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The Gr (eatest Fourth Test Study Guide To Ever Exist…
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May the fates smile upon thee…
Gene expression
• How did scientists first elucidate the gene-protein relationships?
Early evidence indicated most genes specify the structure of proteins
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Believed it to be an “inborn error of metabolism)
1940s- George beadle and Edward tatum: looked at mutations affecting genes
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Wanted to mutate the organism
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Obtained a wide number of mutants
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Linus pauling (1949): a single gene mutagen is responsible for sickle cell anemia
What is RNA?
o link between DNA and proteins
o polymer of nucleotides
o usually single stranded (DNA-2, RNA-1)
o employs ribose sugar at 2’ position (H in DNA)
o uses the nucleotide uracil rather than thymine
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DNA serves as template
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§ Proceeds in the 5’ à 3’ direction
§ Upstream: toward the 5’ end of mRNA (or 3’ end of template DNA
strand)
§ Downstream: toward the 3’ end of mRNA (or the 5’ end of template DNA
strand)
§ RNA polymerases bind to a promoter INITIATES TRANSCRIPTION
• Non-transcribed nucleotide signal region
• Diff genes have diff promoters
• Unwinds DNA and initiates transcription
• Adds nucleotides at 3’ growing end in the elongation phase
• Continues until a codon is reached
§ mRNA synthesis
• contains more coding than nucleotides
o leader sequence: (5’) non-coding, recognition sequence
o coding sequence: polypeptide message
o trailing sequence: termination sequence
What is translation? The mRNA must now move out of the nucleus via nuclear pores
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• Elongation: AA added to growing polypeptide chain (3’)
o Aminocyl-tRNA recognizes the site A anticocon
o Binds with complementary mRNA codon
o Peptide bond b/w AA at A site and carboxyl group of
proceeding AA (P site)
o Translocation: ribosome moves down mRNA 1 codon
• Termination: final stage of translation
o Employs a release fator: protein which recognizes the stop
codon
§ In many bacteria, transcription and translation are coupled together
§ Unitlize clusters of ribosomes (polysomes)
o What is the fate of mRNA?
§ Pre-mRNA molecule
§ 5’ end capped with modified mucleotide
§ poly-A tail added to 3’ end
§ introns (intervening, non-coding regions) removed {exons (expressed,
coding regions) spliced together}
§ mature mRNA transported into the cytosol
§ translation at ribisome
Why is the Central Dogma not always quite accurate? Retrovirus (reverse transcriptase)
What’s a mutation?
o Changes the nucleotide sequence of DNA
o Organisms have a vast array of DNA repair mechanisms
o Base substituions
§ Change in only a pair of nucletides (GC to AT)
§ Missence mutation: change in AA
§ Nonsense mutation: AA to a stop codon
o Frameshift mutations:
§ One or more nucleotides are inserted or deleted
§ Alters reading frame
o Whole chromosome may be latered
o Barbara mccklintock: transposons (moveable genetic elements)
If you could have a single mutation, what would it be?
What are the sources of mutation?
o Locations:
§ Somatic_ not heritable if it does not ultimately involve germinal tissues
§ Germinal- potentially heretable via sexual reproduction
o Phenotypic effects:
§ Morphological, lethal, conditional, biochemical, resistant
How often do they occur?
o Frequency:

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Relatively rare per gamete or per generation in an individual, bun in larger
pop increaidngly common

