Regulation of cpeCDESTR by Two Sensory Pathways Controlling Complementary Chromatic Adaptation in Fremyella diplosiphon

The development of novel morphologies and the circumstances under which they arise and diversify are major research foci in evolutionary developmental biology. Horned beetles in the genus Onthophagus provide excellent opportunities to address the development of novel morphologies because they exhibit a great diversity of horn structures that lack direct homology to any other insect appendages. Horns develop as epidermal outgrowths from the prothorax and/or head but lack joints, muscle, or nerve tissue. Furthermore, size and location vary dramatically across species and between sexes. My research explores developmental patterning mechanisms responsible for the remarkable diversity in horn expression. Specifically, I am examining how members of two developmental regulatory networks (A: the Hox complex and B: the Appendage Patterning network) regulate the development of beetle horns and horn diversity in two different body regions: the prothorax and the head. A: Within the Hox complex, I focus on a possible role of Sex Combs Reduced (Scr) in horn development. Scr regulates segment identity in posterior head and prothorax across insect orders. Thus far, comparative gene expression studies and larval RNA interference (RNAi) suggest that while Scr maintains a conserved function in traditional body regions, it also plays a significant role in specifying both pupal and adult pronotal horns in a sexand species-specific manner. B: Using similar approaches, I am also investigating the function of other candidate genes that play important roles in appendage development in other insects, including decapentaplegic (dpp) (principal member of the TGF-β pathway) and pangolin (pan) (a transcription factor in the wingless (wg) pathway). These pathways work cooperatively to not only determine where appendages will develop, but also initiate the patterning of the proximal-distal axis in growing appendages. Preliminary results suggest that both dpp and pan RNAi not only have direct effects on the development of traditional appendages, but also impact pronotal horn growth during late larval development. Combined, these results suggest extensive co-option of both Hox and Appendage Patterning gene networks during the development and evolution of Onthophagus beetle horns. Characterization of a chimeric Alphavirus with a heterologous RNA synthetic complex Brian R. Wasik and Richard W. Hardy Department of Biology, Indiana University, Bloomington, IN, 47405 The Alphavirus genus of Togaviridae encompasses a diverse group of arthropodbourne RNA viruses that have varied inflammatory manifestations of disease. During a single-cell replication cycle, Alphavirus RNA synthesis complexes must recognize multiple sequence-specific templates to efficiently generate antigenomes, genomes, and subgenomic RNAs. These complexes are composed of nonstructural polyproteins in various states of proteolytic processing, which in coordination with cellular factors specify the template for RNA synthesis. The consistent component in all manifestations of the RNA synthetic complex is cleaved nsP4, which has been identified as the viral RdRp. Replication complex interactions during an infectious cycle remain poorly understood. We have utilized a reverse genetic screen to genetically identify interactions within the replication complex by generating chimeric Alphaviruses with heterologous viral replication complexes. AURA nsP4 sequence was cloned into a Sindbis (SIN) TOTO1101 background, retaining the nsP3-4 cleavage junction (TOTO-AURAnsP4). We report that transfection of TOTO-AURAnsP4 RNA into mammalian BHK-21 cells resulted in observable cytopathology and the generation of plaque-forming virus. The growth of TOTO-AURAnsP4 virus was delayed relative to wild-type SIN, resulting in a small-plaque phenotype. Subsequent passage of virus resulted in reversions to near wildtype plaque morphology. Sequence analysis of revertant viruses showed no changes in the AURA nsP4 sequence, but rather second-site changes in the subgenomic promoter. These changes suggest that the introduction of a heterologous replication complex has reduced promoter-recognition leading to suppressor changes in the promoter sequence. We report on the characterization of chimera TOTO-AURAnsP4 replication relative to wild-type SIN and AURA virus, and the isolation and characterization of revertants and their implication in replication complex interactions. Conditional Essentiality of the VicK Histidine Kinase During Anaerobic Culture of Streptococcus pneumoniae Kyle J. Wayne, Ho-Ching Tiffany Tsui, and Malcolm E. Winkler The VicRK two-component system of S. pneumoniae is a member of the essential YycFG family found in low G+C gram positive bacteria. Previously we showed that phosphorylated VicR response regulator (RR) is essential because of its strong positive regulation of the pcsB gene, which mediates normal cell division. Little is known about the signals sensed by the VicK histidine kinase (HK). We characterized ΔvicK mutants along with point mutations and domain deletions of VicK. vicK mutants are viable during aerobic growth, presumably due to cross talk. Contrary to a previous report, we found that vicK mutants were inhibited for anaerobic growth in BHI rich medium; therefore, the VicK HK is conditionally essential in pneumococcus. Anaerobic growth of ΔvicK mutants was restored by ectopic expression of vicK or constituitive expression of pcsB. We show that the PAS domain, which sense oxygen in other bacteria, is not required for VicK function anaerobically. We found that the levels of VicK during anaerobic growth are three fold less than that of aerobic culture. This suggests that the levels of the VicRKX operon might also be lower anaerobically, which prevents cross talk that occurs during aerobic culture to phosphorylate VicR. The conditional essentiality of VicK during anaerobic culture is bypassed by two classes of suppressor mutations; high and low growth yield. The genome of a high yield suppressor was sequenced by the Illumina method, and we detected three missense point mutations in the suppressor strain. Most interestingly, we detected a mutation in the PnpR RR. Characterization of the requirements for resistance to the DNA damaging agent 4-nitroquinoline-1-oxide in Escherichia coli Ashley B. Williams and Patricia L. Foster Escherichia coli has two Y-family DNA polymerases, DNA polymerase IV (Pol IV, DinB), and DNA polymerase V (Pol V, UmuDC). While the in vivo functions of Pol V are thought to be well understood, the roles of Pol IV in DNA metabolism are not as clear. Studies of Pol IV function have largely focused on its translesion synthesis (TLS) activity, a damage tolerance mechanism that allows continued cell division when DNA replication forks encounter lesions that block the replicative polymerase. We have characterized the relationships between Pol IV and various DNA repair pathways that confer resistance to the DNA damaging agent 4-nitroquinoline-1-oxide (4-NQO). Reaction of 4-NQO with DNA results base adducts that block the replicative polymerase, but that can be substrates for TLS by Pol IV. The data presented here support the model that Pol IV acts mostly in a pathway requiring nucleotide excision repair, most likely TLS, and that Pol IV-dependent resistance to 4-NQO does not require recombination. April
17,
2009
 2009
GCMS
Symposium;

