bluuebunny/crossref_metadata_2025 huggingface dataset:
The pattern of gene expression in human CD15+myeloid progenitor cells
Authors: Lee Sanggyu , Zhou Guolin , Clark Terry , Chen Jianjun , Rowley Janet D. , Wang San Ming
Published: July 2002

We performed a genome-wide analysis of gene expression in primary human CD15+myeloid progenitor cells. By using the serial analysis of gene expression (SAGE) technique, we obtained quantitative information for the expression of 37,519 unique SAGE-tag sequences. Of these unique tags, (i) 25% were detected at high and intermediate levels, whereas 75% were present as single copies, (ii) 53% of the tags matched known expressed sequences, 34% of which were matched to more than one known expressed sequence, and (iii) 47% of the tags had no matches and represent potentially novel genes. The correct genes were confirmed by application of the generation of longer cDNA fragments from SAGE tags for gene identification (GLGI) technique for high-copy tags with multiple matches. A set of genes known to be important in myeloid differentiation were expressed at various levels and used different spliced forms. This study provides a normal baseline for comparison of gene expression in myeloid diseases. The strategy of using SAGE and GLGI techniques in this study has broad applications to the genome-wide identification of expressed genes.
Assembly of the neutrophil respiratory burst oxidase: A direct interaction between p67
PHOX
and cytochrome
b
558
Authors: Dang Pham My-Chan , Cross Andrew R. , Babior Bernard M.
Published: July 2002

Activation of the phagocyte NADPH oxidase complex requires the
assembly of the cytosolic factors p47
PHOX
,
p67
PHOX
, p40
PHOX
, and Rac1 or Rac2, with the
membrane-bound cytochrome
b
558
. Whereas the
interaction of p47
PHOX
with cytochrome
b
558
is well established, an interaction
between p67
PHOX
and cytochrome
b
558
has never been investigated. We report
here a direct interaction between p67
PHOX
and cytochrome
b
558
. First, labeled p67
PHOX
recognizes a 91-kDa band in specific granules from a normal patient but
not from a cytochrome
b
558
-deficient
patient. Second, p67
PHOX
binds to cytochrome
b
558
that has been bound to nitrocellulose.
Third, GTP-p67
PHOX
bound to glutathione agarose is able to
pull down cytochrome
b
558.
Rac1-GTP or
Rac1-GDP increased the binding of p67
PHOX
to cytochrome
b
558
, suggesting that at least one of the
oxidase-related functions of Rac1 is to promote the interaction between
p67
PHOX
and cytochrome
b
558
.
Flexibility of the Kir6.2 inward rectifier K
+
channel pore
Authors: Loussouarn Gildas , Phillips L. Revell , Masia Ricard , Rose Thierry , Nichols Colin G.
Published: July 2002

Interactions of sulfhydryl reagents with introduced cysteines in
the pore-forming (Kir6.2) subunits of the K
ATP
channel were
examined. 2-Aminoethyl methanethiosulfonate (MTSEA
+
) failed
to modify Cd
2+
-insensitive control-Kir6.2 channels, but
rapidly and irreversibly modified Kir6.2[L164C] (L164C) channels.
Although a single Cd
2+
ion is coordinated by L164C, four
MTSEA
+
“hits” can occur, each sequentially reducing
the single-channel current. A dimeric fusion of control-Kir6.2 and
L164C subunits generates Cd
2+
-insensitive channels,
confirming that at least three cysteines are required for coordination,
but MTSEA
+
modification of the dimer occurs in two hits.
L164C channels were not modified by bromotrimethyl ammoniumbimane
(qBBr
+
), even though qBBr
+
caused
voltage-dependent block (as opposed to modification) that was
comparable to that of MTSEA
+
or 3-(triethylammonium)propyl
methanethiosulfonate (MTSPTrEA
+
), implying that
qBBr
+
can also enter the inner cavity but does not modify
L164C residues. The Kir channel pore structure was modeled by homology
with the KcsA crystal structure. A stable conformation optimally places
the four L164C side chains for coordination of a single
Cd
2+
ion. Modification of these cysteines by up to four
MTSEA
+
(or three MTSPTrEA
+
, or two
qBBr
+
) does not require widening of the cavity to
accommodate the derivatives within it. However, like the KcsA crystal
structure, the energy-minimized model shows a narrowing at the inner
entrance, and in the Kir6.2 model this narrowing excludes all ions. To
allow entry of ions as large as MTSPTrEA
+
or
qBBr
+
, the entrance must widen to >8 Å, but this widening
is readily accomplished by minimal M2 helix motion and side-chain
rearrangement.
Small-molecule antagonists of Myc/Max dimerization inhibit Myc-induced transformation of chicken embryo fibroblasts
Authors: Berg Thorsten , Cohen Steven B. , Desharnais Joel , Sonderegger Corinna , Maslyar Daniel J. , Goldberg Joel , Boger Dale L. , Vogt Peter K.
Published: July 2002

Myc is a transcriptional regulator of the basic helix–loop–helix leucine zipper protein family. It has strong oncogenic potential, mutated or virally transduced forms of Myc induce lymphoid tumors in animals, and deregulated expression of Myc is associated with numerous types of human cancers. For its oncogenic activity, Myc must dimerize with the ubiquitously expressed basic helix–loop–helix leucine zipper protein Max. This requirement for dimerization may allow control of Myc activity with small molecules that interfere with Myc/Max dimerization. We have measured Myc/Max dimerization with fluorescence resonance energy transfer and have screened combinatorial chemical libraries for inhibitors of dimerization. Candidate inhibitors were isolated from a peptidomimetics library. Inhibition of Myc/Max interaction was validated by ELISA and electrophoretic mobility-shift assay. Two of the candidate inhibitors also interfere with Myc-induced oncogenic transformation in chicken embryo fibroblast cultures. Our work provides proof of principle for the identification of small molecule inhibitors of protein–protein interactions by using high-throughput screens of combinatorial chemical libraries.
Novel kingdom-level eukaryotic diversity in anoxic environments
Authors: Dawson Scott C. , Pace Norman R.
Published: July 2002

