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407 Cards in this Set
- Front
- Back
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What is the basic unit of DNA (the last fold of DNA) and how may base pairs does it contain?
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nucleosome… 160-180 bp
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T/F: modification of histone has no effect on gene expression
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FALSE
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Characterize the transcription of DNA
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1. DNA becomes accessible… 2. RNA polymerase associates with DNA… 3. (some proteins in polymerase complex are always seen in transcription some are specific for a particular expression)… 4. enhancer regions can be distal to the site of initiation… 5. enhancer can be more than 100,000 bp away and can control and can control several gene (creates a loop of the DNA)
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What is "transcription" by committee?
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the array of proteins binding to a gene that determines the amount of transcription that gene will undergo, therefore the availability of these proteins regulate gene expression
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T/F: transcription regulation protein work in either a negative or a positive manner
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TRUE
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what can make the DNA more suseptible to translation? (Hint: histones)
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the histones in a nucleosome can be modified by the addition of an actyl, methyl, phosphate or other group
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In the transcription of DNA to mRNA what must be added to the 5' end?
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a 7-MeG "Cap" is added, which indicates it's ready to be processed in translation
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In the transcription of DNA to mRNA what must be added to the 3' end? And what is its purpose?
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a) A poly-A tail is added to the 3' end… b) protect the mRNA from exonases, transcription termination, export from the nucleus, and for translation
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Which pre-mRNA is exported for translation?
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Exons
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Is mRNA always ready for translation upon leaving the nucleus?
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no, sometimes it must be transported to a particular location
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When is translation regulated?
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during splicing and polyadenylation
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In terms of storage why is splicing important
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because one gene can express multiple proteins
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Give an example of mRNA being modified.
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Apolipoprotein B in tissue specific cells allows for mRNA to undergo addition modification… THIS IS RARE
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What is Thalassemia an example of?
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Mutation in genes that codes for aberrant process of RNA
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T/F: mRNA lifetime is random and essentially the same for all mRNA
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false, it is usually determined by the 3' end… in fact aberrant (mutant) can be marked for destruction
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What is Nonsense Mediated Decay?
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When mutant/aberrant mRNA is identified and destroyed
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What is RNAi?
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single stranded RNA about 20 nucleotides long, cut from a longer DS RNA that can regulate translation
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Where does translation of mRNA proceed and what binds to this region?
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5' cap, where intiation factors bind
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What codon is the start codon?
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AUG
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What is IRES?
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It is a rare site of translation initiation, which is sometimes used by viruses to hijack the translation mechanisms of a cell
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What is the function of GTPases such as eIF2, IF2 and EF2?
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They ensure that translation progresses forward.
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Give the 4 steps in utilization of GTPases in translation, which is uni-directional
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1) The protein (eIF2, EF1 or EF2) is complexed with GTP… 2) GAP induces the protein to hydrolyze GTP to GDP… 3) the protein is now complexed GDP with higher affinity than GTP… 4) GEP induces the protein to exchange GDP with GTP.
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Can translation regulation be mediated by GEF?
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Yes, the hrough the modifications of the GEF responsible for recycling of eIF2, translation regulation is mediated.
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Name the 4 types of mutations in the exon sequences that can lead to altered proteins.
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Silent mutations (don't actually alter the protein), missense mutations, nonsense mutations, and frame shift mutations.
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What is a silent mutation?
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When a mutated codon produces the same amino
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What is a missense mutation?
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when mutated codon produces a different amino acid
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What is a nonsense mutation?
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when mutated codon is UAA, UAG or UGA… a stop codon… shortening the protein
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What is framshift mutation?
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when 1 or two nucleotides are inserted or deleted that resets the reading frame, making the down stream protein different… usually fatal
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which type of mutations are used by retroviruses?
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retroviruses use frameshift mutations to produce reverse transcriptase.
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How many amino acids are coded for by the genetic code?
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20
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After accounting for post-transcriptional modification, how many amino acids are found in human proteins?
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<100
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Give 4 examples of post-translational modification to amino acids
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1) proline converted to hydroxyproline in collagen, 2) addition of complex sugars to amino acids in the golgi, 3) the phosphorylation of serine, threonine or tyrosine in cell regulation, 4) the acetylation, methylation or phosphorylation of histones.
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Which protein is usually involved in the folding of the one-dimensional polypeptide chain into a complex 3-D shape?
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Chaperonins…
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What is a good example of the final protein product being produced by the cutting off of part of the polypeptide chain?
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Insulin
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Name 6 generally permanent post translational modification
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hydroxylation, methylation, acetylation, glycosylation, attachment of lipid (seen in membrane), prosthetic groups (e.g., heme)
[A prosthetic group is a nonprotein (non-amino acid) component of a conjugated protein. The prosthetic group may be organic (such as a vitamin, sugar, or lipid) or inorganic (such as a metal ion). Prosthetic groups usually bond covalently to their protein. They often play an important role in the function of many proteins such as enzymes. A good example of a prosthetic group is the heme group in hemoglobin. A protein without its prosthetic group is called an apoprotein, while a protein combined with its prosthetic group is called a holoprotein.] |
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What type of temporary post-translational modification plays a role in turning a cell on and off?
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phosphorylation of serine, threonine, tyrosine, and sometimes histidine
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Name 4 temporary post-translational modifications
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phosphorylation, acetylation, methylation, and the addition of small proteins, such as ubiquitin (which leads to degradation by proteosomes) or SUMO…
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Name 3 largely permanent post-translational modifications.
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Cleavage, Dimerization (or oligomerization), Assembly
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Name three examples of the cleaving of proteins.
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1) membrane or secretory proteins entering the ER have a signal sequence that is cleaved off in the ER… 2) cytoplasmic proteins entering the mitochondria have a targeting sequence removed… 3) cleaving of dangerous molecules, like proteosomes.
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Give an example of the permanent dimerization of a protein.
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hemoglobin
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Give an exmple of a permenent biochemical assembly
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a ribosome being assembled into a complex machine
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What is the pathway of most membrane and secretory proteins?
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ER to Golgi, traveling by vessicles
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What is an NLS and its function?
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Specific nuclear localization signals, which lead proteins to the nuclear pores, where they are transported into the nucleus.
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What is an NES and its function?
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Nuclear Export Signal, which leads to the export of a protein from the nucleus
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what effect do the following amino acid modifications have on gene expression? A) methylation of residue 9, B) methylation transferred from residue 9 to N-terminus and acetylation of residue 9 c) phosphorylation of residue 10 and acetylation of residue 14
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A) methylation of residue 9, --> gene silencing/ herterochromatin… B) methylation transferred from residue 9 to N-terminus and acetylation of residue 9 --> gene expression… c) phosphorylation of residue 10 and acetylation of residue 14 --> gene expression
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What is the main function of DNA?
