9 High-throughput Sequencing Four-color fluorescent dyes have replaced the radioactive label. Reads greater than 800 bp are possible, though 500~700 is more common. Applied Biosystem's ABI Prism TM 3700 six 96-well plates per day 96 X 6 X 800 ~ 0.5 M Amersham Pharmacia's Mega BASE 1000 TM
16 6.2.1 Promoter Elements promoter a binding site in a DNA chain at which RNA polymerase binds to initiate transcription of messenger RNA by one or more nearby structural genes
17 18.104.22.168 RNA polymerases β’: to bind to DNA template β: to link one nucleotide to another α: to hold all subunits together σ: to recognize the specific nucleotide sequences (which is less conserved)
20 22.214.171.124 consensus sequence recognized by the same σ-factor agree for many different genes operon the set of genes with related functions regulatory proteins positive regulator enhance ( 強化 ) negative regulator repress ( 抑制 ), attenuate ( 減弱 )
21 lactose ( 乳糖 ) operon ( 操縱子 ) (in E. coli) beta-galactosidase (z) lactose permease (y) lactose transacetylase (a) One long polycistronic RNA makes all three proteins.
22 126.96.36.199 E. Coli ’ s Lac Operon σ 70 Most efficiently expressed only when a cell’s environment is rich in lactose ( 乳 糖 ) and also poor in glucose ( 葡萄糖 ) lactose combined with negative regulator pLacI gene expressed! glucose positive regulator CRP gene enhanced!
25 6.2.2 Open Reading Frames stop codons UAA, UAG, UGA (1 - 3/64) N = 0.05 N~63 E. Coli average length = 316.8 codons, 1.8% shorter than 60 codons Open Reading Frame (ORF) continuous triplet codons without stop codon
26 start codon AUG E. Coli AUG ~ 83%, UUG ~ 17% How to determine the starting position for translation? start codon Shine-Delgarno sequence A,G-rich region serves as ribosome loading sites E.g., 5’ – AGGAGGT – 3’
31 6.3 GC-Content in Prokaryotic Genomes G/C to A/T relative ratio recognized as a distinguishing attribute of bacterial genomes GC: 25% ~ 75%, wide range GC-content of each bacterial species seems to be independently shaped by mutational biases
32 GC-contents are generally uniform throughout bacteria’s genomes horizontal gene transfer the movement of genetic material between bacteria other than by descent in which information travels through the generations as the cell divides GC-contents reflect the evolutionary history of the bacteria
33 Prokaryotic Gene Density 85%~88% are associated with the coding regions E. Coli 4288 genes, average length 950 bp, separated by 118 bp.
34 Finding genes in prokaryotic genomes is relatively easy. Long open reading frames (>60); Matches to simple promoter sequences; Transcriptional termination signal; Comparisons with the nucleotide sequences of known protein coding regions from other organisms.
35 6.5 Eukaryotic Genomes Differences (to prokaryotic genomes) Internal membrane-bound compartments allows them to maintain a wide variety of chemical environment. eukaryotes Multicellular organisms, each cell type usually has a distinctive pattern of gene expression. relatively little constraint on the size of their genomes gene expressions, more complicated & flexible
37 6.6 Eukaryotic Gene Structure 1000 times harder than finding a needle in a haystack??? Long open reading frames is not appropriated since introns exist.
38 Grail EXP & GenScan Rely on neural network and dynamic programming. prediction < 50%
39 Detecting features include promoter a series of introns/exon boundaries putative ORF with codon usage bias
40 6.6.1 Promoter Elements prokaryotes single RNA polymerase eukaryotes three kinds of RNA polymerases
41 RNA polymerase I, III are needed at fairly constant levels in all eukaryotic cells at all times.
42 RNA polymerase II basal promoter RNA polymerase II initiation complex is assembled and transcription begins. upstream promoter elements protein binding Have been estimated that at least 5 upstream promoter elements are required to uniquely identify the genes.
43 RNA polymerase II does not recognize the basal promoter directly. basal transcription factors TATA-binding protein (TBP) at least 12 TBP-associated factors (TAFs) TATA-box for eukaryotes (-25) 5’ – TATAWAW – 3’ (W= A or T) initiator (Inr) sequence 5’ – YYCARR – 3’ (Y=C or T, R=A or G)
45 Transcription factor differences cause tissue-specific expression of some gene.
46 6.6.2 Regulatory Protein Binding Sites bacteria RNA polymerases have high affinity for promoters. emphasis on negative regulation eukaryotes RNA polymerases II & III do not assemble around promoters very efficiently. additional emphasis on positive regulations
47 Transcription Factors constitutive Do not respond to external signal. regulatory Do respond to external signals. sequence-specific DNA-binding protein
49 6.7 Open Reading Frames Nuclear membrane separates the process of transcription and translation. DNA hnRNA (heterogeneous RNA) mRNA translation capped, spliced, poly-A capped: chemical alteration (e.g., methylation) splicing: removal of introns polyadenylation: ~ 250 A’s at the 3’ end
50 Splicing causes a serious problem for gene recognition algorithm. Do not have to posses the statistically significant long ORFs.
