Download Obtaining information from a cloned gene

Obtaining information from a cloned gene
Objectives:
j
1. What is the biochemical role of the gene?
2. Where and when is the gene expressed (transcribed)?
3. Where and when is the protein made?
Reference:
Westhoff et al.
al Molecular Plant Development: from gene to
plant. Chapter 3:39-65.
Gene sequence
provides clues to its biochemical function
1 Open reading frame (ORF)
1.
A sequence consisting exclusively of triplets that can be translated
into amino acids.
Usually only one reading frame open, i.e. is translated and the
others are blocked by frequent termination signals.
2. BLAST search
BLAST = a computer program that compares the Query [Query =
sequence of interest (mostly protein)] with other sequences from
the database to find sequence identity
identity.
Gene sequence
provides clues to its biochemical function
1. Open reading frame (ORF)
Assignment (handout): Find an ORF for the newly isolated
gene that has been translated in six different reading
g
g frames
5’---Æ3’ (#3 is open) - WHY?
It is the longest, therefore likely correct. In a random case
one would expect on average a STOP codon every 21
amino acid:
(43 = 64 codons total,
total 3 STOP codons => 64/3 ~21)
Gene sequence
provides clues to its biochemical function
2. BLAST search
Query: BEL1
Gene sequence
provides clues to its biochemical function
2. BLAST search
Output:
(a) Extent of sequence identity
S
Score
(bits)
(bit ) – score assigned
i
d tto a match
t hb
between
t
ttwo sequences
E-value – likelihood that this kind of sequence identity is possible
by chance (approaches 0 in high % identity matches).
(b) Identical residues present along the entire length of the protein
or
Clustering of identical residues in one protein segment
Clustering likely identifies functional domains
[Eg transcription factor (homeodomain binds DNA)]
[Eg.
When and where is the gene expressed?
1. RNA gel blot (=northern blot) analysis
www.biochem.arizona.edu
When and where is the gene expressed?
1. RNA gel blot (=northern blot) analysis
Northern blot analysis can detect one mRNA in a mixture of
any number of mRNAs in a tissue
It gives information about:
• When, in which tissue, and how highly is the
gene expressed?
• The
Th size
i off the
th transcript
t
i t
• Transcript accumulation in mutants vs. wild type
When and where is the gene expressed?
1. RNA gel blot (=northern blot) analysis
RNA gel blot analysis of steady state ECR mRNA in wild type Col
Col-0,
0 wild type
Ler and ecr mutant lines shows that:
Both ecr-1 and ecr-3 are transcriptional knockouts
knockouts, as no transcript can be
detected in either line.
The abundance of the ECR transcript
p in the ecr-2 line is comparable
p
to the
wild type.
When and where is the gene expressed?
2 In situ hybridization
2.
Longitudinal sections of the inflorescence
Sections of Arabidopsis tissues hybridized to:
(A) antisense CER6 RNA probe
(B) sense CER6 RNA probe (Control!)
Hybridization is indicated by a purple precipitate produced as a result of
an alkaline phosphatase reaction with the NBT/BCIP substrate.
When and where is the protein made?
1. Protein blot (=western blot) analysis
SBE I in crude embryo extracts
SDS-PAGE
WESTERN
(Antiserum)
WESTERN
(Preimmune
serum)
Western blot analysis can detect one protein in a mixture of any number of
proteins, while giving information about the size of the protein.
This method requires the use of a high-quality antibody directed against a
desired protein.
When and where is the protein made?
2. In situ protein localization (tissue or individual cells)
(a) using an antibody
Expression of DEF and GLO proteins is confined to
the epidermis of floral organs
When and where is the protein made?
2. In situ protein localization (tissue or individual cells)
(b) using GFP or an epitope tag
CaMV 35S
GFP
CER5
NOS
CaMV 35S::GFP::CER5
CaMV 35S
CER5
GFP
CaMV 35S::CER5::GFP
NOS
When and where is the protein made?
Epitope tagging
When and where is the protein made?
2. In situ protein localization (tissue or individual cells)
(b) using GFP or fluorescently labeled antibody to protein tag