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101 Cards in this Set
- Front
- Back
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2 ways new organisms can arise:
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1. Mutatuon
2. Genetic Exchanges |
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3 mechanisms by which genetic exchanges occur
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1. Transformation
2. Transduction 3. Conjugation |
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Transformation?
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Lysis of cell and breaking of chromosomal fragments --> release of fragments into environ --> uptake and incorporation of donor DNA into host
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Transduction?
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Transfer of chromosome fragments by incorporating donor DNA into virus genome or virus heads, infection of other cells by transducing phage/particle, and the transfer of the DNA into the cell and then into the cell chromosome
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Conjugation?
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Transfer of fragments or whole chromosome between male (transfer) and female (recipient) cells.
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Plasmids are generally (essential/nonessential) for growth
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Nonessential
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Plasmids can be transferred from cell to cell by all 3 methods of genetic exchange
T or F? |
True
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Genetic Recombination?
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Breaking and rejoining DNA fragments, especially during genetic exchanges where donor DNA must be incorporated into the host DNA
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3 types of genetic recombination
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1. Homologous: Can involve any region of DNA as long as they share similar/identical nucleotide sequences
2. Site-specific: Two fragments recombine only if certain nucleotide sequences (sites) are recognized by a specific enzyme 3. Illegitimate: Rare recombination of fragments with no similarity of nucleotide sequences |
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Transformation, transduction, and conjugation all depend on ____
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Recombination (some exceptions)
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STREPTOCOCCUS PNEUMONIAE
Colony Morphology and result of infection in bunnies |
1. Smooth, mucoid, capsule --> wild-type --> disease
2. Rough, no capsule --> mutant --> no disease 3. No colonies due to heat-killed wild types --> no disease 4. Mix of heat-killed wild with mutants --> disease |
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How can mutant STREPTOCOCCUS PNEUMONIAE mixed with wild types heat-killed cause disease in rabbits?
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Fragments containing capsule-forming gene undergoes homologous recombination with mutant DNA --> binary fission produces 1 mutant daughter and 1 wild type daughter --> wild type causes disease
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Homologous recombination - General process
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Nick one strand --> generate 3' end on strand --> strand invades other duplex --> hydrogen bonding of complementary nucleotides
* Ligase unites 5' and 3' ends If a single fragment of a strand is used for recombination, we can see a piece of the recipient chromosome be replaced by the fragment and degrade. |
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Rec BCD enzyme?
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Initiates recombination repair in presence of DNA damage from radiation, etc.
Occurs frequently at chi sites Cleaves 3' end of upper strand of duplex, then hydrolyzes the 5' end of the bottom strand --> Generates 3' end with chi site --> coats new single strand forming with Rec A protein to promote homologous pairing and strand exchange = Repair |
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Chi sites
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Small stretch of DNA where recombination occurs frequently. Specifically, it serves to signal Rec BCD action
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What are the 2 types of transduction?
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1. Generalized: Any host genes
2. Specialized: Only host genes near prophage |
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Generalized transduction - 2 steps
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1. Phage grows on first host --> some pick up donor DNA
2. Phages infect recipients --> transfer DNA This all occurs due to virus accidentally taking up host DNA instead of viral DNA --> Once in cell, fragment can undergo homologous recombination with recipient |
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Specialized transduction - 3 Steps
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1. Mix host cells with phage --> Some of the Phages lysogenizes donor
2. Induction --> some phages pick up donor DNA 3. Phage lysogenizes second host --> host now carries new donor DNA |
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LEU+ vs LEU-
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LEU - are mutants requiring leucine to grow
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GAL+ vs GAL-
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GAL + can use galactose as sole carbon source, while GAL- can't use galactose and grow on glucose
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Steps of prophage induction in specialized transduction
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DNA damage --> repressor cleavage --> lytic genes no longer inhibited --> excision (by excisionase) of viral DNA --> prophage loops out--> and lytic growth begins --> progeny forms and lysis of cell
In case of lambda, illegitimate recomb. forms phages with GAL+ DNA (BOP)--> prophage with this DNA packaged and sent to recipient |
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Plasmids can carry genes bestowing:
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1. Antibiotic resistance
2. Ability to transfer resistance genes to new cells by transferring plasmid DNA 3. Mineral uptake 4. Toxin production 5. Hydrocarbon metab. |
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The 3 mechanisms of drug resistance in bacteria
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1. Mutation resulting in altered bacterial protein --> no longer recognizes antibiotic and still performs normal function
Ex: Streptomycin 2. Bacteria acquires new genes that code for enzyme that destroys antibiotic Ex: Penicillinase destroying penicillin 3. New gene for enzyme that pumps antibiotic out of bacterial cells Ex: Tetracycline resistance |
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Shigella dysenteriae facts
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1. Bacillus
2. Negative 3. Dysentery |
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What is Shigella dysenteriae resistant to?
