Test 4 - Final Flashcards
A recipient cell containing donor DNA
Transformant
The direct uptake of exogenous genetic material
Transformation
Describe the two stages of transformation. (very unclear from notes)
- Competence - likely due to changes in the cell wall that increase the likelihood of DNA uptake by the cell. Influenced by type of media, stage of growth, degree of aeration and the secretion of competence factors (aka pheromones).
- DNA uptake - DNA enters the cell. Different for Gram + vs -. (see handout). Results of uptake: 1) Plasmid that circularizes. 2) ssDNA undergoes homologous recombination. 3) The DNA is degraded in the cell.
An culture that will readily uptake DNA.
competent cells
A competence system component that binds to the DNA and pulls it into the cell.
pseudopilus
A sequence on own DNA that codes for receptors for the uptake of exogenous DNA.
uptake sequences
Proteins that activate the rest of the cells to become competent
Competence factors (pheromones)
Two stages of DNA uptake. (Don’t know what each is though???)
- Brief, reversible binding stage
2. Longer, irreversible stage
Describe Gram negative transformation via a transformisome.
A transformasome forms as a buldge of cytoplasmic membrane through the cell wall and dsDNA is taken in. As the dsDNA enters the main cell area, nuclease cuts it into ssDNA. SSB or RecA will coat the ssDNA. At this point it can either circularize (using homologous recombination), or integrate into cell’s genome (using homologous recombination), or be degraded.
Gram positive vs Gram negative transformation
Gram negative - 2 methods, either via pseudopilus or via transformisome.
Gram positive - 1 method, competence factor binds DNA, pore forms in peptidoglycan and a pseudopilus pulls the DNA through the cell wall.
A set of genes that aid in the formation of a pseudopilus
Com genes
An enzyme that cuts dsDNA into ssDNA as it enters the cell during transformation.
nuclease
Describe Induced vs Natural transformation
Induced - Manipulation in the lab. Includes calcium ion induction, electroporation, protoplasting, and heat/cold shock.
Natural - Transformation found in nature. Increases genetic variability. The availability of “self” DNA aids in repair of own damaged DNA and reduces problems associated with foreign DNA.
Nucleotide sequences are exchanged between two similar molecules of DNA.
Homologous recombination
Describe why homologous recombination is beneficial.
Maintain cell viability by repairing damaged DNA.
Provides a basis for genetic diversity.
A group of proteins that act at the Holliday Junction of a crossing over event.
Ruv proteins
An event wherein two similar strands of DNA begin an exchange of sequences.
Crossing over
The outcome or the name of a completed crossing over event.
recombination event
Describe the 3 steps of reaction of RecA in the homologous recombination model.
- Presynaptic binding - coats ssDNA with RecA. Requires ATP. SSB removes secondary structure.
- Synapsis (conjunction) - RecA/ssDNA forms a complex with an elongated dsDNA along its major grove. The sequences move along until homologous sequences are lined up, (homologous allignment). 2 joints can be formed, paranemic or plectonemic.
- Postsynaptic strand exchange (heteroduplex extension) - RecA promotes displacement of dsDNA and replacement with the new strand. Unclear mechanism.
Describe what the RecBCD complex does.
Activates 3’ to 5’ exonuclease to eat away at one strand in preparation for crossing over event. Exonuclease stops at the chi sequence.
Activates 5’ to 3’ exonuclease and helicase to prepare the DS break end for homologous recombination. Exonuclease increases at the chi sequence to create overhang where RecA will attach in preparation for the actual Homologous Recombination event (Depicted in handout Figure 9.6)
proteins of a multifunctional complex that play a key role in homologous recombination model.
Rec BCD protein complex
Describe some ways in which Homologous Recombination can be used as a repair mechanism to stalled or collapsed forks.
Collapsed fork - create a new replication fork by strand invasion homologous recombination. Forms a Holliday Junction that when resolved reforms the replication fork via resolving enzymes.
Stalled fork - formation of reverse replication to create a new strand so that new repaired piece of the strand can be spliced and to restart the fork via branch migration and template switching. (doesn’t make much sense)
Dinner fork - not used in DNA repair.
Holliday model vs Homologous Recombination model.
(do they both occur? it looks like one is a single strand invasion and the other is a double strand crossing over…maybe.)
Holliday - a junction is formed wherein the dsDNA molecules are held together by crossed over strands and Ruv proteins.
Homologous Recombination - Break two DNA molecules in the same region where they are similar and join one to the other in a crossing over event.
A region of complementary bases within a DNA molecule wherein each strand is contributed by one of two parent DNA molecules.
