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to use antibodies to isolate (and detect) a protein from lysed cells or mixture of proteins
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What is the function of immunoprecipitation?
- mixture of radiolabeled proteins is incubated with antibody against protein of interest
- the antibody binds to the specific protein
- collect antigen-antibody complexes by incubation with beads that bind to the antibody
- proteins are dissociated by boiling
- gel electrophoreses, protein will migrate down gel
- expose to film to detect the radio-labeled protein
Describe the process of immunoprecipitation.
detection of protein-protein interaction
What is the purpose of co-immunoprecipitation (co-IP)?
- cell nucleus contains two proteins of interest
- nuclear extraction
- add antibody directed against one of the proteins of interest
- add antibody binding beads
- immunoprecipiate the protiens of interest
- wash and collect immunopreciptated proteins
- Western blot (with Antibody #2) analysis of immunoprecipitated protein of intest
Describe the process of co-IP.
overexpress a protein that may change the cellular function
studies of gene expression, protein localization, and short term activity
What can the introduction of DNA into animal cells allow for?
liposomes (lipid vesicles)
What does transfection use?
viruses harboring clones genes
What does infection use?
- introduce DNA plasmid into cultured animal cells
- high fraction of cells take up and transiently express plasmid DNA
- selection in drug containing medium
- only stably transformed cells form colonies
- stably transformed cells contain plasmid sequences integrated into chromosomal DNA (will be transcribed and translated into protein)
Describe the process of introducing DNA into animal cells?
1) How do mutations in specific sequences affect gene function
2) Does mutant protein product cause a phenotype
What questions does mutagenesis help answer?
- after cloning, scientists often want to mutate gene of interest using mutagenesis
- denature and hybridize with mutagenic oligonucleotide
- DNA synthesis and ligase
- plasmid containing mismatched base at site of mutation
- isolate plasmids after replication
- introduce DNA into culture animal cells
Describe the process of mutagenesis with synthetic oligonucleotides
Yes, not just in cloned genes!
Can we study the function of cloned genes in animals?
What percent of injected embryos integrate the plasmid into their chromosomes
random sites throughout the genome - not at site of normal gene
Where does integration of genes occur?
- microinject plasmid DNA into pronucleus of fertilized egg
- let culture in a dish, circular plasmid gents integrated by repair enzymes
- transfer embryos to foster mothers, multiple generations
Describe the production of transgenic mice
the cloned gene could be normal or mutant (normal version is still there) which can cause problems for analysis
What is a problem with the normal production of transgenic mice
the gene is replaced with mutant (no normal gene is produced)
What is different about homologous recombination in comparison to the normal transgenic production?
- embryonic stem cells carry normal copies of gene on both chromosomes
- undergoes homologous recombination with inactive mutant copy of gene
- ES cell carrying one mutated and one normal gene copy
- ES cell is injected into blastocyst and transfer to foster mother
- offspring carrying one mutated and one normal copy gene
- continue mating until some offspring carry two copies of mutated gene
Describe the production of mutant mice by homologous recombination in ES cells.
less than 1%
How effective is homologous recombination in ES cells?
a naturally occurring defense mechanism discovered in bacteria to cut a viral genome - where the bacteria express RNA complementary to foreign (guide RNAs) and the enzyme Cas9 cuts the DNA
can be used as a much more efficient and effective method in mutating a genome (20-30%)
also has therapeutic applications
What is CRISPR/Cas? What can it be applied to do?
- Cell carrying normal copy of gene is exposed to Cas9 with 2 nuclease cleavage sights
- guide RNA binds to RNA complementary to direct Cas9 where to cut
- target DNA is cleaved - Double stranded break (DSB occurs)
- non-homologous end ligated, bases that were cleaved are lost
Describe how CRISPR can be used to cut out a segment of DNA.
Yes, mutated specific mutation introduced into the target gene and homologous recombination occurs at the cleavage site
Can CRISPR be used to insert a mutated copy of a gene?
analyzing gene function by blocking gene expression in cultured cells
What is gene expression inhibition by antisense RNA or DNA used for?
When was this method developed?
single stranded sequence that is complementary to mRNA or DNA
What is antisense DNA or RNA?
antisense RNA or DNA hybridizes to normal mRNA; blocking protein synthesis
double stranded RNA prevents protein translation
How does gene expression blocking by antisense RNA or DNA occur?
RNA intereference (RNAi)
What is the most generally-used powerful method for blocking gene function?
What does endogenous RNAi involve?
Yes, all cells synthesize antisense RNA and the cell can produce small RNAs that fine tune its own expression levels (although scientists didn't know this occurred naturally at first)
Does RNAi occur naturally?
- Double stranded RNA (70-90 bp) is cleaved by Dicer
- siRNA (21-23 bp) is associated with RISC (unwinds siRNA)
- pairing with target mRNA occurs, RISC/siRNA is recycled
- mRNA is cleaved
Describe the process of RNAi
QUANTITATIVE understanding of the integrated dynamic behavior of complex biology systems and processes
What is the goal of systems biology?
bioinformatics and systems biology
What answers the question: What do we do with the information found in complete genome sequencing?
