bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular identification by “suicide PCR” of
Yersinia pestis
as the agent of Medieval Black Death
Authors: Raoult Didier , Aboudharam Gérard , Crubézy Eric , Larrouy Georges , Ludes Bertrand , Drancourt Michel
Published: July 2002

Medieval Black Death is believed to have killed up to one-third of
the Western European population during the 14th century. It was
identified as plague at this time, but recently the causative organism
was debated because no definitive evidence has been obtained to confirm
the role of
Yersinia pestis
as the agent of plague. We
obtained the teeth of a child and two adults from a 14th century grave
in France, disrupted them to obtain the pulp, and applied the new
“suicide PCR” protocol in which the primers are used only once.
There were no positive controls: Neither
Yersinia
nor
Yersinia
DNA were introduced in the laboratory. A
negative result is followed by a new test using other primers; a
positive result is followed by sequencing. The second and third primer
pair used, coding for a part of the
pla
gene, generated
amplicons whose sequence confirmed that it was
Y. pestis
in 1 tooth from the child and 19/19 teeth from the adults. Negative
controls were negative. Attempts to detect the putative alternative
etiologic agents
Bacillus anthracis
and
Rickettsia prowazekii
failed. Suicide PCR avoids any
risk of contamination as it uses a single-shot primer—its specificity
is absolute. We believe that we can end the controversy: Medieval Black
Death was plague.
Cooperative interactions in the induction of long-term potentiation and depression of synaptic excitation between hippocampal CA3-CA1 cell pairs in vitro.
Authors: Debanne D , Gähwiler B H , Thompson S M
Published: July 2002

The requirement for cooperative interactions between multiple synaptic inputs in the induction of long-term potentiation (LTP) and long-term depression (LTD) has been tested at Schaffer collateral synapses with paired recordings from monosynaptically coupled CA3-CA1 cell pairs in rat hippocampal slice cultures. Tetanization of single presynaptic neurons at 50 Hz (repeated 5-7 times for 300-500 ms each) induced only a transient potentiation (< 3 min) of excitatory postsynaptic potentials (EPSPs). Persistent potentiation (> 15 min) was induced only when single presynaptic action potentials were synchronously paired with directly induced postsynaptic depolarizing pulses (repeated 50-100 times). Tetanus-induced potentiation of extracellularly evoked EPSPs lasting > 4 min could only be obtained if the EPSP was > 4 mV. Because unitary EPSP amplitudes average approximately 1 mV, we conclude that high-frequency discharge must occur synchronously] in 4-5 CA3 cells for LTP to be induced in a common postsynaptic CA1 cell. Asynchronous pairing of presynaptic action potentials with postsynaptic depolarizing current pulses (preceding each EPSP by 800 ms) depressed both naive and previously potentiated unitary EPSPs. Likewise, homosynaptic LTD of unitary EPSPs was induced when the presynaptic cell was tetanized at 3 Hz for 3 min, regardless of their amplitude (0.3-3.2 mV). Homosynaptic LTD of extracellularly evoked Schaffer collateral EPSPs < 4 mV could be induced if no inhibitory postsynaptic potential was apparent, but was prevented by eliciting a large inhibitory postsynaptic potential or by injection of hyperpolarizing current in the postsynaptic cell. We conclude that cooperative interactions among multiple excitatory inputs are not required for induction of homosynaptic LTD of unitary EPSPs.
Epstein-Barr virus vectors for gene delivery to B lymphocytes.
Authors: Robertson E S , Ooka T , Kieff E D
Published: July 2002

Basic research in Epstein-Barr virus (EBV) molecular genetics has provided means to maintain episomes in human cells, to efficiently deliver episomes with up to 150 kbp of heterologous DNA to human B lymphocytes, and to immortalize human B lymphocytes with EBV recombinants that can maintain up to 120 kbp of heterologous DNA. Episome maintenance requires an EBV nuclear protein, EBNA1, whereas immortalization of cells with EBV recombinants requires EBNA1, EBNA2, EBNA3A, EBNA3C, EBNALP, and LMP1. EBV-derived vectors are useful for experimental genetic reconstitution in B lymphocytes, a cell type frequently used in establishing repositories of human genetic deficiencies. The ability of EBV-infected cells to establish a balanced state of persistence in normal humans raises the possibility that cells infected with EBV recombinants could be useful for genetic reconstitution, in vivo.
Impact of vaccination on the spatial correlation and persistence of measles dynamics.
Authors: Bolker B M , Grenfell B T
Published: July 2002

The onset of measles vaccination in England and Wales in 1968 coincided with a marked drop in the temporal correlation of epidemic patterns between major cities. We analyze a variety of hypotheses for the mechanisms driving this change. Straightforward stochastic models suggest that the interaction between a lowered susceptible population (and hence increased demographic noise) and nonlinear dynamics is sufficient to cause the observed drop in correlation. The decorrelation of epidemics could potentially lessen the chance of global extinction and so inhibit attempts at measles eradication.
Corticotropin-releasing factor-binding protein ligand inhibitor blunts excessive weight gain in genetically obese Zucker rats and rats during nicotine withdrawal
Authors: Heinrichs Stephen C. , Lapsansky Jeannette , Behan Dominic P. , Chan Raymond K. W. , Sawchenko Paul E. , Lorang Marge , Ling Nicholas , Vale Wylie W. , De Souza Errol B.
Published: July 2002

