehensive understanding of human biology. As a result of the explosion in nucleic acid sequence information and the concurrent development of methods for high-throughput functional characterization of gene products, the genomic revolution also promises to provide a new paradigm for drug discovery, enabling the identification of molecular drug targets in a significant number of human diseases. This molecular view of diseases has contributed to the importance of combining primary sequence data with three-dimensional structure and has increased the awareness of computational homology modeling and its potential to elucidate protein function. In particular, when important proteins or novel therapeutic targets are identified—like the family of protein tyrosine phosphatases (PTPs) (reviewed in reference 53)—a structure-function classification of such protein families becomes an invaluable framework for further advances in biomedical science. Here, we present a comparative analysis of the structural relationships among vertebrate PTP domains and provide a comprehensive resource for sequence analysis of phosphotyrosine-specific PTPs.
690 citations
TL;DR: In many sexually dimorphic species, a mechanism is required to ensure equivalent levels of gene expression from the sex chromosomes, and in mammals, such dosage compensation is achieved by X-chromosome inactivation, a process that presents a unique medley of biological puzzles.
Abstract: In many sexually dimorphic species, a mechanism is required to ensure equivalent levels of gene expression from the sex chromosomes. In mammals, such dosage compensation is achieved by X-chromosome inactivation, a process that presents a unique medley of biological puzzles: how to silence one but not the other X chromosome in the same nucleus; how to count the number of X's and keep only one active; how to choose which X chromosome is inactivated; and how to establish this silent state rapidly and efficiently during early development. The key to most of these puzzles lies in a unique locus, the X-inactivation centre and a remarkable RNA — Xist — that it encodes.
647 citations
TL;DR: An increased AtSERK1 level is sufficient to confer embryogenic competence in culture and demonstrate its role during establishment of somatic embryogenesis in culture.
Abstract: We report here the isolation of the Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (AtSERK1) gene and we demonstrate its role during establishment of somatic embryogenesis in culture. The AtSERK1 gene is highly expressed during embryogenic cell formation in culture and during early embryogenesis. The AtSERK1 gene is first expressed in planta during megasporogenesis in the nucleus of developing ovules, in the functional megaspore, and in all cells of the embryo sac up to fertilization. After fertilization, AtSERK1 expression is seen in all cells of the developing embryo until the heart stage. After this stage, AtSERK1 expression is no longer detectable in the embryo or in any part of the developing seed. Low expression is detected in adult vascular tissue. Ectopic expression of the full-length AtSERK1 cDNA under the control of the cauliflower mosaic virus 35S promoter did not result in any altered plant phenotype. However, seedlings that overexpressed the AtSERK1 mRNA exhibited a 3- to 4-fold increase in efficiency for initiation of somatic embryogenesis. Thus, an increased AtSERK1 level is sufficient to confer embryogenic competence in culture.
TL;DR: These studies identify CED-12/ELMO as an upstream regulator of Rac1 that affects engulfment and cell migration from C. elegans to mammals.
TL;DR: Protein tyrosine phosphatases, the enzymes that dephosphorylate tyrosyl phosphoproteins, were initially believed to be few in number and serve a 'housekeeping' role in signal transduction, but work indicates that this is totally incorrect.
TL;DR: It is reported that methylation of histone H3 lysine 9 on the inactive X chromosome occurs immediately after Xist RNA coating and before transcriptional inactivation of X-linked genes.
TL;DR: The past year has witnessed refinements in models of spliceosome assembly pathways and in the understanding of how splicing factors of the serine/arginine-rich protein family function.
TL;DR: In vivo studies indicate that synaptic activity promotes dendritic arbor elaboration at early stages of brain development and at later stages of development, synaptic activity stabilizes dendrite structure.
TL;DR: This work considers the role that transposons play in establishing methylation patterns and the epigenetic consequences of their perturbation in plants and filamentous fungi.
Abstract: Plants and filamentous fungi share with mammals enzymes responsible for DNA methylation. In these organisms, DNA methylation is associated with gene silencing and transposon control. However, plants and fungi differ from mammals in the genomic distribution, sequence specificity, and heritability of methylation. We consider the role that transposons play in establishing methylation patterns and the epigenetic consequences of their perturbation.
TL;DR: It is shown here that inappropriate exon skipping can be reproduced in vitro, and results from disruption of a splicing enhancer in the coding sequence, arguing against a nuclear reading-frame scanning mechanism for NAS.
