Genomes

1. Genomes

2. Figure 17.7 Synthetic Cells

3. 17 Genomes • 17.1 How Are Genomes Sequenced? • 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • 17.3 What Have We Learned from Sequencing Eukaryotic Genomes? • 17.4 What Are the Characteristics of the Human Genome? • 17.5 What Do the New Disciplines of Proteomics and Metabolomics Reveal?

4. 17 Genomes No other mammal shows as much phenotypic variation as dogs. The Dog Genome Project sequences entire genomes of different breeds and identifies genes that control specific traits, such as size. Opening Question: What does dog genome sequencing reveal about other animals?

5. 17.1 How Are Genomes Sequenced? • Genome sequencing: determine the nucleotide base sequence of an entire genome. • The information is used to: • Compare genomes of different species to trace evolutionary relationships • Compare individuals of the same species to identify mutations that affect phenotypes

6. 17.1 How Are Genomes Sequenced? • Identify genes for particular traits, such as genes associated with diseases • The Human Genome Project was proposed in 1986 to determine the normal sequence of all human DNA. • Methods used were first developed to sequence prokaryotes and simple eukaryotes.

7. 17.1 How Are Genomes Sequenced? • To sequence an entire genome, the DNA is first cut into millions of small, overlapping fragments. • Then many sequencing reactions are performed simultaneously.

8. 17.1 How Are Genomes Sequenced? • High-throughput sequencing uses miniaturization techniques, principles of DNA replication, and polymerase chain reaction (PCR). • It is fully automated, rapid, and inexpensive.

9. Figure 17.1 DNA Sequencing

10. 17.1 How Are Genomes Sequenced? • DNA is cut into small fragments physically or using enzymes. • The fragments are denatured using heat, separating the strands. • Short, synthetic oligonucleotides are attached to each end of each fragment, and these are attached to a solid support.

11. 17.1 How Are Genomes Sequenced? • Fragments are amplified by PCR. • Sequencing: • Universal primers, DNA polymerase, and the 4 nucleotides (dNTPs, tagged with fluorescent dyes) are added. • One nucleotide is added to the new DNA strand in each cycle, and the unincorporated dNTPs are removed.

12. 17.1 How Are Genomes Sequenced? • Fluorescence color of the new nucleotide at each location is detected with a camera. • Fluorescent tag is removed and the cycle repeats.

13. 17.1 How Are Genomes Sequenced? • Then the sequences must be put together. • The DNA sequence fragments, called “reads,” are overlapping, so they can be aligned.

14. 17.1 How Are Genomes Sequenced? • Example: Using a 10 bp fragment, cut three different ways: • TG, ATG, and CCTAC • AT, GCC, and TACTG • CTG, CTA, and ATGC • The correct order is ATGCCTACTG.

15. Figure 17.2 Arranging DNA Fragments

16. 17.1 How Are Genomes Sequenced? • The field of bioinformatics was developed to analyze DNA sequences using complex mathematics and computer programs.

17. Figure 17.3 The Genomic Book of Life

18. 17.1 How Are Genomes Sequenced? • Genome sequence information is used in two research fields: • Functional genomics—sequence information is used to identify functions of various parts of genomes: • Open reading frames—gene coding regions

19. 17.1 How Are Genomes Sequenced? • Amino acid sequences, deduced from sequences of open reading frames • Regulatory sequences, such as promoters and terminators. • RNA genes • Other noncoding sequences

20. 17.1 How Are Genomes Sequenced? • Comparative genomics: comparison of a newly sequenced genome with sequences from other organisms. • This provides more information about functions of sequences and can be used to trace evolutionary relationships.

21. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • The first life forms to be sequenced were the simplest viruses with small genomes. • The first complete genome sequence of a free-living cellular organism was for the bacterium Haemophilus influenzae in 1995.

22. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • Bacterial and archaeal genomes are: • Small, and usually organized into a single chromosome • Compact—85% is coding sequences • Usually do not have introns • Have plasmids, which may be transferred between cells

23. Table 17.1

24. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • Functional genomics: • H. influenzae chromosome has 1,727 open reading frames. • When it was first sequenced, only 58% coded for proteins with known functions. • Since then, the roles of many other proteins have been identified.

25. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • Highly infective strains of H. influenzae have genes for surface proteins that attach the bacterium to the human respiratory tract. • These surface proteins are now a focus of research on treatments for H. influenzae infections.

26. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • Comparative genomics: • M. genitalium lacks enzymes to synthesize amino acids, so it must obtain them from the environment. • E. coli has 55 genes that encode transcriptional activators, whereas M. genitalium has only 7—a relative lack of control over gene expression.

27. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • Genome sequencing provides insights into microorganisms that are important in agriculture and medicine. • Surprising relationships between organisms suggests that genes may be transferred between different species.

28. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • Rhizobium bacteria form symbiotic relationships with plants. The bacteria fix N into forms useable by plants. • Sequencing has identified genes involved in successful symbiosis, and may broaden the range of plants that can form these relationships.

29. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • E. coli strain O157:H7 causes illness in humans. • 1,387 genes are different from those in the harmless strains of this bacterium, but are also present in other pathogenic bacteria, such as Salmonella. • This suggests genetic exchange among species.

30. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • Severe acute respiratory syndrome (SARS) was first detected in southern China in 2002 and rapidly spread in 2003. • Isolation and sequencing of the virus revealed novel proteins that are possible targets for antiviral drugs or vaccines.

31. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • Genome sequencing of organisms involved in global ecological cycles: • Some bacteria produce methane, a greenhouse gas, in cow stomachs. • Others remove methane from the air. • Understanding the genes involved in methane production and consumption may help us slow the progress of global warming.

32. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • Traditionally, microorganisms have been identified by culturing them in the laboratory. • Now, PCR and DNA analysis allow microbes to be studied without culturing.

33. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • DNA can also be analyzed directly from environmental samples. • Metagenomics—genetic diversity is explored without isolating intact microorganisms. • Sequencing is used to detect presence of known microbes and previously unidentified organisms.

34. Figure 17.4 Metagenomics

35. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • It is estimated that 90% of the microbial world has been “invisible” to biologists and is only now being revealed by metagenomics. • The increased knowledge of the microbial world will improve our understanding of ecological processes and better ways to manage environmental problems.

36. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • Transposable elements (transposons) are DNA segments that can move from place to place in the genome or to a plasmid. • If a transposable element is inserted into the middle of a gene, it will be transcribed, and result in abnormal proteins.

37. Figure 17.5 DNA Sequences That Move (A)

38. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • Composite transposons: transposable elements located near one another will transpose together and carry the intervening DNA sequence with them. • Genes for antibiotic resistance can be multiplied and transferred between bacteria in this way, via plasmids.

39. Figure 17.5 DNA Sequences That Move (B)

40. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • Certain genes are present in all organisms (universal genes); and some universal gene segments are present in many organisms. • This suggests that a minimal set of DNA sequences is common to all cells.

41. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • Efforts to define a minimal genome involve computer analysis of genomes, the study of the smallest known genome (M. genitalium), and using transposons as mutagens. • Transposons can insert into genes at random; the mutated bacteria are tested for growth and survival, and DNA is sequenced.

42. Figure 17.6 Using Transposon Mutagenesis to Determine the Minimal Genome

43. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • M. genitalium can survive in the laboratory with only 382 functional genes. • One goal of the research is to design new life forms for specific purposes, such as cleaning up oil spills.

44. 17.2 What Have We Learned from Sequencing Prokaryotic Genomes? • An artificial genome has been created and inserted into bacterial cells. • The entire genome of Mycoplasma mycoides was synthesized, then transplanted into empty cells of Mycoplasma capricolum. • The new cell’s genome had extra sequences, so it was a new organism: Mycoplasma mycoides JCV1-syn.1.0.

45. Figure 17.7 Synthetic Cells

46. Working with Data 17.1: Using Transposon Mutagenesis to Determine the Minimal Genome • In the experiment to create a synthetic genome and determine the minimum set of genes necessary for survival, transposon mutagenesis was used with Mycoplasma genitalium, which had the smallest known genome.

47. Working with Data 17.1: Using Transposon Mutagenesis to Determine the Minimal Genome • Growth of M. genitalium strains with gene insertions (intragenic) was compared with strains with insertions in noncoding regions (intergenic).

48. Working with Data 17.1: Using Transposon Mutagenesis to Determine the Minimal Genome • Question 1: Explain these data in terms of genes essential for growth and survival. Are all of the genes in M. genitalium essential for growth? • If not, how many are essential? • Why did some of the insertions in intergenic regions prevent growth?

49. Working with Data 17.1: Using Transposon Mutagenesis to Determine the Minimal Genome • Question 2: • If a transposon inserts into the following regions of a gene, there might be no effect on the phenotype. • Explain in each case: a. near the 3′ end of a coding region b. within a gene coding for rRNA • How does this affect your answer to Question 1?

50. 17.3 What Have We Learned from Sequencing Eukaryotic Genomes? • There are major differences between eukaryotic and prokaryotic genomes: • Eukaryotic genomes are larger and have more protein-coding genes. • Eukaryotic genomes have more regulatory sequences. Greater complexity requires more regulation.