What is a transcriptome?

A transcriptome is a collection of all the gene transcripts

present in a given cell.
A transcriptome represents the very small percentage of the
genetic code that is transcribed into RNA molecules.
As each gene may produce many different types of mRNA
molecules, so the transcriptome is much more complex than
the genome that encodes it.
What is a proteome?
The entire complement of proteins in a given biological
organism or system at a given time, i.e. the protein
products of the genome.
A cellular proteome is the collection of proteins found in a
particular cell type under a particular set of environmental
conditions.
A complete proteome, can be conceptualized as the
complete set of proteins from all of the various cellular
proteomes. This is very roughly the protein equivalent of
the genome.
Analysis of a Transcriptome

• Analysis of mRNA transcription profile of a genome

• Uses, ESTs, SAGE, differential display, microarray
technologies
• Computational and experimental
• Pattern analysis of large numbers of genes identifies
functionally linked genes
• Identification of expression profiles of genes can
reveal information about novel functions
• Global analysis, not single gene approach
Differential Display Identification of plunc, a Novel Gene
Expressed in Embryonic Palate, Nasal Epithelium, and
Adult Lung. J. Biol. Chem. 1999 274: 13698-13703.
This paper described the isolation of a novel gene Palate, lung and nasal
epithelium clone (Plunc) with a unique expression profile in the
nasopharynx and upper airways. No other genes have been described with
a similar distribution in the respiratory tract. Plunc might be a useful marker
of epithelial cells in these regions
The principles of differential display - one of a
number of ways to study differential gene
expression
Starting Hypothesis. Plunc may serve as a useful
marker for cells of the epithelium of the upper
airways.

A functional genomics study with no prior functional

information

• The gene may be a lineage marker for tracheal development

• It may be related to the development of tracheal abnormalities
• The gene may be useful for targeting transgenes to the upper
airways and nasopharynx
• Plunc may be a useful protein for lung disease diagnosis
Plunc is a member of a distantly related gene family
localized on Chromosome 20

Extensive database analysis identified a number of

distantly related genes located on Chromosome 20. The
first clue came from a Unigene entry which described an
EST cluster which encoded a potential protein 31%
identical to plunc over a 150 amino acid region

Some of these seem to be homologues of proteins from

other species - we have found these in mice, rats, pigs and
cows
EST Projects were planned;

• To identify and catalog transcripts of expressed

(known and novel) genes
• To obtain patterns of expression
• To explore transcripts and splicing variation
• To give an indication of transcript abundance
(expression level)?
Expressed Sequence Tags - ESTs

• Short sequence tags

• Generated from 5’ and 3’ end sequences from cDNA
clones
• Large numbers of sequences analysed
• Can sequence all clones, or selected set
• Try to remove high frequency clones
• Sequences often fragments, internal to transcript
EST analysis

• Sequences assembled into larger contigs of

sequence data representing single transcripts
• Possible because of sequences of partial
clones/fragments
• Data give global analysis of expression pattern of
genes in a specific tissue type
• Important for identification of transcripts, splicing
patterns, alternative sequences
Isolation and characterisation of human plunc

• Plunc cDNAs isolated by searching EST databases

(also found rat and bovine sequences)
• Plunc genomic clones isolated by PCR using the
Promoter Finder system (Clontech)
• Expression tested in total human RNA by RT-PCR
and Northern blotting and in cell lines following
stimulation with cytokines
• Expression by in situ hybridization
 LPLUNC2 LPLUNC3 LPLUNC4 SPLUNC2 SPLUNC3 PLUNC LPLUNC1 RYF3

20Kb
The PLUNC gene family share similar sized exons

The table gives the exon sizes for the human PLUNCs genes along with
those of BPI, LBP, PLTP and CETP.
2005 version - more ESTs and better genomic predictions
Sequenced 50,00 lung derived Ests to generate a lung
transcriptome.

