Anthony A Ferrante

Union Biometrica Inc.; Somerville MA, United States of America

Anthony A Ferrante et al. (2001) International C. elegans Meeting "Automated Sorting of C. elegans Muv Mutants According to Pseudovulva Number"

Russell Gershman, Anthony A Ferrante, W Peter Hansen, Steve Alam, Peter Kalutkiewicz

[International C. elegans Meeting, 2001]

The UBI COPAS BIOSORT system disperses and maintains nematodes in liquid suspension, straightens them, and flows them one-by-one through a laser beam. The system analyzes and sorts each animal according to both size and fluorescence without harm. For this study the system was modified such that positional information was retained as the nematodes were scanned along their longitudinal axis. Rather than averaging the fluorescence signal over the length of the animal, the signal was measured at approximately 100 points along the length of adult C. elegans . Profiling algorithms were developed that determined the location and the number of fluorescent features of the animals. The prototype was used to characterize and sort multivulva (Muv) C. elegans according to the number of vulvae. The Muv strains MT309 lin-15 and MT388 lin-12 (both obtained from the C. elegans Genetics Center, University of Minnesota, St. Paul), as well as wild-type strain N2, were stained using NEMASELECT Male/Herma stain (Union Biometrica, Inc.) to mark the location of their vulvae. Electronic profiles were collected with the new signal processing electronics and single nematodes were deposited in individual wells of a 96-well plate. Nematodes and their associated profiles were visually compared. Close correlation between the number of stained vulva and the number of fluorescence peaks in the profiles was observed. The prototype software accurately scored and sorted the animals according to vulva number.

Anthony A Ferrante et al. (2002) East Coast Worm Meeting "Check out my Profile! Isolation of mutants with altered str-1 expression levels using automated, high-sensitivity fluorescence profiling"

Britta Moellers, Bruce Holcombe, Steve Alam, Vance Chang, Anthony A Ferrante

[East Coast Worm Meeting, 2002]

Autofluorescence background, the natural fluorescence from biological materials, reduces sensitivity of detection of localized fluorescence in multicellular organisms. For example in the case of a transgenic C. elegans that expresses GFP in two cells, that fluorescence can be masked by the autofluorescence of the remaining 1000 somatic cells. We have solved this problem with an add-on to our COPAS analysis and sorter technology that allows the instrument to measure and store the pattern of fluorescence along the length of the animal. That information was used to restrict fluorescence analysis to the region of the animal that contained the fluorescent cells thus improving the signal to autofluorescence ratio more than 20-fold. We used the system to perform a genetic screen in which we isolated mutants of PY1089 (kindly provided by Piali Sengupta), a transgenic C. elegans that expresses GFP in the AWB sensory neurons under control of the str-1 promoter. Without the Profiler technology the COPAS system was unable to distinguish PY1089 from N2 wild type animals, and it could not be used in a genetic screen for mutants with reduced GFP expression. With the addition of Profiler we were able to clearly distinguish PY1089 from N2 and were further able to sort animals that had either reduced or increased GFP expression in the sensory neurons. F2 progeny of mutagenized PY1089 were analyzed and sorted using the COPAS BIOSORT with prototype Profiler hardware. First, 100 animals were run and their peak fluorescence intensities were determined. That information was used to set a range of normal peak heights in the Profiler. The Profiler was instructed to dispense those animals whose peak heights were outside of the normal range, either higher or lower, one per well into 96-well plates. Mutants were confirmed both by regrowing the sorted animals and running them through the Profiler a second time and visually by microscopy. Approximately 50,000 animals from 8 separate pools were screened and 1,000 were dispensed to wells in the first pass. We have retested 96 lines from the sorted animals to date. We have isolated 4 independent mutant lines with decreased GFP expression, 3 independent lines with increased GFP expression and 2 lines with a mix of increased, wild-type, and decreased GFP expression. We are in the process of further characterizing the mutants by fluorescence microscopy and we will further characterize the mutants for their chemotactic response to 2-nonanone, which is dependent on proper AWB neuronal function.

