BIO Assignment 2 2011

   

Assignment 2 - Search, retrieve and annnotate

Note

This assignment is currently active. All changes will be announced on the course mailing list.

Introduction

Baker's yeast, Saccharomyces cerevisiae, is perhaps the most important model organism since it is a eukaryote that has been studied genetically and biochemically in great detail for many decades and it is easily manipulated with high-throughput experimental methods. We will use information from this model organism to study the conservation of function and sequence in other fungi whose genomes have been completely sequenced. This and the following assignments will revolve around a transcription factor that plays an important role in the regulation of the cell cycle: Mbp1, a key component of the MBF complex (Mbp1/Swi6) that regulates gene expression at the crucial G1/S-phase transition of the mitotic cell cycle and has been shown to bind to the regulatory regions of more than a hundred target genes.

One would assume that such control machinery would be conserved in other fungi and it will be your task in these assignments to collect evidence whether related molecular machinery is present in some of the newly sequenced fungal genomes.

(If you need to brush up on the concepts mentioned above, you could study the corresponding chapter in Lodish's Molecular Cell Biology. It is not strictly necessary to understand the details of the yeast cell-cycle to complete the assignments, but highly recommended, if this all is to make some sense.)

In this particular assignment you will go on a search and retrieve mission for information and annotation of Mbp1 homologues in a fungal genome, using common public databases and Web resources.

Preparation, submission and due date

Read carefully. Be sure you have understood all parts of the assignment and cover all questions in your answers! Sadly, we always get assignments back in which important aspects have simply been overlooked and marks are unnecessarily lost. Sadly, we always get assignments back in which important aspects have simply been overlooked and marks are unnecessarily lost. If you did not notice that the above sentence was repeated, you are not reading carefully enough.

Prepare a Microsoft Word document with a title page that contains:

• your full name
• your Student ID
• your e-mail address
• the organism name you have been assigned (see below)

Follow the steps outlined below. You are encouraged to write your answers in short answer form or point form, like you would document an analysis in a laboratory notebook. However, you must

• document what you have done,
• note what Web sites and tools you have used,
• paste important data sequences, alignments, information etc.

If you do not document the process of your work, we will deduct marks. Try to be concise, not wordy! Use your judgement: are you giving us enough information so we could exectly reproduce what you have done?

Write your answers into separate paragraphs and give each its title. Save your document with a filename of: A2_{lastname}.{firstname}.doc (for example my first assignment would be named: A2_steipe.boris.doc - and don't include the brackets this time, please!)

Finally e-mail the document to [boris.steipe@utoronto.ca] before the due date.

Your document must not contain macros. Please turn off and/or remove all macros from your Word document; we will disable macros, since they pose a security risk.

With the number of students in the course, we have to economize on processing the assignments. Thus we will not accept assignments that are not prepared as described above. If you have technical difficulties, contact me.

The due date for the assignment is Monday, October 23. at 10:00 in the morning.

Grading

Don't wait until the last day to find out there are problems! Assignments that are received past the due date will have one mark deducted at the first minute of every twelve hour period past the due date. Assignments received more than 5 days past the due date will not be assessed.

Marks are noted below in the section headings for of the tasks. A total of 10 marks will be awarded, if your assignment answers all of the questions. A total of 2 bonus marks (up to a maximum of 10 overall) can be awarded for particularily interesting findings, or insightful comments. A total of 2 marks can be subtracted for lack of form or for glaring errors. The marks you receive will

• count directly towards your final marks at the end of term, for BCH441 (undergraduates), or
• be divided by two for BCH1441 (graduates).

   

Retrieve

   

The Genome (1 mark)

Access the Organism list to retrieve an organism name for this assignment. Navigate to the NCBI homepage at http://www.ncbi.nlm.nih.gov . Enter the systematic name into the search field, select the taxonomy database and identify the organism that you have been assigned.

 

• Record the taxonomy ID for the species or strain(s) that are associated with this organism name

Return to the the NCBI home-page and navigate to the "Genomic Biology" section, continue with the link for Fungi under the Genome Projects Database. This should take you to a tabular view of ongoing and completed fungal genome sequencing projects. Find your organism name in this table. There may be one or more sequencing projects associated with this organism.

 

• Decide which project is the most suitable one for analysis and record your decision. Report the strain and Taxonomy-ID for this organism.

Click on the organism name to navigate to the Genome Project information page.

 

• Comment briefly on the status of the data you are working with: include information such as whether the entire genome is available or only a partial sequence? How many chromosomes does this genome have? What is the status of its genome assembly and annotation? Has the mitochondrial genome been sequenced as well?

 

APSES domain transcription factors (1 mark)

Mbp1 is a large multidomain protein; it binds DNA through a small domain called the APSES domain and many organisms have more than one transcription factor that has a domain homologous to other APSES domains. In the assignments, we will analyse how these APSES domains have evolved, to obtain a perspective on the evolution of regulatory systems in general. Accordingly we should first define an APSES domain sequence and then use it to find all its relatives in each target organism.

