© 2013 Pearson Education, Inc. Outline 26.1 Mapping the Human Genome 26.2A Trip Along a Chromosome 26.3Mutations and Polymorphisms 26.4Recombinant DNA

© 2013 Pearson Education, Inc.

Outline

26.1 Mapping the Human Genome

26.2 A Trip Along a Chromosome

26.3 Mutations and Polymorphisms

26.4 Recombinant DNA

26.5 Genomics: Using What We Know


Goals1. What is the working draft of the human genome and the

circumstances of its creation? Be able to describe the genome mapping projects and the major accomplishments of their working drafts.

2. What are the various segments along the length of the DNA in a chromosome? Be able to describe the double helix and base pairing in DNA.

3. What are mutations? Be able to define mutations, identify what can cause them, and also identify their possible results.

4. What are polymorphisms and single nucleotide polymorphisms (SNPs) and how can identifying them be useful?

Be able to define polymorphisms and SNPs, and explain the significance of knowing the locations of SNPs.

5. What is recombinant DNA? Be able to define recombinant DNA and explain how it is used for production of proteins by bacteria.

• What does the future hold for uses of genomic information? Be able to provide an overview of the current and possible future applications of the human genome map.



• A genomic map is a physical representation of landmarks in a genome and where they are with respect to one another.

• Mapping the genes on a eukaryotic chromosome is no easy feat; the nucleotides that code for proteins (the exons) are interrupted by noncoding nucleotides (the introns).

• There is neither spacing between “words” in the genetic code, nor any “punctuation.”



• Two organizations led the effort to map the human genome: the Human Genome Project (a collection of 20 groups at not-for-profit institutes and universities) and Celera Genomics (a commercial biotechnology company).

• The Human Genome Project created a series of maps of finer and finer resolution.

• Celera fragmented DNA and then relied on instrumental and computer-driven techniques to establish the sequence.



• In 2001, 90% of the human genome sequence had been mapped in 15 months instead of the originally anticipated four years.

• By October 2004, 99% of the genome was sequenced and declared to be 99.999% accurate.

• The mapped sequence correctly identifies almost all known genes, allowing researchers to rely on highly accurate sequence information.



Human Genome Project Strategy• A genetic map was generated, showing the

physical location of markers, identifiable DNA sequences known to be inherited.

• The physical map refined the distance between markers to about 100,000 base pairs.

• To proceed to a map of finer resolution, a chromosome was cut into large segments and multiple copies of the segments were produced.

• The overlapping clones, which covered the entire length of the chromosome, were arranged in order to produce the next level of map.



Human Genome Project Strategy

• In the next step, each clone was cut into 500 base-pair fragments, and identity and order of bases in each fragment was determined.

• In the final step, all 500 base-pair sequences were assembled into a completed nucleotide map of the chromosome.


26.1 Mapping the Human GenomeCelera Genomics Project Strategy

• In what has come to be known as the shotgun approach, Celera broke the human genome into fragments without identifying the origin of any given fragment.

• The fragments were copied many times to generate many clones of each area of the genome; ultimately they were cut into 500-base long pieces and modified with fluorescently labeled bases that could be sequenced by high-speed machines.

• The sequences were reassembled by identifying overlapping ends. This monumental task was carried out using the world’s largest nongovernmental supercomputing center.



• At both ends of every chromosome are specialized regions of DNA called telomeres.

• Each telomere is a long, noncoding series of a repeating sequence of nucleotides.

• Telomeres act as “endcaps,” or “covers,” protecting the ends of the chromosome from accidental changes that might alter the more important DNA coding sequences.

• Telomeres also prevent the DNA ends from fusing to the DNA in other chromosomes or to DNA fragments.



• As the DNA in each chromosome is duplicated in preparation for cell division, the two copies remain joined together at a constricted point in the middle of the chromosome.

• This is the centromere.

• The duplicated chromosomes bound together at the centromere are known as sister chromatids.



• As the DNA in each chromosome is duplicated in preparation for cell division, the two copies remain joined together at a constricted point in the middle of the chromosome.

