bluuebunny/crossref_metadata_2025 huggingface dataset:
Instability of repetitive DNA sequences: The role of replication in multiple mechanisms
Authors: Bzymek Malgorzata , Lovett Susan T.
Published: July 2002

Rearrangements between tandem sequence homologies of various lengths are a major source of genomic change and can be deleterious to the organism. These rearrangements can result in either deletion or duplication of genetic material flanked by direct sequence repeats. Molecular genetic analysis of repetitive sequence instability in
Escherichia coli
has provided several clues to the underlying mechanisms of these rearrangements. We present evidence for three mechanisms of RecA-independent sequence rearrangements: simple replication slippage, sister-chromosome exchange-associated slippage, and single-strand annealing. We discuss the constraints of these mechanisms and contrast their properties with RecA-dependent homologous recombination. Replication plays a critical role in the two slipped misalignment mechanisms, and difficulties in replication appear to trigger rearrangements via all these mechanisms.
RNA hairpins in noncoding regions of human brain and
Caenorhabditis elegans
mRNA are edited by adenosine deaminases that act on RNA
Authors: Morse Daniel P. , Aruscavage P. Joseph , Bass Brenda L.
Published: July 2002

Adenosine deaminases that act on RNA (ADARs) constitute a family of RNA-editing enzymes that convert adenosine to inosine within double-stranded regions of RNA. We previously developed a method to identify inosine-containing RNAs and used it to identify five ADAR substrates in
Caenorhabditis elegans
. Here we use the same method to identify five additional
C. elegans
substrates, including three mRNAs that encode proteins known to affect neuronal functions. All 10 of the
C. elegans
substrates are edited in long stem-loop structures located in noncoding regions, and thus contrast with previously identified substrates of other organisms, in which ADARs target codons. To determine whether editing in noncoding regions was a conserved ADAR function, we applied our method to poly(A)
+
RNA of human brain and identified 19 previously unknown ADAR substrates. The substrates were strikingly similar to those observed in
C. elegans
, since editing was confined to 3′ untranslated regions, introns, and a noncoding RNA. Also similar to what was found in
C. elegans
, 15 of the 19 substrates were edited in repetitive elements. The identities of the newly identified ADAR substrates suggest that RNA editing may influence many biologically important processes, and that for many metazoa, A-to-I conversion in coding regions may be the exception rather than the rule.
Size exclusion chromatography does not require pores
Authors: Brooks Donald E. , Haynes Charles A. , Hritcu Doina , Steels Bradley M. , Müller Werner
Published: July 2002

Separation of macromolecules on the basis of their molecular weight by size exclusion chromatography has long been considered to be caused by the geometry-dependent partition of macromolecules between a continuous phase and the porous interior of a gel or cross-linked bead. The volume of a pore accessible to a solute is limited by its relative dimensions, so larger molecules will have access to a smaller volume and will remain in a bead for a shorter time than smaller solutes. Our recent alternate picture proposes that the partition coefficient can be calculated from a thermodynamic model for the free energy of mixing of the solute with the gel phase. Size-dependent exclusion caused by the unfavorable entropy of mixing associated with the partition is predicted; the magnitude of the effect is modified by enthalpic interactions between the solute and the gel phase. This concept is extended here to describe the partition of macromolecules into a layer of terminally attached polymer chains grafted onto a solid bead. Both simple mean field and self-consistent field theory calculations predict size-dependent entropic exclusion. Experimental results obtained with neutral polymer chains grafted onto solid polystyrene latex beads confirm the predictions.
The steroid receptor coactivator SRC-3 (p/CIP/RAC3/AIB1/ACTR/TRAM-1) is required for normal growth, puberty, female reproductive function, and mammary gland development
Authors: Xu Jianming , Liao Lan , Ning Guang , Yoshida-Komiya Hiromi , Deng Chuxia , O'Malley Bert W.
Published: July 2002

Steroid receptor coactivator-3 (SRC-3) is a coactivator of nuclear receptors in the SRC family as assayed
in vitro
. Here, we show that mouse SRC-3 is expressed in a tissue-specific fashion and distributed mainly in the oocytes, mammary glands, hippocampus, olfactory bulb, smooth muscle, hepatocytes, and vaginal epithelium. Genetic disruption of
SRC-3
in mice results in a pleiotropic phenotype showing dwarfism, delayed puberty, reduced female reproductive function, and blunted mammary gland development. Hormonal analysis indicates that SRC-3 plays a role in both the growth hormone regulatory pathway and the production of estrogen, which may explain the observed phenotypes. These results suggest that the physiological role of SRC-3 is different from that of SRC-1 and prove the diversity among coactivator family members.
Tetracycline-inducible systems for
Drosophila
Authors: Stebbins Michael J. , Urlinger Stefanie , Byrne Gerry , Bello Bruno , Hillen Wolfgang , Yin Jerry C. P.
Published: July 2002