Sequenced 1000 Ests from each normalised library to

give semi quantitative data
It is important to understand that the number of ESTs
may reflect level of expression or how may ESTs have
been sequence for each expressing tissue!
The role of alternative splicing in the generation of
additional PLUNC proteins.

Recent genome-wide analyses of alternative splicing

indicate that 40–60% of human genes have alternative
splice forms, suggesting that alternative splicing is one of
the most significant components of the functional
complexity of the human genome.

Gives rise to more proteins from a set number of genes

The role of alternative splicing in the generation of
additional PLUNC proteins.

Recent genome-wide analyses of alternative splicing

indicate that 40–60% of human genes have alternative
splice forms, suggesting that alternative splicing is one of
the most significant components of the functional
complexity of the human genome.

Some of the PLUNC genes appear to undergo alternative

splicing - For example SPLUNC3

Our data is not in agreement with the Genome

sequencers/annotators!
Types of alternative splicing and possible effects on
protein.
All appear to be utilised in SPLUNC3
Computational identification of alternative splicing.
Experimental analysis of alternative splicing.

a, Alternative splicing can be verified by RT–PCR using primers that flank

the alternatively spliced region. The relative abundance of different isoforms
in various tissue sample can be assessed from gel. b, High-throughput
identification of alternative splicing can be carried by using microarrays. The
microarray probes would consist of exon–exon junction sequence, as
different alternative splice forms will have different exon–exon junctions.
Ensembl - both mouse and human is not quite with it
when we consider the SPLUNC3 gene
Mouse data is:

Peptide : ENSMUSP00000028984
Peptide sequence Peptide properties
>ENSMUSP00000028984
MHLLWRLLVLLLGLLALPSALPKQPWPGLTKAHKDGRSTLARIIAQGLLKLNAEGRIQSMRLLDRLNVSG
TVAPGMVGWLIGGMNFQQQQEISINITNVQLDCGGIQMAFPKEWFSANITLEFDIEFKLPFNSNIIKTHA
CMGLTAESWLEKDEFGRRELVMGRCRMEPSSGGASMSTEETSPKMKHFLHNLRESLGKVIPNLVESQVCP
LIGEILRQLDVKLLKGLV

Such a product does not give a 2 Cys containing PLUNC

peptide!
Human SPLUNC3 exists as a number of specific mRNA
isoforms in testes RNA.

RT-PCR was performed using

multiple primers and cDNA from
human testes (SPLUNC3 was not
found in nasal and tracheal tissue
by northern analysis).
So far 3 of the products would
encode ORFs
Genomic DNA

Wt SPLUNC3
+

+
Mouse SPlunc3 transcripts are also found in Testes
(and tracheal) RNA

RT-PCR analysis also shows the existence of multiple mRNA

isoforms (more still to be sequenced)

Trachea

Testes

Wt mSPLUNC3
Increased BPI expression
was found by microarray
analysis.
This is the first indication of
non-neutrophil expression of
BPI

Are PLUNCs co-expressed?

Analysis of the Proteome

• Analysis of protein expression profile

• Using yeast-2-hybrid, 2-D gels and Mass
Spectroscopy: not a computational analysis
• Total expression patterns
• Post-translational modifications
• Identification of candidate disease changes, drug
targets, developmental biology
• Global analysis: can link to single proteins
 Proteomic studies have identified PLUNC proteins
in a number of biological fluids

Used simple 2D gel analysis followed by peptide identification

2D analysis of nasal lavage fluid
SPLUNC1 levels are elevated in smokers

Such proteomic studies are increasingly used are

screening tools
Saliva from different sources
contains different proteins
Used both 2D gels and LC-MS
Numbers of proteins identified by proteomic analysis is
far less genes identified in array studies
Proteomic studies can give us information on post translational
modifications of proteins
Used LC-MS and selected glycoslylated Salivary proteins
Saliva is heavily glycosylated and such proteins can be
selectively captured
I hope that it is clear that use of transcriptomic and
proteomic tools is of significant importance from the
smallest lab based projects up to the major genome
mapping efforts.

However it is always important to understand that

there is still lots of biology to be done !