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storage of genetic information
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What 2 general consideration are there that address gene express on the DNA level with respect to factors?
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Transcription factors interact with:1) DNA and chromatin structure associated with DNA… and 2) recruit RNA polymerase II and it associated factors
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T/F: it is common for transcription factors to initiate transcription on "closed" chromatin on its own.
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FALSE, chromatin is first opened up by modification to histones.
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What is "HAT"? And its function
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Histone acetyl transferase, which modify histones and open chromatin for transcription
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What is the best explanation for the mechanism by which chromatin opens?
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cooperative opening of chromatin by chromatin remodeling factors (e.g., HAT) and transcription factors
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What role do histone tails play in opening up chromatin?
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they influence the accessibility and the binding ability of DNA
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What is the role of the TATA box? What is the role of the TATA-box Binding Protein?
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a) it serves as the locator for the site of initiation transcription… b) TBP bends the DNA and helps position the RNA polymerase to initiate transcription
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Describe the 4 steps of the binding sequence of the constitutive transcription factors. (Hint: start with TBP)
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1) TBP and associated TAFs (TFIID) bind to DNA… 2) TFIIB binds to TFIID & DNA… 3) RNA polymerase II binds to the factors & DNA… 4) Elongation factors binda dn transcription commences
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Give an example of the constiutive transcription factors recruited by the DNA binding proteins
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Leucine Zipper, which holds to proteins together
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what are the two parts of the transcription initiation complex?
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1) a binding domain that interacts with DNA and 2) an activation domain that interacts with proteins
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What is the relationship between an enhancer and an activator? How many enhancer regions are there on a gene… and how many activators are in a transcription factor complex
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the enhancer is the region of DNA upstream or downstream where proteins bind to effect transcription… while the activator is the protein that binds to the enhancer region… b) 1 to many of both
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Do transcription factors only bind up stream?
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No… they can bind both upstream and downstream
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Name 3 examples of the mechanisms of gene repression.
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1. Competitve DNA binding… 2) Masking the activation surface… 3) direct interaction with the general transcription factors.
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Without the presence of an injury, what is suspected in an enlarged spleen in children?
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ß-Thalassemia Major
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What is the cause of ß-Thalassemia Major?
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Insufficient ß globin molecules … transcription factors are often to blame
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Give 2 mutational examples of ß-thalassemia caused by an insufficient transcription
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1) Cis-mutation… 2) mutation in the mRNA cleavage signal, which results in an extended globin mRNA and this leads to a shortened mRNA globin lifespan
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Give 7 examples for activating a transcription factor.
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1) protein synthesis… 2) ligand binding… 3) Protein phosphorylation… 4) addition of a second subunit… 5) Unmasking (e.g., release of an inhibitor via phosphorylation)… 6) Stimulation of nuclear entry (e.g., release of an inhibitor)…7) release form membrane
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Trace the steps of gene express activation via JAK/STAT.
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Interferon activate JAK --> receptor dimerizes --> phosphorylation of STAT (which is a transcription factor) STAT will now migrate into the nucleus to bind specific sequences to activate transcription
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What is hnRNA?
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pre-mRNA, before m7G is added to 5', adenylation and splicing
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What happens to the RNA polymerase II shortly after initiation? What terminus binds the RNA polymerase II?
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a) it is phosphorylated and then binds proteins used in RNA processing… b) C-terminal domain
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What does adding a 7-methyl G "Cap" do to the 5' end?
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it gives the mRNA a 3' OH at both ends
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What 4 purposes does the m7G Cap serve?
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1) It is the signature of the Poll II transcript… 2) It protects the mRNA from 5' nucleases… 3) Serves as a signal for mRNA export… 4) Serves as a signal for subsequent translation.
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How is the 3' end of mRNA formed?
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cleavage followed by the adenylation… not by the termination fo the transcript
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What is the function of snRNA?
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splicing of RNA… intron removing… where the RNA carries out the catalytic steps
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What are the 3 key region of an intron?
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5' splice site, 3' splice site and the branch point
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What is a spliceosome?
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it is a protein that splices exons together
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What are the 2 steps in mRNA splicing?
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1) Lariat formation and 5' splice site cleavage… 2) 3' splice stie cleavage and joining of the two exon sequences
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What happens to the snRNP and the Lariat?
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the lariat is degraded and the snRNP is recycled
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Where is the Glycine receptor (GlyR∂2) an important inhibitor and what does a mutation in this receptor cause?
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CNS: Startle response
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What is believed as the source ofalternative splicing?
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differential binding of different proteins
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T/F: all thalassemia mutations are in the coding regions (exons)
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false some are in introns
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What is the problem is splicing a in a intron with 19 nucleotides?
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It is not divisable by 3 and will thus be out of the reading frame
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Why do you see more Apo48 in the intestines than in the liver?
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the intestine has more cytidine deaminease,
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What is the mechanism for alternatively forming Apo48 form instead of Apo100 form of protein?
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Cytidine deaminase converts a C, via deaminated, to a U, which results in a stop codon so Apo40, which is shorter than Apo100, is formed
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What is the function of Apo100 and Apo48?
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Apo48 transports from the intestines to the tissues, while Apo100 delivers cholesterol to cell via LDL receptors.
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Which is the first ribosomal subunit that binds to mRNA?
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40s
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What are the 2 key initiation factors in translation.
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1)eIF4, which binds the CAP, and signifies the RNA is to translated… 2) eIF2, which binds the Met tRNAi and the 40s subunit and GTP
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What inactivates eIF4? How?
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eIF4-BP, via phosphorylation of eIF4-BP… which in not seen in tumor cells.
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What is the mechanism employed by some viruses that allows for the translation of it's mRNA instead of the host's?
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Viral DNA has no CAP, instead a little protein called Vpg. Instead it uses IRES, which permits the inititation process of the cells (IRES), which permits intitation at an internal methionine
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What happens to GTP bound to eIF2
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GTP is hydrolyzed to GDP… and the eIF2-GDP is inactive and protein synthesis slows... and it becomes necessary for a special factor to replace GDP with GTP
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what is eLF-2B?
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the special factor that replaces eIF2-GDP with GTP
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With repsect to inactive eIF-2, what does stress (e.g., the lack of Fe, or viruses) cause?
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the phosphorylation of eIF2-GDP and blocks reactivation
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When do cell need transferin receptors, in high or low iron concentration?
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Low Fe
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when do cells need ferritin in high or low iron concentration?
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high fe
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what is ferritin?
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an iron storage protein
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what do transferrin receptors do?
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they transfer iron into red blood cells in order to produce heme
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With respect to ferritin RNA, what happens in times of HIGH intracellular [Fe]?
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the IRE-BP does not bind to RNA and Ferritin mRNA is translated to make more ferritin
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With respect to ferritin RNA, what happens in times of LOW intracellular [Fe]?