Since their inception, tetracycline (Tet)-inducible systems have become the method of choice for transgenic research. The Tet-Off systems have a number of advantages, including robust target induction using a relatively benign effector molecule. However, use of the Tet-On system has been fraught with difficulties, including high background expression in the absence of effector molecules and inconsistent gene induction. Recently, secondgeneration Tet-On transactivators (TAs) have been described. In HeLa cells, they are far more efficient than the original reverse TA protein, and they exhibit lower background activity in the absence of effectors. Here we examine the most promising TA in transgenic
Drosophila
and characterize its
in vivo
properties. We report that low levels of doxycycline, when added to normal fly food, efficiently and rapidly induce target transgenes in adults, larvae, and embryos. This TA is superior to all other Tet-On proteins, and its performance is comparable to that of the widely used Tet-Off TA. In addition, combining the improved Tet-On TA with the Gal4-UAS (upstream-activating sequence) system produces robust, spatially restricted, temporally controlled transgene induction. Because this Tet-On TA is significantly more efficient than previous ones used in
Drosophila
, it is also possible to modulate gene induction by controlling the dosage of the antibiotic in the food.
True optical resolution beyond the Rayleigh limit achieved by standing wave illumination
Authors: Frohn Jan T. , Knapp Helmut F. , Stemmer Andreas
Published: July 2002

During the last decade, various efforts have been undertaken to enhance the resolution of optical microscopes, mostly because of their importance in biological sciences. Herein, we describe a method to increase the resolution of fluorescence microscopy by illuminating the specimen with a mesh-like interference pattern of a laser source and electronic postprocessing of the images. We achieve 100-nm optical resolution, an improvement by a factor of more than 2 compared with standard fluorescence microscopy and of 1.5 compared with confocal scanning.
Evidence for a circulating islet cell growth factor in insulin-resistant states
Authors: Flier Sarah N. , Kulkarni Rohit N. , Kahn C. Ronald
Published: July 2002

Insulin resistance is a feature of many common disorders including obesity and type 2 diabetes mellitus. In these disorders, the β-cells compensate for the insulin resistance for long periods of time with an increase in secretory capacity, an increase in β-cell mass, or both. To determine whether the β-cell response might relate to a circulating growth factor, we have transplanted normal islets under the kidney capsule of normoglycemic insulin-resistant mice with two different models of insulin resistance: lean mice that have a double heterozygous deletion of the insulin receptor and insulin receptor substrate-1 (DH) or the obese, hyperglycemic
ob
/
ob
mice. In the grafts transplanted into both hosts, there was a marked increase in β-cell mitotic activity and islet mass that was comparable with that observed in the endogenous pancreas. By contrast, islets of the DH mouse transplanted into normal mice showed reduced mitotic index. These data suggest the insulin resistance is associated with a circulating islet cell growth factor that is independent of glucose and obesity.
Ordered membrane insertion of an archaeal opsin
in vivo
Authors: Dale Heather , Angevine Christine M. , Krebs Mark P.
Published: July 2002

The prevailing model of polytopic membrane protein insertion is based largely on the
in vitro
analysis of polypeptide chains trapped during insertion by arresting translation. To test this model under conditions of active translation
in vivo
, we have used a kinetic assay to determine the order and timing with which transmembrane segments of bacterioopsin (BO) are inserted into the membrane of the archaeon
Halobacterium salinarum
. BO is the apoprotein of bacteriorhodopsin, a structurally well characterized protein containing seven transmembrane α-helices (A-G) with an N-out, C-in topology.
H. salinarum
strains were constructed that express mutant BO containing a C-terminal His-tag and a single cysteine in one of the four extracellular domains of the protein. Cysteine translocation during BO translation was monitored by pulse–chase radiolabeling and rapid derivatization with a membrane-impermeant, sulfhydryl-specific gel-shift reagent. The results show that the N-terminal domain, the BC loop, and the FG loop are translocated in order from the N terminus to the C terminus. Translocation of the DE loop could not be examined because cysteine mutants in this region did not yield a gel shift. The translocation order was confirmed by applying the assay to mutant proteins containing two cysteines in separate extracellular domains. Comparison of the translocation results with
in vivo
measurements of BO elongation indicated that the N-terminal domain and the BC loop are translocated cotranslationally, whereas the FG loop is translocated posttranslationally. Together, these results support a sequential, cotranslational model of archaeal polytopic membrane protein insertion
in vivo
.
CD1c molecules broadly survey the endocytic system
Authors: Sugita Masahiko , van der Wel Nicole , Rogers Rick A. , Peters Peter J. , Brenner Michael B.
Published: July 2002

The ability of antigen-presenting cells to sample distinct intracellular compartments is crucial for microbe detection. Major histocompatibility complex class I and class II molecules sample the cytosol or the late endocytic compartment, allowing detection of microbial peptide antigens that arise in distinct intracellular compartments. In contrast, CD1a and CD1b molecules mediate the presentation of lipid and glycolipid antigens and differentially sample early recycling endosomes or late endocytic compartments, respectively, that contain distinct sets of lipid antigens. Here, we show that, unlike the other CD1 isoforms or major histocompatibility complex molecules that each sample restricted only intracellular compartments, CD1c is remarkable in that it distributes broadly throughout the endocytic system and is expressed in both recycling endosomes and late endocytic compartments. Further, in contrast to CD1b, which requires an acidic environment to function, antigen presentation by CD1c was able to overcome dependence on vesicular acidification. Because CD1c is expressed on essential antigen-presenting cells, such as epidermal Langerhans cells (in the absence of CD1b), or on B cells (without CD1a or -b), we suggest that CD1c molecules allow a comprehensive survey for lipid antigens throughout the endocytic system even in the absence of other CD1 isoforms.
Sexually antagonistic coevolution of a postmating-prezygotic reproductive character in desert
Drosophila
Authors: Knowles L. Lacey , Markow Therese A.
Published: July 2002

