The Simple Chordate \u3cem\u3eCiona intestinalis\u3c/em\u3e Has a Reduced Complement of Genes Associated with Fanconi Anemia

Fanconi anemia (FA) is a human genetic disease characterized by congenital defects, bone marrow failure, and increased cancer risk. FA is associated with mutation in one of 24 genes. The protein products of these genes function cooperatively in the FA pathway to orchestrate the repair of DNA interstrand cross-links. Few model organisms exist for the study of FA. Seeking a model organism with a simpler version of the FA pathway, we searched the genome of the simple chordate Ciona intestinalis for homologs of the human FA-associated proteins. BLAST searches, sequence alignments, hydropathy comparisons, maximum likelihood phylogenetic analysis, and structural modeling were used to infer the likelihood of homology between C. intestinalis and human FA proteins. Our analysis indicates that C. intestinalis indeed has a simpler and potentially functional FA pathway. The C. intestinalis genome was searched for candidates for homology to 24 human FA and FA-associated proteins. Support was found for the existence of homologs for 13 of these 24 human genes in C. intestinalis. Members of each of the three commonly recognized FA gene functional groups were found. In group I, we identified homologs of FANCE, FANCL, FANCM, and UBE2T/FANCT. Both members of group II, FANCD2 and FANCI, have homologs in C. intestinalis. In group III, we found evidence for homologs of FANCJ, FANCO, FANCQ/ERCC4, FANCR/RAD51, and FANCS/BRCA1, as well as the FA-associated proteins ERCC1 and FAN1. Evidence was very weak for the existence of homologs in C. intestinalis for any other recognized FA genes. This work supports the notion that C. intestinalis, as a close relative of vertebrates, but having a much reduced complement of FA genes, offers a means of studying the function of certain FA proteins in a simpler pathway than that of vertebrate cells

Proteomic responses to elevated ocean temperature in ovaries of the ascidian \u3cem\u3eCiona intestinalis\u3c/em\u3e

Ciona intestinalis, a common sea squirt, exhibits lower reproductive success at the upper extreme of the water temperatures it experiences in coastal New England. In order to understand the changes in protein expression associated with elevated temperatures, and possible response to global temperature change, we reared C. intestinalis from embryos to adults at 18°C (a temperature at which they reproduce normally at our collection site in Rhode Island) and 22°C (the upper end of the local temperature range). We then dissected ovaries from animals at each temperature, extracted protein, and measured proteomic levels using shotgun mass spectrometry (LC-MS/MS). 1532 proteins were detected at a 1% false discovery rate present in both temperature groups by our LC-MS/MS method. 62 of those proteins are considered up- or down-regulated according to our statistical criteria. Principal component analysis shows a clear distinction in protein expression pattern between the control (18°C) group and high temperature (22°C) group. Similar to previous studies, cytoskeletal and chaperone proteins are upregulated in the high temperature group. Unexpectedly, we find evidence that proteolysis is downregulated at the higher temperature. We propose a working model for the high temperature response in C. intestinalis ovaries whereby increased temperature induces upregulation of signal transduction pathways involving PTPN11 and CrkL, and activating coordinated changes in the proteome especially in large lipid transport proteins, cellular stress responses, cytoskeleton, and downregulation of energy metabolism

The Simple Chordate Has a Reduced Complement of Genes Associated with Fanconi Anemia

Fanconi anemia (FA) is a human genetic disease characterized by congenital defects, bone marrow failure, and increased cancer risk. FA is associated with mutation in one of 24 genes. The protein products of these genes function cooperatively in the FA pathway to orchestrate the repair of DNA interstrand cross-links. Few model organisms exist for the study of FA. Seeking a model organism with a simpler version of the FA pathway, we searched the genome of the simple chordate Ciona intestinalis for homologs of the human FA-associated proteins. BLAST searches, sequence alignments, hydropathy comparisons, maximum likelihood phylogenetic analysis, and structural modeling were used to infer the likelihood of homology between C. intestinalis and human FA proteins. Our analysis indicates that C. intestinalis indeed has a simpler and potentially functional FA pathway. The C. intestinalis genome was searched for candidates for homology to 24 human FA and FA-associated proteins. Support was found for the existence of homologs for 13 of these 24 human genes in C. intestinalis. Members of each of the three commonly recognized FA gene functional groups were found. In group I, we identified homologs of FANCE, FANCL, FANCM, and UBE2T/FANCT. Both members of group II, FANCD2 and FANCI, have homologs in C. intestinalis. In group III, we found evidence for homologs of FANCJ, FANCO, FANCQ/ERCC4, FANCR/RAD51, and FANCS/BRCA1, as well as the FA-associated proteins ERCC1 and FAN1. Evidence was very weak for the existence of homologs in C. intestinalis for any other recognized FA genes. This work supports the notion that C. intestinalis , as a close relative of vertebrates, but having a much reduced complement of FA genes, offers a means of studying the function of certain FA proteins in a simpler pathway than that of vertebrate cells

The FANCD2 NLS mutants fail to correct the ICL sensitivity of FA-D2 patient cells.

