bluuebunny/crossref_metadata_2025 huggingface dataset:
A functional role for
Tsix
transcription in blocking
Xist
RNA accumulation but not in X-chromosome choice
Authors: Stavropoulos Nicholas , Lu Naifung , Lee Jeannie T.
Published: July 2002

In female mammals, up-regulation of
Xist
triggers X-chromosome inactivation in cis. Up-regulation is inhibited by sequences 3′ to
Xist
contained within the antisense locus,
Tsix
. Inhibition could depend on transcription of
Tsix
and/or on DNA elements therein. Here we test the role of
Tsix
transcription by augmenting the duration and strength of
Tsix
expression. We find that
Tsix
hypertranscription is sufficient to block
Xist
RNA accumulation in a cis-limited manner. We propose that
Tsix
transcription is necessary to restrict
Xist
activity on the future active X and, conversely, that
Tsix
repression is required for
Xist
RNA accumulation on the future inactive X. We also find that
Tsix
hypertranscription does not affect X-chromosome choice. Thus, choice is mediated by elements within
Tsix
that are independent of promoter activity.
Compaction of single DNA molecules induced by binding of integration host factor (IHF)
Authors: Ali B. M. Jaffar , Amit Roee , Braslavsky Ido , Oppenheim Amos B. , Gileadi Opher , Stavans Joel
Published: July 2002

We studied the interaction between the integration host factor (IHF), a major nucleoid-associated protein in bacteria, and single DNA molecules. Force–extension measurements of λ DNA and an analysis of the Brownian motion of small beads tethered to a surface by single short DNA molecules, in equilibrium with an IHF solution, indicate that: (
i
) the DNA–IHF complex retains a random, although more compact, coiled configuration for zero or small values of the tension, (
ii
) IHF induces DNA compaction by binding to multiple DNA sites with low specificity, and (
iii
) with increasing tension on the DNA, the elastic properties of bare DNA are recovered. This behavior is consistent with the predictions of a statistical mechanical model describing how proteins bending DNA are driven off by an applied tension on the DNA molecule. Estimates of the amount of bound IHF in DNA–IHF complexes obtained from the model agree very well with independent measurements of this quantity obtained from the analysis of DNA–IHF crosslinking. Our findings support the long-held view that IHF and other histone-like proteins play an important role in shaping the long-scale structure of the bacterial nucleoid.
TOUCHSTONE: An
ab initio
protein structure prediction method that uses threading-based tertiary restraints
Authors: Kihara Daisuke , Lu Hui , Kolinski Andrzej , Skolnick Jeffrey
Published: July 2002

The successful prediction of protein structure from amino acid sequence requires two features: an efficient conformational search algorithm and an energy function with a global minimum in the native state. As a step toward addressing both issues, a threading-based method of secondary and tertiary restraint prediction has been developed and applied to
ab initio
folding. Such restraints are derived by extracting consensus contacts and local secondary structure from at least weakly scoring structures that, in some cases, can lack any global similarity to the sequence of interest. Furthermore, to generate representative protein structures, a reduced lattice-based protein model is used with replica exchange Monte Carlo to explore conformational space. We report results on the application of this methodology, termed TOUCHSTONE, to 65 proteins whose lengths range from 39 to 146 residues. For 47 (40) proteins, a cluster centroid whose rms deviation from native is below 6.5 (5) Å is found in one of the five lowest energy centroids. The number of correctly predicted proteins increases to 50 when atomic detail is added and a knowledge-based atomic potential is combined with clustered and nonclustered structures for candidate selection. The combination of the ratio of the relative number of contacts to the protein length and the number of clusters generated by the folding algorithm is a reliable indicator of the likelihood of successful fold prediction, thereby opening the way for genome-scale
ab initio
folding.
Self-organization of a propulsive actin network as an evolutionary process
Authors: Maly Ivan V. , Borisy Gary G.
Published: July 2002

The leading edge of motile cells is propelled by polymerization of actin filaments according to a dendritic nucleation/array treadmilling mechanism. However, little attention has been given to the origin and maintenance of the dendritic array. Here we develop and test a population–kinetics model that explains the organization of actin filaments in terms of the reproduction of dendritic units. The life cycle of an actin filament consists of dendritic nucleation on another filament (birth), elongation by addition of actin subunits and, finally, termination of filament growth by capping protein (death). The regularity of branch angle between daughter and mother filaments endows filaments with heredity of their orientation. Fluctuations of branch angle that become fixed in the actin network create errors of orientation (mutations) that may be inherited. In our model, birth and death rates depend on filament orientation, which then becomes a selectable trait. Differential reproduction and elimination of filaments, or natural selection, leads to the evolution of a filament pattern with a characteristic distribution of filament orientations. We develop a procedure based on the Radon transform for quantitatively analyzing actin networksin situand show that the experimental results are in agreement with the distribution of filament orientations predicted by our model. We conclude that the propulsive actin network can be understood as a self-organizing supramolecular ensemble shaped by the evolution of dendritic lineages through natural selection of their orientation.
Soft-tissue characters in higher primate phylogenetics
Authors: Gibbs Sally , Collard Mark , Wood Bernard
Published: July 2002

