bluuebunny/crossref_metadata_2025 huggingface dataset:
Copy-choice recombination mediated by DNA polymerase III holoenzyme from Escherichia coli.
Authors: Canceill D , Ehrlich S D
Published: July 2002

Formation of deletions by recombination between short direct repeats is thought to involve either a break-join or a copy-choice process. The key step of the latter is slippage of the replication machinery between the repeats. We report that the main replicase of Escherichia coli, DNA polymerase III holoenzyme, slips between two direct repeats of 27 bp that flank an inverted repeat of approximately equal 300bp. Slippage was detected in vitro, on a single-stranded DNA template, in a primer extension assay. It requires the presence of a short (8 bp) G+C-rich sequence at the base of a hairpin that can form by annealing of the inverted repeats. It is stimulated by (i) high salt concentration, which might stabilize the hairpin, and (ii) two proteins that ensure the processivity of the DNA polymerase III holoenzyme: the single-stranded DNA binding protein and the beta subunit of the polymerase. Slippage is rather efficient under optimal reaction conditions because it can take place on >50% of template molecules. This observation supports the copy-choice model for recombination between short direct repeats.
Interaction of calcineurin with a domain of the transcription factor NFAT1 that controls nuclear import.
Authors: Luo C , Shaw K T , Raghavan A , Aramburu J , Garcia-Cozar F , Perrino B A , Hogan P G , Rao A
Published: July 2002

The nuclear import of the nuclear factor of activated T cells (NFAT)-family transcription factors is initiated by the protein phosphatase calcineurin. Here we identify a regulatory region of NFAT1, N terminal to the DNA-binding domain, that controls nuclear import of NFAT1. The regulatory region of NFAT1 binds directly to calcineurin, is a substrate for calcineurin in vitro, and shows regulated subcellular localization identical to that of full-length NFAT1. The corresponding region of NFATc likewise binds calcineurin, suggesting that the efficient activation of NFAT1 and NFATc by calcineurin reflects a specific targeting of the phosphatase to these proteins. The presence in other NFAT-family transcription factors of several sequence motifs from the regulatory region of NFAT1, including its probable nuclear localization sequence, indicates that a conserved protein domain may control nuclear import of all NFAT proteins.
Altered surfactant function and structure in SP-A gene targeted mice.
Authors: Korfhagen T R , Bruno M D , Ross G F , Huelsman K M , Ikegami M , Jobe A H , Wert S E , Stripp B R , Morris R E , Glasser S W , Bachurski C J , Iwamoto H S , Whitsett J A
Published: July 2002

The surfactant protein A (SP-A) gene was disrupted by homologous recombination in embryonic stem cells that were used to generate homozygous SP-A-deficient mice. SP-A mRNA and protein were not detectable in the lungs of SP-A(-/-) mice, and perinatal survival of SP-A(-/-) mice was not altered compared with wild-type mice. Lung morphology, surfactant proteins B-D, lung tissue, alveolar phospholipid pool sizes and composition, and lung compliance in SP-A(-/-) mice were unaltered. At the highest concentration tested, surfactant from SP-A(-/-) mice produced the same surface tension as (+/+) mice. At lower concentrations, minimum surface tensions were higher for SP-A(-/-) mice. At the ultrastructural level, type II cell morphology was the same in SP-A(+/+) and (-/-) mice. While alveolar phospholipid pool sizes were unperturbed, tubular myelin figures were decreased in the lungs of SP-A(-/-) mice. A null mutation of the murine SP-A gene interferes with the formation of tubular myelin without detectably altering postnatal survival or pulmonary function.
Enhancement of sensorimotor connections by conditioning-related stimulation in Aplysia depends upon postsynaptic Ca2+.
Authors: Murphy G G , Glanzman D L
Published: July 2002

Classical conditioning of Aplysia's siphon-withdrawal reflex is thought to be due to a presynaptic mechanism-activity-dependent presynaptic facilitation of sensorimotor connections. Recent experiments with sensorimotor synapses in dissociated cell culture, however, provide an alternative cellular mechanism for classical conditioning-Hebbian long-term potentiation (LTP) of sensorimotor connections. Induction of Hebbian LTP of these connections is mediated by activation of N-methyl-D-aspartate-related receptors and requires the postsynaptic elevation of intracellular Ca2+. To determine whether the enhancement of sensorimotor synapses during classical conditioning in Aplysia-like LTP of sensorimotor synapses in culture-also depends upon the elevation of postsynaptic Ca2+, we carried out experiments involving the cellular analog of classical conditioning of siphon withdrawal. We examined changes in the strength of monosynaptic siphon sensorimotor connections in the abdominal ganglion of Aplysia following paired presentations of sensory neuron activation and tail nerve shock. This training regimen resulted in significant enhancement of the monosynaptic sensorimotor excitatory postsynaptic potential, as compared with the sensorimotor excitatory postsynaptic potential in preparations that received only test stimulation. Infusing the motor neuron with 1,2-bis(2-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid, a specific chelator of intracellular Ca2+, prior to paired stimulation training blocked this synaptic enhancement. Our results implicate a postsynaptic, possibly Hebbian, mechanism in classical conditioning in Aplysia.
Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha.
Authors: Hwang C S , Mandrup S , MacDougald O A , Geiman D E , Lane M D
Published: July 2002

Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage inhibition patterns (footprinting) of the ob gene promoter revealed that recombinant C/EBP alpha, as well as a nuclear factor present in fully differentiated 3T3-L1 adipocytes, but present at a much lower level in preadipocytes, protects the same region between nucleotides -58 and -42 relative to the transcriptional start site. Electrophoretic mobility-shift analysis using nuclear extracts from adipose tissue or 3T3-L1 adipocytes and an oligonucleotide probe corresponding to a consensus C/EBP binding site at nucleotides -55 to -47 generated a specific protein-oligonucleotide complex that was supershifted by antibody against C/EBP alpha. Probes corresponding to two upstream consensus C/EBP binding sites failed to generate protein-oligonucleotide complexes. Cotransfection of a C/EBP alpha expression vector into 3T3-L1 cells with a series of 5' truncated ob gene promoter constructs activated reporter gene expression with all constructs containing the proximal C/EBP binding site (nucleotides -55 to -47). Mutation of this site blocked transactivation by C/EBP alpha. Taken together, these findings implicate C/EBP alpha as a transcriptional activator of the ob gene promoter and identify the functional C/EBP binding site in the promoter.
Half tetrad analysis in alfalfa using multiple restriction fragment length polymorphism markers.
Authors: Tavoletti S , Bingham E T , Yandell B S , Veronesi F , Osborn T C
Published: July 2002

A maximum likelihood approach of half tetrad analysis (HTA) based on multiple restriction fragment length polymorphism (RFLP) markers was developed. This procedure estimates the relative frequencies of 2n gametes produced by mechanisms genetically equivalent to first division restitution (FDR) or second division restitution and simultaneously locates the centromere within a linkage group of RFLP marker loci. The method was applied to the diploid alfalfa clone PG-F9 (2n = 2x = 16) previously selected because of its high frequency of 2n egg production. HTA was based on four RFLP loci for which PG-F9 was heterozygous with codominant alleles that were absent in the tetraploid tester. Models including three linked and one unlinked RFLP loci were developed and tested. Results of the HTA showed that PG-F9 produced 6% FDR and 94% second division restitution 2n eggs. Information from a marker locus belonging to one linkage group was used to more precisely locate the centromere on a different linkage group. HTA, together with previous cytological analysis, indicated that in PG-F9, FDR 2n eggs are likely produced by diplospory, a mechanism common among apomictic species. The occurrence of FDR 2n eggs in plant species and their importance for crop evolution and breeding is discussed together with the potential applicability of multilocus HTA in the study of reproductive mutants.
Programmed cell death: a way of life for plants.
Authors: Greenberg J T
Published: July 2002

Cell death in higher plants has been widely observed in predictable patterns throughout development and in response to pathogenic infection. Genetic, biochemical, and morphological evidence suggests that these cell deaths occur as active processes and can be defined formally as examples of programmed cell death (PCD). Intriguingly, plants have at least two types of PCD, an observation that is also true of PCD in animals [Schwartz, L. M., Smith, W.W., Jones, M. E. E. & Osborne, B. A. (1993) Proc. Natl. Acad. Sci. USA 90, 980-984]. Thus, in plants, PCD resembles either a common form of PCD seen in animals called apoptosis or it resembles a morphologically distinct form of cell death. The ubiquitous occurrence and necessity of PCD for plant development and defense suggest that the underlying mechanisms of regulation and execution of these processes merit further examination.
Development of an
in vitro
mRNA decay system for
Escherichia coli
: Poly(A) polymerase I is necessary to trigger degradation
Authors: Ingle Caroline A. , Kushner Sidney R.
Published: July 2002

Using a novel
Escherichia coli in vitro
decay system
in which polysomes are the source of both enzymes and mRNA, we
demonstrate a requirement for poly(A) polymerase I (PAP I) in mRNA
turnover. The
in vitro
decay of two different mRNAs
(
trxA
and
lpp
) is triggered by the
addition of ATP only when polysomes are prepared from a strain carrying
the wild-type gene for PAP I (
pcnB
+
). The
relative decay rates of these two messages are similar
in
vitro
and
in vivo
. Poly(A) tails are formed on
both mRNAs, but no poly(A) tails are detected on the 3′ end of mature
23S rRNA. The size distribution of poly(A) tails generated
in
vitro
, averaging 50 nt in length, is comparable to that
previously reported
in vivo
. PAP I activity is
associated exclusively with the polysomes. Exogenously added PAP I does
not restore mRNA decay to PAP I
−
polysomes, suggesting
that,
in vivo
, PAP I may be part of a multiprotein
complex. The potential of this
in vitro
system for
analyzing mRNA decay in
E. coli
is discussed.
A
CDKN2
-like polymorphism in
Xiphophorus
LG V is associated with UV-B-induced melanoma formation in platyfish–swordtail hybrids
Authors: Nairn Rodney S. , Kazianis Steven , McEntire Brenda B. , Della Coletta Luis , Walter Ronald B. , Morizot Donald C.
Published: July 2002