• This is the centromere. • The duplicated chromosomes

bound together at the centromere are known as sister chromatids.



One Genome To Represent Us All?

• Using the DNA of a single individual to represent the entire human genome is a bad idea.

• To avoid this, the path chosen by both genome mapping groups was to employ DNA from a group of anonymous individuals.

• In the Human Genome Project, researchers collected blood (female) or sperm (male) samples from a large number of donors of diverse backgrounds.

• The Celera project relied on anonymous donors of European, African, American (North, Central, South), and Asian ancestry.

• As a result, one of the most frequently asked questions about the human genome, “Whose DNA was sequenced?” can never truly be answered.



• Each new cell starts life with a long stretch of telomeric DNA.

• Some of this repeating sequence is lost with each cell division, so that as the cell ages, the telomere gets shorter and shorter.

• A very short telomere is associated with senescence.

• Continuation of shortening beyond this stage is associated with DNA instability and cell death.

• Telomerase adds telomeres to DNA. It is active during embryonic development. In adults, telomerase is only active in germ cells.



• There is widespread speculation that telomere shortening plays a role in aging.

• Some support for this concept comes from experiments with mice whose telomerase activity has been “knocked out” (in genetic research vernacular).

• These mice age prematurely, and if they become pregnant, their embryos do not survive.

• The majority of cancer cells are known to contain active telomerase, which is thought to confer immortality on these tumor cells.

• Current research suggests that it is the genes responsible for regulating telomerase expression that are altered in cancer cells. There are ongoing experiments on the consequences of telomerase inactivation on cancer cells.



Noncoding DNA• There are noncoding promoter sequences, which are

regulatory regions of DNA that determine which of its genes are turned on.

• Only the genes needed by any individual cell will be activated in that cell.

• Current data suggests that only about 2% of all DNA in the human genome actually codes for protein.

• The human genome has much more noncoding DNA than do the genomes known for other organisms.

• The function of noncoding DNA remains to be discovered, and the debate over its role continues.



Genes• The nucleotides of a single gene are not consecutive along a stretch

of DNA, having coding segments (exons) that alternate with noncoding segments (introns).

• Chromosome 22 was the first to have all of its nonrepetitive DNA sequenced and mapped.

• The chromosome map identified 49 million bases containing 693 genes, with an average of 8 exons and 7 introns per gene.

• Chromosome 22 carries genes known to be associated with the immune system as well as congenital heart disease, schizophrenia, leukemia, cancers, and many other genetically-related conditions.

• The map also revealed several hundred previously unknown genes.

• With the signal (exon) to noise (intron) ratio being so low (meaning more noise to hide the signal) in the human genome, it will be challenging to completely identify all the coding sequences present.



• An error in base sequence that is carried along during DNA replication is called a mutation.

• Mutation commonly refers to variations in DNA sequence found in a very small number of individuals of a species.

• Some mutations result from spontaneous and random events.

• Others are induced by exposure to a mutagen—an external agent that can cause a mutation.

• Viruses, chemicals, and ionizing radiation can all be mutagenic.







• Polymorphisms are variations in the nucleotide sequence of DNA that are common within a given population.

• Most polymorphisms are simply differences in the DNA sequence between individuals due to geographical and ethnic differences and are part of the biodiversity exhibited by life on earth.

• The vast majority of polymorphisms seen have neither advantageous nor deleterious effects, some have been shown to give rise to various disease states.



FIGURE 26.2 A human chromosome map



• The replacement of one nucleotide by another in the same location along the DNA sequence is a single-nucleotide polymorphism.

• The biological effects of SNPs range from negligible to normal variations such as those in eye or hair color, to genetic diseases.

• In addition to producing a change in the identity of an amino acid, a SNP might specify the same amino acid (for example, changing GUU to GUC, both of which code for valine), or it might terminate protein synthesis by introducing a stop codon.



• An international team of scientists is compiling a catalog of SNPs.

• As of 2010, over 5 million SNPs had been recorded. Their frequency is roughly one SNP for about every 2000–5000 bases, with many of them in coding regions.

