Homework
Curious about how to answer these questions? Want to see an example of a homework answer? See this page for an example homework answer.
Database Questions
Somewhere out on the Internet is a database of restriction enzymes.
a. Where is it located? What is the URL for the database file that could be used with the GCG software?
b. What does a typical entry look like for the restriction enzyme file that is formatted for use with the MacVector program?
c. How is the database (formatted for MacVector) organized?
1. What is the delimiter between individual restriction enzyme entries? How does the computer (or you) know when the information from one restriction enzyme stops and another one starts?
2. Is this format similar to the format used by any other database? Which one?
Literature Search Questions
1) Select a protein and find the entries for this protein in the GenBank DNA database, the SwissProt database, and the PDB Protein database. List the attributes or features that are common to the databases and those which are unique to each.
2) How many secreted proteins have been discovered in humans? Explain what database you used, and what keywords you used to do the search.
Friday, October 19, 2007
Bioinformatics Workshop 3
Literature Workshop
In this workshop, we will be exploring how to search for sequence information using various web sites.
Web Resources
NCBI
Visit the NCBI website at http://www.ncbi.nlm.nih.gov
You have different options for searching for sequence information by querying the sequence annotation.
ENTREZ
PUBMED
OMIM
and several other databases
They are all linked together with links into the sequence databases.
When you do a search, you have to first ask yourself these questions
What information are you looking for?
What database would have that information?
Can you restrict your search to certain fields?
Try searching for literature about human growth hormone.
What MeSH term should you be using for that molecule?
What Database should you be searching?
Often, the best way to find something is to first, do some searches, assign some limits, then view the "history" and combine some queries. You can also do this using the Preview/Index option.
PDB
What is the unique identifying code for a protein structure of Lysozyme? You will find lots of lysozymes. Just pick one.
http://www.rcsb.org
SRS at EBI
http://srs.ebi.ac.uk
Search for Human Growth Hormone using the SRS search program.
How does the SRS search program differ from the NCBI search program that you used today.
In this workshop, we will be exploring how to search for sequence information using various web sites.
Web Resources
NCBI
Visit the NCBI website at http://www.ncbi.nlm.nih.gov
You have different options for searching for sequence information by querying the sequence annotation.
ENTREZ
PUBMED
OMIM
and several other databases
They are all linked together with links into the sequence databases.
When you do a search, you have to first ask yourself these questions
What information are you looking for?
What database would have that information?
Can you restrict your search to certain fields?
Try searching for literature about human growth hormone.
What MeSH term should you be using for that molecule?
What Database should you be searching?
Often, the best way to find something is to first, do some searches, assign some limits, then view the "history" and combine some queries. You can also do this using the Preview/Index option.
PDB
What is the unique identifying code for a protein structure of Lysozyme? You will find lots of lysozymes. Just pick one.
http://www.rcsb.org
SRS at EBI
http://srs.ebi.ac.uk
Search for Human Growth Hormone using the SRS search program.
How does the SRS search program differ from the NCBI search program that you used today.
Bioinformatics Workshop 2
Sequence Database Workshop
Downloading files from the Internet using your Web browser
Start up your web browser and go to this URL where you can download files using ftp (File Transfer Protocol).
ftp://ftp.ncbi.nih.gov
Find the "gbrel.txt" file and look at it
Do not click on any other file. These are multi-Gigabyte database files and you don't want to download them.
This gbrel.txt file contains the release information for the GenBank database.
Pay attention to the
Size (number of sequences, number of nucleotides, number of species)
Divisions (The database is not a single file, but a collection of files)
In the next part of the workshop, we will be downloading data from sequence databases.
Data Conversion
1. Go to the NCBI Web site
http://www.ncbi.nlm.nih.gov
2. In the Nucleotide database, find the accession number, and download this sequence
Homo sapiens hemoglobin beta chain mRNA complete cds.
There are many hemoglobin sequences in the database. You need to find the specific one that has this description line.
Examine the sequence. Anything look strange for a mRNA sequence?
3. Convert sequences to FASTA format. Why do we need to do this?
4. Translate the RNA into Protein
translate
At what nucleotide should you start the translation?
