GenomeGenome--level understanding of level understanding of functional microbial system (II)functional microbial system (II)

Guangyi Wang, Ph.D.POST103B

guangyi@hawaii.edu

Spring, 2007

http://www.soest.hawaii.edu/marinefungi/OCN403webpage.htm

Review of Last Lecture• Terminology (should know them)• Classification based on system attributes,

relationship to other scientific disciplines, and objects (can you define marine functional genomics??).

Historic Perspective of Genomics• Genomics is relatively new 

field of biological inquiry, starting with the proposal to map and sequence the human genome in 1885 and 1986.– In 1988, the human genome 

committee commissioned by the NAS and NRC released report  to endorse sequencing human genome in 15 years at a cost of $200 millions per year.

– In 1995, the whole genome sequence of (~1.8 Mb) for Haemophilus influenzae Rd, a free‐living microbe, was published by the Institute for Genomic Research (TIGR).

– Since then, more than 200 microbial genomes have been fully sequenced, and several hundred microbial genome sequencing projects are under way (see the left table).

Historic Perspective (cont.)

The time line of five major methodologies in genomics research (McKusick, 1997)

Historic Perspective (cont.)

DeLong, 2005, Nature Rev. Microbiol.

Historic Perspective (cont.)

DeLong, 2005, Nature Rev. Microbiol.

• The first sequenced marine microorganism was that of a marine archaeon, Methanocaldococcusjannaschii, isolated from hydrothermal vents.

• Now environmental genomics approaches have lead us into a new era of ocean genomics research, with thousands of new marine microbial species described.

Historic Perspective (cont.)Sorcerer II Expedition

www.sorcerer2.expedition.org

Environmental shotgun sequencing of Sargasso sea resulted 1,800 new speceis and 1.2 million genes

Sorcerer II Expedition (cont.)• Pumped200 liters of seawater from the

depth of about 5 feet.• Filtered with a series of 3 size-paper

filters.• Stored in -80 C.

• Back to USA, environmental DAN was extracted.

• The long strands of DNA was broken into millions of fragments by forcing DNA solution through a pinhole nozzle under high pressure.

• Fragment DNA copied, broken into smaller pieces, tagged with fluorescent dyes corresponding to A, C, T, and G.

• Electrical charge carries the pieces through capillaries – tubes about the width of a human hair.

Sorcerer II Expedition (cont.)• DNA pieces arrange themselves by

length as they move through the capillaries.

• A laser then scan the ends of each tagged piece, creating rough sequences called ‘reads’

• Computer algorithms assemble longer DNA sequences by joining segments with overlapping stretches of code.

• Comparison of 16S rRAN genes resulted in known and new species.

• Further DNA sequence annotation identify novel genes and metabolic pathways.

Challenges of Studying Functional Genomics

• Goals of functional genomics– Use genome sequence information and related genome technologies;– Link sequence with functions and phynotype;– Understand how biological systems (ecosystems) at different levels

functions in nature;– Explore biological systems for the maximum benefit of our society

(biotechnology).• Challenges

– Defining gene function (theory vs reality).• Theory vs reality• Can be misleading or incorrect due to many reasons.• Complicated relationships between genes and enzymes (one to one; one to

may, many to one).– Identifying and characterizing the molecular machine of life.

• Interaction and complicated living system• How do proteins confer on cells their capabilities, structure, and higher-

order properties?

• Challenges (cont.)– Delineating gene regulatory networks

• Gene number difference may not explain the huge phenotypic difference of different types of cells, tissues, organs, and living organisms.

– Understanding of biological systems beyond individual cells.• Microorganisms interact each other and with their environments to

produce more complex living systems, such as populations, communities, ecosystems, and the biosphere.

• One challenge of bring genomes to life is to use genomic sequence to understand the consequences of genetic capacity and interactions at spatial scales ranging from microns to continent (ecosystems).

• Challenges (cont.)– Understanding of biological systems beyond individual cells.

• How do the functional properties of different organisms interact to yield the total functionality and stability of community ecosystems?

