Genómica Bibliografía recomendada funcional - UdelaR · Genómica funcional Tecnología ¾Bioinformática ¾Robótica ¾Espectrometría de masas MS Transcriptoma y genoma ¿Cómo

GenómicaGenómicafuncionalfuncional

Esta clase se focalizó en los parásitos helmintos, particularmente en Schistosoma

Bibliografía recomendada

Lectura imprescindible:Artículo de RevisiónKnox DP. Technological advances and genomics in metazoanparasites. Int J Parasitol. 2004 34:139

Se incluyen los pdfs de los ejemplos incluídos en la clase, para las personas que tengan un interés especial, pero los resúmenes y las figuras más relevantes están en la presentación.

Genómicafuncional

Post-genómica

ADNADN GenomaGenoma

ARNmARNm TranscriptomaTranscriptoma

ORFORF ORFeomaORFeomaSec. 1ariaSec. 1aria

Proteína Proteína ProteomaProteomaFuncionalFuncional

InteractomaInteractoma

MetabolomaMetaboloma

Biología de sistemasBiología de sistemas

Organismos Organismos ModeloModelo

GenómicaGenómica funcional funcional PostPost--genómicagenómica

Helmintos parásitosPlatelmintosNematodos

gruposfuncionales

de genes

Artículo de Revisión: Knox DP. Technological advances andgenomics in metazoan parasites. Int J Parasitol. 2004 34:139

Molecular Phylogeny of

Metazoa

GenómicaGenómica funcionalfuncional

TecnologíaTecnología

BioinformáticaBioinformática

RobóticaRobótica

Espectrometría de masas Espectrometría de masas MSMS

Transcriptoma y genoma¿Cómo analizar las bases de datos para obtener información funcional?

Identificación de genes, ORFs, dominios proteicos, sintenia, etc.Data miningGene discoveryGenómica comparada

¿Qué nos puede servir para el diseño de drogas antiparasitarias y vacunas?

Comparar genomas de mamíferos con helmintos, helmintos entre sí, etc.

Buscar genes específicos de grupos filogenéticos de patógenos.

GenómicafuncionalADNADN GenomaGenoma














Perfiles de expresión génica

El transcriptoma refleja un momento particular de un grupo celular (o un organismo)

ESTs. Secuencias de ARNmmRNA abundance classes (Okamuro & Goldberg)Superabundant15-90% of mRNA mass<10 structural gene transcripts>5000 molecules per cell per sequenceAbundant50-75% of mRNA mass~200-1000 structural gene transcripts (5% of diversity)500-2500 molecules per cell per sequenceRare/complex<25% of mRNA mass; individual seqs <0.01%95% of mRNA diversity1-10 molecules per cell per sequence

Perfiles de expresión génicaEl transcriptoma refleja un momento particular de un

grupo celular (o un organismo), o un estadio.

Northern blot & RT-PCR – genes individualesMicroarreglos (microarrays)SAGE Análisis seriado de la expresión génicaMPSS Massively Parallel Signature Sequencing Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nature Biotechnology 18, 630 - 634 (2000) Brenner S et al

Differential displayRepresentational differencesSSH suppresive substractive hybridization

Comparar resistentes y sensibles a las drogas tratados y no tratados

Microarreglos Microarrays

Gender-associated gene expression in two related strains of Schistosoma japonicum

Jennifer M. Fitzpatrick , Maria Vang Johansen, David A. Johnston, David W. Dunne, Karl F.HoffmannMolecular & Biochemical Parasitology 136 (2004) 191–209

