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Construction of an improved host strain for two hybrid screening

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1502-1503
Nucleic
Acids
Research2,
1994,
Vol.
22,
No.
8
-
/
994
(A/)rid
UTuic'vrsitv
Pre(.Ss
Construction
of
an
improved
host
strain
for
two
hybrid
screening
H.E.Feilotter,
G.J.Hannon,
C.J.Ruddell
and
D.Beach*
Cold
Spring
Harbor
Laboratory,
1
Bungtown
Road.
Cold
Spring
Harbor.
NY
11724.
USA
Received
February
16,
1994;
Revised
and
Accepted
March
11,
1994
The
two
hybrid
approach
is
a
genetic
screen
for-
investigating
protein-protein
interactions
(1).
It
has
proven
useful
both
for
testing
interaction
between
known
proteins
and
for
identifying
previously
unknown
proteins
which
interact
with
a
protein
of
interest.
Several
recent
reviews
have
outlined
the
principles
underlying
this
approach
and
have
catalogued
its
successes
(see
2
for
example).
Using
a
two-hybrid
technique
involves
the
co-expression
of
two
hybrid
proteins
in
a
yeast
cell.
Sequences
codino
for
the
first
protein
(the
target)
are
fused
in
frame
to
those
encoding
the
DNA-
binding
domain
of
the
yeast
GAL4
transcriptional
factor.
Sequences
coding
for
the
second
protein
are
fused
to
those
encoding
activation
domain
of
GAL4.
A
functional
GAL4
activator
is
created
when
the
DNA
binding
and
activation
domains
come
together
as
a
result
of
physical
association
between
the
two
hybrids.
GAL4
activity
is
assayed
using
a
reporter
gene
which
depends
on
GAL4
for
transcription
(1).
Though
initially
used
to
test
for
interactions
between
known
proteins,
the
two
hybrid
approach
has
since
been
extended
to
large
scale
screens
in
which
the
target
gene,
fused
to
the
DNA-binding
domain
of
GAL4.
is
tested
against
a
library
of
genomic
or
cDNA
sequences
fused
to
a
transcriptional
activation
domain.
Interacting
proteins
are
selected
by
co-transformation
of
the
target
and
the
library
fusions
into
an
appropriate
host
yeast
strain
(3.4,5).
The
host
for
such
screens
is
the
budding
yeast,
Saccharomnc
es
cerevisiae.
For
use
in
two
hybrid
screening.
host
strains
typically
carry
mutations
which
ensure
that
endogenous
GAL4
is
absent.
In
addition,
GAL80,
whose
product
normally
inhibits
GAL4
function,
is
mutated
so
as
to
avoid
a
requirement
for
galactose
in
the
growth
medium.
The
strains
also
carry
auxotrophic
markers,
typically
leucine
and
tryptophan,
for
selection
of
cells
carrying
the
DNA-binding
and
activation
domain
vectors.
Finally.
these
strains
contain
one
or
more
reporter
genes
which
are
transcriptionally
dependent
on
the
reconstitution
of
functional
GAL4.
A
number
of
such
strains
have
been
constructed
(see
2
for
review).
The
earliest
of
these,
GGY
1::
171
(1)
depended
solely
on
the
transcription
of
a
lacZ
reporter
gene
to
indicate
physical
interaction
between
two
proteins.
While
useful,
screening
for
i3-
galactosidase
alone
was
too
tedious
for
use
in
searching
high-
complexity
libraries
for
interacting
proteins.
The
addition
of
a
second
reporter
gene
in
the
form
of
a
GALI-driven
HIS3
gene
increased
the
utility
of
the
screen,
since
interactince
clones
could
be
identified
by
a
growth
selection
on
medium
lacking
histidine.
Also,
the
use
of
two
reporter
genes
had
the
added
advantage
of
decreasing
the
background
of
false
positives
(2).
Available
versions
of
such
host
strains
(e.g.
YPB2
(2))
require
the
addition
of
the
antimetabolite
3-amino-12,.4-triazole
(3-AT)
(6)
to
the
growth
medium
to
increase
the
stringency
of
selection
since
these
strains
are
phenotypically
prototrophic
foi
histidine
due
to
the
use
of
a
leaky
HIS3
alleles.
Our
early
experiments
suggested
that
the
use
of
3-AT
in
the
selective
mecdia
had
a
number
of
disadvantauces.
First,
it
greatly
Trable
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the
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makinJ
s,trans
carryin
nK
it
prototrophic
tol
Liracul.
Table
2.
