BIO Assignment Week 2
Assignment for Week 2
Scenario, Databases, Search and Retrieve
Note! This assignment is currently inactive. Major and minor unannounced changes may be made at any time.
Concepts and activities (and reading, if applicable) for this assignment will be topics on next week's quiz.
Contents
The Scenario
Baker's yeast, Saccharomyces cerevisiae, is perhaps the most important model organism. 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; the assignments are an exercise in model-organism reasoning: the transfer of knowledge from one, well-studied organism to others.
This and the following assignments will revolve around a transcription factor that plays an important role in the regulation of the cell cycle: Mbp1 is a key component of the MBF complex (Mbp1/Swi6). This complex 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. It is therefore a DNA binding protein that acts as a control switch for a key cellular process.
One would speculate that such central control machinery would be conserved in other fungi and it will be your task in these assignments to collect evidence whether related molecular components are present in some of the newly sequenced fungal genomes. Throughout the assignments we will use freely available tools to conduct bioinformatics investigations of sequences, structures and relationships that may ultimately answer questions such as:
- Do related proteins exist in other organisms?
- What functional features can we detect in the related proteins?
- Do we have evidence that they may bind to similar sequence motifs?
- Do we believe they may function in a similar way?
Task:
Access the information page on Mbp1 at the Saccharomyces Genome Database and read the summary paragraph on the protein's function!
(If you would like to brush up on the concepts mentioned above, you could study the corresponding chapter in Lodish's Molecular Cell Biology and./or read Nobel laureate Paul Nurse's review of the key concepts of the eukaryotic cycle. It is not strictly necessary to understand the details of the yeast cell-cycle to complete the assignments, but it's obviously more satisfying to work with concepts that actually make some sense.)
For reference, this is the FASTA formatted sequence of Mbp1 from Saccharomyces cerevisiae:
>gi|6320147|ref|NP_010227.1| Mbp1p [Saccharomyces cerevisiae S288c]
MSNQIYSARYSGVDVYEFIHSTGSIMKRKKDDWVNATHILKAANFAKAKRTRILEKEVLKETHEKVQGGF
GKYQGTWVPLNIAKQLAEKFSVYDQLKPLFDFTQTDGSASPPPAPKHHHASKVDRKKAIRSASTSAIMET
KRNNKKAEENQFQSSKILGNPTAAPRKRGRPVGSTRGSRRKLGVNLQRSQSDMGFPRPAIPNSSISTTQL
PSIRSTMGPQSPTLGILEEERHDSRQQQPQQNNSAQFKEIDLEDGLSSDVEPSQQLQQVFNQNTGFVPQQ
QSSLIQTQQTESMATSVSSSPSLPTSPGDFADSNPFEERFPGGGTSPIISMIPRYPVTSRPQTSDINDKV
NKYLSKLVDYFISNEMKSNKSLPQVLLHPPPHSAPYIDAPIDPELHTAFHWACSMGNLPIAEALYEAGTS
IRSTNSQGQTPLMRSSLFHNSYTRRTFPRIFQLLHETVFDIDSQSQTVIHHIVKRKSTTPSAVYYLDVVL
SKIKDFSPQYRIELLLNTQDKNGDTALHIASKNGDVVFFNTLVKMGALTTISNKEGLTANEIMNQQYEQM
MIQNGTNQHVNSSNTDLNIHVNTNNIETKNDVNSMVIMSPVSPSDYITYPSQIATNISRNIPNVVNSMKQ
MASIYNDLHEQHDNEIKSLQKTLKSISKTKIQVSLKTLEVLKESSKDENGEAQTNDDFEILSRLQEQNTK
KLRKRLIRYKRLIKQKLEYRQTVLLNKLIEDETQATTNNTVEKDNNTLERLELAQELTMLQLQRKNKLSS
LVKKFEDNAKIHKYRRIIREGTEMNIEEVDSSLDVILQTLIANNNKNKGAEQIITISNANSHA
I have highlighted the protein's APSES domain (also known as a KilA-N domain), which is the DNA binding element of the sequence. Of course, such coloring is not part of the actual FASTA file which contains only a header and sequence letters.
Choosing YFO (Your Favourite Organism)
The first task is to choose a species in which to conduct your explorations.
Many fungal genomes have been sequenced and more are added each year. For the purposes of the course assignments, we need a species
- that has transcription factors with APSES domains;
- whose genome has been completely sequenced;
- for which records exist in the RefSeq database, NCBI's unique sequence collection.