Recent research has cast doubt on the reliability of bones and
teeth for reconstructing phylogenetic relationships among higher
primate species and genera. Herein, we investigate whether this problem
is confined to hard tissues by examining the utility of higher primate
soft-tissue characters for reconstructing phylogenetic relationships at
low taxonomic levels. We use cladistic methods to analyze 197
soft-tissue characters for the extant hominoids and then compare the
resulting phylogenetic hypotheses with the group's consensus molecular
phylogeny, which is widely considered to be accurate. We show that the
soft-tissue characters yield robust phylogenetic hypotheses that are
compatible with the molecular phylogeny. Given the strength of the
evidence for molecular phylogeny, these results indicate that, unlike
craniodental hard-tissue characters, soft tissues are reliable for
reconstructing phylogenetic relationships among higher primate species
and genera. Thus, in higher primates at least, some types of
morphological data are more useful than others for phylogeny
reconstruction.
Alu
-mediated inactivation of the human CMP-
N
-acetylneuraminic acid hydroxylase gene
Authors: Hayakawa Toshiyuki , Satta Yoko , Gagneux Pascal , Varki Ajit , Takahata Naoyuki
Published: July 2002

Inactivation of the CMP-
N
-acetylneuraminic acid
hydroxylase gene has provided an example of human-specific genomic
mutation that results in a widespread biochemical difference between
human and nonhuman primates. We have found that, although a region
containing a 92-bp exon and an
Alu
Sq element in the
hydroxylase gene is intact in all nonhuman primates examined, the same
region in the human genome is replaced by an
Alu
Y
element that was disseminated at least one million years ago. We
propose a mechanistic model for this
Alu
-mediated
replacement event, which deleted the 92-bp exon and thus inactivated
the human hydroxylase gene. It is suggested that
Alu
elements have played potentially important roles in genotypic and
phenotypic evolution in the hominid lineage.
LEAFY COTYLEDON2encodes a B3 domain transcription factor that induces embryo development
Authors: Stone Sandra L. , Kwong Linda W. , Yee Kelly Matsudaira , Pelletier Julie , Lepiniec Loïc , Fischer Robert L. , Goldberg Robert B. , Harada John J.
Published: July 2002

TheArabidopsis LEAFY COTYLEDON2 (LEC2) gene is a central embryonic regulator that serves critical roles both early and late during embryo development.LEC2is required for the maintenance of suspensor morphology, specification of cotyledon identity, progression through the maturation phase, and suppression of premature germination. We cloned theLEC2gene on the basis of its chromosomal position and showed that the predicted polypeptide contains a B3 domain, a DNA-binding motif unique to plants that is characteristic of several transcription factors. We showed thatLEC2RNA accumulates primarily during seed development, consistent with our finding that LEC2 shares greatest similarity with the B3 domain transcription factors that act primarily in developing seeds, VIVIPAROUS1/ABA INSENSITIVE3 and FUSCA3. Ectopic, postembryonic expression ofLEC2in transgenic plants induces the formation of somatic embryos and other organ-like structures and often confers embryonic characteristics to seedlings. Together, these results suggest that LEC2 is a transcriptional regulator that establishes a cellular environment sufficient to initiate embryo development.
Manifold anomalies in gene expression in a vineyard isolate of
Saccharomyces cerevisiae
revealed by DNA microarray analysis
Authors: Cavalieri Duccio , Townsend Jeffrey P. , Hartl Daniel L.
Published: July 2002

Genome-wide transcriptional profiling has important applications in
evolutionary biology for assaying the extent of heterozygosity for
alleles showing quantitative variation in gene expression in natural
populations. We have used DNA microarray analysis to study the global
pattern of transcription in a homothallic strain of
Saccharomyces cerevisiae
isolated from wine grapes in a
Tuscan vineyard, along with the diploid progeny obtained after
sporulation. The parental strain shows 2:2 segregation (heterozygosity)
for three unlinked loci. One determines resistance to trifluoroleucine;
another, resistance to copper sulfate; and the third is associated with
a morphological phenotype observed as colonies with a ridged surface
resembling a filigree. Global expression analysis of the progeny with
the filigreed and smooth colony phenotypes revealed a greater than
2-fold difference in transcription for 378 genes (6% of the genome). A
large number of the overexpressed genes function in pathways of amino
acid biosynthesis (particularly methionine) and sulfur or nitrogen
assimilation, whereas many of the underexpressed genes are amino acid
permeases. These wholesale changes in amino acid metabolism segregate
as a suite of traits resulting from a single gene or a small number of
genes. We conclude that natural vineyard populations of
S.
cerevisiae
can harbor alleles that cause massive alterations in
the global patterns of gene expression. Hence, studies of expression
variation in natural populations, without accompanying segregation
analysis, may give a false picture of the number of segregating genes
underlying the variation.
Life on carbon monoxide: X-ray structure of
Rhodospirillum rubrum
Ni-Fe-S carbon monoxide dehydrogenase
Authors: Drennan Catherine L. , Heo Jongyun , Sintchak Michael D. , Schreiter Eric , Ludden Paul W.
Published: July 2002

