Quiz 3 information Flashcards
What are non-coding RNA’s
- ncRNA
- RNA not translated into a protein
- exact number in human genome is unknown
- known as “junk” dna, but may actually serve a purpose
- protect genomes from foreign nucleic acids
- guide DNA synthesis or genome rearrangement
What are the different classes of ncRNAs, and what are their cellular processes?
- RNA maturation: snRNAs, snoRNAs
- protein synthesis: rRNAs, tRNAs,
- gene expression regulation: miRNAs lncRNAs
most ncRNAs operate as….
- RNA protein complexes
ex: ribosomes, snRNPS, snoRNPs, telomerase, miRNAs and lncRNAs
many ncRNAs exploit…
- the power of base pairing to selectively bind and act on other nucleic acids
What are the two broad classes of regulatory RNAs
- small RNAs
miRNAs
siRNAs
piRNAs - long noncoding RNA (lncRNA)
-
-
What are dsRNA produced from
- endogenous genes (miRNA)
- other DNA sequences (siRNA)
- exogenous sources
describe the mechanism of regulation by small RNAs
- enzyme called a Dicer cuts the dsRNA into fragments
- fragments bound by RISC, denatures
- produces a single strand, guide strand, (biologically active), and passenger strand (degrades)
- guide strand directs one of three gene silencing processes
What are the three gene silencing processes of the guide strand
- complex uses complementary base pairing to attach the guide strand to mRNA, destroys
- RISC-guide RNA binds to complementary mRNAs, prevents translation
- complex directs chromatin-modifying enzymes to the nucleus, silence transcription of target genes
What are miRNAs
- cleaved from precursors of dsRNA
- transcribed from loci that are independent of target
- transcribed by RNA poly II, produce transcripts that fold back to produce dsRNA
Describe creation of miRNA
- primary microRNA forms a double stranded stem with free ends on one side and single-stranded loop on the other
- in animals, drosha enzyme cuts pri-miRNA near middle of stem to produce two segments
- one segment: precursor miRNA… transported to cytoplasm, Dicer removes terminal loop to generate fragments
- RISC binds to fragments, processing to produce miRNA
- miRNA (RISC guide strand) binds to target mRNA in the 3’ UTR
- will either silence gene or if sufficient base pairing, RNase of Argonaute cuts mRNA and miRNA
What is RISC
- multi-protein complex, contains a protein of argonaute family
Descibe small interfering RNAs
- siRNAs
- complementary to RNAs
- regulate gene expression through RNAi
- siRNAs can be introduced artificially for research
What is one of the roles of siRNAs
- chromatin modification
- RNA-induced transcriptionally silencing through chromatin modification
( dont need to know the specific steps)
Describe Piwi interacting RNAs
- piRNAs
- small ncRNA… bind to members of piwi protein to play regulatory roles
- expressed in tissue specific manner
- abnormally expressed in cancer
Describe the functions of piwiRNAs
- transcriptional/post-transcriptional control
- control and silence transposable elements to protect the genome
- regulation of genetic elements in germ line cells
Describe RNAi
- RNA interference
- RNAi silences gene expression transcriptionally or post transcriptionally
- carried out by miRNA or siRNA
- evolved by helping organisms protect genomes against mutational effects of transposable genetic elements
- is a research tool
What are other triggers of RNAi
- parasitic nucleotide sequences
double stranded RNA (dsRNA) of viral organ
aberrant transcripts from repeptitive sequences in the genome such as transposons - foreign DNA
- shRNA (short hairpin RNA)
Describe RNAi as a research tool
- used to silence genes in order to study function
- short hairpin RNA (shRNA)
artificial, act as an miRNA to downregulate genes - effective in medicine, downregulate genes where OE causes disease (cancer)
describe long-noncoding RNA
-lncRNA
- common in mammals
- little or no protein coding potential
- poorly conserved through evolution
- may be classified into five types based on their relationship with protein-coding genes
what are the different classificiations of lncRNA
- sense
- antisense
- intronic
- intergenic
- divergent
What are the functions of lncRNAa
- cell differentiation
- chromatin organizing
- regulation of gene expression (upregulate protein exp by downregulating miRNA)
- miRNA sponge (sucks up miRNA… cant do its job)
What are circular RNAs?
