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MCRO 251: MICROBIAL GENETICS
Eukaryotic Chromosome
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DNA molecule tightly wound around histone proteins
Located in the nucleus
Vary in number from a few to hundreds
Can occur in pairs (diploid) or singles (haploid)
Appear linear
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Bacterial Chromosome
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Condensed and secured by means of histone-like proteins
Single, circular chromosome
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Regulatory Gene
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•control gene expression
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Purines
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•(A and G)
•Pure agony
•Two rings structures
•Larger
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Pyrimidines
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•(C and T)
•T --> U in RNA
•One ring structure
•Smaller
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Central Dogma
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Genetic information flows from DNA to RNA to protein
The master code of DNA is used to synthesize an RNA molecule (transcription)
The information in the RNA is used to produce proteins (translation)
Exceptions: RNA viruses and retroviruses
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Replication
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DNA --> DNA
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Transcription
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DNA --> RNA
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Translation |
RNA --> Protein
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Reverse Transcription
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DNA --> RNA
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Semiconservative Replication
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•Each strand acts as a template
•One old strand pairs with a new one
•Allows accurate replication
•5’ to 3’ in direction
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Helicase
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Unzipping the DNA helix
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Primase
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Synthesizing an RNA primer
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DNA Polymerase III
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Adding bases to the new DNA chain; proofreading the chain for mistakes
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DNA Polymerase I
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Removing primer, closing gaps, repairing mismatches
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Ligase
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Final binding of nicks in DNA during synthesis and repair
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Topoisomerases I and II
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Supercoiling and untangling
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Elongation and Termination of Daughter Molecules
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•As replication proceeds, the newly produced double strand loops down
•DNA polymerase I removes RNA primers and replaces them with DNA
•When the forks come full circle and meet, ligases move along the lagging strand
•Begin initial linking of the fragments
•Complete synthesis and separation of the two circular daughter molecules
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Origin of replication
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•Short sequence
•Rich in A and T
•Held together by only two H bonds rather than three
•Less energy is required to separate the two strands
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RNA
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•Single stranded molecule
•Helical form
•Contains uracil instead of thymine
•The sugar is ribose
•Many functional types, from small regulatory pieces to large structural ones
•Only mRNA is translated into a protein molecule
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Messenger RNA
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•A transcript of a structural gene or genes in the DNA
•Synthesized by a process similar to synthesis of the leading strand during DNA replication
•The message of this transcribed strand is later read as a series of triplets (codons)
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Transfer RNA
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•Uniform in length (75-95 nucleotides long)
•Molecule has a cloverleaf structure that then folds into a complex, 3-D helix
•Bottom loop of the cloverleaf exposes a triplet (the anticodon) that designates the specificity of the _?_ and complements mRNA’s codons
•At the opposite end of the molecule is a binding site for the amino acid that is specific for that anticodon
•For each of the 20 amino acids there is at least one specialized type of _?_ to carry it
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Initiation of Translation
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•mRNA molecule leaves DNA transcription site
•mRNA transported to ribosomes in the cytoplasm
•Ribosomal subunits are specifically adapted to assembling and forming sites to hold the mRNA and tRNAs
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Redundancy
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•a particular amino acid can be coded for by more than a single codon
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Wobble |
•in many cases, only the first two nucleotides are required to encode the correct amino acid- thought to permit some variation or mutation without altering the message
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Termination Codon
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•UAA, UAG, and UGA
•Often called nonsense codons
•Do not code for a tRNA
•When reached, a special enzyme breaks the bond between the final tRNA and the finished polypeptide chain, releasing it from the ribosome
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Amplification in Expression
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•Multiple mRNA can be started from a single promoter
•Multiple ribosomes can bind to a single mRNA
•Many proteins can be made simulaneously
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eukaryotic location of transcription and translation
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transcription and processing-nucleus
translation-cytoplasm
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Eukaryotic Transcription & Translation
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•Start codon is also AUG, but it codes for a different form of methionine
•mRNAs code for just one protein
•The presence of the DNA in the nucleus means that transcription and translation cannot be simultaneous
•mRNA must pass through pores in the nuclear membrane and be carried to the ribosomes in the cytoplasm for translation
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Introns |
•sequences of bases that do not code for protein
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Exons |
•coding regions that will be translated into protein
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Split Gene
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•requires further processing before translation
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Operons |
Collections of genes organized by prokaryotes
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Inducible
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the operon is turned on by the substrate of the enzyme for which the structural genes code
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Repressible
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contain genes coding for anabolic enzymes; several genes in a series are turned off by the product synthesized by the enzyme
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Regulatory Proteins