A crystal structure of the anaerobic Ni-Fe-S carbon monoxide dehydrogenase (CODH) from
Rhodospirillum rubrum
has been determined to 2.8-Å resolution. The CODH family, for which the
R. rubrum
enzyme is the prototype, catalyzes the biological oxidation of CO at an unusual Ni-Fe-S cluster called the C-cluster. The Ni-Fe-S C-cluster contains a mononuclear site and a four-metal cubane. Surprisingly, anomalous dispersion data suggest that the mononuclear site contains Fe and not Ni, and the four-metal cubane has the form [NiFe
3
S
4
] and not [Fe
4
S
4
]. The mononuclear site and the four-metal cluster are bridged by means of Cys
531
and one of the sulfides of the cube. CODH is organized as a dimer with a previously unidentified [Fe
4
S
4
] cluster bridging the two subunits. Each monomer is comprised of three domains: a helical domain at the N terminus, an α/β (Rossmann-like) domain in the middle, and an α/β (Rossmann-like) domain at the C terminus. The helical domain contributes ligands to the bridging [Fe
4
S
4
] cluster and another [Fe
4
S
4
] cluster, the B-cluster, which is involved in electron transfer. The two Rossmann domains contribute ligands to the active site C-cluster. This x-ray structure provides insight into the mechanism of biological CO oxidation and has broader significance for the roles of Ni and Fe in biological systems.
Condensed complexes, rafts, and the chemical activity of cholesterol in membranes
Authors: Radhakrishnan Arun , Anderson Thomas G. , McConnell Harden M.
Published: July 2002

Epifluorescence microscopy studies of mixtures of phospholipids and
cholesterol at the air–water interface often exhibit coexisting liquid
phases. The properties of these liquids point to the formation of
“condensed complexes” between cholesterol and certain
phospholipids, such as sphingomyelin. It is found that monolayers that
form complexes can incorporate a low concentration of a ganglioside
G
M1
. This glycolipid is visualized by using a fluorescently
labeled B subunit of cholera toxin. Three coexisting liquid phases are
found by using this probe together with a fluorescent phospholipid
probe. The three liquid phases are identified as a phospholipid-rich
phase, a cholesterol-rich phase, and a condensed complex-rich phase.
The cholera toxin B labeled ganglioside G
M1
is found
exclusively in the condensed complex-rich phase. Condensed complexes
are likely present in animal cell membranes, where they should
facilitate the formation of specialized domains such as rafts.
Condensed complexes also have a major effect in determining the
chemical activity of cholesterol. It is suggested that this chemical
activity plays an essential role in the regulation of cholesterol
biosynthesis. Gradients in the chemical activity of cholesterol should
likewise govern the rates and direction of intracellular intermembrane
cholesterol transport.
Using flexible loop mimetics to extend Φ-value analysis to secondary structure interactions
Authors: Ferguson Neil , Pires José Ricardo , Toepert Florian , Johnson Christopher M. , Pan Yong Ping , Volkmer-Engert Rudolf , Schneider-Mergener Jens , Daggett Valerie , Oschkinat Hartmut , Fersht Alan
Published: July 2002

Chemical synthesis allows the incorporation of nonnatural amino acids into proteins that may provide previously untried probes of their folding pathway and thermodynamic stability. We have used a flexible thioether linker as a loop mimetic in the human yes kinase-associated protein (YAP 65) WW domain, a three-stranded, 44-residue, β-sheet protein. This linkage avoids problems of incorporating sequences that constrain loops to the extent that they significantly change the nature of the denatured state with concomitant effects on the folding kinetics. An NMR solution structure shows that the thioether linker had little effect on the global fold of the domain, although the loop is apparently more dynamic. The thioether variants are destabilized by up to 1.4 kcal/mol (1 cal = 4.18 J). Preliminary Φ-value analysis showed that the first loop is highly structured in the folding transition state, and the second loop is essentially unstructured. These data are consistent with results from simulated unfolding and detailed protein-engineering studies of structurally homologous WW domains. Previously, Φ-value analysis was limited to studying side-chain interactions. The linkers used here extend the protein engineering method directly to secondary-structure interactions.
Constructing, verifying, and dissecting the folding transition state of chymotrypsin inhibitor 2 with all-atom simulations
Authors: Li Lewyn , Shakhnovich Eugene I.
Published: July 2002

Experimentally, protein engineering and φ-value analysis is the method of choice to characterize the structure in folding transition state ensemble (TSE) of any protein. Combining experimental φ values and computer simulations has led to a deeper understanding of how proteins fold. In this report, we construct the TSE of chymotrypsin inhibitor 2 from published φ values. Importantly, we verify, by means of multiple independent simulations, that the conformations in the TSE have a probability of ≈0.5 to reach the native state rapidly, so the TSE consists of true transition states. This finding validates the use of transition state theory underlying all φ-value analyses. Also, we present a method to dissect and study the TSE by generating conformations that have a disrupted α-helix (α-disrupted states) or disordered β-strands 3 and 4 (β-disrupted states). Surprisingly, the α-disrupted states have a stronger tendency to fold than the β-disrupted states, despite the higher φ values for the α-helix in the TSE. We give a plausible explanation for this result and discuss its implications on protein folding and design. Our study shows that, by using both experiments and computer simulations, we can gain many insights into protein folding.
Evidence that androgens are the primary steroids produced by
Xenopus laevis
ovaries and may signal through the classical androgen receptor to promote oocyte maturation
Authors: Lutz L. B. , Cole L. M. , Gupta M. K. , Kwist K. W. , Auchus R. J. , Hammes S. R.
Published: July 2002

Steroid-induced maturation of
Xenopus
oocytes has long served as a model for studying meiosis. Progesterone has been considered the relevant steroid controlling maturation, perhaps through interactions with classical progesterone receptors. In this study, we provide evidence that androgens, rather than progesterone, are the physiologic mediators of
Xenopus
oocyte maturation. Androgens were equal or more potent activators of maturation
in vitro
relative to progesterone and were significantly more abundant in the serum and ovaries of β-human chorionic growth hormone-stimulated frogs. Androgen action appeared to be mediated by classical androgen receptors (ARs) expressed in oocytes, as androgen-induced maturation and signaling was specifically attenuated by AR antagonists. Interestingly, we found that progesterone was rapidly converted to the androgen androstenedione in isolated oocytes by the enzyme CYP17, suggesting that androgens may be promoting maturation even under conditions typical for “progesterone-mediated” maturation assays. Androgens are thought to play an important role in ovarian development as well as pathology, and signaling through the AR may prove to be a major regulatory mechanism mediating these processes.
DNA packaging and ejection forces in bacteriophage
Authors: Kindt James , Tzlil Shelly , Ben-Shaul Avinoam , Gelbart William M.
Published: July 2002

