Lecture 15students(2)

Lecture 15students(2) - Lecture 15 Genes, muta0ons and...

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: Lecture 15 Genes, muta0ons and genomes How Does the Polymerase Chain Reac0on Amplify DNA? Copies of DNA sequences can be made by the polymerase chain reac/on (PCR) technique. PCR is a cyclical process: •  DNA fragments are denatured by hea0ng •  Primers, plus dNTPs and DNA polymerase are added •  New DNA strands are synthesized Figure 13.22 The Polymerase Chain Reac0on How Does the Polymerase Chain Reac0on Amplify DNA? PCR results in many copies of the DNA fragment—referred to as amplifying the sequence. The base sequence at the 3′ end of the DNA fragment must be known. Complementary primers, about 15–30 bases long, are made in the laboratory. How Does the Polymerase Chain Reac0on Amplify DNA? An ini0al problem with PCR was its temperature requirements. The heat needed to denature the DNA destroyed most DNA polymerases. A DNA polymerase that does not denature at high temperatures (90°C) was taken from a hot springs bacterium, Thermus aqua2cus. What Are Muta0ons Gene0c muta0ons are changes in the nucleo0de sequences of DNA that are passed on to the next genera0on. Muta0ons may or may not have a phenotypic effect. What Are Muta0ons Muta0ons occur in two types: •  Soma/c muta/ons occur in soma0c (body) cells—passed on by mitosis but not to sexually produced offspring •  Germ line muta/ons—occur in germ line cells, the cells that give rise to gametes. A gamete passes a muta0on on at fer0liza0on What Are Muta0ons At the molecular level, muta0ons or altera0ons in the nucleo0de sequence are in two categories: •  A point muta/on—results from the gain, loss, or subs0tu0on of a single nucleo0de •  Chromosomal muta/ons are more extensive—may change the posi0on or cause a DNA segment to be duplicated or lost What Are Muta0ons Muta0ons have different phenotypic effects: •  Silent muta/ons •  Loss of func/on muta/ons •  Gain of func/on muta/ons •  Condi/onal muta/ons Figure 15.1 Muta0on and Phenotype What Are Muta0ons Point muta0ons change single nucleo0des. They can be due to errors in replica0on or to environmental mutagens. Point muta0ons in the coding regions of DNA usually cause changes in the mRNA, but may not affect the protein. Point Muta0ons Point Muta0ons What Are Muta0ons Chromosomal muta0ons: •  Dele/ons— •  Duplica/ons— •  Inversions— •  Transloca/ons Figure 15.4 Chromosomal Mutations (C,D) What Are Muta0ons Muta0ons are caused in two ways: •  Spontaneous muta/ons—occur with no outside influence, and are permanent •  Induced muta/ons—are due to an outside agent, a mutagen Figure 15.6 5 ­Methylcytosine in DNA Is a “Hot Spot” for Muta0ons What Are Muta0ons •  Radia2on damages DNA: Ionizing radia0on such as X rays creates free radicals—highly reac0ve—can change bases, break sugar phosphate bonds UV radia0on (from sun or tanning beds) is absorbed by thymine, causing it to form covalent bonds with adjacent nucleo0des —disrupts DNA replica0on What Are Muta0ons Muta0ons can be ar0ficial or natural: •  Human ­made chemicals (e.g., nitrites) or naturally occurring substances (e.g., molds) •  Radia0on from nuclear reac0ons, bombs, or from the sun What Are Muta0ons Muta0ons have benefits: •  Provide the raw material for evolu0on in the form of gene0c diversity Diversity may benefit the organism immediately—if muta0on is in soma0c cells Or may cause an advantageous change in offspring What Are Muta0ons? Possible costs of muta0ons: •  Some germ line and soma0c cell muta0ons are harmful or lethal Public health policy includes bans on ozone ­deple0ng chemicals and on cigarefe smoking, which cause muta0ons that lead to cancer How Are DNA Molecules and Muta0ons Analyzed? Molecular gene2cs studies DNA changes that lead to specific protein changes. Bacteria have developed a way to fight viruses that inject genes into the host cell so that it can produce more viruses. Bacteria use restric/on enzymes to cleave DNA at specific sequences into smaller noninfec0ous fragments. How Are DNA Molecules and Muta0ons Analyzed? Enzymes break bonds—restric/on diges/on —at a specific sequence called a recogni/on sequence or restric/on site. To protect the host cell: Methylases add methyl groups to restric0on sites on