Genomics

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Genomics

• Chapter 18

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Mapping Genomes

• Maps of genomes can be divided into 2 types
• -Genetic maps
• -Abstract maps that place the relative
  location of genes on chromosomes based on
  recombination frequency
• -Physical maps
• -Use landmarks within DNA sequences, ranging
  from restriction sites to the actual DNA sequence

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Physical Maps

• Distances between landmarks are measured in
  base-pairs
• -1000 basepairs (bp) 1 kilobase (kb)
• Knowledge of DNA sequence is not necessary
• There are three main types of physical maps
• -Restriction maps
• -Cytological maps
• -Radiation hybrid maps

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Physical Maps

• Restriction maps
• -The first physical maps
• -Based on distances between restriction sites
• -Overlap between smaller segments can be used to
  assemble them into a contig
• -Continuous segment of the genome

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Physical Maps

• Cytological maps
• -Employ stains that generate reproducible
  patterns of bands on the chromosomes
• -Divide chromosomes into subregions
• -Provide a map of the whole genome, but at low
  resolution
• -Cloned DNA is correlated with map using
  fluorescent in situ hybridization (FISH)

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Physical Maps
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Physical Maps

• Radiation hybrid maps
• -Use radiation to fragment chromosomes randomly
• -Fragments are then recovered by fusing
  irradiated cell to another cell
• -Usually a rodent cell
• -Fragments can be identified based on banding
  patterns or FISH

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Physical Maps

• Sequence-tagged sites
• -An STS is a small stretch of DNA that is unique
  in the genome
• -Only 200-500 bp
• -Boundary is defined by PCR primers
• -Identified using any DNA as a template
• -STSs essentially provide a scaffold for
  assembling genome sequences

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Genetic Maps

• Genetic maps are measured in centimorgans
• -1 cM 1 recombination frequency
• Linkage mapping can be done without knowing the
  DNA sequence of a gene
• -Limitations
• 1. Genetic distance does not directly
  correspond to actual physical distance
• 2. Not all genes have obvious phenotypes

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Genetic Maps

• Most common markers are short repeat sequences
  called, short tandem repeats, or STR loci
• -Differ in repeat length between individuals
• -13 form the basis of modern DNA fingerprinting
  developed by the FBI
• -Cataloged in the CODIS database to identify
  criminal offenders

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Genetic Maps

• Genetic and physical maps can be correlated
• -Any cloned gene can be placed within the genome
  and can also be mapped genetically

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Genetic Maps

• All of these different kinds of maps are stored
  in databases
• -The National Center for Biotechnology
  Information (NCBI) serves as the US repository
  for these data and more
• -Similar databases exist in Europe and Japan

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Whole Genome Sequencing

• The ultimate physical map is the base-pair
  sequence of the entire genome

-Requires use of high-throughout automated
sequencing and computer analysis
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Whole Genome Sequencing

• Sequencers provide accurate sequences for DNA
  segments up to 800 bp long
• -To reduce errors, 5-10 copies of a genome are
  sequenced and compared
• Vectors use to clone large pieces of DNA
• -Yeast artificial chromosomes (YACs)
• -Bacterial artificial chromosomes (BACs)
• -Human artificial chromosomes (HACs)
• -Are circular, at present

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Whole Genome Sequencing

• Clone-by-clone sequencing
• -Overlapping regions between BAC clones are
  identified by restriction mapping or STS analysis
• Shotgun sequencing
• -DNA is randomly cut into smaller fragments,
  cloned and then sequenced
• -Computers put together the overlaps
• -Sequence is not tied to other information

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The Human Genome Project

• Originated in 1990 by the International Human
  Genome Sequencing Consortium
• Craig Venter formed a private company, and
  entered the race in May, 1998
• In 2001, both groups published a draft sequence
• -Contained numerous gaps

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The Human Genome Project

• In 2004, the finished sequence was published as
  the reference sequence (REF-SEQ) in databases
• -3.2 gigabasepairs
• -1 Gb 1 billion basepairs
• -Contains a 400-fold reduction in gaps
• -99 of euchromatic sequence
• -Error rate 1 per 100,000 bases

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Characterizing Genomes

• The Human Genome Project found fewer genes than
  expected
• -Initial estimate was 100,000 genes
• -Number now appears to be about 25,000!
• In general, eukaryotic genomes are larger and
  have more genes than those of prokaryotes
• -However, the complexity of an organism is not
  necessarily related to its gene number

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Characterizing Genomes
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Finding Genes

• Genes are identified by open reading frames
• -An ORF begins with a start codon and contains
  no stop codon for a distance long enough to
  encode a protein
• Sequence annotation
• -The addition of information, such as ORFs, to
  the basic sequence information

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Finding Genes

• BLAST
• -A search algorithm used to search NCBI
  databases for homologous sequences
• -Permits researchers to infer functions for
  isolated molecular clones
• Bioinformatics
• -Use of computer programs to search for genes,
  and to assemble and compare genomes