Molecular evolutionary studies of eukaryotes have relied on a sparse collection of gene sequences that do not represent the full range of eukaryotic diversity in nature. Anaerobic microbes, particularly, have had little representation in phylogenetic studies. Such organisms are the least known of eukaryotes and probably are the most phylogenetically diverse. To provide fresh perspective on the natural diversity of eukaryotes in anoxic environments and also to discover novel sequences for evolutionary studies, we conducted a cultivation-independent, molecular phylogenetic survey of three anoxic sediments, including both freshwater and marine samples. Many previously unrecognized eukaryotes were identified, including representatives of seven lineages that are not specifically related to any known organisms at the kingdom-level and branch below the eukaryotic “crown” radiation of animals, plants, fungi, stramenopiles, etc. The survey additionally identified new sequences characteristic of known ecologically important eukaryotic groups with anaerobic members. Phylogenetic analyses with the new sequences enhance our understanding of the diversity and pattern of eukaryotic evolution.
Cys-328 of IscS and Cys-63 of IscU are the sites of disulfide bridge formation in a covalently bound IscS/IscU complex: Implications for the mechanism of iron-sulfur cluster assembly
Authors: Kato Shin-ichiro , Mihara Hisaaki , Kurihara Tatsuo , Takahashi Yasuhiro , Tokumoto Umechiyo , Yoshimura Tohru , Esaki Nobuyoshi
Published: July 2002

IscS and IscU from
Escherichia coli
cooperate with each other in the biosynthesis of iron-sulfur clusters. IscS catalyzes the desulfurization of
l
-cysteine to produce
l
-alanine and sulfur. Cys-328 of IscS attacks the sulfur atom of
l
-cysteine, and the sulfane sulfur derived from
l
-cysteine binds to the Sγ atom of Cys-328. In the course of the cluster assembly, IscS and IscU form a covalent complex, and a sulfur atom derived from
l
-cysteine is transferred from IscS to IscU. The covalent complex is thought to be essential for the cluster biogenesis, but neither the nature of the bond connecting IscS and IscU nor the residues involved in the complex formation have been determined, which have thus far precluded the mechanistic analyses of the cluster assembly. We here report that a covalent bond is formed between Cys-328 of IscS and Cys-63 of IscU. The bond is a disulfide bond, not a polysulfide bond containing sulfane sulfur between the two cysteine residues. We also found that Cys-63 of IscU is essential for the IscU-mediated activation of IscS: IscU induced a six-fold increase in the cysteine desulfurase activity of IscS, whereas the IscU mutant with a serine substitution for Cys-63 had no effect on the activity. Based on these findings, we propose a mechanism for an early stage of iron-sulfur cluster assembly: the sulfur transfer from IscS to IscU is initiated by the attack of Cys-63 of IscU on the Sγ atom of Cys-328 of IscS that is bound to sulfane sulfur derived from
l
-cysteine.
Targeted inactivation of
CTNNB1
reveals unexpected effects of β-catenin mutation
Authors: Chan Timothy A. , Wang Zhenghe , Dang Long H. , Vogelstein Bert , Kinzler Kenneth W.
Published: July 2002

Inactivating mutations of the adenomatous polyposis coli gene (
APC
) or activating mutations of the β-catenin gene (
CTNNB1
) initiate colorectal neoplasia. To address the biochemical and physiologic effects of mutant β-catenin, we disrupted either the mutant or wild-type
CTNNB1
allele in a human colorectal cancer cell line. Cells with only wild-type β-catenin had decreased colony-forming ability when plated at low density, although their growth was similar to that of parental cells when passaged under routine conditions. Immunohistochemistry and cell-fractionation studies suggested that mutant β-catenin activity was distinguished primarily by cellular localization and not by protein degradation. Surprisingly, we found mutant β-catenin bound less well to E-cadherin than did wild-type β-catenin, and the membranous localization of wild-type and mutant β-catenin was accordingly distinct. These findings pose several challenges to current models of APC/β-catenin function.
Conversion of a maltose receptor into a zinc biosensor by computational design
Authors: Marvin Jonathan S. , Hellinga Homme W.
Published: July 2002

We have demonstrated that it is possible to radically change the
specificity of maltose binding protein by converting it into a zinc
sensor using a rational design approach. In this new molecular sensor,
zinc binding is transduced into a readily detected fluorescence signal
by use of an engineered conformational coupling mechanism linking
ligand binding to reporter group response. An iterative progressive
design strategy led to the construction of variants with increased zinc
affinity by combining binding sites, optimizing the primary
coordination sphere, and exploiting conformational equilibria.
Intermediates in the design series show that the adaptive process
involves both introduction and optimization of new functions and
removal of adverse vestigial interactions. The latter demonstrates the
importance of the rational design approach in uncovering cryptic
phenomena in protein function, which cannot be revealed by the study of
naturally evolved systems.
Intron evolution as a population-genetic process
Authors: Lynch Michael
Published: July 2002