• The SNP catalog has been used to locate SNPs responsible for total color blindness, one type of epilepsy, and susceptibility to breast cancer.

• It is hoped that this information will inspire the development of new treatments for diseases.



• The cataloging of SNPs has ushered in the era of genetic medicine.

• The SNP catalog may allow physicians to predict for an individual the potential age at which inherited diseases will become active, their severity, and their reactions to various types of treatment.

• The therapeutic course will be designed to meet the distinctive genomic profile of the person.



• Using recombinant DNA technology, it is possible to cut a gene out of one organism and splice it into (recombine it with) the DNA of a second organism.

• Bacteria provide excellent hosts for recombinant DNA.

• Bacterial cells contain part of their DNA in small circular pieces called plasmids, each of which carries just a few genes.



• The ease of isolating and manipulating plasmids plus the rapid replication of bacteria create ideal conditions for production of recombinant DNA and the proteins whose synthesis it directs.

• The plasmid is cut open with a restriction endonuclease or restriction enzyme, that recognizes a specific sequence.



Serendipity and the Polymerase Chain Reaction• Kary Mullis figured out that combining DNA polymerase, target DNA,

nucleoside triphosphates, and short synthetic nucleotide chains (oligonucleotides) in just the right way would massively amplify the target.

• The polymerase chain reaction (PCR) is now carried out automatically by instruments in every molecular biology lab. In 1993, Mullis shared the Nobel Prize in chemistry for this work.

• The reaction is carried out in three steps: heating the sample to cause the helix to unravel into single strands, adding primers complementary to the target, and extending the primers with DNA polymerase.

• The reactants are combined in a closed container and the temperature cycled from about 90 °C for Step 1, to about 50 °C for Step 2, and to about 70 °C for Step 3. The temperature cycle requires only a few minutes and can be repeated over and over again for the same mixture.

• Automation of the PCR was made possible by the discovery of a heat-stable polymerase (Taq polymerase) isolated from a bacterium that lives in hot springs.



• Recombinant DNA is produced by cutting the two DNA segments to be combined with the same restriction endonuclease. The result is DNA fragments with complementary sticky ends.

• The two are mixed in the presence of a DNA ligase enzyme that joins them together by re-forming their phosphodiester bonds, reconstituting the now-altered plasmid.



• The altered plasmid is inserted back into a bacterial cell where the normal processes of transcription and translation synthesize the protein encoded by the inserted gene.

• Bacteria multiply rapidly; there are soon a large number of them, all containing the recombinant DNA and all manufacturing the protein encoded by the recombinant DNA.

• Huge numbers of the bacteria can be put to work as a protein factory.



• One hurdle is getting the recombinant plasmid back into a bacterium.

• Host organisms may modify the protein: yeast cells attach carbohydrates to various amino acids.

• The protein of interest must be isolated from endotoxins—potentially toxic natural compounds found inside the host organism.



• Even small amounts of endotoxins can lead to serious inflammatory responses, so rigorous purification and screening protocols are necessary.

• Proteins manufactured in this manner have already reached the marketplace, including human insulin, human growth hormone, and blood clotting factors for hemophiliacs.

• A major advantage of this technology is that large amounts of these proteins can be made, thus allowing their practical therapeutic use.



DNA Fingerprinting

• DNA fingerprinting relies on variations between two or more DNA samples.

• The repetitive patterns used in DNA fingerprinting are variable number tandem repeats (VNTRs), short DNA sequences that are repeated multiple times. For any given VNTR, the number of copies of the repeated sequence varies between individuals.

• The probability of a match with someone other than the correct individual is estimated at 1 in 1.5 billion.

• There are two common techniques used for DNA fingerprinting today: the restriction fragment length polymorphism (RFLP) approach and the polymerase chain reaction (PCR) method.

• RFLP relies on use of a restriction endonuclease (an enzyme used to cut DNA) that recognizes and cuts sequences on either side of a given VNTR.