Rapid divergence in postmating-prezygotic characters suggests that selection may be responsible for generating reproductive barriers between closely related species. Theoretical models indicate that this rapid divergence could be generated by a series of male adaptations and female counteradaptations by means of sexual selection or conflict, but empirical tests of particular mechanisms are generally lacking. Moreover, although a male–female genotypic interaction in mediating sperm competition attests to an active role of females, molecular or morphological evidence of the female's participation in the coevolutionary process is critically needed. Here we show that postmating-prezygotic variation among populations of cactophilic desert
Drosophila
reflects divergent coevolutionary trajectories between the sexes. We explicitly test the female's role in intersexual interactions by quantifying differences in a specific postmating-prezygotic reproductive character, the insemination reaction mass, in two species,
Drosophila mojavensis
and
Drosophila arizonae
. A series of interpopulation crosses confirmed that population divergence was propelled by male–female interactions, a prerequisite if the selective forces derive from sexual conflicts. An association between the reaction mass and remating and oviposition behavior argues that divergence has been propelled by sexually antagonistic coevolution, and potentially has important implications for speciation.
Circadian rhythms of female mating activity governed by clock genes in
Drosophila
Authors: Sakai Takaomi , Ishida Norio
Published: July 2002

The physiological and behavioral activities of many animals are restricted to specific times of the day. The daily fluctuation in the mating activity of some insects is controlled by an endogenous clock, but the genetic mechanism that controls it remains unknown. Here we demonstrate that wild-type
Drosophila melanogaster
display a robust circadian rhythm in the mating activity, and that these rhythms are abolished in
period
- or
timeless
-null mutant flies (
per

01

and
tim

01

). Circadian rhythms were lost when rhythm mutant females were paired with wild-type males, demonstrating that female mating activity is governed by clock genes. Furthermore, we detected an antiphasic relationship in the circadian rhythms of mating activity between
D. melanogaster
and its sibling species
Drosophila simulans
. Female- and species-specific circadian rhythms in the mating activity of
Drosophila
seem to cause reproductive isolation.
Reelin molecules assemble together to form a large protein complex, which is inhibited by the function-blocking CR-50 antibody
Authors: Utsunomiya-Tate Naoko , Kubo Ken-ichiro , Tate Shin-ichi , Kainosho Masatsune , Katayama Eisaku , Nakajima Kazunori , Mikoshiba Katsuhiko
Published: July 2002

Reelin is a key mediator of ordered neuronal alignment in the brain. Here, we demonstrate that Reelin molecules assemble with each other to form a huge protein complex both
in vitro
and
in vivo
. The Reelin–Reelin interaction clearly is inhibited by the function-blocking anti-Reelin antibody, CR-50, at a concentration known to inhibit Reelin function. This assembly is mediated by electrostatic interaction of the CR-50 epitope region. Recombinant CR-50 epitope fragments spontaneously constitute a soluble, string-like homopolymer with a regularly repeated structure composed of more than 40 monomers. Mutated Reelin, which lacks the CR-50 epitope region, cannot form a homopolymer and fails to induce efficient tyrosine phosphorylation of Disabled 1 (Dab1), which should occur to transduce the Reelin signal. These data suggest that Reelin exerts its biological function by composing a large protein assembly driven by the CR-50 epitope region, proposing a novel model of the Reelin signaling in neurodevelopment.
Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase
Authors: Cravatt Benjamin F. , Demarest Kristin , Patricelli Matthew P. , Bracey Michael H. , Giang Dan K. , Martin Billy R. , Lichtman Aron H.
Published: July 2002

The medicinal properties of marijuana have been recognized for centuries, but clinical and societal acceptance of this drug of abuse as a potential therapeutic agent remains fiercely debated. An attractive alternative to marijuana-based therapeutics would be to target the molecular pathways that mediate the effects of this drug. To date, these neural signaling pathways have been shown to comprise a cannabinoid receptor (CB
1
) that binds the active constituent of marijuana, tetrahydrocannabinol (THC), and a postulated endogenous CB
1
ligand anandamide. Although anandamide binds and activates the CB
1
receptor
in vitro
, this compound induces only weak and transient cannabinoid behavioral effects
in vivo
, possibly a result of its rapid catabolism. Here we show that mice lacking the enzyme fatty acid amide hydrolase (FAAH
−/−
) are severely impaired in their ability to degrade anandamide and when treated with this compound, exhibit an array of intense CB
1
-dependent behavioral responses, including hypomotility, analgesia, catalepsy, and hypothermia. FAAH
−/−
-mice possess 15-fold augmented endogenous brain levels of anandamide and display reduced pain sensation that is reversed by the CB
1
antagonist SR141716A. Collectively, these results indicate that FAAH is a key regulator of anandamide signaling
in vivo
, setting an endogenous cannabinoid tone that modulates pain perception. FAAH may therefore represent an attractive pharmaceutical target for the treatment of pain and neuropsychiatric disorders.
Insulin alters heterogeneous nuclear ribonucleoprotein K protein binding to DNA and RNA
Authors: Ostrowski J. , Kawata Y. , Schullery D. S. , Denisenko O. N. , Higaki Y. , Abrass C. K. , Bomsztyk K.
Published: July 2002