Debate over the mechanisms responsible for the phylogenetic and genomic distribution of introns has proceeded largely without consideration of the population-genetic forces influencing the establishment and retention of novel genetic elements. However, a simple model incorporating random genetic drift and weak mutation pressure against intron-containing alleles yields predictions consistent with a diversity of observations: (
i
) the rarity of introns in unicellular organisms with large population sizes, and their expansion after the origin of multicellular organisms with reduced population sizes; (
ii
) the relationship between intron abundance and the stringency of splice-site requirements; (
iii
) the tendency for introns to be more numerous and longer in regions of low recombination; and (
iv
) the bias toward phase-0 introns. This study provides a second example of a mechanism whereby genomic complexity originates passively as a “pathological” response to small population size, and raises difficulties for the idea that ancient introns played a major role in the origin of genes by exon shuffling.
Identification of a PDZ-domain-containing protein that interacts with the scavenger receptor class B type I
Authors: Ikemoto Mamoru , Arai Hiroyuki , Feng Dongdong , Tanaka Kazumi , Aoki Junken , Dohmae Naoshi , Takio Koji , Adachi Hideki , Tsujimoto Masafumi , Inoue Keizo
Published: July 2002

The scavenger receptor class B type I (SR-BI) mediates the selective uptake of cholesteryl esters from high-density lipoprotein (HDL) and cholesterol secretion into bile in the liver. In this study, we identified an SR-BI-associated protein from rat liver membrane extracts by using an affinity chromatography technique. This protein of 523 amino acids contains four PDZ domains and associates with the C terminus of SR-BI by using its N-terminal first PDZ domain. Therefore, we denoted this protein as CLAMP (
C
-terminal
l
inking
a
nd
m
odulating
p
rotein). CLAMP was located mostly in the sinusoidal membranes, whereas SR-BI was detected in both sinusoidal and canalicular membranes. After the solubilization of the liver membranes with Triton X-100, SR-BI was immunoprecipitated with anti-CLAMP monoclonal antibody, suggesting the association of these proteins
in vivo
. By coexpressing SR-BI with CLAMP in Chinese hamster ovary cells, we observed (
i
) the increase in the expression level of SR-BI, (
ii
) the reduction in the deacylation rate of the cholesteryl esters taken up from HDL, and (
iii
) the change in the intracellular distribution of fluorescent lipid 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine percholate taken up from HDL. Taken together, these data suggest that CLAMP, a four-PDZ-domain-containing protein, is associated with SR-BI in the liver sinusoidal plasma membranes and may modulate the intracellular transport and metabolism of cholesteryl esters taken up from HDL.
A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution
Authors: Ostedgaard Lynda S. , Baldursson Olafur , Vermeer Daniel W. , Welsh Michael J. , Robertson Andrew D.
Published: July 2002

Phosphorylation of the regulatory (R) domain initiates cystic fibrosis transmembrane conductance regulator (CFTR) Cl
−
channel activity. To discover how the function of this domain is determined by its structure, we produced an R domain protein (R8) that spanned residues 708–831 of CFTR. Phosphorylated, but not unphosphorylated, R8 stimulated activity of CFTR channels lacking this domain, indicating that R8 is functional. Unexpectedly, this functional R8 was predominantly random coil, as revealed by CD and limited proteolysis. The CD spectra of both phosphorylated and nonphosphorylated R8 were similar in aqueous buffer. The folding agent trimethylamine
N
-oxide induced only a small increase in the helical content of nonphosphorylated R8 and even less change in the helical content of phosphorylated R8. These data, indicating that the R domain is predominantly random coil, may explain the seemingly complex way in which phosphorylation regulates CFTR channel activity.
Involvement of a human gene related to the
Drosophila spen
gene in the recurrent t(1;22) translocation of acute megakaryocytic leukemia
Authors: Mercher Thomas , Coniat Maryvonne Busson-Le , Monni Richard , Mauchauffé Martine , Khac Florence Nguyen , Gressin Lætitia , Mugneret Francine , Leblanc Thierry , Dastugue Nicole , Berger Roland , Bernard Olivier A.
Published: July 2002

The recurrent t(1;22)(p13;q13) translocation is exclusively
associated with infant acute megakaryoblastic leukemia. We have
identified the two genes involved in this translocation. Both genes
possess related sequences in the Drosophila genome. The chromosome 22
gene (
m
egakaryocytic
a
cute
l
eukemia,
MAL
) product is predicted to be
involved in chromatin organization, and the chromosome 1 gene
(
o
ne
t
wenty-
t
wo,
OTT
) product is related to the Drosophila split-end
(spen) family of proteins. Drosophila genetic experiments identified
spen as involved in connecting the Raf and Hox pathways. Because almost
all of the sequences and all of the identified domains of both OTT and
MAL proteins are included in the predicted fusion protein, the OTT-MAL
fusion could aberrantly modulate chromatin organization, Hox
differentiation pathways, or extracellular signaling.
Quantitative prediction of NF-κB DNA– protein interactions
Authors: Udalova Irina A. , Mott Richard , Field Dawn , Kwiatkowski Dominic
Published: July 2002

We describe a general method based on principal coordinates analysis to predict the effects of single-nucleotide polymorphisms within regulatory sequences on DNA–protein interactions. We use binding data for the transcription factor NF-κB as a test system. The method incorporates the effects of interactions between base pair positions in the binding site, and we demonstrate that such interactions are present for NF-κB. Prediction accuracy is higher than with profile models, confirmed by crossvalidation and by the experimental verification of our predictions for additional sequences. The binding affinities of all potential NF-κB sites on human chromosome 22, together with the effects of known single-nucleotide polymorphisms, are calculated to determine likely functional variants. We propose that this approach may be valuable, either on its own or in combination with other methods, when standard profile models are disadvantaged by complex internucleotide interactions.
Kinesin molecular motors: Transport pathways, receptors, and human disease
Authors: Goldstein Lawrence S. B.
Published: July 2002