Elevation of the neuropeptide corticotropin-releasing factor (CRF)
in the brain is associated with a reduction of food intake and body
weight gain in normal and obese animals. A protein that binds CRF and
the related peptide, urocortin, with high affinity, CRF-binding protein
(CRF-BP), may play a role in energy homeostasis by inactivating members
of this peptide family in ingestive and metabolic regulatory brain
regions. Intracerebroventricular administration in rats of the
high-affinity CRF-BP ligand inhibitor, rat/human CRF (6-33), which
dissociates CRF or urocortin from CRF-BP and increases
endogenous brain levels of “free” CRF or urocortin
significantly blunted exaggerated weight gain in Zucker obese subjects
and in animals withdrawn from chronic nicotine. Chronic administration
of CRF suppressed weight gain nonselectively by 60% in both Zucker
obese and lean control rats, whereas CRF-BP ligand inhibitor treatment
significantly reduced weight gain in obese subjects, without altering
weight gain in lean control subjects. Nicotine abstinent subjects, but
not nicotine-naive controls, experienced a 35% appetite suppression
and a 25% weight gain reduction following acute and chronic
administration, respectively, of CRF-BP ligand inhibitor. In marked
contrast to the effects of a CRF-receptor agonist, the CRF-BP ligand
inhibitor did not stimulate adrenocorticotropic hormone secretion or
elevate heart rate and blood pressure. These results provide support
for the hypothesis that the CRF-BP may function within the brain to
limit selected actions of CRF and/or urocortin. Furthermore, CRF-BP
may represent a novel and functionally selective target for the
symptomatic treatment of excessive weight gain associated with obesity
of multiple etiology.
Chloroplast DNA evidence of colonization, adaptive radiation, and hybridization in the evolution of the Macaronesian flora.
Authors: Francisco-Ortega J , Jansen R K , Santos-Guerra A
Published: July 2002

Most evolutionary studies of oceanic islands have focused on the Pacific Ocean. There are very few examples from the Atlantic archipelagos, especially Macaronesia, despite their unusual combination of features, including a close proximity to the continent, a broad range of geological ages, and a biota linked to a source area that existed in the Mediterranean basin before the late Tertiary. A chloroplast DNA (cpDNA) restriction site analysis of Argyranthemum (Asteraceae: Anthemideae), the largest endemic genus of plants of any volcanic archipelago in the Atlantic Ocean, was performed to examine patterns of plant evolution in Macaronesia. cpDNA data indicated that Argyranthemum is a monophyletic group that has speciated recently. The cpDNA tree showed a weak correlation with the current sectional classification and insular distribution. Two major cpDNA lineages were identified. One was restricted to northern archipelagos--e.g., Madeira, Desertas, and Selvagens--and the second comprised taxa endemic to the southern archipelago--e.g., the Canary Islands. The two major radiations identified in the Canaries are correlated with distinct ecological habitats; one is restricted to ecological zones under the influence of the northeastern trade winds and the other to regions that are not affected by these winds. The patterns of phylogenetic relationships in Argyranthemum indicate that interisland colonization between similar ecological zones is the main mechanism for establishing founder populations. This phenomenon, combined with rapid radiation into distinct ecological zones and interspecific hybridization, is the primary explanation for species diversification.
Cell density regulates cellular reversal frequency in Myxococcus xanthus.
Authors: Shi W , Ngok F K , Zusman D R
Published: July 2002

Myxococcus xanthus is a Gram-negative bacterium that aggregates to form fruiting bodies when nutrients are limiting. Previous studies showed that the frz mutants that are defective in chemotaxis exhibited irregular and infrequent patterns of cellular reversal. In contrast, wild-type cells, when examined individually, reverse relatively frequently, about once every 6 min. It is not known how the change of reversal frequency effects cellular aggregation during fruiting body formation in M. xanthus. In this study, we stained cells with a tetrazolium dye so that we could track the reversal frequencies of single cells and cells in groups. We found that developmental cells in large groups reverse much less than cells in small groups or as single cells. This reduced cellular reversal frequency is related to the frz signal transduction system and correlated with the methylation of FrzCD (a methyl-accepting chemotaxis protein). Cells containing a mutation in the frz genes or in the genes required for social motility do not respond in this way. The reduction in cellular reversals as developmental cells accumulate in groups suggests a simple hypothesis for the aggregation of cells into discrete mounds during fruiting body formation. We also found that M. xanthus cells glide with equal frequency in the forward or reverse directions, indicating that cells do not contain a "head" or "tail."
Cloning and characterization of a functional human homolog of
Escherichia coli
endonuclease III
Authors: Aspinwall Richard , Rothwell Dominic G. , Roldan-Arjona Teresa , Anselmino Catherine , Ward Christopher J. , Cheadle Jeremy P. , Sampson Julian R. , Lindahl Tomas , Harris Peter C. , Hickson Ian D.
Published: July 2002

