Fot this tutorial we will be looking at a gene for auxin found in the plant Arabidopsis Thaliana.

This gene was located by searching Entrez for records related to auxins in plants.  The gene is located in a clone from chromosome 2 in Arabidopsis thaliana.  As shown, this clone contains 114,144 bp. A number of genes are located within this clone. One of these genes codes for a putative auxin-binding protein. It is 801 bp long, and is spans the bases 63391 - 62591.
 

xxx	LOCUS U78721 114144 bp DNA linear PLN 27-FEB-2002 DEFINITION Arabidopsis thaliana chromosome 2 clone T1B8 map TEn5. ACCESSION U78721 VERSION U78721.3 GI:20198308 KEYWORDS HTG. SOURCE thale cress. ORGANISM Arabidopsis thaliana

The Features Table for the putative auxin-binding gene provides information on the hnRNA, and the locations of the Introns and Exons. It also shows the actual CDS of the mature mRNA, and the protein translation as derived from the CDS.

Click on the ""REVIEW" button to review Introns and Exons. Open the image to the left ..... it will help you understand the key points below! The third line in the Features Table (with numbers in red) refers to the heterogenous RNA (hnRNA).  The sequence of the hnRNA corresponds to the complement of the sequence given in this record.  The gene extends from base 63391 to 62591, so the gene is 801 bp long inclusive. Prove to yourself that it is not 800 bp long! However the gene has 3 exons which total to 603 bp long inclusive. exon-1 extends from base 63391 to 63248, and is 144 bp long. exon-2 extends from base 63135 to 62943, and is 193 bp long. exon-3 extends from base 62856 to 62591, and is 266 bp long. This means that there are 2 introns which must be 198 bp long in total. intron-1 (between exon-1 and exon-2) extends from 63247 to 63136 and must be 112 bp long. intron-2 (between exon-2 and exon-3) extends from 62942 to 62857 and must be 86 bp long. Why does it appear that intron-2 is before intron-1? The fifth line of the Features Table shows the actual coding sequences (CDS) in the mature processed mRNA excluding the 5' UTRs and 3' UTRs. the coding sequence of exon-1 begins at base 63338 - not 63391! This means that there must be a 53 bp 5' UTR. the coding sequence of exon-3 ends at 62820 - not 62591. This means there must be 229 bp of 3' UTR! The eleventh line of the Feature Table shows the amino acid translation, given from the N-terminal to C-terminal ends. The protein begins with methionine. The amino acids coded in exon-1 are colored blue. The amino acids coded in exon-2 are colored pink. The amino acids coded in exon-3 are colored blue.

The ORIGIN Table shows the original DNA sequence which was submitted to GenBank. The section of the tutorial below explains how the annotation provided in the Features Table for the putative auxin-binding gene correspond with the original base sequence.

Open the image to the left ..... it will help you understand the key points below! This is the actual genomic sequence of the gene for this auxin-regulated protein. The full sequence of the hnRNA - including 5' and 3' UTR's, and the two introns - is shown bounded in rose in smaller fonts. Genbank always gives sequences in the 5'--->3' direction. However the coding sequence for this gene is the complement of this sequence, and must therefore be read in the 3' --> 5' direction (from bottom right to upper left)! It has been color coded to illustrate the structure of the locus as given in the annotation above:  blue=exon-1 and exon-3;  pink=exon-2;  gold (small font)= intervening sequences; rose (small font)= 5' and 3; UTRs;   "gu" and "ag" splice signals at the 5' and 3' ends of the introns = green letters inside yellow boxes. The start codon and stop codon are underlined. NOTE the following! As shown in the annotation, the gene begins at base 63391 with the 5' UTR. As shown in the annotation, the coding sequence begins at base 63338 with the "tac" codon. The complements is "atg" - the methionine start codon! As shown in the annotation, intron-1 begins with base 63247. The RNA complement of "ca" is "gu" the conserved doublet at the 5' end of an intron. As shown in the annotation, intron-1 ends with base 63136. The RNA complement of "tc" is "ag" the conserved doublet at the 5' end of an intron. As shown in the annotation, the coding sequence ends at base 62820 with the triplet "act". The RNA complement of "act" is "uga" - one of the three stop codons! Not only are the coding sequences split, but even individual  codons may be split between two exons! The splice site between exon-1 and exon-2 occurs in the middle of the triplet codon for glycine! The last full codon of exon-1 is "ttt" (complement is "aaa" the codon for lysine).  The next codon is "cct" (RNA complement  is "ggu", the codon for glycine.) The first base of "cct" is at the 3' end of exon-1, but the second and third bases are at the 5' end of exon-2!  The splice site between exon-2 and exon-3 occurs in the middle of the triplet codon for tryptophan! The last full codon of exon-2 is "cta" (RNA complement is "gau" the codon for aspartic acid).  The next codon is "acc" (complement  is "ugg", the codon for tryptophan.) The first and second bases of "acc" are at the 3' end of exon-2, but the third base is at the 5' end of exon-3. The image to the left shows the nucleotide sequence, in register with, the translation as given above in the Feature Table. It begins with the start codon at the 5' end of exon-1, indicates the two splice sites and ends with the stop codon at the 3' end of exon-3.

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