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the IRE-BP bind mRNA dn prevents translation of ferritin
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With respect to transferrin receptor RNA, what happens in times of HIGH intracellular [Fe]?
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the IRE-BP does not bind RNA and the transferrin receptor RNA quickly degrades
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With respect to transferrin receptor RNA, what happens in times of LOW intracellular [Fe]?
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IRE-BP binds the TR mRNA and extends the mRNA lifetime
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What is NMD and what are its effects on translation?
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Nonsense-Mediated Decay: a nonsense mutation causes a stop codon mid-frame, which is sensed and NMD causes the rapid degradation of the mRNA, which safegards against the production of aberrant proteins
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What is RNAi and what is its role?
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Interferring RNA, which are RNA that control gene expression by inhibiting translation of mRNA, by causing rapid degradation of mRNA, and by causing modification of chromatin
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What is RISC and what is its role?
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RNA-induced silencing complex, which takes up RNAi… it recognizes the target mRNA bearing a complementary strand and cleaves it… the RISC is recycled
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T/F TGP is a protein that is required for the transcription of most genes.
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TRUE
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T/F prokaryotes can acomplish both transcription and translation simultaneously
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TRUE
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When is gene express regulated? How does this provide a method for reducing storage needs of DNA
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a) during polyadenylation and splicing… b) One gene can code for more than one protein
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Can mRNA be modified?
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yes, in RNA editin, but it's rare, e.g., to produce different version of apolipoprotein B
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Which end of of the DNA strand (5' or 3') contains the sequence that regulation protein lifetime duration?
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3'
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What is the function of NMD?
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Nonsense Medicate Decay identifies and destroys aberrant mRNA
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a)What is the function of RNAi b) how is it derived? c) How is this being used clinically?
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a) RNAi regulates gene expression at translation or at mRNA degradation… b) RNAi is a ~20 ss nucleotide RNA cut from a larger sequence by 'dicer'… c) it's not but research is being done to find a therapeutic use for it.
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If two monozygotic twins demonstrate 100% concordance what type of genetic inheritance is this?
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completely genetic
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If two dizygotic twins demonstrate 50% concordance, what type of genetic inheritance is this
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completely genetic
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If concordance is greater among monozygotic twins than dizygotic twins, what does this say about whether or not this is a genetic trait?
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there is a component of genetics with the trait
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If the concordance for MZ=DZ what type of genetic inheritance is this?
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totally environmental
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If pyloric stenosis shows concordance for MZ=22% and DZ=2%, is there a significant genetic component?
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yes
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Which of the following has a genetic component? Cleft lip & palate, pyloric stenosis, schizophrenia, insulin dependent diabetes melitus
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all
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What do multiple miscarriages and retardation suggest?
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chromosomal abnormalitites
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What do earlier age of onset and increasing severity with generation suggest? (anticipation… males)
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trinucleotide repeat disease
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What is a compound heterozygote?
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2 different mutant alleles at the same loci, from mom and dad
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If affected individuals are seen in every generation, what does this suggest?
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Dominant inheritance
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What is the probability of the child of affected and unaffected heterozygous parents having an affected child?
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50%
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What term does the following define? The range of phenotypic expression seen among family members carrying the same mutation.
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Variable expressivity
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What term does the following define? Individuals carry the disease gene but don not show clinical symptoms (all or none)
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Reduced penetrance
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What type of inheritance is demonstrated with Marfans?
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autosomal dominant with reduced penetrance
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What type of inheritance does Huntington's disease demonstrate?
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Autosomal dominance with age dependent penetrance…trinucleotide repeat (CAG)
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If no male to male transmission occurs, what type of inheritance is suggested and the disease is rarely expressed in females? Why is there no male to male inheritance?
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X-linked recessive… because males get their X from their mothers… and females get two Xs
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What is the chance that the daughter of an affected male of an X-recessive inheritance will be a carrier?
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100%
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What are the odds that carrier females of an X-linked recessive disease will transmit an allele to their… a) son… b) daughter
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both are 50%
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If there is an affected in each generation, both males and females are affected, but there are no male to male transmissions and all of the daughters of an affected male are affected, what type of inheritance is suspected?
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X-linked dominance
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In X-linked dominant, who are more likely to be affected, males or females, why?
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Females are 2X as likely to be affected, because they receive an X from mom and an X from dad
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What is the probability that an affected male will have an affected… a) son… b) daughter
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a) son - zero… b) daughter 100%
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In X-linked dominance inheritance, which gender is more likely to be mildly affected, why?
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females, because they have X-inactivation (mosaic), while males can't switch to another X because they have only one X
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What is anticiaption and which gender is likely to produce this phenomenon?
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a)progressively earlier onset and increasing severity with sucessive generations… b) males
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For Huntington's what are the respective penetrance following CAG repeats? <27 CAG… 27-35 CAG… 36-39 CAG… > 39 CAG
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<27 CAG: Normal… 27-35 CAG: premutation… 36-39 CAG: reduced penetrance… > 39 CAG: complete penetrance
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What is a polymorphism as compared to a mutant allele?
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Polymorphism is an allele that exhibits many forms (at least two different types in the population), while a mutant differs from both wild-type and polymorphic alleles (can be used to indicate diseased gene)
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What does hemizygous mean?
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males having an X and a Y
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What is incomplete dominance?
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when an intermediate for a trait exists between homozygous and heterozygous phenotypes
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What is codomince?
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When the phenotype for both alleles is expressed
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In sickle cell anemia for a heterozyous individual, what type of inheritance is seen with respect to each allele?
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the normal allele is incompletely dominant… the abnormal allele is incompletely recessive… it manifests as a recessive trait
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With resect to inheritance, what is it called when less than what is required for normal function of a normal protein causes disese due to mutation?
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haploinsufficient: when a mutation lowers the amount of gene product below a critical level needed for normal activity
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What is it called when the abnormal allele interferes with the function of the normal allele?
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Dominant negative effect
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What is it called when the mutant allele is enhanced in one or more of its normal properties? Give two examples.
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simple gain of function… Huntington's and dwarfing condition achondroplasia
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Contrast the following 1) Allelic heterogeneity, 2) locus heterogeneity and, 3) genetic heterogeneity
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1) Allelic Heterogeneity is a different mutation at the same loci, 2) locus heterogeneity are mutations at different loci, 3) Genetic Allelic encompasses both heterogeneity and locus heterogeneity
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Give two examples of locus heterogeneity.
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1) Retinitis pigmentosa (x-linked, autosomal recessive and autosomal dominant forms… and sometimes with retardation) 2) Ehlers-Danlos syndrome [connective tissue collagen being fragile or too elastic] ( x-linked, autosomal recessive and autosomal dominant forms)
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T/F Allelic heterogeneity is defined by a mutation at a given locus that results in a clinically indistinguishable or similar disorders.