Kinesin molecular motor proteins are responsible for many of the major microtubule-dependent transport pathways in neuronal and non-neuronal cells. Elucidating the transport pathways mediated by kinesins, the identity of the cargoes moved, and the nature of the proteins that link kinesin motors to cargoes are areas of intense investigation. Kinesin-II recently was found to be required for transport in motile and nonmotile cilia and flagella where it is essential for proper left-right determination in mammalian development, sensory function in ciliated neurons, and opsin transport and viability in photoreceptors. Thus, these pathways and proteins may be prominent contributors to several human diseases including ciliary dyskinesias, situs inversus, and retinitis pigmentosa. Kinesin-I is needed to move many different types of cargoes in neuronal axons. Two candidates for receptor proteins that attach kinesin-I to vesicular cargoes were recently found. One candidate,
sunday driver
, is proposed to both link kinesin-I to an unknown vesicular cargo and to bind and organize the mitogen-activated protein kinase components of a c-Jun N-terminal kinase signaling module. A second candidate, amyloid precursor protein, is proposed to link kinesin-I to a different, also unknown, class of axonal vesicles. The finding of a possible functional interaction between kinesin-I and amyloid precursor protein may implicate kinesin-I based transport in the development of Alzheimer's disease.
UL40-mediated NK evasion during productive infection with human cytomegalovirus
Authors: Wang Eddie C. Y. , McSharry Brian , Retiere Christelle , Tomasec Peter , Williams Sheila , Borysiewicz Leszek K. , Braud Veronique M. , Wilkinson Gavin W. G.
Published: July 2002

Human cytomegalovirus (HCMV) exploits a range of strategies to evade and modulate the immune response. Its capacity to down-regulate MHC I expression was anticipated to render infected cells vulnerable to natural killer (NK) attack. Kinetic analysis revealed that during productive infection, HCMV strain AD169 first enhanced and then inhibited lysis of primary skin fibroblasts by a CD94/NKG2A+NKG2D+ILT2+NK line. The inhibition of cytotoxicity against strain AD169-infected fibroblasts was abolished by prior treatment of targets or effectors with anti-MHC I and anti-CD94 monoclonal antibodies, respectively, implying a CD94/HLA-E-dependent mechanism. An HCMV strain AD169, UL40 deletion mutant could not inhibit CD94/NKG2A+NK killing against skin fibroblasts. The contribution of UL40 to evasion of primary NK cells then was tested in a system where targets and effectors were MHC-matched. Primary NK cells activated with IFNα as well as cultured primary NK cell lines showed increased killing against ΔUL40-infected fibroblasts compared with AD169-infected targets. This effect was abrogated by depletion of CD94+cells. These findings demonstrate that HCMV encodes a mechanism of evasion specifically targeted against a proportion of CD94+NK cells and show that this system functions during a productive infection.
Probabilistic clustering of sequences: Inferring new bacterial regulons by comparative genomics
Authors: van Nimwegen Erik , Zavolan Mihaela , Rajewsky Nikolaus , Siggia Eric D.
Published: July 2002

Genome-wide comparisons between enteric bacteria yield large sets of conserved putative regulatory sites on a gene-by-gene basis that need to be clustered into regulons. Using the assumption that regulatory sites can be represented as samples from weight matrices (WMs), we derive a unique probability distribution for assignments of sites into clusters. Our algorithm, “PROCSE” (probabilistic clustering of sequences), uses Monte Carlo sampling of this distribution to partition and align thousands of short DNA sequences into clusters. The algorithm internally determines the number of clusters from the data and assigns significance to the resulting clusters. We place theoretical limits on the ability of any algorithm to correctly cluster sequences drawn from WMs when these WMs are unknown. Our analysis suggests that the set of all putative sites for a single genome (e.g.,
Escherichia coli
) is largely inadequate for clustering. When sites from different genomes are combined and all the homologous sites from the various species are used as a block, clustering becomes feasible. We predict 50–100 new regulons as well as many new members of existing regulons, potentially doubling the number of known regulatory sites in
E. coli.
The target of rapamycin (TOR) proteins
Authors: Raught Brian , Gingras Anne-Claude , Sonenberg Nahum
Published: July 2002

Rapamycin potently inhibits downstream signaling from the target of rapamycin (TOR) proteins. These evolutionarily conserved protein kinases coordinate the balance between protein synthesis and protein degradation in response to nutrient quality and quantity. The TOR proteins regulate (i) the initiation and elongation phases of translation, (ii) ribosome biosynthesis, (iii) amino acid import, (iv) the transcription of numerous enzymes involved in multiple metabolic pathways, and (v) autophagy. Intriguingly, recent studies have also suggested that TOR signaling plays a critical role in brain development, learning, and memory formation.
Role of
Lkb1
, the causative gene of Peutz–Jegher's syndrome, in embryogenesis and polyposis
Authors: Jishage Kou-ichi , Nezu Jun-ichi , Kawase Yosuke , Iwata Takamitsu , Watanabe Miho , Miyoshi Akio , Ose Asuka , Habu Kiyoshi , Kake Takei , Kamada Nobuo , Ueda Otoya , Kinoshita Michiko , Jenne Dieter E. , Shimane Miyuki , Suzuki Hiroshi
Published: July 2002

