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Chapter 19 : The Sequencing Project

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Abstract:

This chapter aims at obtaining the complete sequence of the Philadelphia 1 strain of , derived from the original 1976 outbreak that established the clinical entity known as Legionnaires' disease. The GC content of is ~38% over most of its length. From the genomic sequence so far (more than 85% of the sequence is currently contained in contigs greater than 1 kb), 3,000 open reading frames (ORF) were covered, about 2,000 of which are complete, and on the basis of homology to genes from other prokaryotes, more than 1,100 putative genes in were identified. Genes in Philadelphia 1 can be compared with genes in other pathogenic organisms. The amino acid sequence can guide structural and functional analyses of enzymes and other proteins of the organism. The authors used knockout strategies in the past to assess the function of genes discovered in the course of the sequencing project; and this continues to be their preferred method for functional analysis.

Citation: Qu X, Morozova I, Chen M, Kalachikov S, Chen J, Park H, Georghiou A, Asamani G, Feder M, Rineer J, Greenberg J, Goldsberry C, Rzhetsky A, Fischer S, Zhang P, Cayanis E, Russo J, Segal G, Shuman H, DeJong P. 2002. The Sequencing Project, p 97-104. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch19

Key Concept Ranking

Legionella pneumophila
0.5691044
Legionella rubrilucens
0.5408843
0.5691044
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Figures

Image of FIGURE 1
FIGURE 1

Two-pronged sequencing approach. The method of sequencing is diagrammed in this cartoon. DNA isolated from grown to confluence on charcoal-impregnated agar plates were either partially digested with RI and cloned into the pBACe3.6 vector (left side of figure) or sheared by nebulization and directly cloned into the pUC19 sequencing vector (right side). In the former case, BACs were assembled by the STS mapping approach (see text for details), and BAC pairs from across the genome were individually sheared and subcloned into pUC19. Sequencing was primed from the M13–21 and reverse promoters in the pUC19 vector. BAC ends were also sequenced using T7 and SP6 promoters as extension primers. All sequences were pooled and assembled together.

Citation: Qu X, Morozova I, Chen M, Kalachikov S, Chen J, Park H, Georghiou A, Asamani G, Feder M, Rineer J, Greenberg J, Goldsberry C, Rzhetsky A, Fischer S, Zhang P, Cayanis E, Russo J, Segal G, Shuman H, DeJong P. 2002. The Sequencing Project, p 97-104. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch19
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Image of FIGURE 2
FIGURE 2

Website contig display. Graphical representation of one of the larger contigs in the genome project at this time (66,060 base pairs), it contains the major region of genes (grouped below approximately the third quartile and the beginning of the fourth quartile of the upper bar). Other genes within this contig include homologs to enzymes of the pentose phosphate pathway and sugar catabolism on the left side of the map, and purine ribonucleotide biosynthetic enzymes on the far right. Details on this type of image are provided in the text, and the reader is urged to explore the actual web page to better appreciate its utility and functionality.

Citation: Qu X, Morozova I, Chen M, Kalachikov S, Chen J, Park H, Georghiou A, Asamani G, Feder M, Rineer J, Greenberg J, Goldsberry C, Rzhetsky A, Fischer S, Zhang P, Cayanis E, Russo J, Segal G, Shuman H, DeJong P. 2002. The Sequencing Project, p 97-104. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch19
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Image of FIGURE 3
FIGURE 3

gene comparisons among legionellaceae. (A) Hybridization-based comparison of the gene. DNA from various isolates of (shown with bolder bars and representing 13 different serogroups) and several other species of (lighter bars) were digested with RI, transferred to nylon membranes, hybridized with a probe for the gene from the Philadelphia 1 isolate of and washed at reduced stringency (0.5× SSC/0.1% SDS). Strong hybridization signals were elicited by all the isolates and by (double black bar). (B) Sequence comparison of the gene. A small portion of the sequence of the homolog (near the Ν terminus) in different isolates and species. The top sequence occurs in the Bellingham 1, Bloomington Chicago 8, and Concord 3 strains of the second pattern occurs in the 797-PA-H and another isolate in serogroup 11 of the middle pattern is found in Philadelphia 1, Togus 1, and Chicago 2 isolates of the fourth sequence is seen in the Los Angeles 1, IN-23-G1-C2 and Leiden 1 strains of and interestingly, the last pattern is present in L. Dallas 1, in Lansing and in WA-270A-C2. The less common forms of the bases are shown in small bold letters. The codons that exist in different forms and give rise to altered amino acids are also shown in bold, as are the variant amino acids themselves. Many additional base and amino acid differences that distinguish these five families exist elsewhere within the gene coding sequence.

Citation: Qu X, Morozova I, Chen M, Kalachikov S, Chen J, Park H, Georghiou A, Asamani G, Feder M, Rineer J, Greenberg J, Goldsberry C, Rzhetsky A, Fischer S, Zhang P, Cayanis E, Russo J, Segal G, Shuman H, DeJong P. 2002. The Sequencing Project, p 97-104. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch19
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References

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