We calculate the forces required to package (or, equivalently, acting to eject) DNA into (from) a bacteriophage capsid, as a function of the loaded (ejected) length, under conditions for which the DNA is either self-repelling or self-attracting. Through computer simulation and analytical theory, we find the loading force to increase more than 10-fold (to tens of piconewtons) during the final third of the loading process; correspondingly, the internal pressure drops 10-fold to a few atmospheres (matching the osmotic pressure in the cell) upon ejection of just a small fraction of the phage genome. We also determine an evolution of the arrangement of packaged DNA from toroidal to spool-like structures.
Cryptic vicariance in the historical assembly of a Baja California Peninsular Desert biota
Authors: Riddle Brett R. , Hafner David J. , Alexander Lois F. , Jaeger Jef R.
Published: July 2002

We use analyses of phylogeographic population structure across a
suite of 12 mammalian, avian, amphibian, and reptilian species and
species-groups to assess the role of Late Miocene to Pleistocene
geological history in the evolution of a distinct Baja California
Peninsular Desert biota. Comparative examination of phylogroup
distributions provides support for previously hypothesized vicariant
events produced by: a middle Pleistocene midpeninsular seaway, a late
Pliocene northward transgression of the Sea of Cortéz, and a
Pliocene seaway across the southern peninsular Isthmus of La Paz. Most
of this phylogeographic architecture is cryptically embedded within
widespread taxonomic species and species-groups, such that the unique
evolutionary history of the Peninsular Desert has been obscured and
ignored. The Peninsular Desert can no longer be considered a subset of
the Sonoran Desert—it is a separate regional desert with its own
unique evolutionary history, ecological arena, and conservation value.
Identification of brain-specific and imprinted small nucleolar RNA genes exhibiting an unusual genomic organization
Authors: Cavaillé Jérôme , Buiting Karin , Kiefmann Martin , Lalande Marc , Brannan Camilynn I. , Horsthemke Bernhard , Bachellerie Jean-Pierre , Brosius Jürgen , Hüttenhofer Alexander
Published: July 2002

We have identified three C/D-box small nucleolar RNAs (snoRNAs)
and one H/ACA-box snoRNA in mouse and human. In mice, all four
snoRNAs (MBII-13, MBII-52, MBII-85, and MBI-36) are exclusively
expressed in the brain, unlike all other known snoRNAs. Two of the
human RNA orthologues (HBII-52 and HBI-36) share this expression
pattern, and the remainder, HBII-13 and HBII-85, are prevalently
expressed in that tissue. In mice and humans, the brain-specific
H/ACA box snoRNA (MBI-36 and HBI-36, respectively) is intron-encoded
in the brain-specific serotonin 2C receptor gene. The three human C/D
box snoRNAs map to chromosome 15q11–q13, within a region implicated in
the Prader–Willi syndrome (PWS), which is a neurogenetic disease
resulting from a deficiency of paternal gene expression. Unlike other
C/D box snoRNAs, two snoRNAs, HBII-52 and HBII-85, are encoded in a
tandemly repeated array of 47 or 24 units, respectively. In mouse the
homologue of HBII-52 is processed from intronic portions of the tandem
repeats. Interestingly, these snoRNAs were absent from the cortex of a
patient with PWS and from a PWS mouse model, demonstrating their
paternal imprinting status and pointing to their potential role in the
etiology of PWS. Despite displaying hallmarks of the two families of
ubiquitous snoRNAs that guide 2′-O-ribose methylation and
pseudouridylation of rRNA, respectively, they lack any telltale rRNA
complementarity. Instead, brain-specific C/D box snoRNA HBII-52 has
an 18-nt phylogenetically conserved complementarity to a critical
segment of serotonin 2C receptor mRNA, pointing to a potential role in
the processing of this mRNA.
IscR, an Fe-S cluster-containing transcription factor, represses expression of
Escherichia coli
genes encoding Fe-S cluster assembly proteins
Authors: Schwartz Christopher J. , Giel Jennifer L. , Patschkowski Thomas , Luther Christopher , Ruzicka Frank J. , Beinert Helmut , Kiley Patricia J.
Published: July 2002

IscR (
i
ron-
s
ulfur
c
luster
r
egulator) is encoded by an ORF
located immediately upstream of genes coding for the
Escherichia
coli
Fe-S cluster assembly proteins, IscS, IscU, and IscA. IscR
shares amino acid similarity with MarA, a member of the
MarA/SoxS/Rob family of transcription factors. In this study, we
found that IscR functions as a repressor of the
iscRSUA
operon, because strains deleted for
iscR
have increased
expression of this operon. In addition,
in vitro
transcription reactions established a direct role for IscR in
repression of the
iscR
promoter. Analysis of IscR by
electron paramagnetic resonance showed that the anaerobically isolated
protein contains a [2Fe-2S]
1+
cluster. The Fe-S cluster
appears to be important for IscR function, because repression of
iscR
expression is significantly reduced in strains
containing null mutations of the Fe-S cluster assembly genes
iscS
or
hscA
. The finding that IscR
activity is decreased in strain backgrounds in which Fe-S cluster
assembly is impaired suggests that this protein may be part of a novel
autoregulatory mechanism that senses the Fe-S cluster assembly status
of cells.
Voltage gating and permeation in a gap junction hemichannel.
Authors: Trexler E B , Bennett M V , Bargiello T A , Verselis V K
Published: July 2002