Testine,
of
HF7c
Plasi$d
AA
Plasmidi
B2
His
blo,LC
pGBT95
pGBT9
pCLI
pGBT9-SNF
s
pGAD-GH5
PGAD-GH
pGADGH-SNF4'
pGBT9-SNF
I
pGADGH-SNF4
PIlasnmids
contalinilng
the
DNA-hinding
domaini
of
GAL4.
-Plasmids
contllinine,
thc
activation
domainlfa
o
GAL4.
'Abilitv
of
trnistormants
to
grows
on
micdill
LIckin
12
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tr1
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foll
,
lotsine
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dtJ
ass\s
bIloC,
wh
it
C
'~rc
1.
2.
(,ref.
7.
Fhi
ls
PLaSi
L(t
c
nItains
the
G.AL4
Inci.
l
CG.
H.
this
lLhi
LoIpLb.
daLtal.
*
To
whom
correspondence
should
be
addressed
Nucleic
Acids
Research,
1994,
Vol.
22,
No.
8
1503
increased
the
time
required
for
growth
selection
of
positive
clones.
Second,
it
restricted
the
subset
of
clones
obtained
in
the
two-hybrid
screen.
Therefore,
we
have
developed
a
modified
two-
hybrid
screening
strain
which
we
have
called
HF7c.
This
strain
contains
a
GAL4-dependent
HIS3
reporter
gene
which,
due
to
its
promoter,
is
not
leaky
and
is,
therefore,
phenotypically
auxotrophic
for
histidine.
Thus
HF7c
does
not
require
the
use
of
the
histidine
antimetabolite.
In
addition,
HF7c
carries
a
lacZ
reporter
gene
from
YBP2.
The
first
step
in
the
construction
of
HF7c
was
the
isolation
of
a
strain
identical
to
YPB3
except
that
it
carried
the
MATce
allele.
This
was
accomplished
by
selecting
progeny
from
a
cross
of
parental
strains
YPB3
and
YM4
136
(see
Table
1),
which
were
prototrophic
for
uracil
(indicating
the
presence
of
the
HIS3
construct
integrated
at
the
LYS2
locus)
and
auxotrophic
for
histidine
(indicating
the
presence
of
the
gal4-542
mutation).
Outcrosses
to
tester
MATa
and
MATct
haploid
strains
allowed
selection
of
the
desired
mating
type.
These
progeny
were
then
crossed
to
the
strain
YPB2
(see
Table
1).
The
desired
progeny
would
carry
the
HIS3
allele
from
YPB2
and
the
lacZ
gene
from
YPB2.
The
tighter
HIS3
allele
was
selected
on
the
basis
of
lack
of
any
growth
on
plates
lacking
histidine.
The
presence
of
the
lacZ
gene
was
confirmed
by
transformation
of
the
GAL4
gene
into
those
strains
displaying
a
tight
his-phenotype.
Those
which
gave
a
positive
result
in
an
assay
for
3-galactosidase
activity
were
further
characterized.
HF7c
was
the
one
among
these
which
gave
the
fastest
growth,
highest
transformation
efficiency
and
greatest
3-galactosidase
activity.
HF7c
is
also
auxotrophic
for
tryptophan
and
leucine,
allowing
selection
of
library
and
target
plasmids.
The
complete
genotype
is
given
in
Table
1.
We
have
subjected
HF7c
to
the
following
tests
to
ensure
that
it
conforms
to
all
the
requirements
for
use
in
two
hybrid
screens.
1)
Either
the
GAL4
gene
or
a
control
vector
was
introduced
into
HF7c,
and
transformants
were
tested
for
growth
on
media
lacking
histidine
and
3-galactosidase
activity
(Table
2).
2)
HF7c
was
co-transformed
with
a
pair
of
proteins
previously
shown
to
interact
in
the
two
hybrid
system:
the
SNF]
gene
fused
to
the
DNA-binding
domain
of
GAL4
and
the
SNF4
gene
fused
to
the
GAL4
activation
domain.
The
transformants
were
selected
by
their
ability
to
grow
on
medium
lacking
both
tryptophan
and
leucine
and
were
then
tested
for
expression
of
both
the
HIS3
and
lacZ
genes
(Table
2).
3)
To
determine
whether
HF7c
was
suitable
for
large
scale
screening,
we
co-transformed
the
strain
with
the
human
cdk2
gene
fused
to
the
DNA-binding
domain
of
GAL4
and
a
HeLa
cDNA
library
fused
to
the
activation
domain.