AbstractHost inflammatory responses directed against eggs laid by sexually-mature Schistosoma japonicum female worms instigate lesionformation and associated clinical pathologies during infection. To identify parasite gene transcripts that associate with egg productionand to characterise sexually-mature adult gene expression profiles of two related Chinese strains, S. japonicum cDNA microarrays werefabricated using 457 ESTs originating from three parasite developmental stages. Twenty-two female-associated and 8male-associated genetranscripts were identified in the adult Anhui strain whereas 21 female-associated and 7 male-associated gene transcripts were revealedin the adult Zhejiang strain. RT-PCR analysis, in situ enzyme localisation studies and enzymatic assays confirmed the cDNA microarrayresults, and importantly, provided information previously unappreciated in schistosome conjugal biology. Specifically, our novel findingsinclude the female-specific expression of genes putatively involved in haemoglobin digestion and eggshell formation including extracellularsuperoxide dismutase, two histidine-rich proteins, a large blood–brain barrier amino acid transporter and two tyrosinase orthologues. Incontrast, transcripts involved in mechanical support (actin), cytoskeletal infrastructure (e.g. dynein light chain 3 and myosin regulatorylight chain) and tegumental biology (e.g. TM4SF and Sj25) were more highly represented in adult male schistosomes. Together these dataestablish a transcriptional basis for adult schistosome labour division and expands the list of novel S. japonicum gender-associated genetranscripts that may be considered targets for improved intervention strategies.

Fig. 7. Gene expression and enzymatic activity oftyrosinase-like 1 and 2 (SjTYR1/SjTYR2) are higher in adultfemale schistosomes than in adult male schistosomes.

Fig. 2. Quality control analyses of total RNA, cDNA and cDNA microarrays revealed each reagent was optimally prepared and suitable for the confidentdetection of S. japonicum gender-associated gene transcripts. (A) Five hundred nanograms of total S. japonicum RNA used for cDNA microarray analysisfrom both strains of parasite (An: Anhui strain; Zh: Zhejiang strain) and from each gender was separated on a 1.2% denaturing formaldehyde gel.18S rRNA bands are clearly visible in each sample, with minimal apparent degradation. (B) Empirical determination of optimal PCR cycle numberfor cDNA amplification by ts-PCR. Fifteen cycles (vertical dashed line) generated the maximum yield whilst remaining on the linear portion of theamplification curve and thus this cycle number was applied to all S. japonicum material. Sybr® Green (Molecular Probes) staining of PCR productsseparated on a 2% agarose gel was visualised by digital photography and pixel intensity quantified using NIH software. (C) S. japonicum EST cDNAmicroarrays were fabricated from M13f/r amplified PCR products printed onto -amino-silane-coated glass slides. Each cDNA microarray consisted of457 S. japonicum DNA elements (and 278 negative controls) printed in duplicate and arranged in a 12 × 4 sub-array format. Each sub-array consistedof 32 elements (arranged in a 8 × 4 format). The cDNA microarrays were hybridised and washed as described in Section 2. Analysis of all sub-arrays(numbers 7 and 8 are depicted here as representative of the whole cDNA microarray) gave consistent and reproducible gender-associated patterns of geneexpression. Experiments 1–3 were hybridised with independent biological replicates of Anhui strain adult worm material where female S. japonicumcDNA was labelled with Cy3-dCTP and male S. japonicum cDNA with Cy5-dCTP. Experiment 4 displays hybridisation with Cy3-dCTP-labelled femaleZhejiang strain S. japonicum cDNA and Cy5-dCTP-labelled Zhejiang strain male S. japonicum cDNA, whereas in experiment 5 the dyes were reversed(from experiment 4). (D) Venn diagram illustration of all ESTs which passed initial filtering criterion (signal intensity >1 standard deviation above themean negative control intensity in one channel) for biologically-replicated (Anhui strain (An)) and technically-replicated experiments (Zhejiang strain(Zh)). A total of 388 cDNAs passed this initial filtering criterion in all of the biologically-replicated experiments and 339 cDNAs passed in all of thetechnically-replicated experiments.

(C)

SAGE & MPSSSAGE & MPSS

Serial Analysis of Gene Expression

Massively Parallel Signature Sequencing

Start from mRNA (euks)Generate a short sequence tag (9-21 nt)

for each mRNA ‘species’ in a cell

Restriction digest double-stranded cDNA with a 4-base cutter “anchoring enzyme”; bind to streptavidin coated beads

GGATGCATGXXXXXXXXXXCCTACGTACXXXXXXXXXX

AAAATTTT AAAA

TTTTGTAC

AAAATTTT

AAAATTTT

GGATGCATGOOOOOOOOOOCCTACGTACOOOOOOOOOO

GGATGCATGXXXXXXXXXXOOOOOOOOOOCATGCATCCCCTACGTACXXXXXXXXXXOOOOOOOOOOGTACGTAGG

CATGGTAC1 CATG

GTAC2

1 2

Generate cDNA primed withbiotin-oligo(dT)

Divide pool in half & ligate to different linkers (1 or 2),both of which have a restriction site for the “tagging enzyme”

Restriction digest with a Type IIS restriction enzyme, which recognizes the linker sequences and cuts downstream in a sequence independent fashion; fill-in 5’ overhang to blunt ends.