Repair of oxidative damage to DNA bases is essential to prevent mutations and cell death. Endonuclease III is the major DNA glycosylase activity in
Escherichia coli
that catalyzes the excision of pyrimidines damaged by ring opening or ring saturation, and it also possesses an associated lyase activity that incises the DNA backbone adjacent to apurinic/apyrimidinic sites. During analysis of the area adjacent to the human tuberous sclerosis gene (
TSC2
) in chromosome region 16p13.3, we identified a gene,
OCTS3
, that encodes a 1-kb transcript. Analysis of
OCTS3
cDNA clones revealed an open reading frame encoding a predicted protein of 34.3 kDa that shares extensive sequence similarity with
E. coli
endonuclease III and a related enzyme from
Schizosaccharomyces pombe
, including a conserved active site region and an iron/sulfur domain. The product of the
OCTS3
gene was therefore designated hNTH1 (human endonuclease III homolog 1). The hNTH1 protein was overexpressed in
E. coli
and purified to apparent homogeneity. The recombinant protein had spectral properties indicative of the presence of an iron/sulfur cluster, and exhibited DNA glycosylase activity on double-stranded polydeoxyribonucleotides containing urea and thymine glycol residues, as well as an apurinic/apyrimidinic lyase activity. Our data indicate that hNTH1 is a structural and functional homolog of
E. coli
endonuclease III, and that this class of enzymes, for repair of oxidatively damaged pyrimidines in DNA, is highly conserved in evolution from microorganisms to human cells.
In vivo
analysis of the functional domains of the
Drosophila
splicing regulator RBP1
Authors: Heinrichs Volker , Baker Bruce S.
Published: July 2002

The
Drosophila
splicing factor RBP1 participates together with TRA and TRA-2 in the regulation of alternative splicing of
doublesex
(
dsx
) pre-mRNA. It does so by recognizing RBP1 RNA target sequences in the
dsx
pre-mRNA. RBP1 belongs to the Ser–Arg-rich (SR) protein family of splicing factors, which have in common a N-terminal RNA recognition motif-type RNA binding domain, a Gly-rich region, and a C-terminal SR domain. Using a tissue culture transfection assay, we demonstrate that the Gly residues within the Gly-rich domain, the ribonucleoprotein motifs within the RNA recognition motif RNA binding domain, and the SR domain are required for regulation of
dsx
splicing by RBP1
in vivo
. Furthermore, using a two-hybrid system, we show protein–protein interactions between RBP1 and itself and between RBP1 and TRA-2. The SR domain and the Gly residues within the Gly-rich domain of RBP1 were found to be involved in these protein–protein interactions. Our results suggest that RBP1 and TRA-2 function in regulation of
dsx
splicing by forming a complex.
Cyclic AMP and its receptor protein negatively regulate the coordinate expression of cholera toxin and toxin-coregulated pilus in
Vibrio cholerae
Authors: Skorupski Karen , Taylor Ronald K.
Published: July 2002

Insertion mutations in two
Vibrio cholerae
genes,
cya
and
crp
, which encode adenylate cyclase and the cyclic AMP (cAMP) receptor protein (CRP), respectively, derepressed the expression of a chromosomal cholera toxin (CT) promoter–
lacZ
fusion at the nonpermissive temperature of 37°C. In the classical biotype strain O395, the
crp
mutation increased the production of both CT and toxin-coregulated pilus (TCP)
in vitro
under a variety of growth conditions not normally permissive for their expression. The most dramatic increase in CT and TCP was observed with the
crp
mutant in Luria–Bertani (LB) medium pH 8.5, at 30°C. El Tor biotype strains differ from classical strains in that they do not produce CT or TCP when grown in LB media. Incorporation of the
crp
mutation into El Tor strain C6706 permitted production of these proteins in LB medium pH 6.5, at 30°C. In the infant mouse cholera model, the
crp
mutation decreased colonization in both biotypes at least 100-fold relative to the wild-type strains. The data presented here suggest a model whereby cAMP–CRP negatively regulates the expression of CT and TCP in both classical and El Tor biotypes under certain environmental conditions and also influences pathogenesis by regulating other processes necessary for optimal growth
in vivo
.
Molecular evolution of angiosperm mitochondrial introns and exons
Authors: Laroche Jérôme , Li Peng , Maggia Laurent , Bousquet Jean
Published: July 2002

Numbers of substitutions per site for 15 protein-coding genes and six introns of the plant mitochondria were estimated to compare modes and tempos of evolution between exons and introns, and numbers of insertions–deletions per site also were investigated in introns. Intra-gene homogeneity of numbers of substitutions per site was assessed further among different taxa and between mitochondrial and nuclear paralogs translocated from the mitochondrial genome. Gene-to-gene differences in numbers of substitutions per site were found to be higher for nonsynonymous than synonymous sites, and this could be due to differential selection if mutation rate is assumed constant for the genome. Some mitochondrial genes have evolved as fast as chloroplast genes, thus faster than previously thought. For
cox
I, relative rate tests showed that woody taxa evolved slower than annuals at synonymous sites. Generation time, population size, and speciation rate are likely factors involved in this rate heterogeneity. Introns were less constrained than their adjacent exons for both overall numbers of substitutions per site and indels, but, on average, overall numbers of substitutions per site for introns were similar to numbers of synonymous substitutions per site for exons. Correlations were generally high between numbers of substitutions and numbers of indels per site for the same intron. Mitochondrial genes transferred to the nucleus had an accelerated rate of substitution per site, which was most significant at synonymous sites. These differences between paralogs in two different genomes are likely the result of different mutation rates.
Transitions in the coupling of transcription and nucleotide excision repair within RNA polymerase II-transcribed genes of
Saccharomyces cerevisiae
Authors: Tijsterman Marcel , Verhage Richard A. , van de Putte Pieter , Jong Judith G. Tasseron-de , Brouwer Jaap
Published: July 2002