• With PCR, primers are directed towards regions of the DNA that are known to contain variations. These are amplied about 30 times (at 4 minutes per cycle) so that in two hours more than a billion copies are produced. These fragments can then be separated according to size, stained, and compared against other samples.



• Genomics is the study of whole sets of genes and their functions.

• The study of bacterial genomics has been instrumental in linking the three domains of life—Archaea (formerly archeabacteria), Bacteria, and Eukarya—to one another from an evolutionary standpoint.

• The study of bacterial genomics is giving us a better understanding of how bacteria cause disease, it is also helping in the development of new therapies.

• Plant genomics is enhancing the value and utility of agricultural crops.

• The genomic study of farm animals is improving their health and availability.



Genetically Modified Plants and Animals• The mapping and study of plant and animal genomes

can greatly accelerate our ability to generate crop plants and farm animals with desirable characteristics and lacking undesirable ones.

• Some genetically modified crops are planted in large quantities in the United States.– Each year millions of tons of corn are destroyed by the

European corn borer. To solve this problem, a bacterial gene (from Bacillus thuringiensis, Bt) has been transplanted into corn. The gene causes the corn to produce a toxin that kills the caterpillars.

– Soybeans genetically modified to withstand herbicides are also widely grown. The soybean crop remains unharmed when the surrounding weeds are killed by the herbicide.







Genetically Modified Plants and Animals• Tests are under way with genetically modified coffee

beans that are caffeine-free, potatoes that absorb less fat when they are fried, and “Golden Rice,” a yellow rice that provides the vitamin A desperately needed in poor populations where insufficient vitamin A causes death and blindness.

• Fish farming is an expanding industry as natural populations of fish diminish. There are genetically engineered salmon that can grow to marketable size in one-half the time of their unmodified cousins.

• Similar genetic modifications are anticipated for other varieties of fish, and there is the prospect of cloning leaner pigs.



Genetically Modified Plants and Animals• Will genetically modified plants and animals

intermingle with natural varieties and cause harm to them?

• Should food labels state whether the food contains genetically modified ingredients?

• Might unrecognized harmful substances enter the food supply?

• These questions and have led to the establishment of the Non-GMO Project, the goal of which is to offer consumers a non-GMO choice for organic and natural products.



Gene Therapy• Gene therapy is based on the premise that a

disease-causing gene can be corrected or replaced by inserting a functional, healthy gene.

• The most clear-cut expectations for gene therapy lie in treating monogenic diseases.

• The focus has been on using viruses as vectors, the agents that deliver therapeutic quantities of DNA directly into cell nuclei.

• The Food and Drug Administration (FDA) has, as of July 2011, not yet approved any human gene therapy product for sale.



A Personal Genomic Survey• If a patient lacks an enzyme needed for a drug’s

metabolism or has a monogenic defect, therapies could be individually tailored.

• In cancer therapy, understanding the genetic differences between normal cells and tumor cells could assist in chemotherapy.

• Genetic screening of infants might permit the use of gene therapy to eliminate the threat of a monogenically-based disease, or a lifestyle adjustment for an individual with SNPs that predict a susceptibility to a disease that results from combinations of genetic and environmental influences.



Snips and Chips• Our understanding of SNPs is already at work in

screening implemented by DNA chips. Different individuals may have no effect from a drug, the expected effect, or a greater-than-normal response to the drug.

• Genomic screening can determine whether particular polymorphisms are linked to a patient’s ability to respond to the medication.

• Once such connections have been established, screening could be a diagnostic test carried out by a DNA chip in a doctor’s office.

• DNA chip screening has already revealed genetic variations responsible for two types of pediatric leukemia which require quite different therapies.



Snips and Chips• A DNA chip is a solid support bearing large numbers of

short, single-stranded bits of DNA of known composition. • The DNA is organized for a particular type of screening. • A sample is labeled with a fluorescent tag and applied to

the chip. • During an incubation period, sample DNA and chip DNA

with complementary nucleic acid sequences will bond to each other.

• After excess sample DNA is washed away, the fluorescence remaining on the chip is read to discover where the bonding has occurred and thus, what DNA variations are present in the sample DNA.



