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Exam IV Review

Page history last edited by Christopher Korey 14 years, 11 months ago


This is an interactive review sheet that encourages you to interact with the material and draw out the critical information.  There are several ways to contribute:


  • Add an important vocabulary word and its definition to the list from a specific lecture.  These are anything you feel is essential to the understanding of the material including the names and functions of proteins, types of chemical bonds, names of processes, etc.

  • Add an answer to one of the concept questions

  • Edit and expand on another student's posting

  • Add a link to another website, a video on You Tube, or any other teaching material on the web that you think enhances your understanding of the material.


As I stated in class, 8 points of your exam grade is dependent on your participation in this dialog about the material.  I will use the following rubric for grading: 3 points for adding and defining one vocabulary word, 3 points for participating in the answering of one of the concept questions, 2 points for editing and expanding on another student's posting (edits must be more than just removing a word or adding a word, it must have an impact on the material discussed)


Contributions to this review must be completed by 5 p.m. on April 21st (the Tuesday before the exam)


Topic 14 Eukaryotic Transcription


Important Vocabulary:

TATA Box-A sequence of DNA (T,A,T,A,A/T,A,A/T) located in the promotor region of eukaryotic genes and recognized by TATA binding protein (TBP), one of the many transcription factors involved in eukaryotic transcription.


TATA binding protein (TBP):  A general transcription factor that binds to DNA and kinks it to form a platform where RNA polymerase can bind.


RNA Polymerase I - This polymerase, also called Pol I, is located in the nucleolus of eukaryotic cells.  It transcribes 18S and 28S ribosomal RNA (rRNA).  It is the only enzyme that transcribes ribosomal RNA.  The rRNAs will form the major RNA sections of the ribosome.


RNA Polymerase II - This polymerase, also called Pol II, transcribes messenger RNA (mRNA) from the template DNA during transcription.  It requires a number of general transcription factors to help it bind to promoters on the template DNA and begin transcription.  The general transcription factors that help Pol II bind to the DNA include the TBP (tata binding protein), TFIID, TFIIA, TFIIB, and TFIIF.  TFIIH and ATP bind to Pol II to open the transcription bubble.  Pol II transcribes mRNA that will later be translated into amino acid sequences that will form proteins.


RNA Polymerase III- transcribes DNA in order to synthesize 5s rRNA, tRNAs, and other small RNAs found throughout the nucleus and cytosol.


Poly A Polymerase-  A polymerase that adds stretches of adenines to the 3' end of mRNA using ATP. Approximately 200 adenines are added, and no primer is required for this addition. The tail that is formed provides stability and is required for efficient translation.


Poly A Tail- A region added at the end of the RNA sequence (after the 3' untranslated region). The tail is not encoded for in the DNA sequence; however, there is a consensus sequence (usually AAUAAA) which functions as a signal to make a tail. The actual adenines are not encoded in the DNA; they are added later by the Poly A Polymerase enzyme, and the tail provides mRNA stability (functions similarly to the cap).


TFIIH:  Uses its helicase activity to help polymerase II gain access to the template.  Also responsible for phosphorylating the CTD of Pol II, which is important for the Pol to disengage from the other TF's and begin transcription.


TFIID:  General transcription factor that recognizes the promoter and helps to recruit the RNA polymerase. Also includes TBD in its complex which is the TATA binding protein.



Important Concepts:


What are the different roles of Pol I, II, and III?

Pol I, Pol II, and Pol III are Polymerases. Pol I is located in the nucleolus and it transcribes 18S and 28S rRNA. Pol II makes mRNA and is found in the nucleus NOT the nucleolus. Pol III works on 5S rRNA, tRNA, and small non-coding RNA. Pol III is located in the nucleus NOT the nucleolus.


What are General Transcription Factors?

General transcription factors (GTFs) help polymerase II to recognize and bind with the promoter region of eukaryotic genes, and therefore, they play an active role in the initiation of eukaryotic transcription. The GTFs involved in the initiation stage are TFIIA, TFIIB, TFIID, TFIIE, TFIIF & TFIIH. Each GTF has a specific role in helping the polymerase II to recognize, bind, and begin transcription of the DNA strand. Many of the GTFs bind with polymerase II to form the preinitiation complex which is able to recognize and bind to the DNA template strand in order to form the closed complex. Some of the GTFs then help to "melt" the DNA, and TFIIH uses ATP to form a transcription bubble which begins the open phase of initiation and leads to the phosphorylation of the C-terminal domain.