The molecular mechanism of transcription-coupled nucleotide excision repair in eukaryotes is poorly understood. The identification of the dual role of basal transcription factor TFIIH in DNA repair and transcription provided a plausible link between both processes. However, TFIIH is not part of the elongating transcription complex, suggesting that additional components are required to recruit TFIIH when RNA polymerase II (RNAPII) stalls at the site of DNA damage. Previously, we have shown that the yeast Rad26 protein is involved in transcription-coupled DNA repair. This paper describes the differential contribution of the Rad26 protein to efficient removal of UV-induced cyclobutane pyrimidine dimers (CPDs) from transcribed DNA. Two distinct regions within the transcribed strand of RNAPII-transcribed genes are identified that differ in their requirement for the
RAD26
gene product. Using high-resolution repair analysis, we determined the
in vivo
repair kinetics of cyclobutane pyrimidine dimers positioned around the transcription initiation site of RNAPII-transcribed genes
RPB2
and
URA3
. Although transcription-coupled repair is severely reduced in
rad26
mutants, lesions positioned in a small region immediately downstream of transcription initiation are efficiently removed in the absence of Rad26. The observed transition in repair characteristics is abrupt and in excellent agreement with the region where TFIIH dissociates from RNAPII
in vitro
, strongly suggesting an inverse correlation between TFIIH association and Rad26 requirement. These data suggest that a transcription repair coupling factor (Rad26/CSB) is required for efficient repair only during the elongating stages of RNAPII transcription.
On the relationship between synaptic input and spike output jitter in individual neurons
Authors: Maršálek Petr , Koch Christof , Maunsell John
Published: July 2002

What is the relationship between the temporal jitter in the arrival times of individual synaptic inputs to a neuron and the resultant jitter in its output spike? We report that the rise time of firing rates of cells in striate and extrastriate visual cortex in the macaque monkey remain equally sharp at different stages of processing. Furthermore, as observed by others, multiunit recordings from single units in the primate frontal lobe reveal a strong peak in their cross-correlation in the 10–150 msec range with very small temporal jitter (on the order of 1 msec). We explain these results using numerical models to study the relationship between the temporal jitter in excitatory and inhibitory synaptic input and the variability in the spike output timing in integrate-and-fire units and in a biophysically and anatomically detailed model of a cortical pyramidal cell. We conclude that under physiological circumstances, the standard deviation in the output jitter is linearly related to the standard deviation in the input jitter, with a constant of less than one. Thus, the timing jitter in successive layers of such neurons will converge to a small value dictated by the jitter in axonal propagation times.
Prospero is a panneural transcription factor that modulates homeodomain protein activity
Authors: Hassan Bassem , Li Ling , Bremer Kirsten A. , Chang Weiru , Pinsonneault Julia , Vaessin Harald
Published: July 2002

The panneural protein Prospero is required for proper differentiation of neuronal lineages and proper expression of several genes in the nervous system of
Drosophila
. Prospero is an evolutionarily conserved, homeodomain-related protein with dual subcellular localization. Here we show that Prospero is a sequence-specific DNA-binding protein with novel sequence preferences that can act as a transcription factor. In this role, Prospero can interact with homeodomain proteins to differentially modulate their DNA-binding properties. The relevance of functional interactions between Prospero and homeodomain proteins is supported by the observation that Prospero, together with the homeodomain protein Deformed, is required for proper regulation of a Deformed-dependent neural-specific transcriptional enhancer. We have localized the DNA-binding and homeodomain protein-interacting activities of Prospero to its highly conserved C-terminal region, and we have shown that the two regulatory capacities are independent.
A nonnatural transcriptional coactivator
Authors: Nyanguile Origène , Uesugi Motonari , Austin David J. , Verdine Gregory L.
Published: July 2002

In eukaryotes, sequence-specific DNA-binding proteins activate gene expression by recruiting the transcriptional apparatus and chromatin remodeling proteins to the promoter through protein-protein contacts. In many instances, the connection between DNA-binding proteins and the transcriptional apparatus is established through the intermediacy of adapter proteins known as coactivators. Here we describe synthetic molecules with low molecular weight that act as transcriptional coactivators. We demonstrate that a completely nonnatural activation domain in one such molecule is capable of stimulating transcription
in vitro
and
in vivo
. The present strategy provides a means of gaining external control over gene activation through intervention using small molecules.
Subunit structure of the mammalian exocyst complex
Authors: Kee Yun , Yoo Jin-San , Hazuka Christopher D. , Peterson Karen E. , Hsu Shu-Chan , Scheller Richard H.
Published: July 2002

The exocyst is a protein complex required for the late stages of secretion in yeast. Unlike the SNAREs (SNAP receptors), important secretory proteins that are broadly distributed on the target membrane, the exocyst is specifically located at sites of vesicle fusion. We have isolated cDNAs encoding the rexo70, rsec5, and rsec15 subunits of the mammalian complex. The amino acid sequences encoded by these genes are between 21% and 24% identical to their yeast homologs. All three genes are broadly expressed and multiple transcripts are observed for rexo70 and rsec15. Characterization of cDNAs encoding the 84-kDa subunit of the mammalian complex revealed a novel protein. mAbs were generated to the mammalian rsec6 subunit of the exocyst complex. rsec6 immunoreactivity is found in a punctate distribution at terminals of PC12 cell processes at or near sites of granule exocytosis.
Multiple receptors for HLA-G on human natural killer cells
Authors: Mandelboim O. , Pazmany L. , Davis D. M. , Valés-Gómez M. , Reyburn H. T. , Rybalov B. , Strominger J. L.
Published: July 2002

