JCS044651 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Confirmation of copy number of CRN12 gene in Leishmania donovani genome. Genomic DNA isolated from L. donovani is digested with PstI (lane 1) and SacI (lane 2), and subjected to southern blotting. (A) Ethidium bromide stained gel; (B) Southern blot of ‘A’ probed for CRN12. Unlike SacI, PstI restriction site is internal to the CRN12 gene. Accordingly, two bands in PstI and single band in SacI-digested samples, confirm single copy of CRN12 gene in L. donovani genome. M_r, DNA ladder.

• Supplemental Figure S2 -

  Fig. S2. Analyses of CRN12 gene deletion and protein expression. (A) Southern blot analysis of genomic DNA of Coro^+/+ (wild type) and Coro^+/− (single allele deletion mutant) cells. (a) Ethidium bromide-stained gel of genomic DNA digested with SacI. (b,c) Southern blots probed with digoxin-labeled CRN12 and Neo gene probes, respectively, showing replacement of one of the CRN12 alleles with Neo gene cassette. Lane 1, Coro^+/+ cells; lane 2, Coro^+/− cells. (B) Confirmation of Neo cassette integration at the CRN12 locus by PCR using primers P1 and P2 showing amplification of 2.1 kb DNA band. (C) Western blot analysis of Coro^+/+ and Coro^+/− cells using anti- CRN12 and anti Leishmania actin antibodies. Lane 1, Coro^+/+ cells; lane 2, Coro^+/− cells. (D) Densitometric analysis of CRN12 bands normalized with the respective actin bands showing ∼50% decrease in CRN12 band intensity in Coro^+/− cells (2) when compared with Coro^+/+ cells (1). Values shown are means of three independent determinations ±s.d. (E) Southern blot analysis of SacI-digested genomic DNA of Coro^+/+ cells (lane 1), Coro^+/− cells (lane 2) and 10 doubly selected Coro^−/− clones (lanes 3-12). EtBr, DNA loading control; Coro, probed for CRN12; Neo, probed for neomycin; Hyg, probed for hygromycin. (F), (G) and (H) PCR analyses for the presence of CRN12 gene (1.5 kb), integration of Neo cassette at the CRN12 locus (2.1 kb) and integration of Hyg cassette at the CRN12 locus (2.2 kb) in 10 Coro^−/− clones (lanes 1-10) with a Coro^+/+ control (lane 11). (I) Western blot analysis of the Coro^−/− mutants recovered from the liquid cultures at different time periods after transfection showing undetectable amounts of CRN12 during day 15 to day 21, and reappearance of CRN12 expression on day 24 to day 36. (J) PCR analysis (23 thermal cycles) for the presence of CRN12 gene in the transfected cell populations recovered from the liquid cultures at different time periods. Lanes 1-4, amplification of CRN12 gene, actin gene, neomycin resistance gene and hygromycin resistance gene, respectively, in the genomic DNA isolated from the cell population collected on the day 18; lanes 5 and 6, amplification of CRN12 and actin, respectively, in the genomic DNA isolated on day 24 cells. (K) PCR analysis of the cell population collected from the liquid culture on the day 36 after transfection. Lane 1, amplification of CRN12 gene; lane 2, amplification of 2.1 kb DNA confirming integration of Neo cassette at the CRN12 locus; lane 3, amplification of 2.2 kb DNA, confirming integration of Hyg cassette at the CRN12 locus. M_r, DNA ladder.

• Supplemental Figure S3 -

  Fig. S3. Analyses of growth and bipolar cell formation in Coro^−/− cell population recovered from liquid cultures after 36 days. White triangles, growth curve; black circles, appearance of bipolar cells. Western blot using anti-CRN12 and anti-actin antibodies, and densitometic quantitation of CRN12 (normalized with actin) in Coro^+/+ and Coro^−/− cells are given in the inset. Values shown are means of three independent determinations ±s.d.

• Supplemental Figure S4 -

  Fig. S4. Confocal immunofluorescence microscopy image of a tetra-polar Coro^+/− cell labeled with anti-α,β-tubulin antibodies and DAPI to mark microtubules, and nuclei and kinetoplasts, respectively. This image clearly shows four flagella, four nuclei and four kinetoplasts corresponding to four cells that have still not separated. Such cells corresponded to ∼4% in the Coro^+/− log phase cultures.

• Supplemental Figure S5 -

  Fig. S5. (A) Effect of ‘rhizoxin’ on the emergence of bipolar morphology in Coro^+/− cells. White diamonds, without treatment; black triangles, rhizoxin treatment after emergence of the bipolar cells on day 6 (marked by arrow); black circles, rhizoxin treatment on day 0. (B) Growth curves of the cells in (A). Values shown are means of three independent determinations±s.d.

• Supplemental Figure S6 -

  Fig. S6. Unusual mode of the daughter cell separation in the bipolar cells. Positive ends labeled with YL1/2 antibodies clearly demarcate the zones of daughter cell corsets. Several bipolar cells showing such patterns are presented in a series of possible course of daughter cell separation. Bar, 5 µm.

• Supplemental Table S1 - Adobe PDF