Chapter 27: Genomics Fundamentals Notes

27.1 Mapping the Human Genome

• Genetics: Study of genes, heredity, and the control of gene expression.
• Genomics: Study of entire sets of genes and their functions at the organism level.
• Epigenetics: Study of how behaviors and environment cause changes affecting gene work, reversible and without DNA sequence alteration; global control of gene expression.
• A gene is a segment of DNA that directs the synthesis of a single polypeptide.
• Gene expression refers to the frequency or timing of protein creation based on gene instructions.

Human Genome Project

• Genomics: The study of whole sets of genes and their functions.
  • Human genome contains 3 billion base pairs.
  • Averages 130,400,000 base pairs.
  • Approximately 20,500 genes, averaging 890 genes per chromosome.
• Gene Size:
  • Ranges from approximately 10-15 kb, but varies greatly.
    • Tyrosine tRNA gene: ~0.2kb
    • Dystrophin gene: ~2500kb (28,000 bases)
• Total bases in genes calculation:
  • 28,000 \text{ bases } \times 20,500 \text{ genes } = 574,000,000 \text{ bases}
  • 3,000,000,000 - 574,000,000 = 2,426,000,000 \text{ leftover bases}
• A genetic map is a physical representation of landmarks in a genome, showing their relative positions.
  • Initial genetic disease studies involved identifying co-inherited landmarks with the disease gene.
  • This provided information about the chromosome location and general area of the gene.
• The Human Genome Project was launched in 1990 by a collaboration of 20 groups at not-for-profit institutes and universities led by the NIH.

Human Genome Project Strategy

• Generated a genetic map showing the physical location of markers, identifiable DNA sequences known to be inherited.
• Physical map refined the distance between markers to ~100,000 base pairs.
• A chromosome was cut into large segments and multiple copies (clones) of the segments were produced.
• Overlapping clones were arranged to cover the entire length of the chromosome.
• Each clone was cut into 500 base-pair fragments to determine the identity and order of bases.
• All 500 base-pair sequences in the chromosome were assembled into a completed nucleotide map.

Celera Genomics Project Strategy

• Started in 1995 by Celera Genomics, a commercial biotechnology company, for sequencing the human genome.
• Employed a shotgun approach, breaking the human genome into fragments without identifying their origin.
• Fragments were copied to generate many clones, cut into approximately 500-base-long pieces, and modified with fluorescently labeled bases for sequencing.
• The sequences were reassembled by identifying overlapping ends, using a large supercomputing center.

Project Completion and Accuracy

• In 2001, 90% of the human genome sequence was mapped in 15 months.
• By October 2004, 99% of the genome was sequenced with 99.999% accuracy.
• The mapped sequence identifies almost all known genes.

27.2 DNA and Chromosomes

• Understanding DNA structure provides insight into the biotechnology revolution from the Human Genome Project (HGP).

Telomeres and Centromeres

• Telomeres: Specialized DNA regions at both chromosome ends.
  • Composed of a long, noncoding series of repeating nucleotide sequences, (TTAGGG)_n.
  • Act as "endcaps" to protect chromosome ends from alterations and prevent fusion with other DNA.
• Each new cell starts with approximately 8000 bp of telomeric DNA.
• Telomeres shorten by 50-250 bases with each cell division, an elderly person may have only 1500 bp.
• Short telomeres are associated with senescence.
• Continued shortening leads to DNA instability and cell death.
• Telomerase increases telomere length and is active during embryonic development and in adult germ cells.
• Cancer cells often contain active telomerase, thought to confer immortality.
• Research focuses on genes regulating telomerase expression in cancer cells and the effects of telomerase inactivation.

Centromeres

• Centromere: Constricted point in the middle of a chromosome where duplicated DNA copies remain joined during cell division.
  • Duplicated chromosomes bound at the centromere are called sister chromatids.

Noncoding DNA

• Only approximately 1.5% of the genome codes for proteins.
• Noncoding promoter sequences regulate gene activation.
  • Individual cells only activate necessary genes; approximately 2000 of 20,500 genes are expressed in a given cell.
• Noncoding DNA may facilitate DNA folding within the nucleus or play a role in evolution.
• The function of noncoding DNA is still under investigation.