Next, I would like to assign species from this list randomly to each student, but I'd also like to avoid having to make a fresh table of assignments every year.
Here is R code to accomplish this:
Task:
- Read, try to understand and then execute the following R-code.
pickSpecies <- function(ID) {
# this function randomly picks a fungal species
# from a list. It is seeded by a student ID. Therefore
# the pick is random, but reproducible.
# first, define a list of species:
Species <- c(
"Ajellomyces dermatitidis (AJEDE)",
"Arthroderma gypseum (ARTGY)",
"Ashbya gossypii (ASHGO)",
"Aspergillus clavatus (ASPCL)",
"Aspergillus flavus (ASPFL)",
"Botryotinia fuckeliana (BOTFU)",
"Candida glabrata (CANGL)",
"Chaetomium globosum (CHAGL)",
"Clavispora lusitaniae (CLALU)",
"Coccidioides immitis (COCIM)",
"Coprinopsis cinerea (COPCI)",
"Debaryomyces hansenii (DEBHA)",
"Gibberella zeae (GIBZE)",
"Kluyveromyces lactis (KLULA)",
"Komagataella pastoris (KOMPA)",
"Laccaria bicolor (LACBI)",
"Lachancea thermotolerans (LACTH)",
"Lodderomyces elongisporus (LODEL)",
"Magnaporthe oryzae (MAGOR)",
"Malassezia globosa (MALGL)",
"Meyerozyma guilliermondii (MEYGU)",
"Nectria haematococca (NECHA)",
"Neosartorya fischeri (NEOFI)",
"Paracoccidioides brasiliensis (PARBR)",
"Penicillium chrysogenum (PENCH)",
"Puccinia graminis (PUCGR)",
"Pyrenophora teres (PYRTE)",
"Scheffersomyces stipitis (SCHST)",
"Schizophyllum commune (SCHCO)",
"Phaeospheria nodorum (PHANO)",
"Schizosaccharomyces japonicus (SCHJA)",
"Sclerotinia sclerotiorum (SCLSC)",
"Talaromyces stipitatus (TALST)",
"Trichophyton rubrum (TRIRU)",
"Uncinocarpus reesii (UNCRE)",
"Vanderwaltozyma polyspora (VANPO)",
"Verticillium albo-atrum (VERAL)",
"Yarrowia lipolytica (YARLI)",
"Zygosaccharomyces rouxii (ZYGRO)"
)
l <- length(Species) # number of elements in the list
set.seed(ID) # seed the random number generator
# with the student ID
i <- runif(1, 0, 1) # pick one random number between 0 and 1
i <- l * i # multiply with number of elements
i <- ceiling(i) # round up to nearest integer
choice <- Species[i] # pick the i'th element from list
return(choice)
}
- Execute the function
pickSpecies()
with your student ID as its parameter. Example:
> pickSpecies(991234567)
[ 1] "Candida glabrata (CANGL)"
- Note down the species name and its five letter abbreviation. Use this species whenever this or future assignments refer to YFO.
Keeping a notebook
NCBI databases
Let us explore some of the offerings of the NCBI that can contribute to our objective of studying a particular gene in a newly sequenced organism.
The NCBI administers some of the world's most important databases, such as GenBank. In this section you should
- Explore the NCBI Web site, familiarize yourself with its key databases and explore the resources to become confident that you will find information that you are looking for.
- Follow a protein's annotations into PubMed and familiarize yourself with PubMed's query syntax.
- Explore the Entrez search page, and learn how to limit queries and restrict searches
Entrez
Task:
Remember to document your activities.
- Access the NCBI website at http://www.ncbi.nlm.nih.gov/ Look for the site-map and browse the contents of this large site; find which databases and services are hosted here. Expect to spend at least half an hour to familiarize yourself with the site.
- Access the Map viewer (under the Genomes section of the Databases division). Click on the link under Saccharomyces cerevisiae (Build 2.1) for a whole genome view, then click on the icon for chromosome IV for a more detailed view. Enter the region between 340,000 and 380,000 in the "Region shown" fields on the left. How many genes does this region contain? How many of these are protein genes?
- Click on MBP1 to follow the link to its Entrez Gene page. Study the contents of the page. If you are not clear what the sections show you, click on one of the question marks. If you are still not clear, ask on our mailing list.