This
same
screen
had
been
previously
carried
out
in
our
laboratory
using
the
host,
YPB2,
and
genes
encoding
several
interacting
proteins
had
been
identified
(7).
The
same
genes
were
among
those
identified
using
HF7c.
However,
because
HF7c
does
not
require
the
use
of
3-AT,
the
time
required
from
the
start
to
the
finish
of
such
a
screen
was
considerably
less
than
with
YPB2.
Transformants
were
visible
as
small
colonies
as
early
as
three
days
following
transformation,
whereas
this
took
up
to
two
week
with
YPB2.
HF7c
allows
a
sensitive
primary
selection
based
on
histidine
expression,
and
a
secondary
selection
based
upon
color
detection.
Criteria
for
the
selection
of
those
potential
positives
which
should
be
characterized
further
has
been
discussed
elsewhere
(2,
8).
These
assays
indicate
that
HF7c
is
a
suitable
host
for
large
scale
screens
using
the
two
hybrid
approach
and
should
be
useful
to
the
many
researchers
currently
using
this
technique
to
study
protein-protein
interactions.
ACKNOWLEDGEMENTS
The
authors
wish
to
thank
P.Bartel
and
M.Johnston
for
their
gifts
of
strains.
REFERENCES
1.
Fields,S.
and
Song,O.
(1989)
Nature
340,
245-246.
2.
Bartel,P.
(1993)
In
Hartley,D.A.
(ed.),
Cellular
Interactions
in
Development:
A
Practical
Approach.
Oxford
University
Press,
Oxford,
pp.
153
-
179.
3.
Chien,C.-T.,
Bartel,P.,
Stemglanz,R.
and
Fields,S.
(1991)
Proc.
Natl.
Acad.
Sci.
USA
88,
9578-9582.
4.
Dalton,S.
and
Treisman,R.
(1992)
Cell
68,
597-612.
5.
Moehle,C.M.
and
Hinnebusch,A.G.
(1991)
Mol.
Cell.
Biol.
11,
2723
-2735.
6.
Yocum,R.R.,
Hanley,S.,
West,R.
and
Ptashne,M.
(1984)
Mol.
Cell.
Biol.
4,
1985-1998.
7.
Hannon,G.J.,
Demetrick,D.
and
Beach,D.
(1993)
Genes
Dev.
7,
2378
-2391.
8.
Hannon,G.J.,
Zhu,L.
and
Holz,A.
(1994)
CLONTECHniques
9,
1-4.
... The HF7c strain of S. cerevisiae used in this screen carries two reporter genes, the his3 and lacZ genes, both of which contain a UAS that is regulated by GAL4 (Fig 5.2) (Feilotter et al., 1994). In the case of his3, the UAS and the minimal promoter originate from the native Chapter 5 ...
... GALl promoter region. For lacZ^ the UAS is a synthetic 17'mer and its minimal promoter is from the yeast cytochrome C l {cycl) gene, which is a weak promoter and so weak protein interactions may not be observed unless a highly sensitive p-galactosidase assay is employed (Feilotter et al., 1994; Yeast protocols handbook, Clontech). ...
... In HF7c, this entire region has been replaced as described above, resulting in tight regulation of the his3 reporter in this strain. Furthermore, HF7c yeast lacks leu2 and trpl genes, providing auxotrophic markers for selection o f cells containing the DNA binding and activation vectors (Feilotter et al., 1994). ...
Thesis
Tumour necrosis factor-[alpha] (TNF-[alpha]) is known to induce changes in endothelial cell morphology and paracellular permeability, but the mechanisms have not been extensively characterised. The purpose of this study was to establish the effects of TNF-[alpha] on human umbilical vein endothelial cell (HUVECs) paracellular permeability and tight junctions and relate these responses to changes in the actin cytoskeleton. TNF-[alpha] caused progressive changes to HUVECs over 24 h. TNF-[alpha] induced RhoA activation, myosin light chain phosphorylation, cortical F-actin thickening and the formation of tiny inter-cellular gaps within 10 min, A small increase in permeability accompanied these changes. By 24 h, TNF-[alpha] caused stress fibre formation, cell elongation and extensive gap formation. Occludin and JAM-A were lost from the tight junctions, ZO-1 was partially redistributed and permeability was increased. RhoA and ROCK inhibition prevented TNF-[alpha]-induced changes in F-actin and cell morphology, but ROCK inhibition did not re-establish the junctional localisation of ZO-1, nor did it prevent increased permeability. Myosin light chain kinase inhibition had no impact on TNF-[alpha]- induced stress fibres, cell elongation or permeability at 24 h. These results indicate that the TNF-[alpha]-induced morphological and cytoskeletal changes are not solely responsible for increased permeability and that signalling to the tight junction proteins may be more important for TNF-[alpha] regulation of barrier function. To identify potential interacting partners of occludin, a yeast two-hybrid experiment was performed using the C-terminus of occludin. The transcriptional co-activator protein, Ski-interacting protein (SKIP) and the Ser/Thr protein kinases, casein kinase I[epsilon] (CKI[epsilon]) and UNC-51 like kinase-1 (ULK-1) were identified in this screen. These novel interactions may be important for occludin regulation and function. During the course of these studies, adenoviruses were used to introduce genes into HUVECs. An inhibitory effect of control adenoviruses on TNF- -induced cytoskeletal changes and permeability was observed, suggesting that adenovirus binding and/or entry could modulate endothelial cell behaviour and responses.