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1. Sulfonamides
2. Streptomycin 3. Chloramphenicol 4. Neomycin |
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NEISSERIA GONORRHOEAE facts
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1. Coccus
2. Negative 4. Gonorrhea Resistant to penicillin and streptomycin Sensitive to Ceftriaxone |
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Insertion sequences
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1. Short
2. Code for only one gene - Transposase 3. Transposes to multiple sites in DNA 4. No phenotype seen as long as no genes are interrupted |
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Insertion sequence structure
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Inverted repeat -- Transposase coding region -- inverted repeat -- duplicated sequence
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Transposons
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1. Transpose to multiple sites
2. Carry antibiotic resistance genes 3. Bracketed by inverted insertion sequences |
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Integrons
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1. Gene capture element
2. Code for info necessary to insert host gene into itself - Integrase to move gene - Attachment site to put gene into 3. Have promoter to express captured gene 4. Can contain multiple captured genes expressed by promoter |
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Super integron
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First termed for vibrio cholerae --> integron contained 179 inserted genes
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How are integrons associated with DNA mobility?
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They can be located on transposons and plasmids --> because of this, when they catch genes, they can be transferred by way of transposons and conjugative plasmids
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Conjugative transposons
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1. Move from donor to recipient cells
2. Can move to different or identical sites 3. Code for integration, excision (from cell holding it), conjugation, and antibiotic resistance 4. Excise --> single strand transfers to recipient --> circularizes --> copied into double strand --> integrated into new host chromosome |
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Genomic island
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1. Linear
2. Have integration and transfer enzymes 3. Integrate in 3' ends of tRNA genes, boundaries are direct repeating sequences 4. Difference in G and C content from host genome indicates origin in another genus 5. Can cause rapid evolution |
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4 types of genomic islands
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1. Pathogenicity islands: Toxin genes, adhesins (pili) in uropathogenic e. colo, iron uptake (plague)
2. Ecological islands: Phenol catabolism 3. Symbiosis island: Nitrogen fixation 4. Saprophytic islands: Iron uptake - e. coli growing in intestine |
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4 Pathogenicity islands of uropathogenic e. coli.
*Most common cause of bladder/kidney infections |
1. PAI - I: Codes for hemolysin
2. PAI - II: Codes for Hemolysin, adhesin 3. PAI - III: Codes for adhesin, iron uptake 4. PAI - IV: Iron uptake |
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F Factor
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Bestows fertility in bacteria --> F+ = male/donor
F- = Female/Recipient Double-stranded DNA with an origin of transfer spot. Codes for replication and transfer genes |
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F+
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Have sex pili
Duplicate their F factor in fission Transfer F factor with conjugation bridge --> one strand is transferred here --> replication reforms double strand in recipient AND donor |
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How is the F Factor transfered?
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By a conjugation bridge --> single strand travels into recipient after being nicked at origin (Ori T)
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What is a HFR?
How formed? |
High frequency recombinant
F integrates into chromosome --> F+ becomes HFR |
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What is transferred by HFR?
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Chromosome fragments of donor (HFR) to recipient (F-)
Fragment can then undergo homologous recombination with recipient chromosome |
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Can a HFR excise the F Factor out again or is this a permanent change?
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It can precisely excise the F Factor out to reproduce the F+ status.
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What is a F' male?
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It is a male cell that went from HFR to F' by excising the F Factor but by illegitimate recombination.
In other words, in going back to F+, some chromosomal genes are taken along with the F Factor. This causes a deletion from the chromosome due to the gene moving into the F Factor |
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What is the result of a mating between F' and F-?
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Both cells are F', but the recipient may become a partial diploid due to having more than one copy of a gene
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What does Pro+ entail?
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Able to produce own proline
In the examples seen for the F Factor cells, the F Factor would carry the wild (P+) gene. F+/STR-S can't grow on Gluc+Strep plate, F-/STR-R can't grow, either. But by conjugation, the F- becomes F+/STR-R, allowing growth on Gluc+Step plate |
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6 Genetic Engineering techniques
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1. Transformation by plasmids
2. Cutting of restriction sites 3. Molecular cloning - Foreign DNA into vectors --> generates hybrid DNA moleculres 4. Determination of DNA sequences 5. Synthesis of DNA strands from predetermined sequences 6. PCR - Gene amplification |
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What does DNA Methylase do?