Abstract: Point mutations can generate defective and sometimes harmful proteins. The nonsense-mediated mRNA decay (NMD) pathway minimizes the potential damage caused by nonsense mutations. In-frame nonsense codons located at a minimum distance upstream of the last exon-exon junction are recognized as premature termination codons (PTCs), targeting the mRNA for degradation. Some nonsense mutations cause skipping of one or more exons, presumably during pre-mRNA splicing in the nucleus; this phenomenon is termed nonsense-mediated altered splicing (NAS), and its underlying mechanism is unclear. By analyzing NAS in BRCA1, we show here that inappropriate exon skipping can be reproduced in vitro, and results from disruption of a splicing enhancer in the coding sequence. Enhancers can be disrupted by single nonsense, missense and translationally silent point mutations, without recognition of an open reading frame as such. These results argue against a nuclear reading-frame scanning mechanism for NAS. Coding-region single-nucleotide polymorphisms (cSNPs) within exonic splicing enhancers or silencers may affect the patterns or efficiency of mRNA splicing, which may in turn cause phenotypic variability and variable penetrance of mutations elsewhere in a gene.
TL;DR: It is suggested that CAF-1 plays a critical role in the organization of SAM and RAM during postembryonic development by facilitating stable maintenance of gene expression states.
TL;DR: The Distributed Annotation System (DAS) allows sequence annotations to be decentralized among multiple third-party annotators and integrated on an as-needed basis by client-side software.
Abstract: Background
Currently, most genome annotation is curated by centralized groups with limited resources. Efforts to share annotations transparently among multiple groups have not yet been satisfactory.
TL;DR: The differential antagonism between a negative and two positive regulators exemplifies how inclusion of an alternative exon can be modulated.
TL;DR: It is shown that synaptic transmission from mushroom body neurons is required during memory retrieval but not during acquisition or storage, and it is proposed that the hebbian processes underlying olfactory associative learning reside in mushroom body dendrites or upstream of the mushroom body and that the resulting alterations in synaptic strength modulate mushroom body output during memory retrieved.
Abstract: Surgical, pharmacological and genetic lesion studies have revealed distinct anatomical sites involved with different forms of learning. Studies of patients with localized brain damage and work in rodent model systems, for example, have shown that the hippocampal formation participates in acquisition of declarative tasks but is not the site of their long-term storage1,2. Such lesions are usually irreversible, however, which has limited their use for dissecting the temporal processes of acquisition, storage and retrieval of memories3,4. Studies in bees and flies have similarly revealed a distinct anatomical region of the insect brain, the mushroom body, that is involved specifically in olfactory associative learning5,6. We have used a temperature-sensitive dynamin transgene, which disrupts synaptic transmission reversibly and on the time-scale of minutes7, to investigate the temporal requirements for ongoing neural activity during memory formation. Here we show that synaptic transmission from mushroom body neurons is required during memory retrieval but not during acquisition or storage. We propose that the hebbian processes underlying olfactory associative learning reside in mushroom body dendrites or upstream of the mushroom body and that the resulting alterations in synaptic strength modulate mushroom body output during memory retrieval.
TL;DR: It is proposed that ubiquitylation regulates TAD function by serving as a dual signal for activation and activator destruction, demonstrating that activator ubiquitylated is essential for transcriptional activation.
Abstract: The ability of transcriptional activation domains (TADs) to signal ubiquitin-mediated proteolysis suggests an involvement of the ubiquitin-proteasome pathway in transcription. To probe this involvement, we asked how ubiquitylation regulates the activity of a transcription factor containing the VP16 TAD. We show that the VP16 TAD signals ubiquitylation through the Met30 ubiquitin-ligase and that Met30 is also required for the VP16 TAD to activate transcription. The requirement for Met30 in transcription is circumvented by fusion of ubiquitin to the VP16 activator, demonstrating that activator ubiquitylation is essential for transcriptional activation. We propose that ubiquitylation regulates TAD function by serving as a dual signal for activation and activator destruction.
TL;DR: Evidence is provided that chromatin immunoprecipitation can be used to identify E2F-regulated promoters which contain both consensus and nonconsensus binding sites and it is shown that not all E 2F- regulated promoters show identical expression profiles.