Peutz–Jeghers syndrome (PJS) is a dominantly inherited human disorder characterized by gastrointestinal hamartomatous polyposis and mucocutaneous melanin pigmentation. LKB1 (STK11) serine/threonine kinase is the product of the causative gene of PJS, which has been mapped to chromosome 19p13.3. However, several studies have produced results that are not consistent with a link between
LKB1
gene mutation and PJS. We constructed a knockout gene mutation of
Lkb1
to determine whether it is the causative gene of PJS and to examine the biological role of the
Lkb1
gene.
Lkb1
−/−
mice died
in utero
between 8.5 and 9.5 days postcoitum. At 9.0 days postcoitum,
Lkb1
−/−
embryos were generally smaller than their age-matched littermates, showed developmental retardation, and did not undergo embryonic turning. Multiple gastric adenomatous polyps were observed in 10- to 14-month-old
Lkb1
+/−
mice. Our results indicate that functional
Lkb1
is required for normal embryogenesis and that it is related to tumor development. The
Lkb1
+/−
mouse is suitable for studying molecular mechanism underlying the development of inherited gastric tumors in PJS.
PapD-like chaperones provide the missing information for folding of pilin proteins
Authors: Barnhart Michelle M. , Pinkner Jerome S. , Soto Gabriel E. , Sauer Frederic G. , Langermann Sol , Waksman Gabriel , Frieden Carl , Hultgren Scott J.
Published: July 2002

A fundamental question in molecular biology is how proteins fold into domains that can serve as assembly modules for building up large macromolecular structures. The biogenesis of pili on the surface of Gram-negative bacteria requires the orchestration of a complex process that includes protein synthesis, folding via small chaperones, secretion, and assembly. The results presented here support the hypothesis that pilus subunit folding and biogenesis proceed via mechanisms termed donor strand complementation and donor strand exchange. Here we show that the steric information necessary for pilus subunit folding is not contained in one polypeptide sequence. Rather, the missing information is transiently donated by a strand of a small chaperone to allow folding. Providing the missing information for folding, via a 13-amino acid peptide extension to the C-terminal end of a pilus subunit, resulted in the production of a protein that no longer required the chaperone to fold. This mechanism of small periplasmic chaperone function described here deviates from classical hsp60 chaperone-assisted folding.
A gene family required for human germ cell development evolved from an ancient meiotic gene conserved in metazoans
Authors: Xu Eugene Yujun , Moore Frederick L. , Pera Renee A. Reijo
Published: July 2002

The
Deleted in AZoospermia
(
DAZ
) genes encode potential RNA-binding proteins that are expressed exclusively in prenatal and postnatal germ cells and are strong candidates for human fertility factors. Here we report the identification of an additional member of the
DAZ
gene family, which we have called
BOULE
. With the identification of this gene, it is clear that the human
DAZ
gene family contains at least three members:
DAZ,
a Y-chromosome gene cluster that arose 30–40 million years ago and whose deletion is linked to infertility in men;
DAZL,
the “father” of
DAZ,
a gene that maps to human chromosome 3 and has homologs required for both female and male germ cell development in other organisms; and
BOULE,
a gene that we propose is the “grandfather” of
DAZ
and maps to human chromosome 2. Human and mouse
BOULE
resemble the invertebrate meiotic regulator
Boule
, the proposed ortholog of
DAZ
, in sequence and expression pattern and hence likely perform a similar meiotic function. In contrast, the previously identified human
DAZ
and
DAZL
are expressed much earlier than
BOULE
in prenatal germ stem cells and spermatogonia;
DAZL
also is expressed in female germ cells. These data suggest that homologs of the
DAZ
gene family can be grouped into two subfamilies (
BOULE
and
DAZL
) and that members of the
DAZ
family evolved from an ancestral meiotic regulator,
Boule,
to assume distinct, yet overlapping, functions in germ cell development.
A cellular mechanism for targeting newly synthesized mRNAs to synaptic sites on dendrites
Authors: Steward Oswald , Worley Paul F.
Published: July 2002

Long-lasting forms of activity-dependent synaptic plasticity involve molecular modifications that require gene expression. Here, we describe a cellular mechanism that mediates the targeting newly synthesized gene transcripts to individual synapses where they are locally translated. The features of this mechanism have been revealed through studies of the intracellular transport and synaptic targeting of the mRNA for a recently identified immediate early gene called activity-regulated cytoskeleton-associated protein
Arc
.
Arc
is strongly induced by patterns of synaptic activity that also induce long-term potentiation, and
Arc
mRNA is then rapidly delivered into dendrites after episodes of neuronal activation. The newly synthesized
Arc
mRNA localizes selectively at synapses that recently have been activated, and the encoded protein is assembled into the synaptic junctional complex. The dynamics of trafficking of
Arc
mRNA reveal key features of the mechanism through which synaptic activity can both induce gene expression and target particular mRNA transcripts to the active synapses.
v-Crk activates the phosphoinositide 3-kinase/AKT pathway in transformation
Authors: Akagi Tsuyoshi , Shishido Tomoyuki , Murata Kazutaka , Hanafusa Hidesaburo
Published: July 2002

v-Crk induces cellular tyrosine phosphorylation and transformation of chicken embryo fibroblasts (CEF). We studied the molecular mechanism of the v-Crk-induced transformation. Experiments with Src homology (SH)2 and SH3 domain mutants revealed that the induction of tyrosine phosphorylation of cellular proteins requires only the SH2 domain, but both the SH2 and SH3 domains are required for complete transformation. Analysis of three well defined signaling pathways, the mitogen-activated protein kinase (MAPK) pathway, the Jun N-terminal kinase (JNK) pathway, and the phosphoinositide 3-kinase (PI3K)/AKT pathway, demonstrated that only the PI3K/AKT pathway is constitutively activated in v-Crk-transformed CEF. Both the SH2 and SH3 domains are required for this activation of the PI3K/AKT pathway in CEF. We also found that the colony formation of CEF is strongly induced by a constitutively active PI3K mutant, and that a PI3K inhibitor, LY294002, suppresses the v-Crk-induced transformation. These results strongly suggest that constitutive activation of the PI3K/AKT pathway plays an essential role in v-Crk-induced transformation of CEF.
Rescue of a
Wnt
mutation by an activated form of LEF-1: Regulation of maintenance but not initiation of
Brachyury
expression
Authors: Galceran Juan , Hsu Shu-Chi , Grosschedl Rudolf
Published: July 2002

