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luciferase for yeast?

francis at NCBI.NLM.NIH.GOV francis at NCBI.NLM.NIH.GOV
Fri Oct 6 12:32:35 EST 1995

> Wolfgang  Fecke <wf14 at columbia.edu> wrote:

> Does anyone have experience with the luciferase gene in yeast? 

> From: Donal Hickey (dhickey at maeve.genetics.uottawa.ca) 

> We have used luciferase as a reporter gene in yeast cells; 
> see PNAS 91:11109-11112. 

In addition, if you look up 'yeast' and 'luciferase' in Entrez you can
see some 30 abstracts (none of which are the above reference, because
'luciferase' is not mentioned in Donal's paper).  If you bind this to
titles only, then you have 4.  I attach these 4 abstracts below.  There
might be something for you there.

Entrez is available from our anonymous FTP site if you are interested
(ncbi.nlm.nih.gov) and runs on most platforms.  Either via the network
(free, once you are connected), WWW (at this URL http://www.ncbi.nlm.nih.gov/)
or at a minimal cost for the CD-ROM version.

Send some mail to info at ncbi.nlm.nih.gov if you want more info.



| B.F. Francis Ouellette  
| GenBank
| francis at ncbi.nlm.nih.gov   

FEBS Lett 368: 435-440  (1995) [95361918]

Conserved ATPase and luciferase refolding activities between bacteria
and yeast Hsp70 chaperones and modulators.

E. J. Levy, J. McCarty, B. Bukau & W. J. Chirico

Department of Anatomy and Cell Biology, State University of New York
Health Science Center at Brooklyn 11203, USA.

We have reconstituted an ATP-dependent protein folding machinery using
purified yeast cytosolic proteins. The S. cerevisiae Hsp70 Ssa1p and
the DnaJ homolog Ydj1p refolded denatured firefly luciferase. In E.
coli, efficient refolding of luciferase requires the Hsp70 DnaK and two
modulators, DnaJ and GrpE, that synergistically stimulate its ATPase
activity. Exchanging DnaJ homologs between the S. cerevisiae and E.
coli systems revealed that their ability to stimulate Hsp70 ATPase
activity was conserved. In contrast, GrpE further stimulated only
DnaK's ATPase activity. Efficient refolding of luciferase by Ssa1p and
DnaJ, but not by DnaK and Ydj1p, suggests that a compatible Hsp70/DnaJ
homolog pair can act as a protein folding machinery.

MeSH Terms:
  Enzyme Activation
  Escherichia coli/*metabolism
  Heat-Shock Proteins 70/*metabolism
  *Protein Folding
  Saccharomyces cerevisiae/*metabolism
  Support, Non-U.S. Gov't
  Support, U.S. Gov't, Non-P.H.S.

  Adenosinetriphosphatase (EC
  Luciferase (EC 1.13.12.-)
  Heat-Shock Proteins 70

New Biol 4: 157-65  (1992) [92207909]

The carboxyl-terminal tripeptide serine-lysine-leucine of firefly
luciferase is necessary but not sufficient for peroxisomal import in

B. Distel, S. J. Gould, T. Voorn-Brouwer, M. van der Berg, H. F. Tabak
& S.  Subramani

E.C. Slater Institute for Biochemical Research, Academical Medical
Centre, University of Amsterdam, The Netherlands.

Firefly luciferase is imported into peroxisomes in insects, mammals,
plants, and yeast, which implies that the mechanism of protein
translocation into peroxisomes has been conserved during eukaryotic
evolution. The carboxyl- terminal tripeptide serine-lysine-leucine in
luciferase acts as a peroxisomal import signal in mammalian cells. We
have investigated whether this tripeptide is also involved in
translocation of firefly luciferase into peroxisomes in yeast
(Saccharomyces cerevisiae). We show by gene fusion experiments that the
carboxyl-terminal 104 amino acids of luciferase can direct a
heterologous protein to yeast peroxisomes. Luciferase mutant proteins
were tested for their ability to be imported into yeast peroxisomes in
vivo. We demonstrate that mutations in the carboxyl-terminal
serine-lysine-leucine tripeptide abolish translocation of the protein
into yeast peroxisomes. However, when a passenger protein was tagged at
its carboxyl terminus with this tripeptide the fusion protein did not
go to peroxisomes. These results indicate that, in yeast, the
tripeptide is necessary but not sufficient for peroxisomal import.