The genetic basis of spontaneous melanoma formation in spotted
dorsal (
Sd
)
Xiphophorus
platyfish–swordtail hybrids has been studied for decades, and is
adequately explained by a two-gene inheritance model involving a
sex-linked oncogene,
Xmrk
, and an autosomal tumor
suppressor,
DIFF
. The
Xmrk
oncogene
encodes a receptor tyrosine kinase related to
EGFR
; the
nature of the
DIFF
tumor suppressor gene is unknown. We
analyzed the genetic basis of UV-B-induced melanoma formation in
closely related, spotted side platyfish–swordtail hybrids, which carry
a different sex-linked pigment pattern locus,
Sp
. We
UV-irradiated spotted side
Xiphophorus
platyfish–swordtail backcross hybrids to induce melanomas at
frequencies 6-fold higher than occur spontaneously in unirradiated
control animals. To identify genetic determinants of melanoma
susceptibility in this UV-inducible
Xiphophorus
model,
we genotyped individual animals from control and UV-irradiated
experimental regimes using allozyme and DNA restriction fragment length
polymorphisms and tested for joint segregation of genetic markers with
pigmentation phenotype and UV-induced melanoma formation. Joint
segregation results show linkage of a
CDKN2
-like DNA
polymorphism with UV-B-induced melanoma formation in these hybrids. The
CDKN2
-like polymorphism maps to
Xiphophorus
linkage group V and exhibits recombination
fractions with
ES1
and
MDH2
allozyme
markers consistent with previous localization of the
DIFF
tumor suppressor locus. Our results indicate that
the
CDKN2
-like sequence we have cloned and mapped is a
candidate for the
DIFF
tumor suppressor gene.
Normal host prion protein (PrP
C
) is required for scrapie spread within the central nervous system
Authors: Brandner Sebastian , Raeber Alex , Sailer Andreas , Blättler Thomas , Fischer Marek , Weissmann Charles , Aguzzi Adriano
Published: July 2002

Mice devoid of PrP
C
(
Prnp
o/o
) are resistant to scrapie and do
not allow propagation of the infectious agent (prion).
PrP
C
-expressing neuroectodermal tissue grafted into
Prnp
o/o
brains but not the surrounding
tissue consistently exhibits scrapie-specific pathology and allows
prion replication after inoculation. Scrapie prions administered
intraocularly into wild-type mice spread efficiently to the central
nervous system within 16 weeks. To determine whether PrP
C
is required for scrapie spread, we inoculated prions intraocularly into
Prnp
o/o
mice containing a PrP-overexpressing
neurograft. Neither encephalopathy nor protease-resistant PrP
(PrP
Sc
) were detected in the grafts for up to 66 weeks.
Because grafted PrP-expressing cells elicited an immune response that
might have interfered with prion spread, we generated
Prnp
o/o
mice immunotolerant to PrP and
engrafted them with PrP-producing neuroectodermal tissue. Again,
intraocular inoculation did not lead to disease in the PrP-producing
graft. These results demonstrate that PrP is necessary for prion spread
along neural pathways.
Retinoic acid alters photoreceptor development
in vivo
Authors: Hyatt George A. , Schmitt Ellen A. , Fadool James M. , Dowling John E.
Published: July 2002

Application of exogenous retinoic acid (RA) to zebrafish during the
initial stages of photoreceptor differentiation results in a precocious
development of rod photoreceptors and an inhibition of cone
photoreceptor maturation. The acceleration of rod differentiation is
observed initially within the ventral retina 3 days after
fertilization, following 24 hr of RA application, and within the dorsal
retina 4 days after fertilization, following 48 hr of RA application.
The differentiation of rods was impeded significantly when the
synthesis of endogenous retinoic acid was inhibited by
citral prior to the initial stage of rod differentiation. RA-treated
embryos labeled for bromodeoxyuridine (BrdU) uptake revealed that RA
exerts its effect on a postmitotic cell population within the
developing retina. During normal development in zebrafish, rod
differentiation is most robust within the ventral retina, a region
previously shown to be rich in RA. Our data suggest that the RA
signaling pathway is involved in the differentiation and maturation of
both the rod and cone photoreceptors within the developing zebrafish
retina.
Addition of destabilizing poly(A)-rich sequences to endonuclease cleavage sites during the degradation of chloroplast mRNA
Authors: Lisitsky Irena , Klaff Petra , Schuster Gadi
Published: July 2002

In this work, we report the posttranscriptional addition of
poly(A)-rich sequences to mRNA in chloroplasts of higher plants.
Several sites in the coding region and the mature end of spinach
chloroplast
psbA
mRNA, which encodes the D1 protein of
photosystem II, are detected as polyadenylylated sites. In eukaryotic
cells, the addition of multiple adenosine residues to the 3′ end of
nuclear RNA plays a key role in generating functional mRNAs and in
regulating mRNA degradation. In bacteria, the adenylation of several
RNAs greatly accelerates their decay. The poly(A) moiety in the
chloroplast, in contrast to that in eukaryotic nuclear encoded and
bacterial RNAs, is not a ribohomopolymer of adenosine residues, but
clusters of adenosines bounded mostly by guanosines and rarely by
cytidines and uridines; it may be as long as several hundred
nucleotides. Further analysis of the initial steps of chloroplast
psbA
mRNA decay revealed specific endonuclease cleavage
sites that perfectly matched the sites where poly(A)-rich sequences
were added. Our results suggest a mechanism for the degradation of
psbA
mRNA in which endonucleolytic cleavages are
followed by the addition of poly(A)-rich sequences to the upstream
cleavage products, which target these RNAs for rapid decay.
Dimerization of Ste5, a mitogen-activated protein kinase cascade scaffold protein, is required for signal transduction
Authors: Yablonski Deborah , Marbach Irit , Levitzki Alexander
Published: July 2002

The mitogen-activated protein kinase cascade of the
Saccharomyces cerevisiae
pheromone response pathway is
organized on the Ste5 protein, which binds each of the kinases of the
cascade prior to signaling. In this study, a structure–function
analysis of Ste5 deletion mutants uncovered new functional domains of
the Ste5 protein and revealed that Ste5 dimerizes during the course of
normal signal transduction. Dimerization, mediated by two regions in
the N-terminal half of Ste5, was first suggested by intragenic
complementation between pairs of nonfunctional Ste5 mutants and was
confirmed by using the two-hybrid system. Coimmunoprecipitation of
differently tagged forms of Ste5 from cells in which the pathway has
been activated by Ste5 overexpression further confirmed dimerization. A
precise correlation between the biological activity of various Ste5
fragments and dimerization suggests that dimerization is essential for
Ste5 function.
Cell cycle-regulated generation of single-stranded G-rich DNA in the absence of telomerase
Authors: Dionne Isabelle , Wellinger Raymund J.
Published: July 2002