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True, but not completely true… these disorders can also be very different
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Give an example of allelic heterogeneity.
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Some mutations in the RET gene can cause Hirschsprung disease (colonic motility/severe constipation) or multiple endocrine neoplasia (dominantly inherited cancer) or both
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What are the 4 types of mutation?
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missense, nonsense, splicing, and frameshift
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Of the 4 types of mutation, which 2 commonly account for Loss of Function mutations?
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Nonsense and frameshift, which makes sense if you consider these changes will stop and/or shorten the RNA length… thus loss of function would be probable
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How are loss of function disorders usually inherited?
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autosomal recessive, however some dominant diseases manifest in loss of function.
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What is a dominant negative mutation?
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Seen in protein multimeric complexes, a mutant gene can interact with the wild-type gene rendering the normal gene non-functional
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What is it called when an altered protein product (due to mutation) produces a new property? How is this type of mutation usually inherited.
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Gain of Function… Autosomal dominant
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In a novel protein, what type of mutation serves to point to functionally important residues on a protein?
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missense
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What is in vitro and in vivo?
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in vitro: in cell culture… in vivo: in animals
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What types of experiments are done first when working with novel genes?
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Descriptive: to determine where in the cell the normal protein is express and how this pattern may change in mutant proteins… Also microarrays can be used comparatively between subjects
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For a loss of function mutation, what will a knock-out mouse produce?
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disease
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For a gain of function mutation, what will a knock-out mouse provide?
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clues to the normal function of the gene in question by not producing the phenotype and giving evidence about the mechanism involved
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What is a knock-out mouse and how is it used?
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it is a mouse where the human mutation is added which will result in an expression of the human gene of interest… it can be used to check the efficacy of a novel treatment
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How does one determine function of a novel gene? (3 things)
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clues from: sequence analysis, mutations, and experimental models
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what 3 things are used for sequence analysis?
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1) homology searches (function or mutant phenotype), 2) domain and motif searches (specific characteristics, i.e., phosphorylation glycosylation), 3) Gene families (family members become candidates for similar diseases)
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what 3 things are comparative genomics used for?
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1) determine evolutionary relationship, 2) identification of regulatory elements using sequence conservation in non-coding regions, 3) Gene identification
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What 3 clues do you use to determine the function of a novel gene?
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Clues from: 1) sequence analysis, 2) Mutations, 3) Experimental models
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identify the following mutations as either a substitution, Deletion, Insertion, or duplication: 1) nonsense, 2) frameshift, 3) missense, 4) loss of codon, 5) TOM GET TOM GET
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1) nonsense = substitution, 2) Frameshift = deletion, 3) missense = substitution, 4) loss of codon=deletion, 5) frameshift=deletion
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What is a missense mutation
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Substitution: Where a mutation causes a change in residue, but its still a residue.
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What is a nonsense mutation?
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Substitution: where the codon does not represent an actual amino acid (
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What is a frameshift mutation?
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Deletion or Insertion: where an insertion or deletion causes a mis-reading in the codon for an amino acid, usually causes termination of the transcription/translation
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Name the 3 types of Loss of Function mutation.
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1) Truncation, 2) Haploinsufficiency, 3) Dominant Negative
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What is a truncation loss of function mutation? Give an example.
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Gross inactivation due to nonsense or frameshift… Parkinson's
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Is a truncation loss of function usually dominant or recessive?
|
recessive
|
|
|
What is a haploinsufficiency loss of function mutation? Give an example
|
1) protein below a critical level… 2) Waardenburg's syndrom is a loss of function in the Pax3 gene that leads to (deafness),
|
|
|
Is haploinsufficiency usually dominant or recessive?
|
dominant
|
|
|
What is a Dominant negative loss of function mutation? Give an example
|
1) mutant protein poisoning the normal protein… 2) early onset dystonia.. (a movement disorder)
|
|
|
What is a Gain of Function mutation? Give an example.
|
Altered protein w/ a new property… Huntington's disease
|
|
|
What does a loss of function in the Pax3 gene lead to? What types of mutation cause this?
|
Haploinsufficiency -> Waardenburg's syndrom (deafness)… b) nonsense and frameshift (+/-)
|
|
|
What types of mutation would be nontruncating
|
missense
|
|
|
Huntington's disease is a ____ of Function disease. What is its mechanism?
|
Gain… CAGn repeat expansion that codes for a polyglutamin tract that promotes aggregations that may kill the cell
|
|
|
What are the 3 things you use to characterize the gene product?
|
1) Expression Pattern, 2) Cellular localization, 3) intracellular localization
|
|
|
Which 2 tools do you use to characterize the gene product EXPRESSION PATTERN?
|
Northern Blot (mRNA) and Western blot (protein)
|
|
|
Which 2 tools do you use to characterize the gene product by CELLULAR LOCALIZATION (Spatial patterns in tissues or cells)?
|
1) in situ hypridization (mRNA), 2) immunohistochemistry (protein)
|
|
|
What is immunohistochemistry used for?
|
cellular localization of proteins
|
|
|
What is in situ hybridization used for?
|
cellular localization of mRNA activity
|
|
|
What is Northern blot used for?
|
mRNA degree of expression and which tissues
|
|
|
What is Western Blot used for?
|
protein degree of express and which tissues are expressing
|
|
|
Which 2 tools do you use to characterize the gene product via INTRACELLULAR LOCALIZATION (Localization within a cell) ?
|
1) cell fragmentation (protein), 2) immunofluorescence microscopy (protein)
|
|
|
What is cell fragmentation used for?
|
to characterize gene expression of a protein inside the cell
|
|
|
What is immunofluorescence microscopy used for?
|
to characterize gene expression of a protein inside the cell (e.g., expression of wt and mt torsinA in CAD cells)
|
|
|
What is "in situ" hybridization" used for?
|
cellular localization of mRNA (spatial patterns of expressed mRNA within tisues or cells)
|
|
|
What is a immunohistochemistry assay used for?
|
cellular localization (to determine the spatial patterns of expressed proteins in tissues or cells)
|
|
|
What type of mouse does the following scenario represent? Destroy normal function in mouse, if disease is due to loss of function this should produce an animal model of the disease
|
Knock-out mouse
|
|
|
What type of mouse does the following scenario represent? Destroy normal function in mouse, if disease is due to gain of function then we should be able to insert the human disease mutation in the mouse and produce the human disease.
|
knock-in
|
|
|
When and how is a Transgenic Mouse Model used?
|
When? it is used to test Gain of Function mutations where a single copy of a gene is sufficient to cause the disease… How? Insertion fo of the human gene at random
|
|
|
What 2 things can explain why a mouse model does not express phenotype or partial phenotype?
