A PPLICATION OF M OLECULAR B IOLOGY T ECHNIQUES IN A STROBIOLOGY IN A STROBIOLOGY R ICCARDO S. G ATTA Ph.D. Molecular Genetics, I.C.G.E.B. New Delhi, Regd Pharmacist J ULIAN C HELA- F LORES The Abdus Salam International Centre for Theoretical Physics Strada Costiera 11; Trieste, Italy and Instituto de Estudios Avanzados, Caracas 1015A, Venezuela CLICKtoadvance

I NTRODUCTION C LASSIC M OLECULAR B IOLOGY APPROACH TO R ECOGNISE M OLECULAR B IOSIGNATURES, A PPLY THIS IN AN I SOLATED E NVIRONMENT. & S ELECT T ARGET, S ELECT T ECHNIQUE, A DAPT... CLICKtoadvance

H AEKEL ( ) W OESE (1980s) S EARCH FOR L IFE IN THE U NIVERSE D ARWIN ( ) A NCESTOR M ODERN ? K NOWN U NKNOWN ? ANAXIMANDER (VI BCE) DEMOCRITUS ( BCE) T he O rigin O BSERVATIONS - E VOLUTION M ACROSCOPIC M ICROSCOPIC M OLECULAR D IVERGENT C ONVERGENT E VOLUTION M ODELS OF P ARALLEL L AST A NCESTOR U NIVERSAL C OMMON CLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvance CLICKtoadvance CLICKtoadvance

Yes E VOLUTION & N ATURAL S ELECTION UNIVERSAL C ONCEPTS ? No continue… L IFE …? (…and skip next slide) IF CLICKtoadvanceCLICKtoadvance

Structure LIFE AS A T ARGET I DENTIFY & Q UANTIFY Complex Structures Chemistry Emergent Chirality LIFE Complexity Energy Transduction Polymers Replication Specific Interactions Driving Forces Molecular / Cellular Metabolism More Complexity More Disequilibrium Waste Yes E VOLUTION & N ATURAL S ELECTION UNIVERSAL C ONCEPTS ? No continue… L IFE …? (…and skip next slide) IF CLICKtoadvanceCLICKtoadvanceCLICKtoadvance CLICKtoadvance CLICKtoadvance

Figure from Labfrontier Corporation REPLICATION: DNA DNA TRANSLATION: mRNA Protein TRANSCRIPTION: DNA mRNA C ENTRAL D OGMA OF M OLECULA R B IOLOGY Crick, 1958 DO NOT code for : DNARNA PROTEINS … to apply only to present-day organisms, and not to events in the remote past, such as the origin of life or the origin of the code. Crick, 1970 CLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvance

K v K ir Ca v Na v Early K channels Voltage-gated channels P HANEROZOIC 570 Myo - present P ROTEROZOIC 2.5 Gyo – 570 Myo A RCHEAN 4.6 – 2.5 Gyo ~800 million years ago Ancient Proteins, membrane channels T IME / E VOLUTION Modern Proteins Example of divergence from LUCA at Check-Point Intervals Check-Point Intervals For: Homology with known molecules Na Ca K E VOLUTIONARY C RITERIA Moczydlowski, 1998 Homology with known molecules Where can we seek homology? BUT CLICKtoadvance CLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvance

1: 2: Homology in: BLUE 2, Joan Miro. 1: INDIVIDUAL ELEMENTS COMPOSITION 8, Vassily Kandinsky. 2: BUILD UP (CONSTRUCTION) CLICKtoadvanceCLICKtoadvance CLICKtoadvance

DECONSTRUCT IDENTIFY BASIC ELEMENTS EUROPA FROM: NASA, JPL, UA, UC. 3: THE PARADIGM: 2: BUILD UP (CONSTRUCTION) FIRST THEN CLICKtoadvance CLICKtoadvanceCLICKtoadvance

S AMPLE A NALYSIS A SSESS ENVIRONMENTAL CONDITIONS, L EGITIMISE FURTHER ASSAYS, D IRECT CHOICE OF PLAUSIBLE ASSAYS, I NDICATE OPTIMAL CONDITIONS FOR ASSAYS. E NVIRONMENTAL S AMPLE A NALYSIS P RELIMINARY A NALYSIS CLICKtoadvance

