Biology Reference
In-Depth Information
mosome 17p11.2 (Fig. 6.1) (Liang et al. 1998). Two consanguineous fami-
lies from India with profound congenital deafness were also found to be
linked to genetic markers in the 17p11.2 region, suggesting that there are
multiple alleles of
DFNB3
(Liang et al. 1998).
3.11.1 Identifying
DFNB3
by Functional Cloning of Mouse
Shaker 2
The map position of
DFNB3
on human 17p11.2 predicted a homologue,
shaker 2
(
sh-2
), in the syntenic region of mouse chromosome 11 (Friedman
et al. 1995). Homozygous
sh-2
mice are deaf and exhibit circling behavior.
shaker 2
was mapped in a 500-meiosis cross to a critical region of 0.2 cM
(~400 to 800 kb of DNA) (Liang et al. 1998). A physical map of this region
was constructed using Bacterial Artificial Chromosomes (BACs). In Sally
Camper's laboratory, each BAC was individually injected into the pronu-
cleus of homozygous
sh-2
fertilized eggs, and the eggs were then transferred
to a foster mother. One transgenic offspring did not circle, and responded
to sound with a normal Preyer's reflex. This mouse had been injected with
BAC425p24. The mouse was mated and correction of the
shaker 2
pheno-
type was correlated with transmission of BAC425p24 in the germline of his
offspring. The location of
sh-2
was thus refined from ~400-800 kb to just
140 kb, the insert size of BAC425p24 (Probst et al. 1998).
The DNA sequence of BAC425p24 was analyzed using two com-
puter programs that detect probable protein-coding regions of genes
(exons) (GRAIL, http://compbio.ornl.gov/Grail-bin/EmptyGrailForm and
GENSCAN, http://CCR-081.mit.edu/GENSCAN.html). These analyses
predicted two genes encoded in BAC425p24, one of which is a novel uncon-
ventional myosin that was designated myosin XV (
Myo15
). It was already
known that mutations of other unconventional myosins could cause hearing
loss (Adato et al. 1997; Avraham et al. 1997; Weil et al. 1997) and, there-
fore,
Myo15
was a strong candidate for
sh-2
.
3.11.2 The
Shaker 2
Locus Encodes
Myo15
, an Unconventional Myosin
Myo15
has 66 exons with several splice isoforms (Liang et al. 1999) (Fig.
6.4). Each of the 66 exons was screened for mutations in the two known
alleles of
shaker 2
:
sh-2
and
sh-2
J
. The
sh-2
allele has a G-to-A transition in
the motor domain of myosin XV that substitutes tyrosine for a highly con-
served cysteine (Cope et al. 1996; Probst et al. 1998). The
sh-2
J
allele was
shown to have a deletion of the last 6 exons of
Myo15
(Anderson et al.
2000).
Some clues about the role of myosin XV in the inner ear can be obtained
from light- and electron-microscopic studies of tissues from
shaker 2
and
shaker 2
J
mutant mice. Hair cells was present, but the stereocilia on the
inner and outer hair cells were very short, approximately 1/10 of the normal
length (Probst et al. 1998). The stereocilia of the inner hair cells are essen-