Current models of telomere replication predict that due to the
properties of the polymerases implicated in semiconservative
replication of linear DNA, the two daughter molecules have one end that
is blunt and one end with a short 3′ overhang. Telomerase is thought to
extend the short 3′ overhang to produce long single-stranded overhangs.
Recently, such overhangs, or TG
1–3
tails, were shown to
occur on both telomeres of replicated linear plasmids in yeast.
Moreover, indirect evidence suggested that the TG
1–3
tails
also occurred in a yeast strain lacking telomerase. We report herein a
novel in-gel hybridization technique to probe telomeres for
single-stranded DNA. Using this method, it is shown directly that in
yeast strains lacking the
TLC1
gene encoding the yeast
telomerase RNA, TG
1–3
single-stranded DNA was generated on
chromosomal and plasmid telomeres. The single-stranded DNA only
appeared in S phase and was sensitive to digestion with a
single-strand-specific exonuclease. These data demonstrate that during
replication of telomeres, TG
1–3
tails can be generated in
a way that is independent of telomerase-mediated strand elongation. In
wild-type strains, these TG
1–3
tails could subsequently
serve as substrates for telomerase and telomere binding proteins on all
telomeres.
Effects of protein calorie malnutrition on tuberculosis in mice
Authors: Chan John , Tian Yu , Tanaka Kathryn E. , Tsang Ming S. , Yu Keming , Salgame Padmini , Carroll Dinah , Kress Yvonne , Teitelbaum Rachel , Bloom Barry R.
Published: July 2002

Infectious diseases and malnutrition represent major burdens afflicting millions of people in developing countries. Both conditions affect individuals in industrialized nations, particularly the aged, the HIV-infected, and people with chronic diseases. While malnutrition is known to induce a state of immunodeficiency, the mechanisms responsible for compromised antimicrobial resistance in malnourished hosts remain obscure. In the present study, mice fed a 2% protein diet and developing protein calorie malnutrition, in contrast to well-nourished controls receiving a 20% protein diet, rapidly succumbed to infection withMycobacterium tuberculosis. Malnourished mice exhibited a tissue-specific diminution in the expression of interferon γ, tumor necrosis factor α, and the inducible form of nitric oxide synthase in the lungs, but not the liver. The expression of these molecules critical to the production of mycobactericidal nitrogen oxides was depressed in malnourished animals in the lungs specifically at early times (<14 days) after infection. At later times, levels of expression became comparable to those in well-nourished controls, although the bacillary burden in the malnourished animals continued to rise. Nevertheless, urinary and serum nitrate contents, an index of total nitric oxide (NO) productionin vivo, were not detectably diminished in malnourished, mycobacteria-infected mice. In contrast to the selective and early reduction of lymphokines and the inducible form of nitric oxide synthase in the lung, a marked diminution of the granulomatous reaction was observed in malnourished mice throughout the entire course of infection in all tissues examined (lungs, liver, and spleen). Remarkably, the progressively fatal course of tuberculosis observed in the malnourished mice could be reversed by restoring a full protein (20%) diet. The results indicate that protein calorie malnutrition selectively compromises several components of the cellular immune response that are important for containing and restricting tuberculous infection, and suggest that malnutrition-induced susceptibility to some infectious diseases can be reversed or ameliorated by nutritional intervention.
High-resolution mapping and isolation of a yeast artificial chromosome contig containingfw2.2: A major fruit weight quantitative trait locus in tomato
Authors: Alpert Kevin B. , Tanksley Steven D.
Published: July 2002

A high-resolution physical and genetic map of a major fruit weight quantitative trait locus (QTL),fw2.2, has been constructed for a region of tomato chromosome 2. Using an F2nearly isogenic line mapping population (3472 individuals) derived fromLycopersicon esculentum(domesticated tomato) ×Lycopersicon pennellii(wild tomato),fw2.2has been placed near TG91 and TG167, which have an interval distance of 0.13 ± 0.03 centimorgan. The physical distance between TG91 and TG167 was estimated to be ≤ 150 kb by pulsed-field gel electrophoresis of tomato DNA. A physical contig composed of six yeast artificial chromosomes (YACs) and encompassingfw2.2was isolated. No rearrangements or chimerisms were detected within the YAC contig based on restriction fragment length polymorphism analysis using YAC-end sequences and anchored molecular markers from the high-resolution map. Based on genetic recombination events,fw2.2could be narrowed down to a region less than 150 kb between molecular markers TG91 and HSF24 and included within two YACs: YAC264 (210 kb) and YAC355 (300 kb). This marks the first time, to our knowledge, that a QTL has been mapped with such precision and delimited to a segment of cloned DNA. The fact that the phenotypic effect of thefw2.2QTL can be mapped to a small interval suggests that the action of this QTL is likely due to a single gene. The development of the high-resolution genetic map, in combination with the physical YAC contig, suggests that the gene responsible for this QTL and other QTLs in plants can be isolated using a positional cloning strategy. The cloning offw2.2will likely lead to a better understanding of the molecular biology of fruit development and to the genetic engineering of fruit size characteristics.
Transcription of tufA and other chloroplast-encoded genes is controlled by a circadian clock in Chlamydomonas.
Authors: Hwang S , Kawazoe R , Herrin D L
Published: July 2002