Members of the LEF-1/TCF family of transcription factors have been implicated in mediating a nuclear response to Wnt signals by association with β-catenin. Consistent with this view, mice carrying mutations in either the
Wnt3a
gene or in both transcription factor genes
Lef1
and
Tcf1
were previously found to show a similar defect in the formation of paraxial mesoderm in the gastrulating mouse embryo. In addition, mutations in the
Brachyury
gene, a direct transcriptional target of LEF-1, were shown to result in mesodermal defects. However, direct evidence for the role of LEF-1 and Brachyury in Wnt3a signaling has been limiting. In this study, we genetically examine the function of LEF-1 in the regulation of
Brachyury
expression and in signaling by Wnt3a. Analysis of the expression of
Brachyury
in
Lef1
−/−
Tcf1
−/−
mice and studies of
Brachyury
:
lacZ
transgenes containing wild type or mutated LEF-1 binding sites indicate that
Lef1
is dispensable for the initiation, but is required for the maintenance of
Brachyury
expression. We also show that the expression of an activated form of LEF-1, containing the β-catenin activation domain fused to the amino terminus of LEF-1, can rescue a
Wnt3a
mutation. Together, these data provide genetic evidence that
Lef1
mediates the Wnt3a signal and regulates the stable maintenance of
Brachyury
expression during gastrulation.
A second, substrate-dependent site of protein import into chloroplasts
Authors: Reinbothe Steffen , Mache Regis , Reinbothe Christiane
Published: July 2002

Chloroplasts must import a large number of proteins from the cytosol. It generally is assumed that this import proceeds for all stromal and thylakoid proteins in an identical manner and is caused by the operation of two distinctive protein import machineries in the outer and inner plastid envelope, which form the general import site. Here we show that there is a second site of protein translocation into chloroplasts of barley, tobacco,
Arabidopsis thaliana
, and five other tested monocotyledonous and dicotyledonous plant species. This import site is specific for the cytosolic precursor of the NADPH:protochlorophyllide (Pchlide) oxidoreductase A, pPORA. It couples Pchlide synthesis to pPORA import and thereby reduces the actual level of free Pchlide, which, because of its photodynamic properties, would be destructive to the plastids. Consequently, photoprotection is conferred onto the plant.
Eavesdropping and animal conflict
Authors: Johnstone Rufus A.
Published: July 2002

Fights between pairs of animals frequently take place within a wider social context. The displays exchanged during conflict, and the outcome of an encounter, are often detectable by individuals who are not immediately involved. In at least some species, such bystanders are known to eavesdrop on contests between others, and to modify their behavior toward the contestants in response to the observed interaction. Here, I extend Maynard Smith's well known model of animal aggression, the Hawk–Dove game, to incorporate the possibility of eavesdroppers. I show that some eavesdropping is favored whenever the cost of losing an escalated fight exceeds the value of the contested resource, and that its equilibrium frequency is greatest when costs are relatively high. Eavesdropping reduces the risk of escalated conflict relative to that expected by chance, given the level of aggression in the population. However, it also promotes increased aggression, because it enhances the value of victory. The net result is that escalated conflicts are predicted to occur more frequently when eavesdropping is possible.
Novel folded protein domains generated by combinatorial shuffling of polypeptide segments
Authors: Riechmann Lutz , Winter Greg
Published: July 2002

It has been proposed that the architecture of protein domains has evolved by the combinatorial assembly and/or exchange of smaller polypeptide segments. To investigate this proposal, we fused DNA encoding the N-terminal half of a β-barrel domain (from cold shock protein CspA) with fragmented genomic
Escherichia coli
DNA and cloned the repertoire of chimeric polypeptides for display on filamentous bacteriophage. Phage displaying folded polypeptides were selected by proteolysis; in most cases the protease-resistant chimeric polypeptides comprised genomic segments in their natural reading frames. Although the genomic segments appeared to have no sequence homologies with CspA, one of the originating proteins had the same fold as CspA, but another had a different fold. Four of the chimeric proteins were expressed as soluble polypeptides; they formed monomers and exhibited cooperative unfolding. Indeed, one of the chimeric proteins contained a set of very slowly exchanging amides and proved more stable than CspA itself. These results indicate that native-like proteins can be generated directly by combinatorial segment assembly from nonhomologous proteins, with implications for theories of the evolution of new protein folds, as well as providing a means of creating novel domains and architectures
in vitro
.
Gene expression profiling of clear cell renal cell carcinoma: Gene identification and prognostic classification
Authors: Takahashi Masayuki , Rhodes Daniel R. , Furge Kyle A. , Kanayama Hiro-omi , Kagawa Susumu , Haab Brian B. , Teh Bin Tean
Published: July 2002