5. Convert the protein back to RNA (reverse translation or back translation)
backtranslate
What Codon Preference Table should you use? Why do you even need a Codon Preference Table?
Did you get the same nucleotide sequence you started with?
We have software that can answer this question.
Using LALIGN, compare these two nucleotide sequences. We will discuss this program more in an upcoming lecture.
Downloading files from the Internet using your Web browser
Start up your web browser and go to this URL where you can download files using ftp (File Transfer Protocol).
ftp://ftp.ncbi.nih.gov
Find the "gbrel.txt" file and look at it
Do not click on any other file. These are multi-Gigabyte database files and you don't want to download them.
This gbrel.txt file contains the release information for the GenBank database.
Pay attention to the
Size (number of sequences, number of nucleotides, number of species)
Divisions (The database is not a single file, but a collection of files)
In the next part of the workshop, we will be downloading data from sequence databases.
Data Conversion
1. Go to the NCBI Web site
http://www.ncbi.nlm.nih.gov
2. In the Nucleotide database, find the accession number, and download this sequence
Homo sapiens hemoglobin beta chain mRNA complete cds.
There are many hemoglobin sequences in the database. You need to find the specific one that has this description line.
Examine the sequence. Anything look strange for a mRNA sequence?
3. Convert sequences to FASTA format. Why do we need to do this?
4. Translate the RNA into Protein
translate
At what nucleotide should you start the translation?
5. Convert the protein back to RNA (reverse translation or back translation)
backtranslate
What Codon Preference Table should you use? Why do you even need a Codon Preference Table?
Did you get the same nucleotide sequence you started with?
We have software that can answer this question.
Using LALIGN, compare these two nucleotide sequences. We will discuss this program more in an upcoming lecture.
Bio-informatics Workshop 1
These series of workshops are those given to us in our bioinformatics class by Prof Lee Kozar, who is also the director of CMGM at Stanford!
Downloading files from the Internet using your Web browser
Start up your web browser and go to this URL
ftp://ftp.ncbi.nih.gov
Find the "gbrel.txt" file and look at it
This file contains the release information for the GenBank database.
Pay attention to the
Size (number of sequences, number of nucleotides, number of species)
Divisions (The database is not a single file, but a collection of files)
In the next class, we will analyze it more fully and learn how to download specific sequences.
Downloading files from the Internet using your Web browser
Start up your web browser and go to this URL
ftp://ftp.ncbi.nih.gov
Find the "gbrel.txt" file and look at it
This file contains the release information for the GenBank database.
Pay attention to the
Size (number of sequences, number of nucleotides, number of species)
Divisions (The database is not a single file, but a collection of files)
In the next class, we will analyze it more fully and learn how to download specific sequences.
Free software to read sequencing data
These links were given to us by Dr. B after completion of our sequencing experiments.
Chromas sequence viewing software:
chromas11-32.exe (118.426 Kb)
Chromas Lite:
chromaslite201.exe (215.945 Kb)
Link to get sequence scanner:
http://www.appliedbiosystems.com/support/software_community/free_ab_software.cfm
Chromas sequence viewing software:
chromas11-32.exe (118.426 Kb)
Chromas Lite:
chromaslite201.exe (215.945 Kb)
Link to get sequence scanner:
http://www.appliedbiosystems.com/support/software_community/free_ab_software.cfm
An example of a good summary and critical analysis
This was posted by Dr Claudia Stone as a template for us to follow.
Lammich et al. (2004) Expression of the Alzheimer protease BACE1 is suppressed via its 50-untranslated region.
1) Summary:
· Specific questions
o The entire study focused around whether the 5’-untranslated region of BACE1 mRNA was responsible for translational repression of the BACE1 protein, and specifically what characteristics of the 5’UTR are responsible for the observed repression.
Theoretical context
It is assumed that the cause of Alzheimer’s disease is linked to the collection of the amyloid β-peptide (Aβ).
This is created by the actions of two proteases on the membrane amyloid precursor protein.
γ- secretase
β- secretase
Also known as BACE1
Previous studies.
Vassar, 2002
Mice with targeted deletion of BACE1 do not produce any Aβ.
