![-FGENESH [Find Genes] (HMMs)
-FGENESH_C (Similarity based)
-FGENESH+ (Combination of ab initio &similarity
based)
-MZEF [Michael Zhang’s Exon Finder](QDA)
HMMs
-GENSCAN (Fifth order HMMs); combination of
hexamer frequencies with coding signals;probability
score P>0.5
-HMMgene (Conditional Maximum Likelihood);
combination of ab initio & homology-basedalgorithm](https://image.slidesharecdn.com/gene-180525203814/85/Gene-prediction-and-expression-19-320.jpg)
![ Homology-based-
Exonstructures and sequencesof related speciesare
highly conserved.
Comparison of homologous sequences derived from
cDNAor ExpressedSequenceTags (ESTs).
-GenomeScan (Combination of GENSCANprediction
results with BLASTXsimilaritysearches)
-EST2Genome (Intron-exon boundaries); Comparison
of an ESTsequence with agenomic DNAsequence
-SGP-1 [Syntenic Gene Prediction] (Similar to EST2)
-TwinScan (gene-finding server; similar to
GenomeScan)](https://image.slidesharecdn.com/gene-180525203814/85/Gene-prediction-and-expression-20-320.jpg)
Gene prediction and expression
- 2. Gene: • Asequence of nucleotides coding for protein. CentralDogma: • Proposed in 1958 by Francis Crick. • Hepostulated that all possibleinformation transferred, are not viable. • Hepublished apaper in 1970. CODONS: • Discovered by Sydney Brenner and Francis Crickin 1961. • In every triplet of nucleotides, each codoncodesfor one amino acid in aprotein.
- 3. DNA RNA PROTEIN PHENOTYPE 2 4 cDNA 1 3 1. TRANSCRIPTION 2. TRANSLATION 3. GENE EXPRESSION 4. REVERSETRANSCRIPTION
- 4. DEfiniTION • It is aprerequisite for detailed functionalannotation of genesand genomes. • It candetect location of ORFs(Open Reading Frames), structures of introns andexons. • It describes all the genescomputationally withnear 100% accuracy. • It canreduce the amount ofexperimental verification work required.
- 5. TYPES • Abinitio- gene signals, intron splice, transcription factor binding site, ribosomal binding site, poly- adenylation site, triplet codon structure and gene content. • Homology- significant matches of query sequence with sequence of knowngenes. • Probabilistic models like Markov model or Hidden Markov Models (HMMs). Abinitio-based Homology- based
- 6. Translation Protein Splicing mRNA Cap- -Poly(A) Transcription pre-mRNA Cap- -Poly(A) Genomic DNA Stop codon GT AG exon intron Splice sites Donor site Acceptor site SEQUENCE SIGNALS Start codon Exonsare usually shorter thanintrons.
- 7. Prokaryoticgene prediction • Geneprediction is easier in microbialgenomes. • Smaller genomes, high gene density, very few repetitive sequence, more sequenced genomes. • Start codon is ATG. • Ribosomal binding site/Shine Dalgarno sequence.
- 8. Openreadingframes • A sequence defined by in-frame start and stop codon, which in turn defines aputative amino acid sequence. • Agenome of length n is comprised of (n/3)codons. • Stop codons break genome into segments between consecutive stop codons. • Thesub-segments of these that start from the Start codon (ATG)areORFs. • DNA is translated in all six possible frames, three frames forward and three reverse. ATG TGA Genomic Sequence Open reading frame
- 10. Probabilisticmodels • Statistical description of agene. • Markov Models &Hidden Markov Models. • Usedto distinguish oligonucleotide distributions in the coding regions from those for non-coding regions. • Probability of distribution of nucleotides inDNA sequence depends on the order k. • Typesof order- zero,first and second. • Order , gene canpredicted more accurately.
