1. Genetics: From Genes to Genomes
PowerPoint to accompany
Genetics: From Genes to Genomes
Third Edition
Hartwell ● Hood ● Goldberg ● Reynolds ● Silver ● Veres
Chapter 10
Prepared by Malcolm Schug
University of North Carolina Greensboro
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

2. Reconstructing the Genome
Through
Genetic and Molecular Analysis
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

3. Outline of Chapter 10 Challenges and strategies of genome analysis
Genome size
Features to be analyzed
Problems with DNA polymorphisms
Development of whole-genome maps
Insights emerging from complete genome sequencing
Number and type of genes
Extent of repeated sequences
Genome organization and structure
Evolution by lateral gene transfer
High throughput tools for analyzing genomes and their protein products
DNA sequencers
DNA arrays
Mass spectrophotometers
Two paradigm changes propelled by whole-genome sequences and new tools of genome analysis
Systems biology
Predictive and preventative medicine
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

4. The genomes of living organisms vary enormously in size.
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

5. Genomicists look at two basic features of genomes: sequence and polymorphism.
Major challenges to determine sequence of each chromosome in genome and identify many polymorphisms:
How does one sequence a 500 Mb chromosome 600 bp at a time?
How accurate should a genome sequence be?
DNA sequencing error rate is about 1% per 600 bp.
How does one distinguish sequence errors from polymorphisms?
Rate of polymorphism in diploid human genome is about 1 in 500 bp.
Repeat sequences may be hard to place.
Unclonable DNA cannot be sequenced.
Up to 30% of genome is heterochromatic DNA that can not be cloned
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

6. Divide and conquer strategy meets most challenges.
Chromosomes are broken into small overlapping pieces and cloned.
Ends of clones sequenced and reassembled into original chromosome strings
Each piece is sequenced multiple times to reduce error rate.
10-fold sequence coverage achieves a rate of error less than 1/10,000.
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

7. Figure 10.2
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Fig. 10.2

8. Techniques for mapping and cloning
Library of DNA fragments 500 – 1,000,000 bp
Insert into one of a variety of vectors
Hybridization
Location of a particular DNA sequence within the library of fragments
PCR amplification
Direct amplification of a particular region of DNA ranging from 1 bp to > 20kb
DNA sequencing
Automated DNA sequencer using Sanger method determines sequences 600 bp at a time.
Computational tools
Programs for identifying matches between a particular sequence and a large population of previously sequenced fragments
Programs for identifying overlaps of DNA fragments
Programs for estimating error rates
Programs for identifying genes in chromosomal sequences
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

9. Making a large scale linkage map
Types of DNA polymorphisms used for large-scale mapping:
Single nucleotide polymorphisms (SNPs) – 1/500 – 1/1000 bp across genome
Simple sequence repeats (SSRs) – 1/20-1/40 kb across genome
2-5 nucleotides is repeated 4-50 or more times.
Most SNPs and SSRs have little or no effect on the organism.
Serve as DNA markers across the chromosomes
Must be able to rapidly identify and assay in populations from 100s to 1000s of individuals
Figure 10.3
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Fig. 10.3

10. Genome wide identification of genetic markers
Initial genetic maps used SSRs which are highly polymorphic.
Identified by screening DNA libraries with SSR probes
Amplified by PCR and length differences assayed
SNPs – millions more recently identified by comparison of orthologous regions of cDNA clones from different individuals
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

11. Paralogous – arise by duplication within same species
Homologous – genes with enough sequence similarity to be related somewhere in evolutionary history
Orthologous – genes in two different species that arose from the same gene in the two species’ common ancestor
Paralogous – arise by duplication within same species
Orthologous genes are always homologous, but homologous genes are not always orthologous.
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

12. SNPs and SSRs for genome coverage
Until recently, maps were constructed from about 500 SSRs evenly spaced across genome (1 SSR every 6 Mb).
SNPs provide more than 500,000 DNA markers across the genome.
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

13. Genome wide typing of genetic markers
Two-stage assay for simple sequence repeats
PCR amplification
Size separation
Figure 10.4
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Fig. 10.4

14. Long range physical maps: karyotypes and genomic libraries position markers on chromosomes.
Overlapping DNA fragments ordered and oriented that span each of the chromosomes
Based on direct analysis of DNA rather than recombination on which linkage maps are based
Chart actual number of bp, kb, or Mb that separate a locus from its neighbors
Linkage vs. physical maps
1 cM = 1 Mb in humans
1 cM = 2 Mb in mice
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

15. Vectors used for clone large inserts for physical mapping
YACs (yeast artificial chromosomes)
Insert size 100-1,000,000 Mb
BACs (bacterial artificial chromosomes)
Insert size 50 – 300 kb
More stable and easier to purify from host DNA than YACs
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