These mice show no any overt phenotype making BACE1 an ideal drug target.
Fukumoto et al, 2002; Holsinger et al, 2002; Yang et al, 2003
BACE1 protein levels are significantly upregulated in the brains of AD patients compared with non-AD controls.
Yasojima et al, 2001; Holsinger et al, 2002; Preece et al, 2003
These increased BACE1 levels corresponded to unchanged mRNA levels.
Suggests that post-transcriptional mechanisms are at play.
Why pinpoint the 5’UTR as the key?
The structure of this segment is:
446 nucleotides long
GC content of 77%
Three uORFs
All of these characteristics are assumed to be important for the inhibition of translation.
Importance of these questions
Determining mechanisms for the regulation of the BACE1 protein, especially at the translational level, would give ideal targets for future therapy in the progression and prevention of Alzheimer’s disease.
Key experiments with results
Determined whether the 5’ UTR may affect the expression of BACE1.
Expression vectors encoding the ORF of BACE1 alone, with the 5’UTR, or with the 3’UTR were transiently transfected into human embryonic kidney HEK293 cells.
Detection done via immunoblotting of the cell lysate.
Showed the presence of the 5’UTR greatly reduced BACE1 protein levels.
Determined whether the BAE1 5’ UTR could inhibit the expression of a similar downstream open reading frame different from BACE1.
Vectors encoding luciferase with or without the 5’UTR, or an empty control vector, were expressed in HEK293 cells.
Luciferase activity was measured in cell lysates.
Luciferase activity was greatly reduced in cells containing the 5’UTR of BACE1.
Proved that the 5’UTR lowered BACE1 protein levels by selectively reducing the translation of BACE1.
Vectors encoding BACE1, with or without the 5’UTR or empty control vector, were expressed in HEK293 cells.
BACE1 protein was measured by immunoblotting cell lysates.
mRNA levels were measured via northern blotting.
The presence of the 5’UTR had no significant effect on mRNA levels, while simultaneously showing lowered BACE1 protein levels.
Additionally demonstrated that the 5’UTR represses the expression of BACE1 at the translational level.
In vitro-transcribed BACE1 mRNA, with and without the 5’UTR, were translated in a nuclease-treated rabbit reticulocyte lysate.
BACE1 protein was detected without the 5’UTR, however was not observed when using the 5’UTR.
Determined whether the high GC content of the long 5’UTR is sufficient for repressing BACE1 expression or whether the uORFs and their encoded short peptides are required
Mutated the start codon of three uORFs from ATG to ATA
Mutated BACE1 plasmids were transfected into HEK293-APP cells.
BACE1 protein levels were measured in the cell lysate by immunoblotting.
Combined mutations of upstream ATGs showed a slight but significant increase in BACE1 expression compared with single mutations
Reveals that the uORFs account for only partial repression of BACE1 expression
Investigated the effect of several deletion mutants of the 5’UTR of BACE 1 with lowered GC content.
Mutations occurred either at nucleotides 1-223, 224-446, or 1-390 and the expression of BACE1 protein was measured.
Showed that both the 5’- and the 3’half of the UTR have a strong inhibitory effect, with it being more pronounced at the 5’ end.
2) Critical Analysis
The article is a systematic progression of the author’s ideas and logic in the development of the study. The reader is given a clear background discussion to serve as the introduction to the topic and why the author has chosen to formulate such a study. Each experiment is explained in the results and discussion section, and reasoning is given unto why the next experiment should be carried out. Additionally, each experiment is explained concisely, with the necessary specifics laid out in the methods section, allowing the reader to follow the thought process of the author and constantly anticipate the next direction of the study.
There is more than enough evidence supporting the author’s claim that the 5’UTR is responsible for repressing the translation of BACE1 without the need to repress transcription. Most of the experiments were designed to test this hypothesis explicitly, and even when it had been proven with an experiment, the study goes one step further by confirming it with an additional experiment.
