Download Neuronal specific and NGF inducible expression directed by the

A94
Biochemical Society Transactions (1999) 27
Preferential degradation of IKBa associated with NF-KB
but reassociation of NF-KB with free IKBa before
nuclear translocation.
Lin Yang, Franco Carlotti and Eva E. Qwarnstrom
Division of Molecular and Genetic Medicine, University of
Sheffield Medical School, Royal Hallamshire Hospital, Glossop
Road, Sheffield, S10 2JF
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NF-KB transcription factors consist of homo- or hetero-dimers of
a family of five related subunits. In unstimulated cells, these
dimers are retained in an inactive, cytoplasmic form by interaction
with a related family of inhibitory subunits, IKB. During immune
and inflammatory responses, the action of a variety of factors at
the cell surface induces the degradation of IKB and consequent
activation of the transcription factor. To study in detail the
mechanism of this activation, we have constructed fusions of
EGFP with IKBa and monitored their degradation in single cells
in response to IL- 1p stimulation.
By co-transfection with relA, we show that the subcellular
distribution of IKBa is dependent on its expression level as a
consequence of titration of endogenous NF-KB. Further,
degradation of IKBa is most enhanced in a concentration
dependent manner if it is complexed with NF-KB. Using cyan
and yellow variants of EGFP fused respectively with relA and
IKBa, we demonstrate molecular interaction in transfected cells
by FRET and analyse the simultaneous degradation of the
inhibitor and nuclear translocation of the transcription factor. So
far, this analysis shows that significant IKBa degradation occurs
before NF-KB nuclear translocation is initiated. This indicates
that, whilst IKBa is only degraded when complexed with NF-KB,
free NF-KB rapidly associates with excess free IKBa rather than
translocating to the nucleus.
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NF-KB activation in single living cells -Analysis of antiapoptosis and kinetics of activation by IL-1p.
Franco Carlotti, Lin Yang, Steven K. Dower and Eva E.
Qwarnstrom
Division of Molecular and Genetic Medicine, University of
Sheffield Medical School, Royal Hallamshire Hospital, Glossop
Road, Sheffield, S10 2JF
Signal transduction in mammalian cells is classically illustrated as
linear arrangements of receptors, protein kinase cascades and
effector molecules. However, it is becoming clear that there is
extensive cross talk between pathways resulting in a complex web
of interactions. A property of such webs is that small input
differences can generate large differences in response.
Consequently, biochemical methods which depend on
characterisation of the mean response of the population may not
accurately reflect the response of individual cells. Therefore, we
have been developing methods using GFP fusion proteins to study
the activation of NF-KB by interleukin I p in single living cells.
We will present data demonstrating activation of NF-KB by
transient transfection of GFP fusions and confocal microscopy.
This reveals wide heterogeneity in cellular expression level, with
the highest expressors (which provide most of the signal in
biochemical analyses) having aberrant properties. Analysis of the
kinetics of activation reveals concentration dependence with
maximum nuclear transport peaking at ca. 50 molecules per
second for ca. 20 fold overexpression. Further, we have analysed
the anti-apoptotic function of the transfected protein and find this
correlates with intermediate expression levels. The expression
level at which anti-apoptotic function peaks corresponds with that
for nuclear import and we are currently investigating the possible
titration of a common limiting factor.
Transcriptional Regulation of the Serotonin
Transporter Gene. CE Fiskerstrand, ,E Lovejoy, JP
Quinn
Dept. Vet Pathology., University of Edinburgh, EH9 1QH
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Serotonin re-uptake by pre-synaptic neurons is facilitated by
the Serotonin Transporter, 5-HTT. Dysregulation of 5-HTT
has long been associated with psychiatric and behavioral
disorders, some of which can be treated with selective
serotonin re-uptake inhibitors (SSFU). A polymorphic region
within the non-coding intron 2 of the 5-HTT gene has been
shown to correlate with a predisposition to affective disorders.
This polymorphism is a variable number tandem repeat
(VNTR) consisting of 9 to 12 repeats of a 16-1 7 base pair
consensus sequence, and it is this variation in number which
correlates with the susceptibility. We have established by
electrophoretic mobility shift analyses (EMSA) that 17bp
oligonucleotides representing individual repeat sequences
from within the VNTR can bind a number of different
complexes in a specific manner. Slight variation in the
sequence of the repeats results in them preferentially binding
distinct factors. This suggests the VNTR may regulate
transcription of the 5-HTT gene. Transient transfection
experiments using embryonic stem cells indicate that the 5HTT VNTR can function as an enhancer in vitro when linked
to a reporter gene. Differential expression is supported by
different number of repeats (e.g. 10 compared with 12
copies). This suggests that the copy number may have
differential effects on gene expression in vivo, and secondly
that the specific sequence of the repeats themselves may
impart further regulation.
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Neuronal specific and NGF inducible expression directed
by the Preprotachykinin-A promoter delivered by an adenoassociated viral vector. Leslev Gerrard, Patrick T. Harrison,
Robert G. Dalziel, and John P. Quinn. Veterinary Pathology,
University of Edinburgh, EH9 1QH
The proximal preprotachykinin (PPT) promoter (-865 to
+447) has activity restricted to neurons. Neurons are generally
refractory to all the common transfection procedures for
introduction of DNA into cells. This has hindered our analysis of
PPT promoter activity, as reporter gene constructs had to be
manually microinjected into primary cultures of neurons. This is
very laborious, time consuming and a technically demanding
procedure. However, a major paradox with these microinjection
studies was that the PPT promoter (spanning -3500 to +500)
remains unresponsive to NGF although multiple NGF responsive
elements within this fragment have been demonstrated in PC 12
cells. NGF is a characterized regulator of PPT in vivo. We
suggested that the lack of growth factor responsiveness was due to
the stimulation of stress-activated pathways during the
microinjection of neurons. Consistent with t h s all the individual
elements which were NGF responsive in PC 12 cells although
active in neurons were not further regulated by exposure to NGF
after microinjection. We have generated an adeno-associated virus
vector that uses the PPT promoter (-865 to +92) to support reporter
gene expression. We demonstrate that this virus has a neuronal
specific expression pattern. Moreover, it is shown for the first time
that the PPT promoter is nerve growth factor inducible in neurons.
This virus will be a useful tool to I ) modify neuronal phenotype by
expressing therapeutic molecules or antisense nucleic acid and 2)
dissect the signal transduction pathways that regulate promoter
function in vivo. We will present data on the stimulus induction of
this promoter fragment in response to various stimuli in primary
cultures of neurons