Gap junction channels are formed by members of the connexin gene family and mediate direct intercellular communication through linked hemichannels (connexons) from each of two adjacent cells. While for most connexins, the hemichannels appear to require an apposing hemichannel to open, macroscopic currents obtained from Xenopus oocytes expressing rat Cx46 suggested that some hemichannels can be readily opened by membrane depolarization [Paul, D. L., Ebihara, L., Takemoto, L. J., Swenson, K. I. & Goodenough, D. A. (1991), J. Cell Biol. 115, 1077-1089]. Here we demonstrate by single channel recording that hemichannels comprised of rat Cx46 exhibit complex voltage gating consistent with there being two distinct gating mechanisms. One mechanism partially closes Cx46 hemichannels from a fully open state, gammaopen, to a substate, gammasub, about one-third of the conductance of gammaopen; these transitions occur when the cell is depolarized to inside positive voltages, consistent with gating by transjunctional voltage in Cx46 gap junctions. The other gating mechanism closes Cx46 hemichannels to a fully closed state, gammaclosed, on hyperpolarization to inside negative voltages and has unusual characteristics; transitions between gammaclosed and gammaopen appear slow (10-20 ms), often involving several transient substates distinct from gammasub. The polarity of activation and kinetics of this latter form of gating indicate that it is the mechanism by which these hemichannels open in the cell surface membrane when unapposed by another hemichannel. Cx46 hemichannels display a substantial preference for cations over anions, yet have a large unitary conductance (approximately 300 pS) and a relatively large pore as inferred from permeability to tetraethylammonium (approximately 8.5 angstroms diameter). These hemichannels open at physiological voltages and could induce substantial cation fluxes in cells expressing Cx46.
Isolation and characterization of two human transcription factor IIH (TFIIH)-related complexes: ERCC2/CAK and TFIIH.
Authors: Reardon J T , Ge H , Gibbs E , Sancar A , Hurwitz J , Pan Z Q
Published: July 2002

Transcription factor IIH (TFIIH) is a multisubunit protein complex essential for both the initiation of RNA polymerase class II (pol II)-catalyzed transcription and nucleotide excision repair of DNA. Recent studies have shown that TFIIH copurifies with the cyclin-dependent kinase (cdk)-activating kinase complex (CAK) that includes cdk7, cyclin H, and p36/MAT1. Here we report the isolation of two TFIIH-related complexes: TFIIH* and ERCC2/CAK. TFIIH* consists of a subset of the TFIIH complex proteins including ERCC3 (XPB), p62, p44, p41, and p34 but is devoid of detectable levels of ERCC2 (XPD) and CAK. ERCC2/CAK was isolated as a complex that exhibits CAK activity that cosediments with the three CAK components (cdk7, cyclin H, and p36/MAT1) as well as the ERCC2 (XPD) protein. TFIIH* can support pol II-catalyzed transcription in vitro with lower efficiency compared with TFIIH. This TFIIH*-dependent transcription reaction was stimulated by ERCC2/CAK. The ERCC2/CAK and TFIIH* complexes are each active in DNA repair as shown by their ability to complement extracts prepared from ERCC2 (XPD)- and ERCC3 (XPB)-deficient cells, respectively, in supporting the excision of DNA containing a cholesterol lesion. These data suggest that TFIIH* and ERCC2/CAK interact to form the TFIIH holoenzyme capable of efficiently assembling the pol II transcription initiation complex and directly participating in excision repair reactions.
The tumor necrosis factor-inducible zinc finger protein A20 interacts with TRAF1/TRAF2 and inhibits NF-kappaB activation.
Authors: Song H Y , Rothe M , Goeddel D V
Published: July 2002

TRAF1 and TRAF2 form an oligomeric complex that associates with the cytoplasmic domains of various members of the tumor necrosis factor (TNF) receptor superfamily. TRAF2 action is required for activation of the transcription factor NF-kappaB triggered by TNF and the CD40 ligand. Here we show that TRAF1 and TRAF2 interact with A20, a zinc finger protein, whose expression is induced by agents that activate NF-kappaB. Mutational analysis revealed that the N-terminal half of A20 interacts with the conserved C-terminal TRAF domain of TRAF1 and TRAF2. In cotransfection experiments, A20 blocked TRAF2-mediated NF-kappaB activation. A20 also inhibited TNF and IL-1-induced NF-kappaB activation, suggesting that it may inhibit NF-kappaB activation signaled by diverse stimuli. The ability of A20 to block NF-kappaB activation was mapped to its C-terminal zinc finger domain. Thus, A20 is composed of two functionally distinct domains, an N-terminal TRAF binding domain that recruits A20 to the TRAF2-TRAF1 complex and a C-terminal domain that mediates inhibition of NF-kappaB activation. Our findings suggest a possible molecular mechanism that could explain A20's ability to negatively regulate its own TNF-inducible expression.
Negative electrostatic surface potential of protein sites specific for anionic ligands.
Authors: Ledvina P S , Yao N , Choudhary A , Quiocho F A
Published: July 2002