The study loses the flow of direct evidence during the discussion that the “GC-rich region of the 5’UTR forms a constitutive transition barrier, which may prevent the ribosome from efficiently translating the BACE1 mRNA.” The author states that the 5’UTR repression is functioning because of either the high GC content, or because of the uORFs. An experiment is conducted that refutes the idea of the uORFs, however the author immediately states that it must because of the GC content creating a tightly folded secondary structure. Although computer modeling (MFOLD program) of the 5’UTR shows that it’s free energy is sufficient for inhibiting translation, no subsequent testing of this ribosome blocking theory is carried out. An additional experiment is carried out which shows that substituting certain regions of the GC-rich sections of the 5’UTR does indeed increase expression of the BACE1 protein. My belief is that the author is using the previous studies of Wood et al, 1996 and Clemens & Bommer, 1999 as support for the ribosome assumption, but without direct reference.
I agree with the author that these studies are important. The mere fact that there are over 24 million cases of dementia worldwide with around 60% due to AD, shows that identifying a specific mechanism and target for therapy could benefit many individuals. Because the specific, and probably varied, cause of the disease remains undiscovered, the ability to block a mechanism this far downstream would negate may factors that reside earlier, such as at the chromosomal level. Thus a treatment designed at this point could be applied to many patients regardless of disease origin.
Lammich et al. (2004) Expression of the Alzheimer protease BACE1 is suppressed via its 50-untranslated region.
1) Summary:
· Specific questions
o The entire study focused around whether the 5’-untranslated region of BACE1 mRNA was responsible for translational repression of the BACE1 protein, and specifically what characteristics of the 5’UTR are responsible for the observed repression.
Theoretical context
It is assumed that the cause of Alzheimer’s disease is linked to the collection of the amyloid β-peptide (Aβ).
This is created by the actions of two proteases on the membrane amyloid precursor protein.
γ- secretase
β- secretase
Also known as BACE1
Previous studies.
Vassar, 2002
Mice with targeted deletion of BACE1 do not produce any Aβ.
These mice show no any overt phenotype making BACE1 an ideal drug target.
Fukumoto et al, 2002; Holsinger et al, 2002; Yang et al, 2003
BACE1 protein levels are significantly upregulated in the brains of AD patients compared with non-AD controls.
Yasojima et al, 2001; Holsinger et al, 2002; Preece et al, 2003
These increased BACE1 levels corresponded to unchanged mRNA levels.
Suggests that post-transcriptional mechanisms are at play.
Why pinpoint the 5’UTR as the key?
The structure of this segment is:
446 nucleotides long
GC content of 77%
Three uORFs
All of these characteristics are assumed to be important for the inhibition of translation.
Importance of these questions
Determining mechanisms for the regulation of the BACE1 protein, especially at the translational level, would give ideal targets for future therapy in the progression and prevention of Alzheimer’s disease.
Key experiments with results
Determined whether the 5’ UTR may affect the expression of BACE1.
Expression vectors encoding the ORF of BACE1 alone, with the 5’UTR, or with the 3’UTR were transiently transfected into human embryonic kidney HEK293 cells.
Detection done via immunoblotting of the cell lysate.
Showed the presence of the 5’UTR greatly reduced BACE1 protein levels.
Determined whether the BAE1 5’ UTR could inhibit the expression of a similar downstream open reading frame different from BACE1.
Vectors encoding luciferase with or without the 5’UTR, or an empty control vector, were expressed in HEK293 cells.
Luciferase activity was measured in cell lysates.
Luciferase activity was greatly reduced in cells containing the 5’UTR of BACE1.
Proved that the 5’UTR lowered BACE1 protein levels by selectively reducing the translation of BACE1.
Vectors encoding BACE1, with or without the 5’UTR or empty control vector, were expressed in HEK293 cells.
BACE1 protein was measured by immunoblotting cell lysates.
mRNA levels were measured via northern blotting.
The presence of the 5’UTR had no significant effect on mRNA levels, while simultaneously showing lowered BACE1 protein levels.
Additionally demonstrated that the 5’UTR represses the expression of BACE1 at the translational level.
In vitro-transcribed BACE1 mRNA, with and without the 5’UTR, were translated in a nuclease-treated rabbit reticulocyte lysate.
BACE1 protein was detected without the 5’UTR, however was not observed when using the 5’UTR.