Blunt end ligate pool 1 to pool 2, and PCR amplify with primers specific to linker sequences 1 and 2

Restriction digest with same anchoring enzyme (above); concatenate ditags and ligate to cloning/sequencing vector

1 2

AAAATTTT AAAA

TTTTGTAC

Tag 1 Tag 2

Ditag

Ditag-----CATGXXXXXXXXXXOOOOOOOOOOCATGXXXXXXXXXXOOOOOOOOOOCATG--------GTACXXXXXXXXXXOOOOOOOOOOGTACXXXXXXXXXXOOOOOOOOOOGTAC----

Ditag

Tag 1 Tag 2 Tag 3 Tag 4

MPSSMPSS

SAGE • Described by Velculescu et al. (1995)• Originally 9 bp tags, now LongSAGE 21 bp• 10-50 tags in a clone• Only requires a sequencer (and some time)

MPSS• Proprietary technology; published 2000• Generates 17 nt “signature sequence”• Collects >1,000,000 signatures per sample• Requires 2 µg of mRNA and $$

An evaluation of serial analysis of gene expression (SAGE) in the parasitic nematode, Haemonchus contortusP. J. SKUCE*, R. YAGA, F. A. LAINSON and D. P. KNOX Parasitology (2005), 130, 553–559.This study evaluated a relatively new molecular technique, serial analysis of gene expression (SAGE), as a tool for quantifying gene expression in the ovineabomasal nematode Haemonchus contortus for which there is relatively limited (y20% gene coverage) sequence information. SAGE technology generates data that are both qualitative and quantitative and, as such, compliments other functional genomics approaches such as EST analysis and micro-array. Prior toembarking on large-scale comparisons, the present study was initiated to establish (i) how well SAGE and EST data taken from the same life-cycle stage wouldcompare, (ii) how easily SAGE tags could be assigned to genes given that the genome sequence is not available and (iii) whether it would be possible to extendthe sequences of the SAGE tags to facilitate their identification. Of 2825 tag sequences analysed from adults harvested 28 days post-infection, the identity of theencoding gene could be ascribed to 63% of the tags. The relative abundance of these genes, arbitrarily categorized on the basis of function,was comparable withthat of an EST dataset also from adults (n=2317). In addition, tag sequences could be readily extended and thereby identi.ed using a tag-based primer andReverse Transcription-PCR.

Manipulación genéticaInterferencia de ARN (RNAi) Gene knock-down

ARN antisentido (antisense RNA)

“Complementación” de mutantes de C. elegans con genes de nematodos.

Transfección de genes

Aptámeros ácidos nucleicos y peptídicos

Transposon-based techniques in functional genomics

¿Cómo entran los àcidos nucleicos a las células o parásitos?Electroporación, alimentación (feeding) Microinyección, Balística, Bacterias o virus








How does RNAi work?Genetic and biochemical data indicate a possible two-step mechanism for RNA interference (RNAi): an initiation step and an effector step. a | In the first step, input double-stranded (ds) RNA is processed into 21–23-nucleotide 'guide sequences'. Whether they are single- or double-stranded remains an open question. An RNA amplification step (shaded box) has been suggested on the basis of the unusual properties of the interference phenomenon in whole animals, but this has not been reproduced definitively in vitro. b | The guide RNAs are incorporated into a nuclease complex, called the RNA-induced silencing complex (RISC), which acts in the second effector step to destroy mRNAs that are recognized by the guide RNAs through base-pairing interactions. We also suggest the incorporation of an active mechanism to search for homologous mRNAs. (Endo, endonucleolytic nuclease; exo, exonucleolytic nuclease; recA, homology-searching activity related to E. coli recA.) Animated online http://www.nature.com/nrg/journal/v2/n2/fig_tab/nrg0201_110a_F1.html