Determination of the crystal structure of an "open" unliganded active mutant (T141D) form of the Escherichia coli phosphate receptor for active transport has allowed calculation of the electrostatic surface potential for it and two other comparably modeled receptor structures (wild type and D137N). A discovery of considerable implication is the intensely negative potential of the phosphate-binding cleft. We report similar findings for a sulfate transport receptor, a DNA-binding protein, and, even more dramatically, redox proteins. Evidently, for proteins such as these, which rely almost exclusively on hydrogen bonding for anion interactions and electrostatic balance, a noncomplementary surface potential is not a barrier to binding. Moreover, experimental results show that the exquisite specificity and high affinity of the phosphate and sulfate receptors for unions are insensitive to modulations of charge potential, but extremely sensitive to conditions that leave a hydrogen bond donor or acceptor unpaired.
The root of the universal tree and the origin of eukaryotes based on elongation factor phylogeny.
Authors: Baldauf S L , Palmer J D , Doolittle W F
Published: July 2002

The genes for the protein synthesis elongation factors Tu (EF-Tu) and G (EF-G) are the products of an ancient gene duplication, which appears to predate the divergence of all extant organismal lineages. Thus, it should be possible to root a universal phylogeny based on either protein using the second protein as an outgroup. This approach was originally taken independently with two separate gene duplication pairs, (i) the regulatory and catalytic subunits of the proton ATPases and (ii) the protein synthesis elongation factors EF-Tu and EF-G. Questions about the orthology of the ATPase genes have obscured the former results, and the elongation factor data have been criticized for inadequate taxonomic representation and alignment errors. We have expanded the latter analysis using a broad representation of taxa from all three domains of life. All phylogenetic methods used strongly place the root of the universal tree between two highly distinct groups, the archaeons/eukaryotes and the eubacteria. We also find that a combined data set of EF-Tu and EF-G sequences favors placement of the eukaryotes within the Archaea, as the sister group to the Crenarchaeota. This relationship is supported by bootstrap values of 60-89% with various distance and maximum likelihood methods, while unweighted parsimony gives 58% support for archaeal monophyly.
Surface protein phosphorylation by ecto-protein kinase is required for the maintenance of hippocampal long-term potentiation.
Authors: Chen W , Wieraszko A , Hogan M V , Yang H A , Kornecki E , Ehrlich Y H
Published: July 2002

During the induction of long-term potentiation (LTP) in hippocampal slices adenosine triphosphate (ATP) is secreted into the synaptic cleft, and a 48 kDa/50 kDa protein duplex becomes phosphorylated by extracellular ATP. All the criteria required as evidence that these two proteins serve as principal substrates of ecto-protein kinase activity on the surface of hippocampal pyramidal neurons have been fulfilled. This phosphorylation activity was detected on the surface of pyramidal neurons assayed after synaptogenesis, but not in immature neurons nor in glial cells. Addition to the extracellular medium of a monoclonal antibody termed mAb 1.9, directed to the catalytic domain of protein kinase C (PKC), inhibited selectively this surface protein phosphorylation activity and blocked the stabilization of LTP induced by high frequency stimulation (HFS) in hippocampal slices. This antibody did not interfere with routine synaptic transmission nor prevent the initial enhancement of synaptic responses observed during the 1-5 min period immediately after the application of HFS (the induction phase of LTP). However, the initial increase in the slope of excitatory postsynaptic potentials, as well as the elevated amplitude of the population spike induced by HFS, both declined gradually and returned to prestimulus values within 30-40 min after HFS was applied in the presence of mAb 1.9. A control antibody that binds to PKC but does not inhibit its activity had no effect on LTP. The selective inhibitory effects observed with mAb 1.9 provide the first direct evidence of a causal role for ecto-PK in the maintenance of stable LTP, an event implicated in the process of learning and the formation of memory in the brain.
Molecular characterization of the mosquito vitellogenin receptor reveals unexpected high homology to the Drosophila yolk protein receptor.
Authors: Sappington T W , Kokoza V A , Cho W L , Raikhel A S
Published: July 2002

The mosquito (Aedes aegypti) vitellogenin receptor (AaVgR) is a large membrane-bound protein (214 kDa when linearized) that mediates internalization of vitellogenin, the major yolk-protein precursor, by oocytes during egg development. We have cloned and sequenced two cDNA fragments encompassing the entire coding region of AaVgR mRNA, to our knowledge the first insect VgR sequence to be reported. The 7.3-kb AaVgR mRNA is present only in female germ-line cells and is abundant in previtellogenic oocytes, suggesting that the AaVgR gene is expressed early in oocyte differentiation. The deduced amino acid sequence predicts a 202.7-kDa protein before posttranslational processing. The AaVgR is a member of the low density lipoprotein receptor superfamily, sharing significant homology with the chicken (Gallus gallus) VgR and particularly the Drosophila melanogaster yolk protein receptor, in spite of a very different ligand for the latter. Distance-based phylogenetic analyses suggest that the insect VgR/yolk protein receptor lineage and the vertebrate VgR/low density lipoprotein receptor lineage diverged before the bifurcation of nematode and deuterostome lines.
How photons start vision.
Authors: Baylor D
Published: July 2002

