When expressed under control of the Punc - 7S promoter, UNC - 7L (Punc - 7S:: unc - 7L [M121L]-RRB-
rescued unc - 7 (e5) forward locomotion; though much of the signal remained associated with cell bodies, some puncta were seen localized to AVB: B motor neuron gap junctions (Figure 10D).
A fully -
rescuing unc - 7 construct (Figure 3B) extended to the Xho I site (nucleotide 15,574) and included a frameshift generated by filling in the Sal I site (nucleotide 14,412).
Our genomic unc - 9:: gfp construct was found to only partially
rescue unc - 9 mutants.
Together these studies suggest that Punc - 7S drives expression of UNC - 7S in a subset of neurons (including AVA and AVB, but not motor neurons) that can effect rescue of forward locomotion, and promoter sequences upstream of exon 2 drive additional expression of UNC - 7S in motor neurons or possibly other neurons required to fully
rescue all unc - 7 locomotory defects.
(B) Fully
rescuing unc - 7 min plasmid expressing UNC - 7S and UNC - 7SR.
An unc - 9:: gfp expression cassette was made by introduction of GFP (afmx) from the partially
rescuing unc - 9:: gfp genomic construct (above).
First, the genomic region represented by the unc - 9:: gfp construct was shown to fully
rescue unc - 9 (fc16) animals, indicating that this region is sufficient to encode a functional unc - 9 product.
M121 was mutated to L in unc - 7SΔ1S; this construct (unc - 7SΔ1S - M121L; Figure 3F) plus F56B12 failed to
rescue unc - 7, suggesting that UNC - 7L does not have rescuing activity.
Not exact matches
(B) Pcex - 1::
unc - 7S:: gfp (AVA, AVD interneurons)
rescues forward locomotion but does not concentrate in puncta near B cell bodies.
Lane 1, wild type (N2); lanes 2 and 3,
unc - 7 (e5)
rescued with
unc - 7S:: gfp + cosmid F56B12, expected to express all isoforms; lane 4,
unc - 7 (e5)
rescued with
unc - 7SΔ1S + F56B12, predicted to express
UNC - 7L:: and SR:: GFP.
One
rescued line, EH536, was maintained and used for genetic crosses involving
unc - 9 (fc16).
Phenotypic
rescue of
unc - 7 by various constructs was assessed by tapping transformed animals on the nose or tail and observing whether or not smooth sinusoidal waves were propagated.
A shorter version of
unc - 7 min lacked promoter sequences upstream of exon 2 (
unc - 7S:: gfp; Figure 3C) and
rescued forward but not backward locomotion, suggesting that promoter sequences in intron 3 (Punc - 7S; Figure 3C) drive a subset of
UNC - 7S expression sufficient to
rescue only forward movement.
We will refer to the predicted products of the
unc - 7
rescuing transcript as
UNC - 7S with the understanding that
UNC - 7SR is also translated.
When co-injected with F56B12, the
unc - 7S:: gfp construct
rescued both forward and reverse locomotion.
Like
unc - 7S:: gfp, this construct
rescued forward locomotion in the absence of cosmid F56B12, and we postulated that M121 in exon IV (the canonical innexin start site) might be used to generate a shorter but functional
UNC - 7S - like product (
UNC - 7SR).
(D)
UNC - 7L expressed in interneurons (Punc - 7S::
unc - 7L [M121L]-RRB-
rescues forward locomotion in
unc - 7 (e5) and localizes to puncta near B motor neuron cell bodies.
To demonstrate that mutation of M121 did not lead to loss of an essential protein function, exon 1S was added back to allow for expression of
UNC - 7S (M121L), and this construct (
unc - 7S - M121L; Figure 3G) fully
rescued.
The
unc - 9:: gfp construct partially
rescued uncoordinated locomotion in
unc - 9 (fc16)-- hermaphrodites exhibit wild - type forward movement interrupted with bouts of spastic kinking.
Using
unc - 9 mutants and heterologous promoters, we showed that expression of
UNC - 9 in B class motor neurons is necessary to
rescue the localization of
UNC - 7S expressed in AVB.
To address this, genetic mosaic analysis of
rescue of forward locomotion by the
unc - 7S construct was carried out.
Using anti-GFP antibodies, two major protein bands, not present in wild - type, were detected in
unc - 7 (e5) animals
rescued with
unc - 7S:: GFP + F56B12 (Figure 3H).
The shortest genomic region capable of
rescuing forward and backward locomotion defects in
unc - 7 mutants encompassed the coding region of
unc - 7 and 1 kb of promoter sequence upstream of exon 2 (
unc - 7 min; Figure 3B).
Psra - 11::
unc - 7S:: gfp expressed in AVB did not
rescue forward locomotion in e5 animals; the Pcex - 1::
unc - 7s: gfp construct, expressed in AVA and AVD but not AVB, did, however,
rescue e5 forward locomotion.
Other strains used included CB1377 daf - 6 (e1377) X and CW129
unc - 9 (fc16) X. For transformation
rescue, SP1531 ncl - 1 (e1865)
unc - 36 (e251) III was sometimes used; the ncl - 1 gene was incidental in these cases.
In animals
rescued with
unc - 7SΔ1S + F56B12, predicted to express
UNC - 7SR:: GFP but not
UNC - 7S:: GFP, only the smaller band was detected.
Therefore,
unc - 7 locomotion defects can be
rescued by either
UNC - 7S or
UNC - 7SR, but
UNC - 7L can not substitute for this function (although it may have a subtle role or function in another process).
It was surprising that re-establishment of AVB: B motor neuron gap junctions was not implicated in the
rescue of
unc - 7 (e5) forward locomotion by
unc - 7S.
unc - 7 transformation
rescue.
(C) Co-localization (arrow) in an
unc - 7 (e5) animal
rescued for forward locomotion with
unc - 7S construct (Figure 3C, minus GFP).