What is the function of TFIID?

TFIID is protein that is made up of a complex of subunits, including the TATA binding protein (TBP). TFIID helps to position the polymerase II over the transcriptional promoter region. The TBP unit of the complex is what is responsible for identifying the promoter region since it specifically binds with the TATA box sequence located within the promoter. Without the TFIID, the polymerase is unable to recognize the promoter region, and therefore, transcription can not begin. The TBP also helps the DNA to "melt" during the formation of the transcription bubble by bending the DNA. This bending facilitates the breaking of the relatively weak bonds between the Ts and As that are prevalent in the TBP's binding sequence (TATA box).


What are the many roles of Polymerase II C-terminal domain phosphorlyation?

One of the roles of C-terminal domain phosphorylation is to move the RNA Polymerase II away from the initiation site to begin transcription.  TFIIH, an important general transcription factor, is a kinase that phosphorylates the C-terminal domain of the RNA Polymerase II. 


Another role of C-terminal domain phosphorylation is capping the 5' end of the mRNA.  The cap-synthesizing and cap-binding complex bind to the C-terminal domain to cap the 5' end.  The cap-synthesizing complex removes the gamma phosphate from the C-terminal domain and replaces it with a GMP.  The cap is important in helping the mRNA bind to the ribosome during translation.


What is unique about transcription in Eukaryotes?

In Eukaryotes, as opposed to Prokaryotes, transcription is not coupled to translation, although it is coupled to RNA processing (also new). The reason that transcription is not coupled to translation in eukaryotes is due to the presence of the nucleus.  In prokaryotes there is no nucleus so translation can begin as transcription is still finishing, however, in eukaryotes there is a nucleus separating the two processes so the cell has very different ways of going about these processes.  The information in the DNA isolated in the nucleus must find a way to communicate to the rest of the cell.  RNA copies the information and leaves through the nuclear pores and then translation occurs outside of the nucleus.  Not only is there a 5' cap added to the newly made mRNA, but there is a polyA tail added afterward. Addtionally, the mRNA is not the final step before translation-the introns have to be spliced out of the pre mRNA, making the final, mature mRNA.  Also in eukaryotes, trancription occurs in the nucleus of the cell.


Topic 15 Eukaryotic Transcriptional Regulation I


Important Vocabulary:


Activators: Activators bind to the enhancer to stabilize polymerase. They are less structured and "Sticky" to be able to hold on to DNA. Jobs of activators include: recruiting Pol II, recruiting general transcription factors, and stabilization.


 Activators often have two domains, the one side being the sticky side which binds to the DNA and the other side is the activation domain.


HATs:  Histone Acetyl-transferases.  They are a family of proteins that catalyze the formation of an amide bond between the epsilon-amino group of an amino-terminal lysine residue of one of the core histone proteins (H2A, H2B, H3 and H4).  These are activators that increase acetylation, increase DNA availability, and increase transcription.


Enhancers: similar to operators, can bind repressors and activators (DNA sequences)


HDACs: (Histone Deacetylases)-  A repressor that decreases acetylation causing a decrease in the availability of DNA, and therefore a decrease in transcription.  HDACs remove the acetyl groups from the ends of the histone tails that neutralize the positive nature of the histone tails.  Without this positive character, the histone tails would not be attracted to the negatively charged DNA, and the DNA would be released from the histone, opening the coiled DNA and allowing access to that portion of DNA.  Removal of the acetyl groups returns the positive nature to the histone tails, wrapping the DNA back around the histone and closing that portion of DNA from trasncription.


7-methyl-G: 5'-5' connection of the CAP. Protects mRNA and provides stability as well as promotes ribosomal binding.


Chromatin-Remodeling Complex:  Involved in chromatin remodeling; uses ATP hydrolysis to move nucleosomes


Important Concepts:


How do activators function in Eukaryotes?  Are there differences when compared to Prokaryotic activators? Activators bind and stabilize polymerase. They work with polymerase bringing everything needed down to the promoter. Occurs in 3 steps: (1) recruitment of pol II, (2) recruitment of general transcription factors, (3) stabilizatio of polymerase.  Activators recognize a sequence in DNA, the binding domain. The activation domains is used to activate transcription.


How do we know that DNA binding and activation are separable components of Activators?

We know that DNA binding and activation are separable because we have shown this in experiments.  The activating and binding domains of activators can be mixed-and-matched.  The binding region alone bound to DNA will not initiate transcription. If you create an activator that has the binding domain for LexA and the activator for Gal4, the activator will bind to the LexA site, but will activate the Gal4 gene (lac z). 