... Haploid S. cerevisiae MATa strain CG-1945 or AH109 and MATα strain Y187 (Harper et al, 1993;Feilotter et al, 1994;James et al, 1996) were used for single transformations (Dohmen et al, 1991) with bait or prey constructs, respectively (all based on original vectors from Clontech Laboratories; for further plasmid details, see Supplemental Table 1). After selection on either −Trp (prey) or −Leu (bait) plates, single colonies were picked and grown as liquid culture. ...
Preprint
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Proteins ZC3HC1 and TPR are construction elements of the nuclear pore complex (NPC)-attached nuclear basket (NB). NB-location of ZC3HC1 depends on TPR already occurring NPC-anchored, whereas additional TPR polypeptides are appended to the NB by ZC3HC1. The current study examined the molecular properties of ZC3HC1 that enable it to bind to the NB and TPR. We report the identification and definition of a nuclear basket-interaction domain (NuBaID) of Hs ZC3HC1 comprising two similarly built modules, both essential for the binding to the NB's NPC-anchored Hs TPR. Furthermore, we describe such a bimodular construction as evolutionarily conserved and exemplify the kinship of Hs ZC3HC1 by the NB- and Dd TPR-interacting homolog of Dictyostelium discoideum and by characterizing protein Pml39 as the ZC3HC1 homolog in Saccharomyces cerevisiae . Among several properties shared by the different species' homologs, we unveil the integrity of the bimodular NuBaID of Sc Pml39p as being essential for binding to the yeast's NBs and its TPR homologs Sc Mlp1p and Sc Mlp2p, and we further present Pml39p as enabling interlinkage of Mlp1p subpopulations. In addition to phyla-specific features, we delineate the three species' common NuBaID as the characterizing structural entity of a one-of-a-kind protein found not in all but likely most taxa of the eukaryotic realm.
... plasmids and assessing growth on quadruple deficient plates. Back-transformation using a 488 modified prey plasmid coding for the third PDZ domain of PSD93 was performed in HF7C yeast 489 (Feilotter et al., 1994). (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...
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Full-text available
The palmitoyl acyltransferase (PAT) ZDHHC14 is highly expressed in the hippocampus and is the only PAT predicted to bind Type I PDZ domain-containing proteins. However, ZDHHC14’s neuronal roles are unknown. Here, we identify the PDZ domain-containing Membrane-associated Guanylate Kinase (MaGUK) PSD93 as a direct ZDHHC14 interactor and substrate. PSD93, but not other MaGUKs, localizes to the Axon Initial Segment (AIS). Using lentiviral-mediated shRNA knockdown in rat hippocampal neurons, we find that ZDHHC14 controls palmitoylation and AIS clustering of PSD93 and also of Kv1 potassium channels, which directly bind PSD93. Neurodevelopmental expression of ZDHHC14 mirrors that of PSD93 and Kv1 channels and, consistent with ZDHHC14’s importance for Kv1 channel clustering, loss of ZDHHC14 decreases outward currents and increases action potential firing in hippocampal neurons. To our knowledge, these findings identify the first neuronal roles and substrates for ZDHHC14 and reveal a previously unappreciated role for palmitoylation in control of neuronal excitability. Impact Statement ZDHHC14 controls palmitoylation and axon initial segment targeting of PSD93 and Kv1-family potassium channels, events that are essential for normal neuronal excitability.
... Positive clones were confirmed by re-transformation of yeast with purified bait and individual 'hit' plasmids and assessing growth on quadruple deficient plates. Back-transformation using a modified prey plasmid coding for the third PDZ domain of PSD93 was performed in HF7C yeast (Feilotter et al., 1994). ...
