Download Chapter 21: Genomics I: Analysis of DNA and Transposable Elements

Chapter 21: Genomics I: Analysis of DNA and Transposable Elements
Student Learning Objectives
Upon completion of this chapter you should be able to:
1. Understand the concept of genomic analysis and the forms that it may take.
2. Distinguish between the three forms of chromosome mapping: cytogenetic, linkage,
and physical mapping.
3. Understand the procedures for determining the location of a gene on a chromosome.
4. Know the different types of molecular markers used in genome analysis.
5. Understand the different methods used to sequence genomes.
6. Know the goals of the Human Genome Project.
7. Distinguish between transposable elements and their mechanisms of transposition.
21.1 Overview of Chromosome Mapping
Overview
In this chapter we will begin to look at the genome of an organism as a whole. The study
of the genome is called genomics. It can be divided into two main types: structural genomics,
which aims to elucidate the organization and sequences of genes with a species’ genome; and
functional genomics (Chapter 22), which examines the roles of these genetic sequences. This first
section provides an introduction to the three mapping strategies used to determine locations of
genes and other DNA segments along a chromosome. Notice that each of the methods uses
different terms to describe the location of the gene.
The three mapping strategies are: 1) cytogenetic (or cytological) mapping, which
involves microscopy studies of chromosomes; 2) linkage (or genetic) mapping, which involves
crosses among organisms that are heterozygous for two or more genes; and 3) physical mapping,
which involves cloning DNA and then computing the distances between genes and other DNA
regions in numbers of nucleotide base pairs.
Outline of Key Terms
Genome
Genomics
Functional genomics
Proteomics
Mapping
Cytogenetic mapping
Linkage mapping
Physical mapping
Genetic (chromosome) map
Locus (loci)
Focal Points

A comparison of cytogenetic, linkage, and physical maps (Figure 21.1)
Exercises and Problems
For the following questions, use the following key.
a. Cytogenetic mapping
b. Linkage mapping
c. Physical mapping
_____
_____
_____
_____
_____
1. Measures map distances in centiMorgans
2. Measures map distances in basepairs
3. Measures map distances in map units
4. Maps genes relative to band locations
5. Also called genetic mapping
21.2 Cytogenetic Mapping Via Microscopy
Overview
As mentioned earlier, cytogenetic mapping relies on the localization of gene sequences
within chromosomes that are viewed microscopically. This strategy is commonly used in
eukaryotes, which have much larger chromosomes that those found in bacteria. Eukaryotic
chromosomes can be distinguished from one another in three main ways: size, centromere
position, and banding patterns (see Chapter 8, Figure 8.1 for review). Discrete banding patterns
for each chromosome can be obtained by treating chromosomal preparations with particular dyes.
Cytogeneticists can then use these banding patterns to describe specific regions along
chromosomes. This section explores methods used to generate cytogenetic maps, and focuses on
one of the most common techniques: fluorescence in situ hybridization (Figure 21.2).
Outline of Key Terms
In situ hybridization
Fluorescence in situ hybridization (FISH)
Chromosome painting
Focal Points

The technique of fluorescence in situ hybridization (FISH) (Figure 21.2)
Exercises and Problems
Complete the following sentences with the most appropriate term(s):
1. The term ____ indicates that the procedure is conducted on chromosomes being held in place.
2. The method of ____ uses computer-imaging to assign each fluorescent probe a different color.
3. Banding patterns on a chromosome are commonly produced by staining with the dye ______.
4. The most common dye used in FISH is abbreviated ______.
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21.3 Linkage Mapping Via Crosses
Overview
This section examines linkage mapping, which relies on the frequency of recombinant
offspring to determine distances between chromosome sites along the same chromosome. In
chapter 6, we had discussed how testcrosses are used to map relative locations of genes on a
chromosome via detection of allelic differences in the offspring. Here, we will focus on the use of
molecular markers to map genes.
The common types of molecular markers are presented in Table 21.1. This section begins
with a discussion of restriction fragment length polymorphisms (RFLPs), and how they are used
to generate RFLP maps. The lod score method (pages 504-505) is a statistical test that determines
the likelihood of linkage between two RFLPs. The section ends with a discussion of
microsatellites: their identification via PCR primers (Figure 21.5), and their use in genetic
analyses of human pedigrees (Figure 21.6).
Outline of Key Terms
Monomorphic
Founder
Microsatellites
Simple sequence repeats (SSRs)
Short tandem repeats (STRs)
Sequence tagged site (STS)
Molecular marker
Polymorphic
Restriction fragment length
polymorphisms (RFLPs)
Lod score method
Focal Points