What problems do nucleosomes cause for transcription?

Nucleosomes cause problems for transcription because they have DNA tightly wrapped around their protein core.  The nucleosome has a "+" charge which holds onto the "-" charged DNA backbone.  This attraction between the two creates a very tight hold on the DNA which prevents polymerase from transcribing the DNA.  In order for this problem to be solved, the polymerase displaces the DNA from the nucleosome and forms a closed loop.  This occurs when the HATs cause acetylation of the histone therefore makes the "+" charge weaker and loosens nucleosomes hold on the DNA.  This loop causes torsion on the DNA and causes it to supercoil.  This allows the nucleosome to keep contact with the DNA behind the RNA polymerase which allows the RNA polymerase to transcribe the DNA without interference.


How do histone tail modifications, particularly acetylation, change chromatin structure?Helicase / Exam IV Review

Addition of acetyl groups to the histone tail adjusts DNA to activate transcription.  The alteration is read to change how the chain is transcribed.  Sine the Lysines have a positive charge on the histone tails, they can hold the negative backbone.  By adding acetyl groups to the histone tail, there is a decrease in the positive charge.  This causes the histone tail to loosen its grip on the DNA, making it more accessible for DNA.


What are the functions of Histone Acetylases and Deacetylases?

Histone acetylases adds acetyl groups which decreases the positive charge causing the histone do detach more from the negatively charged DNA backbone.  This causes the DNA to be more accessible for transcription because acetyl-lysine combinations serve as platforms for proteins, and there is more room between the histone and DNA for proteins to interact.  Histone deacetylase on the other hand acts as an indirect repressor removing the acetyl groups causing the DNA to bind tighter to the histone reducing the amount of transcription because the proteins can't attach. 


How do Repressors work?

Repressors work by a mechanism of competition.  They are proteins that bind to specific sites on DNA and prevent transcription of nearby genes. Repressors either interfere with RNAP binding…the transition from the closed to the open RNAP-DNA complex…or by binding of required activators. 


Topic 16 Eukaryotic Transcriptional Regulation II


Important Vocabulary:


Prader-Willi Syndrome (PWS) - An inherited developmental disorder caused by a eukaryotic imprinting error. Symptoms include but are not limited to hypogonadism, almond shaped eyes, thin upper lip, rapid weight gain within the first six years, and feeding problems. PWS is caused by a gene missing on part of chromosone 15. Normally your parent each pass down a copy of this chromosome. Most patients with PWS are missing the genetic material on part of their father's chromosome. The remaining patients have two copies of their mother's chromosome 15.


Maintenace methylases: In development, when DNA is replicated, the DNA formed is hemimethylated. In order to continue silencing the genes in somatic cells, the maintenance methylase puts methyl groups onto the daughter strand where the corresponding mother strand is methylated.


Imprinting: monoallelic expression of biallelic genes, where one of the two alleles is repressed leaving the expression of the gene completely dependent on either the maternally or paternally inherited gene.


Stem Cell Research: Embryonic cells are termed stem cells because they are at a stage where they can turn into any type of cell needed.  Scientists are very interested in these types of cells because if they could be stimulated to turn into a certain kind of cell needed, then we could use them to replace damaged cells in the body.  There is much controversy about using embryonic cells in this research because the of the sticky situation of trying to determine when a human life actually begins.  This has caused much interest in trying to figure out if a normal adult cell could be unprogramed to become a stem cell again.


Histone Deacetylase:  Removes acetyl groups and restores the positive charge to the histone, allowing the histones to bind tightly with the DNA, thereby preventing transcription, and turning the gene off. 


Important Concepts:


What is the difference between Euchromatin and Heterochromatin?

Euchromatin are areas of DNA where genes are being actively transcribed.  They are less dense areas of chromatin because the histone tails have been acetylated. 

     Heterochromatin is characterized by a complete repression of transcription.  These densely packed areas of chromatin are usually found around centromeres, telomeres, and silent genes.  The heterochromatin is less accessible to externally added proteins than euchromatin. Here there are fully silenced regions of DNA with the complete shut down of any expression and characterized by a great deal of methylation.  


What kind of Modifications of Histones and DNA are associated with active vs. silent chromatin?

For gene silencing, the Histone tails are methylated at particular amino acids. The HP1 protein binds to the methyl groups, attracting HDACs and compacting chromatin. The DNA is methylated at CG nucleotides which attracts MeCP2.  This will attract HDACs and deacetylate histone tails to compact chromatin.