Heteroduplex
Describe how we know that certain enzymes play a role in Homologous Recombination and repair. (question he said in class was a good test question so I made a card.)
We can detect which enzymes get turned on then mutate the genes for those enzymes. If we do a deletion mutation which inactivates the enzyme, and no repair occurs, we will know that that specific enzyme must have been involved.
What 3 things were key to the discovery and emergence of the first recombinant DNA technology experiments? Describe why each is important.
- Plasmids - Found while trying to find reasons for antibiotic resistance.
- Ligase - Found while trying to figure out how DNA is replicated. Puts open SS ends back together after use of EcoR1.
- Restriction Endonucleases - Found while trying to figure out purpose of DNA methylation. EcoR1 creates open SS ends that can be put back together with ligase.
Side note - go to chapter 10 in the book. It is much more clear than his notes and has some color to the figures he used in his handouts. The book was more clear on homologous recombination.
Did you get your book out?…did you?… … … you didn’t.
What properties does a vector need in order to transfer genes?
Able to enter host
Able to replicate in host (ori region)
Selectable marker genes for identification (resistance)
A gene transfer vector that can accept large DNA fragments
λ phage
A gene transfer vector with unique promoters that aid in DNA sequencing.
M13 phage
A gene transfer vector that contains antibiotic resistance genes, an ori site, polylinkers, etc…
(give an example)
Plasmids. pBR322
Describe the method for the use of insertional inactivation for the identification of clones.
- Cut and anneal a plasmid with foreign DNA. Results in re-annealed original plasmid, or desired plasmid containing foreign DNA inserted into and inactivating the tetracycline resistant gene.
- Cause a transformation event.
- Grow cells on Ampicillin, all cells will grow.
- Replica plate cells on Tetracycline, cells containing foreign DNA will not grow due to insertional inactivation.
- Isolate colonies that grew on ampicillin but not on tetracycline. These contain the cloned, foreign DNA.
Describe the α-complementation system (aka Xgal or lacZ system).
- Ligate and anneal a lacZ-containing plasmid with foreign DNA. Either get re-annealed original plasmid, or plasmid with foreign DNA causing insertional inactivation of lacZ region.
- Plate on agar containing Ampicillin so only transformants will grow.
- Agar also contains IPTG and X-gal.
- Cells with functional lacZ - lacZ induced by IPTG to cleave Xgal, which makes the colony blue.
- Cells with inactivated lacZ will not be induced and white colonies will grow. These contain the desired, cloned, foreign DNA.
A sulfur analog of lactose
IPTG
A portion of genetic sequence inserted into a plasmid which contains unique restriction enzyme cutting sites for use in inserting foreign DNA.
Multiple polylinker molecule
Describe the power of metagenomics and the use of PCR in microorganism identification.
Plating a sample of mud from the Great Salt Lake and run the Xgal lab protocol on it. It will return plasmids with foreign 16s sequences inserted into it. If you take the same mud sample and harvest all of the DNA from it, run a PCR, and send it off to be sequenced, you can potentially receive hundreds of 16s sequences that did not grow on the plate. This is precisely how we know that there are so many microbes out there that cannot be grown in the lab but that do exist.
A type of vector, engineered in E. coli, used for transferring genes into more complex eukaryotic cells such as yeast.
Shuttle Vector
Uses a reporter gene connected to an inducible promoter site to examine the promoter’s regulation of that gene.
Expression Vector
we used Xgal as the inducible promoter and IPTG to promote it. Blue colonies showed expression of the gene
Different organisms and enzymes that cut the same site but may generate a different cut.
Isoschizomers
Cuts at palindromic sequences
Type II Restriction Endonuclease
Method to determine where a RE cuts DNA relative to other RE.
Restriction Mapping
joins blunt ended DNA fragments
T4 phage ligase
Method of joining blunt end DNA into a plasmid form by adding multiple poly A and poly T tails to the separate strands then joining the tails together.
Homopolymer tailing
The use of a T4 ligase to join two blunt ends of DNA together.
Blunt End Ligation
Used to ensure palindromic sequences are inserted during blunt end ligation. (So we can cut the plasmid again once it is made)
Linker or Polylinker
DNA created by reverse transcriptase activity
cDNA
Describe the process of cDNA creation.
- Primer annealed to mRNA
- DNA copy made with reverse transcriptase
- Alkali treatment removes mRNA
- DNA polymerase extends hook back into complementary strand
- Treat with S1 nuclease to remove hook
A list of cDNA within a cell composed of coding regions that are searchable. Used in looking for genes or proteins of interest.
cDNA library
Describe ways to detect recombinant molecules
Insertional inactivation - Inactivate one of 2 resistance genes and grow on two different antibiotic media to see which has the genes inserted.