- Follow the link to PubMed for this gene. You should find (at least) 27 publications. Click on the History tab to find the index of the query that got you here (eg. "#4"). Now search for those papers in your query that were published in 2008: enter #4 AND 2008[DP] into the search field and click "Go". Make yourself familiar with the Search field descriptions and tags (in particular [DP], [AU], [TI], and [TA]), how you use the History to combine searches, and the use of AND, OR, NOT and brackets.
- Back at the MapViewer pager, click on pr in the same row as the MBP1 gene to find a list of GenPept (protein) records for this gene. Follow the link to the RefSeq record for this protein: NP_010227. This is a flat-file record for the Mbp1 gene. Study the fields and the format. Then use the "Display" option in the header to show this protein sequence in a FASTA format, choose "send to ... Text" to get only the FASTA format. Make sure you understand the difference between GenBank/GenPept and RefSeq, between GI number, accession and locus (refer to the lecture slides as soon as they are posted).
- Click on MBP1 to follow the link to its Entrez Gene page. Study the contents of the page. If you are not clear what the sections show you, click on one of the question marks. If you are still not clear, ask on our mailing list.
- In the header bar of the MapViewer, click on the link to Entrez. Enter mbp1 into the search field of the Entrez page and click "GO".
- Increase the relevance of returned items by restricting your search to a particular organism. Access and read the Help pages for Entrez and make sure you understand how to use limits and how to search in search field indexes. You will already have encountered similar concepts when you visited PubMed.
- Enter: mbp1 AND "saccharomyces cerevisiae"[organism] into the Entrez search field and click "GO". Click on the CoreNucleotide link of the results.
- The RefSeq record listed in the results contains the entire yeast chromosome IV (1.5 Mbp) which you probably don't want to explore unless you actually want to. The result is correct, since mbp1 is one of the 787 genes annotated on that chromosome, but perhaps not what we had in mind when we queried for a nucleotide sequence of the mbp1 gene. Check the results for a different record that contains only the mbp1 gene's (full-length) nucleotide sequence. There are (as of this writing) two such records. Explore either one of the two, these are nucleotide sequences in the GenBank flat file format.
Sequence retrieval
Cross-reference
Structure search
Visit the RCSB PDB website at http://www.pdb.org/ , explore the database and familiarize yourself with its contents.
- Look for the "Getting started" page and explore the page.
- Explore the links on the "Education" page to see where you might fill in gaps in your knowledege of structural molecular biology, such as the Biological Units tutorial; read up on one or two the excellent molecule of the month articles, such as the TATA binding protein (July 2005).
- From the homepage, search for the yeast Mbp1 protein (by keyword) and explore the information that is available in one of the entries that was retrieved.
Structure retrieval
Visualize in VMD
VMD
Task:
- Access the VMD page.
- Install the program as per the instructions in the section: "Installing VMD".
- In the tutorial section work through
- Part 1 (Introduction), and
- Part 2 (Working with a single molecule).
Stereo vision (1 mark):=
Task:
Access the Stereo Vision tutorial and practice viewing molecular structures in stereo.
Practice at least ...
- two times daily,
- for 3-5 minutes each session,
Keep up your practice throughout the course. Stereo viewing will be required in the final exam, but more importantly, it is a wonderful skill that will greatly support any activity of yours related to structural molecular biology. Practice with different molecules and try out different colours and renderings.
Note: do not go through your practice sessions mechanically. If you are not making any progress with stereo vision, contact me so we can help you on the right track.
R
The R statistics environment and programming language is an exceptionally well engineered, free (as in free speech) and free (as in free beer) platform for data manipulation and analysis. The number of functions that are included by default is large, there is a very large number of additional, community-generated analysis modules that can be simply imported from dedicated sites (e.g. the Bioconductor project for molecular biology data), or via the CRAN network, and whatever function is not available can be easily programmed. The ability to filter and manipulate data to prepare it for analysis is an absolute requirement in research-centric fields such as ours, where the strategies for analysis are constantly shifting and prepackaged solutions become obsolete almost faster than they can be developed. Besides numerical analysis, R has very powerful and flexible functions for plotting graphical output.
R is not a main focus of the course, but an important tool I would like you to pick up "on the side".
Task:
- Access the R tutorial on this site.
- Work through the sections Installation, User interface, and Packages.