For gene activation, demethylation of DNA and Acetylation of histone tails.  The acetylation of the tails loosens the grip of nucleosomes on DNA allowing for access to the DNA.


What is imprinting and how does it work?

Imprinting is the turning on or off of parental genes in offspring.  Some genes are inherited only from the mother, while others are inherited only from the father.  Imprinting occurs through DNA methylation.  When methylation occurs near the promoter of a gene, transcription of this gene is inhibited.  In the case of imprinting, offspring usually inherit normal (potentially functional) copies of the gene from both parents; however, only one copy is expressed because the other is silenced. Imprinting is an alteration by chemicals, not a change in the DNA sequence.


How does methylation ensure the inheritance of gene activation states?

As chromosomes pass through the male and female germlines they must acquire some imprint to signal a difference between paternal and maternal alleles in the developing organism. In order for this to be done correctly, some mechanism must be able to distinguish between the two different inherited alleles. Allele-specific DNA methylation is a key component in maintaining the imprinted status and to ensure that certain genes are activated abnd others are not. When methylation occurs, the chromatin is highly compacted, acetylation is decreased and certain genes have been silenced in order for only the desired genes to be expressed.


Why would you want to reprogram the chromatin organization of a cell?

In some organs genes are silenced where they are left on in other organs. In stem cell research the cells are blank with only the master imprint on them. The cells that are designated for certain regions of the body silence some genes and turn on others. If you erase all the chromatin organization you can activate or silence any gene so that it can become any cell type wanted.


Topic 17 RNA Structure, Ribozymes, and Regulatory RNA


Important Vocabulary:

Ribozyme- These are the enzymes made of RNA that catalyze the hydrolysis of the RNA phosphodiester bonds.  The ribozyme can either cleave itself or another molecule of RNA. The discovery of riboenzymes was innovative becuase it was originally thought that only proteins catalyzed reactions. A key example is the Hammerhead Riboenzyme. The Hammerhead Ribozyme is a structure with three stems. RNA cleaves itself when it folds into this shape.


miRNA- microRNAs are single stranded RNA molecules that are 20-21 nucleotides long that function in gene regulation. miRNAs are encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (non-coding RNA). miRNA is complimentary to a part of one or more messenger RNAs (mRNAs). 


siRNA - small interfering RNAs, produced invitro and added to cells, consist of 20-25 nucleotide-long double-stranded RNA molecules which interfere with the expression of a gene.


Drosha- is an RNAse III enzyme responsible for initiating the process of microRNA. Drosha interacts with RISC (RNA-induced silencing complex) to induce cleavage of mRNA.  It is involved in the first cleavage, and leaves a 3' overhang.


Dicer- RNAse which further processes microRNA from Drosha to GUIDE RNA (AKA mature miRNA) which becomes single stranded from the dicer which induces the second cleavage that releases the mature miRNA which then joins with the RISC complex or gets amplified.



Alkali hydrolysis - a chemical process in which a molecule is cleaved into two parts by the addition of a molecule of water in a solution with a pH greater than 7.  The 2-prime OH acts as a nucleophile which makes the RNA extremely unstable and breaks into two molecules.


Hammerhead Ribozymes- small self-cleaving RNA found in several of the viroids associated with plant RNA viruses and other species. Hammerheads replicate through a rolling circle mechanism which catalyze the sequence-specific cleavage of RNA phosphodiester bonds due to the three-dimensional structure that they form. The secondary structure resembles a hammerhead giving them their name.


RISC complex- (RNA-induced Silencing Complex) obtains ss guide RNA (siRNA) and uses it as a template for recognizing complementary mRNA and degrades them, resulting in decreases levels of protein translation. When it finds this matching RNA, it activates the RNase and cleaves the RNA. It also has a PAZ domain that binds the 3 prime end of RNA and an AGO2 protein, an endonuclease, which mediates mRNA cleavage.


Important Concepts:

What makes RNA different than DNA?

RNA is different than DNA in several ways. The main primary difference is that RNA is single stranded in nature while DNA is double stranded. The nitrogenous base Uracil is commonly found in RNA (instead of Thymine). RNA utilizes a ribose ring sugar featuring a 2' OH group (which enables many of the chemical reactions RNA undergoes). In contrast, DNA utilizes a deoxyribose sugar. Also, RNA features 5' --> 3' polarity.  RNA also can fold up on itself forming a stem loop and can act a lot like a protein.  There are many different types of RNA such as transfer, messenger, micro, etc. with only one type of DNA.  RNA also contains non watson and crick pairing where two Us can pair up.  RNA's flexibility and functions make it unique and even though it can transcribe DNA they are very different molecules. 


