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Toward a protein–protein interaction map between the yeast prot...
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关于蛋白质的研究,对蛋白质的分类的认识和预测。
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Paper alert 169
In vitro cloning of complex mixtures of DNA on microbeads:
physical separation of differentially expressed cDNAs.
Brenner S, Williams SR, Vermaas EH, Storck T, Moon K,
McCollum C, Mao JI, Luo S, Kirchner JJ, Eletr S et a/.: froc
Nat/ Acad Sci USA 2000, 97:1665-l 670.
l
a Significance: It is possible to compare the level of tran-
scription of genes in two experimental conditions by
segregating cDNA clones on microbeads that can be sepa-
rated using a fluorescence activated cell sorter. The beads
that contain a desired gene can then be harvested for further
characterization. Because no prior information about the
cloned molecules is required, this process is useful for
organisms whose genome sequence databases are incom-
plete or nonexistent.
Findings: The authors describe a method for cloning nucleic
acid molecules onto the surfaces of microbeads. Using com-
binatorial chemistry, the authors have developed a set of 16
million 32-mer tags with similar hybridization characteristics.
A subset of these sequences are attached to approximately
160,000 mRNA molecules and each uniquely tagged cDNA
is amplified by PCR. The resulting library is hybridized to
microbeads that each carry approximately 10s strands com-
plementary to one of the tags. The vast excess of tags
compared to cDNAs ensures that each microbead will only
carry one gene. As about 10s copies of each molecule are
collected on each microbead, these are comparable to bio-
logically derived cDNA clones. To comparatively measure
transcript abundance in two tissues, microbeads bearing
clones that are expressed differently in each of the two
libraries were separated using a fluorescence-activated cell
sorter. The microbeads can be released for further character-
ization (e.g. DNA sequencing).
Toward a protein-protein interaction map of the budding
yeast: A comprehensive system to examine two-hybrid
interactions in all possible combinations between the yeast
proteins. Ito T, Tashiro K, Muta S, Ozawa R, Chiba T,
Nishizawa M, Yamamoto K, Kuhara S, Sakaki Y: froc Nat/ Acad
Sci USA 2000, 97:1143-l 147.
l
Significance: The systematic identification of protein-protein
interactions is an important step in our understanding of living
organisms. The approach described by these authors may pro-
vide many clues to understanding how various cellular functions
are integrated. For example, one of the protein-protein interac-
tions uncovered may help to explain a currently unsolved
mechanism for the connection between the distinct steps of
vesicular transport.
Findings: The authors describe a comprehensive system that
allowed them to examine two-hybrid interactions between all of
the possible combinations of proteins of Saccharomyces cere-
visiae. All of the yeast open reading frames were individually
cloned into both a ‘bait’ and a ‘prey’ expression cassette. The
bait constructs contain a DNA-binding domain and were
expressed in a MATa strain. The activation domain fusions
(‘prey’) were expressed in a MATa strain. The clones were
divided into pools, each containing 96 clones. These bait and
prey clone pools were systematically mated with each other,
and the transformants subjected to strict selection for the acti-
vation of three reporter genes. Examination of approximately
4 x 10s different combinations, constituting approximately 10%
of the total number of combinations to be tested, revealed 163
independent two-hybrid interactions, more than half of which
are entirely novel.
lntrachromosomal homologous recombination in
Arabidopsis induced by a maize transposon. Xiao Y-L,
Peterson T: Mol Gen Genet 2000, 263:22-29.
l
Significance: The spontaneous frequency of intrachromoso-
mal homologous recombination in plants is very low. The
incidence of recombination increases more than lOOO-fold if
homologous sequences are separated by a Ds transposon and
an AC transposase is present. This high level of recombination
may provide a useful means for homology-dependent gene
manipulations in plants.
Findings: The ability of a transposon to induce recombination
was directly tested using transgenic Arabidopsis plants cany-
ing a construct in which overlapping segments of the bacterial
uidA gene were separated by a Ds transposon. Homologous
recombination events were scored by staining for p-glu-
curonidase activity (encoded by uidA). As previously reported
for the maize P locus, the recombination frequency increased in
the presence of AC. Surprisingly, the recombination frequency
was much higher than previously reported for other double
stand break (DSB)-inducing agents such as chemicals or the
HO endonuclease from yeast. Whether recombination is stimu-
lated solely by DSB induction through Ds excision or also by
other properties of AC is currently unknown.
Integrated cytogenetic map of chromosome arm 4s of
A. thaliana: Structural organization of heterochromatic
knob and centromere region. Fransz PF, Armstrong S, de
Jong JH, Parnell LD, van Drunen C, Dean C, Zabel P,
Bisseling T, Jones GH: Cell 2000,100:367-376.
AND
The complete sequence of a heterochromatic island from a
higher eukaryote. McCombie WR, de la Bastide M,
Habermann K, Parnell L, Dedhia N, Gnoj L, Schutz K, Huang E,
Spiegel L, Yordan C et a/.: Cell 2000, 100:377-386.
l
0 Significance: The structural organization of euchromatin
and heterochromatin are important determinants of chrome-.
some function. So far, the genome of Arabidopsis is not well
characterized at the cytogenetic level. A cytogenetic map for
one chromosome arm has been established and condensa-
tion properties of euchromatin and heterochromatin were
studied. This analysis led to the discovery of a heterochro-
matic knob, whose sequence, functional and structural
properties were characterized.
Findings: An integrated cytogenetic and molecular map
was generated for chromosome arm 4s using fluorescent in
situ hybridization on meiotic chromosomes and chromatin
fibers. Spatial and temporal chromatin condensation pat-
terns during meiotic prophase were strikingly different
between euchromatin and heterochromatic segments.
Centromeric and pericentromeric regions were found to be
functionally and structurally different. In some ecotypes, a
heterochromatic knob was identified that probably resulted
from an inversion involving pericentromeric sequences. The
heterochromatic knob is characterized by low gene density,
low levels of recombination, and a low incidence of new.
transposon insertions. It consists of long tandem repeats
and a large number of transposons, both of which are
heavily methylated.
Structural domains and matrix attachment regions along
colinear chromosomal segments of maize and sorghum.
Tikhonov AP, Bennetzen JL, Avramova ZV: Plant Cell 2000,
12:24g-264.
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