Use the NCBI Entrez system to search for the string "apses" in the "Conserved Domains" database and access the entry for the APSES domain. You should find a number of aligned sequences on that page, each with their own GI identifier.

 

• Identify the two sequences that come from Saccharomyces cerevisiae (the Mbp1 and Swi4 APSES domains).

Access the Genbank record for the Saccharomyces cerevisiae Mbp1 protein.

 

• Obtain the FASTA sequence for the protein, save it and paste it into your assignment.

Working from the APSES domain alignment in CDD, define the sequence of the entire APSES domain in the Mbp1 protein.

 

• Save the sequence in FASTA format (with an appropriate header) and paste it into your assignment. Comment if and how it is different from the sequence you find on the CDD page.

Navigate back to the Genome Project Database table for fungi and click on the "B" link next to your organism. This takes you to a page with a BLAST search form. Run a BLAST search with the Saccharomyces cervisiae Mbp1 protein sequence.

 

• Record the parameters you have used for the search and the relevant search results.

• List the accession numbers and names of all putative homologues. How many are there? How many (if any) do you expect? What do you conclude ?

Run a second BLAST search using only the Saccharomyces cervisiae Mbp1 APSES domain sequence.

 

• Record the parameters you have used for the search and the relevant search results.

• List the accession numbers and names of all putative homologues. How many are there? Are the results different from your previous search? How? What do you conclude ?

 

(Please contact me immediately in case you cannnot find any significant alignments - you cannot continue with the assignment if you get stuck at this point.)

 

Align

   

Sequences and accession numbers (1 mark)

Retrieve the entire protein sequences for those significant hits that you have found with the APSES domain search in your organism. The easist way to do this is to click on the links on the BLAST results page. The NCBI does most of their internal cross-referencing with GI numbers, however these are less useful fro crossreferencing to other databases.

 

Find and record

• the GI number
• the GenPept accession number(s) (NCBI),
• the RefSeq identifier(s) if available,
• and the Uniprot accession number(s) (EBI)
• and as always, report what you have done to find this information.

You can find these database identifiers in some cases when the appropriate cross-references have been entered into the annotations. Alternatively you can run running a BLAST search with a sequence to find the exact same sequence in the other database. Depending on what you are looking for, either search at the NCBI or at the EBI. But (!) restrict the search to your assigned organism! You will have to figure out how to do that and of course report the parameters you have used. I know that this BLAST method appears to be a maximally inefficient way to retrieve a cross-reference for a sequence. However, frequently the databases are simply not providing the appropriate cross-references and the detour through a BLAST search is the only practical way to get them. Most unfortunate.

Discovering a significantly better (or at least significantly more interesting) way to obtain the cross-references and being the first one to post it on the mailing list probably will merit a bonus point.

Sequence alignment (1 mark)

Retrieve the FASTA sequence of the protein in your organism that you have found to be most similar to yeast Mbp1.

Use an online tool to generate an optimal full-length alignment (BLAST does not generate optimal alignments!) between this protein sequence and S. cerevisiae Mbp1. You have to figure out where to find a Web service that does such alignments, which algorithm to use and to define reasonable parameters.

 

• Report your procedure and your alignment and comment on its quality. Is the protein a full-length homologue of Mbp1?

Analyse

   

Sequence annotation (2 marks)

Annotate the amino acid sequence of your organism's Mbp1 homologue with the following online tools (most of these can be found via links on http://www.expasy.org/ ):

 

• predicted molecular weight
• presence of transmembrane helices (TMpred or TMHMM)
• presence of internal repeats
• presence of signal sequences (SignalP 3.0)
• prediction for localization of the protein (PSORT II - make sure you use the right psort program!)
• prediction of functional motifs and patterns (ScanProsite or InterproScan)
• RPS BLAST

Briefly state for each analysis your procedure, the importnat results (if not obvious from the output), what the results mean, and whether your results are consistent with your expectations about this protein.

 

Homologous structure (1 mark)

DNA binding site (3 marks)

The Mbp1 APSES domain has been shown to bind to DNA and the residues involved in DNA binding have been characterized. (Taylor et al. (2000) Biochemistry 39: 3943-3954) . In particular the residues between 50-74 have been proposed to comprise the DNA recognition motif.

 

• Generate a RasMol image (wall-eyed stereo view) that clearly shows the Mbp1 DNA recognition domain residues distinctly coloured.

• Generate a second RasMol image that shows the secondary structure of the domain in question. Paste the images and the RasMol commands you have used into your assignment.

DNA binding interfaces are expected to comprise a number of positively charged amino acids, that might form salt-bridges with the phosphate backbone.

 

• Report whether this is the case here and which residues might be included?

• Do the DNA binding residues form a contiguous surface that is compatible with a binding interface?

• Consider the surface exposed residues that could form part of the DNA binding interface of Mbp1 (i.e. the cationic residues you have described above and the exposed sidechains inbetween): are they conserved in your protein?

 

[End of assignment]

If you have any questions at all, don't hesitate to mail me at boris.steipe@utoronto.ca or post your question to the Course Mailing List