Abstract: We have taken a new approach to the identification of E2F-regulated promoters. After modification of a chromatin immunoprecipitation assay, we cloned nine chromatin fragments which represent both strong and weak in vivo E2F binding sites. Further characterization of three of the cloned fragments revealed that they are bound in vivo not only by E2Fs but also by members of the retinoblastoma tumor suppressor protein family and by RNA polymerase II, suggesting that these fragments represent promoters regulated by E2F transcription complexes. In fact, database analysis indicates that all three fragments correspond to genomic DNA located just upstream of start sites for previously identified mRNAs. One clone, ChET 4, corresponds to the promoter region for beclin 1, a candidate tumor suppressor protein. We demonstrate that another of the clones, ChET 8, is strongly bound by E2F family members in vivo but does not contain a consensus E2F binding site. However, this fragment functions as a promoter whose activity can be repressed by E2F1. Finally, we demonstrate that the ChET 9 promoter contains a consensus E2F binding site, can be activated by E2F1, and drives expression of an mRNA that is upregulated in colon and liver tumors. Interestingly, the characterized ChET promoters do not display regulation patterns typical of known E2F target genes in a U937 cell differentiation system. In summary, we have provided evidence that chromatin immunoprecipitation can be used to identify E2F-regulated promoters which contain both consensus and nonconsensus binding sites and have shown that not all E2F-regulated promoters show identical expression profiles.
TL;DR: A set of discriminant functions that can recognize structural and compositional features such as CpG islands, promoter regions and first splice-donor sites are presented and it is shown by cross-validation that the program could predict 86% of the first exons with 17% false positives.
Abstract: The identification of promoters and first exons has been one of the most difficult problems in gene-finding. We present a set of discriminant functions that can recognize structural and compositional features such as CpG islands, promoter regions and first splice-donor sites. We explain the implementation of the discriminant functions into a decision tree that constitutes a new program called FirstEF. By using different models to predict CpG-related and non-CpG-related first exons, we showed by cross-validation that the program could predict 86% of the first exons with 17% false positives. We also demonstrated the prediction accuracy of FirstEF at the genome level by applying it to the finished sequences of human chromosomes 21 and 22 as well as by comparing the predictions with the locations of the experimentally verified first exons. Finally, we present the analysis of the predicted first exons for all of the 24 chromosomes of the human genome.
TL;DR: The aim of high-quality annotation is to identify the key features of the genome — in particular, the genes and their products.
Abstract: The genome sequence of an organism is an information resource unlike any that biologists have previously had access to. But the value of the genome is only as good as its annotation. It is the annotation that bridges the gap from the sequence to the biology of the organism. The aim of high-quality annotation is to identify the key features of the genome - in particular, the genes and their products. The tools and resources for annotation are developing rapidly, and the scientific community is becoming increasingly reliant on this information for all aspects of biological research.
TL;DR: An electroporation technique for targeting gene transfer to individual cells in intact tissue that will allow unprecedented spatial and temporal control over gene delivery and protein expression is reported.
TL;DR: WormBase is a web-based resource for the Caenorhabditis elegans genome and its biology that builds upon the existing ACeDB database of the C.elegans genome by providing data curation services, a significantly expanded range of subject areas and a user-friendly front end.
Abstract: WormBase (http://www.wormbase.org) is a web-based resource for the Caenorhabditis elegans genome and its biology. It builds upon the existing ACeDB database of the C.elegans genome by providing data curation services, a significantly expanded range of subject areas and a user-friendly front end.
TL;DR: DNA transposons in plants appear to be regulated by chromatin remodeling, and gene silencing and paramutation are also regulated by DDM1, providing support for the proposition that epigenetic silencing is related to transposon regulation.
Abstract: Robertson's Mutator transposable elements in maize undergo cycles of activity and then inactivity that correlate with changes in cytosine methylation. Mutator-like elements are present in the Arabidopsis genome but are heavily methylated and inactive. These elements become demethylated and active in the chromatin-remodeling mutant ddm1 (Decrease in DNA Methylation), which leads to loss of heterochromatic DNA methylation. Thus, DNA transposons in plants appear to be regulated by chromatin remodeling. In inbred ddm1 strains, transposed elements may account, in part, for mutant phenotypes unlinked to ddm1. Gene silencing and paramutation are also regulated by DDM1, providing support for the proposition that epigenetic silencing is related to transposon regulation.
TL;DR: Experiments show that the cytoskeleton plays a critical role in the regulation of various cellular processes linked to transformation including proliferation, contact inhibition, anchorage-independent cell growth, and apoptosis.