- 11. Genecontent and length distribution of prokaryotic genes TYPICAL ATYPICAL Ranges from100 to 500amino acids with a nucleotide distribution typical ofthe organism. Shorter or longer with different nucleotidestatistics. Genes tend toescape detection when typical gene modelis used.
- 12. Genefindingprogramsin prokaryotes • Theprograms are based on HMM/IMM. GeneMark.hmm (microbial genomes) Glimmer (UNIX program from TIGR). Computation involves two steps viz. model building & gene prediction. FGENESB (bacterial sequences). It uses Vertibi algorithm & linear discriminant analysis(LDA). RBSfinder- Searches from ribosomal binding site or shine dalgarno sequence for prediction of translation initiation site.
- 13. Sensitivity Ability to include correct predictions. It is the fraction of known genescorrectlypredicted. Specificity Ability to exclude incorrect predictions. It is the fraction of predicted genes that correspond to true genes. Both are the proportion of true signals.
- 14. Eukaryoticgeneprediction • Genomes are much larger than prokaryotes(10Mbp to 670 Gbp). • Low gene density. • Spacebetween genesis very large and rich in repetitive sequences & transposableelements. • Splitting of genesby intervening noncodingsequences (introns) and joining of coding sequences(exons).
- 15. • Splice junctions follow GT-AGrule. • An intron at the 5’ splice junction hasaconsensus motif GTAAGTand that at 3’ endNCAG. exon 1 exon 2 • Geneshave ahigh density of CGdinucleotides near the transcription start site. Thisregion is CpGisland. It helps to identify the transcription initiation site of an eukaryotic gene. • Somepost-transcriptional modification occur with the transcript to become mature mRNAviz. Capping, Splicing and Polyadenylation. Acceptor Site Donor Site GT AG
- 16. o CAPPING: Occurs at the 5’ end of the transcript. It involves methylation at the initial residue of the RNA. o SPLICING: Processof removal of intronsand joining of exons. It involves alargeRNA-protein complex called spliceosome. o POLYADENYLATION:Addition of astretch ofAs (~250) at the 3’ end of the RNA.Theprocessis accomplished by poly-Apolymerase.
- 17. Genefindingprogramsin EUkaryotes • Three categories of algorithms Ab Initiobased- It joins the exonsin correct order.Twosignals-> a) Genesignals: asmall pattern within the genomic DNAincluding putative splice sites, start and stop sites of transcription or translation, branchpoints, transcription factor binding sites, recognizable consensus sequences. b) Genecontent: aregion of genomic DNAincluding nucleotide and amino acid distribution, Synonymous codon usageand hexamer frequencies.
- 18. Neural network based algorithm -Composed of network of mathematicalvariables. -Multiple layers like input, output and hiddenlayers. -GRAIL (Splice junctions, start and stop codons, poly-A sites, promoters and CpGislands). It scansthe query sequence with windows of variable lengths &scores. Discriminant analysis -Linear Discriminant Analysis (LDA) represents 2D graph of coding signals vs. all possible 3’ splice site positions; adiagonal line. -Quadratic DiscriminantAnalysis (QDA)represents quadratic function; acurved line. -FGENES (LDA)
- 19. -FGENESH [Find Genes] (HMMs) -FGENESH_C (Similarity based) -FGENESH+ (Combination of ab initio &similarity based) -MZEF [Michael Zhang’s Exon Finder](QDA) HMMs -GENSCAN (Fifth order HMMs); combination of hexamer frequencies with coding signals;probability score P>0.5 -HMMgene (Conditional Maximum Likelihood); combination of ab initio & homology-basedalgorithm
- 20. Homology-based- Exonstructures and sequencesof related speciesare highly conserved. Comparison of homologous sequences derived from cDNAor ExpressedSequenceTags (ESTs). -GenomeScan (Combination of GENSCANprediction results with BLASTXsimilaritysearches) -EST2Genome (Intron-exon boundaries); Comparison of an ESTsequence with agenomic DNAsequence -SGP-1 [Syntenic Gene Prediction] (Similar to EST2) -TwinScan (gene-finding server; similar to GenomeScan)