16. How to determine order of clones across genome
Overlapping inserts help align cloned fragments.
Bottom-up approach – overlapping sequences of tens of thousands of clones determined by restriction site analysis or sequence tag sites (STSs)
Top-down approach – insert is hybridized against karyotype of entire genome.
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

17. Identifying and isolating a set of overlapping fragments from a library
Two approaches:
Linkage maps used to derive a physical map
Set of markers less than 1 cM apart
Use markers to retrieve fragments from library by hybridization.
Construct contigs – two or more partially overlapping cloned fragments.
Chromosome walk by using ends of unconnected contigs to probe library for fragments in unmapped regions
Physical mapping techniques:
Direct analysis of DNA
Overlapping clones aligned by restriction mapping
Sequence tag segments (STSs)
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

18. Physical mapping by analysis of STSs Bottom-up approach
Figure 10.5
Fig. 10.5
Each STS represents a unique segment of the genome amplified by PCR.
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

19. Human Karyotype
(a) Complete set of human chromosomes stained with Giemsa dye shows bands.
(b) Ideograms show idealized banding pattern.
Figure 10.5 a, b
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Fig a, b

20. Chromosome 7 at three levels of resolution
Figure 10.5 c
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Fig c

21. FISH protocol for top-down approach
Figure 10.8
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Fig. 10.8

22. Sequence maps show the order of nucleotides in a cloned piece of DNA.
Two strategies for sequence human genome:
Hierarchical shotgun approach
Whole-genome shotgun approach
Shotgun – randomly generated overlapping insert fragments:
Fragments from BACs
Fragments from shearing whole genome
Shearing DNA with sonication
Partial digestion with restriction enzymes
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

23. Hierarchical shotgun strategy Used in publicly funded effort to sequence human genome
Shear 200 kb BAC clone into ~2 kb fragments
Sequence ends 10 times
Need about 1700 plasmid inserts per BAC and about 20,000 BACs to cover genome
Data form linkage and physical maps used to assemble sequence maps of chromosomes
Significant work to create libraries of each BAC and physically map BAC clones
Figure 10.9
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Fig. 10.9

24. Whole-genome shotgun sequencing Private company Celera used to sequence whole human genome.
Whole genome randomly sheared three times
Plasmid library constructed with ~ 2kb inserts
Plasmid library with ~10 kb inserts
BAC library with ~ 200 kb inserts
Computer program assembles sequences into chromosomes.
No physical map construction
Only one BAC library
Overcomes problems of repeat sequences
Figure 10.10
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Fig

25. Limitations of whole genome sequencing
Some DNA can not be cloned.
e.g., heterochromatin
Some sequences rearrange or sustain deletions when cloned.
Future large genome sequencing will use both shotgun approaches.
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

26. Sequencing of the human genome
Most of draft took place during last year of project.
Instrument improvements – 500,000,000 bp/day
Automated factory-like production line generated sufficient DNA to supply sequencers on a daily basis.
Large sequencing centers with instruments – 150,000,000 bp/day
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

27. Integration of linkage, physical, and sequence maps
Provides check on the correct order of each map against other two
SSR and SNP DNA linkage markers readily integrated into physical map by PCR analysis across insert clones in physical map
SSR, SNP (linkage maps), and STS markers (physical maps) have unique sequences 20 bp or more, allowing placement on sequence map.
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

28. Changes in biology, genetics and genomics from human genome sequence
Genetics parts list
Speeds gene-finding and gene-function analysis
Sequence identification in second organism through homology
Gene function in one organism helps understand function in another for orthologous and paralogous genes
Genes often encode one or more protein domains
Allows guess at function of new protein by comparison of protein sequence in databases of all known domains
Ready access to identification of known human polymorphism
Speeds mapping of new organisms by comparison
e.g., mouse and human have high similarity in gene content and order
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

29. Major insights from human and model organism sequences
Approximately 25,000 human genes
Genes encode noncoding RNA or proteins.
Repeat sequences are > 50% of genome.
Distinct types of gene organization:
Gene families
Gene rich regions
Combinatorial strategies amplify genetic information and increase diversity.
Evolution by lateral transfer of genes from one organism to another
Males have twofold higher mutation rate than females.
Human races have very few unique distinguishing genes.
All living organisms evolve from a common ancestor.
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

30. Conserved segments of syntenic blocks in human and mouse genomes
Figure 10.12
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Fig

31. Noncoding RNA genes
Transfer RNAs (tRNAs) – adaptors that translate triplet code of RNA into amino acid sequence of proteins
Ribosomal RNAs (rRNAs) – components of ribosome
Small nucleolar RNAs (snoRNAs) – RNA processing and base modification in nucleolus
Small nuclear RNAs (sncRNAs) - spliceosomes
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