Epigenetics

• Epigenetic mechanisms are influenced by development, environmental factors, chemicals, drugs, aging, and diet.
• DNA methylation: Methyl groups tag DNA, activating or repressing genes, and can affect health.
• Histones: Proteins around which DNA winds for compaction.
• Histone modification: Binding of epigenetic factors to histone "tails" alters DNA wrapping and gene availability.
• These factors can influence health, increasing the risk of cancer, autoimmune diseases, mental disorders, and diabetes.

27.3 Mutations and Polymorphisms

• Mutation: An error in base sequence during DNA replication.
  • Refers to DNA sequence variations in a small number of individuals within a species.
  • Table 27.1. Types of Mutations
    • Point Mutations
      • Silent: A single base change that specifies the same amino acid
      • Missense: A change that specifies a different amino acid
      • Nonsense: A change that produces a stop codon
    • Frameshift
      • Insertion: Addition of one or more bases
      • Deletion: Loss of one or more bases
• Error rate of replication is approximately 1 in 1 billion.
• Mutations can result from spontaneous events or exposure to mutagens, such as viruses, chemicals, and ionizing radiation

Polymorphisms

• Polymorphisms: Common variations in DNA nucleotide sequences within a population, reflecting biodiversity due to geographical and ethnic differences.
• The majority have neutral effects, but some can lead to disease.
• Table 27.1. Some Common Hereditary Diseases, Their Causes, and Their Prevalence
  • Phenylketonuria (PKU)
  • Albinism
  • Tay-Sachs disease
  • Cystic fibrosis
  • Sickle-cell anemia

Single-Nucleotide Polymorphism (SNP)

• Single-nucleotide polymorphism (SNP): Replacement of one nucleotide at a specific DNA sequence location.
  • Biological effects of SNPs range from negligible to variations (eye or hair color) to genetic diseases.
  • SNPs are the most common source of human variation.
  • Can alter amino acid identity, have no effect, or terminate protein synthesis.
• SNPs occur roughly every 300 nucleotides, including in coding regions.
• SNP catalogs help predict individual disease risks.
  • Have been used to locate SNPs responsible for abnormal conditions, including total color blindness, epilepsy, and breast cancer susceptibility.
  • The National Human Genome Research Institute maintains a SNP catalog with over 147 million SNP entries (as of June 2015).
• The SNP catalog may allow physicians to predict the potential age at which inherited diseases will become active, their severity, and their reactions to various types of treatment.
• Services such as Ancestry.com and 23 and Me use SNP analysis to determine ancestry and test for some diseases.

Worked Example 27.1

• The severity of a mutation in a DNA sequence that changes a single amino acid in a protein depends on the type of amino acid replaced and the nature of the new amino acid.
• Exchange of a an amino acid with a small nonpolar side chain for another with the same type of side chain (e.g., glycine for alanine) or exchange of amino acids with very similar side chains (e.g., serine for threonine) might have little effect.
• Conversion of an amino acid with a nonpolar side chain to one with a polar, acidic, or basic side chain could have a major effect because the side-chain interactions that affect protein folding may change. Some examples of this type include exchanging threonine, glutamate, or lysine for isoleucine. In hemoglobin, a single replacement of glutamic acid with a valine leads to sickle-cell anemia.

27.4 Recombinant DNA

• Recombinant DNA: DNA containing two or more segments not found together in nature.
  • Technology that predates the Human Genome Project.
  • Built upon in progress in all aspects of genomics.
• Recombinant DNA technology enables cutting a gene from one organism and splicing it into the DNA of another organism.