|
1) Differences in pathways (mouse may have alternative pathways), 2) Genetic modifiers
|
|
|
A) How was Huntingtin's disease confirmed NOT to be a loss of function? B) How was the disease confirmed as a gain of function?
|
A) a knock mice die in utero… thus, when they lack the gene they are not viable.. B) knock-in mice with the human gene developed inclusions consisten with the human diease HD.
|
|
|
Which model oranism is used for developmental and neurologic studies?
|
C. elegans
|
|
|
Which model is used to study developmental pathways?
|
Drosophila
|
|
|
which models are used to screen for suppressors and enhancer of mutant phenotypes?
|
worms and flies
|
|
|
What 3 things can the human genome sequence be used for?
|
Functional genomics, pharmacogenomics, and whole genome association studies.
|
|
|
What is functional genomics?
|
large scale investigation of gene function
|
|
|
What is pharmacogenomics used for?
|
To determine how an individual's genetic inheritance affects the body's response to a drug
|
|
|
What are whole genome association studies used for?
|
Using SNPs, they identify genes involved in complex traits
|
|
|
What is used to predict survival probabilities?
|
functional genomics
|
|
|
How can pharmacogenetics be used?
|
using SNPs, we can correlate the efficacy of the drug in trials with the SNPs to predict.
|
|
|
Lecture 11
|
|
|
|
What 4 things caues and maintain genetic variation?
|
Mutation, gene flow, selection, and drift over evolutionary time
|
|
|
When did the mutations that are responsible for many common human genetic disease arise (or underwent expansion in frequency)?
|
relatively recently in human evolution
|
|
|
What is the Hardy-Weinberg law used for?
|
to quantify geneotype frequency and allele frequency in a population… it can also be used to assess the risk of genetic disease
|
|
|
What proves that genetic frequencies do not change from one generation to the next?
|
Hardy-Weinberg law
|
|
|
The Hardy-Weinberg law is based on which 6 assumptions?
|
large population, random mating, no novel mutation, lack of selection, lack of genetic drift and land of gene flow
|
|
|
Which word characterizes the point at which a particular allele becomes the only allele at its locus in a population - the frequency of the allele.
|
Fixation
|
|
|
What is purifying selection?
|
Selection against alleles that have harmful phenotypic effects, which leads to their loss from the population
|
|
|
What is diversitying selection?
|
Natural selection that favors extreme over intermediate phenotypes
|
|
|
What is balanced selection
|
A selection regime that results in the maintenance of two or more alleles at a locus in a population (e.g. overdominance)
|
|
|
What is genetic drift?
|
Random fluctuations in gene frequencies, most evident in small populations
|
|
|
What is gene flow
|
the movement of genes from one population to another
|
|
|
What is assortive mating?
|
Where sexually reproducing organisms tend to mate with others that are like themselves
|
|
|
what causes a SNP?
|
nucleotide substitution
|
|
|
Give 4 examples of mutation that can bring about genetic variability.
|
Nucleotide substitution (SNP), 2) Insertion/deletion, insertion of a transposable element, or chromosomal rearrangement (tranlocations, inverstion or duplications)
|
|
|
According to Kimura's neutral mutation theory, what are the 3 possible effects of mutations?
|
advantageous, neutral or deleterious
|
|
|
Rank Kimura's mutation in order of least common to most.
|
neutral > deleterious > advantageous
|
|
|
What does it mean when the frequency of an allele in a population = 1?
|
This is Fixation… it is the only allele in the population
|
|
|
What is the fixation (for new alleles) equation for a nearly neutral mutations, where s=~0 (s= selective advantage)?
|
P = 1/(2N)=q… where P= probability of fixation… and q= for a new allele… thus, the probability of fixation is equal to the frequence of the new allele when there is no selectivce advantage for the new allele
|
|
|
If there is selective advantage in a new allele (advantageous mutation), is it likely to undergo fixation? Does the same apply to deleterious mutation?
|
a) no… for example if a mutation increases fitness by 1%, the probability would increase by 2%… b) yes, it has a low but finite probablity of fixation
|
|
|
A) What is the time to fixation for advantageous mutation of about 1%? B) And time to fixation for neutral mutations?
|
A) ~40,000 years… B) 800,000 years
|
|
|
What does a larger ancestrial population suggest about the diversity of a given population… and why?
|
A large ancestrial population would result in greater current diversity within a population, because the larger the population the more time is needed for fixation
|
|
|
What is purifying selection?
|
selection that results in favoring the predominant allele
|
|
|
Name the mechanisms of Drift
|
1. Random fluctionations in allelic segregation… 2. Also founder effect and bottle neck will cause this.
|
|
|
what 2 things can account for allele frequencies being substantially different from one population to another?
|
Selection and drift
|
|
|
What effects do new mutation have on genetic variability in a population?
|
Increase variability
|
|
|
What effects do new loss of alleles by selection or drift have on genetic variability in a population?
|
decrease variability
|
|
|
give the hardy-weinberg equation
|
p + q = 1… p^2 + 2pq + q^2… where p=dominant and q=recessive… (note: p + q = 1… and p=AA, 2pq=AB and q=BB)
|
|
|
Given a the total number of people in a population (N) and the total number of a homozygous individuals (Ho) in a population, and the number of heterozygotes (He) in a population ( determine the frequency of both alleles
|
p= (2Ho + He)/ N… q=1-p
|
|
|
Given p, what is the frequency of heterozygotes in a population?
|
q=1-p… thus, 2pq
|
|
|
How do you calculate carrier frequency for an autosomal recessive mutation from Disease incidence?
|
q^2=disease incidence… thus, q= square root of q^2… thus, p=1-q… Therefore: carriers = 2pq= 2 X q X (1-q)
|
|
|
How do you determine the probability that the mating of two individuals will produce a child with a recessive autosomal disorder? Where one person has a 1 degree relative that is affected and the other has an estimated ethnic risk (carrier freq).
|
If the relative is a sib, and neither parent is affected: that person has a 1/3 chance of being a carrier. The other person has an assigned risk (carrier freq). These combined are the risk of both being… 1/3 X carrier freq X 1/4… the 1/4 is the probability of a recessive from two heterozygotes.
|
|
|
How do you determine the risk of two people having a child with an autosomal recessive disorder if neither has a family history of the disease, but both belong to a ethnic group that expresses this mutation?
|
[(carrier frequency)^2] X 1/4
|
|
|
what are the chances that 2 caucasian will marry each other?
|
(0.043)^2
|
|
|
What are the the problems with genetic testing?
|
reliability and cost… so probability is sometimes used
|
|
|
How would you find the total frequency of affected individuals for a dominantly inherited disease, given the frequency of unaffecteds (p^2)
|
p = square root of P^2… q=1-p… 2pq = heterozygous affected… thus: number of affected would be = (1-p)^2 + 2p(1-p)
|
|
|
How do you find the allele frequency of X-linked recessive disorders for: females and males?