- circRNA
- ncRNA … presence of covalent bond linking 3’ and 5; ends
- formed by exon skipping to produce lariat structure
- more stable than mRNA
What are the functions of circRNAs
- acting as a sponge of miRNA
- regulating transcription
- regulating RNA splicing
- interacting with RNA binding proteins
What are the three tissue culture techniques
- transformation
- transfection
- transduction
WHat is transformation
- process of converting a normal cell into a cancer type cell
What is transfection
- introducing DNA into mammalian cells
what is transduction
- gene is introduced into a cell by a viral vector
Describe the steps in animal cell culture/tissue culture
- tissues minced for culturing
- disaggregation by use of enzyme
- cells inoculated in fresh culture medium
- confluent culture cell separated using enzymatic disaggregation… single layer of cells
ENDS WITH EITHER: - cryopreservation = cryopreservation of cells for further use
- subculturing or passaging
normal mammalian cells show:
- anchorage dependence
- density-dependent inhibition
cell viability assay
- MTT assay : biochemical assay measures metabolic activity of cells
- measures single points in time, not measuring over a period of time
- ability for cells to proliferate
how to identify oncogenes by cell transformation assays
- DNA of interest is isolated and introduced into a standard cell line called NIH/T3T (mouse fibroblast cells) by….
- 1) chemically transformed mouse fibroblasts
- 2) extract DNA
- 3) transfect using calcium phosphate co-precipitation procedure
- 4) add transfected cells to normal mouse fibroblasts
- 5) formation of a focus of morphologically transformed cells
- 6) inject transformed cells into mouse host… tumor grows
What are NIH/3T3 cells
- spontaneously immortalized (do not turn off) cells
- show anchorage dependence and density-dependent inhibition
What is the “focus” (foci)?
- cluster of transformed cells growing amid a surrounding monolayer of normal cells in culture
ways to measure RNA levels?
- microarray and RNAseq (transcriptomic approach)
- northern blot and qPCR (gene specific)
- RT-qPCR
What is RT-qPCR
Reverse transcriptase PCR
- reverse transcription to generate cDNA followed by qPCR using real time PCR machine to determine RNA level (use RNA as a template to make DNA)
- method used to quantify level of specific RNA in a cell
PCR review
- polymerase chain reaction
- rapidly make millions of copies of a specific DNA sample
- 1) DNA denaturing by heat
- 2) annealing of DNA primers synthesized to flank target seq
- 3) extension by DNA polymerase
what is qPCR
- quantitative PCR…. carried out with real time PCR machine
- monitors amplification of target DNA molecule during PCR in real time… quantitatively how much DNA is present in starting material
- allows you to see how much DNA is made in each cycle…
What is the difference between RT-PCR and RT-qPCR
- RT-PCR = reverse transcription to generate cDNA followed by PCR
- RT-qPCR = reverse transcription to generate cDNA followed by quantitative PCR using real time machine to determine RNA level
types of evidence
- correlative: compare 2 events/conditions and show a correlation
- loss of function: inhibit function of a gene
- gain of function: add a gene
how are gain-of-function tests carried out?
- transfect a cell with a plasmid that will over express the gene of interest (up-regulation)
- introduce molecule of interest itself (typically RNA) into the cell so that its levels are substantially higher
How are loss-of-function tests carried out
- down regulation
- delete gene from chromosome (KO)
- reduce expression using RNAi
- reduce activity using inhibitors of enzyme activity
- use of antibodies to block protein function
describe forward genetics
- create mutants and screen for diverging phenotypes
- look for the gene involved in this defect
- analyze the function of the gene
(mechanism/phenotype –> gene indentification)
describe reverse genetics
- isolate a specific gene and generate a mutant allele to find out what phenotype will be mutated
(gene ID –> pehnotype/mechanism identification)
How is mutagenesis carried out?
first: pick an organism and a mutagen
types of mutations:
- insertional/disruption mutant
- conditional mutants
What are insertional/disruption mutants
- use a technique to insert random DNA into the coding sequence to disrupt the sequence
- in other words, insertions that generate “loss of function”
What are conditional mutants
- creating mutant in essential gene, useful for studying genes encoding essential function
- treat cells with mutagen and grow at low temperature, they will not be strong but will still be functional
- ## shift them to a higher temperature and they will stop growing, the protein will fall apart. can then study the impacts of this mutagen.
describe the difference of mutagenesis with haploid/diploid
- diploids require added steps for a full null knockout of a gene
steps of forward genetics:
- Mutagenesis: saturation mutagenesis (mutations randomly generated)
- screen for desired phenotype
- identify mutant gene
- analyze function of gene
describe step 3 of forward genetics
- identify mutant gene (actual DNA seq)
- methods depend on the organism and mutagenesis procedure… there are multiple approaches
- clonsed/localized DNA is sequenced
- libraries are often used in the process
What is a genomic library?