Levels of mRNA for the chloroplast-encoded elongation factor Tu (tufA) showed a dramatic daily oscillation in the green alga Chlamydomonas reinhardtii, peaking once each day in the early light period. The oscillation of tufA mRNA levels continued in cells shifted to continuous light or continuous dark for at least 2-3 days. Run-off transcription analyses showed that the rate of tufA transcription also peaked early in the light period and, moreover, that this transcriptional oscillation continued in cells shifted to continuous conditions. The half-life of tufA mRNA was estimated at different times and found to vary considerably during a light-dark cycle but not in cells shifted to continuous light. Light-dark patterns of transcription of several other chloroplast-encoded genes were examined and also found to persist in cells shifted to continuous light or dark. These results indicate that a circadian clock controls the transcription of tufA and other chloroplast-encoded genes.
gamma/delta and other unconventional T lymphocytes: what do they see and what do they do?
Authors: Kaufmann S H
Published: July 2002

T lymphocytes recognize specific ligands by clonally distributed T-cell receptors (TCR). In humans and most animals, the vast majority of T cells express a TCR composed of an alpha chain and a beta chain, whereas a minor T-cell population is characterized by the TCR gamma/delta. Almost all of our knowledge about T cells stems from alpha/beta T cells and only now are we beginning to understand gamma/delta T cells. In contrast to conventional alpha/beta T cells, which are specific for antigenic peptides presented by gene products of the major histocompatibility complex, gamma/delta T cells directly recognize proteins and even nonproteinacious phospholigands. These findings reveal that gamma/delta T cells and alpha/beta T cells recognize antigen in a fundamentally different way and hence mitigate the dogma of exclusive peptide-major histocompatibility complex recognition by T cells. A role for gamma/delta T cells in antimicrobial immunity has been firmly established. Although some gamma/delta T cells perform effector functions, regulation of the professional and the nonprofessional immune system seems to be of at least equal importance. The prominent residence of gamma/delta T cells in epithelial tissues and the rapid mobilization of gamma/delta T cells in response to infection are consistent with such regulatory activities under physiological and pathologic conditions. Thus, although gamma/delta T cells are a minor fraction of all T cells, they are not just uninfluential kin of alpha/beta T cells but have their unique raison d'être.
Modulation of promoter occupancy by cooperative DNA binding and activation-domain function is a major determinant of transcriptional regulation by activators in vivo.
Authors: Tanaka M
Published: July 2002

Binding of transcriptional activators to a promoter is a prerequisite process in transcriptional activation. It is well established that the efficiency of activator binding to a promoter is determined by the affinity of direct interactions between the DNA-binding domain of an activator and its specific target sequences. However, I describe here that activator binding to a promoter is augmented in vivo by the effects of two other determinants that have not been generally appreciated: (i) the number of activator binding sites present in a promoter and (ii) the potency of activation domains of activators. Multiple sites within a promoter can cooperatively recruit cognate factors regardless of whether they contain an effective activation domain. This cooperativity can result in the synergistic activation of transcription. The second effect is the enhancement of activator binding to a promoter by the presence of activation domains. In this case, activation domains are not simply tethered to the promoter by the DNA-binding domain but instead assist the DNA-binding domain being tethered onto the promoter. This effect of activation domains on DNA binding is instrumental in determining how potent activators can induce steep transcriptional increases at low concentrations.
7-Chloro-3-methyl-3,4-dihydro-2
H
-1,2,4-benzothiadiazine
S
,
S
-dioxide: A partial modulator of AMPA receptor desensitization devoid of neurotoxicity
Authors: Impagnatiello F. , Oberto A. , Longone P. , Costa E. , Guidotti A.
Published: July 2002

In cerebellar granule neurons of neonatal rats micromolar concentrations of 7-chloro-3-methyl-3,4-dihydro-2
H
-1,2,4-benzothiadiazine
S
,
S
-dioxide (IDRA-21) and cyclothiazide, two negative modulators of the spontaneous agonist-dependent rapid desensitization of α-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA)-gated ion channels, facilitate AMPA receptor function by increasing the content of free cytosolic Ca
2+
as measured by single-cell fura-2 acetoxymethyl ester (Fura-2) Ca
2+
-dependent fluorescence and intracellular Na
+
measured with the sodium-binding bezofuran isophthalate acetoxymethyl ester fluorescence indicator. IDRA-21 increases intracellular Na
+
transient with a threshold (5 μM) that is ≈10 times higher and has an intrinsic activity significantly lower than that of cyclothiazide. By virtue of its low intrinsic activity, IDRA-21 elicits a free cytosolic Ca
2+
transient increase that is shorter lasting than that elicited by cyclothiazide even when the drug is left in contact with cultured granule cells for several minutes. Additionally, while dose dependently, 5–25 μM cyclothiazide in the presence of AMPA is highly neurotoxic, IDRA-21 (up to 100 μM) is devoid of neurotoxicity. The neurotoxicity elicited by cyclothiazide persists in the presence of dizocilpine (an antagonist of
N
-methyl-
d
-aspartate-selective glutamate receptors) but is blocked by 2,3-dihydroxy-6-nitrosulfamoylbenzo[
f
]quinoxaline (a competitive AMPA receptor antagonist) and the 1-(aminophenyl)-4-methyl-7,8-methylendioxy-5
H
-2,3-benzodiazepine (GYKI 52466; a noncompetitive AMPA receptor antagonist). Since the doses of IDRA-21 that enhance cognitive processes in rats and monkeys are several orders of magnitude lower than those required to elicit marginal neurotoxicity in cultured neurons, it can be surmised that IDRA-21 is a potent cognition-enhancing drug virtually devoid of neurotoxic liability because it acts as a partial negative allosteric modulator of AMPA receptor desensitization.
Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein
Authors: Yun Dae-Jin , Zhao Yuan , Pardo José M. , Narasimhan Meena L. , Damsz Barbara , Lee Hyeseung , Abad Laura R. , D’Urzo Matilde Paino , Hasegawa Paul M. , Bressan Ray A.
Published: July 2002