What do large-scale experimental approaches (genome wide- studies) form the basis of?
What uses single gene and protein experiments to understand individual molecules and pathways?
What uses genome and proteome wide experiments to understand integrate cell processes?
What is a genome-wide functional analysis?
screening for any cellular property testable in high throughput format (ex: changes in gene expression, cell signaling cell morphology, etc.)
What does genome-wide RNAi screening allow for?
- each well in an assay contains siRNA against an individual gene
- inoculate with cells
- incubate and allow for cell growth
- well in which siRNA blocked cell growth or viability will appear differently/not grow
Describe the genome-wide RNAi screening for cell growth and viability
which genes are involved in survival and growth
What questions does a genome-wide RNA screen answer?
How have genome sizes changed over evolutionary time?
1) Structure of genes (intergenic DNA)
2) Introns - intragenic DNA
3) Repetitive sequences
4) Gene duplications and pseudogenes
What are the components of DNA in higher eukaryotes?
sequences between genes that play a very important role in gene regulation
What is intergenic DNA?
What are many intergenic DNA strands transcribed into?
RNA sequences that code for proteins
What are exons?
RNA sequences that get transcribed but get spliced out before translation
What are introns?
introns - within a gene - DNA sequences that do not code for an amino acid
What is intragenic DNA?
What were used in the identification of introns?
- adenovirus mRNA was hybridized to DNA
- using microscopy and biochemical techniques, they found that there were unmatched areas/ some DNA strands were missing in the RNA
How were introns discovered?
untranslated regions; usually found in first and last exons
What are UTRs? Where are they usually found?
untranslated regions, Start Codon, translated regions
What does the first exon have?
UTRs, translated, stop codon
What does the last exon have?
ENCYclopedia Of DNA Elements, database showing the number of bases
What is the ENCODE database?
coding sequence < total exon sequence < intron sequence
Are there more base pairs in the total exon sequence or intron sequence or codon sequence?
How many exons are in the average human genome?
What percentage of the bacterial genome is protein-coding sequence? What percent is intron sequence?
What percentage of the human genome is protein coding sequences? What percent is intron sequence?
regulatory roles - transcription and alternative splicing
What important role do introns play?
non-coding RNA; they are spliced
What do some introns encode? What happens to most of them?
How did introns become a part of eukaryotic genes?
when one gene produces more than one mature RNA with different combinations of exons
What are alternative transcripts?
How many mRNAs per gene can there be for human pre-mRNAs
sequential order (Exon 1, Exon 2)
While the exons in each alternative transcript varies, what order are they always in?
What are enzymes in alternative splicing code based on?
How does the cell make slight changes/fine tune in protein functions
What type of repetitive sequences make up approximately 10% of the genome?
What make up hundreds to thousands of repeats that emerged as a part of genome evolution and relate to chromosomes' higher order organization (eg: ACAAACT)
What fraction of the genome do transposons make up?
What are single units repeats that have a gene structure (5' end, 3' end and coding sequence) and are scattered throughout the genes?
genome structure; viral integration
What do transposons help with? How did they emerge?
1) transcription into mature mRNA
2) reverse transcriptase produces retrotransposon DNA
3) Integration into chromosomal DNA - two copies
millions of years
How did retrotransposons end up in the chromosome? How long did this take?
- enzyme to copy itself - will not code for the virus again because the sequence was mutated and integrated
What do retrotransposons produce?
evolutionary role in gene rearrangements and regulation of gene expression (not necessarily on purpose)
What are the roles of transposable elements?
- transfer of DNA sequence by unequal cross-over or DNA replication error
- reverse transcription: processed pseudogenes
What are some methods for duplication of segments of DNA?
How many pseudogens are in the human genome?
non-functional gene copies that have acquired many mutations (do not produce protein)
What are pseudogens?
unequal crossing over leads to multiple copies of a gene (embryonic, psuedogenes, fetal and adult)
How do gene families emerge?
gene replication of processed pseudogenes own genome, using its own reverse transcriptase
What is gene duplication by reverse transcription?
- reverse transcription and integration into new chromosomal site on host genome
- adds mechanisms to prevent transcription (doesn't have transcription coding sequences and some are in regulatory regions)
- results in processed pseudogene (double stranded DNA identical to mRNA)
How are processed pseudogenes formed?
Are most pseudogenes processed or unprocessed?
- growth factor genes is transcribed and spliced
- mRNA is reverse transcribed and integrated into genome
- double stranded copy is inserted into retrotransposon resulting in abnormal growth factor gene expression
How are retrogenes that determine short legs in dog breeds transposed?
processed pseudogene integrated into retrotransposon, leading to abnormal expression
What is a retrogene?
When was the launch of the human genome initiative?
2001 - dideoxy sequencing
When was the first human genome sequencing drafted? What was used?
When were thousands of individual genomes sequenced (ENCODE)
No, approximately all vertebrates have the same number of genes
Do some vertebrates have more genes than others?
What percent of genes are common to human, mouse, and rat