What are the unique folding properties of single stranded RNA?

RNA has the power to fold in a very specialized fashion. For example, RNA bends to form hairpin loops to terminate transcription. A specific example of a hairpin loop is a Tetraloop.  They are 4 bases that are common in rRNA, and often cap double helices.  RNA also has the ability to undergo Non-Watson Crick Pairing which could allow typically non-pairing bases such as G-U to pair together. Triple base pairing is also possible (U-A-U). 


What is mechanism by which RNA undergoes alkali hydrolysis?

When RNA folds up on itself, it allows for alkali hydrolysis.  The 2'-OH group on the RNA can cleave the RNA.


What are Ribozymes and how do they work?

Ribozymes are enzymes made of RNA that catalyze the hydrolysis of phosphodiester bonds in RNA which allows the RNA to be cleaved based on base pairing. An example is the hammerhead ribosome.


What is the difference between miRNA and siRNA?

miRNA (micro) are pieces of sequence found in the genome that are encoded.  siRNA (small interfering) are pieces of sequence made and synthesized in the lab, and added to cells in vitro.


Where are miRNAs coded for?

miRNAs are found in both coding and noncoding regions where folding is found.


Why are miRNAs generally about 20-21 nucleotides long?

The miRNA matures for the pre-miRNA in two steps.  The first steps starts with a secoundary structure that is formed by the pre-microRNA this is made up of the basal segments, lowers stem (11bp), upper stem (about 22 bp) and the termanial loop.  Drosha recognizes this structure is the first on to cleave at +1 and -2 site at the begaining of the upper stem and leaves a 3' overhang.  Drosha releases this part of the RNA from the big chain of RNA.  Then Dicer comes and cleaves at the end of the upper steam by the termanl loop which reases the mature miRNA which becomes the guide and sent off to the risc complex.   This is why the miRNA is 20-21 base paires becuse the upper stem is about 22 basepairs and the shape always has to occure so Drosha and  Dicer can recoginze and cut the RNA.


Why can the RNases Drosha and Dicer process all miRNAs? Because these enzymes recognize the stem loop structure or the miRNA's secondary structure, thus a specific sequence is not needed so all miRNAs with this structure can be processed by Drosha and Dicer.


How does RISC function?- The RISC complex with a bound siRNA recognizes complementary messenger RNA (mRNA) molecules and degrades them, resulting in dramatically decreased levels of protein translation and turns off the gene.


RNA-induced silencing complex (RISC)- is a multiprotein complex that incorporates one strand of a small interfering (siRNA). RISC uses the siRNA as a template for recognizing complementary mRNA. When it finds a complementary strand, it activates RNase and cleaves the RNA. This process is important in gene regulation.


Topic 18 RNA Splicing


Important Vocabulary:


A great video on RNA splicing with spliceosome function:  http://vcell.ndsu.edu/animations/mrnasplicing/movie.htm


snRNPs- small nuclear ribonucleoproteins. A complex made of both snRNA (small nuclear RNA) and protein.  The snRNA works as the spliceosome which is the most important part of this complex, while the protein facilitates the function of the snRNA.


U1 snRNA- marks 5' junction with complementary RNA strand on intron


U2 snRNA- prepares branch point "A"


Exon- a mature RNA molecule that is left after trans- or cis-splicing.  Contains a 5' and a 3' untranslated regions (UTR) and also contains codons that will be in the final mRNA. 


Intron- A DNA region in a gene that is not translated into a protein.  It is cut (or spliced) out of the gene, leaving only the exons to be translated.


ESE- Exon Sequence Enhancers are sequences in the exon bound by SR Proteins and function to attract U1 snRNP and help recruit U2 snRNP


SR Proteins- Serine- and Arganine-rich RNA binding proteins


Exon splicing inhancers-  in the exon sequince, bound by SR proteins. Ensure correct juncture is made during splicing, by bringing U1 snRNP from 5' to U2 snRNP.


Splicing repressor - convert and inhibits splicosomes (splicing machinery) from binding, so that mRNA remains unspliced.


Self-splicing - When introns fold up and facilitate chemstritry without snRNP. 2' OH attacts 5' splice site forming a larriat. The 3' OH of the 5' exons uses transesterification to bind to 3' splice site joining exons together. Example Tetrahymena.