Common types of molecular markers (Table 21.1)
RFLP analysis of chromosomal DNA from three different individuals (Figure 21.4)
Mapping with microsatellites (Figures 21.5 and 21.6)
Exercises and Problems
For questions 1 to 8, match the term to its correct definition.
_____ 1.
Molecular marker
_____ 2.
Polymorphism
_____ 3.
Lod score method
_____ 4.
Monomorphic
_____ 5.
Founder
_____ 6.
Sequence tagged site
_____ 7.
RFLP
_____ 8.
Microsatellite
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a.
b.
c.
d.
e.
f.
g.
h.
A genetic segment that varies among members of the population.
Genome sites where the distance between two restriction site varies among different people.
Determines the likelihood of linkage between two markers.
Also called simple sequence repeats.
The original member of a population that possessed a disease.
A general term for a segment of DNA that is found at a specific location on a chromosome.
A sequence of DNA that is amplified by a single set of DNA primers.
A genetic segment that is identical among all members of the population.
21.4 Physical Mapping Via Cloning
Overview
As the name implies, physical mapping involves determining the exact location of a gene
on a chromosome. There are a variety of methods that may be used to construct a physical map,
and the major types are introduced here. The section first examines the different types of artificial
chromosomes that have been used as vectors for cloning large DNA inserts. These include BACs,
YACs, PACs, and cosmids. You should familiarize yourself with how scientists can use these
vectors to create contigs, which are basically collections of clones (See Figures 21.7 and 21.8).
The section also examines the technique of chromosome walking, which enables the cloning of a
gene that is close to a known marker (Refer to Figure 21.12).
Outline of Key Terms
Positional cloning
Chromosome walking
Subcloning
Contig
Artificial chromosome
Yeast artificial chromosome (YAC)
Bacterial artificial chromosome
(BAC)
P1 artificial chromosome (PAC)
Cosmid
Focal Points





The construction of a contig (Figure 21.7)
The use of genetic markers to align a contig (Figure 21.8)
The use of a YAC vector in DNA cloning (Figure 21.9)
A correlation of chromosome maps (Figure 21.11)
The technique of chromosome walking (Figure 21.12)
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Exercises and Problems
For questions 1 to 7, complete the following sentences with the most appropriate term(s):
1. ________ are cloning vectors that contain two telomeres and a centromere.
2. ________ are cloning vectors derived from F factors.
3. Cloning of a gene based upon its mapped position along a chromosome is called ________.
4. A hybrid between a plasmid and phage  is called a ________.
5. ________ are cloning vectors derived from bacteriophages chromosomes.
6. A collection of clones is called a ________.
7. ________ is a technique that involves a series of subclones to isolate the gene to a specific
region.
21.5 Genome Sequencing Projects
Overview
The final section of this chapter covers genome sequencing projects, which are
worldwide research endeavors aimed at determining the entire genome sequence of species.
Shotgun sequencing is the most efficient way to carry out such huge projects, and its various
types are discussed. The section also introduces the Human Genome Project, which represents
one of the most important undertakings in the study of genetics. Its goal, to map the human
genome, was completed in 2006, but the analysis of the material may take decades. As students of
genetics, you should be familiar with the goals of this project. The section ends by discussing
recent innovations in DNA sequencing that have made the process faster and cheaper.
Outline of Key Terms
Next-generation sequencing technologies
High-throughput sequencing
Pyrosequencing
Sequencing by synthesis (SBS)
Genome sequencing projects
Human genome project
Comparative genomics
Shotgun sequencing
Hierarchial shotgun sequencing
Whole-genome shotgun sequencing
Double-barrel shotgun sequencing
Focal Points




Two approaches of shotgun sequencing of genomes (Figure 21.14)
Goals of the Human Genome Project (page 514)
Examples of genomes that have been sequenced (Table 21.3)
Pyrosequencing: a newer high-throughout sequencing technology (Figure 21.15)
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Exercises and Problems
For questions 1 to 5, complete the following sentences with the most appropriate term(s).
1. The genome of the bacterium ________ was the first to be sequenced.
2. The ability to rapidly sequence large amounts of DNA is often referred to as ________.
3. In ______, DNA fragments to be sequenced are randomly generated from larger DNA pieces.
4. _______ genomics uses information from genome projects to understand the genetic variation
among different populations.
5. The general term ________ refers to methods involving the identification of each nucleotide
immediately after its incorporation into a DNA strand by DNA polymerase.
21.6 Transposition
Overview
The last section of this chapter examines the phenomenon of transposition. Transposable
elements (TEs), also called transposons, have sometimes been referred to as “jumping genes”
because they are inherently mobile. They were discovered by Barbara McClintock in the 1950s
through her classic studies with corn. Since then, geneticists have discovered many different
types of TEs in organisms as diverse as bacteria, fungi, plants, and animals.
The text first explores the three main mechanisms by which transposons may move
within the genome (Figure 21.16), and the common structure or organization of transposable
elements (Figure 21.17). You should study these carefully before you move onto the next topics,
which include detailed discussions of simple transposition (Figures 21.18 and 21.19) and
retrotransposition (Figures 21.20 and 21.21). The chapter concludes with a discussion of the
importance of transposons on mutation and evolution (pages 522-525).
Outline of Key Terms
LTR retrotransposons
Long-terminal repeats (LTRs)
Non-LTR retrotransposons
Target-site primed reverse
transcription (TPRT)
Autonomous elements
Nonautonomous elements
Reverse transcriptase
Integrase
Long interspersed elements (LINEs)
Short interspersed elements (SINEs)
Selfish DNA hypothesis
Exon shuffling
Hybrid dysgenesis
Transposition
Transposable elements (TEs)
Transposons
Transposition mechanisms
Simple (conservative) transposition
Replicative transposition
Retrotransposition
Retroelements
Retrotransposons
Retroposons
Direct repeats (DRs)
Insertion sequences
Inverted repeats (IRs)
Transposase
Composite transposons
Replicative transposons
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Focal Points