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Genome Organization

• Genomes consist of two main regions
• -Coding DNA
• -Contains genes than encode proteins
• -Noncoding DNA
• -Regions that do not encode proteins

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Coding DNA in Eukaryotes

• Four different classes are found
• -Single-copy genes Includes most genes
• -Segmental duplications Blocks of genes copied
  from one chromosome to another
• -Multigene families Groups of related but
  distinctly different genes
• -Tandem clusters Identical copies of genes
  occurring together in clusters
• -Also include rRNA genes

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Noncoding DNA in Eukaryotes

• Each cell in our bodies has about 6 feet of DNA
  stuffed into it
• -However, less than one inch is devoted to
  genes!
• Six major types of noncoding human DNA have been
  described

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Noncoding DNA in Eukaryotes

• Noncoding DNA within genes
• -Protein-encoding exons are embedded within much
  larger noncoding introns
• Structural DNA
• -Called constitutive heterochromatin
• -Localized to centromeres and telomeres
• Simple sequence repeats (SSRs)
• -One- to six-nucleotide sequences repeated
  thousands of times

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Noncoding DNA in Eukaryotes

• Segmental duplications
• -Consist of 10,000 to 300,000 bp that have
  duplicated and moved
• Pseudogenes
• -Inactive genes

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Noncoding DNA in Eukaryotes

• Transposable elements (transposons)
• -Mobile genetic elements
• -Four types
• -Long interspersed elements (LINEs)
• -Short interspersed elements (SINEs)
• -Long terminal repeats (LTRs)
• -Dead transposons

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Noncoding DNA in Eukaryotes
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Expressed Sequence Tags

• ESTs can identify genes that are expressed
• -They are generated by sequencing the ends of
  randomly selected cDNAs
• ESTs have identified 87,000 cDNAs in different
  human tissues
• -But how can 25,000 human genes encode three to
  four times as many proteins?
• -Alternative splicing yields different
  proteins with different functions

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Alternative Splicing
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Variation in the Human Genome

• Single-nucleotide polymorphisms (SNPs) are sites
  where individuals differ by only one nucleotide
• -Must be found in at least 1 of population
• Haplotypes are regions of the chromosome that are
  not exchanged by recombination
• -Tendency for genes not to be randomized is
  called linkage disequilibrium
• -Can be used to map genes

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Genomics

• Comparative genomics, the study of whole genome
  maps of organisms, has revealed similarities
  among them
• -For example, over half of Drosophila genes have
  human counterparts
• Synteny refers to the conserved arrangements of
  DNA segments in related genomes
• -Allows comparisons of unsequenced genomes

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Genomics
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Genomics

• Organellar genomes
• -Mitochondria and chloroplasts are descendants
  of ancient endosymbiotic bacterial cells
• -Over time, their genomes exchanged genes with
  the nuclear genome
• -Both organelles contain polypeptides encoded
  by the nucleus

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Genomics

• Functional genomics is the study of the function
  of genes and their products
• DNA microarrays (gene chips) enable the
  analysis of gene expression at the whole-genome
  level
• -DNA fragments are deposited on a slide
• -Probed with labeled mRNA from different
  sources
• -Active/inactive genes are identified

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Genomics

• Transgenics is the creation of organisms
  containing genes from other species (transgenic
  organisms
• -Can be used to determine whether
• -A gene identified by an annotation program is
  really functional in vivo
• -Homologous genes from different species have
  the same function

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Proteomics

• Proteomics is the study of the proteome
• -All the proteins encoded by the genome
• The transcriptome consists of all the RNA that is
  present in a cell or tissue

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Proteomics

• Proteins are much more difficult to study than
  DNA because of
• -Post-translational modifications
• -Alternative splicing
• However, databases containing the known protein
  structural motifs exist
• -These can be searched to predict the structure
  and function of gene sequences

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Proteomics
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Proteomics

• Protein microarrays are being used to study large
  numbers of proteins simultaneously
• -Can be probed using
• -Antibodies to specific proteins
• -Specific proteins
• -Small molecules
• The yeast two-hybrid system has generated
  large-scale maps of interacting proteins

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Applications of Genomics

• The genomics revolution will have a lasting
  effect on how we think about living systems
• The immediate impact of genomics is being seen in
  diagnostics
• -Identifying genetic abnormalities
• -Identifying victims by their remains
• -Distinguishing between naturally occurring and
  intentional outbreaks of infections

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Applications of Genomics
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Applications of Genomics

• Genomics has also helped in agriculture

-Improvement in the yield and nutritional
quality of rice
-Doubling of world grain production in last 50
years, with only a 1 cropland increase
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Applications of Genomics

• Genome science is also a source of ethical
  challenges and dilemmas
• -Gene patents
• -Should the sequence/use of genes be freely
  available or can it be patented?
• -Privacy concerns
• -Could one be discriminated against because
  their SNP profile indicates susceptibility to a
  disease?