Molecular & Biochemical Parasitology (2005)

AbstractSchistosoma mansoni is an important flatworm parasite of man that has remained intractable to experimental analyses of gene function.We have developed an approach for using dsRNA to target schistosome transcripts for RNA interference, and used it to address the roleof cathepsin B (SmCB1), a cysteine protease that has been proposed to play a central role in hemoglobin digestion in the schistosome gut.Electroporation of 3 h old larval schistosomes with SmCB1-specific dsRNA (SmCB1-dsRNA) resulted in a greater than 10-fold reduction inSmCB1 transcript levels that persisted for >20 days. RNAi mediated reductions in transcript levels led to associated reductionsin SmCB1enzyme activity. Schistosomes treated with SmCB1-dsRNA were viable and developed intestinal heme pigmentation indicative of hemoglobindigestion, but showed significant growth retardation when compared to control parasites, indicating that SmCB1 function is not essential forhemoglobin digestion but is necessary for normal parasite growth. This effect on growth was apparent when parasites were maintained inculture or introduced into mammalian hosts. The report sheds new light on the role of SmCB1 and provides a template for using RNAi toexamine gene function in the mammal-parasitic stages of schistosomes during early development in vitro and in vivo.© 2005 Elsevier B.V. All rights reserved.

Fig. 4. RNAi induced by introduction of dsRNA at 3 h has effects that persist for at least 3 weeks in vivo. (A) Examples of schistosomes recovered by portal veinperfusion 3 weeks following electroporation with either L-dsRNA (left) or SmCB1-dsRNA (right) and intramuscular injection into mice. (B) Population quantitation of parasite size 3 weeksfollowing dsRNA treatment (*p < 0.005). Results are fromone experiment. This experiment was repeated twicewith similar results.

SummaryPlanarians have been a classic model system for thestudy of regeneration, tissue homeostasis, and stemcell biology for over a century, but they have not histhetorically been accessible to extensive genetic maawaitnipulation. Here we utilize RNA-mediated geneticinterference (RNAi) to introduce large-scale gene in-hibition studies to the classic planarian system. 1065genes were screened. Phenotypes associated withthe RNAi of 240 genes identify many specific defectsin the process of regeneration and define the majorcategories of defects planarians display followinggene perturbations. We assessed the effects of inhibstemiting genes with RNAi on tissue homeostasis in intactanimals and stem cell (neoblast) proliferation in Ambiology pivputated animals identifying candidatestem cell, re- metazogeneration,and homeostasis regulators. Our study demonstrates the great potentialof RNAi for the systematic exploration of gene function in understudied organisms and establishes planarians as a powerful Mumodel for the molecular genetic study of stem cells, regeneration, and tissue homeostasis.








En los genomas eucariotas se encuentran unidades transcripcionalessimples o complejas.

La complejidad está dada por el procesamiento diferencial (intrones y otrosEs posible obtener varios polipéptidos diferentes a partir de un mismo gen.

ORFeoma

Organismos modelo

Closing in on the C. elegansORFeome by cloningTWINSCAN predictions.Chaochun Wei,Philippe Lamesch, Manimozhiyan Arumugam,JenniferRosenberg,Ping Hu, Marc Vidal, Michael R. BrentGenome Research 15:577–582 (2005)








Proteómica funcionalExpresión y caracterización de conjuntos grandes de proteínas

Proteómica estructuralCristalografía y difracción de RX de conjuntos grandes de proteínasNMR

Proteomics• The study of the expression, location, interaction, function

and structure of all the proteins in a given cell or organism Expressional

• Proteomics• 2D or Capillary Electrophoresis, protein chips• Mass Spectrometry, Laser induced fluorescence

• Functional Proteomics• Mass Spectrometry, micro-assays, protein chips• Yeast or Bacterial 2-hybrid systems

• Structural Proteomics• High throughput X-ray crystallography• High throughput NMR spectroscopy

ProteómicaPAGE 2D – MS

LC – MS Capillary scale multidimensional liquid chromatography

Integración de datos de transcriptoma y proteoma.Integrating gene and protein expression data: pattern analysis and profile mining.Cox B, Kislinger T, Emili A. Methods. (2005) 35(3):303-14.