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Full-text available
The palmitoyl acyltransferase (PAT) ZDHHC14 is highly expressed in the hippocampus and is the only PAT predicted to bind Type I PDZ domain-containing proteins. However, ZDHHC14's neuronal roles are unknown. Here, we identify the PDZ domain-containing Membrane-associated Guanylate Kinase (MaGUK) PSD93 as a direct ZDHHC14 interactor and substrate. PSD93, but not other MaGUKs, localizes to the Axon Initial Segment (AIS). Using lentiviral-mediated shRNA knockdown in rat hippocampal neurons, we find that ZDHHC14 controls palmitoylation and AIS clustering of PSD93 and also of Kv1 potassium channels, which directly bind PSD93. Neurodevelopmental expression of ZDHHC14 mirrors that of PSD93 and Kv1 channels and, consistent with ZDHHC14's importance for Kv1 channel clustering, loss of ZDHHC14 decreases outward currents and increases action potential firing in hippocampal neurons. To our knowledge, these findings identify the first neuronal roles and substrates for ZDHHC14 and reveal a previously unappreciated role for palmitoylation in control of neuronal excitability.
... A cDNA library, representing 10 6 independent EcoRI-XhoI cDNA clones, was prepared in the Hybrizap vector (Stratagene) from the mRNA of wild-type apices, ‫5.0ف‬ cm long, containing the second and third shoot segments. The HF7c yeast line was used as the host because its growth on the histidine (ϪHis) selective medium is completely suppressed (Feilotter et al., 1994). Procedures and controls were as recommended by the manufacturer (Stratagene). ...
... The yeast two-hybrid strain HF7C (Feilotter et al., 1994) was cotransformed with pGADGH (empty vector or pGADGH-RCR1 mutants) and pGBT9 (empty vector or pGBT9-CHS5 [1-261]). Cells were grown to saturation in synthetic growth media (-Leu,-Trp) and 10-fold dilutions were spotted on synthetic growth media (-Leu,-Trp) and synthetic growth media (-Leu,-Trp,-His) supplemented with 2 mM 3-amino-1,2,4-triazole. ...
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Rsp5, the Nedd4 family member in yeast, is an E3 ubiquitin ligase involved in numerous cellular processes, many of which require Rsp5 to interact with PY-motif containing adaptor proteins. Here, we show that two paralogous transmembrane Rsp5 adaptors, Rcr1 and Rcr2, are sorted to distinct cellular locations: Rcr1 is a plasma membrane (PM) protein, whereas Rcr2 is sorted to the vacuole. Rcr2 is delivered to the vacuole using ubiquitin as a sorting signal. Rcr1 is delivered to the PM by the exomer complex using a newly uncovered PM sorting motif. Further, we show that Rcr1, but not Rcr2, is up-regulated via the calcineurin/Crz1 signaling pathway. Upon exogenous calcium treatment, Rcr1 ubiquitinates and down-regulates the chitin synthase Chs3. We propose that the PM-anchored Rsp5/Rcr1 ubiquitin ligase-adaptor complex can provide an acute response to degrade unwanted proteins under stress conditions, thereby maintaining cell integrity.
Thesis
Muskelin is a novel intracellular protein involved in cell responses to the adhesion modulating matrix component, Thrombospondin-1 (TSP-1). The muskelin polypeptide sequence contains six motifs with homology to the tandem kelch motifs, initially identified in the Drosophila Kelch protein, a component of ring canals. The focus of the thesis project has been to identify the molecular interactions of muskelin by use of a number of approaches including the yeast dihybrid system, copprecipitation, pharmacological agents and gel overlays. The two hybrid approach did not prove to be appropriate, because expression of muskelin had toxic effects on yeast cells. Treatment of cell extracts with a panel of pharmacological agents, to target components of cell signalling pathways, had no effect on the distribution of muskelin between detergent soluble (cytosol and membranes) and insoluble (cytoskeleton) fractions. Using a panel of cell lines including skeletal myoblasts, a set of proteins migrating with apparent molecular weights between 45-57 kDa were specifically detected upon muskelin overlays of SDS-polyacrylamide gels. These proteins did not correspond to the most abundant proteins within cell extract. Interaction of muskelin with these proteins was detectable throughout myoblast fusion into myotubes. Fascin, tubulin and actin were investigated as potential candidates for the proteins detected by muskelin gel overlay. A combination of muskelin and fascin overlays of cell extracts demonstrated that muskelin did not directly bind fascin. Muskelin did not interact with purified tubulin and actin in gel overlay assays and cosedimentation studies revealed that muskelin does not interact direct with microtubules or microfilaments. The most prominant interaction of muskelin in a gel overlay was with a 45 kDa protein of pI 6.0. This interaction was enhanced during myoblast fusion. Apparent postranslational modification of the 45 kDa protein was detected by muskelin overlay of extracts resolved on 2-Dimensional IEF/SDS-PAGE gels. A two step approach was developed to isolate the 45 kDa protein by using a strong anion exchange column to enrich for the 45 kDa protein, followed by 2D gel anaKsis. Identifying muskelin's binding partners will enhance understanding of its functional role.