Anthony A Ferrante et al. (2002) Mid-west Worm Meeting "Check out my Profile! Isolation of chemotaxis defective mutants with altered str-1 expression levels using automated, high-sensitivity fluorescence profiling"

Bruce Holcombe, Peter Van Osta, Vance Chang, Jennifer Kean, Britta Moellers, Anthony A Ferrante, Steve Alam, Gregory O'Connor

[Mid-west Worm Meeting, 2002]

Autofluorescence background, the natural fluorescence from biological materials, reduces sensitivity of detection of localized fluorescence in multicellular organisms. For example in the case of a transgenic C. elegans that expresses GFP in two cells, that fluorescence can be masked by the autofluorescence of the remaining 1000 somatic cells. We have solved this problem with an add-on to our COPAS analysis and sorter technology that allows the instrument to measure and store the pattern of fluorescence along the length of the animal. That information was used to restrict fluorescence analysis to the region of the animal that contained the fluorescent cells thus improving the signal to autofluorescence ratio more than 20-fold. We used the Profiler system to perform a genetic screen in which we isolated mutants of PY1089 (kindly provided by Piali Sengupta), a transgenic C. elegans that expresses GFP in the AWB sensory neurons under control of the str-1 promoter. Proper expression of the odorant receptor, STR-1, in the AWB sensory neurons is required for appropriate chemotactic response to the repulsive odorant 2-nonanone. Without the Profiler technology the COPAS system was unable to distinguish PY1089 from N2 wild type animals, and it could not be used in a genetic screen for mutants with reduced GFP expression. With the addition of Profiler we were able to clearly distinguish PY1089 from N2 and were further able to sort animals that had either reduced or increased GFP expression in the sensory neurons. F2 progeny of mutagenized PY1089 were analyzed and sorted using the COPAS BIOSORT with prototype Profiler hardware. First, 100 animals were run and their peak fluorescence intensities were determined. That information was used to set a range of normal peak heights in the Profiler. The Profiler was instructed to dispense those animals whose peak heights were outside of the normal range, either higher or lower, one per well into 96-well plates. Mutants were confirmed by regrowing the sorted animals and running them through the Profiler a second time. Approximately 50,000 animals from 8 separate pools were screened and 1,000 were dispensed to wells in the first pass. We have retested 109 lines from the sorted animals to date. We have isolated 5 independent mutant lines with decreased GFP expression, 5 independent lines with increased GFP expression and 2 lines with a mix of increased, wild-type, and decreased GFP expression. So far, we have tested 5 mutant lines for their chemotactic response to 2-nonanone. Of these one line has demonstrated defective chemotaxis

Anthony A Ferrante et al. (2002) West Coast Worm Meeting "Check out my Profile! Isolation of chemotaxis defective mutants with altered str-1 expression levels using automated, high-sensitivity fluorescence profiling."

Gregory O'Connor, Steven Alam, Bruce Holcombe, Anthony A Ferrante, Jennifer Kean, Peter Van Osta, Vance Chang, Britta Moellers

[West Coast Worm Meeting, 2002]

Autofluorescence background, the natural fluorescence from biological materials, reduces sensitivity of detection of localized fluorescence in multicellular organisms. For example in the case of a transgenic C. elegans that expresses GFP in two cells, that fluorescence can be masked by the autofluorescence of the remaining 1000 somatic cells. We have solved this problem with an add-on to our COPAS analysis and sorter technology that allows the instrument to measure and store the pattern of fluorescence along the length of the animal. That information was used to restrict fluorescence analysis to the region of the animal that contained the fluorescent cells thus improving the signal to autofluorescence ratio more than 20-fold. We used the Profiler system to perform a genetic screen in which we isolated mutants of PY1089 (kindly provided by Piali Sengupta), a transgenic C. elegans that expresses GFP in the AWB sensory neurons under control of the str-1 promoter. Proper expression of the odorant receptor, STR-1, in the AWB sensory neurons is required for appropriate chemotactic response to the repulsive odorant 2-nonanone. Without the Profiler technology the COPAS system was unable to distinguish PY1089 from N2 wild type animals, and it could not be used in a genetic screen for mutants with reduced GFP expression. With the addition of Profiler we were able to clearly distinguish PY1089 from N2 and were further able to sort animals that had either reduced or increased GFP expression in the sensory neurons.