What types of mutation did we discuss?
o Forward mutations by far most common
o Reverse very, very rare: difficult to discern from forward mutaations representing
diff pathwat but restoring original phenotype
o Most mutations deleterious (CAUSE DAMAGE)
o Non-deleterious mutations usually produce minor phenotypic changes
o Accumulations of slight changes may lead to major changes
o Mutagent agents: UV light, radiation, very low/high temps, chemicals, age of
organs
o Inversions: switching of pieves of a chromosome
o Translocations: pieves swapped b/w 2 chromsoeomes
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Many primitive groups with high base
#s (low ploidy levels extinct)
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g
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CAN BE TURNED OFF
o How are they similar?
What are some examples of positive gene regulators?
o Positive regulators:
§ Stimulate transcription via activator proteins
• Lac operon uses both negative and positive regulation
• Recognizes absence of glucose
• Much cheaper to use available glucose rather than making
additional enzymes such as B-galactosidase (GALACTOSE)
• When glucose levels are low, stimulates the binding of cAMP to
CAP
• Causes allosteric shift (CHANGE IN SHAPE/FUNCTION)
• Allows RNA polymerase to bind efficiently
• At times, groups of operons may be controlled by a single
regulator (regulon)
What is a posttranscriptional control mechanism?
o Regulation after transcription
o Translational controls: regulate rate of mRNA translation
§ Up to 1000x rate for some genes
• Use differential ribiosme binding
o Posttransitional controls: activate or inactivate one or more existing enzymes
How do eukaryotes regulate their genes?
o Not organized into operons
o Many genes inducible only at certain times in the life of the organism (temporal
regulation)
o Others under tissue-specific regulation
o Gene regulation occurs at the levels of:
§ Transcription, mRNA processing, translation, the protein product

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How do eukaryotic promoters (NEEDED FOR TRANSCRIPTION) work (the non-Don
King ones)?
o Transcription initiation site: site where transcription begins
o TATA box: promoter sequence ca
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p
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p
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Why are genomic libraries so important?
o Genome: sum total of all the genetic info in cell
o Genomic library: collection of DNA fragments that represent all the DNA in the
genome
§ Contained in a collection of recombinant bacterial cells
§ Useful for study of individual genes
o Chromosome library: all the DNA fragments in a particular chromsome
o Several steps for generating both types of libraries:
§ Cleave DNA of interest into fragments
§ Produce recombinant (ARTIFICIAL) DNA
§ Insert plasmids via transformation into competent cells
• Low concentrations of plasmids so multiple genes not incorporated
§ Plate out bacterial colonies
o How are they constructed?
§ What’s the deal with cDNA?
§ Complementary DNA
• Complement to mRNA
• Used to construct valuable cDNA libraries
PCR: wonderful thing or tool of the Eldar Ones?
o Polymerase chain reaction
o Developed in 1985 by kary mullis
o Revolutionized molecular bio
o 4 main components:
§ small amt of double-stranded DNA
§ DNA precursors
§ Specific nucleic acid primers
§ Taq DNA polymerase
o PCR is denatured
o Primers attach to primer-binding site on each DNA strand
o Each strand acts as template for DNA synthesis
o How does DNA sequencing work?
§ Began in the 1970s from sanger and gilber
§ Developed the chain termination method
§ Employs dideoxynucleotides (lacks a 3’ hydroxyl group)
§ May use actual radio-labeled gels or automated sequencer
o Gel electrophoresis?
§ Method for separating certain macromolecules

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§ Add lithium bromide (fluoresces under UV light)
What are some of the excellent uses for DNA technology?
o Medical and pharmacology
§ Genetic testing: to determine if certain mutations are present
§ Gene therapy: use of specific DNA to treat genetic disease
§ Pharmaceuticals: human insulin
§ Tissue eengineering: growing cels in tissues in culture
§ Develop recombinant vaccines
o DNA typing:
§ DNA fingerprinting or profiling
§ Used in forensic pursuits, identify cell lineages, track populations
§ Employs PCR and gel electrophoresis
• Polymorphisms: population level detectible variation
• Short tandem repeats: repetitive DNA up to 200 bp
o Transgenic organisms
§ Organisms with foreign DNA
§ Often via injection of DNA into embryonic stem cells (recently fertizlied
cell)
§ Genetic targeting: researcher designated inactivation of a single gene
§ Typically introduced into knockout mice lineages
§ Mutagenesis screening: treated with a mutagen and studies
§ “pharm” organisms: used to produce some human desired product
§ transgenic crops: can synthesize their own fertilizers and can provde own
anti-herbovry compounds
o industrial:
§ manufacturing proteins using bacterial, fungal, and mammalian cells as
factories
§ strain improvement for existing bioprocesses
§ development of new strains for new bioprecesses
o agricultural:
§ introduction of desirable traits onto agriculturally important animals and
plants
• bovine growth hormone
• nitrogen fixation capabilities
• plant resistence to env stresses
o social:
Human Genome
What is a genome?
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an organism's complete set of DNA, including all of its genes

