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The NOS problem
In many situations involving screening for lethal mutations, a phenotype is followed and then a
sequence change is identified that is proposed to be responsible for the phenotype. Pat presented a
real-life scenario in class in which Regulski et al. generated a homozygous lethal line (NOSC) that
contained a lesion in the NOS (Nitric oxide synthase) locus that was shown to weaken NOS activity,
which led them to conclude that NOS function is essential. Yakubovich et al., however, came up with
evidence supporting that the lethality in NOSC was due not to the lesion within the NOS locus, but was in
fact due to a closely associated mutation in another gene, supporting that NOS function is not essential.
In this review I will guide you through the findings of both labs.
Regulski et al, 2004
How did this lab come up with mutant NOSC, and why did they decide that it was a homozygous lethal
allele of NOS?
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First they generated a 20kb deletion of the NOS locus, which also included 5 flanking genes. This
was called Df(2L)69F. In this review I’ll write it as Df(69F) as Pat did in lecture.
Second they went through a screen for NOS point mutants, and they utilized Df(69F) to screen
for lethality. FIGURE 1
o They isolated 32 lethal mutations falling into 5 complementation groups.
They further supported that one of these mutants, which they called NOSC (actually dNOSC), had
a homozygous lethal allele of NOS (and thus that NOS is essential) by doing the following:
o They backcrossed 5 times and found that NOSC still had lethality, indicating that the
lethality is not due to a “secondary mutation”outside of the NOS locus.
o They sequenced the NOSC genomic DNA and found a single point mutation within the
NOS locus that caused a structurally important amino acid substitution.
 They saw that NOSC flies homozygous for the point mutation died during late
embryonic and early larval stages, which suggested that this point mutation
significantly reduces the enzymatic function of NOS to non-viable levels (again,
suggesting that NOS is essential)
They showed that NOSC indeed had a null mutation of NOS by showing that NOSC had reduced
levels of NOS enzymatic activity in several in vitro assays:
o They assayed NOS activity in protein extracts from NOSC heads
o They expressed the mutant NOS protein in bacteria and assayed NOS activity
Yakubovich et al, 2010
Through more critically testing the lethality of NOSC, this lab showed that the lethality of the NOSC line is
not due to the mutation in the NOS locus, and indeed that NOS is not an essential gene.
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They reintroduced wild type NOS (by expressing NOS cDNA via a ubiquitous promoter) to NOSC
but this did not rescue the lethality associated with the NOSC chromosome
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Caveats: constitutive expression of NOS cDNA might not mimic the spatially and
temporally controlled endogenous expression of NOS; this might be a reason it doesn’t
rescue. Pat says that since the expression of NOS cDNA doesn’t even modify the stage
of lethality, this shows that the lethality must be due to another gene.
Since Regulski, et al. found their NOS lethal by crossing to a deficiency that also deleted 5
flanking ORFs, it was possible that they had a lethal mutation in another gene within that
deficiency region, linked such that backcrossing didn’t get rid of it. They therefore tried to see if
they could isolate viable recombinant chromosomes that retained the NOSC mutation (that is,
they wanted to see if the lethality of NOSC was really due to the point mutation within NOS or to
a closely associated mutant). Figure 2
o They did indeed isolate viable recombinant chromosomes that retained the NOSC
mutation.
o Also, they calculated the Map Units from the # of recombinant chromosomes and found
that the NOS point mutation was roughly .08 Map Units away from the lethal allele.
 recombinant chromosomes/total chromosomes = (3*2)/7500 = .08
o They sequenced these viable NOSC mutants and found that they still contained the NOS
locus point mutation of the original NOSC mutants.
They also generated a new deficiency line, NOS15, containing another NOS deletion and found
that, while it had lowered NOS activity in in vitro assays, it was homozygous viable
Lastly, they found that when they stained tissues of NOS15 for by-products of NOS activity, they
still got wild-type levels, suggesting the possibility of a redundancy in NO production not
attributed to NOS itself (which might explain why NOS is not essential)
What can we learn from all this? While Reguslki, et al. showed that the sequence change in the NOS
locus caused weakened NOS activity, they failed to show that it was responsible for the homozygous
lethality of the NOSC line. Lesson: they should have tested the lethality, as Yakubovich et al. did, in
addition to determining whether NOS activity was perturbed, before they could determine whether the
lesion within NOS was responsible for the lethality of NOSC!