Thesis
Mirror is a homeodomain transcription factor in Drosophila melanogaster. The mirror gene is part of the Iroquois complex (Iro-C) which also contains araucan and caupolican. The Iro-C genes have important roles in the development and patterning of the eye and wing imaginal discs as well as the oocyte and embryo. The Iroquois gene products are the founding members of the IRO family of proteins which are characterised by a highly conserved homeodomain of the TALE class (three amino acid loop extension). IRO proteins have been identified in organisms from C. elegans to humans and seem to have conserved roles in developmental patterning. To discover more about how Mirror functions as a transcription factor, the yeast-two-hybrid system was used to screen a Drosophila embryonic cDNA library for proteins that interact with Mirror. Several putative interactors were identified including two known transcription factors and a chromatin-remodelling factor as well as a number of previously uncharacterised gene products. A subset of the confirmed yeast-two-hybrid interactors have been investigated for biological relevance by comparing their expression patterns and mutant phenotypes to those of mirror as well as through genetic interactions. Two of the putative Mirror-binding proteins have been studied in detail, the novel forkhead associated (FHA) domain containing protein CG1135 and chromodomain helicase/ATPase DNA-binding protein 1 (CHD1). A P element insertion allele of CG1135 was shown to interact genetically with mirror alleles. Characterisation of the CG1135 phenotype revealed a putative role in cell proliferation or survival. The function of CHD1 in Drosophila is not known. In order to characterise the role of CHD1 and investigate any interactions with Mirror, a putative dominant negative version was generated and analysed. Studies of the localisation of CHD1 on polytene chromosomes indicate that CHD1 may be associated with transcriptional elongation.
Thesis
The Serum Response Element (SRE) is a promoter element important in the transcriptional regulation of many immediate early and muscle specific genes. Serum Response Factor (SRF) and Ternary Complex Factor (TCF) form a complex at the c-fos SRE. TCF is phosphorylated on MAP Kinase activation resulting in activation of transcription. In addition, a TCF independent pathway, dependent on RhoA signalling and depletion of G-actin levels, activates transcription through SRF. The sensitivity of SRF target genes to inhibitors of the actin pathway correlates with the presence of a TCF binding site adjacent to the SRE. c-fos and egr-1 promoters have a TCF binding site adjacent to the SRE and are insensitive to the actin pathway, vinculin and actin promoters do not have a TCF binding site and are sensitive to the actin pathway. In this study vinculin has been used to investigate whether the presence of a TCF binding site is sufficient to render the SRE insensitive to the actin pathway. At the vinculin promoter a TCF site alone is not sufficient to reduce the sensitivity to the actin pathway inhibitor, Latrunculin B. This finding is in contrast to studies using the c-fos promoter, which can be made sensitive to the actin pathway by the removal of the TCF binding site (Murai and Treisman, 2002). The promoter context of the SRE may therefore be important in rendering a promoter insensitive to the actin pathway. However, targeting an altered DNA binding specificity TCF molecule to the SRE can inhibit the actin pathway at the vinculin promoter. In addition, a 1-hybrid screen was conducted to identify factors capable of interacting with SRF, to allow identification of the SRF co-activator of the actin pathway. A clone was identified that is not the co-activator of the actin pathway, but which may have a role in SRF mediated transcriptional activation.
