Table of Contents:

1.	What is the scope of information available at NCBI on cystic fibrosis in humans?
2.	Besides the cystic fibrosis transmembrane conductance regulator gene (CFTR), what other genes are associated with cystic fibrosis in humans and what is their relationship to the disease?
3.	Nocturnal asthma is associated with what gene in humans? What are the RefSeq codes for this gene's mRNA and protein sequences? On the GenBank accession pages, data can be displayed in different formats. What is the difference between default and FASTA formats for these sequence files? How can these RefSeq codes be used to search for similar sequences in other species? What are the results of such a search?
4.	Are there any solved protein crystal structure(s) for the nocturnal asthma gene. Does the structure include the transmembrane segments? Are the found structure protein and the nocturnal asthma protein closely enough related to believe the results?
5.	Find proteins that are known to contribute to pulmonary artery hypertension and determine if animal models exist in which the disease can be studied. Can a full length dog protein sequence be found?
6.	Are there knockout mice available to study the AGPAT6 gene? How would you order one of these cell lines?

#1	What is the scope of information available at NCBI on cystic fibrosis in humans?

Hints:

Do a All Databases search at NCBI (http://www.ncbi.nlm.nih.gov). Then repeat the search narrowing the returned hits to human.

Answer: Lots of data to be explored.

#1 step by step instructions

1.	Go to http://www.ncbi.nlm.nih.gov,



enter cystic fibrosis in the for box



	and click Go.
2.	The returned Entrez page is organized with literature matches in the top box, sequence based information in the middle one and NLM's resources in the bottom box. Items of possible interest are denoted by numbers in a white box next to a topic title.




[search was run on 2/1/2008]
These results will change with time as more information is generated on the topic.

There are 151 OMIM entries [catalog of human genes and genetic disorders].

OMIM background information with links off to help and frequently asked questions


http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM

The sequence based information section contains data on cystic fibrosis from all species.

Some of these topics are sensitive to the addition of species information to the search query. OMIM and MeSH are examples of this.

3.	Return to the previous window. To restrict the sequence data to that from humans, by adding homo sapiens to the current cystic fibrosis in the Search across databases box



and click Go.



4.	The number of matches for nucleotide, protein, gene topics have decreased, but, there still are a very large number of items to sift through.

#2	Besides the cystic fibrosis transmembrane conductance regulator gene (CFTR), what other genes are associated with cystic fibrosis in humans and what is their relationship to the disease?

Hints:

Perform an Entrez Gene search (http://www.ncbi.nlm.nih.gov) to find the other genes and their function or relationship to the disease.

Possible answers:

S100A8	-	cystic fibrosis antigen
TGFB1	-	mutations modify severity of pulmonary disease in cystic fibrosis patients
	-	protein expression correlates with portal tracts showing histological abnormalities associated with cystic fibrosis liver disease
GOPC	-	CFTR binding
ADRB2	-	2002 polymorphisms contribute to clinical severity and disease progressionin cystic fibrosis 2005 - transfected beta3 not beta2-adrenergic receptors regulates CFTR activity via new pathway
SLC9A3R1	-	6/2007 plays a role in the turnover of CFTR at the cell surface
	-	5/2007 modulation of the expression of CFTR protein partners, like NHE-RF1, can rescue sequence-deleted CFTR activity.
ABCB1	-	study to see how the common cystic fibrosis mutation might disturb transmembrane segments of the protein using ABCB1 as a model ABCB1 expression increases ATP release in respiratory cystic fibrosis cells potential clinical benefits discussed

#2 step by step instructions

1.	Go to http://www.ncbi.nlm.nih.gov,



change the Search option from All Databases to Gene using the pull down menu,



enter homo sapiens cystic fibrosis in the for box



and click Go.





	Click on at least five diverse hits below the CFTR gene, finding out their relationship to cystic fibrosis. Ignore any gene that doesn't have a NCBI Reference Sequences (RefSeq) section.
	Here is what one of the results pages looks like.







	Check out the Gene References into Function section of the Bibliography part of the page, if it exists. No hits, click on the PUBMED links in this section and scan through the titles for mention of cystic fibrosis.
	The given example (S100A8) had 4 pages of references and the cystic fibrosis ones were on the last page.
	If no papers are listed with cystic fibrosis in the description, check out the OMIM link on the side of the page or the links in the GeneOntology section.