How do we analyze such things?
o Cytogenetics: study of chromosomes and their role in inheritance (CYTOROLE)
o Karyotyping: studies of individual chromosomes in the nucleus (NUKLEUS)
§ Enables researchers to indentify chromosome adnormalities
o Pedigree (family tree): transmission of genetic traits over generations
§ Imp for making informed medical decisions

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Why do we care to undertake such endeavors?
o ca
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23-30,000 genes
o still have yet to understand of sequence the myriad number of diff alleles
o bioinformatics: the storage, retrieval, and comparison of DNA within and among
species (BIO-SPECIES
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Fatal in animals
§ Aneuploidy: abnormalities resulting from the presence/absence of a single
chromosome
• Much more common
• Disomy: wild type (2 copies)
• Monosomy: 1 copy
• Trisomy: 3 copies
§ Meiotic nondisjunction: sister chromatids or homologous chromosomes
fail to tear apart in meiosis
o Down’s syndrome
§ Among most common chromosomal abnormalities
§ Trisomic for chromosome 21
§ Results in genetic imbalance
§ Affetcted by mothers age
o Sex chromosome aneuploidy
§ Tolerated more than non-sex
§ Klinfelter syndrome: males XXY (unusually tall, female breasts, small
testes)
§ Turner syndrome: XO (develop as femals, but internal and external sex
features are underdeveloped
o Translocations: chromosome fragments attach to non-homologous chromosome
o May lead to deletions

o Fragile sites: places where part of the chromatid appears to be attached by a thin
thread of DNA
o Single-gene mutations:
§ Almost universally only recovered in homozygotes
§ 3 categories:
• autosomal recessive disorder
o phenylketonuria
o inability to convert the AA phenylalanine to tyrosine
o avoid meat, fish, dairy, breads, nuts
o may be toxic of hetro- or homozygous babies
o sickle cell anemia, cystic fibrosis
• autosomal dominant disorder
o huntingtons disease
• x-linked recessive: hemophilia
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Population Genetics
What is a population?
o Individuals of the same species that live in a particular place at the same time
o Population genetics: study of genetic variability within a pop, and the forces
acting upon it
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o Involves phenotype, genotype and allele frequencies
o Know the differences between genotypic, phenotypic and allelic frequencies
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Increases genetic variability
acted on by natural selection
§ Genetic drift: random change in allele freqs of a small pop
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Changes it cause are usually not adaptive
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o What are the chief mechanisms of microevolution?
§ Gene flow: the movement of alleles cause by migration of individuals
between pops
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Operates on an
organisms phenotype
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• Stabilizing: selection favors the mean
• Directional: favors one Phenotypic extreme
• Disruptive: favors two or more phenotypic extremes
What leads to genetic variation?
o Cause by 2 forces: mutations and sexual reproduction
§ Genetic polymorphism: presence in a pop of 2 or more alleles per locus
o Modes of balanced polymorphsims:
§ Heterozygous advantage: selection of heterozygous forms over
homozygotes
§ Freq dependent selection: fitness of a phenotype depends on how freq It
appears in the pop
o Neutral variation: background, non-selective variation
o Varitation occurs in individuals as well as diff pops (geographic variation)

o Cline: gradual change in a species phenotype and genotypic freqs from and env
gradient



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