Thesis
Research described in the first part of this thesis was focused on studying the control of post-transcriptional initiation events in yeast. A transcriptional run-on assay in permeabilised yeast cells was used to study the distribution of RNA polymerase II (Pol II) complexes before and after activation of GAL1 gene by the Gal4 activator. Polymerases were found engaged on the template at the 5' end before activation, but only appeared at the 3' end after activation. Mutations of the Pol II CTD, the CTD kinase Kin28, and the holoenzyme subunit, Srb2, all inhibited formation of 3' polymerases in response to an activator. However, these mutations did not inhibit establishment of polymerases at the 5' end. The differences between 3' and 5' Pol II ternary complexes suggest that they represent qualitatively distinct "activated" and "non-activated" forms of polymerase. The results implicate CTD phosphorylation in a switch from "non-activated" transcription, that is confined to the 5' end, to an "activated" mode that traverses the length of the gene. Second part of the thesis was focused on studying transcriptional activation of c-fos mediated by the serum response element (SRE). A ternary complex with the transcription factors SRF (serum response factor) and TCP (ternary complex factor) is formed at the SRE. Functional RhoA is required for serum or LPA activated transcription at the SRE in a SRF dependent TCF independent manner. In order to isolate factors that directly target SRF in response to RhoA, two yeast screens were performed. The first screen used SRF bound at the SRE, and resulted in isolation of Zhx 1, a protein containing 5 homeoboxes and 2 zinc fingers. Results show that the SRF DNA binding domain is sufficient for interaction with Zhx1. Moreover, stable complexes with SRF and Zhx266-620 can be detected in vitro. However, Zhx1 does not activate SRE containing reporters in a mammalian system. The second screen used SRF bound at the Gal4 operator. This screen resulted in isolation of Sp4, a protein containing 3 zinc fingers. Results show that Sp4312-784 can interact with full length SRF but the SRF DNA binding domain is not sufficient for this interaction. Sp4312-784 can also cause a modest decrease in transcriptional activation of SRE containing reporters in mammalian cells.
Article
Full-text available
We present the DNA sequence of a 914-base pair fragment from Saccharomyces cerevisiae that contains the GAL1-GAL10 divergent promoter, 140 base pairs of GAL10 coding sequence, and 87 base pairs of GAL1 coding sequence. From this fragment, we constructed four pairs of GAL1-lacZ and GAL10-lacZ fusions on various types of yeast plasmid vectors. On each type of vector, the fused genes were induced by galactose and repressed by glucose. The response of a GAL1-lacZ fusion to gal4 and gal80 regulatory mutations was similar to the response of intact chromosomal GAL1 and GAL10 genes. A set of deletions that removed various portions of the GAL10 regulatory sequences from a GAL10-CYC1-lacZ fusion was constructed in vitro. These deletions defined a relatively guanine-cytosine-rich region of 45 base pairs that contained sequences necessary for full-strength galactose induction and an adjacent guanine-cytosine rich 55 base pairs that contained sequences sufficient for weak induction.
Article
We used a yeast genetic screen to isolate cDNAs that encode a protein, SRF accessory protein-1 (SAP-1), that is recruited to the c-fos serum response element (SRE) as part of a ternary complex that includes serum response factor (SRF). SAP-1 requires DNA-bound SRF for ternary complex formation and makes extensive DNA contacts to the 5′ side of SRF, but does not bind DNA autonomously. Ternary complex formation by SAP-1 requires only the DNA-binding domain of SRF, which can be replaced by that of the related yeast protein MCM1. We isolated cDNAs encoding two forms of SAP-1 protein, SAP-1a and SAP-1b, which differ at their C termini. Both SAP-1 proteins contain three regions of striking homology with the elk-1 protein, including an N-terminal ets domain. Ternary complex formation by SAP-1 requires both the ets domain and a second conserved region 50 amino acids to its C-terminal side. SAP-1 has similar DNA binding properties to the previously characterized HeLa cell protein .
Article
We describe a method that detects proteins capable of interacting with a known protein and that results in the immediate availability of the cloned genes for these interacting proteins. Plasmids are constructed to encode two hybrid proteins. One hybrid consists of the DNA-binding domain of the yeast transcriptional activator protein GAL4 fused to the known protein; the other hybrid consists of the GAL4 activation domain fused to protein sequences encoded by a library of yeast genomic DNA fragments. Interaction between the known protein and a protein encoded by one of the library plasmids leads to transcriptional activation of a reporter gene containing a binding site for GAL4. We used this method with the yeast SIR4 protein, which is involved in the transcriptional repression of yeast mating type information. (i) We used the two-hybrid system to demonstrate that SIR4 can form homodimers. (ii) A small domain consisting of the C terminus of SIR4 was shown to be sufficient to mediate this interaction. (iii) We screened a library to detect hybrid proteins that could interact with the SIR4 C-terminal domain and identified SIR4 from this library. This approach could be readily extended to mammalian proteins by the construction of appropriate cDNA libraries in the activation domain plasmid.