HLA-G is the putative natural killer (NK) cell inhibitory ligand expressed on the extravillous cytotrophoblast of the human placenta. Killing of the class I negative human B cell line 721.221 by NK cells is inhibited by the expression of HLA-G. This inhibition is dependent on a high level of HLA-G expression. In the present study, the nature of the receptors that mediate the inhibition has been studied with 140 NK cell lines from two donors and 246 NK clones from 5 donors by blocking the inhibition using monoclonal antibodies against the known NK inhibitory receptors: CD158a, CD158b, and CD94. Both CD94 and the two CD158 proteins can function as receptors, although the former clearly predominates. In many cases, a combination of antibodies to these receptors is required to achieve maximal reversal of inhibition. Moreover, in at least one-third of the NK cells that are inhibited by HLA-G, these antibodies alone or in combination do not reverse inhibition, strongly suggesting the existence of a third major unidentified receptor for HLA-G.
Coordination dynamics of biological zinc “clusters” in metallothioneins and in the DNA-binding domain of the transcription factor Gal4
Authors: Maret Wolfgang , Larsen Kjeld S. , Vallee Bert L.
Published: July 2002

The almost universal appreciation for the importance of zinc in metabolism has been offset by the considerable uncertainty regarding the proteins that store and distribute cellular zinc. We propose that some zinc proteins with so-called zinc cluster motifs have a central role in zinc distribution, since they exhibit the rather exquisite properties of binding zinc tightly while remaining remarkably reactive as zinc donors. We have used zinc isotope exchange both to probe the coordination dynamics of zinc clusters in metallothionein, the small protein that has the highest known zinc content, and to investigate the potential function of zinc clusters in cellular zinc distribution. When mixed and incubated, metallothionein isoproteins-1 and -2 rapidly exchange zinc, as demonstrated by fast chromatographic separation and radiometric analysis. Exchange kinetics exhibit two distinct phases (
k
fast
≃ 5000 min
−1
·M
−1
;
k
slow
≃ 200 min
−1
·M
−1
, pH 8.6, 25°C) that are thought to reflect exchange between the three-zinc clusters and between the four-zinc clusters, respectively. Moreover, we have observed and examined zinc exchange between metallothionein-2 and the Gal4 protein (
k
≃ 800 min
−1
·M
−1
, pH 8.0, 25°C), which is a prototype of transcription factors with a two-zinc cluster. This reaction constitutes the first experimental example of intermolecular zinc exchange between heterologous proteins. Such kinetic reactivity distinguishes zinc in biological clusters from zinc in the coordination environment of zinc enzymes, where the metal does not exchange over several days with free zinc in solution. The molecular organization of these clusters allows zinc exchange to proceed through a ligand exchange mechanism, involving molecular contact between the reactants.
Cell proliferation and hair tip growth in the
Arabidopsis
root are under mechanistically different forms of redox control
Authors: Sánchez-Fernández Rocío , Fricker Mark , Corben Liz B. , White Nick S. , Sheard Nicki , Leaver Christopher J. , Van Montagu Marc , Inzé Dirk , May Mike J.
Published: July 2002

We provide evidence that the tripeptide thiol glutathione (GSH) participates in the regulation of cell division in the apical meristem of
Arabidopsis
roots. Exogenous application of micromolar concentrations of GSH raised the number of meristematic cells undergoing mitosis, while depletion of GSH had the opposite effect. A role for endogenous GSH in the control of cell proliferation is also provided by mapping of GSH levels in the root meristem using the GSH-specific dye monochlorobimane and confocal laser scanning microscopy. High levels of GSH were associated with the epidermal and cortical initials and markedly lower levels in the quiescent center. The mechanisms controlling cell division could also be triggered by other reducing agents: ascorbic acid and dithiothreitol. Our data also reveal significant plasticity in the relationship between the trichoblast cell length and the hair it subtends in response to alterations in intracellular redox homeostasis. While mechanisms that control trichoblast elongation are influenced by nonspecific redox couples, root hair tip growth has a more specific requirement for sulfhydryl groups. The responses we describe here may represent the extremes of redox control of root plasticity and would allow the root to maintain exploration of the soil under adverse conditions with minimal cell divisions and root hair production or capitalize on a favorable environment by production of numerous long hairs. Redox sensing of the environment and subsequent redox-dependent modulation of growth and development may be crucial components in the strategies plants have evolved for survival in a fluctuating environment.
A chloroplast processing enzyme functions as the general stromal processing peptidase
Authors: Richter Stefan , Lamppa Gayle K.
Published: July 2002

A highly specific stromal processing activity is thought to cleave a large diversity of precursors targeted to the chloroplast, removing an N-terminal transit peptide. The identity of this key component of the import machinery has not been unequivocally established. We have previously characterized a chloroplast processing enzyme (CPE) that cleaves the precursor of the light-harvesting chlorophyll
a/b
binding protein of photosystem II (LHCPII). Here we report the overexpression of active CPE in
Escherichia coli
. Examination of the recombinant enzyme
in vitro
revealed that it cleaves not only preLHCPII, but also the precursors for an array of proteins essential for different reactions and destined for different compartments of the organelle. CPE also processes its own precursor in trans. Neither the recombinant CPE nor the native CPE of chloroplasts process a preLHCPII mutant with an altered cleavage site demonstrating that both forms of the enzyme are sensitive to the same structural modification of the substrate. The transit peptide of the precursor of ferredoxin is released by a single cleavage event and found intact after processing by recombinant CPE and a chloroplast extract as well. These results provide the first direct demonstration that CPE is the general stromal processing peptidase that acts as an endopeptidase. Significantly, recombinant CPE cleaves in the absence of other chloroplast proteins, and this activity depends on metal cations, such as zinc.
Genetic analysis of a mammalian wound-healing trait
Authors: McBrearty Beth Ann , Clark Lise Desquenne , Zhang Xiang-Ming , Blankenhorn Elizabeth P. , Heber-Katz Ellen
Published: July 2002