M OLECULAR T ARGETS DNA HIGHLY CONSERVED MOTIFS RNA TATAAA, Shine-Dalgarno, rRNA sub-types, shifts in pyrimidine/purine ratios… TRANSLATIONAL - ENZYMATIC messenger RNA, inhibitory RNA… Protein STRUCTURAL SCAFFOLDS Other: conserved charged residues, essential enzymes, functionally divergent… superfamilies traceable to LUCA… chirality… special fatty acids or sugar residues… lipids, peptidolipids, lipopolysaccharides, conserved charged residues, essential enzymes, functionally divergent… superfamilies traceable to LUCA… CLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvance

PRIMER Taqman R Applied Biosystems M OLECULAR R ECOGNITION T ECHNIQUES PCR DNA POLYMERASE CHAIN REACTION PROBE FRAGMENT RESTRICTION FRAGMENT LENGTH POLYMORPHISM RIBOTYPING of rDNA (5S, 16S, 23S etc. rRNA genes) PHYLOGENETIC STUDIES, DIAGNOSTICS… RIBOSOMAL GENE SEQUENCES RFLP RFLPs LARGE FRAGMENTS (1,000 – 20,000 kb) FRAGMENT 2D gel PULSE FIELD GEL ELECTROPHORESISPFGE SMALLER FRAGMENTS (10 – 1000 kb) RELATIONSHIP - BACTERIAL STRAINS RESOLUTION IN 2D WHOLE GENOME From: Hernandez-Rivas and Scherf, 1997 GENOME SIZE – PHYLOGENETIC STUDIES… From: Pillai SD B A FLUORESCENCE IN SITU HYBRIDISATION VERSATILE TECHNIQUE SH F I National Human Genome Research Institute (NHGRI) Darryl Leja DENATURE & LABEL WITH FLUORESCENT DYE HYBRIDISE PULSE FIELD GEL ELECTROPHORESIS RESTRICTION FRAGMENT LENGTH POLYMORPHISM FLUORESCENCE IN SITU HYBRIDISATION PROBE SINGLE NUCLEOTIDE POLYMORPHISMS (SNP) EXPRESSED SEQUENCE TAGS (EST) CLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvance

SEQUENCING M OLECULAR R ECOGNITION T ECHNIQUES DNA ONCE THE TARGET SEQUENCE IS IDENTIFIED RT-PCR RNA REAL TIME REVERSE TRANSCRIPTION PCR DIFFERENTIAL DISPLAY REVERSE TRANSCRIPTION PCR SINGLE CELL mRNA RESOLUTION RNA EXPRESSION PROFILES NORTHERN BLOT WESTERN BLOT SOUTHERN BLOT BLOTTING proteins Protein PROBES: fluorescent technology From: Alberts, Bray, Johnson, Lewis, Raff, Roberts, Walter c 1999 ANTIBODIES, mono-poly… green fluorescent protein… labeling environment… limited number of conformations… CLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvance

Nikon Eclipse TE2000 Gatta, ICGEB New Delhi MOL-BIO LAB USE: AS A LIGHT MICROSCOPE: for cell counting, etc. IN FLUORESCENCE MODE: to highlight transfection efficiency... MINIMAL SAMPLE PREPARATION, BASIC TECHNIQUE, APPLICATIONS MAY BE LIMITLESS... FLUORESCENCE TECHNIQUES – IMAGE ENHANCING MetaMorph ® Universal Imaging Corp. Olympus Inc. Differential Interference Contrast (DIC), (optic ganglion tissue from rat retina) Fluorescence illumination: -diazonium salt fluorochrome (fast blue). Phase Contrast, (3T3 fibroblast monolayer, NIH) Fluorescence illumination: -4',6-diamidino-2-phenylindole (DAPI), Remove some out-of-focus effects: Digital deconvolution. - phase rings on fluorite objective. A NALYTICAL D EVICES FLUORESCENCE TECHNIQUES CLICKtoadvance CLICKtoadvance CLICKtoadvance CLICKtoadvanceCLICKtoadvanceCLICKtoadvanceCLICKtoadvance