• With accumulating genomics information and technologies, we can link subcellular metabolic processes with the functional performance of biological communities.

– Computational challenges.• Use what essentially amounts to a “part catalogue” and synthesize those parts

using computer models that simulate biological cellular functions.• Synthesize the knowledge of cellular-level processes through computational

modeling and simulations to understand population-, community- and ecosystem-level dynamics for new knowledge.

– Multidisciplinary collaborations.• Forming a large and efficient team is in itself a challenge.

Scope and General Approaches• Structural Genomics (today’s reading assignment)

– The disruption and manipulation of genome structure• Turning off the function of a gene by disrupting a gene or inactivating genes

(future lectures) for understanding of gene function– Protein structure determination and prediction (3D structure and

computation approaches).• Transcriptomics (DNA microarray)

– The comprehensive analysis of transcripts (mRNA)• Correlation of gene expression and function, why?

– Gene expression in specific cells under specific condition related to the fitness of the organism.

– Fine-tuned regulatory works controls the time, location, and level of gene products.• Connection between gene expression and physiological states.• Guilt by association

– Gene expressed together may function together.• Importance of transcriptional regulation• Technological advances.

Scope and General Approaches (cont.)

• Proteomics– Why important?

• Proteins- active reagents in cells;• Prediction of an ORF may not necessarily meant the existence of a function

protein;• Postranscriptional regulation can be detected using gene expression profiling;• Protein decay occurs in cells;• Protein modification occur after translation;• It is difficult to predict the cellular location of a protein;• Protein-protein interaction can only detected in protein level-approaches.

– Three main categories• Protein identification and analysis;• Proteome-wide differential display of proteins under various conditions (2 D

gel, MS, and protein array).• Protein-protein interactions (a two-hybrid system, phage display, ribosome

display, protein array, and MS).

Scope and General Approaches (cont.)• Metabolomics

– What is the metabolomics ?• "systematic study of the unique chemical fingerprints that specific cellular

processes leave behind" - specifically, the study of their small-molecule metabolite profiles (Daviss, 2005).

– Elucidation of gene function by analysis of the metabolome has several advantages:

• Metabolites also constitute functional entities within the cells;• The total complement of metabolites highly related to physiological,

developmental, or pathological state of cell, tissue, organ, or organism (medical diagnosis);

• The number of metabolite is far fewer than that of gene or gene products (e.g. Saccharomyces cerevisiae - ~6,000 protein-encoding genes vs fewer than 600 low molecular weight intermediates).

– Two main approaches to understand gene function vs metabolic analyses:• Identification of the biochemical reactions catalyzed by enzymes encoded by

genes of unknown functions;• Functional analysis by corespones (many gene responsible for the production or

degradation of one metabolites).

Scope and General Approaches (cont.)

• Metabolomics (cont.)

the measurement of co-response coefficient profiles can reveal the site of action of the product of a silent S. cerevisiae gene, even when deletion of that gene has no measurable effect on yeast growth rate (Raamsdonk et al, 2001, Nature Biotech).

Common Tools for Genomics• General introduction

• General introduction (cont.)

• BLAST (Basic Local Alignment Search Tool)

• BLAST (Basic Local Alignment Search Tool) (cont.)

1) Nucleotide-nucleotide blast (blastn)

Uncultured bacterium clone Fitz2-2 DQ256530

1) Nucleotide-nucleotide blast (blastn) (cont.)

1) Nucleotide-nucleotide blast (blastn) (cont.)

1) Nucleotide-nucleotide blast (blastn) (cont.)

1) Nucleotide-nucleotide blast (blastn) (cont.)

2) Protein-protein blast (blastp)

cytochrome c oxidase from Oscarella carmela (EF081250)

Summary• Historical perspective of genomics (should know

the Sorcerer II Expedition!)• Challenges of studying functional genomics

– Knowing the challenges helps you to identify potential problems

– Knowing basic challenges• Scopes and general approaches

– Structural genomics– Transcriptomics– Proteomics– Metabolomics

• Common genomics tools and their application