Genomics

Chapter 18

2

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.

3

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 (constructed use restriction enzymes)

-Cytological maps (chromosome-banding pattern)

-Radiation hybrid maps (using radiation to fragment chromosomes)

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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 MapsCytological 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)

6

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.

7

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

8

Genetic Maps

Genetic and physical maps can be correlated:

-Any cloned gene can be placed within the genome and can also be mapped genetically.

9

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

10

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.

11

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

12

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

16

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.

17

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.

19

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.

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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.

22

Noncoding DNA in Eukaryotes

Noncoding DNA within genes:-Protein-encoding exons (less than 1.5%) are embedded within much larger noncoding introns (about 24%).

Structural DNA:-Called constitutive heterochromatin;-Localized to centromeres and telomeres.

Simple sequence repeats (SSRs):-One- to six-nucleotide sequences repeated thousands of times. (SSRs can arise from DNA replication errors. About 3%).

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Noncoding DNA in Eukaryotes

Segmental duplications:

-Consist of 10,000 to 300,000 bp that have duplicated and moved either within a chromosome or to a nonhomologous chromosome.

Pseudogenes:

-Inactive genes that may have lost function because of mutation.

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Noncoding DNA in Eukaryotes

Transposable elements (transposons)

-Mobile genetic elements

- Able to move from one location on a chromosome to another.

-Four types:

-Long interspersed elements (LINEs) (21%)

-Short interspersed elements (SINEs) (13%)

-Long terminal repeats (LTRs) (8%)

-Dead transposons (3%)

TOTAL OF 45% OF THE GENOME!!!!

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Genomics

Comparative genomics, the study of whole genome maps of organisms, has revealed similarities among them:

-Over half of Drosophila genes have human counterparts;- Humans and mouse: only 300 genes that have no counterparts in the genome.

Synteny refers to the conserved arrangements of DNA segments in related genomes;

-Allows comparisons of unsequenced genomes.

26Genomic Alignment (Segment Rearrangement)

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Rice

Sugarcane

Corn

Wheat

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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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Proteomics

Proteomics is the study of the proteome:-All the proteins encoded by the genome.- A single gene can code for multiple proteins using alternative splicing.

Although all the DNA in a genome can be isolated from a single cell, only a portion of the proteome is expressed in a single cell or tissue.

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 exist:

-These can be searched to predict the structure and function of gene sequences.

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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?