The interaction of the multimodular heterogeneous nuclear ribonucleoprotein (hnRNP) K protein with many of its protein and nucleic acid partners is regulated by extracellular signals. Acting as a docking platform, K protein could link signal-transduction pathways to DNA- and RNA-directed processes such as transcription, mRNA processing, transport, and translation. Treatment of hepatocyte culture with insulin increased K protein tyrosine phosphorylation. Insulin altered K protein interaction with RNA and DNA
in vitro.
Administration of insulin into mice had similar effects on K protein in liver. Coimmunoprecipitations of RNA with K protein revealed preferential
in vivo
K protein binding of a subset of transcripts, including the insulin-inducible c-
fos
mRNA. These results suggest a class of insulin pathways that signal nucleic acid-directed processes that involve K protein.
Bacterial and yeast chaperones reduce both aggregate formation and cell death in mammalian cell models of Huntington's disease
Authors: Carmichael Jenny , Chatellier Jean , Woolfson Adrian , Milstein César , Fersht Alan R. , Rubinsztein David C.
Published: July 2002

Huntington's disease (HD) is an autosomal dominant neurodegenerative condition caused by expansions of more than 35 uninterrupted CAG repeats in exon 1 of the huntingtin gene. The CAG repeats in HD and the other seven known diseases caused by CAG codon expansions are translated into long polyglutamine tracts that confer a deleterious gain of function on the mutant proteins. Intraneuronal inclusions comprising aggregates of the relevant mutant proteins are found in the brains of patients with HD and related diseases. It is crucial to determine whether the formation of inclusions is directly pathogenic, because a number of studies have suggested that aggregates may be epiphenomena or even protective. Here, we show that fragments of the bacterial chaperone GroEL and the full-length yeast heat shock protein Hsp104 reduce both aggregate formation and cell death in mammalian cell models of HD, consistent with a causal link between aggregation and pathology.
Phenotypic diversity and ecosystem functioning in changing environments: A theoretical framework
Authors: Norberg Jon , Swaney Dennis P. , Dushoff Jonathan , Lin Juan , Casagrandi Renato , Levin Simon A.
Published: July 2002

Biodiversity plays a vital role for ecosystem functioning in a
changing environment. Yet theoretical approaches that incorporate
diversity into classical ecosystem theory do not provide a general
dynamic theory based on mechanistic principles. In this paper, we
suggest that approaches developed for quantitative genetics can be
extended to ecosystem functioning by modeling the means and variances
of phenotypes within a group of species. We present a framework that
suggests that phenotypic variance within functional groups is linearly
related to their ability to respond to environmental changes. As a
result, the long-term productivity for a group of species with high
phenotypic variance may be higher than for the best single species,
even though high phenotypic variance decreases productivity in the
short term, because suboptimal species are present. In addition, we
find that in the case of accelerating environmental change, species
succession in a changing environment may become discontinuous. Our work
suggests that this phenomenon is related to diversity as well as to the
environmental disturbance regime, both of which are affected by
anthropogenic activities. By introducing new techniques for modeling
the aggregate behavior of groups of species, the present approach may
provide a new avenue for ecosystem analysis.
When genetic distance matters: Measuring genetic differentiation at microsatellite loci in whole-genome scans of recent and incipient mosquito species
Authors: Wang Rui , Zheng Liangbiao , Touré Yeya T. , Dandekar Thomas , Kafatos Fotis C.
Published: July 2002

Genetic distance measurements are an important tool to differentiate field populations of disease vectors such as the mosquito vectors of malaria. Here, we have measured the genetic differentiation between
Anopheles arabiensis
and
Anopheles gambiae
, as well as between proposed emerging species of the latter taxon, in whole genome scans by using 23–25 microsatellite loci. In doing so, we have reviewed and evaluated the advantages and disadvantages of standard parameters of genetic distance,
F

ST

,
R

ST

, (δμ)
2
, and
D
. Further, we have introduced new parameters,
D′
and
D

K

, which have well defined statistical significance tests and complement the standard parameters to advantage.
D′
is a modification of
D
, whereas
D

K

is a measure of covariance based on Pearson's correlation coefficient. We find that
A. gambiae
and
A. arabiensis
are closely related at most autosomal loci but appear to be distantly related on the basis of
X
-linked chromosomal loci within the chromosomal
Xag
inversion. The M and S molecular forms of
A. gambiae
are practically indistinguishable but differ significantly at two microsatellite loci from the proximal region of the
X
, outside the
Xag
inversion. At one of these loci, both M and S molecular forms differ significantly from
A. arabiensis
, but remarkably, at the other locus,
A. arabiensis
is indistinguishable from the M molecular form of
A. gambiae
. These data support the recent proposal of genetically differentiated M and S molecular forms of
A. gambiae
.
Crystal structure of the quorum-sensing protein LuxS reveals a catalytic metal site
Authors: Hilgers Mark T. , Ludwig Martha L.
Published: July 2002

The ability of bacteria to regulate gene expression in response to
changes in cell density is termed quorum sensing. This behavior
involves the synthesis and recognition of extracellular, hormone-like
compounds known as autoinducers. Here we report the structure of an
autoinducer synthase, LuxS from
Bacillus subtilis
, at
1.6-Å resolution (
R
free
= 0.204;
R
work
= 0.174). LuxS is a homodimeric
enzyme with a novel fold that incorporates two identical tetrahedral
metal-binding sites. This metal center is composed of a
Zn
2+
atom coordinated by two histidines, a cysteine, and a
solvent molecule, and is reminiscent of active sites found in several
peptidases and amidases. Although the nature of the autoinducer
synthesized by LuxS cannot be deduced from the crystal structure,
features of the putative active site suggest that LuxS might catalyze
hydrolytic, but not proteolytic, cleavage of a small substrate. Our
analysis represents a test of structure-based functional assignment.
An acquired and a native penicillin-binding protein cooperate in building the cell wall of drug-resistant staphylococci
Authors: Pinho Mariana G. , de Lencastre Hermínia , Tomasz Alexander
Published: July 2002