To better understand the molecular mechanisms that underlie the
tumorigenesis and progression of clear cell renal cell carcinoma
(ccRCC), we studied the gene expression profiles of 29 ccRCC tumors
obtained from patients with diverse clinical outcomes by using 21,632
cDNA microarrays. We identified gene expression alterations that were
both common to most of the ccRCC studied and unique to clinical
subsets. There was a significant distinction in gene expression profile
between patients with a relatively nonaggressive form of the disease
[100% survival after 5 years with the majority (15/17 or 88%)
having no clinical evidence of metastasis] versus patients with a
relatively aggressive form of the disease (average survival time 25.4
months with a 0% 5-year survival rate). Approximately 40 genes most
accurately make this distinction, some of which have previously been
implicated in tumorigenesis and metastasis. To test the robustness and
potential clinical usefulness of this molecular distinction, we
simulated its use as a prognostic tool in the clinical setting. In 96%
of the ccRCC cases tested, the prediction was compatible with the
clinical outcome, exceeding the accuracy of prediction by staging.
These results suggest that two molecularly distinct forms of ccRCC
exist and that the integration of expression profile data with clinical
parameters could serve to enhance the diagnosis and prognosis of ccRCC.
Moreover, the identified genes provide insight into the molecular
mechanisms of aggressive ccRCC and suggest intervention strategies.
α-Synuclein occurs in lipid-rich high molecular weight complexes, binds fatty acids, and shows homology to the fatty acid-binding proteins
Authors: Sharon Ronit , Goldberg Matthew S. , Bar-Josef Ifat , Betensky Rebecca A. , Shen Jie , Selkoe Dennis J.
Published: July 2002

α-Synuclein (αS) is a 140-residue neuronal protein that forms insoluble cytoplasmic aggregates in Parkinson's disease (PD) and several other neurodegenerative disorders. Two missense mutations (A53T and A30P) are linked to rare forms of familial PD. The normal function of αS is unknown, and cultured cell systems that model its modification from soluble monomers to aggregated forms have not been reported. Through a systematic centrifugal fractionation of mesencephalic neuronal cell lines and transgenic mouse brains expressing wild-type or A53T human αS, we observed unusual, previously unrecognized species of αS that migrate well above the 17-kDa monomeric form in denaturing gels. Incubation at 65°C of high-speed cytosols from cells or brains revealed a modified αS species migrating at ≈36 kDa and an extensive higher molecular mass αS-reactive smear. Extraction of the cytosols with chloroform/methanol or with a resin (Lipidex 1000) that binds fatty acids resulted in a similar pattern of higher molecular mass αS forms. On the basis of this effect of delipidation, we reexamined the primary structure of αS and detected a motif at the N and C termini that is homologous to a fatty acid-binding protein signature. In accord, we found that purified human αS binds oleic acid, with an apparent
K
d
of 12.5 μM. We also observed an enhanced association of A53T αS with microsomal membranes in both mesencephalic cells and transgenic mouse brains. We conclude that αS has biochemical properties and a structural motif that suggest it is a novel member of the fatty acid-binding protein family and may thus transport fatty acids between the aqueous and membrane phospholipid compartments of the neuronal cytoplasm.
Activation of β-major globin gene transcription is associated with recruitment of NF-E2 to the β-globin LCR and gene promoter
Authors: Sawado Tomoyuki , Igarashi Kazuhiko , Groudine Mark
Published: July 2002

The mouse β-globin gene locus control region (LCR), located upstream of the β-globin gene cluster, is essential for the activated transcription of genes in the cluster. The LCR contains multiple binding sites for transactivators, including Maf-recognition elements (MAREs). However, little is known about the specific proteins that bind to these sites or the time at which they bind during erythroid differentiation. We have performed chromatin immunoprecipitation experiments to determine the recruitment of the erythroid-specific transactivator p45 NF-E2/MafK (p18 NF-E2) heterodimer and small Maf proteins to various regions in the globin gene locus before and after the induction of murine erythroleukemia (MEL) cell differentiation. We report that, before induction, the LCR is occupied by small Maf proteins, and, on erythroid maturation, the NF-E2 complex is recruited to the LCR and the active globin promoters, even though the promoters do not contain MAREs. This differentiation-coupled recruitment of NF-E2 complex correlates with a greater than 100-fold increase in β-major globin transcription, but is not associated with a significant change in locus-wide histone H3 acetylation. These findings suggest that the β-globin gene locus exists in a constitutively open chromatin conformation before terminal differentiation, and we speculate that recruitment of NF-E2 complex to the LCR and active promoters may be a rate-limiting step in the activation of β-globin gene expression.
Discrepancy between ELISPOT IFN-γ secretion and binding of A2/peptide multimers to TCR reveals interclonal dissociation of CTL effector function from TCR-peptide/MHC complexes half-life
Authors: Rubio-Godoy Verena , Dutoit Valerie , Rimoldi Donata , Lienard Danielle , Lejeune Ferdy , Speiser Daniel , Guillaume Philippe , Cerottini Jean-Charles , Romero Pedro , Valmori Danila
Published: July 2002