Process

• Bacteria are excellent hosts for recombinant DNA as they contain one large circular DNA with all its genes.
  • Bacterial cells may contain small circular pieces known as plasmids which carry a few genes, can be passed between bacteria, and often carry antibiotic resistance genes.
  • Plasmids replicate through base-pairing.
• Plasmids from Escherichia coli are hosts for recombinant DNA.
• The ease of isolating and manipulating plasmids coupled with E. coli's rapid replication create ideal conditions for production of recombinant DNA and the proteins encoded by it.
• The plasmid is cut open with a restriction endonuclease or restriction enzyme at a specific sequence, making the cut at the same spot on both strands of the double-stranded DNA when read in the same 5’ to 3’ direction.
• This results in unpaired bases, known as sticky ends because they are available to match up with complementary base sequences.
• A gene fragment cut from human DNA is inserted into a plasmid.
  • The gene and plasmid are cut with the same restriction enzyme, and phosphodiester bonds are re-formed using ligase.
• The altered plasmid is inserted back into a bacterial cell.
  • Transcription and translation processes synthesize the protein encoded by the inserted gene.
  • Bacteria multiply rapidly, resulting in a large number containing the recombinant DNA and producing the protein.

Hurdles

• Technical hurdles exist before a protein made this way can be used commercially.
  • The recombinant plasmid must be inserted into a bacterium.
  • Host organisms may modify the protein, by glycosylation for example.
  • The protein must be isolated from endotoxins inherent to the host organism.

Applications

• Proteins such as human insulin, human growth hormone, and blood clotting factors for hemophiliacs are being manufactured and have reached the market place.
• A major advantage of this technology is that large amounts of these proteins can be made, thus allowing their practical therapeutic use.

27.5 Genomics: Using What We Know

Genetically Modified Plants and Animals

• Mapping and studying plant and animal genomes accelerates the development of crop plants and farm animals with desirable characteristics.
• Some genetically modified crops are planted in large quantities in the United States.
  • This solves problems such as corn crops lost to the European corn borer by transplanting a bacterial gene from Bacillus thuringiensis (Bt) into corn, which causes the corn to produce a toxin that kills the caterpillars.
• Tests are underway 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 is rich in vitamin A and provides the vitamin A desperately needed in poor populations where insufficient vitamin A causes death and blindness.

Concerns

• Ecological
  • Will genetically modified plants and animals intermingle with natural varieties and cause harm to them?
• Labeling
  • Should food labels state whether the food contains genetically modified ingredients?
• Health
  • Might unrecognized harmful substances enter the food supply?
• The Non-GMO Project was established to offer consumers non-GMO choices for organic and natural products.

Gene Therapy

• Gene therapy aims to correct or replace disease-causing genes by inserting functional, healthy genes, and has the clearest expectations for monogenic diseases.
• Focuses on using nonpathogenic viruses as vectors to deliver therapeutic DNA into cell nuclei for lifelong elimination of inherited diseases.

Challenges and Advances

• Expectations currently outweigh achievements, the FDA has not approved any human gene therapy product for sale as of 2014.
• Early vectors, such as AAV, caused allergic reactions.
• CRISPR-Cas9 allows the targeted modification of specific sequences in living cells.

FDA Considering CRISPR Therapy for Sickle Cell Disease

• FDA is considering CRISPR therapy for sickle cell disease.
  • Average life expectancy of 45 years, compared to 77 in the USA.
  • Single base change in the globin gene.
• Proposed treatment regimen:
  • Extract patient’s stem cells, treat with CRISPR to modify the globin gene, and return cells to the patient.
• Clinical Trial Result
  • 39 of 40 patients “had no vaso-occlusive crisis” (blocked blood vessel)
• Potential Concern
  • Off-target modification of the DNA.

Personal Genomic Survey

• Individualized therapies can be designed, such as:
  • If a patient lacks an enzyme needed for a drug’s metabolism or has a monogenic defect.
  • In cancer therapy, understanding the genetic differences between normal cells and tumor cells could assist in chemotherapy, immune cell therapy, and CAR-T cells.
• 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.

Bioethics

• The ethical and social implications of genomics are a major concern.
• The ELSI (Ethical, Legal, and Social Implications) program of the National Human Genome Research Institute addresses these issues.
• Key questions include:
  • 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?
  • Should we get every newborn’s full genetic sequence?