Characterisation of a cluster of genes encoding Theileria annulata AT hook DNA-binding proteins and evidence for localisation to the host cell nucleus
David G. Swan1,*,
Rowena Stern1,
Sue McKellar1,
Kirsten Phillips2,
Chris A. L. Oura1,
Tülin Ilhan Karagenc3,
Laura Stadler1 and
Brian R. Shiels1
1
Department of Veterinary Parasitology, University of Glasgow, Bearsden Road,
Glasgow, G61 1QH, UK
2
Department of Molecular Recognition, The Hannah Institute, Mauchline Road, Ayr
KA6, Scotland, UK
3
Adnan Menderes University, Faculty of Veterinary Medicine, Department of
Parasitology, Isikli, Aydin, Turkey
*
Author for correspondence (e-mail:
d.swan{at}vet.gla.ac.uk
)
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Fig. 1. Restriction map and schematic of the TashAT cluster.
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Fig. 2. Sequence analysis of TashAT1 and TashAT3 and their comparison with TashAT2.
(A) Sequence of the predicted translation products of TashAT1 and
TashAT3. The potential signal sequence is in italics. The AT hook
DNA-binding domain is in bold and the putative transcriptional transactivation
domain is underlined. Where the sequence of TashAT1 differs from that of
TashAT3 is shown above the relevant sequence of TashAT3. The asterisks mark
the end of each sequence. (B) A comparison of TashAT2 with TashAT3. Identical
residues are shown in bold. The numbers at the end of the comparison denote
the region which is 100% identical between the two sequences. A gap generated
by the analysis in the sequence of TashAT2 is shown by a series of dashes. (C)
A comparison of the AT hook motifs of TashAT1 and TashAT3 with those of the
HMGI(Y) protein. Rows 1-4 are the AT hook motifs of TashAT1/3. Rows 5-7 are
those of HMGI(Y). Accession Numbers for the TashAT cluster:
TashAT1, AJ291829; TashAT2, AJ132045; TashAT3,
AJ291830.
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Fig. 3. Indirect Immunofluorescence assays using antisera to TashAT1/3. (A-E)
Analysed using anti-TashAT1/3 antiserum; (F) Analysed using pre-immune serum.
The cells used were BL20 (A), TBL20 (B), D7B12 (C); D7 at 37°C (D), D7 at
41°C for 7 days (E), D7B12 (F). Scale bar: 10 µm.
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Fig. 4. (A) Northern analysis of T. annulata-infected and uninfected cell
lines probed with the entire TashAT3 gene. (B) The same northern
blots probed with 2P3 (large subunit rRNA of T. annulata). DIY,
Diyarbakir; Hid, Hiderseyh; P, passage number; Pi, piroplasms; Sp,
sporozoite.
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Fig. 5. Western analysis of cell and nuclear extracts using anti-TashAT1/3
antiserum. (1) D7B12 total cell extract. (2) D7B12 host nuclear fraction. (3)
D7B12 parasite nuclear fraction. (4) BL20 nuclear fraction.
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Fig. 6. DNA-binding analysis of TashAT2. (A) Electrophoretic mobility shift assays
carried out with the radiolabelled PCR product obtained after each round of
binding to the GST-TashAT fusion protein. Lanes 1-4 represent cycles 1-4. Lane
5 represents cycle 4 and lane 6 represents the major band from cycle 4 excised
and PCR amplified. (B) DNA sequence obtained after excising and PCR amplifying
the band from lane 6 and sub cloning into pGEM7ZF. The TAAAT/ATTTA motif is
shown in bold.
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© The Company of Biologists Ltd 2001
gt11 clone expressing a fragment of
TashAT1 bearing the AT hook motif. Panels C and D show binding of
CAT1M2 and CAT3, respectively, to a