the cell’s own DNA. The restric0on enzyme passes over the methylated sequence but cuts the unmethylated viral DNA. Figure 15.7 What are restric0on envading Bacteria Fight I nzymes? Viruses by Making Restric0on Enzymes How Are DNA Molecules and Muta0ons Analyzed? Bacterial restric0on enzymes can be isolated from the cells that make them. Restric0on enzyme diges0on is used in the laboratory to iden0fy and analyze point muta0ons. DNA fragments can then be amplified using PCR. How Are DNA Molecules and Muta0ons Analyzed? DNA fragments cut by enzymes can be separated by gel electrophoresis. A mixture of fragments is placed in a well in a semisolid gel and an electric field is applied across the gel. Nega0vely charged DNA fragments move towards posi0ve end. Smaller fragments move faster than larger ones. How Are DNA Molecules and Muta0ons Analyzed? DNA fragments separate and give three types of informa0on: •  The number of fragments •  The sizes of the fragments •  The rela2ve abundance of the fragments, indicated by the intensity of the band Figure 15.8 Separa0ng Fragments of DNA by Gel Electrophoresis (Part 1) Figure 15.8 Separa0ng Fragments of DNA by Gel Electrophoresis (Part 2) How Are DNA Molecules and Muta0ons Analyzed? Aler separa0on on a gel a specific DNA sequence can be found with a single ­stranded probe. The gel region can be cut out and the DNA fragment removed. The purified DNA can be analyzed by sequence or amplified and used experimentally. How Are DNA Molecules and Muta0ons Analyzed? DNA fingerprin/ng for iden0fica0on uses restric0on diges0on and gel electrophoresis. It works best with highly polymorphic sequences—having mul0ple alleles that are likely to differ between individuals. How Are DNA Molecules and Muta0ons Analyzed? Two types of polymorphisms: Single nucleo/de polymorphisms (SNPs): Inherited varia0ons involving a single base— point muta0ons Short tandem repeats (STRs): Short repe00ve sequences occurring side by side on chromosomes, usually in noncoding regions Figure 15.9 DNA Fingerprin0ng with Short Tandem Repeats How Are DNA Molecules and Muta0ons Analyzed? Example of DNA fingerprin0ng: In 1918, Tsar Nicholas II and his family were reportedly executed and their bodies burned during the Communist revolu0on. A gravesite was discovered in1991. DNA fingerprin0ng of the bone fragments revealed that they were a family and were related to living descendants of the Tsar. Figure 15.10 DNA Fingerprin0ng of the Russian Royal Family How Are Genomes Sequenced? The Human Genome Project was proposed in 1986 to determine the normal sequence of all human DNA. The publicly funded effort was aided and complemented by privately funded groups. Methods used were first developed to sequence prokaryotes and simple eukaryotes. How Are Genomes Sequenced? To sequence an en0re genome, the DNA is first cut into fragments about 500 base pairs (bp) long. The haploid human genome has about 3.3 billion bp, resul0ng in 6 million fragments. The fragment sequences are put together using larger, overlapping fragments. How Are Genomes Sequenced? Example: Using a 10 bp fragment, cut three different ways̶ TG, ATG, and CCTAC AT, GCC, and TACTG CTG, CTA, and ATGC The correct order is ATGCCTACTG. How Are Genomes Sequenced? The field of bioinforma/cs was developed to analyze DNA sequences using complex mathema0cs and computer programs. How Are Genomes Sequenced? The shotgun sequencing method cuts DNA into smaller, overlapping fragments that are cloned and sequenced. Computers are used to search for overlapping markers. This approach is much faster and cheaper. Figure 17.2 Sequencing DNA (Part 1) Figure 17.2 Sequencing DNA (Part 2) How Are Genomes Sequenced? High ­throughput sequencing methods are used to sequence large genomes. They involve amplifica0on of DNA templates by PCR and physical binding of template DNA to a solid surface or to microbeads. Thousands or millions of sequencing reac0ons are run at once: Massively parallel DNA sequencing. How Are Genomes Sequenced? Genome sequence informa0on is used to iden0fy: •  Open reading frames, or coding regions •  Amino acid sequences of proteins •  Regulatory sequences •  RNA genes • Other noncoding sequences How Are Genomes Sequenced? In compara2ve genomics, newly sequenced genomes are compared