The blanket resistance of methicillin-resistant
Staphylococcus aureus
to all β-lactam antibiotics—which had such a devastating impact on chemotherapy of staphylococcal infections—is related to the properties of the key component of this resistance mechanism: the “acquired” penicillin-binding protein (PBP)-2A, which has unusual low affinity for all β-lactam antibiotics. Until now, the accepted model of resistance implied that in the presence of β-lactam antibiotics in the surrounding medium, PBP2A must take over the biosynthesis of staphylococcal cell wall from the four native staphylococcal PBPs because the latter become rapidly acylated and inactivated at even low concentrations of the antibiotic. However, recent observations indicate that this model requires revision. Inactivation of the transglycosylase domain, but not the transpeptidase domain, of PBP2 of
S. aureus
prevents expression of β-lactam resistance, despite the presence of the low-affinity PBP2A. The observations suggest that cell-wall synthesis in the presence of β-lactam antibiotics requires the cooperative functioning of the transglycosylase domain of the native staphylococcal PBP2 and the transpeptidase domain of the PBP2A, a protein imported by
S. aureus
from an extra species source.
Immune mimicry in malaria:Plasmodium falciparumsecretes a functional histamine-releasing factor homologin vitroandin vivo
Authors: MacDonald Susan M. , Bhisutthibhan Jamaree , Shapiro Theresa A. , Rogerson Stephen J. , Taylor Terrie E. , Tembo Madalitso , Langdon Jacqueline M. , Meshnick Steven R.
Published: July 2002

ThePlasmodium falciparumtranslationally controlled tumor protein (TCTP) is a homolog of the mammalian histamine-releasing factor (HRF), which causes histamine release from human basophils and IL-8 secretion from eosinophils. Histamine, IL-8, and eosinophils have been reported to be elevated in patients with malaria. This study was undertaken to determine whether malarial TCTP is found in the plasma of malaria-infected patients and to determine whether it has HRF biologic activity. Malarial TCTP was found in lightly infected human volunteers and in heavily infected Malawian children, but not in uninfected patients. Recombinant malarial TCTP, like HRF, stimulated histamine release from basophils and IL-8 secretion from eosinophilsin vitro. Whereas malarial TCTP was less active than HRF, the concentrations that were effectivein vitrocould be achievablein vivo. These data suggest that malarial TCTP, present in human plasma during a malarial illness, may affect host immune responsesin vivo.
Coupled two-way clustering analysis of gene microarray data
Authors: Getz Gad , Levine Erel , Domany Eytan
Published: July 2002

We present a coupled two-way clustering approach to gene microarray
data analysis. The main idea is to identify subsets of the genes and
samples, such that when one of these is used to cluster the other,
stable and significant partitions emerge. The search for such subsets
is a computationally complex task. We present an algorithm, based on
iterative clustering, that performs such a search. This analysis is
especially suitable for gene microarray data, where the contributions
of a variety of biological mechanisms to the gene expression levels are
entangled in a large body of experimental data. The method was applied
to two gene microarray data sets, on colon cancer and leukemia. By
identifying relevant subsets of the data and focusing on them we were
able to discover partitions and correlations that were masked and
hidden when the full dataset was used in the analysis. Some of these
partitions have clear biological interpretation; others can serve to
identify possible directions for future research.
Telomere fusions caused by mutating the terminal region of telomeric DNA
Authors: McEachern Michael J. , Iyer Shilpa , Fulton Tracy Boswell , Blackburn Elizabeth H.
Published: July 2002

Mutations in the template region of a telomerase RNA gene can lead
to the corresponding sequence alterations appearing in newly
synthesized telomeric repeats. We analyzed a set of mutations in the
template region of the telomerase RNA gene (
TER1
) of the
budding yeast
Kluyveromyces lactis
that were predicted
to lead to synthesis of mutant telomeric repeats with disrupted binding
of the telomeric protein Rap1p. We showed previously that mutating the
left side of the 12-bp consensus Rap1p binding site led to immediate
and severe telomere elongation. Here, we show that, in contrast,
mutating either the right side of the site or both sides together leads
initially to telomere shortening. On additional passaging, certain
mutants of both categories exhibit telomere–telomere fusions. Often,
six new Bal-31-resistant, telomere repeat-containing bands appeared,
and we infer that each of the six
K. lactis
chromosomes
became circularized. These fusions were not stable, appearing
occasionally to resolve and then reform. We demonstrate directly that a
linear minichromosome introduced into one of the fusion mutant strains
circularized by means of end-to-end fusions of the mutant repeat
tracts. In contrast to the chromosomal circularization reported
previously in
Schizosaccharomyces pombe
mutants
defective in telomere maintenance, the
K. lactis
telomere fusions retained their telomeric DNA repeat sequences.
Neuronal death enhanced by
N
-methyl-
d
-aspartate antagonists
Authors: Ikonomidou Chrysanthy , Stefovska Vanya , Turski Lechoslaw
Published: July 2002