Recent studies have elucidated how the absorption of a photon in a rod or cone cell leads to the generation of the amplified neural signal that is transmitted to higher-order visual neurons. Photoexcited visual pigment activates the GTP-binding protein transducin, which in turn stimulates cGMP phosphodiesterase. This enzyme hydrolyzes cGMP, allowing cGMP-gated cationic channels in the surface membrane to close, hyperpolarize the cell, and modulate transmitter release at the synaptic terminal. The kinetics of reactions in the cGMP cascade limit the temporal resolution of the visual system as a whole, while statistical fluctuations in the reactions limit the reliability of detection of dim light. Much interest now focuses on the processes that terminate the light response and dynamically regulate amplification in the cascade, causing the single photon response to be reproducible and allowing the cell to adapt in background light. A light-induced fall in the internal free Ca2+ concentration coordinates negative feedback control of amplification. The fall in Ca2+ stimulates resynthesis of cGMP, antagonizes rhodopsin's catalytic activity, and increases the affinity of the light-regulated cationic channel for cGMP. We are using physiological methods to study the molecular mechanisms that terminate the flash response and mediate adaptation. One approach is to observe transduction in truncated, dialyzed photoreceptor cells whose internal Ca2+ and nucleotide concentrations are under experimental control and to which exogenous proteins can be added. Another approach is to observe transduction in transgenic mouse rods in which specific proteins within the cascade are altered or deleted.
Star scientists and institutional transformation: Patterns of invention and innovation in the formation of the biotechnology industry
Authors: Zucker Lynne G. , Darby Michael R.
Published: July 2002

The most productive (“star”) bioscientists had intellectual
human capital of extraordinary scientific and pecuniary value for some
10–15 years after Cohen and Boyer’s 1973 founding discovery for
biotechnology [Cohen, S., Chang, A., Boyer, H. & Helling, R. (1973)
Proc. Natl. Acad. Sci. USA
70, 3240–3244]. This
extraordinary value was due to the union of still scarce knowledge of
the new research techniques and genius and vision to apply them in
novel, valuable ways. As in other sciences, star bioscientists were
very protective of their techniques, ideas, and discoveries in the
early years of the revolution, tending to collaborate more within their
own institution, which slowed diffusion to other scientists. Close,
bench-level working ties between stars and firm scientists were needed
to accomplish commercialization of the breakthroughs. Where and when
star scientists were actively producing publications is a key predictor
of where and when commercial firms began to use biotechnology. The
extent of collaboration by a firm’s scientists with stars is a
powerful predictor of its success: for an average firm, 5 articles
coauthored by an academic star and the firm’s scientists result in
about 5 more products in development, 3.5 more products on the market,
and 860 more employees. Articles by stars collaborating with or
employed by firms have significantly higher rates of citation than
other articles by the same or other stars. The U.S. scientific and
economic infrastructure has been particularly effective in fostering
and commercializing the bioscientific revolution. These results let us
see the process by which scientific breakthroughs become economic
growth and consider implications for policy.
A neurotransmitter transporter encoded by the
Drosophila
inebriated gene
Authors: Soehnge Holly , Huang Xi , Becker Marie , Whitley Penn , Conover Diana , Stern Michael
Published: July 2002

Behavioral and electrophysiological studies on mutants defective in
the
Drosophila
inebriated (
ine
) gene
demonstrated increased excitability of the motor neuron. In this paper,
we describe the cloning and sequence analysis of
ine
.
Mutations in
ine
were localized on cloned DNA by
restriction mapping and restriction fragment length polymorphism (RFLP)
mapping of
ine
mutants. DNA from the
ine
region was then used to isolate an
ine
cDNA.
In
situ
hybridization of
ine
transcripts to
developing embryos revealed expression of this gene in several cell
types, including the posterior hindgut, Malpighian tubules, anal plate,
garland cells, and a subset of cells in the central nervous system. The
ine
cDNA contains an open reading frame of 658 amino
acids with a high degree of sequence similarity to members of the
Na
+
/Cl
−
-dependent neurotransmitter
transporter family. Members of this family catalyze the rapid reuptake
of neurotransmitters released into the synapse and thereby play key
roles in controlling neuronal function. We conclude that
ine
mutations cause increased excitability of the
Drosophila
motor neuron by causing the defective
reuptake of the substrate neurotransmitter of the
ine
transporter and thus overstimulation of the motor neuron by this
neurotransmitter. From this observation comes a unique opportunity to
perform a genetic dissection of the regulation of excitability of the
Drosophila
motor neuron.
Cocrystal structure of YY1 bound to the adeno-associated virus P5 initiator
Authors: Houbaviy Hristo B. , Usheva Anny , Shenk Thomas , Burley Stephen K.
Published: July 2002

Ying–Yang 1 protein (YY1) supports specific, unidirectional
initiation of messenger RNA production by RNA polymerase II from two
adjacent start sites in the adeno-associated virus P5 promoter, a
process which is independent of the TATA box-binding protein (TBP). The
2.5-Å resolution YY1-initiator element cocrystal structure reveals
four zinc fingers recognizing a YY1-binding consensus sequence.
Upstream of the transcription start sites protein–DNA contacts involve
both strands and downstream they are virtually restricted to the
template strand, permitting access to the active center of RNA
polymerase II and ensuring specificity and directionality. The observed
pattern of protein–DNA contacts also explains YY1 binding to a
preformed transcription bubble, and YY1 binding to a DNA/RNA hybrid
analog of the P5 promoter region containing a nascent RNA transcript. A
model is proposed for YY1-directed, TBP-independent transcription
initiation.
Transposon Tc1-derived, sequence-tagged sites in
Caenorhabditis elegans
as markers for gene mapping
Authors: Korswagen Hendrik C. , Durbin Richard M. , Smits Miriam T. , Plasterk Ronald H. A.
Published: July 2002