with sequences from other organisms. This can give informa0on about the func0ons of sequences, and is used to trace evolu0onary rela0onships. What Have We Learned from Sequencing Prokaryo0c Genomes? The first life forms to be sequenced were the simplest viruses with rela0vely small genomes. The first complete genomic sequence of a free ­living cellular organism was for the bacterium Haemophilus influenzae, in 1995. What Have We Learned from Sequencing Prokaryo0c Genomes? Func/onal genomics assigns func0ons to the products of genes. H. influenzae chromosome has 1,738 open reading frames. When it was first sequenced, only 58 percent coded for proteins with known func0ons. Since then the roles of many other proteins have been iden0fied. Figure 17.5 Func0onal Organiza0on of the Genome of H. influenzae What Have We Learned from Sequencing Prokaryo0c Genomes? Transposable elements are DNA sequences 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. What Have We Learned from Sequencing Prokaryo0c Genomes? Longer transposable elements (up to 5,000 bp) carry addi0onal genes and are called transposons. Some0mes these DNA regions contain a gene for an0bio0c resistance. What Have We Learned from Sequencing Prokaryo0c Genomes? Genome sequencing provides insights into microorganisms that cause human diseases and reveal new methods to combat them. Sequencing also reveals rela0onships between pathogenic organisms, sugges0ng that genes may be transferred between different strains. What Have We Learned from Sequencing Prokaryo0c Genomes? Tradi0onally, microorganisms have been iden0fied by culturing them in the laboratory. Now, PCR and DNA analysis allow microbes to be studied without culturing. What Have We Learned from Sequencing Prokaryo0c Genomes? DNA can also be analyzed directly from environmental samples. Metagenomics̶gene0c diversity is explored without isola0ng intact organisms. Shotgun sequencing is used to detect presence of known microbes, as well as heretofore uniden0fied organisms. Figure 17.7 Metagenomics What Have We Learned from Sequencing Prokaryo0c Genomes? It is es0mated that 90 percent of the microbial world has been invisible to biologists and is only now being revealed by metagenomics. What Have We Learned from Sequencing Prokaryo0c 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. What Have We Learned from Sequencing Prokaryo0c 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. What Have We Learned from Sequencing Eukaryo0c Genomes? There are major differences between eukaryo0c and prokaryo0c genomes: •  Eukaryo2c genomes are larger and have more protein ­coding genes •  Eukaryo2c genomes have more regulatory sequences. Greater complexity requires more regula0on What Have We Learned from Sequencing Eukaryo0c Genomes? •  Much of eukaryo2c DNA is noncoding, including introns, gene control sequences, and repeated sequences •  Eukaryotes have mul2ple chromosomes; each must have an origin of replica0on (ori), a centromere, and a telomeric sequence at each end What Have We Learned from Sequencing Eukaryo0c Genomes? The nematode, Caenorhabdi2s elegans: A millimeter ­long soil roundworm. It has a transparent body made up of about 1,000 cells, yet has complex organ systems. It has about 3.5 0mes as many protein ­coding genes as do yeasts. What Have We Learned from Sequencing Eukaryo0c Genomes? The fruit fly, Drosophila melanogaster: The fruit fly has ten 0mes more cells and is more complex than C. elegans. It has a larger genome but fewer coding genes than C. elegans. Figure 17.9 Func0ons of the Eukaryo0c Genome What Have We Learned from Sequencing Eukaryo0c Genomes? The thale cress, Arabidopsis thaliana: The genomes of some plants are huge, but A. thaliana has a much smaller genome. Many of the genes found in animals have homologs in plants, sugges0ng a common ancestor. What Have We Learned from Sequencing Eukaryo0c Genomes? But some genes are unique to plants, such as those involved in photosynthesis, water transport, assembly of the cell wall, and making molecules for defense against microbes and herbivores. Figure 17.10 Plant Genomes What Have We Learned from Sequencing Eukaryo0c Genomes? Eukaryotes have closely related genes called gene families. These arose over evolu0onary 0me when different copies