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What is mutation rate?
The number of events that produce mutated alleles per locus per generation.
If 4 of 100,000 births show a mutation from a recessive to a dominant allele, we've ctually sampled 200,000 genes because we have two copies of each gene. So the mutation rate would be 4/200,000 or 2/100,000.
To ensure that the mutant phenotype can be measured, it must:
Never be produced by recessie alleles
Always be fully expressed and completely penetrant so that mutant individuals can be indentified
Have clearly established paternity
Never be produced by nongeneric agents such as drus or infection and be produced by a dominantly inherited mutation of only one gene
Focused on environmental causes of mutations
Ames and his test
Identified mutagenic agents but the disadvantage to this test was that he used bacteria, which is a different metabolic pathway than is found in rats or humans. Sonce the bacteria doesn't have that metabolic product, just abecause a chemical passes the Ames test, doesn't necessarily mean that it is not a mutanogen
Mutations that cause the substitution of one amino acied for another in a protein.
- CTC-->CAC (glutamic acid-->valine) (sickle cell anemia)
Mutations that change a termination codon into one that codes for an amino acid. Such mutations produce elongated protiens.
- UAA-->AAA (removal of termination codon
Mutations that change an amino acid specifying a codon to one of the three termination codons. This shortens the protein product.
- AAA-->UAA (termination codon)
Mutational events in which a number of bases (other than multiples of three) are added to or removed from DNA, causing a shift in the codon reading frame.
- Insertion: CAT CAT CAT GCA T
- Deletion: CAT CAT CAC AT
A form of mutation associated with the expansion in copy number of a nucleotide triplet in or near a gene
How many trinucleotide repeats are there in Fragile X syndrome?
CGC - 230
How many trinucleotide repeats are there in Myotonic Dystrophy?
CTG - 50 to 2,000 repeats
How many trinucleotide repeats are there in Huntington's Disease?
CAG - 42 to 100
Onset of a generic disorder at earlier ages and with increasing severity in successive generations
Examples of DNA repair genes
UV damage - thymine dimers (excision repair)
Xeroderma Pigmentosum - thymine dimer defect
Bloom Syndrome - DNA ligase defect
Genomic Imprinting: Reversible alterations to the genome
Activate certain genes and deactivate other genes. Sometimes, genes that are not necessary are activated and start influencing changes. This results in chance for mutations and an increase in the chance for cancer.
Fetal tissue mutations
A cell when it has the ability to give rise to an entire organism. Occurs during the 16 cell stage.
Genetically identical molecules, cells, or organisms all derived from a single ancestor.
Reproductive cloning and therapeutic cloning
The two types of stem cell cloning
Why stem cell clones are not exact replicas
- 1. telomeres of chromosomes of donor nuclei are shorter
- 2. genomic imprinting when chromosomes are passed via germ line
- 3. somatic cells accumulate mutations
- 4. X-inactivation pattern
- 5. mitochondria is from recipient cell not donor cell
- 6. coat color differences-pigmented cells move about differently
- 7. environmental factors during embryonic/fetal development: nutrition (methylation) stress, exposure to environmental diseases
Why stem cell cloning fails
- 1. Meiosis not involved (meiosis in the female completes fertilization)
- 2. Diploid nucleus is plunked into oocyte cytoplasm where signals direct it to do what a female secondary oocyte tends to do (a.) shed half of chromosomes as polar body--hapoid--lethal and (b.) replicates DNA--tetraploid--lethal
Bioethical issues of stem cell cloning
- 1. Violation of rights of early-stage embryos versus violation of rights of individuals who might benefit from such therapy
- 2. 2001 USA legislation outlaw creating or selling "any embryo produced by human cloning"
Steps of cloning genes
- 1. isolate plasmid DNA and human DNA
- 2. cut both DNA's with the same restriction endonuclease--resulting in DNA fragments
- 3. combine both DNA's--cut ends re-associate or anneal
- 4. seal gaps with DNA ligase
- 5. creates recombinant DNA molecules composed of human DNA and vector DNA
- 6. transfer vector into bacteria
- 7. at each bacterial cell division, the plasmid is replicated resulting in many copies or clones of the DNA insert
- 8. lyse bacteria--extract recombinant plasmids
- 9. cloned human DNA can be released from plasmid with same restriction enzyme used for cloning
- 10. cloned DNA can be used directly, put in expression vector and transfected into cells
Human genomic library
- 1. A collection of clones that contain all of the genetic information in an individual--cut all DNA of donor cell--put in cloning vector (bacteriophage as an example)
- 2. Retrieve gene of interest ("needle in hay stack") by using DNA probe
- 3. DNA probe--a synthetic piece of labeled DNA that is complementary to part of gene of interest
Extract mRNA from cells, reverse transcribe to single stranded cDNA. DNA polymerase to give double-stranded cDNA, cut, put in cloning vector
Polymerase Chain Reaction (PCR)--1986
A method for amplifying DNA segments using cycles of denaturation, annealing to primers, and DNA-polymerase-directed DNA synthesis.
- 1. DNA amplified by heating to break hydrogen bonds, yielding single strnded DNA
- 2. Short nucleotide sequences act as primers for DNA replication added
- 3. Enzyme, DNA polymerase, begins t primers and synthesizes a DNA strand complementary to the region between the primers, a proces called "primer extension"
- 10 cycles = 1,024 copies 30 cycles = 1,073,741,820 copies
A method for transferring DNA fragments from a gel to a membrane filter, developed by Edwin Souther for use in hybridization experiments
Used to find diferences in notmal and mutant alleles, identify related genes in other organisms, and study gene evolution
Used for identifying proteins
Uses same process in Southern Blotting only for proteins
Used for identifying RNA
Don't have to digest enzymes to study RNA like you would in Southern blots to study DNA
A series of techniques in which DNA fragments are linked to self-replicating vectors to create recombinant DNA molecules, which are replicated in a host cell.
