Table II. hZW10 and N1 insertion mutants... | Rockefeller University Press

Table II.

hZW10 and N1 insertion mutants assayed for kinetochore localization and hZwint-1 interaction

hZW10 mutant	Insertion site (amino acid)	Insertion sequence (amino acid)^{^a}	Kinetochore localization	hZwint-1 yeast two-hybrid interaction	β-Galactosidase assay (± one SD)^{^b}
A	43	CGRIS	Positive	Positive	341 ± 173
B	61	VRPHQ	Positive	Negative	1.3 ± 0.3
C	90	CGRTG	Positive	Positive	333 ± 114
D	106	CGRIL	Positive	Positive	ND
E	131	CGRKY	Positive	Positive	ND
F	191	LRPQP	Positive	Positive	ND
G	208	CGRNL	Positive	Positive	ND
H	210	VRPHT	Positive	Positive	ND
I	226	VRPHS	Positive	Positive	ND
J	248	LRPQL	Negative	Negative	ND
K	335	VRPHW	Positive	Positive	ND
L	375	NAAAL	Positive	Positive	ND
M	421	CGRNN	Positive	Positive	ND
N	467	DAAAL	Positive	Positive	ND
O	475	TAAAP	Positive	Positive	ND
P	536	LRPHQ	Negative	Negative	9.9 ± 9.6
Q	563	CGRSL	Positive	Positive	ND
R	583	CGRRR	Negative	Negative	2.4 ± 0.7
S	607	SAAAA	Positive	Positive	ND
T	608	MRPHR	Positive	Positive	ND
U	654	CGRMG	Negative	Negative	3.7 ± 1.6
V	663	CGRTE	Negative	Negative	2.2 ± 0.9
W	666	CGRIG	Negative	Negative	1.6 ± 0.7
X	671	CGRTL	Negative	Negative	2.1 ± 1.7
Y	682	LRPQL	Positive	Positive	ND
Z	686	CGRIC	Negative	Negative	1.4 ± 0.8
Aa	712	CGRKE	Positive	Positive	ND
a	149	SMRPQ	Positive	N/A	N/A
b	210	TECGR	Positive	N/A	N/A
c	235	CGRIG	Positive	N/A	N/A
d	283	LVFKH	Positive	N/A	N/A
e	289	PCLNT	Positive	N/A	N/A
f	303	QMRPH	Positive	N/A	N/A
g	331	AAALG	Positive	N/A	N/A
h	334	CLNTN	Positive	N/A	N/A
i	390	NAAAL	Positive	N/A	N/A
j	454	LRPHQ	Positive	N/A	N/A
k	520	HVFKH	Positive	N/A	N/A
l	541	CFGIH	Positive	N/A	N/A
m	567	LFKHL	Positive	N/A	N/A
n	640	CLMNW	Negative	N/A	N/A
o	658	AAALN	Negative	N/A	N/A
p	675	TVFKH	Negative	N/A	N/A
q	681	LLRPQ	Positive	N/A	N/A

hZW10 mutant	Insertion site (amino acid)	Insertion sequence (amino acid)^{^a}	Kinetochore localization	hZwint-1 yeast two-hybrid interaction	β-Galactosidase assay (± one SD)^{^b}
A	43	CGRIS	Positive	Positive	341 ± 173
B	61	VRPHQ	Positive	Negative	1.3 ± 0.3
C	90	CGRTG	Positive	Positive	333 ± 114
D	106	CGRIL	Positive	Positive	ND
E	131	CGRKY	Positive	Positive	ND
F	191	LRPQP	Positive	Positive	ND
G	208	CGRNL	Positive	Positive	ND
H	210	VRPHT	Positive	Positive	ND
I	226	VRPHS	Positive	Positive	ND
J	248	LRPQL	Negative	Negative	ND
K	335	VRPHW	Positive	Positive	ND
L	375	NAAAL	Positive	Positive	ND
M	421	CGRNN	Positive	Positive	ND
N	467	DAAAL	Positive	Positive	ND
O	475	TAAAP	Positive	Positive	ND
P	536	LRPHQ	Negative	Negative	9.9 ± 9.6
Q	563	CGRSL	Positive	Positive	ND
R	583	CGRRR	Negative	Negative	2.4 ± 0.7
S	607	SAAAA	Positive	Positive	ND
T	608	MRPHR	Positive	Positive	ND
U	654	CGRMG	Negative	Negative	3.7 ± 1.6
V	663	CGRTE	Negative	Negative	2.2 ± 0.9
W	666	CGRIG	Negative	Negative	1.6 ± 0.7
X	671	CGRTL	Negative	Negative	2.1 ± 1.7
Y	682	LRPQL	Positive	Positive	ND
Z	686	CGRIC	Negative	Negative	1.4 ± 0.8
Aa	712	CGRKE	Positive	Positive	ND
a	149	SMRPQ	Positive	N/A	N/A
b	210	TECGR	Positive	N/A	N/A
c	235	CGRIG	Positive	N/A	N/A
d	283	LVFKH	Positive	N/A	N/A
e	289	PCLNT	Positive	N/A	N/A
f	303	QMRPH	Positive	N/A	N/A
g	331	AAALG	Positive	N/A	N/A
h	334	CLNTN	Positive	N/A	N/A
i	390	NAAAL	Positive	N/A	N/A
j	454	LRPHQ	Positive	N/A	N/A
k	520	HVFKH	Positive	N/A	N/A
l	541	CFGIH	Positive	N/A	N/A
m	567	LFKHL	Positive	N/A	N/A
n	640	CLMNW	Negative	N/A	N/A
o	658	AAALN	Negative	N/A	N/A
p	675	TVFKH	Negative	N/A	N/A
q	681	LLRPQ	Positive	N/A	N/A

The assay of direct interactions between hZwint-1 and hZW10 insertion 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.

The exact amino acid insertion sites in hZW10 and hZW10^N1 5-aa insertion mutants were determined by sequencing.

The hZwint-1 interaction with hZw10 insertion mutants were assayed as outlined in Table I.

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