MMG 301, Lec. 23 GenomicsQuestions for today:1. How are microbial genome sequences determined?2. What can we learn from the E. coli genome?3. What is known about other microbial genomes?Genome Sequence Analyses: The process“Shotgun approach”-- Begin with a pure culture (WHY?)-- Isolate DNA (chromosome and plasmid) phenol/chloroform extractionethanol precipitation-- Fragment DNANebulizer to shear DNA randomly ORUse several restriction endonuclease (that cut at only a few sites)e.g., NotI from Nocardia otitidas-caviarumGC↓GGCCGCCGCCGG↑CG8-cutter; thus, (1/4)^8 = 1/65,472 bases~76 fragments for E. coli chromosome-- insert the fragments into a “vector”Bacteriophage such as LambdaPlasmid such as M13mp18(Random DNA fragment)M13mp18carries lacZencoding β-galactosidase-- Place the vector into a host strain-- Identify colonies containing inserted DNA:When its gene is interrupted β-galactosidase is inactive. X-gal (a galactose-like compound called 5-bromo—4-chloro-3-indolyl-β-D-galacto-pyranoside) is not converted to a blue dyeNo insertInsert present(antibiotic resistancemay also be used)-- Each colony contains a single DNA insert, now present in many copies (~1 million cells/colony)-- The plasmids present in each colony are isolatedGel electrophoresisCentrifugation-- Automated sequencing by Sanger methodSeparate strandsAdd primer matching portion of vectorAdd dATP, dGTP, dCTP, dTTPSplit into 4 samplesAdd dideoxynucleotides at low conc.Add polymerase, run polyacrylamide gelConcern: 32P is ahazard!-- Alternative sequencing method: only need a single reaction tubeddNTPs fluorescently labeled-- Assemble information from randomly sequenced fragments into a contiguous sequenceRequires extensive computational effortNeed to find sequence overlaps(puzzle)Direction of fragments unknownAny one stretch of DNA may be sequenced many (~10) times: shotgunKeep sequencing fragments until completeThe entire process can be highly automated using roboticsThis approach does not use radioactivityCan sequence ~500 bases at a time![These eventually form a circle]“Chromosome Walking” approach: -- Most steps are identical-- Difference is that one purposely seeks out overlapping fragments so as to “walk out” from the first sequence. -- This is useful for filling in gaps in small genomes or for determining very large genomes(1) Sequence the initial fragment(2) Make primers matching sequences on each end(3) Seek out other fragments that match the primers and obtain their sequences(4) Deduce overlaps, then repeat-- Identify “Open Reading Frames” (ORFs) NotUse the genetic code (Table 7.3) Universal!Consider all 6 possible reading framesLook for likely start codons (AUG = Met)Note stop codons and “codon usage”One gene is derived from a single reading frame in prokaryotesWithin a chromosome, all 6 reading frames are usedCodon usage:Leu = CUU, CUC, CUA, CUG, UUA, UUG; E. coliprefers 2Neisseriameningitidesgenome (fromSalyers & Whitt)The E. coligenome-- Determined in 1997 (this is recent history!)-- Single, circular molecule-- 4,639,221 base pairs for one lab strain ***-- 4,288 ORFs ***-- 62% are identified: 38% unknown!metabolism (~21%)structure (5%)transport (~10%)replication (~3%)transcription (~1%)translation (~5%)regulation (~9%)other known (~8%)This is anabbreviatedversion of theE. coligenomeWhat does the map show?Genes involved in biosynthesis are easy to study. A mutation of that gene prevents synthesis of the product, but addition of the amino acid (or other missing component) allows growth.trpABCDE: operon of genes encoding enzymes in a pathway for Trp biosynthesis. Genes are italicized.TrpA (not italics) is the protein encoded by trpA.Not all enzymes in a pathway are encoded by adjacent genes; e.g., argR, argG, argD vs. argHBCE.oriC: origin of replication100 min and Hfr strains: It takes 100 min to transfer the chromosome to another cell by conjugation (lec 26). The position of genes can be shown by interrupted mating.lamB (also called malB) and gal operon: important to bacteriophage Lambda infection (lec. 25)lac operon: used for growth on lactose. The regulation of this operon is well known (lec. 24)The sequence of E. coli from strain O157:H7 (a pathogen) was also sequenced: 5.5 Mb (1632 newORFs), with 4.1 Mb highly conserved. Some new genes present, some now absent, and various types of rearrangements observed! ~131 proteins thought to be connected to pathogenicity or virulence.Other microbial genomes(Bacteria, Archaea, Eukarya) (Don’t memorize this list: for illustration only)1995: the field of genomics began!Haemophilus influenzae: First bacterial genome, pathogen, ~1700 ORFsMycoplasma genitalium: STD, smallest genome, 470 ORFs1996Synechocystis sp.: Cyanobacterium (phototroph)Methanococcus jannaschii: First archaealgenome and first methanogenMycoplasma pneumoniae: Pathogen1997Saccharomyces cerevisiae: First eukaryote, 12,100,000 bp, 6034 ORFs; only 2 ½ times the number made by E. coli!Helicobacter pylori: Cause of ulcersEscherichia coli:Archaeoglobus fulgidusBacillus subtilis: Important in food industry, spore formerMethanobacterium thermoautotrophicum: 60°CBorrelia burgdorferi: Cause of Lyme disease, linear chromosome1998Aquafex aeolicus Pyrococcus horikoshii: Extreme thermophileMycobacterium tuberculosis: TuberculosisTreponema pallidum: SyphillusRickettsia prowazekiiChlamydia trachomatisSTD1999Helicobacter pylori: Second strainAeropyrum pernixChlamydia pneumoniaeThermotoga maritimeDeinococcus radiodurans: Radiation resistant2000Campylobacter jejuniChlamydia trachomatisChlamydia pneumoniaeNeisseria meningitidis: Recall deaths at MSUNeisseria meningitidis: (another strain)Vibrio cholerae: CholeraXylelia fastidiosaPseudomonas aeruginosa: Bioremediation (40 cmpds); Cystic fibrosis (lungs); burn patientsArabidopsis thaliana: First plantMore recentlyHomosapiens: 3,300,000,000 bp (=900 books); 30-35,000 ORFs; 23 linear chromosomesMany more microbesFor a complete compilation of known sequences, see http://www.tigr.orgThe Institute of Genomic ResearchGeneral comments about prokaryotic genomes:95 completed prokaryotic genomes as of 2/25/03 (79 Bacteria and 16 Archaea)~200 genomes in progress (not counting private industries)Focus on pathogens, thermophiles, and microbes with unique physiology (phototrophs, alkalophiles, halophiles)Can do entire genome in single day using a room of instruments!General Comments about Eukaryal genomes:Eukaryotic microbes:
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