1. BIOINFORMATICS
Ayesha M. Khan
Spring 2013
Lec-8

2. GENE PREDICTION/GENE FINDING
The vast amount of raw sequence data generated because of advancement in sequencing technology needs biological interpretation
Known as ‘annotation’
To find genes and determine their functions
At present, the annotation of most human genes is based on cDNA sequence data.
Lec-8

3. Protein coding genes Prokaryotic Eukaryotic
No introns, simpler regulatory features
Eukaryotic
Exon-intron structure
Complex regulatory features
Many different types of RNA exist: tRNA, rRNA, miRNA, siRNA, snoRNA, snRNA
Lec-8

4. Coding sequence
Actual region of DNA that is translated to form proteins. While the ORF may contain introns as well, the CDS refers to those nucleotides that can be divided into codons which are actually translated into amino acids by the ribosomal translation machinery. In prokaryotes the ORF and the CDS are the same.
Lec-8

5. What is gene prediction?
Which region codes for a protein?
Which DNA strand is used to encode the gene?
Where does the gene start and end?
Where are the exon-intron boundaries in eukaryotes?
Where (optionally) are the regulatory sequences for that gene?
The characterization of genomic features using computational and experimental methods is called gene prediction or annotation.
Lec-8

6. Computational methods of gene prediction
Computational gene finding is a process of:
Identifying common phenomena in known genes
Building a computational framework/model that can accurately describe the common phenomena
Using the model to scan uncharacterized sequence to identify regions that match the model, which become putative genes
Test and validate the predictions
Lec-8

7. Biological overview of ‘gene’
Gene: defined as a segment of DNA that contains the necessary information to produce a functional product, usually a protein.
DNA (or RNA in some viruses)
Promoter: controls the activity of a gene
Coding sequence: determines what the gene produces
Core promoter-minimal portion of the promoter required to initiate transcription properly
Proximal promoter-tends to contain primary regulatory elements ; serves as a binding site for specific transcription factors
ORF -Open reading frame
Starts with ATG (start codon) though not always
Terminates with TAA, TAG or TGA (stop codons)
Promoter is the regulatory region of the DNA located upstream (towards the 5’ region) of a gene. This provides a control point for regulated gene transcription. It contains specific DNA sequences that are recognized by proteins known as transcription factors. These factors bind to the promoter sequences recruiting RNA polymerase, the enzyme that synthesizes RNA from the coding region of the gene.
Lec-8

8. Structure of eukaryotic gene
In general -25 to -30 from the start, a consensus sequence is found called TATA or Hogness box,  which acts as first recognition sequence for the assembly of RNA-pol complex, without which enzymes won’t assemble.  The start point itself is bracketed by a set of sequences; hence this region is called InR box or InR sequence. In the upstream region of the TATA box, at a distance there several sequence boxes such as GC box GGGCGG at -90, a CAAT box at -75 and an eight base Octamer box.  The number of such sequence and the position from the start or TATA box varies.  Besides the binding of RNA polymerase complex to TATA box, other factors binding to specific sequence boxes increase the efficiency of transcription. 
Lec-8

9. Methods of gene prediction
Look for something that looks like an already known gene (homology)
Extrinsic/Homology method
Look for something that matches statistical patterns common to all genes (ab initio)
Intrinsic/Ab initio method
Combining homology and ab initio
Hybrid method
Lec-8

10. Extrinsic/Homology Method
Based on sequence similarity of query sequence with annotated genes present in databases.
It is known that only approx. half of the genes can be found by homology to other known genes or proteins.
Based on the following principles:
Coding regions evolve slower than non-coding regions, i.e. local sequence similarity can be used as a gene finder
Homologous sequences reflect a common evolutionary origin and possibly a common gene structure.
Standard pair-wise comparison methods can be used (BLAST or Smith-Waterman)
Include gene syntax information (start/stop codons etc.)
Useful to confirm predictions inferred by other methods
Lec-8

11. Intrinsic/Ab initio Method
Predicts genes based on statistical properties of the given DNA sequence.
Statistical patterns inside and outside of the gene regions as well as typical patterns at their boundaries.
Lec-8

12. Features for gene prediction in eukaryotes
Signal sensors
Content sensors (extrinsic and intrinsic content sensors)
Signal sensors
Evaluates fixed-length features in DNA
Signals: splice sites, start/stop codon, branch points, promoters and terminators of transcription, polyadenylation sites, ribosomal-binding sites, topoisomerase II binding sites, various transcription factor-binding sites etc.
These are measures that try to detect the presence of the functional sites specific to a gene.
The basic signal sensor is a simple consensus sequence or an expression that describes a consensus sequence along with allowable variations.
Use of weight matrices
Lec-8

13. Features for gene prediction in eukaryotes (contd.)
Content sensors
Evaluates variable length features which extend from one signal to another
They classify a DNA region into different types, e.g. coding vs non-coding
Extrinsic content sensor
These sensors perform similarity searching between a genomic sequences region and a protein or DNA sequence present in a database.
Basic tools needed for similarity searching, i.e. BLAST, FASTA etc.
Intragenomic and Intergenomic comparisons
Intra-genomic comparisons provide useful information regarding multigenic families, representing a huge percentage of the existing genes
Intergenomic or cross-species comparisons identify orthologous genes without a preliminary knowledge about them
Lec-8

14. Features for gene prediction in eukaryotes (contd.)
Intrinsic content sensor
Based on statistical models of the nucleotide frequencies and dependencies present in codon structure
Use of MM
CpG islands (regions which often mark the beginning of genes where frequency of CG is not as low as it is in the rest of the genome)
Sensors for repetitive DNA (e.g. ALU sequences)
In mammalian genomes, CpG islands are typically 300-3,000 base pairs in length, and have been found in or near approximately 40% of promoters of mammalian genes. About 70% of human promoters have a high CpG content. Given the frequency of GC two-nucleotide sequences, the number of CpG dinucleotides is much lower than would be expected.
Alu element/sequence is a short stretch of DNA originally characterized by the action of the Alu (Arthrobacter luteus) restriction endonuclease.
Lec-8

15. Gene prediction tools Software based on ab initio methods
GENSCAN, FGENESH, GeneMark.hmm, Glimmer,
Genie, GeneID
Software based on similarity-based methods
GeneWise, SYNCOD, ORFgene2, EbEST
Lec-8