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induction and repression occur because of the activity of _?_
•these either inhibit transcription (negative control) or promote transcription (positive control) their activity is modulated by inducers, corepressors and inhibitors
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Operon
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•the sequence of bases coding for one or more polypeptides along with the promoter and operator or activator binding sites
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Lac Operon
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•Regulates lactose metabolism in Escherichia coli
•Three important features:
•The regulator
•The control locus
--Promoter
--Operator
•Structural locus
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Regulator |
a gene that codes for a protein capable of repressing the operon [a repressor]
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Promoter |
recognized by RNA polymerase
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Operator |
a sequence that acts as an on/off switch for transcription
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Structural Locus
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•Three genes each coding for a different enzyme needed to catabolize lactose
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Neg. Control
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•presence of regulatory protein (repressor) at regulatory site (operator) decreases mRNA synthesis
•repressor proteins
•exist in active and inactive forms
•inducers and corepressors alter activity of repressor
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Pos. Control
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•presence of a regulatory protein (activator protein) at a regulatory region promotes transcription
•e.g., lactose operon
•regulated by catabolite activator protein (CAP) and cyclic AMP (cAMP)
•In absence of glucose, CAP is active and promotes transcription of operons used for catabolism of other sugars
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Wild Type
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a microorganism that exhibits a natural, non-mutated characteristic
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Mutation |
change in nucleotide sequence
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Mutant Strain
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•when a microorganism bears a mutation
•Useful for tracking genetic events
•Unraveling genetic organization
•Pinpointing genetic markers
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Point Mutation
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•involve addition, deletion, or substitution of single bases
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Missense Mutation
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any change in the code that leads to placement of a different amino acid
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Nonsense Mutation
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changes a normal codon into a stop codon
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Silent Mutation
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•alters a base but does not change the amino acid and thus has no effect
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Back-Mutation
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•when a gene that has undergone mutation reverses to its original base composition
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Frameshift Mutation
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•mutations that occur when one or more bases are inserted into or deleted from a newly synthesized DNA strand
•Changes the reading frame of the mRNA
•Nearly always result in a nonfunctional protein
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Mutations from Electromagnetic Radiation
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•X-rays, gamma rays nick DNA
•UV light causes T-T dimers to form
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Spontaneous Tautomers Mutation
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•during replication
•Mis-pairing between bases (defies base pairing rules)
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Mutations from Chemicals
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•Analogs of bases
•Base-modifying chemicals
•Nitrosoguanidine, nitrous acid
•Intercalators insert between bases
•Cause frameshift mutations
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Bruce Ames
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Measured mutagen strength
•Salmonella typhimurium used to test mutagens
•His- mutant strain grown in the absence of histidine
•Look for reversions to His+
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DNA Photolyase
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•DNA that has been damaged by UV radiation
•Restored by photoactivation or light repair
•light-sensitive enzyme
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Excision Repair
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•Remove mutations by a series of enzymes
•Remove incorrect bases and add correct one
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Recombination
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•when one bacterium donates DNA to another bacterium
ØThe end result is a new strain different from both the donor and the original recipient
ØBacterial plasmids and gene exchange
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Recombinant Organism
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•Any organism that contains (and expresses) genes that originated in another organism
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Hfr Strains
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•High frequency of recombination
•Integrated F factor (episome)
•Conjugal transfer
•Chromosomal genes introduced
•Incorporation of new genes into the chromosome
•These were used to “map” the relative positions of genes before DNA sequencing became so easy
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Resistance Factors/Plasmids
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•bear genes for resisting antibiotics
•Can confer multiple resistance to antibiotics to a strain of bacteria
•can also carry resistance to heavy metals or for synthesizing virulence factors
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Transformation
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•Griffith’s experiment demonstrated that DNA released from a killed cell can be acquired by a live cell
•Nonspecific acceptance by a bacterial cell
•Facilitated by special DNA-binding proteins on the cell wall
•Competent cells
•Useful for certain types of recombinant DNA technology
needs naked DNA
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Competent Cells
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•capable of accepting genetic material
•Useful for certain types of recombinant DNA technology
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Generalized Transduction
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•Involves a lytic phage
•Infection as usual
•Mistaken packaging of a host gene
•Defective phage
•One in a million odds
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Specialized Transduction
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•Lysogenic phage
•Inserts as prophage
•Aberrant excision
•Pick up adjacent gene
•Defective phage
•One in a million odds
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Conjugation
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needs cell contact
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Transduction
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involves bacteriophage
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Transposons
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•Jumping genes
•Mobile genetic elements
•Move from place to place in the genome, plasmids, and viral genomes
•Disrupt genes when they land
•May mobilize other genes (like antibiotic resistance)
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