Bioethics• The ELSI program of the National Human Genome

Research Institute deals with the Ethical, Legal, and Social Implications of human genetic research such as:– Who should have access to personal genetic information and

how will it be used?– Who should own and control genetic information?– Should genetic testing be performed when no treatment is

available?– Are disabilities diseases? Do they need to be cured or

prevented?– Preliminary attempts at gene therapy are exorbitantly

expensive. Who will have access to these therapies? Who will pay for their use?

– Should we re-engineer the genes we pass on to our children?


Chapter Summary

1. What is the working draft of the human genome and the circumstances of its creation?

• The Human Genome Project, an international consortium of not-for-profit institutions, and Celera Genomics, a for-profit company, have both announced completion of working drafts of the human genome. With the exception of large areas of repetitive DNA, the DNA base sequences of all chromosomes have been examined.

• The Human Genome Project utilized a series of progressively more detailed maps to create a collection of DNA fragments with known location. Celera began by randomly fragmenting all of the DNA without first placing it within the framework of a map. In both groups the fragments were cloned, labeled, ordered, and the individual sequences assembled by computers.

• The results of the two projects are generally supportive of each other. There are about three billion base pairs and 20,000–25,000 genes in the human genome, each able to direct the synthesis of more than one protein. The bulk of the genome consists of noncoding, repetitive sequences. About 200 of the human genes are identical to those in bacteria.


Chapter Summary, Continued

2. What are the various segments along the length of the DNA in a chromosome?

• Telomeres, which fall at the ends of chromosomes, are regions of noncoding, repetitive DNA that protect the ends from accidental changes. At each cell division, the telomeres are shortened, with significant shortening associated with senescence and death of the cell.

• Telomerase, the enzyme that lengthens telomeres, is typically inactivated in adult cells, but becomes reactivated in cancer cells.

• Centromeres are the constricted regions of chromosomes that form during cell division and also carry noncoding DNA.

• Exons are the protein coding regions of DNA and together make up the genes that direct protein synthesis. The repetitive noncoding segments of DNA are of either no function or unknown function.



3. What are mutations?

• A mutation is an error in the base sequence of DNA that is passed along during replication.

• Mutations arise by random error during replication but may also be caused by ionizing radiation, viruses, or chemical agents (mutagens).

• Mutations can cause inherited diseases and the tendency to acquire others.


Chapter Summary, Continued4. What are polymorphisms and single nucleotide

polymorphisms (SNPs) and how can identifying them be useful?

• A polymorphism is a variation in DNA that is found within a population. An SNP is the replacement of one nucleotide by another.

• The result might be the replacement of one amino acid by another in a protein, no change because the new codon specifies the same amino acid, or the introduction of a stop codon.

• Many inherited diseases are known to be caused by SNPs, but they can also be beneficial or “neutral”.

• Understanding the location and effect of SNPs is expected to lead to new therapies.



5. What is recombinant DNA? • Recombinant DNA is produced by joining DNA

segments that do not normally occur together. • A gene from one organism is inserted into the

DNA of another organism. Recombinant DNA techniques can be used to create large quantities of a particular protein. The gene of interest is inserted into bacterial plasmids (small, extrachromosomal circular DNA).

• Bacteria carrying these plasmids then serve as factories for the synthesis of large quantities of the encoded protein.



6. What does the future hold for uses of genomic information?

• Mapping the human genome holds major promise for applications in health and medicine.

• Drugs can be precisely chosen based on a patient’s own DNA, thereby avoiding drugs that are ineffective or toxic for that individual. Perhaps one day inherited diseases will be prevented or cured by gene therapy.

• By genetic modification of crop plants and farm animals, the productivity, marketability, and health benefits of these products can be enhanced.

• Progress in each of these areas is bound to be accompanied by controversy and ethical dilemmas.

Documents

© 2013 Pearson Education, Inc. Outline 26.1 Mapping the Human Genome 26.2A Trip Along a Chromosome 26.3Mutations and Polymorphisms 26.4Recombinant DNA