Wound healing of mammalian tissue is an essential process in the maintenance of body integrity. The general mechanism of wound healing usually studied in adult mammals is repair, in contrast to the regeneration seen in more primitive vertebrates. We recently have discovered that MRL/MpJ mice, unlike all other strains of mice tested, undergo rapid and complete wound closure that resembles regeneration. Specifically, through-and-through surgical ear hole wounds close without scarring in <4 weeks with normal gross and microanatomic architecture, including chondrogenesis. We also demonstrated that this healing is a heritable trait in inbred mice. In this study, we present results pertaining to its genetic control in progeny segregating for this phenotype. To identify the genetic loci that control the wound closure process, a genome-wide scan was performed on (MRL/MpJ-
Fas
lpr
× C57BL/6)F2 and backcross populations. In the primary screens of these populations, quantitative trait loci that control the extent of wound closure were detected on chromosomes 8, 12, and 15 and at two separate locations on chromosome 13. Evidence of further genetic control of healing was found on chromosome 7. All alleles that contribute to full wound closure are derived from the MRL/MpJ-
Fas
lpr
parent except for the quantitative trait locus on chromosome 8, which is derived from C57BL/6.
Tracking molecular evolution of photosynthesis by characterization of a major photosynthesis gene cluster from
Heliobacillus mobilis
Authors: Xiong Jin , Inoue Kazuhito , Bauer Carl E.
Published: July 2002

A DNA sequence has been obtained for a 35.6-kb genomic segment from
Heliobacillus mobilis
that contains a major cluster of photosynthesis genes. A total of 30 ORFs were identified, 20 of which encode enzymes for bacteriochlorophyll and carotenoid biosynthesis, reaction-center (RC) apoprotein, and cytochromes for cyclic electron transport. Donor side electron-transfer components to the RC include a putative RC-associated cytochrome
c
553
and a unique four-large-subunit cytochrome
bc
complex consisting of Rieske Fe-S protein (encoded by
petC
), cytochrome

b
6

(
petB
), subunit IV (
petD
), and a diheme cytochrome
c
(
petX
). Phylogenetic analysis of various photosynthesis gene products indicates a consistent grouping of oxygenic lineages that are distinct and descendent from anoxygenic lineages. In addition,
H. mobilis
was placed as the closest relative to cyanobacteria, which form a monophyletic origin to chloroplast-based photosynthetic lineages. The consensus of the photosynthesis gene trees also indicates that purple bacteria are the earliest emerging photosynthetic lineage. Our analysis also indicates that an ancient gene-duplication event giving rise to the paralogous
bchI
and
bchD
genes predates the divergence of all photosynthetic groups. In addition, our analysis of gene duplication of the photosystem I and photosystem II core polypeptides supports a “heterologous fusion model” for the origin and evolution of oxygenic photosynthesis.
Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating proton flux
Authors: Shimizu Shigeomi , Eguchi Yutaka , Kamiike Wataru , Funahashi Yoshimitsu , Mignon Alexandre , Lacronique Virginia , Matsuda Hikaru , Tsujimoto Yoshihide
Published: July 2002

We and others have recently shown that loss of the mitochondrial membrane potential (Δψ) precedes apoptosis and chemical-hypoxia-induced necrosis and is prevented by Bcl-2. In this report, we examine the biochemical mechanism used by Bcl-2 to prevent Δψ loss, as determined with mitochondria isolated from a cell line overexpressing human Bcl-2 or from livers of Bcl-2 transgenic mice. Although Bcl-2 had no effect on the respiration rate of isolated mitochondria, it prevented both Δψ loss and the permeability transition (PT) induced by various reagents, including Ca
2+
, H
2
O
2
, and
tert
-butyl hydroperoxide. Even under conditions that did not allow PT, Bcl-2 maintained Δψ, suggesting that the functional target of Bcl-2 is regulation of Δψ but not PT. Bcl-2 also maintained Δψ in the presence of the protonophore SF6847, which induces proton influx, suggesting that Bcl-2 regulates ion transport to maintain Δψ. Although treatment with SF6847 in the absence of Ca
2+
caused massive H
+
influx in control mitochondria, the presence of Bcl-2 induced H
+
efflux after transient H
+
influx. In this case, Bcl-2 did not enhance K
+
efflux. Furthermore, Bcl-2 enhanced H
+
efflux but not K
+
flux after treatment of mitochondria with Ca
2+
or
tert
-butyl hydroperoxide. These results suggest that Bcl-2 maintains Δψ by enhancing H
+
efflux in the presence of Δψ-loss-inducing stimuli.
Helioseismology: Probing the interior of a star
Authors: Demarque P. , Guenther D. B.
Published: July 2002

Helioseismology offers, for the first time,
an opportunity to probe in detail the deep interior of a star (our
Sun). The results will have a profound impact on our understanding not
only of the solar interior, but also neutrino physics, stellar
evolution theory, and stellar population studies in astrophysics.
Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning
Authors: Liu Yao-Guang , Shirano Yumiko , Fukaki Hidehiro , Yanai Yukihiro , Tasaka Masao , Tabata Satoshi , Shibata Daisuke
Published: July 2002