OK257

Caenorhabditis elegans

Jacques Pecreaux et al. (2006) European Worm Meeting "Mechanical Oscillations during Asymmetric Spindle Positioning Require a Threshold Number of Active Cortical Force Generators"

Jonathon Howard, Frank Julicher, Stephan W. Grill, Anthony A. Hyman, Karsten Kruse, Jens-Christian Roper, Jacques Pecreaux

[European Worm Meeting, 2006]

Jacques Pecreaux1, Jens-Christian Rper2, Karsten Kruse3, Frank Julicher3, Anthony A. Hyman1, Stephan W. Grill, Jonathon Howard1 Background. Asymmetric division of the C. elegans zygote is due to the posterior-directed movement of the mitotic spindle during metaphase and anaphase. During this movement along the anterior-posterior axis, the spindle oscillates transversely. A theoretical analysis indicates that oscillations might occur as a result of the concerted action of many cortical force generators that pull on astral microtubules in a tug-of-war situation. This model predicts a threshold of motor activity below which no oscillations occur. Results: We have tested the existence of a threshold by using RNA interference to gradually reduce the levels of GPR-1 and GPR-2 that are involved in the G-protein-mediated regulation of the force generators. We found an abrupt cessation of oscillations as expected if the activity drops below a threshold. Furthermore, we could account for the complex choreography of the mitotic spindle - the precise temporal coordination of the build-up and die-down of the transverse oscillations with the posterior displacement - by a gradual increase in the processivity of the force generators during metaphase and anaphase. Conclusions: The agreement between our results and modeling suggests that the same motor machinery underlies two different spindle motions in the embryo: the equal and opposite motors on each side of the AP axis drive oscillations whereas the imbalanced motors in the two halves of the embryo drive posterior displacement.

Anthony Rogers et al. (2006) European Worm Meeting "WormBase: Recent and Future Developments"

Anthony Rogers, The WormBase Consortium

[European Worm Meeting, 2006]

Anthony Rogers1, The WormBase Consortium1234. WormBase is a database concerned primarily with providing current and comprehensive information on the genome and genetics of Caenorhabditis elegans. As more nematode worm genomes are being sequenced WormBase attempts to make this data available to the scientific community in a convenient and familiar fashion. We are constantly developing and evaluating new analyses and tools to help users make the most of this wealth of data. Recent developments have included the incorporation of external data sets such as InParanoid, COGS and TreeFam, Vancouver fosmid mappings and nematode EST clusters from NEMBASE and Nematode Net, amongst others.. As well as adding new data and features, our recent user survey indicated that some of the things weve been doing for a long time are still important. Correct gene structures were a high priority for the majority of respondents. We constantly review these in light of new data and user submissions. As more worm genome sequences become available they will give us more valuable comparative data for determining gene structures and other genomic sequence features. WormBase will play a central role in making the new genome data available in a timely manner. We will also be taking advantage of the COMPARA system developed for EnsEMBL to analyse and display this multispecies data.. To increase the accessibility of this data we have developed WormMart, a BioMart based data-warehouse including an easy-to-use wizard style query tool. The functionality of GBrowse has been enhanced, giving the ability to create genetic and physical maps. Web site speed has been an issue in the past and we have addressed this by restructuring the web site code, hardware and data management strategies. The establishment of a second European mirror in the UK should be of particular help to conference delegates.