Strains of the yeastSaccharomyces cerevisiaediffer in their sensitivities to tobacco osmotin, an antifungal protein of the PR-5 family. However, cells sensitive to tobacco osmotin showed resistance to osmotin-like proteins purified from the plantAtriplex nummularia, indicating a strict specificity between the antifungal protein and its target cell. A member of a gene family encoding stress proteins induced by heat and nitrogen limitation, collectively called Pir proteins, was isolated among the genes that conveyed resistance to tobacco osmotin to a susceptible strain. We show that overexpression of Pir proteins increased resistance to osmotin, whereas simultaneous deletion of allPIRgenes in a tolerant strain resulted in sensitivity. Pir proteins have been immunolocalized to the cell wall. The enzymatic digestion of the cell wall of sensitive and resistant cells rendered spheroplasts equally susceptible to the cytotoxic action of tobacco osmotin but not to other osmotin-like proteins, indicating that the cell membrane interacts specifically with osmotin and facilitates its action. Our results demonstrate that fungal cell wall proteins are determinants of resistance to antifungal PR-5 proteins.
Overexpression of an
Arabidopsis thaliana
high-affinity phosphate transporter gene in tobacco cultured cells enhances cell growth under phosphate-limited conditions
Authors: Mitsukawa Norihiro , Okumura Satoru , Shirano Yumiko , Sato Shigeru , Kato Tomohiko , Harashima Satoshi , Shibata Daisuke
Published: July 2002

A higher plant homologue to the high-affinity phosphate transporter gene of yeast (
Saccharomyces cerevisiae
)
PHO84
was isolated from
Arabidopsis thaliana
. Expression of the
Arabidopsis
gene
PHT1
at high levels in tobacco-cultured cells increased the rate of phosphate uptake. The uptake activity attributable to the transgene was inhibited by protonophores, suggesting an H
+
cotransport mechanism of phosphate uptake, and had a
K
m
of 3.1 μM which is within limits characteristic of high-affinity transport mechanisms. These results indicate that
PHT1
encodes a high-affinity phosphate transporter. The transgenic cells exhibited increased biomass production when the supply of phosphate was limited, establishing gene engineering of phosphate transport as one approach toward enhancing plant cell growth.
α-Tocopheryl hydroquinone is an efficient multifunctional inhibitor of radical-initiated oxidation of low density lipoprotein lipids
Authors: Neužil Jiří , Witting Paul K. , Stocker Roland
Published: July 2002

As the oxidation of low density lipoprotein (LDL) lipids may be a key event in atherogenesis, there is interest in antioxidants as potential anti-atherogenic compounds. Here we report that α-tocopheryl hydroquinone (α-TQH
2
) strongly inhibited or completely prevented the (per)oxidation of ubiquinol-10 (CoQ
10
H
2
), α-tocopherol (α-TOH), and both surface and core lipids in LDL exposed to either aqueous or lipophilic peroxyl radicals, Cu
2+
, soybean lipoxygenase, or the transition metal-containing Ham’s F-10 medium in the absence or presence of human monocyte-derived macrophages. The antioxidant activity of α-TQH
2
was superior to that of several other lipophilic hydroquinones, including endogenous CoQ
10
H
2
, which is regarded as LDL’s first line of antioxidant defence. At least three independent activities contributed to the antioxidant action of α-TQH
2
. First, α-TQH
2
readily associated with LDL and instantaneously reduced the lipoprotein’s ubiquinone-10 to CoQ
10
H
2
, thereby maintaining this antioxidant in its active form. Second, α-TQH
2
directly intercepted aqueous peroxyl radicals, as indicated by the increased rate of its consumption with increasing rates of radical production, independent of LDL’s content of CoQ
10
H
2
and α-TOH. Third, α-TQH
2
rapidly quenched α-tocopheroxyl radical in oxidizing LDL, as demonstrated directly by electron paramagnetic resonance spectroscopy. Similar antioxidant activities were also seen when α-TQH
2
was added to high-density lipoprotein or the protein-free Intralipid, indicating that the potent antioxidant activity of α-TQH
2
was neither lipoprotein specific nor dependent on proteins. These results suggest that α-TQH
2
is a candidate for a therapeutic lipid-soluble antioxidant. As α-tocopherylquinone is formed
in vivo
at sites of oxidative stress, including human atherosclerotic plaque, and biological systems exist that reduce the quinone to the hydroquinone, our results also suggest that α-TQH
2
could be a previously unrecognized natural antioxidant.
Tumor necrosis factor (TNF)-mediated kinase cascades: Bifurcation of Nuclear Factor-κB and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2
Authors: Song Ho Yeong , Régnier Catherine H. , Kirschning Carsten J. , Goeddel David V. , Rothe Mike
Published: July 2002