To accelerate gene isolation from plants by positional cloning, vector systems suitable for both chromosome walking and genetic complementation are highly desirable. Therefore, we developed a transformation-competent artificial chromosome (TAC) vector, pYLTAC7, that can accept and maintain large genomic DNA fragments stably in both
Escherichia coli
and
Agrobacterium tumefaciens
. Furthermore, it has the cis sequences required for
Agrobacterium
-mediated gene transfer into plants. We cloned large genomic DNA fragments of
Arabidopsis thaliana
into the vector and showed that most of the DNA fragments were maintained stably. Several TAC clones carrying 40- to 80-kb genomic DNA fragments were transferred back into
Arabidopsis
with high efficiency and shown to be inherited faithfully among the progeny. Furthermore, we demonstrated the practical utility of this vector system for positional cloning in
Arabidopsis
. A TAC contig was constructed in the region of the
SGR1
locus, and individual clones with
ca.
80-kb inserts were tested for their ability to complement the gravitropic defects of a homozygous mutant line. Successful complementation enabled the physical location of
SGR1
to be delimited with high precision and confidence.
A Crm1p-independent nuclear export path for the mRNA-associated protein, Npl3p/Mtr13p
Authors: Liu Yan , Guo Wei , Tartakoff Paola Y. , Tartakoff Alan M.
Published: July 2002

mRNA export involves association of mRNAs with nucleoplasmic proteins, delivery to the nuclear pore complex, translocation to the cytoplasm, and reimport of recycling components. Many yeast mutants inhibit mRNA export, but there is little information concerning the RNA carriers and steps of transport that they affect. The hnRNP/serine-arginine-rich-like protein, Npl3p/Mtr13p, binds poly(A)
+
RNA and shuttles between the nucleus and cytoplasm. Its export accelerates on inhibition of RNA synthesis.
In vivo
tests show that its export requires two proteins with putative leucine-rich nuclear export signals: Gle1p, Mex67p, and several additional nuclear and nuclear pore complex-associated proteins. Surprisingly, a nonnuclear pool of an import factor (the importin α homologue, Srp1p) is also required. Changes in the methylation status of Npl3p do not correlate with its nucleocytoplasmic distribution. A
crm1
mutant that inhibits export of proteins with leucine-rich nuclear export signals and mRNAs does not inhibit Npl3p export. Moreover, several proteins needed for Npl3p export are not needed for export of a typical Crm1p cargo. Thus, Npl3p export requires only a subset of proteins implicated in mRNA export, suggesting that more than one mRNA export path exists. A distinct group of mutants, including a mutation of a member of the importin β superfamily, inhibits Npl3p reimport from the cytoplasm.
Identification of the endonuclease domain encoded by R2 and other site-specific, non-long terminal repeat retrotransposable elements
Authors: Yang Jin , Malik Harmit S. , Eickbush Thomas H.
Published: July 2002

The non-long terminal repeat (LTR) retrotransposon, R2, encodes a sequence-specific endonuclease responsible for its insertion at a unique site in the 28S rRNA genes of arthropods. Although most non-LTR retrotransposons encode an apurinic-like endonuclease upstream of a common reverse transcriptase domain, R2 and many other site-specific non-LTR elements do not (CRE1 and 2, SLACS, CZAR, Dong, R4). Sequence comparison of these site-specific elements has revealed that the region downstream of their reverse transcriptase domain is conserved and shares sequence features with various prokaryotic restriction endonucleases. In particular, these non-LTR elements have a Lys/Arg-Pro-Asp-X
12–14aa
–Asp/Glu motif known to lie near the scissile phosphodiester bonds in the protein–DNA complexes of restriction enzymes. Site-directed mutagenesis of the R2 protein was used to provide evidence that this motif is also part of the active site of the endonuclease encoded by this element. Mutations of this motif eliminate both DNA-cleavage activities of the R2 protein: first-strand cleavage in which the exposed 3′ end is used to prime reverse transcription of the RNA template and second-strand cleavage, which occurs after reverse transcription. The general organization of the R2 protein appears similar to the type IIS restriction enzyme,
Fok
I, in which specific DNA binding is controlled by a separate domain located amino terminal to the cleavage domain. Previous phylogenetic analysis of their reverse transcriptase domains has indicated that the non-LTR elements identified here as containing restriction-like endonucleases are the oldest lineages of non-LTR elements, suggesting a scenario for the evolution of non-LTR elements.
Human papillomavirus type 31 oncoproteins E6 and E7 are required for the maintenance of episomes during the viral life cycle in normal human keratinocytes
Authors: Thomas Jennifer T. , Hubert Walter G. , Ruesch Margaret N. , Laimins Laimonis A.
Published: July 2002

The E6 and E7 oncoproteins of the high-risk human papillomavirus (HPV) types are able to immortalize human keratinocytes
in vitro
and likely contribute to the development of anogenital malignancies
in vivo
. The role of these oncoproteins in the productive viral life cycle, however, is not known. To begin to examine these possible roles, mutations in E6 were introduced in the context of the complete HPV 31 genome. Although transfected wild-type HPV 31 genomes, as well as genomes containing an E6 translation termination linker, an E6 frameshift mutation, and a point mutation in the p53 interacting domain were able to replicate in transient assays, only the wild-type genome was stably maintained as an episome. Interestingly, mutant genomes in either the E6 splice-donor site or splice-acceptor site were reduced in replication ability in transient assays; however, cotransfection of E1 and E2 expression vectors restored this function. In a similar fashion, genomes containing mutant HPV 31 E7 genes, including a translation termination mutant, two Rb-binding site mutants, a casein kinase II phosphorylation site mutant, and a transformation deficient mutant, were constructed. Although transient replication was similar to wild type in all of the E7 mutants, only the casein kinase II mutant had the ability to maintain high copies of episomal genomes. These findings suggest a role for E6 and E7 in the viral life cycle beyond their ability to extend the life span of infected cells.
The
Caulobacter crescentus smc
gene is required for cell cycle progression and chromosome segregation
Authors: Jensen Rasmus B. , Shapiro Lucy
Published: July 2002