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Methylates (adds a methyl group) to DNA nucleic acids --> prevents these from being cleaved
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In the Hemophilis Influenzae extract, containing E. coli phage T7 DNA and Hemo. Influenzae DNA, what do we see with the addition of the Hincll restriction enzyme?
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Phage DNA cut into 40 specific pieces
Hemo. Influen. DNA left alone Suggests the cleaving occurs at specific sites on DNA to produce the 40 pieces. Also suggests restriction enzymes are useful in cleaving foreign matter |
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What can act as a vector in molecular cloning?
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Plasmids, phages, virus
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General procedure for molecular cloning?
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Gene of interest is cleaved --> mixed with a vector --> fragments anneal so target gene is on vector, restriction enzyme that cut gene out is inactivated --> vector transmitted to new organism --> expression and replication of gene
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Dealing with intros in molecular cloning
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They must be spliced out of mRNAs --> reverse transcriptase to form DNA --> use this to obtain gene of interest --> proceed as usual with molecular cloning --> select for cells transformed by process (with hybrid plasmids)
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What properties should a good vector have? Five of them
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1. Small, easily isolated
2. Should have only 1 site for restriction enzyme 3. Restriction enzyme site should be somewhere other than where the essential gene is 4. They should carry a selectable gene/marker, like tetracycline resistance 5. Must be replicons |
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What is a replicon?
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A vector that is able to replicate as the host cell grows and divides
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Process of cDNA cloning
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Process mRNA (Splice out introns) --> Reverse transcriptase to form DNA --> DNA replicated to double strand --> restriction enzyme cuts cDNA
DNA sequence determined --> Double strand denatured --> Primers added to ends --> complementary strands polymerize |
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How HIV provirus replicates
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Chromosome denatured --> HIV DNA primers anneal to chromosome --> polymerization
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Genomics
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Study of all genes and sites within a chromosome of an organism by analyzing the nucleotide sequences of entire chromosome
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Functional genomics
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Study of functions of cell or organism by analysis of nucleotide sequences
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Structural genomics
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Study of 3D shapes of proteins by comparison of amino acid sequences to protein structure databases
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Bioinformatics
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Development of software and algorithms to analyze gene/protein sequences for comparison in databases
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Algorithm
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Rules for procedures for solving mathematical problems, usually involving repetitive operations
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Homology (In genomics)
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Extent of similarity between genes and proteins
- Proteins with similar amino acid sequences often perform similar functions - Genes encoding similar proteins have similar nucleotide sequences |
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Whole genome shotgun sequencing (random)
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Preparing a library of random fragments
Can use small and large inserts --> Need to have at least one copy of every region of chromosome, so the fragments are very redundant in number Short inserts = plasmids Large inserts = Phage lambda, cosmids Arrange overlapping regions by using 3' ends to align and form contigs --> put contigs together to form genome Seal contigs --> identify genes by comparing deduced amino acid sequences to all known protein sequences --> identify tRNA and rRNA genes and operons --> Identify repeat sequences |
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How close gaps between contigs (genomics)
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Synthesize primers corresponding to chromosome's inserts and use them to prime additional sequencing
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Annotation (genomics)
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Find the protein-coding regions, Open Reading Frames --> greater than or equal to 80 contiguous codons with start/stop codons and shine dalgarno
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Orthologous proteins:
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Similar proteins from different species (orthologs)
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Paralogous proteins
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Similar proteins from the same organism (paralogs)
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DNA/Gene/Microarrays and DNA chips
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DNA fragments corresponding to every gene on separate spots on a slide
Can help detect and quantitate mRNA from every gene |
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Functional genomics steps
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1. Form array: Extract chromosomal DNA --> PCR --> affix each fragment to slide
2. Extract RNA from cultures --> synth. cDNA from mRNA by reverse transcr. -Aerobic cultures cDNAs dCTP-Red fluorescent tag -Anaerobic cultures cDNAS dCTP green fluorescent tag 3. Hybridize cDNA to microarrays to quantitate cDNA --> fluorescent intensity is proportional to levels of each cDNA --> this reflects amount of mRNA of each gene --> this correlates expression levels of genes with grown conditions |
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Major results of genomics studies of prokaryotes: Core gene pool vs. flexible gene pool
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Core: Chromosome
- Deal with replication, transcription, translation, glycolysis, cell walls Flexible: Genomic islands, phages (prophages), plasmids, integrons, transposons, insertion sequences - Deal with pathogenecity, drug resistance, conjugation, toxins |
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Proteomics
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Understanding everything about all proteins in an organism
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What is the nucleic acid content of retroviruses?