<p>(<b>A</b>) FA-D2 cells stably expressing LacZ, FANCD2-WT, FANCD2-∆N57, FANCD2-∆N100, FANCD2-3N, or FANCD2-K561R were incubated in the absence or presence of 8 or 16 nM MMC for 24 h and the numbers of chromosome aberrations including gaps and breaks, dicentrics, and complex chromosome aberrations, including radial formations, were scored. Metaphase spreads were analyzed using a Zeiss AxioImager.A1 upright epifluorescent microscope with AxioVision LE 4.6 image acquisition software. At least 80 metaphases were scored per treatment. Error bars represent the standard error of the means. (<b>B</b>) FA-D2 cells stably expressing LacZ, FANCD2-WT and FANCD2-∆N57 were incubated in the absence (NT) or presence of 40 nM MMC for 18 h, and allowed to recover for 0, 4.5 or 7 h. Cells were then fixed, stained with rabbit polyclonal anti-DNA-PK_CS pS2056, and counterstained with DAPI. At least 300 cells were scored for nuclei with > 5 DNA-PK_CS foci. Error bars represent the standard error of the means from two independent experiments. ***, <i>p</i> < 0.001. </p

The FANCD2 NLS is required for the nuclear localization of a subset of FANCI.

<p>(<b>A</b>) FA-D2 patient cells or FA-D2 cells stably expressing FANCD2-WT were incubated in the absence (NT) or presence of MMC for 24 h, fixed, stained with rabbit polyclonal anti-FANCD2 or anti-FANCI antibody and counterstained with phalloidin and DAPI. AF-488, Alexa Fluor 488. (<b>B</b>) FA-D2 cells stably expressing LacZ, FANCD2-WT, FANCD2-∆N57, FANCD2-∆N100, and FANCD2-3N were incubated in the absence (NT) or presence of MMC for 24 h, fixed, and stained with rabbit polyclonal anti-FANCI antibody, and counterstained with phalloidin and DAPI. At least 300 cells were scored for cytoplasmic (Cyto.), nuclear (Nucl.), and both cytoplasmic and nuclear (Both) localization of FANCI. (<b>C</b>) COS-7 cells were transiently transfected with no DNA, FANCI-GFP, FANCI-GFP plus FANCD2-V5-WT, or FANCI-GFP plus FANCD2-V5-∆N57. Whole-cell lysates were immunoprecipitated with anti-V5 or anti-GFP antibodies and immune complexes immunoblotted with anti-GFP and anti-V5 antibodies.</p

The FANCD2 NLS is required for efficient FANCD2 and FANCI monoubiquitination and chromatin association.

<p>(<b>A</b>) FA-D2 cells stably expressing LacZ, FANCD2-WT, FANCD2-K561R, FANCD2-∆N57, FANCD2-∆N100 and FANCD2-3N were incubated in the absence and presence of 250 nM MMC for 18 h, and whole-cell lysates were immunoblotted with antibodies to FANCD2, V5, FANCI and α-tubulin. The FANCD2 and FANCI L/S ratios are the ratios of monoubiquitinated to nonubiquitinated protein, and were calculated by measuring protein band intensities using ImageJ image processing and analysis software (<a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>). (<b>B</b> and <b>C</b>) FA-D2 cells stably expressing FANCD2-WT, FANCD2-∆N57, FANCD2-∆N100 and FANCD2-3N were treated as above and cell pellets were fractionated into soluble (S) and chromatin-associated (C) fractions. Fractions were immunoblotted with antibodies against V5, FANCI, α-tubulin and H2A. W, unfractionated whole cell extract.</p

FANCD2-dependent and -independent mechanisms of FANCI nuclear localization.

<p>We propose that a subset of FANCI (blue) associates with FANCD2 (red) in the cytoplasm, and that the ID2 heterodimer is transported to the nucleus <i>via</i> an importin α/β (brown)-mediated transport mechanism, using the amino terminal FANCD2 NLS (light green). Nuclear ID2 binds to DNA (orange) and is also phosphorylated by the ATM/ATR kinases (dark green). One or both of these events may trigger ID2 complex restructuring, facilitating FANCD2 and FANCI monoubiquitination by FANCL (black), UBE2T (yellow) and the FA core complex (not shown).</p

Proteomic responses to elevated ocean temperature in ovaries of the ascidian Ciona intestinalis

Ciona intestinalis, a common sea squirt, exhibits lower reproductive success at the upper extreme of the water temperatures it experiences in coastal New England. In order to understand the changes in protein expression associated with elevated temperatures, and possible response to global temperature change, we reared C. intestinalis from embryos to adults at 18°C (a temperature at which they reproduce normally at our collection site in Rhode Island) and 22°C (the upper end of the local temperature range). We then dissected ovaries from animals at each temperature, extracted protein, and measured proteomic levels using shotgun mass spectrometry (LC-MS/MS). 1532 proteins were detected at a 1% false discovery rate present in both temperature groups by our LC-MS/MS method. 62 of those proteins are considered up- or down-regulated according to our statistical criteria. Principal component analysis shows a clear distinction in protein expression pattern between the control (18°C) group and high temperature (22°C) group. Similar to previous studies, cytoskeletal and chaperone proteins are upregulated in the high temperature group. Unexpectedly, we find evidence that proteolysis is downregulated at the higher temperature. We propose a working model for the high temperature response in C. intestinalis ovaries whereby increased temperature induces upregulation of signal transduction pathways involving PTPN11 and CrkL, and activating coordinated changes in the proteome especially in large lipid transport proteins, cellular stress responses, cytoskeleton, and downregulation of energy metabolism

A Java Tool for Exploring State Estimation using the Kalman Filter

This paper describes a novel software application that assists in understanding the process of estimating the state of a linear dynamic system based on noisy output signal measurements. An interactive Java tool, based on the Kalman Filter, is described. This consists of two main parts, a simple one-dimensional filter and a multi-dimensional filter tool. The user can set all input parameters through a single interface or by following a series of guided steps. Raw measurement data can be generated automatically or inserted manually by the user and the results of estimating the true system state are then presented as a series of graphs in real-time. The application is described through the use of two simple examples. Such an application could be used to teach signal and systems engineering