TNF-induced activation of the transcription factor NF-κB and the c-jun N-terminal kinase (JNK/SAPK) requires TNF receptor-associated factor 2 (TRAF2). The NF-κB-inducing kinase (NIK) associates with TRAF2 and mediates TNF activation of NF-κB. Herein we show that NIK interacts with additional members of the TRAF family and that this interaction requires the conserved “WKI” motif within the TRAF domain. We also investigated the role of NIK in JNK activation by TNF. Whereas overexpression of NIK potently induced NF-κB activation, it failed to stimulate JNK activation. A kinase-inactive mutant of NIK was a dominant negative inhibitor of NF-κB activation but did not suppress TNF- or TRAF2-induced JNK activation. Thus, TRAF2 is the bifurcation point of two kinase cascades leading to activation of NF-κB and JNK, respectively.
T-strand integration in maize protoplasts after codelivery of a T-DNA substrate and virulence genes
Authors: Hansen Geneviève , Shillito Raymond D. , Chilton Mary-Dell
Published: July 2002

We describe a plant protoplast transformation method that provides transformants with a simple pattern of integration of a foreign gene. The approach is to deliver into plant protoplasts by direct gene transfer the
Agrobacterium
virulence genes
virD1
and
virD2
with or without
virE2
, together with a target plasmid containing a gene of interest flanked by
Agrobacterium
T-DNA border repeat sequences of 25 bp. We present evidence of T-DNA formation in maize protoplasts and its integration into the maize genome. The frequency of VirD1-VirD2-mediated integration events was about 20–35% of the total number of transformants. The addition of
virE2
doubled the transformation efficiency. The method described here is of sufficient efficiency and simplicity to be useful for the production of transgenic plants with single-copy well-defined transgenic inserts.
Sequence variation in the Fanconi anemia gene
FAA
Authors: Levran Orna , Erlich Tamar , Magdalena Neiva , Gregory John J. , Batish Sat Dev , Verlander Peter C. , Auerbach Arleen D.
Published: July 2002

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive syndrome associated with chromosomal instability, hypersensitivity to DNA crosslinking agents, and predisposition to malignancy. The gene for FA complementation group A (
FAA
) recently has been cloned. The cDNA is predicted to encode a polypeptide of 1,455 amino acids, with no homologies to any known protein that might suggest a function for
FAA
. We have used single-strand conformational polymorphism analysis to screen genomic DNA from a panel of 97 racially and ethnically diverse FA patients from the International Fanconi Anemia Registry for mutations in the
FAA
gene. A total of 85 variant bands were detected. Forty-five of the variants are probably benign polymorphisms, of which nine are common and can be used for various applications, including mapping studies for other genes in this region of chromosome 16q. Amplification refractory mutation system assays were developed to simplify their detection. Forty variants are likely to be pathogenic mutations. Seventeen of these are microdeletions/microinsertions associated with short direct repeats or homonucleotide tracts, a type of mutation thought to be generated by a mechanism of slipped-strand mispairing during DNA replication. A screening of 350 FA probands from the International Fanconi Anemia Registry for two of these deletions (1115–1118del and 3788–3790del) revealed that they are carried on about 2% and 5% of the FA alleles, respectively. 3788–3790del appears in a variety of ethnic groups and is found on at least two different haplotypes. We suggest that
FAA
is hypermutable, and that slipped-strand mispairing, a mutational mechanism recognized as important for the generation of germ-line and somatic mutations in a variety of cancer-related genes, including
p53
,
APC
,
RB1
,
WT1
, and
BRCA1
, may be a major mechanism for
FAA
mutagenesis.
Evolutionary specialization of the nuclear targeting apparatus
Authors: Malik Harmit S. , Eickbush Thomas H. , Goldfarb David S.
Published: July 2002

The α- and β-karyopherins (Kaps), also called importins, mediate the nuclear transport of proteins. All α-Kaps contain a central domain composed of eight approximately 40 amino acid, tandemly arranged, armadillo-like (Arm) repeats. The number and order of these repeats have not changed since the common origin of fungi, plants, and mammals. Phylogenetic analysis suggests that the various α-Kaps fall into two groups, α1 and α2. Whereas animals encode both types, the yeast genome encodes only an α1-Kap. The β-Kaps are characterized by 14–15 tandemly arranged HEAT motifs. We show that the Arm repeats of α-Kaps and the HEAT motifs of β-Kaps are similar, suggesting that the α-Kaps and β-Kaps (and for that matter, all Arm and HEAT repeat-containing proteins) are members of the same protein superfamily. Phylogenetic analysis indicates that there are at least three major groups of β-Kaps, consistent with their proposed cargo specificities. We present a model in which an α-independent β-Kap progenitor gave rise to the α-dependent β-Kaps and the α-Kaps.
Loperamide: A positive modulator for store-operated calcium channels?
Authors: Harper Jacquie L. , Shin Yangmee , Daly John W.
Published: July 2002