Glutamate promotes neuronal survival during brain development and
destroys neurons after injuries in the mature brain. Glutamate
antagonists are in human clinical trials aiming to demonstrate
limitation of neuronal injury after head trauma, which consists of both
rapid and slowly progressing neurodegeneration. Furthermore, glutamate
antagonists are considered for neuroprotection in chronic
neurodegenerative disorders with slowly progressing cell death only.
Therefore, humans suffering from Huntington's disease, characterized
by slowly progressing neurodegeneration of the basal ganglia, are
subjected to trials with glutamate antagonists. Here we demonstrate
that progressive neurodegeneration in the basal ganglia induced by
the mitochondrial toxin 3-nitropropionate or in the hippocampus by
traumatic brain injury is enhanced by
N
-methyl-
d
-aspartate antagonists but
ameliorated by α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate
antagonists. These observations reveal that
N
-methyl-
d
-aspartate antagonists may
increase neurodestruction in mature brain undergoing slowly progressing
neurodegeneration, whereas blockade of the action of glutamate at
α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors may be
neuroprotective.
Ouabain, a steroid hormone that signals with slow calcium oscillations
Authors: Aizman Oleg , Uhlén Per , Lal Mark , Brismar Hjalmar , Aperia Anita
Published: July 2002

The plant-derived steroid, digoxin, a specific inhibitor of Na,K-ATPase, has been used for centuries in the treatment of heart disease. Recent studies demonstrate the presence of a digoxin analog, ouabain, in mammalian tissue, but its biological role has not been elucidated. Here, we show in renal epithelial cells that ouabain, in doses causing only partial Na,K-ATPase inhibition, acts as a biological inducer of regular, low-frequency intracellular calcium ([Ca
2+
]
i
) oscillations that elicit activation of the transcription factor, NF-κB. Partial inhibition of Na,K-ATPase using low extracellular K
+
and depolarization of cells did not have these effects. Incubation of cells in Ca
2+
-free media, inhibition of voltage-gated calcium channels, inositol triphosphate receptor antagonism, and redistribution of actin to a thick layer adjacent to the plasma membrane abolished [Ca
2+
]
i
oscillations, indicating that they were caused by a concerted action of inositol triphosphate receptors and capacitative calcium entry via plasma membrane channels. Blockade of ouabain-induced [Ca
2+
]
i
oscillations prevented activation of NF-κB. The results demonstrate a new mechanism for steroid signaling via plasma membrane receptors and underline a novel role for the steroid hormone, ouabain, as a physiological inducer of [Ca
2+
]
i
oscillations involved in transcriptional regulation in mammalian cells.
Exploring the folding landscape of a structured RNA
Authors: Russell Rick , Zhuang Xiaowei , Babcock Hazen P. , Millett Ian S. , Doniach Sebastian , Chu Steven , Herschlag Daniel
Published: July 2002

Structured RNAs achieve their active states by traversing complex,
multidimensional energetic landscapes. Here we probe the folding
landscape of the
Tetrahymena
ribozyme by using a
powerful approach: the folding of single ribozyme molecules is followed
beginning from distinct regions of the folding landscape. The
experiments, combined with small-angle x-ray scattering results, show
that the landscape contains discrete folding pathways. These pathways
are separated by large free-energy barriers that prevent
interconversion between them, indicating that the pathways lie in deep
channels in the folding landscape. Chemical protection and mutagenesis
experiments are then used to elucidate the structural features that
determine which folding pathway is followed. Strikingly, a specific
long-range tertiary contact can either help folding or hinder folding,
depending on when it is formed during the process. Together these
results provide an unprecedented view of the topology of an RNA folding
landscape and the RNA structural features that underlie this
multidimensional landscape.
Role of the calcium-binding protein parvalbumin in short-term synaptic plasticity
Authors: Caillard Olivier , Moreno Herman , Schwaller Beat , Llano Isabel , Celio Marco R. , Marty Alain
Published: July 2002

GABAergic (GABA = γ-aminobutyric acid) neurons from
different brain regions contain high levels of parvalbumin, both in
their soma and in their neurites. Parvalbumin is a slow
Ca
2+
buffer that may affect the amplitude and time course
of intracellular Ca
2+
transients in terminals after an
action potential, and hence may regulate short-term synaptic
plasticity. To test this possibility, we have applied paired-pulse
stimulations (with 30- to 300-ms intervals) at GABAergic synapses
between interneurons and Purkinje cells, both in wild-type (PV+/+)
mice and in parvalbumin knockout (PV−/−) mice. We observed
paired-pulse depression in PV+/+ mice, but paired-pulse facilitation
in PV−/− mice. In paired recordings of connected
interneuron-Purkinje cells, dialysis of the presynaptic interneuron
with the slow Ca
2+
buffer EGTA (1 mM) rescues paired-pulse
depression in PV−/− mice. These data show that parvalbumin potently
modulates short-term synaptic plasticity.
Prevalence of small inversions in yeast gene order evolution
Authors: Seoighe Cathal , Federspiel Nancy , Jones Ted , Hansen Nancy , Bivolarovic Vesna , Surzycki Ray , Tamse Raquel , Komp Caridad , Huizar Lucas , Davis Ronald W. , Scherer Stewart , Tait Evelyn , Shaw Duncan J. , Harris David , Murphy Lee , Oliver Karen , Taylor Kate , Rajandream Marie-Adele , Barrell Bart G. , Wolfe Kenneth H.
Published: July 2002