Cosmids - plasmids that cary the COS site from λ phage. (read handout). The power of this is that the camel DNA used contains both COS sites, one on either side of the desired sequence. It will be cut at both COS sites and the sequence EXACTLY fits into the phage, and NOTHING else.
Another method dealing with cohesive ends that doesn’t make sense to me.
Pulled glass pipette method used to directly insert DNA into cells.
Microinjection
Artificial membranes which fuse to host cell membrane, used in transfer of DNA into host cell.
Liposomes
Method used to shoot DNA into a cell with gas and microprojectiles made of gold covered in charged silicon and DNA.
Gene gun
Used to detect protein production by the use of radioactive antibodies.
Radioactive antibody test
Discovered PCR
Kary Mullis (mullet?)
What is required for a PCR reaction?
Primers
Thermocycler
dNTPs
Taq Polymerase - harvested from hot pots extremophiles
Name 5 uses of PCR.
Make copies of genes for 16s rRNA sequencing Identify rare genes Site directed mutagenesis Environmental surveying DNA sequencing technology
Alteration of an embryo so that when it fully matures, the animal creates a desired protein product. (ex. goat making insulin)
Pharming
The replacement of bad genes in a eukaryotic host, usually human.
Gene therapy
A short DNA tag, added to an ORF, recognized by an antibody probe once it has been transcribed.
Epitope tag
Describe the Sanger Method of DNA sequencing.
Use of PCR to create numerous segments of entire genome. Altered nucleotides (ddNTP) are mixed in with normal nucleotides which terminate the sequences at varying locations when incorporated. Done in 4 separate tubes with one ddNTP per tube. Various PCR sequences are compared and a reading is made by reading the terminal base of each of the PCR products. Doing this with all 4 tubes, and comparing chain lengths and their terminal ends, tells you the entire sequence.
Describe pyrosequencing
Short sequences done in parallel at the same time. Uses pyrophosphate and is read as PCR is occuring. Very fast but like putting a puzzle together at the end. (Find more info about this to clarify)
What is it called where they run the entire genome through a capillary tube with florescent dyes attached to each different type of base. It is ran past a laser and read by the computer.
Is it capillary electrophoresis?
A group of overlapping clones representing a region of DNA, used in assembling of sequenced data.
Contigs
How do you tell where a gene is on a sequence?
Look for an ORF that has a start codon, or a ribosome binding site, and a stop codon. In Eukaryotes, search for exon/intron boundaries.
Describe the two methods for entire genome sequencing.
- Clone by clone approach - create thousands of clones and sequence portions of the genome from each of the clones. Very slow but reliable.
- Whole genome shotgun - generate random pieces of DNA and trying to piece them back together. Very fast but difficult to assemble.
The use of computational tools to acquire, analyze, store, and access DNA and protein sequences
Bioinformatics
The total complement of genetic information of a cell or virus
Genome
total set of proteins encoded by a genome or the total protein complement of an organism.
Proteome
Every protein made under specific conditions
Translatome
The complement of all RNA produced by an organism under a specific set of conditions
Transcriptome
Oligonucleotide for each gene put on a solid matrix, hybridization between mRNA and the specific nucleotide.
Microarrays (gene chips)
Uses of microarrays (gene chips)
Global gene expression
Comparison of genes in closely related organisms
Assess microbial diversity
Assess metabolic methods
Total complement of small molecules and metabolic intermediates produced by a cell or organism, determined by mass spec.
Metabolome
Total set of interactions between proteins or other macromolecules in an organism, network diagrams of proteins with interacting proteins connected.
Interactome
total complement of organisms present in a particular environment, can reveal uncultured or unculturable organisms.
Metagenome
Area of study that maps, sequences, analyzes, and compares genomes.
Genomics
Area of genome-wide study of the structure, function, and regulation of the proteins of an organism
Proteomics
Genomic analysis of pooled DNA or RNA from an environmental sample containing organisms that have not been isolated.
Metagenomics
The determination of gene expression by an organism under a specific set of conditions.
Transcriptomics
Open reading frame
ORF
putting the DNA sequences in the correct order
Genome assembly
process of identifying genes and functional regions on the genome
Annotation
comparison of sequence data to all known sequences
BLAST search
collection of prokaryotic cells associated with another organism, often in reference to humans.
Microbiome
One electrophoresis direction separates proteins by pH, another direction of electrophoresis separates proteins by size.
2D gel electrophoresis
A homologous recombination joint wherein DNA is not entwined
Paranemic (unstable)
A homologous recombination joint that is double helical with free a free end joined
Plectonemic (stable)