We present an approach to map large numbers of Tc1 transposon
insertions in the genome of
Caenorhabditis
elegans
. Strains have been described that contain up to 500
polymorphic Tc1 insertions. From these we have cloned and shotgun
sequenced over 2000 Tc1 flanks, resulting in an estimated set of 400 or
more distinct Tc1 insertion alleles. Alignment of these sequences
revealed a weak Tc1 insertion site consensus sequence that was
symmetric around the invariant TA target site and reads CAYATATRTG. The
Tc1 flanking sequences were compared with 40 Mbp of a
C.
elegans
genome sequence. We found 151 insertions within the
sequenced area, a density of ≈1 Tc1 insertion in every 265 kb. As the
rest of the
C. elegans
genome sequence is obtained,
remaining Tc1 alleles will fall into place. These mapped Tc1 insertions
can serve two functions: (
i
) insertions in or near genes
can be used to isolate deletion derivatives that have that gene
mutated; and (
ii
) they represent a dense collection of
polymorphic sequence-tagged sites. We demonstrate a strategy to use
these Tc1 sequence-tagged sites in fine-mapping mutations.
Detection of cortical activation during averaged single trials of a cognitive task using functional magnetic resonance imaging
Authors: Buckner Randy L. , Bandettini Peter A. , O’Craven Kathleen M. , Savoy Robert L. , Petersen Steven E. , Raichle Marcus E. , Rosen Bruce R.
Published: July 2002

Functional neuroimaging studies in human subjects using positron
emission tomography or functional magnetic resonance imaging
(fMRI) are typically conducted by collecting data over extended
time periods that contain many similar trials of a task. Here methods
for acquiring fMRI data from single trials of a cognitive task are
reported. In experiment one, whole brain fMRI was used to
reliably detect single-trial responses in a prefrontal region within
single subjects. In experiment two, higher temporal sampling of a more
limited spatial field was used to measure temporal offsets between
regions. Activation maps produced solely from the single-trial data
were comparable to those produced from blocked runs. These findings
suggest that single-trial paradigms will be able to exploit the high
temporal resolution of fMRI. Such paradigms will provide experimental
flexibility and time-resolved data for individual brain regions on a
trial-by-trial basis.
Kinetic discrimination in T-cell activation.
Authors: Rabinowitz J D , Beeson C , Lyons D S , Davis M M , McConnell H M
Published: July 2002

We propose a quantitative model for T-cell activation in which the rate of dissociation of ligand from T-cell receptors determines the agonist and antagonist properties of the ligand. The ligands are molecular complexes between antigenic peptides and proteins of the major histocompatibility complex on the surfaces of antigen-presenting cells. Binding of ligand to receptor triggers a series of biochemical reactions in the T cell. If the ligand dissociates after these reactions are complete, the T cell receives a positive activation signal. However, dissociation of ligand after completion of the first reaction but prior to generation of the final products results in partial T-cell activation, which acts to suppress a positive response. Such a negative signal is brought about by T-cell ligands containing the variants of antigenic peptides referred to as T-cell receptor antagonists. Results of recent experiments with altered peptide ligands compare favorably with T-cell responses predicted by this model.
Baculovirus-mediated gene transfer into mammalian cells.
Authors: Boyce F M , Bucher N L
Published: July 2002

This paper describes the use of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a vector for gene delivery into mammalian cells. A modified AcMNPV virus was prepared that carried the Escherichia coli lacZ reporter gene under control of the Rous sarcoma virus promoter and mammalian RNA processing signals. This modified baculovirus was then used to infect a variety of mammalian cell lines. After infection of the human liver cell lines HepG2, >25% of the cells showed high-level expression of the transduced gene. Over 70% of the cells in primary cultures of rat hepatocytes showed expression of beta-galactosidase after exposure to the virus. Cell lines from other tissues showed less or no expression of lacZ after exposure to the virus. The block to expression in less susceptible cells does not appear to result from the ability to be internalized by the target cell but rather by events subsequent to viral entry. The onset of lacZ expression occurred within 6 hr of infection in HepG2 cells and peaked 12-24 hr postinfection. Because AcMNPV is able to replicate only in insect hosts, is able to carry large (>15 kb) inserts, and is a highly effective gene delivery vehicle for primary cultures of hepatocytes, AcMNPV may be a useful vector for genetic manipulation of liver cells.
Molecular mechanism of transmembrane signaling by the aspartate receptor: a model.
Authors: Chervitz S A , Falke J J
Published: July 2002

The aspartate receptor of bacterial chemotaxis is representative of a large class of membrane-spanning receptors found in prokaryotic and eukaryotic organisms. These receptors, which regulate histidine kinase pathways and possess two putative transmembrane helices per subunit, appear to control a wide variety of cellular processes. The best characterized subgroup of the two-helix receptor class is the homologous family of chemosensory receptors from Escherichia coli and Salmonella typhimurium, including the aspartate receptor. This receptor binds aspartate, an attractant, in the periplasmic compartment and undergoes an intramolecular, transmembrane conformational change, thereby modulating the autophosphorylation rate of a bound histidine kinase in the cytoplasm. Here, we analyze recent results from x-ray crystallographic, solution 19F NMR, and engineered disulfide studies probing the aspartate-induced structural change within the periplasmic and transmembrane regions of the receptor. Together, these approaches provide evidence that aspartate binding triggers a "swinging-piston" displacement of the second membrane-spanning helix, which is proposed to communicate the signal across the bilayer.