The depletion of inositol trisphosphate-sensitive intracellular pools of calcium causes activation of store-operated calcium (SOC) channels. Loperamide at 10–30 μM has no effect on intracellular calcium levels alone, but augments calcium levels in cultured cells when SOC channels have been activated. In HL-60 leukemic cells, the apparent positive modulatory effect of loperamide on SOC channels occurs when these channels have been activated after ATP, thapsigargin, or ionomycin-elicited depletion of calcium from intracellular storage sites. Loperamide has no effect when levels of intracellular calcium are elevated through a mechanism not involving SOC channels by using sphingosine. Loperamide caused augmentation of intracellular calcium levels after activation of SOC channels in NIH 3T3 fibroblasts, astrocytoma 1321N cells, smooth muscle DDT-MF2 cells, RBL-2H3 mast cells, and pituitary GH
4
C
1
cells. Only in astrocytoma cells did loperamide cause an elevation in intracellular calcium in the absence of activation of SOC channels. The augmentation of intracellular calcium elicited by loperamide in cultured cells was dependent on extracellular calcium and was somewhat resistant to agents (SKF 96365, miconazole, clotrimazole, nitrendipine, and trifluoperazine) that in the absence of loperamide effectively blocked SOC channels. It appears that loperamide augments influx of calcium through activated SOC channels.
Foreign (M13) DNA ingested by mice reaches peripheral leukocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to mouse DNA
Authors: Schubbert Rainer , Renz Doris , Schmitz Birgit , Doerfler Walter
Published: July 2002

Food-ingested foreign DNA is not completely degraded in the gastrointestinal tract of mice. Phage M13mp18 DNA as a test molecule devoid of homology to mouse DNA was pipette-fed to or added to the food supply of mice. The fate of this foreign DNA in the animals was followed by several methods. In 84 animals, fragments of M13mp18 DNA were detected in the contents of the small intestine, the cecum (until 18 h), the large intestine, or the feces. In 254 animals, M13mp18 DNA fragments of up to 976 bp were found in blood 2–8 h after feeding. In buffer-fed control animals, M13mp18 DNA could not be detected. M13mp18 DNA fragments were traced by PCR in peripheral leukocytes and located by fluorescentin situhybridization in about 1 of 1000 white cells between 2 and 8 h, and in spleen or liver cells up to 24 h after feeding, but not later. M13mp18 DNA could be traced by fluorescentin situhybridization in the columnar epithelial cells, in the leukocytes in Peyer’s patches of the cecum wall, in liver cells, and in B cells, T cells, and macrophages from spleen. These findings suggest transport of foreign DNA through the intestinal wall and Peyer’s patches to peripheral blood leukocytes and into several organs. Upon extended feeding, M13mp18 DNA could be recloned from total spleen DNA into a λ vector. Among about 2.5 × 107λ plaques, one plaque was isolated that contained a 1299 nucleotide pair fragment (nt 4736–6034) of sequence-identified M13mp18 DNA. This fragment was covalently linked to an 80 nt DNA segment with 70% homology to the mouse IgE receptor gene. The DNA from another λ plaque also contained mouse DNA, bacterial DNA, and rearranged λ DNA. Two additional plaques contained M13mp18 DNA fragments of at least 641 (nt 2660–3300) or 794 (nt 4640–5433) nucleotide pairs. The medical and evolutionary implications of these observations may be considerable.
Evidence that eukaryotic triosephosphate isomerase is of alpha-proteobacterial origin
Authors: Keeling Patrick J. , Doolittle W. Ford
Published: July 2002

We have cloned and sequenced genes for triosephosphate isomerase (TPI) from the gamma-proteobacterium
Francisella tularensis
, the green non-sulfur bacterium
Chloroflexus aurantiacus
, and the alpha-proteobacterium
Rhizobium etli
and used these in phylogenetic analysis with TPI sequences from other members of the Bacteria, Archaea, and Eukarya. These analyses show that eukaryotic TPI genes are most closely related to the homologue from the alpha-proteobacterium and most distantly related to archaebacterial homologues. This relationship suggests that the TPI genes present in modern eukaryotic genomes were derived from an alpha-proteobacterial genome (possibly that of the protomitochondrial endosymbiont) after the divergence of Archaea and Eukarya. Among these eukaryotic genes are some from deeply branching, amitochondrial eukaryotes (namely
Giardia
), which further suggests that this event took place quite early in eukaryotic evolution.
Recombinational construction in
Escherichia coli
of infectious adenoviral genomes
Authors: Crouzet Joël , Naudin Laurent , Orsini Cécile , Vigne Emmanuelle , Ferrero Lucy , Le Roux Aude , Benoit Patrick , Latta Martine , Torrent Christophe , Branellec Didier , Denèfle Patrice , Mayaux Jean-François , Perricaudet Michel , Yeh Patrice
Published: July 2002

A two-step gene replacement procedure was developed that generates infectious adenoviral genomes through homologous recombination in
Escherichia coli
. As a prerequisite, a human adenovirus serotype 5 (Ad5)-derived genome was first introduced as a
Pac
I restriction fragment into an incP-derived replicon which, in contrast to ColE1-derivatives (e.g., pBR322 or pUC plasmids), is functional in a
polA
mutant of
E. coli
. Any modification can be introduced at will following two consecutive homologous recombinations between the incP/Ad5 replicon and the ColE1 plasmid. The overall procedure requires only the
in vitro
engineering of the ColE1-derivative by flanking the desired modification with small stretches of identical sequences. In the first step, a cointegrate between the tetracycline-resistant incP/Ad5 replicon and the kanamycin-resistant ColE1-derivative is selected by growing the
polA
host in the presence of both antibiotics. Resolution of this cointegrate is further selected in sucrose growth conditions due to the loss of a conditional suicide marker (the
sacB
gene of
Bacillus subtilis
) present in the ColE1 plasmid, leading to unmodified and modified incP/Ad5 replicons that can be differentiated upon restriction analysis. Consecutive rounds of this two-step cloning procedure allowed the introduction of multiple independent modifications within the virus genome, with no requirement for an intermediate virus. The potential of this procedure is demonstrated by the recovery of several E1E3E4-deleted adenoviruses following transfection of the corresponding
E. coli
-derived genomes in IGRP2 cells.