Streptomyces lividans K+ channel (KcsA), Moczydlowski, Aplysia californica K+ channel (Kv), Miller, Recognition Site A ? Recognition Site B ? Intra- Cellular Extra- Cellular H OW C AN T HEY B E R ECOGNISED ? I ON C HANNELS AS T ARGETS A NTIBODIES R ATIONAL D ESIGN From: Miller, W HOLE / L YSED CELLS, D IABODIES, B IOMIMETIC P ROBES … CLICKtoadvanceCLICKtoadvance

A NALYTICAL D EVICES Potential for Size and Power Optimisation (Figure: Bruker BioSpin AVANCE TM ICE ® ) (Figure: Bruker BioSpin Minispec ® ) (Figure: Haskin Research Group) NMR Hyphenation with other techniques: …other devices: Confocal Microscope, Chromatography (CZE), Spectroscopy (MS, IMS). UnityNOVA Varian Inc. Teepe et al., 2001 Raman IMS SNOM/ATM, Seiko Instruments, Inc SPM200 TM WITek From: MBARI. From: JPL MS CE Micro-Tech Scientific, Inc. A NALYTICAL D EVICES Potential for Size and Power Optimisation Scanning Near-Field Optical Microscope, Atomic Force Microscope. CLICKtoadvanceCLICKtoadvance SNOM/ATM

A NALYTICAL D EVICES From: MBARI. From:Wang J, NMSU LABORATORY-ON-A-CHIP Gourley and Sasaki, 2001 From:Wang J, NMSU Gast and Fiehn, 2003 G ENERAL S TRUCTURE: CE CLICKtoadvanceCLICKtoadvance

Hyphenation with other techniques: Scanning Near-Field Optical Microscope, Atomic Force Microscope. …other devices: Confocal Microscope, Chromatography (CZE), Spectroscopy (MS, IMS). Teepe et al., 2001 IMS From: MBARI. From: JPL MS CE Potential for Size and Power Optimisation µ-Capillary Electrophoresis Ion Mobility Spectrometer macromolecule separation. field investigation of VOCs, chip-based : ITP / CZE, combination with detection : - electrochemical (Φ, Ω, MS etc.), - optical (diode-LIF), - for analysis of sample / environment. Applications: multi-organic component analysis, A NALYTICAL D EVICES FOR A PRELIMINARY ANALYSIS CLICKtoadvanceCLICKtoadvance CLICKtoadvance

(Figure: ©1998 by Alberts, Bray, Johnson, Lewis, Raff, Roberts, Walter Published by Garland Publishing.) (Figure: GeneChip ® Affymetrix) Specific Analysis: Few Observations = more hypotheses Multi-Protein Analysis : More Variables = More DATA A NALYTICAL D EVICES MICRO-ARRAYS / BIO-CHIPS ES Bio-Chip reading techniques: choice of techniques and materials, automation and miniaturisation, - antibodies / fluorescence… - cantilever biosensors, Courtesy of Flavio Robles / PDB NanoReader TM BioForce NanoSciences Daniel T. Colbert Silica from Tethya aurantia, Weaver J. and Morse D - novel detection methods, - surface plasmon resonance, Figure: Biacore ® Corporation Biacore® - new materials… CLICKtoadvanceCLICKtoadvanceCLICKtoadvance

S UMMARY REVIEWED AVAILABLE TECHNIQUES, RATIONALE OF TARGET SELECTION, SELECTION OF ANALYSIS TECHNIQUES. APPLICATION TO ASTROBIOLOGY PARADIGM, F UTURE C HALLENGES M ICROFLUIDIC E NVIRONMENT FOR A SSAYS, P ERMIT M ULTIPLE S IMULTANEOUS A SSAYS (tens of 1000s), S EQUENTIAL N ANO- M ODELING OF P ROBES,... R ETURN M ISSION. SECURE CLICKtoadvance CLICKtoadvanceCLICKtoadvance

A CKNOWLEDGEMENTS Participants of the Seventh Trieste Conference on Chemical Evolution and the Origin of Life the abdus salam international centre for theoretical physics abdus salam international centre for theoretical physics CLICKtoEND

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