1From Mendel to Genomics

• Historically– Identify or create mutations,

follow inheritance– Determine linkage, create

maps

• Now: Genomics– Not just a gene, but as many

genes as may be involved in a process.

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Genomics: The study of genes and their function. Genomics aims to understand the structure of the genome, including mapping genes and sequencing the DNA. Genomics examines the molecular mechanisms and the interplay of genetic and environmental factors in disease.

Genomics:Focus: entire genome, not individual genes Uses recombinant DNA methodsMethodology in place for sequencing entire genomes and

looking at the activity of multiple genes simultaneously

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Genomics includes: Functional genomics -- the characterization of genes and their mRNA and protein products. Structural genomics -- the dissection of the architectural features of genes and chromosomes.

Comparative genomics -- the evolutionary relationships between the genes and proteins of different species.

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4Bioinformatics

• Sequencing creates huge amount of information that must be stored and analyzed

• Bioinformatics is the science of methods for storing and analyzing that information– Melding of computer

science and molecular biology

http://www.swbic.org/products/clipart/images/bioinformatics.jpg

5Sequencing the Human Genome

• Publicly funded consortium– Clone-by-clone method– Create library of clones of entire genome– Order clones using restriction enzyme maps and

various DNA markers– Then sequence each clone

• Craig Venter and private enterprise– Shotgun method– Create library of clones of entire genome– Sequence all the clones– Use supercomputer to determine order

• Sequencing done multiple times to get it right.

Sequencing the Human Genome

• A Huge job– Human DNA has over 3

trillion has pairs (3 x 109)– Much of the technology

had to be invented and improved to do this particular job

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www.achievement.org/.../achievers/col1-005

7Clone-by-clone Shotgun approach

www.yourgenome.org/ intermediate/all/

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Is sequencing a genome the answer?

No, only the beginning of the questions.

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9Annotation: making sense of the sequence

• Looking for regulatory regions, RNA genes, repetitive regions, and protein genes.

• Finding protein genes– Look for ORFs (open reading frames)

• Start codon (ATG), stop codon.• Codons must be “in frame”, distance long enough

– Problems: 3 reading frames x 2 strands, widely spaced genes, introns.

– Help: new software finds TATA box and other elements; codon bias can help• Different codons not used equally in organisms

10Where is the reading frame?

Could start in one of 3 different places.

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Find the start codon. Do all the codons that follow spell out a protein sequence seen before?

12Functional Genomics

• OK you have a sequence. What does the gene do? What is the function of the protein?– Search the databases for similar sequences– Is the sequence similar to sequences for proteins of

known function?– Use computer to search for functional motifs.

• Various proteins that do the same thing have similar structural elements.

• Example: transcription factors that have lecuine zippers bind to DNA

About Human Genome

• The average gene: 3000 bases, but sizes vary greatly– largest known human gene is dystrophin: 2.4 million bases.

• The total number of genes is estimated at 30,000• Almost all (99.9%) nucleotide bases are exactly the

same in all people. • The functions are unknown for over 50% of discovered

genes. • Less than 2% of the genome codes for proteins. • Repeated sequences are at least 50% of genome.

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http://www.ornl.gov/sci/techresources/Human_Genome/project/info.shtml

14Fundamental questions

• Questions can be asked using whole genome information that couldn’t before.– How did genomes evolve?– What is the minimum number of genes necessary

for a free-living organism?

• Much can be learned about the ecology of an organism by genomics and proteomics.– First bacterium sequenced: Mycoplasma genitalium– Lives a parasitic existence, evident from genes.

15Protein function # of genes

Amino acid biosynthesis 0

Purine, pyrimidine, nucleoside and nucleotide metabolism 19

Fatty acid and phospholipid metabolism 8

Biosynthesis of co-factors, prosthetic groups and carriers 4

Central intermediary metabolism 7

Energy metabolism 33

Transport and binding proteins 33

DNA metabolism 29

Transcription 13

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Protein synthesis 90

Protein fate 21

Regulatory functions 5

Cell envelope 29

Cellular processes 6

Other categories 0

Unknown 12

Hypothetical

Database match 168

No database match 6

Total number 483

17Advances in understanding genomes

• Prokaryotic- eubacterial• not all genomes are circular• not all genomes are in one piece• when is a plasmid not a plasmid but a

chromosome?• not all genomes are small• very little wasted space, very few with introns

• Significant quantity of genes organized into operons

18Understanding-2

• Archaeal genomes similar to eubacteria but• have histones, sequence similarities to

eukaryotes, and introns in tRNA genes

• Eukaryotic genomes -wide variations• low gene density, that is few genes per amount

of DNA• introns, more in some (humans) than others• repetitive sequences

19Proteomics

• Proteome: all the proteins an organism makes

• Proteomics: the study of those proteins– Timing of gene expression– Regulation of gene expression– Modifications made to proteins– Functions of the proteins– Subcellular location of proteins

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/3_14d.jpg

20Proteomics: study of proteins

• Proteomics– 30,000 genes, 100,000 different proteins

• must be lots of post translational modifications–>100 different ways of modifying proteins–addition of groups, crosslinking, inteins

• many genes code for proteins of unknown function

– methods of study• 2D gel electrophoresis• Peptide fragments generated with trypsin,

studied by MS

212D gel electrophoresis of proteins

http://www.biochem.mpg.de/en/research/rd/oesterhelt/web_page_list/Proteome_Hasal_cytosolic/absatz_3_bild.gif

Blue and green arrows mark proteins of interest.

Proteins of Halobacterium.

Left to right: pH

Vertical: MW

Spots digested w/ trypsin then studied using mass spec.