Question

Genes in eukaryotes are often organized into
exons and intrans, which require splicing to produce
an mRNA that can be translated. The gene organization is the order of the DNA segments
that comprise the gene starting with the promoter,
the first exon, the first intron, the second
exon, and so on. The interspersed intrans can
make gene identification difficult in eukaryotesparticularly
in higher eukaryotes with many introns
and alternative spliced mRNAs. Prediction
of many genes and their organization has been
based on similarity searches between genomic
sequence and known protein amino acid sequences
and genomic sequence and the corresponding
full-length cDNAs. cDNAs are reversetranscribed
mRNAs and therefore generally do
not contain intron sequences. cDNAs (i.e., copied
DNA) can be considered mRNAs. A comparison of
a genomic sequence (with introns) to its corresponding
cDNAs will reveal where introns begin
and end. Gen Bank will contain the genomic
sequence and the cDNA sequence. To find out the
structure of the gene (i.e., the arrangement of the
exons and intrans) we simply need to perform a
sequence comparison between the genomic sequence
and the cDNA sequence. Shown below is
a genomic sequence from the species C.elegans.
The Basic Local Alignment Sequence Tool (BLAST)
can be used to elucidate part of the gene organization
(arrangement of exons and introns) of a
36 CHAPTER 2 Information Organization and Sequence Databases
genomic sequence. BLAST can be used to
compare genomic DNA sequence with all RNA
sequences (i.e., cDNA sequences) in GenBank .
The top hit of the output will be a sequence
comparison between your sequence (the query
sequence) and the most similar sequence in the
database (subject sequence). Subsequent hits will
display sequence comparisons between the
query sequence and subject sequences that are
increasingly less similar . If all hits have 100%
identity, use the hit with the most extensive
percent coverage to report on. Use the nucleotide
BLAST tool and appropriate databases to
construct a schematic diagram that shows the
arrangement of introns and exons in the genomic
sequence. Remember that the species source of
genomic sequence is Caenorhabditis elegans.
ATTTTTAAAAATGTACAAAATCAAACGCCCTACAA
ATCATGTGTGTGAAGAAGAATAATAACTAACATAT
CTATTTATATTTACCGAATAAATATATATTCATCAAT
TAACCTGAAGAACAAACGATATCGGCTACAGGC
GTCGATCAGTCTCGAATCTAGTAACAACAAGAGAG
CAATACGAAAACCGGTAAATCAATAGGGGGAAGCG
AAACAGTAGGTACAAATTGGAGGGGAAGCACCAA T
ACATTAGGTGGGGGGTACGACTTGAAAAATGAGCT
GATTTTCGAATAGTTAAAGCGATGATCGTGTCCGA
AAAACAGTTCATTTTTCAAGACAACATTGAGACTG
GGAGTACGGGGAAGCTCATTTACGGTGAGAGGAA
TTGGTGAGATCTTTAGAATATGCTTAAGGAGTTGGG
GTGGCTGGAGAAGTTCCTGTAGCCTCCGTGCCGG
GATTCGATGGAGAAGTCGTTGCGGCTGGTCCCTTTT
CCTTCACTGGTGCTGGATCCTTGGCTGGAAGACAT
ATGCGTGGCTTGACAGTCGATGAGGTGCGAGCCGA
CGAGTCCTTGTGAACTTCGTATCTGGAAATATTTTA
CTTAGATAGCAAATACTAAAATTGTAAAATTACC
TCAAAATCTCAGTATCCGGAATGCTCAATTTCTGCT
TCAAAACCTGTCCGATGCGAAGATTGACATCATC
GCGAGTAGCATCACGAGTCCACAAGGAAACCTTGT
CACCCTTTTGACGAACATTCACGACAGCTCCGCAG
ATGTAGTCTCCGTACTCGTCGAATTGCTCTCCAA
CAATAGCCATCAACAGCTCCAACCAGTAGTGATCGA
GCAATTGCGTTCTTCTCTGAAGCTTCTATGATTCAT
TGAATAAAATATATTTCTCAAAACGTACTTGCTT
ATCGACAACAACCAACCAACTGCCACCTTGAACGTT
GTTGACGTCCTCCCACATTGGCTTGATTCCTTCC
TTGAACAAGTAATAATCGGATCCCCAGTTCAATCCT
CCGGCAGACTGAATGTGATTGTACAGCGACCAGA
AGTCCTCGACAGTGTCGAAAAGTGAAACCATCTGGA
AAAAATCGATAAAAGACGTATTTAAAAATCTTCT
ACCTTCAGACAATCCTCCCATTCCTTGTTACGGTCA
GCTTTCAAGTACCAGAGAGCCCAGCTGTACTGGA
GGGGGTGTCTGGTGAGAAGCTCTGGAGGAACTGAAGC
ATCGGACGCATTCACATCGCCGGAAGC TGACAA
TGCTTTGTTTTCCGCTACGGATGTGCTCATTTAGC
TGAAAATAGGTAATATTATATACGATTAGAGCTCG
GAAAACGATAAAATAGAGAAGAGTATGAATTTGGTT
CAAATAACTCGGATTTTATAGGAAATTTTGTTTT
ACTGCACATTTTCGGCTAGTTTCCAAGCTTTTTAGA
TTTTTCAAGTGTAATTGGTAACATCGGGCACAAT
AAATTGATATTAAAGCTTGGAAAACAATAA
In addition to construction of the schematic diagram
answer the following:
a. Give the name and accession numbers of each
distinct mRNA produced from this gene.
b. Give the names and accession numbers of the
protein product(s).
c. Note the numbering of the sequences in the alignments
. Does the database genomic sequence
progress in the same direction as the database
mRNA? In other words is it the same orientation
(see below) :
1 ... ... .. .... ........ ..... ........ 114 = query
61 ..... ........ .... ......... .... .... 98 = subject
or opposite orientation (below):
1 ........ .... ........ ..... ........ 114 = query
98 ... ..... ........ .... ......... .... . 61 = subject
d. Consider the alignment of the query sequence and
the subject sequence. What does the orientation
of the sequences relative to each other tell you
about the sequence that was used as the query
sequence?
e. Give the amino acid sequences separately translated
from each exon sequence of the longest
transcript.
f. How many alternative splice variants are associated
with this genomic sequence? List their accession
numbers .
g. Give the chromosome position numbers that
denote the start and end of the TP53 gene . The
position number is the base number on chromosome
17. Calculate the length of the primary transcript
. Give the lengths , in base pairs, of each exon
and intron

Answer 1

a) The mRNA formed from the gene is Caenorhabditis elegans Eukaryotic translation initiation factor 4E-3 (ife-3). Its has three variants. The accession number for these three mRNA variants are: NM070723.7 , NM_171920.4 , NM_070722.4

b) The protein formed from is Eukaryotic translation factor 4E-3 and the accession number of the protein is O61955.

c) The database of mRNA progress in the opposite direction of the genomic database. For example:

1......................................114 query

98.......................................61 subject

d) The DNA strand which gives rise to mRNA aligns in a reverse manner with the sequence of the gene.