Information organization

Oct 2, 2012Learning objectives-Demonstrate Dotter Program. Understand how information is stored in GenBank. Learn how to read a GenBank flat file. Learn how to search GenBank for information. Understand difference between header, features and sequence. Distinguish between a primary database and secondary database.Homework #2 due today.Homework #3 due Tues. Oct. 9

What is GenBank?

Gene sequence database

Annotated records that represent single contiguous stretches of DNA or RNA-may have more than one coding region.

Generated from direct submissions to the DNA sequence databases from the authors.

Part of the International Nucleotide Sequence Database Collaboration.

http://www.ncbi.nlm.nih.gov/Genbank/genbankstats.html

History of GenBank

Began with Atlas of Protein Sequences and Structures (Dayhoff et al., 1965)In 1986 it shared data with EMBL and in 1987 it shared data with DDBJ.Primary databaseExamples of secondary databases derived from GenBank: UniProt, EST database.GenBank Flat File is a human readable form of a GenBank record.

Transcription

5’

5’

3’

3’Promoter

Coding strand

Template strand

Transcription

initiation site

Transcription

termination site

Protein Coding Sequence (CDS)

5’ untranslated region (5’UTR)

3’ untranslated region (3’UTR)

Downstream (relative to CDS)Upstream (relative to CDS)

DNA

RNA

Translation

Protein

Protein folding

Folded protein

Start of gene End of gene

5’ 3’

1 2 3 4

Transcription

1 2 3 4

DNA

Primary

transcript

Splicing

mRNA

Translation

protein

Intron 1 Intron 2 Intron 3

Transcript splicing

Primary transcript1 2 3 4

Alternative splicing

General Comments on GBFF

Three sections: 1) Header-information about the whole record 2) Features-description of annotations-each

represented by a key. 3) Nucleotide sequence-each ends with // on

last line of record.

DNA-centered

Translated sequence is a feature

Feature Keys

Purpose: 1) Indicates biological nature of sequence 2) Supplies information about changes to

sequencesFeature Key Description

conflict Separate determinations of the same seq. differ rep_origin Origin of replication protein_bind Protein binding site on DNA CDS (Protein) coding sequence

Feature Keys-Terminology

Feature Key Location/Qualifiers

CDS 23..400

/product=“alcohol dehydro.”

/gene=“adhI”

The feature CDS is a coding sequence beginning at base 23 and ending at base 400 that has a product called “alcohol dehydrogenase” and corresponds to the gene called “adhI”.

Feature Keys-Terminology (Cont.)

Feat. Key Location/Qualifiers

CDS join (544..589,688..1032)

/product=“T-cell recep. B-ch.”

/partial

The feature CDS is a partial coding sequence formed by joining the indicated elements to form one contiguous sequence encoding a product called T-cell receptor beta-chain.

Record from GenBank

LOCUS SCU49845 5028 bp DNA PLN 21-JUN-1999

DEFINITION Saccharomyces cerevisiae TCP1-beta gene, partial cds, and

Axl2p (AXL2) and Rev7p (REV7) genes, complete cds.

ACCESSION U49845

VERSION U49845.1 GI:1293613

KEYWORDS .

SOURCE baker's yeast.

ORGANISM Saccharomyces cerevisiae

Eukaryota; Fungi; Ascomycota; Hemiascomycetes; Saccharomycetales;

Saccharomycetaceae; Saccharomyces.

Modification dateGenBank division (plant, fungal and algal)

Coding sequenceAccession number (never changes)

Nucleotide sequence identifier (changes when there is a changein sequence (accession.version))

GeneInfo identifier (changes whenever there is a change)

Word or phrase describing the sequence (not based on controlled vocabulary).Not used in newer records.

Common name for organism

Formal scientific name for the source organism and its lineagebased on NCBI Taxonomy Database

Locus name

Record from GenBank (cont.1)

REFERENCE 1 (bases 1 to 5028)

AUTHORS Torpey,L.E., Gibbs,P.E., Nelson,J. and Lawrence,C.W.

