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. 2003 Dec;185(24):7077-84.
doi: 10.1128/JB.185.24.7077-7084.2003.

Development of a physical and genetic map of the virulent Wolbachia strain wMelPop

Ling V Sun  1 , Markus Riegler, Scott L O'Neill
Affiliations

Development of a physical and genetic map of the virulent Wolbachia strain wMelPop

Ling V Sun et al. J Bacteriol. 2003 Dec.

Abstract

We report here the construction of a physical and genetic map of the virulent Wolbachia strain, wMelPop. This map was determined by ordering 28 chromosome fragments that resulted from digestion with the restriction endonucleases FseI, ApaI, SmaI, and AscI and were resolved by pulsed-field gel electrophoresis. Southern hybridization was done with 53 Wolbachia-specific genes as probes in order to determine the relative positions of these restriction fragments and use them to serve as markers. Comparison of the resulting map with the whole genome sequence of the closely related benign Wolbachia strain, wMel, shows that the two genomes are largely conserved in gene organization with the exception of a single inversion in the chromosome.

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Figures

FIG. 1.
FIG. 1.
PFGE of digested wMelPop genomic DNA. (A) Lane 1, 8- to 48-kb ladder; lane 2, 1-kb ladder; lanes 3 to 9, digested genome; lane 3, ApaI; lane 4, SmaI; lane 5, ApaI and SmaI; lane 6, ApaI and AscI; lane 7, SmaI and AscI; lane 8, ApaI, SmaI, and AscI; lane 9, ApaI, SmaI, and FseI. (B) Lane 1, low-range pulsed-field gel marker; lanes 2 to 9, digested genome; lane 2, ApaI; lane 3, SmaI; lane 4, ApaI and SmaI; lane 5, AscI; lane 6, ApaI and AscI; lane 7, SmaI and AscI; lane 8, FseI and ApaI; lane 9, FseI and SmaI. (C) Lane 1, λ ladder; lane 2, 8- to 48-kb ladder; lanes 3 to 9, digested genome; lane 3, ApaI; lane 4, SmaI; lane 5, ApaI and SmaI; lane 6, ApaI and AscI; lane 7, SmaI and AscI; lane 8, ApaI, SmaI, and AscI; lane 9, ApaI, SmaI, and FseI. (D) Lane 1, λ ladder; lanes 2 to 9, digested genome; lane 2, ApaI; lane 3, SmaI; lane 4, ApaI and SmaI; lane 5, ApaI, AscI, and SmaI; lane 6, ApaI and AscI; lane 7, SmaI and AscI; lane 8, ApaI, SmaI, and FseI; lane 9, ApaI, AscI, and SmaI and FseI; lane 10, yeast chromosomal marker. (E) Lane 1, yeast chromosomal marker; lanes 2 to 4, digested genome; lane 2, AscI; lane 3, FseI; lane 4, AFSF.
FIG. 2.
FIG. 2.
Autoradiographs of Southern blots of PFGE gels of digested wMelPop genome probed with the atpA gene fragment. (A) Lane 1, AscI; lane 2, FseI; lane 3, AFSF. (B) Lane 1, ApaI; lane 2, SmaI; lane 3, ApaI and SmaI; lane 4, SmaI and AscI; lane 5, ApaI and FseI; lane 6, SmaI and FseI. (C) Composite Southern blots. Lane 1, ApaI; lane 2, SmaI; lane 3, ApaI and SmaI; lane 4, ApaI and AscI; lane 5, SmaI and AscI; lane 6, ApaI, SmaI, and AscI; lane 7, ApaI, SmaI, and FseI; lane 8, AFSF. FseI usually does not digest completely, which is indicated by the presence of two labeled DNA fragments in lanes where FseI was used.
FIG.3.
FIG.3.
Autoradiograph of a Southern blot of Digested wMelPop genome probed with trmD gene fragment (harboring SmaI site). Lane 1, ApaI; lane 2, SmaI; lane 3, ApaI and SmaI; lane 4, ApaI and AscI; lane 5, SmaI and AscI; lane 6, ApaI, AscI, and SmaI.
FIG. 4.
FIG. 4.
A physical and genetic map of the wMelPop genome. Arrows point to the location of genes determined by Southern hybridization (see Tables 1 to 3). Short bars indicate probes that contain the indicated restriction site. Numbers indicate the sizes of restriction fragments.
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