Possible answers:

CFM1	-	no RefSeq data (ignored)
CFM2	-	no RefSeq data (ignored)
S100A8	-	cystic fibrosis antigen
TGFB1	-	mutations modify severity of pulmonary disease in cystic fibrosis patients
TGFB1	-	protein expression correlates with portal tracts showing histological abnormalities associated with cystic fibrosis liver disease
GOPC	-	CFTR binding
ADRB2	-	2002 polymorphisms contribute to clinical severity and disease progressionin cystic fibrosis 2005 - transfected beta3 not beta2-adrenergic receptors regulates CFTR activity via new pathway
SLC9A3R1	-	6/2007 results indicate that NHERF1 plays a role in the turnover of CFTR at the cell surface, and that rDeltaF508 CFTR at the cell surface remains highly susceptible to degradation
SLC9A3R1	-	5/2007 modulation of the expression of CFTR (cystic fibrosis transmembrane conductance regulator) protein partners, like NHE-RF1, can rescue sequence-deleted CFTR activity
ABCB1	-	study to see how the common cystic fibrosis mutation might disturb transmembrane segments of the protein using ABCB1 as a model ABCB1 expression increases ATP release in respiratory cystic fibrosis cells potential clinical benefits discussed

Note there is another database that is relevant for getting clinical information, Online Mendelian Inheritance in Man (OMIM or MIM). Althought OMIM can not be searched via an actual sequence, it does allow searching by gene symbol, chromosome location, keywords or other features. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=omim

Notice that gene names can change over time. SLC9A3R1 used to be called EBP50, NHERF, NHERF1 or NHE-RF1.

#3

Nocturnal asthma associated with what gene in humans? What are the RefSeq codes for this gene's mRNA and protein sequences? On the GenBank accession pages, data can be displayed in different formats. What is the difference between default and FASTA formats for these sequence files? How can these RefSeq codes be used to search for similar sequences in other species? What are the results of such a search?

Hints:

Do a Gene search at NCBI (http://www.ncbi.nlm.nih.gov), record the codes. Compare the formats of the mRNA and protein sequences. Run a BLAST search, (http://blast.ncbi.nlm.nih.gov).

Answers:

gene - ADBR2 adrenergic, beta-2-, receptor, surface

RefSeq codes: mRNA Sequence NM_000024
Product (protein) NP_000015

FASTA format is very concise, limited to the actual sequence and an identification line that starts with a > symbol. The default format is very verbose, giving all sorts of reference details about the sequence and a version of the sequence that is more easily read by the user.

BLAST searching allows for different types of data entry including the use of accession codes (such as a RefSeq accession code).

ADBR2 contains the 7tm_1 conserved domain signature which is highly conserved across species.

#3 step by step instructions

1.	Go to http://www.ncbi.nlm.nih.gov,



change the All Databases Search option to Gene using the pull down menu,



enter homo sapiens nocturnal asthma in the for box



	and click Go.
2.	Check the resulting hits to insure that the summary information on the gene mentions that various types of changes in this gene are associated with the disease.



Here are the summary sections of the top three hits.




Only the first one contains a reference to nocturnal asthma.


ADBR2 adrenergic, beta-2-, receptor, surface

3.	Scroll down the page to the NCBI Reference Sequences (RefSeq) section. Record the mRNA sequence and Product (protein) codes:



The required information:


mRNA Sequence NM_000024
Product (protein) NP_000015

4.	Click on the mRNA code to see the data on the actual mRNA sequence data. Scroll down the page taking in the format of the information presented. mRNA sequence link
5.	Scroll back to the top of the page and change the Display option from GenBank to FASTA. The format automatically changes. Note the difference. FASTA format is the sequence format required by many database searching programs. background information on fasta format
6.	Click back to the Entrez Gene page and repeat this process with the protein code. protein link
7.	After noting the difference, click on the NCBI logo at the top of the page.



From the blue navigation bar on the main NCBI page,



click on BLAST.



