Transcrição intrônica antisenso no genoma humano

1. Transcrição intrônicaantisenso no genoma humano Sergio Verjovski-Almeida Departmento de Bioquímica Instituto de Química Universidade de São Paulo

2. The genomic era Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR, Bult CJ, Tomb JF, Dougherty BA, Merrick JM, et al., Science 269:496-512, Jul 28, 1995 The minimal gene complement of Mycoplasma genitalium Fraser CM, Gocayne JD, White O, Adams MD, Clayton RA, Fleischmann RD, Bult CJ, Kerlavage AR, Sutton G, Kelley JM, et al. Science 270: 397-403, Oct 20, 1995 The Sequence of the Human Genome J. Craig Venter, Mark D. Adams, Eugene W. Myers et al., Science 291: 304-1351, Feb 16, 2001 Initial sequencing and analysis of the human genome Lander ES, Linton LM, Birren B, Nusbaum et al., Nature 409: 860-921, Feb 15, 2001

3. Initial sequencing and analysis of the human genome International Human Genome Sequencing Consortium Nature 409, 860-921(15 February 2001) The last quarter of a century has been marked by a relentless drive to decipher first genes and then entire genomes, spawning the field of genomics. The fruits of this work already include the genome sequences of 599 viruses and viroids, 205 naturally occurring plasmids, 185 organelles, 31 eubacteria, seven archaea, one fungus, two animals and one plant. The sequence of the human genome is of interest in several respects. It is the largest genome to be extensively sequenced so far, being 25 times as large as any previously sequenced genome and eight times as large as the sum of all such genomes. It is the first vertebrate genome to be extensively sequenced. And, uniquely, it is the genome of our own species.

4. Initial sequencing and analysis of the human genome International Human Genome Sequencing Consortium Nature 409, 860-921(15 February 2001) We thank Compaq Computer Corporations's High Performance Technical Computing Group for providing a Compaq Biocluster (a 27 node configuration of AlphaServer ES40s, containing 108 CPUs, 216 GB RAM, serving as compute nodes and a file server with one terabyte of secondary storage) to assist in the annotation and analysis. Compaq provided the systems and implementation services to set up and manage the cluster for continuous use by members of the sequencing consortium. Platform Computing Ltd. provided its LSF scheduling and loadsharing software without license fee.

5. Initial sequencing and analysis of the human genome International Human Genome Sequencing Consortium Nature 409, 860-921(15 February 2001)

6. Generating ESTs with low-stringency RT-PCR and non-degenerate primers Genes are ~ 3 % of the human genome The gene and its protein in higher eukaryotes Processing Random Reverse Transcription Poly-dT Reverse Transcription cDNA Cloning Low-stringency RT-PCR poly-dT primed RT

7. Microarray Lab (Dec. 2000) - CAGE IQ-USP . GenIII spotter and scanner (Amersham Pharmacia) . Microarray system program (Amersham Pharmacia) . BioRobotics system (clone replication and rearray) . Clone stocks . Clean Room 10,000 (humidity and temperature control) Bioinformatics Lab (Sept. 2000) . ES40 Workstation, 1 node, 4 processors, 8 Gb RAM, 900 Gb Disc . BlastMachine, Paracel/Celera, 6 nodes, 12 processors, 24 Gb RAM

8. Bioinformatics Lab (Sept. 2000) . BlastMachine, Paracel/Celera, 6 nodes, 12 processors, 24 Gb RAM The Paracel BlastMachine runs parallelized versions of NCBI BLAST, PSI-BLAST, and MegaBLAST. It is an Intel-based cluster, consisting of 6 dual-processor nodes. Each node contains two 933 MHz Intel PIII processors and 2 Gigabytes of memory. The operating system is a proprietary cluster variant of Linux. One dual-processor node acts as the head node, scheduling and controlling jobs. 10 processors are available to run sequence search jobs. Communication between nodes and with the fileserver is via a 100 MHz Ethernet connection. The BlastMachine accelerates the BLAST algorithms by dividing a job across multiple processors. It can divide both the database and the query among groups of processors. The number of pieces a database will be divided into is determined at the time the database is formatted on the BlastMachine. The BlastMachine always divides a given database the same way to insure precise repeatability of results. The BlastMachine has its own queueing and job control system. This system is accessed from a host computer using the client program pb. The system attempts to maximize total throughput; different search jobs can and will run simultaneously. The actual number of processors assigned to any given job varies dynamically with machine load.

9. Bioinformatics Lab (Sept. 2000) . BlastMachine, Paracel/Celera, 6 nodes, 12 processors, 24 Gb RAM

11. The FAPESP-LICR Human Cancer Genome Project A Program for Human Gene Discovery and Complete Sequence Compilation • E. D. Neto et al., Proc. Natl. Acad. Sci. USA 97: 3491-3496, 2000 • Camargoet al., Proc. Natl. Acad. Sci. USA 98: 12103-12108, 2001 • H. Brentani et al., Proc. Natl. Acad. Sci. USA 100:13418-13423, 2003

12. Are the novel transcripts expressed in prostate ?

19. Central Dogma Central Dogma Central Dogma genomic DNA genomic DNA genomic DNA Transcription Transcription Transcription Transcription Transcription Sense Pre Sense Pre Sense Pre - - - mRNA mRNA mRNA regulation New New New New AAAAAA AAAAAA AAAAAA - - - Processing Processing Processing Processing Processing antisense RNA antisense RNA antisense RNA antisense RNA sense RNA sense RNA sense RNA mature sense mRNA mature sense mRNA mature sense mRNA Translation Translation Translation Translation Translation Protein Protein Protein Prote Prote Prote in in in Revised paradigm oncogenesis = mutations in protein-coding genes AND alterations in regulatory non-coding antisense RNAs regulation regulation Paradigm oncogenesis = accumulation of mutations in protein-coding genes that act as oncogenes/tumor suppressors

21. The genomic revolution Complete sequencing of the Human Genome (2001) Sequencing of 5,340,464 human Expressed Sequence Tags (ESTs) Automated DNA sequencing . Sanger dideoxy method . Clone and sequence 500 bp frags Future (Aug 2006): “454” pyrosequencing . whole genomes in 4 hours . no cloning, sequence 100 bp frags High-throughput methods cDNA and oligo microarrays for measuring SAGE, MPSS gene expression

28. verjo@iq.usp.br http://verjo2.iq.usp.br