of genes underwent separate muta0ons. For example: Genes encoding the globin proteins all arose from a single common ancestral gene. Figure 17.11 The Globin Gene Family What Have We Learned from Sequencing Eukaryo0c Genomes? During development, different members of the globin gene family are expressed at different 0mes and in different 0ssues. Hemoglobin of the human fetus contains γ ­ globin, which binds O2 more 0ghtly than adult hemoglobin. What Have We Learned from Sequencing Eukaryo0c Genomes? Many gene families include nonfunc0onal pseudogenes (Ψ), resul0ng from muta0ons that cause a loss of func0on. A pseudogene may simply lack a promoter and thus fail to be transcribed; or a recogni0on site needed for the removal of an intron. What Have We Learned from Sequencing Eukaryo0c Genomes? Eukaryo0c genomes have repe00ve DNA sequences: •  Highly repe//ve sequences̶short sequences (< 100 bp) repeated thousands of 0mes in tandem; not transcribed Short tandem repeats (STRs) of 1–5 bp can be used in DNA fingerprin0ng Figure 17.13 Sequences in the Eukaryo0c Genome What Are the Characteris0cs of the Human Genome? The complete haploid human genome sequence was finished in 2005. Since then, the diploid genomes of several individuals have been sequenced and published. What Are the Characteris0cs of the Human Genome? Some interes0ng facts about the human genome: •  Protein ­coding regions make up less than 2 percent, about 24,000 genes Each gene must code for several proteins, and posfranscrip0onal mechanisms (e.g., alterna0ve splicing) must account for the observed number of proteins in humans What Are the Characteris0cs of the Human Genome? •  An average gene has 27,000 base pairs •  All human genes have many introns •  Over 50 percent of the genome is transposons and other repe00ve sequences What Are the Characteris0cs of the Human Genome? •  97 percent of the genome is the same in all people •  Genes are not evenly distributed over the genome. The Y chromosome has the fewest genes (231); chromosome 1 has the most (2,968) Figure 17.14 Evolu0on of the Genome Proteomics and Metabolomics Reveal? The proteome is the sum total of proteins produced by an organism; it is more complex than the genome. The aim of proteomics is to iden0fy and characterize all of the expressed proteins. Figure 17.17 Proteomics Proteomics and Metabolomics Reveal? Two techniques are used to analyze the proteome: •  Two ­dimensional gel electrophoresis separates proteins based on size and electric charges •  Mass spectrometry iden0fies proteins by their atomic masses Proteomics and Metabolomics Reveal? Comparisons of eukaryo0c proteomes has revealed a common set of about 1,300 proteins that provide the basic metabolic func0ons. Figure 17.18 Proteins of the Eukaryo0c Proteome Proteomics and Metabolomics Reveal? Proteins have different func0onal regions or domains. Proteins that are unique to a par0cular organism are olen just unique combina0ons of domains that exist in other organisms. This reshuffling of the gene2c deck is a key to evolu2on. Proteomics and Metabolomics Reveal? Gene and protein func0on are both affected by the internal and external environments of the cell. Enzyme ac0vi0es affect concentra0ons of their substrates and products, called metabolites. As the proteome changes, so will the abundances of metabolites. Proteomics and Metabolomics Reveal? The metabolome is the quan0ta0ve descrip0on of all of the small molecules in a cell or organism: •  Primary metabolites are involved in normal processes, such as in pathways like glycolysis. Also includes hormones and other signaling molecules Proteomics and Metabolomics Reveal? Secondary metabolites are olen unique to par0cular organisms or groups. Examples include: An0bio0cs made by microbes, and chemicals made by plants for defense against pathogens and herbivores. Proteomics and Metabolomics Reveal? Measuring metabolites involves gas chromatography and high ­performance liquid chromatography, which separate molecules. Mass spectrometry and nuclear magne0c resonance spectroscopy are used to iden0fy them. ...
View Full Document

This note was uploaded on 10/11/2011 for the course BIS 2A taught by Professor Grossberg during the Summer '08 term at UC Davis.

Ask a homework question - tutors are online