Gene order evolution in two eukaryotes was studied by
comparing the
Saccharomyces cerevisiae
genome sequence
to extensive new data from whole-genome shotgun and cosmid sequencing
of
Candida albicans
. Gene order is substantially
different between these two yeasts, with only 9% of gene pairs that
are adjacent in one species being conserved as adjacent in the other.
Inversion of small segments of DNA, less than 10 genes long, has been a
major cause of rearrangement, which means that even where a pair of
genes has been conserved as adjacent, the transcriptional orientations
of the two genes relative to one another are often different. We
estimate that about 1,100 single-gene inversions have occurred since
the divergence between these species. Other genes that are adjacent in
one species are in the same neighborhood in the other, but their
precise arrangement has been disrupted, probably by multiple successive
multigene inversions. We estimate that gene adjacencies have been
broken as frequently by local rearrangements as by chromosomal
translocations or long-distance transpositions. A bias toward small
inversions has been suggested by other studies on animals and plants
and may be general among eukaryotes.
Drug-resistant
Drosophila
indicate glutamate-gated chloride channels are targets for the antiparasitics nodulisporic acid and ivermectin
Authors: Kane Nanci S. , Hirschberg Birgit , Qian Su , Hunt David , Thomas Brande , Brochu Richard , Ludmerer Steven W. , Zheng Yingcong , Smith McHardy , Arena Joseph P. , Cohen Charles J. , Schmatz Dennis , Warmke Jeffrey , Cully Doris F.
Published: July 2002

The fruit fly
Drosophila melanogaster
was used to
examine the mode of action of the novel insecticide and acaricide
nodulisporic acid. Flies resistant to nodulisporic acid were selected
by stepwise increasing the dose of drug in the culture media. The
resistant strain,

glc
1

, is at least 20-fold
resistant to nodulisporic acid and 3-fold cross-resistant to the
parasiticide ivermectin, and exhibited decreased brood size, decreased
locomotion, and bang sensitivity. Binding assays using

glc
1

head membranes showed a marked decrease
in the affinity for nodulisporic acid and ivermectin. A combination of
genetics and sequencing identified a proline to serine mutation (P299S)
in the gene coding for the glutamate-gated chloride channel subunit
DmGluClα. To examine the effect of this mutation on the biophysical
properties of DmGluClα channels, it was introduced into a recombinant
DmGluClα, and RNA encoding wild-type and mutant subunits was injected
into
Xenopus
oocytes. Nodulisporic acid directly
activated wild-type and mutant DmGluClα channels. However, mutant
channels were ≈10-fold less sensitive to activation by nodulisporic
acid, as well as ivermectin and the endogenous ligand
glutamate, providing direct evidence that nodulisporic acid and
ivermectin act on DmGluClα channels.
RNAi-mediated oncogene silencing confers resistance to crown gall tumorigenesis
Authors: Escobar Matthew A. , Civerolo Edwin L. , Summerfelt Kristin R. , Dandekar Abhaya M.
Published: July 2002

Crown gall disease, caused by the soil bacterium
Agrobacterium tumefaciens
, results in significant economic losses in perennial crops worldwide.
A. tumefaciens
is one of the few organisms with a well characterized horizontal gene transfer system, possessing a suite of oncogenes that, when integrated into the plant genome, orchestrate
de novo
auxin and cytokinin biosynthesis to generate tumors. Specifically, the
iaaM
and
ipt
oncogenes, which show ≈90% DNA sequence identity across studied
A. tumefaciens
strains, are required for tumor formation. By expressing two self-complementary RNA constructions designed to initiate RNA interference (RNAi) of
iaaM
and
ipt
, we generated transgenic
Arabidopsis thaliana
and
Lycopersicon esculentum
plants that are highly resistant to crown gall disease development. In
in vitro
root inoculation bioassays with two biovar I strains of
A. tumefaciens
, transgenic
Arabidopsis
lines averaged 0.0–1.5% tumorigenesis, whereas wild-type controls averaged 97.5% tumorigenesis. Similarly, several transformed tomato lines that were challenged by stem inoculation with three biovar I strains, one biovar II strain, and one biovar III strain of
A. tumefaciens
displayed between 0.0% and 24.2% tumorigenesis, whereas controls averaged 100% tumorigenesis. This mechanism of resistance, which is based on mRNA sequence homology rather than the highly specific receptor–ligand binding interactions characteristic of traditional plant resistance genes, should be highly durable. If successful and durable under field conditions, RNAi-mediated oncogene silencing may find broad applicability in the improvement of tree crop and ornamental rootstocks.
Surface organization and nanopatterning of collagen by dip-pen nanolithography
Authors: Wilson Donna L. , Martin Raquel , Hong Seunghun , Cronin-Golomb Mark , Mirkin Chad A. , Kaplan David L.
Published: July 2002

Collagen is a key fibrous protein in biological systems, characterized by a complex structural hierarchy as well as the ability to self-assemble into liquid crystalline mesophases. The structural features of collagen influence cellular responses and material properties, with importance for a wide range of biomaterials and tissue architectures. The mechanism by which fibrillar collagen structures form from liquid crystalline mesophases is not well characterized. We report positive printing of collagen and a collagen-like peptide down to 30–50-nm line widths, using the atomic force microscopy technique of dip-pen nanolithography. The method preserved the triple-helical structure and biological activity of collagen and even fostered the formation of characteristic higher levels of structural organization. The “direct-write” capability of biologically relevant molecules, while preserving their structure and functionality, provides tremendous flexibility in future biological device applications and in proteomics arrays, as well as a new strategy to study the important hierarchical assembly processes of biological systems.
Referred phantom sensations and cortical reorganization after spinal cord injury in humans
Authors: Moore Christopher I. , Stern Chantal E. , Dunbar Carolyn , Kostyk Sandra K. , Gehi Anil , Corkin Suzanne
Published: July 2002