2D Gel Principles

SDSPAGE

Protein ID Protocol

Proteómica

Journal of Proteome Research 2005, 4, 958- 966

Figure 1. Research workflow and verification of tegument purity

by immunoblot analysis. All subsequent procedures that were

followed throughout our research are schematically depicted (for

description see text in Results section). The top inset shows a

transmission electron micrograph of the tegumental outersurface

structure of S. mansoni, whereas the bottom inset shows

the result of the immunoblot analysis of the tegument (T) and

stripped-worm samples (W) for the tegument-specific glucose

transporter polypeptide SGTP4. The picture of the adult schistosome

is derived from the Internet site of the World Health

Organization, www.who.int.

Figure 5. Classification of protein identifications. To gain insightinto the functional and structural properties of the tegumentcompared to the rest of the parasite, all identified proteins wereassigned to functional classes. The relative abundance of identifiedproteins in each class in the respective fractions is shown.Black, white, and gray bars represent the number of proteinsidentified in the tegument, stripped worm and both fractions,respectively. These classes are (1) routing (all proteins involvedin vesicle trafficking such as Rab proteins and clathrins); (2)structural (all proteins that take part in the constitution andorganization of the cytoskeleton); (3) Hsp’s (proteins involved instress response and Hsp-organizing proteins); (4) transporters(integral membrane proteins involved in the transport of solutes,like sugar and ion transporters); (5) protein and nucleotidesynthesis (comprising all proteins present in the nucleus, endoplasmicreticulum and Golgi complex involved in the synthesisand modification of DNA, RNA, and proteins); (6) membraneproteins (class of proteins that are integral membrane proteinsor membrane-associated proteins, not including the transportersassigned to class 4); (7) signaling proteins (proteins involved inintracellular signaling, for example protein kinases and phosphatases);(8) energy (all proteins involved in energy productionand transport, comprising all proteins identified to be residentin mitochondria); (9) peptidases (proteins involved in proteincleavage and degradation); (10) lipid metabolism (such asproteins involved in the synthesis of membrane lipids); (11)proteins involved in oxidative stress (such as thioredoxin); (12)proteins with unknown function; and (13) unique schistosomalproteins with unknown function (containing all proteins that showno similarity with any other protein from other species thanschistosomes in a BLAST search).

Computational Tools for Protein Identification

• PeptIdent– http://us.expasy.org/tools/peptident.html

• Mascot– http://www.matrixscience.com/search_form_select.html

• ProteinProspector– http://prospector.ucsf.edu/

• MOWSE– http://srs.hgmp.mrc.ac.uk/cgi-bin/mowse

• PeptideSearch– http://www.mann.embl-heidelberg.de/

GroupPages/PageLink/peptidesearchpage.html• AACompSim/AACompIdent

– http://www.expasy.ch/tools

Covered in

Lab 6.4

Bioinformatics in proteomics Proteomics• Human proteome estimated to contain

500,000+ proteins• The next “big wave” in bioinformatics

• How to deal with so much data?• How to link structure to function to sequence?• How to show or store temporal and spatial data?• How to use it in drug discovery & development?

Proteomics WorkshopJuly 19 – 24th, 2004

Calgary, Alberta

Metaboloma

Genómica enzimática: Screening de actividades enzimáticas a gran escala.

Peptidómica

Proteínas recombinantes

InteractomaEn los organismos parásitos:

Respuesta del huésped: RNA signature

Otros términos de la era post-genómicaThe Cutting Edge of Proteomics

• Evolution of Proteomes• Structural Genomics• Quantitative Mass Spectrometry and

Protein Chip Technology• Chemical Proteomics• Proteome Scale Analysis of Networks, i.e.,

signal transduction, Y2H experiments

Protein:Protein Interaction Networks

Documents

Genómica Bibliografía recomendada funcional - UdelaR · Genómica funcional Tecnología ¾Bioinformática ¾Robótica ¾Espectrometría de masas MS Transcriptoma y genoma ¿Cómo