32. Protein coding genes generate the proteome.
Proteome – collective translation of 30,000 protein coding genes into proteins
Complexity of proteome increase from yeast to humans.
More genes
Shuffling, increase, or decrease of functional modules
More paralogs
Alternative RNA splicing – humans exhibit significantly more
Chemical modification of proteins is higher in humans.
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

33. Protein coding genes generate the proteome How transcription factor protein domains have expanded in specific lineages
Figure 10.13
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Fig

34. Examples of domain accretions in chromatin proteins
Figure 10.16
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Fig

35. Number of distinct domain architectures in four eukaryotic genomes
Figure 10.14
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Fig

36. Repeat sequences fall into five classes.
Transposon-derived repeats
Processed pseudogenes
SSRs
Segmental duplications of kb
Blocks of repeated sequences at centromere, telomeres and other chromosomal features
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

37. Repeat sequences constitute more than 50% of the genome.
Fig
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Fig

38. Gene organization of genome
Gene families
Closely related genes clustered or dispersed
Gene-rich regions
Functional or chance events?
Gene deserts
Span 144 Mb or 3% of genome
Contain regions difficult to identify?
e.g., big genes – nuclear transcript spans 500 kb or more with very large introns (exons < 1% of DNA)
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

39. Genome has a distinct organization
Genome has a distinct organization. Gene family – olfactory receptor gene family
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

40. Class II region of human major histocompatibility complex contains 60 genes in 700 kb
Figure 10.20
Fig
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

41. Combinatorial strategies
At DNA level – T-cell receptor genes are encoded by a multiplicity of gene segments.
Fig
At RNA level – splicing of exons in different orders
Figure (top) / Figure 10.19a (bottom)
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Fig a

42. Lateral transfer of genes
> 200 human genes may arise by transfer from organisms such as bacteria.
Lateral transfer is direct transfer of genes from one species into the germ line of another.
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

43. Twofold higher mutation rate in males
Comparison of X and Y chromosomes
Same may be true for autosomes, but difficult to measure.
Majority of human mutations arise in males.
Males give rise to more defects, but also more diversity.
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

44. Human races have similar genes.
Genome sequence centers have sequenced significant portions of at least three races.
Range of polymorphisms within a race can be much greater than the range of differences between any two individuals of different races.
Very few genes are race specific.
Genetically, humans are a single race.
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

45. All living organisms are a single race.
All living organisms have remarkably similar genetic components.
Life evolved once and we are descendents of that event.
Analysis of appropriate biological systems in model organisms provides fundamental insight into corresponding human systems.
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

46. In the future, other features of chromosomes will become increasingly important.
Chemical modification of bases
Understand DNA methylation now
Others may be discovered
Interaction of various proteins with chromosome
Three dimensional structure of proteins in nucleus
May determine interactions of chromosomal regions with regions of nuclear envelope
More effective tools need to be developed to examine chromosome features.
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

47. Copyright © The McGraw-Hill Companies, Inc
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

48. High-throughput instruments DNA sequencer
Figure 10.20
Fig
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

49. High-throughput instruments e.g, microarrays
Figure 10.21
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Fig

50. Two color - DNA microarray
Figure 10.22
Fig
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

51. Analysis of genomic and RNA sequences
Quantitative analysis of mRNA levels
Serial analysis of gene expression (SAGE)
Small cDNA tags of 15 bp from 3’ ends of mRNA are linked and sequenced.
Massively parallel signature sequence (MPSS)
Transcriptome – population of mRNAs expressed in a single cell or cell type
MPSS allows identification of most of cell’s rarely expressed mRNAs
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

52. Lynx therapeutics sequencing strategy of MPSS
Figure 10.24
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Fig

53. New approach to studying biological systems has made possible:
Systems Biology – the global study of multiple components of biological systems and their interactions
New approach to studying biological systems has made possible:
Sequencing genomes
High-throughput platform development
Development of powerful computational tools
The use of model organisms
Comparative genomics
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

54. Human Genome Project has changed the potential for predictive/preventive medicine.
Provided access to DNA polymorphisms underlying human variability
Makes possible identification of genes predisposing to disease
Understanding of defective genes in context of biological systems
Circumvent limitations of defective genes
Novel drugs
Environmental controls
Approaches such as stem-cell transplants or gene therapy
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

55. Social, ethical, and legal issues
Privacy of genetic information
Limitations on genetic testing
Patenting of DNA sequences
Society’s view of older people
Training of physicians
Human genetic engineering
Somatic gene therapy – inserting replacement genes
Germ-line therapy – modifications of human germ line
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display