TITLE Cloning and sequence of REV7, a gene whose function is required

for DNA damage-induced mutagenesis in Saccharomyces cerevisiae

JOURNAL Yeast 10 (11), 1503-1509 (1994)

MEDLINE 95176709

REFERENCE 2 (bases 1 to 5028)

AUTHORS Roemer,T., Madden,K., Chang,J. and Snyder,M.

TITLE Selection of axial growth sites in yeast requires Axl2p, a

novel plasma membrane glycoprotein

JOURNAL Genes Dev. 10 (7), 777-793 (1996)

MEDLINE 96194260

Oldest reference first

Medline UID

REFERENCE 3 (bases 1 to 5028)

AUTHORS Roemer,T.

TITLE Direct Submission

JOURNAL Submitted (22-FEB-1996) Terry Roemer, Biology, Yale University,

New Haven, CT, USA

Submitter of sequence (always the last reference)

Record from GenBank (cont.2)

FEATURES Location/Qualifiers

source 1..5028

/organism="Saccharomyces cerevisiae"

/db_xref="taxon:4932"

/chromosome="IX"

/map="9"

CDS <1..206

/codon_start=3

/product="TCP1-beta"

/protein_id="AAA98665.1"

/db_xref="GI:1293614"

/translation="SSIYNGISTSGLDLNNGTIADMRQLGIVESYKLKRAVVSSASEA

AEVLLRVDNIIRARPRTANRQHM"

Partial sequence on the 5’ end. The 3’ end is complete.

There are three parts to the feature key: a keyword (indicates functional group), a location (instruction for finding the feature), and a qualifier (auxiliary information about a feature)

Keys

Location

Qualifiers

Descriptive free text must be in quotations

Start of open reading frame

Database cross-refsProtein sequence ID #

Note: only a partial sequence

Values

Record from GenBank (cont.3) gene 687..3158 /gene="AXL2" CDS 687..3158 /gene="AXL2" /note="plasma membrane glycoprotein" /codon_start=1 /function="required for axial budding pattern of S. cerevisiae" /product="Axl2p" /protein_id="AAA98666.1" /db_xref="GI:1293615"

/translation="MTQLQISLLLTATISLLHLVVATPYEAYPIGKQYPPVARVN. . . “ gene complement(3300..4037) /gene="REV7" CDS complement(3300..4037) /gene="REV7" /codon_start=1 /product="Rev7p" /protein_id="AAA98667.1" /db_xref="GI:1293616"

/translation="MNRWVEKWLRVYLKCYINLILFYRNVYPPQSFDYTTYQSFNLPQ . . . “

Cutoff

Cutoff

Another location

Another location

Coding strand is complementary strand

Record from GenBank (cont.4)

BASE COUNT 1510 a 1074 c 835 g 1609 t

ORIGIN

1 gatcctccat atacaacggt atctccacct caggtttaga tctcaacaac ggaaccattg

61 ccgacatgag acagttaggt atcgtcgaga gttacaagct aaaacgagca gtagtcagct . . .//

RNA

cDNA

DNA protein

DNA databases derived from GenBankcontaining data for a single gene

•Non-redundant (nr)•dbGSS•dbSTS

RNA (cDNA) databases derivedfrom GenBankcontaining data for a single gene•dbEST•UniGene•RefSeq

Protein databases derivedfrom GenBank containingdata for a single gene•Non-redundant (nr)•UniProtKB

Types of primary databases carrying biological infomation

GenBank/EMBL/DDBJ

dbEST-expressed sequence tags-single pass cDNA sequences (high error freq.)

It is non-redundant

PDB-Three-dimensional structure coordinates of biological molecules

PROSITE-database of protein domain/function relationships.

Summary

GenBank-longest running molecular biology database.

Three sections in every GenBank record

Primary databases and secondary databases.

RefSeq-contains unique record for each RNA variant.

UniProtKB-protein centered

Workshop

Do problem 1 in Chapter 2.

Homework

Do problems 2 and 3 in Chapter 2.