To test the hypothesis that cortical remapping supports phantom
sensations, we examined referred phantom sensations and cortical
activation in humans after spinal-cord injury (SCI) at the thoracic
level (T3-T12). Of 12 SCI subjects, 9 reported phantom sensations, and
2 reported referred phantom sensations. In both of these subjects,
referred phantom sensations were evoked by contact in reference zones
(RZ) that were not adjacent in the periphery and were not predicted to
be adjacent in the postcentral gyrus (PoCG), suggesting that
representations separated by centimeters of cortical space were
simultaneously engaged. This finding was supported by functional MRI
(fMRI). In a subject with a T6-level complete SCI, contact in RZ on the
left or right forearm projected referred phantom sensations to the
ipsilateral chest. During fMRI, contact in either forearm RZ evoked
activity in the central PoCG (the position of the forearm
representation) and the medial PoCG (the position of the chest
representation) with ≥1.6 cm of nonresponsive cortex intervening. In
contrast, stimulation in non-RZ forearm and palm regions in this
subject and in lesion-matched SCI subjects evoked central but not
medial PoCG activation. Our findings support a relation between PoCG
activation and the percept of referred phantom sensations. These
results, however, present an alternative to somatotopic cortical
reorganization, namely, cortical plasticity expressed in coactivation
of nonadjacent representations. The observed pattern suggests that
somatotopic subcortical remapping, projected to the cortex, can support
perceptual and cortical reorganization after deafferentation in
humans.
Protein kinase activity of Tel1p and Mec1p, two
Saccharomyces cerevisiae
proteins related to the human ATM protein kinase
Authors: Mallory Julia C. , Petes Thomas D.
Published: July 2002

The
Saccharomyces cerevisiae
proteins Tel1p and
Mec1p are involved in telomere length regulation and cellular responses
to DNA damage. The closest relative of these proteins is the human
A
taxia
T
elangiectasia
M
utated (ATM) protein, a wortmannin-sensitive protein
kinase that primarily phosphorylates serines in an SQ motif. We
constructed yeast strains containing functional epitope-tagged versions
of Tel1p and Mec1p. We showed that immunoprecipitated Tel1p and Mec1p
were capable of
in vitro
phosphorylation of the
mammalian protein PHAS-I (
P
hosphorylated
H
eat and
A
cid
S
table
protein). These activities are sensitive to wortmannin. Tel1p
phosphorylates serine in an SQ motif in PHAS-I. Mutations in the kinase
domains of Tel1p and Mec1p result in loss of
in vitro
kinase activity and the
in vivo
phenotypes associated
with the null
tel1
and
mec1
mutations.
Specificity in transmembrane helix–helix interactions can define a hierarchy of stability for sequence variants
Authors: Fleming Karen G. , Engelman Donald M.
Published: July 2002

The folding, stability, and oligomerization of helical
membrane proteins depend in part on a precise set of packing
interactions between transmembrane helices. To understand the energetic
principles of these helix–helix interactions, we have used
alanine-scanning mutagenesis and sedimentation equilibrium analytical
ultracentrifugation to quantitatively examine the sequence dependence
of the glycophorin A transmembrane helix dimerization. In all cases, we
found that mutations to alanine at interface positions cost free energy
of association. In contrast, mutations to alanine away from the dimer
interface showed free energies of association that are insignificantly
different from wild-type or are slightly stabilizing. Our study further
revealed that the energy of association is not evenly distributed
across the interface, but that there are several “hot spots” for
interaction including both glycines participating in a GxxxG motif.
Inspection of the NMR structure indicates that simple principles of
protein–protein interactions can explain the changes in energy that
are observed. A comparison of the dimer stability between different
hydrophobic environments suggested that the hierarchy of stability for
sequence variants is conserved. Together, these findings imply that the
protein–protein interaction portion of the overall association energy
may be separable from the contributions arising from protein–lipid and
lipid–lipid energy terms. This idea is a conceptual simplification of
the membrane protein folding problem and has implications for
prediction and design.
A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in
Drosophila
Authors: Morin Xavier , Daneman Richard , Zavortink Michael , Chia William
Published: July 2002

In
Drosophila
, enhancer trap strategies allow rapid
access to expression patterns, molecular data, and mutations in trapped
genes. However, they do not give any information at the protein
level, e.g., about the protein subcellular localization. Using the
green fluorescent protein (GFP) as a mobile artificial exon carried by
a transposable P-element, we have developed a protein trap system. We
screened for individual flies, in which GFP tags full-length
endogenous proteins expressed from their
endogenous locus, allowing us to observe their cellular and
subcellular distribution. GFP fusions are targeted to virtually any
compartment of the cell. In the case of insertions in previously known
genes, we observe that the subcellular localization of the fusion
protein corresponds to the described distribution of the
endogenous protein. The artificial GFP exon does not
disturb upstream and downstream splicing events. Many insertions
correspond to genes not predicted by the
Drosophila
Genome Project. Our results show the feasibility of a protein trap in
Drosophila
. GFP reveals in real time the dynamics of
protein's distribution in the whole, live organism and provides useful
markers for a number of cellular structures and compartments.