Three mechanisms of transposition (Figure 21.16)
Common organization of transposable elements (Figure 21.17)
Simple transposition (Figure 21.18)
Retrotransposition of an LTR retrotransposon (Figure 21.20)
Retrotransposition of a non-LTR retrotransposon (Figure 21.21)
Examples of transposable elements (Table 21.4)
Exercises and Problems
For questions 1 to 10, choose the type of transposable element that is indicated by the statement.
Some questions may use more than one answer.
a. replicative transposition
b. simple transposition
c. retrotransposition
_____ 1. The viral like group of these contain long terminal repeats.
_____ 2. “Cut and paste” transposition.
_____ 3. Uses the resolvase enzyme.
_____ 4. Includes a reverse transcriptase enzyme.
_____ 5. Uses an RNA intermediate.
_____ 6. “Copy and paste” transposition.
_____ 7. Contains direct repeats.
_____ 8. Simple transposable elements found in bacteria move by this mechanism.
_____ 9. Contains a transposase gene.
_____ 10. Produces multiple copies of the transposon in the genome.
For questions 11 to 16, match the term with its correct definition.
_____ 11. Selfish DNA hypothesis
_____ 12. Composite transposon
_____ 13. Autonomous transposon
_____ 14. LTR retrotransposon
_____ 15. LINEs and SINEs
_____ 16. Non-LTR retrotransposon
a.
b.
c.
d.
e.
f.
Evolutionarily related to known retroviruses.
May contain genes that are not related to transposon activity.
An evolutionary explanation for the persistence of TEs in the genome.
A transposon that contains all of the genetic information for transposition to occur.
Replicates and integrates via the target-site primed reverse transcriptase.
Repetitive sequences in the DNA that are probably the result of tranposon activity.
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Chapter Quiz
1. Which of the following allows mapping to the precise nucleotide?
a. cytological mapping
b. linkage mapping
c. physical mapping
d. FISH
2. Which of the following is NOT a molecular marker used in linkage mapping?
a. RFLPs
b. PACs
c. STSs
d. AFLPs
e. microsatellites
3. Positions such as 2p4c and 17q1c would be determined by which of the following?
a. FISH
b. PCR
c. chromosome sorting
d. AFLPs
4. Which of the following is NOT a mechanism of transposition?
a. simple
b. replicative
c. conservative
d. retrotransposition
e. choose this answer if all of the above are transposition mechanisms
5. Which of the following represents a hybrid between a plasmid and phage vector?
a. BACs
b. YACs
c. contigs
d. subclones
e. cosmids
6. LTR retrotransposons may contain all of the following EXCEPT
a. reverse transcriptase.
b. direct repeats.
c. long terminal repeats.
d. integrase.
e. resolvase.
7. A segment is called ______ if 99% of the individuals in a population have identical sequences
for the segment.
a. contiguous
b. continuous
c. polymorphic
d. monomorphic
e. homomorphic
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8. Pyrosequencing is an example of which of the following sequencing methods?
a. sequencing by synthesis
b. hierarchial genome shotgun sequencing
c. whole genome shotgun sequencing
d. none of the above
9. A gene is mapped to within 20 map units of a marker. Which of the following was used to
determine this?
a. FISH
b. PFGE
c. linkage mapping
d. cytological mapping
e. physical mapping
10. A sequence-tagged site occurs how many times in the genome?
a. none
b. once
c. several
d. multiple
Answer Key for Study Guide Questions
This answer key provides the answers to the exercises and chapter quiz for this chapter. Answers
in parentheses ( ) represent possible alternate answers to a problem, while answers marked with
an asterisk (*) indicate that the response to the question may vary.
21.1
1. b
2. c
3. b
4. a
5. b
21.2
1. in situ
2. chromosome painting
3. Giemsa
4. DAPI
21.3
1. f
2. a
3. c
4. h
5. e
6. g
7. b
8. d
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21.4
1. Yeast artificial chromosomes (YACs)
2. Bacterial artificial chromosomes (BACs)
3. positional cloning
4. cosmid
5. P1 artificial chromosomes (PACs)
6. contig
7. chromosome walking
21.5
1. Haemophilus influenzae
2. high-throughput sequencing
3. shotgun sequencing
4. Comparative
5. sequencing by synthesis (SBS)
21.6
1. c
2. b
3. a
4. c
5. c
6. a
7. a, b, c
8. b
9. a, b
10. a, c
11. c
12. b
13. d
14. a
15. f
16. e
Quiz
1. c
2. b
3. a
4. c
5. e
6. e
7. d
8. a
9. c
10. b
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