Northern Blot:  Targets RNA by using a DNA or RNA probe.  The probe will match the gene your looking for.  Run RNA on gel.  With this kind of blot you can answer question such as where is the gene expressed and how many different RNA's are made.


Alternative splicing - The process in which more than one combination of exons can be used for one gene. This enables the gene to be multi-functional (ie to have one function in one area of the body, such as the brain, and to have another function in another area, such as the skin).


Important Concepts:


What is splicing?

Splicing is the removal of introns between exons which result in the covalent bonding of exons together.  This process is performed by the spliceosome which makes cuts in the RNA that cause the intron to fold and form a lariat, leaving a free exon.  The lariat is then cut from the other exon and the two exons are joined together with the intron removed.  Splicing occurs much more frequently in Eukaryotes than in Prokaryotes.  Splicing allows for more flexibility in the different types of proteins that can be encoded, which means less genetic material is needed if it can serve multiple purposes.


What sequences are required for the splicing of introns?

In order to splice the introns out of the sequence, the GU-AG rule is followed. This rule indicates that a G-U sequence on the 5' splice site is recognized close to an A-G sequence on the 3' splice site.  A branch site toward the middle of the intron is also required; this sequence contains an adenine that plays a key role in intron removal.


What is the function of U1 and U2 snRNPs?  How is base pairing important for their function?

snRNPs are proteins which combine with pre-mRNA and other proteins to form spliceosomes.  SnRNPs recoginize places along a strand of pre-mRNA and are necessary to remove introns which is why base pairing is important.  They are found in the nuclei of the cell.  U1 and U2 function in specifically interacting with the SMN protein and other proteins to form the SMN complex.  U1's 5' end forms complementary base pairs with the splice junction defining the donor site of the intron which is also why base pairing is important.  Complementary binding also occurs between U2 and the branchpoint sequence of the intron resulting in bulging of unpaired Adenosine on the BPS which causes attack of 5' splice site causing the 1st of 2 transesterification reactions that aid in splicing.


How are self-splicing introns related to the spliceosome?

Self-splicing introns fold and splice the exact same way as the spliceosomes but without using any ribonucleoproteins to facilitate the splicing event.  In the case of the self-splicing, the intron folds up to facilitate the chemistry.  The spliceosome, however, snRNAs fold up and replace the intron to facilitate the binding of the exons.


What is alternative splicing?

Alternative splicing is the RNA splicing mechanism in which the exons of the primary gene transcript, the pre-mRNA, are separated and reconnected to produce alternative ribonucleotide arrangements. These linear combinations then undergo the process of translation where specific and unique sequences of amino acids are specified, resulting in different types of proteins. Alternative splicing allows for the synthesis of a greater variety of proteins. There are several different types of alternative splicing including exon skipping, intron retaining mode, and alternative donor/acceptor site mode.


How do Splicing repressors and Activators work?

Attraction and repulsion of the splicing machinery plays a role in which introns are eventually cut out. Splicing repressors function by binding at the repressor site and then covering/blocking the splicing site, preventing hte spliceosome from binding a particular spot. When repressors are present, no introns are cut out of the sequence. Splicing activators function by binding to the RNA at the splicing enhancer site and attracting the spliceosome to the RNA. This allows for introns to be removed from hte sequence, giving a spliced RNA end product.


How can Northern blots, RT-PCR, and In Situ Hybridization be used to learn about a particular gene’s mRNA and expression pattern?

Northern blotting is used to study gene expression by detection of isolated mRNA in a sample.Levels of mRNA can be quantitatively measured by Northern blotting, a process which involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe complementary to part of or the entire target sequence.. Quantification is done by measuring band strength in an image of a gel. Northern blotting allows the discrimination of alternately spliced transcripts.With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions.

RT-PCR measures mRNA abundance and can produce an absolute measurement such as number of copies of mRNA per nanolitre of homogenized tissue.

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific mRNA sequence in a portion or section of tissue. For ISH sample cells and tissues are usually treated to fix the target transcripts in place and to increase access of the probe. The probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed away. Then, the probe that was labeled with either radio-, fluorescent- or antigen-labeled bases is localized and quantitated in the tissue using either autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes, labeled with radioactivity or the other non-radioactive labels, to simultaneously detect two or more transcripts.

With all of these reactions, if no product is produced, then the gene is not producing mRNA and it will not be expressed.


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