|
1) females: treat as a normal autosomal question… 2) p + q = 1… where p = unaffected and q = affected.
|
|
|
How do you find the allele frequency of X-Linked dominant disorders for females and males?
|
For females: Exactly like dominant disorder, where allele frequency = P^2 +2pq… For Males: p + q = 1
|
|
|
Which population is at increased risk for Tay-Sachs disease and what are the mutations?
|
Ashkenazi Jews: exon 11 insertion and intron 12 splice signal… French-Canadian: 7.6 kb 5' deletion
|
|
|
Which populations are at riskfor Familial Hypecholesterolemia?
|
French-Canadian: promoter + exon 1 deletion… and Lebonese: premature stop at aa 660
|
|
|
What are the exceptions to H-W equilibrium?
|
Exception to random mating, purifying & diversity selection, new mutations, founder effects/ bottleneck, genetic drift (short time in large populations is negligible, but long time in small populations drift is inevitible, and balance selection
|
|
|
What are 3 exceptions to random mating?
|
Stratification, assortive mating, and consanguinuity
|
|
|
Define: Balancing Selection (aka heterozygous advantage)
|
might explain the prescence of recessive disease gene in a population
|
|
|
Name two mutational diseases that gives strong support for balancing selection and and the protection it provides
|
sickle cell anamia, ß-thalassemia: both Malaria
|
|
|
Name a disease that gives some evidence in support of balancing selection hypothesis
|
cystic fibrosis: supports against typhoid and cholera
|
|
|
What 2 disease and the supposed disease they support agains have little evidence that support the balancing selection
|
Hypercholesterolemia and hypertension: both protect against starvation… little support
|
|
|
How do disease alleles persist in humans?
|
If Dominant allele is lethal at birth… then All mutations are new…
|
|
|
Lecture 12
|
|
|
|
What causes Prader-Willis Syndrome?
|
Paternal deletions of 15q11-q13… or Maternal uniparental disomy
|
|
|
What causes Angelman Syndrome?
|
Maternal deletion of 15q11-q13 or paternal uniparental disomy
|
|
|
How do you treat Prader-Willi Syndrome?
|
Growth hormone
|
|
|
What is another imprinting disorder?
|
Beckwith-Wiedeman syndrome
|
|
|
See page 42 of Genetics Syllabus for genomic imprinting pedigree
|
|
|
|
What would you expect a mitochondrial inherited disease to look like?
|
Passed from mother to son or daughter… but not inherited from the father
|
|
|
What is the DNA capacity of BAC and where was it used?
|
150-200 kb… useed to sequence to the human genome.
|
|
|
what is a proteome and how many do humans have?
|
protein coding genes: 3-40 thousand
|
|
|
Are polymorphic genetic markers evenly spaced? How were the markers make?
|
yes, they were made from microsatellite simple tandem repeat sequence such as (CA)n…
|
|
|
What is the function of the microsatellite simple tandem repeat sequences?
|
none, but they are helpful in mapping
|
|
|
Where are PCR primers generated from?
|
PCR primers can be generated from unique sequences flanking the ese sequences.
|
|
|
What do you find at CpG islands?
|
transcription initiaton site of a gens
|
|
|
Where are most rRNA genes?
|
on the p arm (acrocentric)
|
|
|
What do small nuclear RNAs do?
|
they form ribonucleoprotein particles and are important for splicing
|
|
|
What percentage of the genome is exons?
|
5%
|
|
|
what is the typical length of a gene?
|
10-15 kb including exons and introns
|
|
|
what is the average length of an mRNA?
|
2.5 kb
|
|
|
How were mRNA from tissue isolated in the genome?
|
They were reversed transcribed to cDNA and then shotgub subcloned into bacterial vecdtors
|
|
|
What do you call a copy of a gene containg its normal intron/exon structure, but harboring mutations, due to time and lack of selection?
|
unprocessed or pseudogene
|
|
|
Can pseudogenes be expressed as mRNA?
|
yes, but because they don't produce a functional gene they are not translated.
|
|
|
Where do you find pseudogenes? What purpose do they serve?
|
they cluster in the pericentromeric or subtelomeric regions… where they serve as a sink or donor of new genes or dying genes.
|
|
|
Where do psuedogenes come from?
|
they are defective (incomlete) gene copies that result from the reverse trancription of mRNA to a cDNA in a cell followed by its reinsertion randomly into the genome
|
|
|
What percentage of the human genome is made up of transposable elements contain?
|
50%
|
|
|
What do transposable elements comprise?
|
high copy reptetive DNA elements, ie., LINEs and SINEs (long and short interspersed nuclear elements)
|
|
|
Do LINEs function? If so, what is required?
|
a small number are functional and can transpose… b) 2 ORFs one encodes for a reverse transriptase and endonuclease wich make it possible for the element to jump around in the genome
|
|
|
What is an HERV?
|
it's an endogenous human retrovirus… most copies are non-functional
|
|
|
What is the most abundant SINE? Where are they found?
|
Alu (millions of copies)… found in gene rich areas, which suggests a functional role
|
|
|
What are the hazzards associated with LINEs and SINEs?
|
they can interrupt a gene, or unequal crossover between similar SINEs and LINEs can cause chromosome rearrangements like delitions or translocation that result in disease
|
|
|
Why should we care about noncoding junk DNA?
|
they can be used as markers, for example microsatellites which can help identify candidate genes.
|
|
|
What is the advantage of possessing the sequence from other species?
|
can be used as models, identification of similar (conserved genes) and used for tests
|
|
|
What makes us human vs. non-human?
|
?
|
|
|
Are reuglatory regions usually well conserved?
|
Yes
|
|
|
Of the non-coding genes what are the two catagories of genes?
|
pseudogenes and regualtory regions
|
|
|
besides genes, what is in the geneome?
|
junk DNA
|
|
|
Is there is a positive relationship between the number of exons and importance of the gene?
|
no
|
|
|
What is the RB1 gene and what can happen if it's mutated?
|
it's a tumor repressor gene… if mutated it can lead to retinal tumors
|
|
|
What direction is the U16 gene on the retinobastoma gene transcribed?
|
its 5' to 3', but it's coded for on the opposite strand… thus genes can be oriented in forward or reverse direction but they are always transcribed 5' to 3'
|
|
|
Are genes that are related always together? Give an example of close and distal
|
no, some families of are distal… e.g., close: ß-globin… distal: histones
|
|
|
What are referred to as non-coding coserved regions?
|
transcriptional regulatory regions… since these genes are well conserved
|
|
|
What can we use to find whole genes?
|
comparative genomics (genes from different species)
|
|
|
What can be done to determine the function of gene?