MeSH Terms:
  Base Sequence
  Biological Transport
  Cloning, Molecular
  Molecular Sequence Data
  Mutagenesis, Site-Directed/genetics/physiology
  Saccharomyces cerevisiae
  Support, Non-U.S. Gov't
  Support, U.S. Gov't, P.H.S.
  Transformation, Genetic

Gene Symbols:

  Leucine (CAS 7005-03-0)
  Luciferase (EC 1.13.12.-)
  Lysine (CAS 56-87-1)
  Serine (CAS 56-45-1)

Biochemistry 29: 4758-66  (1990) [90304108]

Targeting of cloned firefly luciferase to yeast mitochondria.

C. Aflalo

Department of Chemistry, University of California, San Diego, La Jolla

The firefly luciferase gene (luc) was fused to a 5' fragment of the
70-kDa protein gene (70K) from yeast. The fragment codes for the
N-terminal putative signal sequence which targets and anchors the
70-kDa protein to the cytoplasmic side of the outer membrane in
mitochondria. Two versions of the fusion gene, 70K[232]::luc and
70K[93]::luc (containing 292 and 93 5' codons from 70K, respectively),
were constructed in a bacterial expression plasmid. Both the genes were
expressed in Escherichia coli, and in both cases, bioluminescence
activity was associated with the expression. The 70K[93]::luc gene was
transferred to a yeast-bacteria shuttle vector used to transform
Saccharomyces cerevisiae cells. As a control, the same strain was
transformed with a plasmid including the original luc. With both
transformants, bioluminescence activity was detected in intact cells
and crude extracts. Upon growth on a nonfermentable carbon source and
fractionation, the product of the fusion gene was associated mostly
with mitochondria. In the control transformant, the product of luc was
more delocalized. However, a significant amount remained associated
with isolated mitochondria. No such spontaneous association of purified
luciferase with wild-type mitochondria was observed in vitro. Trypsin
treatment of mitochondria isolated from both transformed strains
indicated that the fusion protein is anchored to the outer membrane and
exposed to the medium while the unfused luciferase retained with the
mitochondria is occluded in a compartment unaccessible to trypsin and
released in the presence of detergent.  The fusion protein retained the
major catalytic properties of the parent firefly luciferase, as
determined in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

MeSH Terms:
  Adenosine Triphosphate/analysis
  Amino Acid Sequence
  Base Sequence
  Cloning, Molecular
  Escherichia coli/genetics
  Molecular Sequence Data
  Recombinant Fusion Proteins/genetics
  Saccharomyces cerevisiae/enzymology/genetics
  Support, Non-U.S. Gov't
  Support, U.S. Gov't, Non-P.H.S.

  DNA (CAS 9007-49-2)
  Adenosine Triphosphate (CAS 56-65-5)
  Luciferase (EC 1.13.12.-)
  Recombinant Fusion Proteins

Gene 81: 349-54  (1989) [90034207]

Active bacterial luciferase from a fused gene: expression of a Vibrio
harveyi luxAB translational fusion in bacteria, yeast and plant cells.

G. Kirchner, J. L. Roberts, G. D. Gustafson & T. D. Ingolia

Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN

The luxA and luxB genes encoding the luciferase from Vibrio harvey were
fused by site-directed mutagenesis so that one polypeptide was encoded
by the fused gene. The fused gene facilitated light production in
Escherichia coli, Saccharomyces cerevisiae, and Nicotiana
plumbaginifolia when the substrates decanal and reduced flavin
mononucleotide were present. The specific activity of the encoded
enzyme is not known. In E. coli and S. cerevisiae cells the light
emission could be measured in viable, intact cells. The luxAB fusion
provides a simple reporter gene for in vivo measurement of promoter
strength in these species and may be useful in other systems as well.

MeSH Terms:
  Base Sequence
  Cells, Cultured
  Cloning, Molecular
  Escherichia coli/genetics
  *Gene Expression
  Saccharomyces cerevisiae/genetics
  Support, Non-U.S. Gov't

  Luciferase (EC 1.13.12.-)

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