Determined whether the high GC content of the long 5’UTR is sufficient for repressing BACE1 expression or whether the uORFs and their encoded short peptides are required
Mutated the start codon of three uORFs from ATG to ATA
Mutated BACE1 plasmids were transfected into HEK293-APP cells.
BACE1 protein levels were measured in the cell lysate by immunoblotting.
Combined mutations of upstream ATGs showed a slight but significant increase in BACE1 expression compared with single mutations
Reveals that the uORFs account for only partial repression of BACE1 expression
Investigated the effect of several deletion mutants of the 5’UTR of BACE 1 with lowered GC content.
Mutations occurred either at nucleotides 1-223, 224-446, or 1-390 and the expression of BACE1 protein was measured.
Showed that both the 5’- and the 3’half of the UTR have a strong inhibitory effect, with it being more pronounced at the 5’ end.
2) Critical Analysis
The article is a systematic progression of the author’s ideas and logic in the development of the study. The reader is given a clear background discussion to serve as the introduction to the topic and why the author has chosen to formulate such a study. Each experiment is explained in the results and discussion section, and reasoning is given unto why the next experiment should be carried out. Additionally, each experiment is explained concisely, with the necessary specifics laid out in the methods section, allowing the reader to follow the thought process of the author and constantly anticipate the next direction of the study.
There is more than enough evidence supporting the author’s claim that the 5’UTR is responsible for repressing the translation of BACE1 without the need to repress transcription. Most of the experiments were designed to test this hypothesis explicitly, and even when it had been proven with an experiment, the study goes one step further by confirming it with an additional experiment.
The study loses the flow of direct evidence during the discussion that the “GC-rich region of the 5’UTR forms a constitutive transition barrier, which may prevent the ribosome from efficiently translating the BACE1 mRNA.” The author states that the 5’UTR repression is functioning because of either the high GC content, or because of the uORFs. An experiment is conducted that refutes the idea of the uORFs, however the author immediately states that it must because of the GC content creating a tightly folded secondary structure. Although computer modeling (MFOLD program) of the 5’UTR shows that it’s free energy is sufficient for inhibiting translation, no subsequent testing of this ribosome blocking theory is carried out. An additional experiment is carried out which shows that substituting certain regions of the GC-rich sections of the 5’UTR does indeed increase expression of the BACE1 protein. My belief is that the author is using the previous studies of Wood et al, 1996 and Clemens & Bommer, 1999 as support for the ribosome assumption, but without direct reference.
I agree with the author that these studies are important. The mere fact that there are over 24 million cases of dementia worldwide with around 60% due to AD, shows that identifying a specific mechanism and target for therapy could benefit many individuals. Because the specific, and probably varied, cause of the disease remains undiscovered, the ability to block a mechanism this far downstream would negate may factors that reside earlier, such as at the chromosomal level. Thus a treatment designed at this point could be applied to many patients regardless of disease origin.
Sunday, October 7, 2007
Summary and Critical Analysis - Sample 1
As part of my Molecular and Cell Biology course work, I have to routinely write summaries and critical analyses of scientific articles. Though not perfect, these few samples may give you an idea of what to put down.
Paper 1: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1299076
Summary and Critical Analysis: Expression of the Alzheimer protease BACE1 is suppressed via its 5’- untranslated region
Summary:
BACE1 or Beta site amyloid precursor protein (APP) cleaving enzyme 1 is a membrane protein involved with the secretory pathway, and hence is localized to endosomes, trans golgi apparatus and the plasma membrane. BACE1 is expressed primarily in the brain and the pancreas. Previous research has shown an interesting link between BACE1 and Alzheimer’s disease. It was observed that Alzheimer’s disease patients expressed elevated levels of BACE1 protease. Alzheimer’s disease is presumed to be caused by the formation and aggregation of amyloid β peptides (Aβ). Studies have shown that Aβ peptides are formed by the cleavage of APP by BACE1. It was also noted that despite elevated protein levels, mRNA levels of BACE1 were not affected in AD patients. In attempt to analyze the cause for an elevation in BACE1 levels among Alzheimer’s disease patients, Lammich et al have proposed that the 5’ untranslated region plays a regulatory role in normal adults. Once conclusively proven that 5’ UTR is the defective portion in Alzheimer’s disease, they contend that a cure for the disease can be attempted.