|
produce KO mice to see if it's essential to life
|
|
|
review page 27
|
|
|
|
What can a centromere be used for in working with DNA?
|
Centromeres are characterized by repeats and can help orient 10 Mb region
|
|
|
What does UTR stand for?
|
untranscribed region at the 5' and 3' regions of a gene
|
|
|
What are unique variations in DNA?
|
polymorphisms… satellites and SNPs, which are not in the coding region and thus harmless, but can effect disease.
|
|
|
How many microsatellites do you find per 50,000 bp?
|
one
|
|
|
Are microsatellite similar in related individuals?
|
no, the are highly variable if you are not related
|
|
|
Are microsatellite evenly spaced?
|
yes… they are used as markers in linkage studies
|
|
|
Can SNPs be important to function?
|
yes… they are single base pair variations that differe from one person to another, within a family… they can be in the gene or in regulatory regions… Anywhere.
|
|
|
What are SNPs used for?
|
positional cloning
|
|
|
At what frequency do you find SNPs per 1000 bp?
|
one… less than 1% of genome… most are found in the coding regions
|
|
|
What's a non-synonymous SNP?
|
codes for a change in codon
|
|
|
Are most SNPs in coding or non coding genes?
|
non coding, but they can still effect disease by effecting regulatory regions
|
|
|
Where are genes located, light or dark regions?
|
light
|
|
|
Where are LINEs located, light or dark regions?
|
dark
|
|
|
Lecture 2
|
|
|
|
When do Chromosomal Disorders have a great impact?
|
before birth
|
|
|
When do single gene disorders have a great impact?
|
early childhood
|
|
|
When do multifactorial disorders have a great impact?
|
later in life in adulthood
|
|
|
What is cytogenics?
|
A method by which chromosomes can be analysed under the microscope… for example to find trisomy
|
|
|
What is a bipartite?
|
its when DNA consists of 2-sister chromatids with exact DNA
|
|
|
When is the reduction stage of meiosis? Why?
|
MI, because the 2-sister chromatids stay together (it's like 2 of the same DNA, so it reduced from a diploid to a haploid)
|
|
|
When does homologous recombination occur?
|
MI: meiotic cross-over pachytene stage) before the sister chromatids separate
|
|
|
In what stage do oocytes become arrested?
|
prophase I, until ovulation
|
|
|
What test is used to check for chromosomal abnormalites?
|
G banding of lymphocytes of blood
|
|
|
What 2 tests are used to detect SUBTLE rearrangements or terminal deletions?
|
Fluorescence in situ hybridzation (FISH)
|
|
|
What information is needed for FISH?
|
you have to have an idea of what abnormality you're looking for, with respect to disease (and or a particular deletion or duplication)
|
|
|
What is the kb size limitation?
|
10-20 kb
|
|
|
Describe FISH protocol
|
a single stranded labeled DNA probe is hybridized to the region of interest
|
|
|
What test can help determine whether or not a trisomy event is due to mother or father contribution?
|
polymorphic microsatellite markers by P"CR approaches, where gel electrophoresis of microsatellite markers on chromosome 21 (3 each) the DS child is run and compared with the mother and father's microsatellite… note that the one that contributed 2 is the contributor of the extra chromosome
|
|
|
What is the frequency for aneuploidies?
|
1/154 live births
|
|
|
Which 3 chromosomes are implicated in trisomy events most common trisomy
|
21 (DS), 18 and 13
|
|
|
What is Turner's?
|
45 X females
|
|
|
What is the cause of an unbalanced translocation?
|
these children are the offspring of partial aneuploidy events of a normal carrier of a BALANCED TRANSLOCATION
|
|
|
What congenital effect does an altered gene dose usually result in?
|
mental retardation
|
|
|
What is a contiguous gene disorder?
|
it is multiple haploinsufficiency caused by the small hemizygous losses of DNA (on the order of 15-30 genes)
|
|
|
What is the usual cause of unbalanced translocation?
|
crossover events between region specific LCR (low copy repeat) on chromosomes.
|
|
|
What is used to determine an genomic disorders caused unbalanced translocation?
|
FISH
|
|
|
When is interphase FISH mapping used ?
|
to detect multiple duplication
|
|
|
What type of deletions are associated with mental retardation?
|
terminal deletions, as they are near the telemers.
|
|
|
When do male gametes undergo prophase I?
|
in adulthood
|
|
|
What is occuring during prophase I?
|
segregation… haploids are created
|
|
|
When does recombination occur?
|
MI
|
|
|
What is the difference between metaphase I and metaphase of meiosis?
|
MI produces haploids that may be different due to cross over… mitosis metaphase produces daughter cells (diploid)
|
|
|
What do defects in meiosis lead to ?
|
aneuploidy
|
|
|
What is the most common cause of mental retardation?
|
aneuploidy
|
|
|
What is the frequency of Down Syndrome?
|
1/800 live births
|
|
|
How many different (not alike) alleles for a given chromosome do you find in a Down Syndrome individual, where non-disjunction occurred in MI?
|
M1 = 3 Types
|
|
|
How many different (not alike) alleles for a given chromosome do you find in a Down Syndrome individual, where non-disjunction occurred in MII?
|
MII = 2 different types
|
|
|
What is a haplotype?
|
the unique order of markers along a chromosome
|
|
|
A child is born with major birth defects (craniofacial, heart, brain), severe developmental delay. What diagnostic tests would you do?
|
1. Do a kayotype oto see if chromosomes are abnormal in sturcture or copy number… 2) If you suspect a specific genetic syndrome, use a molecular cytogenetic probe and perfomr FISH mapping to see the deleted or duplicated region
|
|
|
At what phase in mitosis would you do a FISH test?
|
Metaphase, where the chromosomes are condensed.
|
|
|
What test would you use to detect multiple rearrangements, such as seen in cancer?
|
SKY (spectral karyotyping)
|
|
|
What are genomic disorders caused by?
|
deletions or duplications
|
|
|
What causes VCFS (di George Syndrome)?
|
deletion of 22q11.2
|
|
|
What causes William's syndrome?
|
deletion of part of 7q11.2
|
|
|
Besides learning disabilities and characteristic faces, what do di George people present with?
|
heart defects and immune disorders
|
|
|
What test do you use to diagnose di George or William's syndromes?
|
FISH
|
|
|
What is the mechanism responsible for getting deletions or duplications in the genome?
|
Non-allelic homologous recombination events between chromosomes cause deletions or duplications
|
|
|
Is it just as possible for a deletion or duplication to occure between two different alleles (inter-chromosomal as it is for this to occur on the same chromosome?
|
yes, Intra-chromosomal
|
|
|
Are most parents normal in genomic disorders?
|
yes
|
|
|
Lectures 3 and 4
|
|
|
|
In what order does the protein made (Hint: direction 3-5, C-terminus... or 5-3, N-terminus)
|
5' -> 3' N-terminus
|
|
|
What is the % of introns and exons/
|
exons = 5%… introns = 20%
|
|
|
What percentage of the human genes are alternatively spliced?
|
40%
|
|
|
Which restriction enzyme is blunt?
|
Hpal
|
|
|
What is the average size of a 4 bp long restriction enzyme?
|
256 = 4*4*4*4
|
|
|
Does the restriction enzyme cut uniformaly?
|
no, it's an average
|
|
|
What does a DNA ligase do?
|
it covalently links two pieces of DNA
|
|
|
What happens when you place a restriction enzyme in with DNA and a plasmid and then ligase it?