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All of them have RNA
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Human Herpes virus: nucleic acid content, enveloped?
Also, list the 8 types |
DNA, double strand -- Enveloped
1. Fever blisters above waist (HSV -1) 2. Genital herpes (HSV -2) 3. Varicella zoster: Chicken pox 4. Epstein-Barr virus: infectious mono. 5. Cytomegalo virus: Blindness, birth defects, abortions 6. Roseola: rash in children 7. Works alongside 6 8. Kaposi's sarcoma |
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Vaccinia virus - nucleic acid content, enveloped, diseases
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Double stranded DNA, enveloped, smallpox
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Enterovirus - na content, enveloped, diseases
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Single stranded RNA (+), naked, polio
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Hepatovirus - na content, enveloped, diseases
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Single stranded RNA (+), naked, hepatitis A
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Influenza virus - na content, enveloped, diseases
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SS RNA ( - ), enveloped, influenza
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Lyssavirus - na content, enveloped, diseases
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SS RNA ( - ), enveloped, rabies
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Adsorption
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Specific surface protein for virus connection to host cell (receptor)
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Tissue tropism
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Adsorption by virus only to specific tissue types in host
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What does HIV typically adsorb to?
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Human lymphocytes
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What does polio virus typically adsorb to?
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Receptors in gut, nasopharynx, and anterior horn cells of spinal cord.
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Measles virus adsorbs to...?
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Receptors on most tissues - creates widespread rash
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Fusion (viruses)
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Viral envelope and host cytoplasmic membrane fuse so virion can be released into cell
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Endocytosis (viruses)
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Cell surface protrudes and engulfs to form phagosome for purposes of destroying molecule - not applicable to viruses
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Clathrin-mediated endocytosis
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Virus adsorbs to surface receptors of clathrin protein --> virus engulfed and coated with clathrin (vesicle) --> clathrin removed --> virus fuses with endosome that digests --> virion released
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Which herpes viruses are neurotropic?
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1-3
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Which herpes viruses are lymphotropic?
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4-8
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Oncogene
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Altered host gene carried by virus
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Proto-oncogene
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Normal host gene before picked up by virus
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Which viruses cause cancers?
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Oncovirus/oncogenic viruses
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Another name for tumor
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Neoplasm
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5 types of tumors
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Sarcoma = connective tissue
Lymphoma = lymphocyte Carcinoma = epithelial cells Adenocarcinoma = epithelial cells of glands Leukemia = Blood/ bone marrow |
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3 mechanisms by which viruses cause cancers
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1. Integration into host DNA and hijacking of expression control
2. Viral integration causes overamplification of chromosome's oncogene 3. Translocation of proto-oncogene to new area that causes altered expression |
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Viroids
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Circular, SS RNA molecules. Plant pathogens
No coding regions --> may interfere with host mRNA translation by integration |
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Virusoids
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Circular SS RNA molecules. Code for one or few genes.
Replicated by host cell only if host is infected by a true virus (helper virus) Ex: Hep B is helper to Hep D |
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Prions
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Proteinaceous infectious particles
Malformed nerve cell proteins Cause encephalopathies. Transmitted by eating nerve tissues Ataxia, Mad Cow Disease, dementia, CJD (Inherited) and vCJD (infectious) |
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The leading cause of diarrhea in youth in both first and third world countries
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Rotavirus
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Synthesis of DNA viruses - general steps
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Endocytosis --> chromosome uncoated --> chrom. heads to nucleus where it becomes circular--> transcription --> translation in cytoplasm --> viral proteins to nucleus --> viral DNA replication by viral DNA polym. --> assembly of nucleocapsids --> packaging of materials --> virions exit by exocytosis
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Rolling circle DNA rep. in herpes virus
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Nick DNA circle to form 3' end --> DNA polym. synthesizes from here --> forms discontinuous strands (like in lagging strand formation --> ligation --> cut and packaged
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Synthesis of RNA viruses - general steps
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Endocytosis or fusion --> genome RNA translated --> replication of genomic RNA formed at same time --> polyprotein formed and cleaved into 25 proteins (4 capsid proteins, RNA rep. protein, proteases for cleaving protein assembly) --> packaging of genomic RNA in capsids surrounded by glycoproteins --> release
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Synthesis of retroviruses - general steps
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Fusion --> reverse transcription to form DNA --> integration --> transcription to RNAs for genome and parts of virus --> budding (virus acquired envelope) and release
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