PMID- 18445284
OWN - NLM
STAT- MEDLINE
DCOM- 20080715
LR  - 20211020
IS  - 1471-2180 (Electronic)
IS  - 1471-2180 (Linking)
VI  - 8
DP  - 2008 Apr 29
TI  - Stable transformation of an episomal protein-tagging shuttle vector in the 
      piscine diplomonad Spironucleus vortens.
PG  - 71
LID - 10.1186/1471-2180-8-71 [doi]
AB  - BACKGROUND: Diplomonads are common free-living inhabitants of anoxic aquatic 
      environments and are also found as intestinal commensals or parasites of a wide 
      variety of animals. Spironucleus vortens is a putatively commensal diplomonad of 
      angelfish that grows to high cell densities in axenic culture. Genomic sequencing 
      of S. vortens is in progress, yet little information is available regarding 
      molecular and cellular aspects of S. vortens biology beyond descriptive 
      ultrastructural studies. To facilitate the development of S. vortens as an 
      additional diplomonad experimental model, we have constructed and stably 
      transformed an episomal plasmid containing an enhanced green fluorescent protein 
      (GFP) tag, an AU1 epitope tag, and a tandem affinity purification (TAP) tag. This 
      construct also contains selectable antibiotic resistance markers for both S. 
      vortens and E. coli. RESULTS: Stable transformants of S. vortens grew relatively 
      rapidly (within 7 days) after electroporation and were maintained under puromycin 
      selection for over 6 months. We expressed the enhanced GFP variant, eGFP, under 
      transcriptional control of the S. vortens histone H3 promoter, and visually 
      confirmed diffuse GFP expression in over 50% of transformants. Next, we generated 
      a histone H3::GFP fusion using the S. vortens conventional histone H3 gene and 
      its native promoter. This construct was also highly expressed in the majority of 
      S. vortens transformants, in which the H3::GFP fusion localized to the chromatin 
      in both nuclei. Finally, we used fluorescence in situ hybridization (FISH) of the 
      episomal plasmid to show that the transformed plasmid localized to only one 
      nucleus/cell and was present at roughly 10-20 copies per nucleus. Because S. 
      vortens grows to high densities in laboratory culture, it is a feasible 
      diplomonad from which to purify native protein complexes. Thus, we also included 
      a TAP tag in the plasmid constructs to permit future tagging and subsequent 
      purification of protein complexes by affinity chromatography via a two-step 
      purification procedure. CONCLUSION: Currently, progress in protistan functional 
      and comparative genomics is hampered by the lack of free-living or commensal 
      protists in axenic culture, as well as a lack of molecular genetic tools with 
      which to study protein function in these organisms. This stable transformation 
      protocol combined with the forthcoming genome sequence allows Spironucleus 
      vortens to serve as a new experimental model for cell biological studies and for 
      comparatively assessing protein functions in related diplomonads such as the 
      human intestinal parasite, Giardia intestinalis.
FAU - Dawson, Scott C
AU  - Dawson SC
AD  - Department of Microbiology, 255 Briggs Hall, One Shields Ave,, UC-Davis Davis, CA 
      95616, USA. scdawson@ucdavis.edu
FAU - Pham, Jonathan K
AU  - Pham JK
FAU - House, Susan A
AU  - House SA
FAU - Slawson, Elizabeth E
AU  - Slawson EE
FAU - Cronembold, Daniela
AU  - Cronembold D
FAU - Cande, W Zacheus
AU  - Cande WZ
LA  - eng
GR  - R01 AI054693/AI/NIAID NIH HHS/United States
GR  - A1054693/PHS HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20080429
PL  - England
TA  - BMC Microbiol
JT  - BMC microbiology
JID - 100966981
RN  - 0 (Histones)
RN  - 0 (Protozoan Proteins)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (enhanced green fluorescent protein)
RN  - 147336-22-9 (Green Fluorescent Proteins)
SB  - IM
MH  - Animals
MH  - Cell Nucleus/metabolism
MH  - Diplomonadida/*genetics/metabolism
MH  - Electroporation
MH  - Genetic Vectors/analysis/*genetics
MH  - Green Fluorescent Proteins/*genetics/metabolism
MH  - Histones/genetics/metabolism
MH  - In Situ Hybridization, Fluorescence
MH  - Microscopy, Fluorescence
MH  - Microtubules/metabolism
MH  - Plasmids/analysis/genetics
MH  - Promoter Regions, Genetic
MH  - Protozoan Proteins/genetics/metabolism
MH  - Recombinant Fusion Proteins/genetics/metabolism
MH  - Reverse Transcriptase Polymerase Chain Reaction
MH  - *Transformation, Genetic
PMC - PMC2386477
EDAT- 2008/05/01 09:00
MHDA- 2008/07/17 09:00
PMCR- 2008/04/29
CRDT- 2008/05/01 09:00
PHST- 2007/08/15 00:00 [received]
PHST- 2008/04/29 00:00 [accepted]
PHST- 2008/05/01 09:00 [pubmed]
PHST- 2008/07/17 09:00 [medline]
PHST- 2008/05/01 09:00 [entrez]
PHST- 2008/04/29 00:00 [pmc-release]
AID - 1471-2180-8-71 [pii]
AID - 10.1186/1471-2180-8-71 [doi]
PST - epublish
SO  - BMC Microbiol. 2008 Apr 29;8:71. doi: 10.1186/1471-2180-8-71.