52 GU-AG rule 5’ – GU - ……………………… - AG – 3’ Splicing apparatus scrutinizes sequences lying within introns; do not constrain the information content of exons. introns: > 60 bp; ~ 450 bp in vertebrates
53 6.7.2 Alternative Splicing 20% of human genes have alternative splicing extreme example: 64 different mRNAs Splicing apparatus have small nuclear RNAs (snRNAs) several proteins
59 6.8 GC Content in Eukaryotic Genomes Eukaryotic ORFs are much harder to recognize. Help to detect the functionalities (genes, promoters, codon choices) of genomes.
60 6.8.1 CpG Islands statistical evaluation of the frequencies of GG, GA, GT, GC, …… CpG: 5’ – CG – 3’ p: phosphodiester bond CpG only 20% of the frequency as by chance CpG islands: at the normal level
65 DNA methylation acetylation (CH 3 CO, 乙醯基 ) of histones methylation & histone acetylation high level of gene expression Methylation patterns of DNA are somewhat difficult to determine experimentally.
69 euchromatin less tightly active heterochromatin densely packed inactive
70 6.8.2 Isochores long regions of homogeneous base composition (w.r.t. GC-content) >1M in length GC-content of an isochore is relatively uniform throughout (by sliding window of 1000 bp, difference < 1%). mosaic the genome
71 human genome 5 different classes of isochores L1 (39%), L2 (42%), H1 (46%), H2 (49%), H3 (54%) H3: at least 20 times of the density of genes to L1 |H3| ~ 3% to 5%, contains ~80% of all housekeeping genes. |L1|+|L2| ~ 66%, contain ~ 85% of tissue- specific genes.
73 GC-rich region tends to have low level of methylation; be stored as transcriptionally active euchromatin; have promoter sequence closer the the transcriptional start site; have shorter introns and genes; have short interspersed nuclear elements (SINEs); bias the use of codons.
74 6.8.3 Codon Usage Bias In yeast genome arginine: AGA 48% (CGT, CGC, CGA, CGG, AGG) Codon usage bias can be used to detect exons (reflecting the preference).
75 6.9 Gene Expression Eukaryotic gene recognition algorithms employ Known promoter elements (i.e., TATA and CAAT boxes); CpG islands; Splicing signal associated with introns; ORFs with characteristic codon utilizations; Similarity to the ESTs or genes from other organism.
76 6.9.1 cDNAs and ESTs cDNA (complementary DNAs) cDNAs can be cloned into vectors and maintained as a cDNA library. Fig. 6.11
79 ESTs (expressed sequence tags) cDNAs provide the population of genes being expressed; the mRNA’s relative abundance. reassociation kinetics: R 0 t 1/2
80 6.9.2 Serial Analysis of Gene Expression SAGE Make cDNAs from the sample; Break the cDNAs into small fragments (10~14 nt) by restriction enzymes; Randomly ligate the fragments into longer DNA molecules that are cloned and sequenced; Use computer to recognize the original small fragments and compare them to known transcripts from the organism. Tags reflect the relative abundance of the corresponding transcript.
82 6.10 Transposition DNA transposons insertion sequences retrotransposons
83 6.11 Repetitive Elements tandemly repeated DNA satellite DNA 5~200 bp repeat unit, millions of copies minisatellite < 25 bp microsatellite < 4 bp
84 interspersed repeat LINE (long interspersed nuclear element) SINE (short interspersed nuclear element) DNA finger print
85 6.12 Eukaryotic Gene Density 3000 Mb human genome < 90 M (3%) corresponds to coding sequences. 810Mb (27%) are associated with introns, promoters, and pseudogenes. 2100 Mb (70%) 1680 Mb (56%) unique sequences 420 Mb (14%) repetitive DNA
86 參考資料及圖片出處 1. Fundamental Concepts of Bioinformatics Dan E. Krane and Michael L. Raymer, Benjamin/Cummings, 2003. Fundamental Concepts of Bioinformatics 2. Biochemistry, by J. M. Berg, J. L. Tymoczko, and L. Stryer, Fith Edition, 2001. Biochemistry 3. Biology, by Sylvia S. Mader, 8th edition, McGraw-Hill, 2003. Biology