This week we’ll be reading this review article by Francis et al. 1999
Please read the article closely, answer the following questions (save your answers in a Word document and the copy and paste them here):
Provide the full reference to this article using this format: Author 1 Last Name, First name initials; Author 2 Last Name, First name initials, (Year of publication). Article title. Journal name in italics Volume#(Issue#): start_page:end_page.
Shendelman S, Jonason A, Martinat C, Leete T and Abeliovich A. 2004. DJ-1 is a redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation. Plos Biology 2(11): 1764-1773.
Is this article primary or secondary literature? If secondary literature, is it a meta-analysis? See: how to distinguish primary vs secondary literature.
What is the hypothesis being tested?
What is the experimental approach to testing the hypothesis? What was measured?
What were the important quantitative results?
Explain whether the results support or contradict the hypothesis (based on the discussion section).
What do the results mean in terms of the broader issues that motivated the study (based on the conclusions)?
Don’t use quotes, you need to summarize the findings using your own words.
cloning. Second, it is sensitive. Activities can
be detected in the purified GST-ORF pools
that simply cannot be detected in extracts or
cells, the starting point of both conventional
purification and expression cloning. Because
the GST-ORFs are individually expressed at
high levels and are largely free of extract
proteins after purification, activities can be
measured for hours without competing activ-
ities that destroy the substrate, the product, or
In addition to the conventional use demon-
strated here, this array could be used in two
other ways: (i) to determine the range of poten-
tial substrate proteins for any protein-modifying
enzyme (such as a protein kinase) before genet-
ic or biochemical tests to establish authentic
substrates and (ii) to identify genes encoding
proteins that bind any particular macromole-
cule, ligand, or drug. Thus, one could rapidly
ascribe function to many presently unclassified
yeast proteins, complementing other genomic
approaches to deduce gene function from ex-
pression patterns, mutant phenotypes, localiza-
tion of gene products, and identification of in-
References and Notes
1. H. Simonsen and H. F. Lodish, Trends Pharmacol. Sci.
15, 437 (1994).
2. Plasmid pYEX 4T-1 (Clontech, Palo Alto, CA) was
modified by the addition of a 140-nucleotide recom-
bination domain, 39 of its Eco RI site, linearized within
the recombination domain by restriction digestion,
and cotransformed with a genomic set of reamplified
ORFs that had the same ends as the linearized plas-
mid [ J. R. Hudson Jr. et al., Genome Res. 7, 1169
(1997)] into strain EJ 758 [MATa his3-D200, leu2-
3,112, ura3-52, pep4::URA3], a derivative of JHRY-
20-2Ca (5). Transformants obtained on synthetic
minimal (SD) 2 Ura drop-out plates [F. Sherman,
Methods Enzymol. 194, 3 (1991)] (.100 in all cases,
and more than five times the cut vector in 97% of the
cases) were eluted in batch and saved in 96-well
microtiter plates. The library contains 6080 ORF-
containing strains and 64 strains with vector only.
3. Cell patches were inoculated in SD 2 Ura liquid
medium, grown overnight, reinoculated, and grown
overnight in SD 2 Ura 2 Leu medium, and then
inoculated into 250 ml of SD 2 Ura 2 Leu medium,
grown to absorbance at 600 nm of 0.8, and induced
with 0.5 mM copper sulfate for 2 hours before har-
vest [I. G. Macreadie, O. Horaitis, A. J. Verkuylen,
K. W. Savin, Gene 104, 107 (1991)]. Cells were re-
suspended in 1 ml of buffer [50 mM tris-HCl (pH 7.5),
1 mM EDTA, 4 mM MgCl2, 5 mM dithiothreitol (DT T),
10% glycerol, and 1 M NaCl] containing leupeptin (2
mg/ml) and pepstatin (1 mg/ml), and extracts were
made with glass beads [S. M. McCraith and E. M.
Phizicky, Mol. Cell. Biol. 10, 1049 (1990)], followed
by supplementation with 1 mM phenylmethylsulfo-
nyl fluoride and centrifugation. GST-ORF fusion pro-
teins were purified by glutathione agarose chroma-
tography in buffer containing 0.5 M NaCl, essentially
as described [ J. R
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