The highly conserved SMC (Structural Maintenance of Chromosomes) proteins function in chromosome condensation, segregation, and other aspects of chromosome dynamics in both eukaryotes and prokaryotes. A null mutation in the
Caulobacter crescentus smc
gene is conditionally lethal and causes a cell cycle arrest at the predivisional cell stage. Chromosome segregation in wild-type and
smc
null mutant cells was examined by monitoring the intracellular localization of the replication origin and terminus by using fluorescence
in situ
hybridization. In wild-type cells, the origin is located at the flagellated pole of swarmer cells and, immediately after the initiation of DNA replication in stalked cells, one of the origins moves to the opposite pole, giving a bipolar localization of the origins. The terminus moves from the end of the swarmer cell opposite the origin to midcell. A subpopulation of the
smc
null mutant cells had mislocalized origins or termini, showing that the
smc
null mutation gives DNA segregation defects. Nucleoid morphology was also abnormal. Thus, we propose that the
Caulobacter
chromosomal origins have specific cellular addresses and that the SMC protein plays important roles in maintaining chromosome structure and in partitioning. The specific cell cycle arrest in the
smc
null mutant indicates the presence of a cell cycle checkpoint that senses perturbations in chromosome organization or segregation.
Origin and evolutionary relationships of giant Galápagos tortoises
Authors: Caccone Adalgisa , Gibbs James P. , Ketmaier Valerio , Suatoni Elizabeth , Powell Jeffrey R.
Published: July 2002

Perhaps the most enduring debate in reptile systematics has involved the giant Galápagos tortoises (
Geochelone nigra
), whose origins and systematic relationships captivated Charles Darwin and remain unresolved to this day. Here we report a phylogenetic reconstruction based on mitochondrial DNA sequences from Galápagos tortoises and
Geochelone
from mainland South America and Africa. The closest living relative to the Galápagos tortoise is not among the larger-bodied tortoises of South America but is the relatively small-bodied
Geochelone chilensis
, or Chaco tortoise. The split between
G. chilensis
and the Galápagos lineage probably occurred 6 to 12 million years ago, before the origin of the oldest extant Galápagos island. Our data suggest that the four named southern subspecies on the largest island, Isabela, are not distinct genetic units, whereas a genetically distinct northernmost Isabela subspecies is probably the result of a separate colonization. Most unexpectedly, the lone survivor of the
abingdoni
subspecies from Pinta Island (“Lonesome George”) is very closely related to tortoises from San Cristóbal and Española, the islands farthest from the island of Pinta. To rule out a possible recent transplant of Lonesome George, we sequenced DNA from three tortoises collected on Pinta in 1906. They have sequences identical to Lonesome George, consistent with his being the last survivor of his subspecies. This finding may provide guidance in finding a mate for Lonesome George, who so far has failed to reproduce.
Epiregulin is a potent vascular smooth muscle cell-derived mitogen induced by angiotensin II, endothelin-1, and thrombin
Authors: Taylor David S. , Cheng Xinbo , Pawlowski John E. , Wallace Alison R. , Ferrer Patricia , Molloy Christopher J.
Published: July 2002

Vasoactive GTP-binding protein-coupled receptor agonists such as
angiotensin II (AII), endothelin-1 (ET-1), and α-thrombin (α-Thr)
have been reported to indirectly stimulate vascular smooth muscle cell
(VSMC) proliferation by regulating the expression of one or more
autocrine growth factors. Using ion-exchange, gel-filtration, and
reverse-phase chromatographic purification methods, we isolated a major
mitogenic protein present in AII-stimulated rat aortic smooth muscle
(RASM) cell conditioned medium. Twenty N-terminal amino acids of the
purified peptide were identified, and they had 75% amino acid sequence
identity with mouse epiregulin, an epidermal growth factor
(EGF)-related growth factor. We cloned the cDNA for rat epiregulin to
determine its pattern of expression in G-protein-coupled receptor
agonist-stimulated cells and confirm its activity as a mitogen. After
treatment of RASM cells with AII, ET-1, or α-Thr for 1 h,
induction of two epiregulin transcripts was observed, including a
4.8-kb transcript and a novel transcript of approximately 1.2 kb.
Recombinant rat epiregulin was strongly mitogenic for RASM cells,
stimulating DNA synthesis to levels similar to those induced by serum
or platelet-derived growth factor and approximately 3-fold above that
observed with saturating concentrations of EGF. In addition, epiregulin
caused rapid EGF receptor activation in RASM cells. However, relative
levels of EGF receptor tyrosine phosphorylation stimulated by
epiregulin were less than those induced by EGF or betacellulin. Taken
together, these results indicate that epiregulin is a potent
VSMC-secreted mitogen, induced in common by AII, ET-1, and α-Thr,
that may contribute to VSMC proliferation and vascular remodeling
stimulated by vasoactive agonists.