Transcript Ch 18 - Quia

Genomics
Chapter 18
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 19
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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
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thousands of times
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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Genomics
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Genomics
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Genomics
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Genomics
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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 56
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?
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