- clones of different fragments from a chromosome (clones = books in a library)
What are the types of genomic libraries?
- genomic library
- cDNA library (expression library)
Describe how a genomic library works
- fragments of genomic DNA are created through RE digestion
- fragments are ligated to plasmids
- many plasmids are created with different fragments (library)
Describe cDNA libraries
- isolate RNA
- poly T primers anneal to poly A tail of mRNA
- extend with Reverse transferase to make single strand DNA from RNA
- use PCR
- ligate
- cDNA libraries = copies of RNAs made inside cell
draw the “summary and comparison” diagram
Describe the difference between genomic and cDNA libraries
- genomic: all sequences (genic and intergenic) are represented approximately equally
- cDNA: frequencies reflect levels of gene expression and include only sequences found in mature mRNA (no intron/intergenic seq)
- study the diagram to understand
how can library screening be done
- can be done by complementation or nucleic acid hybridization
Describe complementation library screening
- transform cells with the cDNA library, which is designed so that cDNA sequences are transcribed/translated in the host
- each cell will receive a different cDNA clone from the library
- grow at low temp, then at a high temp
- only colonies containing cDNA clone that can complement cdc2 mutant will grow at high temp
after screening, you are able to identify the clones carrying the gene of interest, so you have the DNA seq. What is the next step?
- gene identification
- when mutant gene (DNA fragment) is isolated, DNA sequence can be analyzed by in silico methods
- gene identity can be inferred by alignment with sequences in a database
- unnknown seq will align with a seq of a similar function
How can you gain more useful results after using alignment of gene sequences?
- alignment with the deduced amino acid sequence
- can compare protein sequences and discover domains
why proteins?
Because a protein’s structure provides the key to understanding the mechanism of its function
describe step 4 of forward genetics?
- analyze the function of the gene:
- characterize mutant phenotype (genes genetic, biochemical/physiological role)
- identify the role of the genes product
- identify interacting and redundant genes (modifier screens to find other genes that play a role in the process of interest/ identify interacting partners)
What are modifier screens
- definition: conducted in order to find genes that play a role in a process of interest through random/selected mutations
-screen for double mutants that can modify phenotype of first mutant - mutagenize a wild type to create many mutations, breed with a mutant carrying the gene of interest
- F1 will produce three mutants, enhancer , suppresser and mutant
- the enhancer and suppressor are important because they the second mutation they carry may have a possible relation to the gene of interest
What is genetic redundancy
- between pathway interactions…
two pathways perform the same essential function, mutation of either may not have consequences. mutation of both may = loss of essential functions
what is enhancement in the modifier screen
- within pathway interactioms
-partial loss-of-function mutations in certain genes can reduce function, but the organism still viable… both components mutated, pathway may become nonfunctional
ex:
C1 WT, C2 WT = essential function
c1 mut, C2 WT = reduced pathway activity… viable
C1 WT, c2 mut = reduced pathway activity… viable
c1 mut, c2 mut = insufficient pathway activity… lethal
what is suppression in modifier screen
- WT = response (if A is active)
- A- (no A) = no response
- A-, B- = response
B- is referred to as the suppressor mutation (2nd mutation that suppresses the effect of the 1st mutation)
Describe the two approaches of reverse genetics
- generate mutant allele and characterize mutant phenotypes
- produce protein (via expression plasmid) and study its function
Describe the ways to generate mutant alleles
- homologous recombination
knockouts/disruptions - CRISPR-cas
- RNAi
knockdowns
describe homologous recombination
clone gene.. choose DNA with marker and insert into the coding sequence..
generate the knockout plasmid construct (disrupt gene on plasmid)
put plasmid inside cell, and it becomes part of the chromosome
describe the working model of homologous recombination with linear DNA molecules
plasmid is linearized, and a double crossover occurs at site 1 and 2 of the plasmid x the target gene on a chromosome
the double crossover results in a replacement of the target gene
describe CRISPR-cas9
describe RNAi in genome editing
- inject RNA into cell to downregulate the target
- transfect/inject dsRNA into cell…
- dicer cuts the dsRNA into fragments of siRNA
- slicer activity of argonaut cleaves target mRNAs that are complementary to siRNA
OR…
- introduce dsRNA virally
transgene
- gene that has been modified in vitro by recombinant DNA technology… introduced into genome thru transformation/transfection
transgenic organism
- organism harboring a transgene