Mihail Sarov et al. (2006) European Worm Meeting "Generic Method for C. elegans Gene Tagging by in vivo Recombineering in E. coli"

Francis Stewart, Mihail Sarov, Anthony Hyman

[European Worm Meeting, 2006]

Mihail Sarov1, Anthony Hyman2, Francis Stewart1. Here we present a fast and generic protocol for production of tagged transgenes for bombardment-based transformation in C. elegans by engineering of genomic BAC clones. The tag is inserted with minimal disturbance to the endogenous genomic context. The gene is then subcloned into an unc-119 based shuttle vector. All steps are done by in vivo recombineering in E. coli. Large DNA sequences can be modified in this way, and the method is applicable for virtually any gene of interest. All regulatory regions from 5 and 3 UTRs, introns etc. are present in the transgenic construct, and are likely to result in a correct expression pattern.. As a proof of principle we used the RPCI-94 Caenorhabditis briggsae BAC library. We GFP tagged and subcloned a relatively large (16 kbp) gene. Transformation efficiency by bombardment was similar to that of the commonly used pAZ132 vector. GFP expression patterns were reproducible and similar to that of a published promoter:GFP reporter construct. To show that the C. briggsae transgene is functional in C. elegans we knocked down the endogenous gene by RNAi. This makes the transgene the only expressed copy of the gene, essentially simulating a knock-in situation. The RNAi induced phenotype was rescued by the transgene in most of the animals.. While the ability to knock down the endogenous gene is a big advantage of the cross-species transgenes, C. elegans clones would still be preferable for most studies. Recently a genomic fosmid library for C. elegans was announced. Our protocol can be directly applied to these clones. The method is fast - tagging and subcloning can be accomplished within a week. The high efficiency of recombination in E. coli makes it possible do all steps in liquid culture, only checking the final construct for correct recombination. We are currently setting up the conditions for 96 well format tagging that would allow us to automatically process a large number of genes in parallel. We aim to establish a library of tagged transgenic constructs covering the whole genome as a community resource.

Chan-Hsien Yeh et al. (2004) East Asia Worm Meeting "Screen for TLK-1-interacting proteins"

Chan-Hsien Yeh, Yi-Chun Wu

[East Asia Worm Meeting, 2004]

Tousled-like kinases (TLKs) constitute a family of serine/threonine kinases conserved in multi-cellular organisms and have important functions during cell-cycle progression. Altering TLK activity in loss or gain-of-function experiments caused aberrant chromosome morphology and arrested nuclear divisions. Human and fly TLKs appear to function in chromosome assembly and nuclear division, at least in part, through phosphorylation of histone H3 and histone chaperone protein Asf-1 (anti-silencing function protein 1) (Li Y. et al, 2001; Carrera P. et al, 2003). Our studies of the tlk-1 gene by RNAi showed that tlk-1 is important for DNA condensation and segregation during mitosis. In tlk-1(RNAi) embryos, mitotic DNA was not condensed normally and DNA segregation defects were observed. The phosphorylation of histone H3 has been implicated in mitotic DNA condensation. However, histone H3 was still phosphorylated in tlk(RNAi) mutants as detected by immuno-staining using the antibody against phospho-histone H3 (Han Z. et al, 2003).To understand how TLK-1 functions, we performed a yeast two-hybrid screen using TLK-1 as bait. In a pilot screen, we obtained TLK-1-interacting proteins with previously identified roles in translation, ubiquitination, signal transduction, cell-cycle regulation or cytoplasmic cargo translocation. We are currently testing the physiological significance of the interactions and performing a large-scale yeast two-hybrid screen for more TLK-1 interacting candidates. In parallel, we are establishing a TLK-1 kinase assay using the TLK-1 protein expressed in the baculovirus system. References 1.Carrera, P., Moshkin, Y.M., Gronke, S., Sillje, H.H.W., Nigg, E.A., Jackle, H., and Karch, F. (2003) Genes & Dev., 17, 2578-2590. 2.Han, Z., Saam J.R., Adams, H.P., Mango S.E., and Schumacher, J.M. (2003) Curr. Biol., 13, 1921-1929. 3.Li, Y., DeFatta, R., Anthony, C., Sunavala, G., and DeBenedetti, A. (2001) Oncogene, 20, 726-738.