Used in agriculture to make transgenic corn plants that are resistant to insects and to give transgenic pigs growth hormones
Used in medicine and pharmacology to make human insulin via bacteria or milk from cows, goats, or sheep
Used in cloning pigs for organ donors (knockout of carbohydrate that is involved in transplant rejection)
Bacterial enzymes that cut DNA at specific sites
Self-replicating DNA molecules that are used to transfer foreign DNA segments between host cells
Refers to the transfer of genes between species by recombinant DNA technology; transgenic organisms have received such a gene
Applications of DNA technology
RFLP (restriction fragment length polymorphism)
Prenatal (as well as postnatal screening for genetic defects)
Nucleotide sequences 14 to 100 base pairs long organized into clusters of varying lengths; used in the construction of DNA fingerprints.
Short tandem repeat (STR)
Short nucleotide sequences 2 to 9 base pairs long otganized into clusters of varying lengths; used in the construction of DNA profiles
The pattern of of STR allele frequencies used to identify individuals
Altered self cells that excape normal growth regulatory controls
1 in 3 will develop cancer in a lifetime
1 in 3 wil develop cancer in a lifetime
1 in 8 women develop breast cancer if the live to be 80
1 in 8 women develop breast cancer if they live to be 80
90% of lung cancer due to tobacco
90% of lung cancer due to tobacco
Stages in cancer
- 1. Initiation
- 2. Promotion
- 3. Progression/metastasis
Initiation stage of cancer
Event that is associated with the first mutation that leads to cancer
Promotion phase of cancer
Altered genes/cells increasing and recruiting mor growth factors, increasing the cells affected by the cancer.
Benign or malignant can be determined at this point
Progression/metastasis stage of cancer
If tumor is malignant, the cells can spread to other parts of the body
Properties of Cancer
- 1. Clonally derived
- 2. Accumulate mutations: display growth regulatory changes
- 3. Movement-metastases
- 4. Altered biochemical pathways
- 5. Disorganized cytoskeleton
- 6. Chromosomal abnormalities
- 1. Chemical--tobacco tar and SENCAR mice
- 2. Virus--(Rous Sacroma virus (Peyton Rous); ERB-B (Bishop and Varmus)
- 3. Translocation--Burkett's lymphoma (c-myc on chromosome #8 is translocated to Ig region on chromosome #14)
Causes of Cancer
- 1. Environment--lung and skin cancer
- 2. Diet--fat: breast, prostate
- 3. Chemical
- 4. DNA and RNA viruses
- 5. Genetics (approximately 10% of cancers)
Tumor suppressor genes
Genes encoding proteins that suppress cell division
Genes that initiate or maintain cell diveision and that may become cancer genes (oncogenes) by mutations
Genes that induce or continue uncontrolled cell proliferation
Genes that regulate cell growth and passage through the cell cycle, for example, tumor suppressor genes
Genes that help maintain the integrity of the genome, for example, DNA repair genes
How to enhance immune response to cancer antigens/immunogens
Antigens (antibody generators)
Molecules carried or produced by microorganisms that initiate antibody production
Any molecule that can bind to a antibody.
Elicits celular and humoral response
A class of proteins produced by B cells that bind to foreign molecules (antigens)and inactivate them.
Made in response to immunogen according to the specificity of the immunogen.
Humoral Branch of Immune System
B cells/plasma cells make and secrete antibody/immunoglobulins
Cellular Branch of Immune System
T helpers enhance immune response; cytotoxic T cells destroy altered self
A type of lymphocyte that matures in the bone marrow and mediates antibody-directed immunity
White blood cells that originate in bone marrow and mediate the immune response
A type of lymphocyte that undergoes maturation in the thyus and mediates cellular immunity
Helper T cell
A lymphocyte that stimulates the production of antibodies by B cells when an antigen is present, and stimulates division of B cells and cytotoxic T cells
Memory Immune Principle
Once primed by immunogen, rapid response to immunogen
Specificity Immune Principles
Reacts specifically with challenging immunogen
Classes of Immunoglobulins
IgM (M as in Morning, Morning is early)
Early response, effective in defense against baceria infections
IgG (Greatest or major immunoglobulin)
Major immunoglobulin, lasts for years; cross placenta
IgA (Secrete out your Ass)
Found in body secretions
IgE (When I have allergies, I sneeze and say Excuse me)
Involved in allergic responses
- 1. Agammaglobulinemias
- 2. Severe-Comined Immunodeficiency (SCID)
- 3. Athymic Nude mice
- 4. HIV--AIDS
Blood Type = Antigens (Type A blood has A antigens, Type O has none)
Antibodies = opposite of blood type (Type A has anti-B Antibodies, Type AB has none, Type o has anti-A and anti-B)
ABO blood types, transfusions and incompatibility
IgE antibody ataches to blast cells in tissues, binds allergen, and cause blast cells to release pharmacological chemicals
Organ Transplants: HLA complex
Immune system recognize incompatible transplants as non-self and destroy the transplants. Must use immunosuppressive drugs to permit transplants to survive
Autoimmunity: defects in he immune response system
Immune system attacks and destroys self
- Multiple Sclerosis
- Type 1 Diabetes
- Rheumatoid Arthrits