In order to study the role of the 5’UTR in BACE1 expression, the authors first characterized the region and conformed that it is 446bp long, has 77% GC content and contains 3 upstream ORFs. They also presume that the 5’UTR plays an important role as a major part is highly conserved in humans, mice and rats. In their initial experiments, the authors transfected HEK293 cells with BACE1 ORF, BACE1 + 5’UTR and BACE1 + 3’UTR. They observed that BACE1 protein levels were significantly less in cells transfected with BACE1 + 5’UTR. 3’UTR did not seem to play a significant role in BACE1 inhibition. In order to determine the regulatory role of the 5’UTR region, they transfected HEK293 cells with the lucferase ORF and luciferase containing the 5’UTR of BACE1. They observed that cells containing the 5’UTR expressed very low levels of luciferase indicating that the 5’UTR indeed played a role in repressing the expression of a downstream ORF.
In the next portion of their study, Lammich et al proposed that the BACE1 5’UTR affected translation and not transcription. In order to prove this, they first studied the levels of BACE1 proteins in transfected HEK293 cells by immunoblotting and also studied the levels of mRNA by northern blotting. They observed that levels of mRNA were not affected even though BACE1 proteins levels in those cells was reduced up to 40 fold. To further confirm their finding, they repeated the experiment in nuclease treated rabbit reticulocyte lysate and obtained similar results.
In order to show that the results obtained were not cell-type dependant, the experiments were repeated in African green monkey COS7 cells and also in human neuroglioma H4 cells. Similar results were obtained in all the cell types confirming that the regulatory effect of the 5’UTR was cell-type independent. In order to prove that the results were not promoter dependent, the experiments were repeated with two other promoters CMV and EF1α and similar results were obtained.
In order to determine the exact portion of the 5’UTR playing a repressive role, they conducted various experiments with different portions of the 5’UTR deleted. They observed that though deletions in the three uORFs showed increased BACE1 levels, the increase were not as significant as when the 5’UTR was completely absent. Hence, Lammich et al propose that the stable secondary structure of the BACE1 5’UTR rich in GC forms many stem loops which inhibit translation in vitro.
Critical Analysis:
CDC estimates that Alzheimer’s disease is a seriously debilitating illness affecting about 4 million Americans. Being a leading cause of dementia in older adults, there is an urgent need to understand and find a cure to Alzheimer’s. In attempting to solve this problem, Lammich et al have indeed chosen a very important issue.
The paper is very concise and to the point, with ample illustrations of results obtained. Though very clear and concise in most parts, I would have appreciated further explanation about Fig 1B, which I found very difficult to analyze.
The author’s use of COS7 cells and H4 cells in addition to HEK293 cells helps reduce the probability of cell-type influence on the results, but I am unsure if they are sufficient to eliminate all possibility of cell-type effects. More experiments are probably required, using other cell types, like pancreatic cells, to confirm the results obtained. Further studies to analyze the actual structure of the 5’UTR and the mechanism of repression of translation would also prove to be very beneficial.
Another key question not answered in this paper is the role of BACE1 in normal brain cells. The authors mention that BACE1 levels are primarily elevated in the brain and in pancreas, and they also mention a possible secretory role for BACE1, which does not explain the higher occurrence of BACE1 in healthy brain cells.
In conclusion, I believe that this paper is very informative but further research is needed to get closer to understanding BACE1 and the 5’UTR of BACE1 and their role in the prevention and cure of Alzheimer’s disease.