Activation of CD8+cytolytic T lymphocytes (CTLs) by antigen is triggered by the interaction of clonotypic αβ T cell receptors (TCRs) with antigenic peptides bound to MHC class I molecules (pMHC complexes). Fluorescent multimeric pMHC complexes have been shown to specifically stain antigen-specific CTLs by directly binding the TCR. In tumor-infiltrating lymphocytes from a melanoma patient we found a high frequency of tyrosinase368–376peptide-specific cells as detected by IFN-γ ELISPOT, without detectable staining with the corresponding A2/peptide multimers. Surprisingly, these T cells were able to lyse tyrosinase368–376peptide-pulsed target cells as efficiently as other specific T cells that were stained by multimers. Analysis of the staining patterns under different conditions of incubation time and temperature revealed that these results were explained by major differences in TCR-multimeric ligand interaction kinetics among the clones. Whereas no direct quantitative correlation between antigenic peptide concentration required for CTL effector functions and equilibrium multimer binding was observed interclonally, the latter was profoundly affected by the kinetics of TCR-ligand interaction. More importantly, our data indicate that similar levels of T cell activation can be achieved by independent CD8+T cell clonotypes displaying different TCR/pMHC complex dissociation rates.
Correspondence analysis applied to microarray data
Authors: Fellenberg Kurt , Hauser Nicole C. , Brors Benedikt , Neutzner Albert , Hoheisel Jörg D. , Vingron Martin
Published: July 2002

Correspondence analysis is an explorative computational method for the study of associations between variables. Much like principal component analysis, it displays a low-dimensional projection of the data, e.g., into a plane. It does this, though, for two variables simultaneously, thus revealing associations between them. Here, we demonstrate the applicability of correspondence analysis to and high value for the analysis of microarray data, displaying associations between genes and experiments. To introduce the method, we show its application to the well-known
Saccharomyces cerevisiae
cell-cycle synchronization data by Spellman
et al.
[Spellman, P. T., Sherlock, G., Zhang, M. Q., Iyer, V. R., Anders, K., Eisen, M. B., Brown, P. O., Botstein, D. & Futcher, B. (1998)
Mol. Biol. Cell
9, 3273–3297], allowing for comparison with their visualization of this data set. Furthermore, we apply correspondence analysis to a non-time-series data set of our own, thus supporting its general applicability to microarray data of different complexity, underlying structure, and experimental strategy (both two-channel fluorescence-tag and radioactive labeling).
Molecular synapomorphies resolve evolutionary relationships of extant jawed vertebrates
Authors: Venkatesh Byrappa , Erdmann Mark V. , Brenner Sydney
Published: July 2002

The evolutionary relationships of gnathostomes (jawed vertebrates), which comprise chondrichthyans (cartilaginous fishes), lobe-finned fishes (coelacanths and lungfishes), tetrapods, and actinopterygians (ray-finned fishes), have been debated for almost a century. Phylogenetic analyses based on fossils, morphology, and molecular sequences have generated different models of relationships that remain unresolved. We identified 13 derived shared molecular markers (synapomorphies) that define clades in the vertebrate lineage and used them to resolve the phylogenetic relationships of extant jawed vertebrates. Our markers include the presence or absence of insertions and deletions in coding sequences, nuclear introns, and alternatively spliced transcripts. The synapomorphies identified by us are congruent with each other and give rise to a single phylogenetic tree. This tree confirms that chondrichthyans are basal to all living gnathostomes, that lungfishes (Dipnoi) are the closest living relatives of tetrapods, and that bichirs (Cladistia) are the living members of the most ancient family of ray-finned fishes. Our study also provides molecular evidence to support the monophyly of living tetrapods and teleosts.
Decreased lipid synthesis in livers of mice with disrupted Site-1 protease gene
Authors: Yang Jian , Goldstein Joseph L. , Hammer Robert E. , Moon Young-Ah , Brown Michael S. , Horton Jay D.
Published: July 2002

Site-1 protease (S1P) cleaves membrane-bound sterol regulatory element-binding proteins (SREBPs), allowing their transcription-stimulating domains to translocate to the nucleus where they activate genes governing lipid synthesis. S1P is a potential target for lipid-lowering drugs, but the effect of S1P blockade in animals is unknown. Here, we disrupt the
S1P
gene in mice. Homozygous germ-line disruptions of
S1P
were embryonically lethal. To disrupt the gene inducibly in liver, we generated mice homozygous for a floxed
S1P
allele and heterozygous for a transgene encoding Cre recombinase under control of the IFN-inducible MX1 promoter. When IFN was produced, 70–90% of
S1P
alleles in liver were inactivated, and S1P mRNA and protein were reduced. Nuclear SREBPs declined, as did mRNAs for SREBP target genes. Cholesterol and fatty acid biosynthesis in hepatocytes declined by 75%. Low density lipoprotein (LDL) receptor mRNA declined by 50%, as did the clearance of
125
I-labeled LDL from plasma, but plasma cholesterol fell, suggesting that LDL production was reduced. These data raise the possibility that S1P inhibitors may be effective lipid-lowering agents, but they suggest that nearly complete inhibition will be required.
Chemical inhibition of the Pho85 cyclin-dependent kinase reveals a role in the environmental stress response
Authors: Carroll Adam S. , Bishop Anthony C. , DeRisi Joseph L. , Shokat Kevan M. , O'Shea Erin K.
Published: July 2002

In addition to its well-established role in responding to phosphate starvation, the cyclin-dependent kinase Pho85 has been implicated in a number of other physiological responses of the budding yeast
Saccharomyces cerevisiae
, including synthesis of glycogen. To comprehensively characterize the range of Pho85-dependent gene expression, we used a chemical genetic approach that enabled us to control Pho85 kinase activity with a cell-permeable inhibitor and whole genome transcript profiling. We found significant phenotypic differences between the rapid loss of activity caused by inhibition and the deletion of the genomic copy of
PHO85
. We demonstrate that Pho85 controls the expression of not only previously identified glycogen synthetic genes, but also a significant regulon of genes involved in the cellular response to environmental stress. In addition, we show that the effects of this inhibitor are both rapid and reversible, making it well suited to the study of the behavior of dynamic signaling pathways.