To whom it may concern,
Re: Dr. S. Gorski, Research Fellowship, 2001 Summary of Progress to Date
I. Project Summary
Programmed cell death (PCD) or apoptosis is a highly conserved and genetically regulated form of cell death that plays important roles in animal development, homeostasis, and disease. Dysregulation of cell death has been linked to a variety of human diseases including cancer. To understand the roles played by PCD in both cancer progression and treatment, I intend to discover and characterize new genes involved in programmed cell death with a long-term view towards identifying potential therapeutic targets. I will employ a functional genomics approach in the model system Drosophila melanogaster, an organism demonstrated previously to share with humans many features of apoptosis including the involvement of known conserved cell death genes. My strategy exploits the wealth of human and Drosophila DNA sequence information that is available publicly, including the essentially complete sequences of the genomes.
My gene discovery effort will employ Serial Analysis of Gene Expression (SAGE) to produce gene expression profiles from three distinct developmental stages of the Drosophila salivary gland. This is an ideal tissue because every cell is programmed to die. Previous studies of the Drosophila salivary gland have identified a stage just prior to apoptosis when certain cell death genes are upregulated and cell survival genes are downregulated, priming the tissue for massive cell death. I will compare the identity and abundance of all transcripts at this “death-priming” stage to all transcripts derived from earlier stages of development in the same tissue. This comparison will yield a large number of differentially expressed genes, potentially involved in apoptosis. Differential expression will be verified by real-time RT-PCR and candidate genes will be subjected subsequently to secondary screening strategies. These will include computer analyses to assess whether highly related sequences exist in the human genome, and in situ hybridization to other Drosophila stages/tissues that undergo apoptosis. The secondary screens will be used to prioritize genes for future detailed functional analyses. This comprehensive DNA sequence-based approach offers a powerful and unique platform for discovery of apoptosis related genes, a necessary first step towards better understanding apoptosis and the roles it plays in cancer.
II. Progress to Date
IIa. Apoptosis assay and RT-PCR studies in the Drosophila salivary gland: To determine the developmental timepoints for salivary gland dissection and to verify the salivary gland apoptosis observations made previously by Jiang et al. (1997, 2000), we conducted an apoptosis assay in salivary glands and initiated gene expression analyses. First, we used the early apoptosis indicator acridine orange to verify that the earliest detectable changes in the salivary glands occur at approximately 12 to13 hrs after puparium formation (APF; Appendix, Figure 1). Death of the gland occurs at approximately 14-15 hrs APF. Second, we designed PCR primers for genes known to be expressed in the salivary gland: one anti-apoptotic gene, Drosophila inhibitor of apoptosis protein-2 (diap-2) and 3 pro-apoptotic genes, reaper, hid, and grim. Using semi-quantitative RT-PCR (Figure 2) and quantitative real-time RT-PCR ( Fig 3), we examined so far the expression levels of reaper, hid, and diap-2 in salivary gland RNA isolated from multiple stages. Consistent with Jiang et al. (1997) we detected increasing levels of reaper and hid expression and decreasing levels of diap2 in 8 to 12 hr APF (@25ºC) salivary glands (Figures 2 and 3). The quantitative real-time RT-PCR was carried out using the SYBR green detection method followed by melting curve analysis to ensure a single major PCR amplification product. This method will be used also in future studies to verify differential gene expression discovered in the SAGE library comparisons (IIc below). IIb. Salivary gland-specific cDNA library and Expressed Sequence Tag (EST) generation:To assess transcript complexity in the salivary gland and to provide a valuable resource for our SAGE experiment analyses, we constructed a salivary gland-specific directional cDNA library from 500 salivary gland pairs encompassing the developmental stages we will use for our SAGE libraries (approximately 8 to 12 hr APF; S. Gorski, S. Chittaranjan, unpublished). This cDNA library will be used to retrieve cDNA sequences of interest not contained already in the Drosophila cDNA sequence databanks. Initially, we obtained 3’ end sequences from 314 cDNAs and compared these to the Drosophila sequence databases (Appendix, Table). This revealed that 13.7% of our sequences matched mitochondrial large ribosomal RNA, a known abundant polyadenylated sequence in Drosophila (Benkel et al. 1988). The next most-abundant cDNAs were recovered at 5.1%, 3%, and the majority at less than 1.0%, indicating complex gene expression in the salivary gland. Interestingly, one of our sequences corresponded to diap-1 (or thread). Like diap-2, the diap-1 gene product is an anti-apoptotic regulator but previous studies by Northern blot analysis failed to detect its presence in the salivary gland (Jiang et al. 1997). This result emphasizes the sensitivity of our sequence-based approach.