- 21. Consensus-based- Combination of results of multiple programsbased on consensus. Improvement of specificity by correctingfalse positives & problem ofoverprediction. Lowered sensitivity & missedpredictons. -GeneComber (Combination of HMMgene& GenScanprediction results) -DIGIT (Combination of FGENESH,GENSCAN& HMMgene)
- 22. GENE EXPRESSION Two steps are required 1. Translation The synthesis of a polypeptide chain using the genetic code on the mRNA molecule as its guide. 1. Transcription The synthesis of mRNA uses the gene on the DNA molecule as a template This happens in the nucleus of eukaryotes
- 23. Types OF RNA Messenger RNA (mRNA) <5% Ribosomal RNA (rRNA) Up to 80% Transfer RNA (tRNA) About 15% In eukaryotes small nuclear ribonucleoproteins (snRNP aka spliceosomes Structural characteristics of RNA molecules Single polynucleotide strand which may be looped or coiled (not a double helix) Sugar Ribose (not deoxyribose) Bases used: Adenine, Guanine, Cytosine and Uracil (not Thymine
- 24. Transcription: The synthesis of a strand of mRNA (and other RNAs) Uses an enzyme RNA polymerase Proceeds in the same direction as replication (5’ to 3’) Forms a complementary strand of mRNA It begins at a promotor site, which signals that the beginning of the gene is near (about 20 to 30 nucleotides away) After the end of the gene is reached, there is a terminator sequence that tells RNA polymerase to stop transcribing NB Terminator sequence ≠ terminator codon RNA POLYMERASE
- 25. Editing the mRNA In prokaryotes, transcribed mRNA goes straight to the ribosomes in the cytoplasm In eukaryotes, freshly transcribed mRNA in the nucleus is about 5000 nucleotides long When the same mRNA is used for translation at the ribosome it is only 1000 nucleotides long The mRNA has been edited The parts which are kept for gene expression are called EXONS (exons = expressed) The parts which are edited out (by spliceosomes) are called INTRONS.
- 26. Translation TRANSLATION Complete protein Polypeptide chain Ribosomes Stop codon Start codon © 2016 Paul Billiet ODWS
- 27. Translation Location: The ribosomes in the cytoplasm that provide the environment for translation The genetic code is brought by the mRNA molecule. © 2016 Paul Billiet ODWS
- 28. An important discovery Retro viruses (e.g. HIV) carry RNA as their genetic information When they invade their host cell they convert their RNA into a DNA copy using reverse transcriptase Thus the central dogma is modified: DNA↔RNAProtein This has helped to explain an important paradox in the evolution of life. Reverse transcriptase © 2016 Paul Billiet ODWS
- 29. The paradox of DNA DNA is a very stable molecule It is a good medium for storing genetic material but… DNA can do nothing for itself It requires enzymes for replication It requires enzymes for gene expression The information in DNA is required to synthesise enzymes (proteins) but enzymes are require to make DNA function Which came first in the origin of life DNA or enzymes? © 2016 Paul Billiet ODWS
- 30. RIBOZYMES: Both genetic and catalytic Certain forms of RNA have catalytic properties RIBOZYMES Ribosomes and spliceosomes are ribozymes RNA could have been the first genetic information synthesizing proteins… …and at the same time a biocatalyst Reverse transcriptase provides the possibility of producing DNA copies from RNA. © 2016 Paul Billiet ODWS
- 31. The ribosome a ribozyme
- 32. REFERENCES http://www.4ulr.com/products/currentprotocols/bioinformatics.html http://proxy.lib.iastate.edu:2103/nrg/journal/v3/n9/full/nrg890_fs.html http://proxy.lib.iastate.edu:2103/nrg/journal/v5/n4/full/nrg1315_fs.html Xiong J.;Essential bioinformatics; QH324.2.X56 2006