Paper 1: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1299076
Summary and Critical Analysis: Expression of the Alzheimer protease BACE1 is suppressed via its 5’- untranslated region
Summary:
BACE1 or Beta site amyloid precursor protein (APP) cleaving enzyme 1 is a membrane protein involved with the secretory pathway, and hence is localized to endosomes, trans golgi apparatus and the plasma membrane. BACE1 is expressed primarily in the brain and the pancreas. Previous research has shown an interesting link between BACE1 and Alzheimer’s disease. It was observed that Alzheimer’s disease patients expressed elevated levels of BACE1 protease. Alzheimer’s disease is presumed to be caused by the formation and aggregation of amyloid β peptides (Aβ). Studies have shown that Aβ peptides are formed by the cleavage of APP by BACE1. It was also noted that despite elevated protein levels, mRNA levels of BACE1 were not affected in AD patients. In attempt to analyze the cause for an elevation in BACE1 levels among Alzheimer’s disease patients, Lammich et al have proposed that the 5’ untranslated region plays a regulatory role in normal adults. Once conclusively proven that 5’ UTR is the defective portion in Alzheimer’s disease, they contend that a cure for the disease can be attempted.
In order to study the role of the 5’UTR in BACE1 expression, the authors first characterized the region and conformed that it is 446bp long, has 77% GC content and contains 3 upstream ORFs. They also presume that the 5’UTR plays an important role as a major part is highly conserved in humans, mice and rats. In their initial experiments, the authors transfected HEK293 cells with BACE1 ORF, BACE1 + 5’UTR and BACE1 + 3’UTR. They observed that BACE1 protein levels were significantly less in cells transfected with BACE1 + 5’UTR. 3’UTR did not seem to play a significant role in BACE1 inhibition. In order to determine the regulatory role of the 5’UTR region, they transfected HEK293 cells with the lucferase ORF and luciferase containing the 5’UTR of BACE1. They observed that cells containing the 5’UTR expressed very low levels of luciferase indicating that the 5’UTR indeed played a role in repressing the expression of a downstream ORF.
In the next portion of their study, Lammich et al proposed that the BACE1 5’UTR affected translation and not transcription. In order to prove this, they first studied the levels of BACE1 proteins in transfected HEK293 cells by immunoblotting and also studied the levels of mRNA by northern blotting. They observed that levels of mRNA were not affected even though BACE1 proteins levels in those cells was reduced up to 40 fold. To further confirm their finding, they repeated the experiment in nuclease treated rabbit reticulocyte lysate and obtained similar results.
In order to show that the results obtained were not cell-type dependant, the experiments were repeated in African green monkey COS7 cells and also in human neuroglioma H4 cells. Similar results were obtained in all the cell types confirming that the regulatory effect of the 5’UTR was cell-type independent. In order to prove that the results were not promoter dependent, the experiments were repeated with two other promoters CMV and EF1α and similar results were obtained.
In order to determine the exact portion of the 5’UTR playing a repressive role, they conducted various experiments with different portions of the 5’UTR deleted. They observed that though deletions in the three uORFs showed increased BACE1 levels, the increase were not as significant as when the 5’UTR was completely absent. Hence, Lammich et al propose that the stable secondary structure of the BACE1 5’UTR rich in GC forms many stem loops which inhibit translation in vitro.
Critical Analysis:
CDC estimates that Alzheimer’s disease is a seriously debilitating illness affecting about 4 million Americans. Being a leading cause of dementia in older adults, there is an urgent need to understand and find a cure to Alzheimer’s. In attempting to solve this problem, Lammich et al have indeed chosen a very important issue.
The paper is very concise and to the point, with ample illustrations of results obtained. Though very clear and concise in most parts, I would have appreciated further explanation about Fig 1B, which I found very difficult to analyze.
The author’s use of COS7 cells and H4 cells in addition to HEK293 cells helps reduce the probability of cell-type influence on the results, but I am unsure if they are sufficient to eliminate all possibility of cell-type effects. More experiments are probably required, using other cell types, like pancreatic cells, to confirm the results obtained. Further studies to analyze the actual structure of the 5’UTR and the mechanism of repression of translation would also prove to be very beneficial.
Another key question not answered in this paper is the role of BACE1 in normal brain cells. The authors mention that BACE1 levels are primarily elevated in the brain and in pancreas, and they also mention a possible secretory role for BACE1, which does not explain the higher occurrence of BACE1 in healthy brain cells.
In conclusion, I believe that this paper is very informative but further research is needed to get closer to understanding BACE1 and the 5’UTR of BACE1 and their role in the prevention and cure of Alzheimer’s disease.
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