The Genom of Homo sapiens.pdf

332 MALEK ET AL.Figure 1. The bacterial two-hybrid system. (a) A protein of interestis fused to λcI and another protein of interest is fused toRNA polymerase α-subunit. λcI binds the operator upstream ofa weak promoter. (b) An interaction between the two proteins ofinterest drives transcription of reporter genes by recruitment ofthe transcriptional machinery.FUNCTIONAL SHOTGUN SEQUENCINGAND ANNOTATIONTo test the approach, we shotgun-sequenced thegenome of Rickettsia sibirica, an intracellular humanpathogen among the spotted fever group Rickettsiae. Thegeneral functional shotgun approach involves cloningrandom fragments from the genome into the pBAIT vector,sequencing to determine clones containing in-framefragments, and transferring these fragments to thepPREY vector to create a proteome library against whichto screen baits of interest (Fig. 2). In this study, randomfragments were cloned in the pBAIT, a modified versionof the pACλcI vector, using a BstXI linker method. Thegenome was sequenced to 12.5x sequence coverage fromthis pBAIT library using a primer designed to allow elucidationof the λcI fusion translation product for each insert.A further 3x coverage was conducted in fosmidpaired-end reads to order and orient the contigs. Thegenome was assembled with the Paracel Genome Assembler (Paracel, Pasadena, California), resulting in onescaffold of seven contigs. Assembly revealed a genomeof ~1,250,021 bp. Gene regions were determined usingstandard HMM-based software and assigned function byBLASTP to the GenBank NR database. In this manner,1,234 protein-coding genes were annotated covering a totalof 324,008 amino acids. Translation of the pBAIT insertsrevealed that 3,932 contained fragments, translatedin-frame with the λcI, matching a portion of an annotatedprotein-coding gene. These in-frame fragments contained599,602 amino acids, representing 278,832 unique aminoacids, or about 1.85x random proteome coverage. At1.85x coverage, 85.3% of the proteome should be covered.In-frame fragments were obtained from 986 distinctORFs with 86% of all amino acids covered at least once.From this set of clones, we were able to select clonesspanning regions of interest for screening against eitherthe sheared genomic Prey library or the shuttled ORFfragment Prey library.SCREENING OF TYPE IV SECRETIONSYSTEM PROTEINSThe Type IV secretion system (T4SS) is a multi-subunitcomplex frequently involved in virulence effectortransport in various gram-negative bacteria. Inter-subunitinteractions have been studied using the yeast two-hybridsystem for the Agrobacterium tumefaciens T4SS (Wardet al. 2002). Prior to screening the entire proteome, we selectedproteins from the R. sibirica T4SS for screeningagainst the prey random genomic library and prey fragmentORF library. The goal was to observe interactionspreviously reported among subunits while obtainingnovel interactions by screening against the entire proteome.Selected fragments used in the screening covered6 known T4SS proteins (Fig. 3).SCREENING RESULTSScreening of multiple fragments from the 6 T4SS proteinsyielded 285 interactions occurring between the 6subunits and 155 distinct proteins (Fig. 4). Of the interactions,162 were observed once, 48 were category-observedmore than once, and 74 were observed more thanonce by different fragments. Of the 162 interactions observedonce, 136 (84%) were observed with more than onesubunit, adding a level of contextual validation to the in-Figure 2. Functional shotgun sequencing. (i) Genomic DNA israndomly sheared and cloned in the pBAIT vector. (ii) Clonesare sequenced, the genome assembled and subsequently annotated.(iii) Clones containing fragments, translated correctly,from open reading frames, are arrayed and the inserts transferredto the pPREY vector to create a library against which to screen.(iv) Clones spanning proteins of interest can be screened againsteither a random library or the cloned, in-frame ORF library.Figure 3. Regions of the type 4 secretion system screened. Proteinsfrom the type 4 secretion system in Rickettsia sibirica areidentified as red arrows. The underlying black lines represent regionsfor which a clone with a properly cloned protein fragmentwas available for screening.