8.	From the main BLAST page, click on protein blastp in the Basic BLAST section.



In the blastp suite page, click on the ? icon at the top of the box in the Enter Query Sequence section to find out about what sort of inputs this form accepts. Clicking the more... link provides additional details.



	After reading the presented information, click on the "?" icon again to close the information block and then enter the protein code into the Enter Query Sequence box. Once this is done, information appears in the Job Title box.



In the Choose Search Set section of the page,and start to enter the term Vertebrata into the Organism box.



As the term is entered, matching Entrez terms start to appear. When enough of the word is entered to find the desired term, select this term from the list.



Clicking the BLAST button at the bottom of the page starts the search.



	If results are to be displayed in a new window, click on the "Show results in a new window" box prior to clicking the BLAST button.
	Protein searches gets around the problem of multiple codons coding for the same amino acid that impacts nucleotide searches. However, depending in the information sought, this is not always possible.
9.	It may take a few seconds for the search to be completed. While waiting, click on the 7tm_1 in the image to find out about the conserved domain that was found in the sequence.



Initial Conserved Domains page.



Clicking on the + symbol at the beginning of the green highlighted line produces the full version of the page.



7tm_1 indicates that the protein being search with contains the transmembrane receptor signature of the rhodopsin family of transmembrane proteins. This signature is located in residues 50 to 326 of the sequence. Close the popup window. Note the length of the query sequence, this may be given on the Query line, the Job Title line or in the conserved domain image. 413 letters Wait for the results page to appear.

Here is a screen shot of the top part of the Blast results page.






10.	Scroll down the results page past the image with its colored horizontal bars to the Sequences producing significant alignments section.
	Scores are based on the length of the query sequence and the size of the database. Short sequences will never produce great scores. To get a E value of 0.0 requires a match of at least 330 characters. A very long sequence could easily have a match this long and still not have a match that covers a significant portion of the query sequence. Always look at the resulting alignment. The mathematics of the process can sometimes result in the strange ordering of hits.
	A hit line gives the database the hit is from, its accession code, a description of the sequence from the database, its Bits score and finally the E value. Hits in the list are ordered their E value, then their Bits score which reflects the length of their actual match. Enough of the description may be given to see what species the match is from.
	Clicking on the link given on the left side of a hit line goes off to the actual sequence information. Clicking on the right side link moves down to the alignment data for that hit.
	Notice that there are over 30 hits with an E value of 0.0 at the top of this list and that the protein code entered is at the top of the list. There are about 130 hits in the list which mention ADBR2, beta-2 adrenergic receptor or variations thereof before sequence description changes to something else. The first 12 hits are all from man with from 0 to 2 mismatches in the alignment. NCBI used to make an effort to remove redundant sequences, but the size of the database increased to such an extent that it was no longer possible to do this quickly enough so that it wouldn't impact the processing of new data. When an accession code begins with XP_, it means that the data is the results of an automated analysis process. This situation usually occurs when a genome sequencing project is first being analyzed. These sequences have not been checked for accuracy and can be much longer or shorter than their homologs from more mature genome studies. These sequences usually have their description start with PREDICTED:. The letter inclosed in colored boxes to the right of the hit line indicate that there is additional information available about that matching sequence elsewhere. A boxed U means that there is Unigene data. A boxed G indicates that there is Entrez Gene data. The boxed S means that there is structural data. Check out some of the hits beyond the 0.0 E values and determine where the match is actually taking place within the query sequence. PREDICTED: similar to beta-2 adrenergic receptor [Monodelphis domestica] Length=404 (opossum) results alignment section 12 - 396 PREDICTED: similar to beta-2 adrenergic receptor, [Gallus gallus] Length=397 (chicken) results alignment section 5 - 396 beta-2 adrenergic receptor [Homo sapiens] Length=275 (man) results alignment section 52- 326 beta-2 adrenergic receptor [Macaca mulatta] Length=275 (rhesus monkey) results alignment section 52 - 326 beta-2 adrenergic receptor [Hylobates concolor] Length=275 (crested gibbon) results alignment section 52 - 326 beta-2 adrenergic receptor [Ateles fusciceps] Length=275 (spider monkey) results alignment section 52 - 326 The match is happening in the 7tm_1 region of the sequence which appears to be highly conserved.