Subsequent to our initial analysis of the 314 cDNAs, we obtained 3’ end sequences from over 7,000 of our salivary gland cDNAs. Preliminary analyses of these ESTs indicate the presence of several apoptosis-related genes. Bioinformatics analyses are underway (S. Gorski, E. Garland, S. Chittaranjan) and include determination of the following: i) number of different genes represented, ii) number of cDNAs not already represented in the Berkeley Drosophila Genome Project (BDGP) cDNA set, iii) number of cDNAs for which there exists predicted transcripts only, iv) number of cDNAs for which there exists no predicted transcript, and v) BLAST comparisons. Importantly, our salivary gland 3’ ESTs are also being used as a resource for correlating SAGE tags to genes (IIc below).
IIc. SAGE library construction: The microSAGE method (B. St. Croix et al., www.sagenet.org; I-SAGE kit, Invitrogen) is being used to construct Drosophila salivary gland SAGE libraries from three developmental stages leading up to apoptosis (approximately 8 hr, 10 hr, and 12 hr APF @ 25C; equivalent to 16, 20 and 23 hr APF @ 18C). The advantage of the microSAGE method, compared to the original SAGE method, is that it requires a significantly reduced amount of starting material. For example, we dissected approximately 20 pairs of salivary glands (opposed to 1,000 pairs) per library to obtain a starting material of 10 ug total RNA. Construction of two libraries, corresponding to 8 hr APF and 10 hr APF, is near completion. Initial sequencing of the 10 hr APF library yielded approximately 10,000 SAGE tags so far, and software developed at the Genome Sequence Centre (S. Zuyderduyn, S. Jones) was used to assess library quality. SAGE tag distribution, tag sequencing accuracy, and number of tags sequenced per clone were examined and are summarized in Figure 4. In addition, preliminary SAGE tag to gene mapping indicated the presence of some known salivary gland transcripts (E. Garland, S. Chittaranjan). We intend to sequence approximately 60,000 SAGE tags per library; subsequent analyses will include determination of SAGE tag frequencies, identification of tags which show significant differential expression between libraries, correlation of SAGE tags to genes, and verification of differential gene expression.
IId. Genetic and phenotypic analyses of a cell death mutant, inxs: In collaboration with J. Rusconi and R. Cagan at Washington University in St. Louis, I am characterizing a Drosophila cell death mutant called inxs. Two mutations in inxs were isolated in a large scale genetic screen for modifiers of the irreC-rst retinal cell death phenotype and the inxs locus was mapped to the third chromosome (Tanenbaum, Gorski, et al. 2000; Gorski and Cagan, unpublished). I have conducted phenotypic analyses of the inxs phenotype and ascertained that inxs has a retinal cell death phenotype on its own. My meiotic mapping resulted in the localization of inxs to the 64-65 region on chromosome 3L, and deficiency mapping further refined the inxs candidate region to 64E-65A. Further genetic complementation tests with deficiencies and existing mutations in the region of interest are in progress. Future goals include the molecular identification of the inxs gene.
IIe. Other training:
i) Microscopy course: I attended the International 3D Microscopy of Living Cells course held at the University of British Columbia, June 2000. The course included theory and practice in live cell techniques, confocal microscopy, and deconvolution.
ii) Supervisory training: I have been assisted in my project by Ms. S. Chittaranjan. Over the past year, I have been responsible for directing the daily activities and performing performance reviews for Ms. Chittaranjan. For the past two months, I have been assisted also by Mr. D. Freeman. Similarly, I am responsible for directing his daily activities and performing performance reviews as required. I was involved in the interview and selection process for both individuals.
iii) I initiated and am responsible for organizing and leading the Genome Sequence Centre’s Functional Genomics lab meeting.
Benkel, B.F., Duschesnay, P., Boer, P.H., Genest, Y., and Hickey, D.A. (1988). Nucleic Acids Res 16, 9880.
Jiang, C., Baehrecke, E. H., and Thummel, C. S. (1997). Development 124, 4673-83.
Jiang, C., Lamblin, A-F.J., Steller, H., and Thummel, C.S. (2000). Molecular Cell 5, 445-455.
Tanenbaum, S.B., Gorski, S.M., Rusconi, J.C., and Cagan, R.L. (2000). Genetics 156, 205-17.