Peter
C.
Zee
 
 Title:
 Effects
of
genetic
background
on
pilA‐swarming
phenotype
in
M.
xanthus
 
 Abstract:
 Responses
in
biological
systems
are
context
dependent.
Context
can
be
due
to
 species
composition,
abiotic
environment,
social
interactions,
etc.

At
the
genetic
 level,
the
effects
of
individual
genes
can
be
dependent
on
the
genetic
background
in
 which
they
are
expressed.

Here,
we
test
for
variable
effects
of
genetic
background
 on
the
phenotypic
expression
of
a
gene
of
known
effect,
pilA,
in
a
lab
evolved
strain
 of
Myxococcus
xanthus.
Strain
E8
evolved
from
a
S‐motility
deficient
ancestor
 (∆pilA)
after
selection
for
increased
surface
swarming.
E8
evolved
a
novel
swarming
 mechanism
that
both
increased
swarming
rate
and
dramatically
altered
swarm
 morphology.

At
each
evolutionary
time
point,
we
performed
transformations
to
re‐ introduce
the
pilA
gene
into
the
genome.

Through
this
approach,
we
are
able
to
 monitor
effects
of
the
changing
genetic
background
on
both
rate
and
morphology
of
 pilA
swarming.

Preliminary
results
show
that
re‐introduction
of
pilA
rescues
the
 ancestral
swarming
to
wildtype
levels,
but
at
late
time
points
strongly
represses
the
 novel
swarming.

Ongoing
work
will
finish
the
transformations
to
achieve
finer
 resolution
of
the
effect
of
pilA
effect
on
a
changing
genetic
background.

Two
 potentially
interesting
results
could
emerge
from
this
study.
A
sharp
decline
in
pilA
 swarming
could
be
associated
with
specific
mutations
in
E8.

Alternatively,
there
 could
be
a
linear
decline
in
swarming
with
additional
mutations.

In
addition
to
 demonstrating
the
importance
of
genetic
background
on
gene
effects,
this
work
has
 potential
implications
for
the
evolution
of
pathways
and
the
role
of
horizontal
gene
 transfer
in
adaptive
evolution.