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recombinant DNA, where the DNA of interest is inserted into the plasmid
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What are preferred properties of cloning vectors?
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minimal size, origin of replication, high yield of DNA, dominant selectable markers (like AMP or tricylin), lots of restriction sites, selection screen for inserts, inducible or high level of expression)
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What is the protocol for Cloning a gene of interest?
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place DNA in container with the vector and Restriction enzyme… 2) add ligase… 3) insert into bacterial cells… 4) grow on selective plate with antibiotic… 5) bacterial replication will reproduce your sequence
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What does forced dimerization of Caspase-8 cause in adiocytes?
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apoptosis
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What is the result transfection with the FAT-ATTAC gene in mice?
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decreased obessity
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How do you make a cDNA library? (5 steps)
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1) mRNA is prepared from the tissue of interest… 2) a complementary copy of DNA (cDNA) is made using oligo-dT as a primer and reverse transcriptase as a primer… 3) The RNA is removed using RNAseH, which digests the RNA in RNA-DNA hybrids… 4) a second strand of DNA is made using DNA polymerase... 5) The ds DNA can now be inserted into a vector
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What is cDNA?
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it's a sequence of DNA made from mRNA
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Why would you make cDNA?
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Since it's made from mRNA you know that it's the gene that was being translated… " cDNA: gene representation is relative to expression level"
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How is a Genomic Library made?
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by purifying DNA from a human tissue or cell line… cutting it with a restriction enzyme… then ligating the resulting fragments into a vector
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What does the relative abundance of cDNA represent?
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the actual presence in the cell
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What is nucleic acid hybridization?
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anealing of two strands of DNA-DNA or DNA-RNA
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How do you screen a DNA fragment by hybridization?
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Lyse phases (vector), and denature DNA --> hybridize to labeled probe
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What does Gel electorphoresis do?
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separates DNA and RNA by size
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What drives the molecules throught an electrophoresis gel?
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current
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Which flow through an electrophoresis gel, large or small particles?
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small
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What is Southern blot used to measure?
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DNA
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What are the 4 steps in Southern blotting?
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1) restriction digestion of DNA… 2) Gel electrophoresis of DNA by size… 3) Transfer to membrane… 4) use a labeled DNA probe for detection
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What is the DNA tranferred to from the agarose gel?
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Ntrocellulose paper
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What is RFLP used for?
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genetic finger printing and paternity cases
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What is a RFLP?
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restriction fragment length polymorphism
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what is a missense mutation? What causes it?
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codes for a different amino acid… substitution
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what is a nonsense mutation? What causes it?
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codes for a stop codon (UAG, UAA, UGA)… substitution
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what is a frameshift mutation? What causes it?
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when all subsequent amino acids are wrong… deletion or insertion… usually fatal
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what is a loss of codon mutation? What causes it?
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an entire amino acid is lost… deletion
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what is a duplication mutation? What causes it?
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when an entire sequence is duplicated… (transposable elements)
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What is a trunctation usually caused by?
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nonsense mutation or frameshift
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Is a truncation usually a dominant disorder or a recessive disorder?
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recessive
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Is a truncation mutation loss or gain of function?
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loss
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What is a dominant negative mutation?
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when an abnormal protein phenotype poisons the other functional protein
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What is haploinsufficiency?
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When 50% of the gene product is insufficient to support normal function
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Is a haploinsuffiency usually recessive or dominant
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dominant
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What is a gain of function mutation?
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where an altered protein has a new property (can be an increase in normal or different)
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LOOK UP GAIN OF FUNCTION
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What does a Northern blot measure?
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RNA expression
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What is the Northern blot protocol?
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Electrophoresis of RNA… 2) transfer to membrane… 3) use a labeled probe for detection
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When is a Northern blot used?
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to check expression of a gene given a stimulous over time… mRNA
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What is the expression of mRNA Acrp30 associated with?
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fat cell protein
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What does the overexpression of adiponectin lead to ?
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increased fat mass
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What would you use to measure expression of a mRNA (or DNA) in a tissue?
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in situ hybridization
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What is the protocol for PCR?
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add DNA of interes… 2) add primers… 3) heat to 90°… 4) cool to aneal… repeat…
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The next question related to the use and protocol of microarrays
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What do each spot of a microarray have on it?
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ssDNA probes for thousands to milions of genes, each spot with a location specific to a particular gene
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After you obtain mRNA from the tissue sources being compared (e.g., treated and untreated with drug… diseased vs. normal), WHAT DO YOU DO WITH THE mRNA NEXT?
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Convert to cDNA and add a fluorescent label for each one (red and green)
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What do you do with the cDNA?
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apply to the chip… the cDNA and the probe will hybridize
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What does it mean when the cDNA binds to the chip?
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The binding corresponds to the expression of that gene being expresson in the tissues (differentially red or green)… a computer will analyize these expressions
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What is the benefit of using microarrays?
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many fragments can be visualized at one time
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What is laser capture microdissection (LCMD)?
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laser cuts and collects cells in eppendorf
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What is a Western blot used for?
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protein analysis
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What is the protocol for SDS-page Western blots?
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1) SDS-page & heat: denatures and normalizes the charge on proteins to NEGATIVE CHARGE (because not all the charges are the same on the same)… 2) place in polyacrylaamide gel-electrophoresis… small protein will travel faster…
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How can you use isoelecdtric focusing to higher resolution of SDS-PAGE?
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it will move along the gel to its isoelectric point… THUS a protein can be analyzed by size and isoelectric focusing… 2-D SDS-PAGE… this is a better purification (resolution)
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How do you vizualize the the proteins in an SDS-PAGE Western blot?
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apply a porous sheet (nitrocellulose) to the gel… apply an antibody for the protein… was away excess proteins (which have not bound to the probe)… incubate with an enzyme linked antibody that binds to the other end of the probe.
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Adiponectin is an example of a use for SDS-PAGE Western blotting. What does high molecular weight of Adiponectin in individuals predict?
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insulin resistance and metabolic syndrome
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What can you use to visualize where a protein is being expressed within a cell?
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Immunofluorescence
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What can you use to sequence protein amino acid sequences?
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mass spectrometry
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