Article
An amino acid limitation in bacteria elicits a global response, called stringent control, that leads to reduced synthesis of rRNA and ribosomal proteins and increased expression of amino acid biosynthetic operons. We have used the antimetabolite 3-amino-1,2,4-triazole to cause histidine limitation as a means to elicit the stringent response in the yeast Saccharomyces cerevisiae. Fusions of the yeast ribosomal protein genes RPL16A, CRY1, RPS16A, and RPL25 with the Escherichia coli lacZ gene were used to show that the expression of these genes is reduced by a factor of 2 to 5 during histidine-limited exponential growth and that this regulation occurs at the level of transcription. Stringent regulation of the four yeast ribosomal protein genes was shown to be associated with a nucleotide sequence, known as the UASrpg (upstream activating sequence for ribosomal protein genes), that binds the transcriptional regulatory protein RAP1. The RAP1 binding sites also appeared to mediate the greater ribosomal protein gene expression observed in cells growing exponentially than in cells in stationary phase. Although expression of the ribosomal protein genes was reduced in response to histidine limitation, the level of RAP1 DNA-binding activity in cell extracts was unaffected. Yeast strains bearing a mutation in any one of the genes GCN1 to GCN4 are defective in derepression of amino acid biosynthetic genes in 10 different pathways under conditions of histidine limitation. These Gcn- mutants showed wild-type regulation of ribosomal protein gene expression, which suggests that separate regulatory pathways exist in S. cerevisiae for the derepression of amino acid biosynthetic genes and the repression of ribosomal protein genes in response to amino acid starvation.
Article
Protein-protein interactions between two proteins have generally been studied using biochemical techniques such as crosslinking, co-immunoprecipitation and co-fractionation by chromatography. We have generated a novel genetic system to study these interactions by taking advantage of the properties of the GAL4 protein of the yeast Saccharomyces cerevisiae. This protein is a transcriptional activator required for the expression of genes encoding enzymes of galactose utilization. It consists of two separable and functionally essential domains: an N-terminal domain which binds to specific DNA sequences (UASG); and a C-terminal domain containing acidic regions, which is necessary to activate transcription. We have generated a system of two hybrid proteins containing parts of GAL4: the GAL4 DNA-binding domain fused to a protein 'X' and a GAL4 activating region fused to a protein 'Y'. If X and Y can form a protein-protein complex and reconstitute proximity of the GAL4 domains, transcription of a gene regulated by UASG occurs. We have tested this system using two yeast proteins that are known to interact--SNF1 and SNF4. High transcriptional activity is obtained only when both hybrids are present in a cell. This system may be applicable as a general method to identify proteins that interact with a known protein by the use of a simple galactose selection.
Article
A two-hybrid protein interaction screen was used to isolate cDNAs encoding human proteins that can interact with human CDK2 in yeast. A new member of the retinoblastoma susceptibility gene family, Rbr-2 (Rb-related), was obtained. The sequence of the Rbr-2 protein shares approximately 50% identify with p107 and homology to Rb within the pocket domain. Several lines of evidence indicate that Rbr-2 is the adenovirus E1A-associated p130. Like Rb and p107, p130Rbr-2 can bind to viral oncoproteins, SV40 large T antigen, and adenovirus E1A through its pocket domain. Although p130Rbr-2 does not bind to CDK2 in vitro, it can interact with cyclins, with a clear preference for D-type cyclins. Because both CDK2 and p130Rbr-2 show affinity for cyclins, we suggest that p130Rbr-2 and CDK2 interacted through a yeast-derived cyclin bridge in the two-hybrid screen. The gene encoding p130Rbr-2 mapped to 16q13, a region of frequent genomic alteration in human tumors.
Article
We used a yeast genetic screen to isolate cDNAs that encode a protein, SRF accessory protein-1 (SAP-1), that is recruited to the c-fos serum response element (SRE) as part of a ternary complex that includes serum response factor (SRF). SAP-1 requires DNA-bound SRF for ternary complex formation and makes extensive DNA contacts to the 5' side of SRF, but does not bind DNA autonomously. Ternary complex formation by SAP-1 requires only the DNA-binding domain of SRF, which can be replaced by that of the related yeast protein MCM1. We isolated cDNAs encoding two forms of SAP-1 protein, SAP-1a and SAP-1b, which differ at their C termini. Both SAP-1 proteins contain three regions of striking homology with the elk-1 protein, including an N-terminal ets domain. Ternary complex formation by SAP-1 requires both the ets domain and a second conserved region 50 amino acids to its C-terminal side. SAP-1 has similar DNA binding properties to the previously characterized HeLa cell protein p62/TCF.
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Cellular Interactions in Development: A Practical Approach
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