hZW10 N- and C-terminal truncation mutants assayed for kinetochore localization and hZwint-1 interaction
| hZW10 construct . | Encoding amino acids . | Kinetochore localization . | hZwint-1 yeast two-hybrid interaction . | β-Galactosidase assay (± one SD) . |
|---|---|---|---|---|
| Full length | 1–779 | Positive | Positive | 187 ± 20 |
| N1 | 52–779 | Positive | Negative | 2.14 ± 1.9 |
| N2 | 75–779 | Negative | Negative | ND |
| N3 | 85–779 | Negative | Negative | ND |
| N4 | 132–779 | Negative | Negative | ND |
| N5 | 215–779 | Negative | Negative | ND |
| N6 | 335–779 | Negative | Negative | ND |
| N7 | 462–779 | Negative | Negative | ND |
| N8 | 556–779 | Negative | Negative | ND |
| N9 | 621–779 | Negative | Negative | ND |
| C1 | 1–772 | Negative | Negative | 3.4 ± 0.9 |
| C2 | 1–698 | Negative | Negative | 2.6 ± 0.6 |
| C3 | 1–560 | Negative | Negative | 2.5 ± 0.8 |
| C4 | 1–496 | Negative | Negative | 1.0 ± 0.8 |
| C5 | 1–410 | Negative | Positive | 26.6 ± 3 |
| C6 | 1–356 | Negative | Positive | 275 ± 33 |
| C7 | 1–318 | Negative | Positive | 132 ± 62 |
| C8 | 1–224 | Negative | Positive | 233 ± 23 |
| C9 | 1–139 | Negative | Positive | 445 ± 128 |
| C10 | 1–82 | Negative | Positive | ND |
| hZW10 construct . | Encoding amino acids . | Kinetochore localization . | hZwint-1 yeast two-hybrid interaction . | β-Galactosidase assay (± one SD) . |
|---|---|---|---|---|
| Full length | 1–779 | Positive | Positive | 187 ± 20 |
| N1 | 52–779 | Positive | Negative | 2.14 ± 1.9 |
| N2 | 75–779 | Negative | Negative | ND |
| N3 | 85–779 | Negative | Negative | ND |
| N4 | 132–779 | Negative | Negative | ND |
| N5 | 215–779 | Negative | Negative | ND |
| N6 | 335–779 | Negative | Negative | ND |
| N7 | 462–779 | Negative | Negative | ND |
| N8 | 556–779 | Negative | Negative | ND |
| N9 | 621–779 | Negative | Negative | ND |
| C1 | 1–772 | Negative | Negative | 3.4 ± 0.9 |
| C2 | 1–698 | Negative | Negative | 2.6 ± 0.6 |
| C3 | 1–560 | Negative | Negative | 2.5 ± 0.8 |
| C4 | 1–496 | Negative | Negative | 1.0 ± 0.8 |
| C5 | 1–410 | Negative | Positive | 26.6 ± 3 |
| C6 | 1–356 | Negative | Positive | 275 ± 33 |
| C7 | 1–318 | Negative | Positive | 132 ± 62 |
| C8 | 1–224 | Negative | Positive | 233 ± 23 |
| C9 | 1–139 | Negative | Positive | 445 ± 128 |
| C10 | 1–82 | Negative | Positive | ND |
The assay of direct interaction between hZwint-1 and hZW10 deletion constructs analyzed with the LexA yeast two-hybrid system using Xgal and the β-galactosidase assay, as well as kinetochore localization, through fluorescence microscopy analysis of EGFP fusion constructs. Yeast colonies were scored for appearance of blue color 2 and 3 d after streaking out. Each combination of interactions was redone in three separate experiments and confirmed for expression of fusion constructs by Western blotting with anti-HA and anti-LexA antibodies (not depicted). The β-galactosidase assay was performed using the β-galactosidase assay kit according to the manufacturer's instructions.