bluuebunny/crossref_metadata_2025 huggingface dataset:
Selection of marker-free transgenic plants using the isopentenyl transferase gene
Authors: Ebinuma Hiroyasu , Sugita Koichi , Matsunaga Etsuko , Yamakado Mikiko
Published: July 2002

We have developed a new plant vector system for repeated transformation (called MAT for multi-auto-transformation) in which a chimeric
ipt
gene, inserted into the transposable element
Ac
, is used as a selectable marker for transformation. Selectable marker genes conferring antibiotic or herbicide resistance, used to introduce economically valuable genes into crop plants, have three major problems: (
i
) the selective agents have negative effects on proliferation and differentiation of plant cells; (
ii
) there is uncertainty regarding the environmental impact of many selectable marker genes; (
iii
) it is difficult to perform recurrent transformations using the same selectable marker to pyramid desirable genes. The MAT vector system containing the
ipt
gene and the
Ac
element is designed to overcome these difficulties. When tobacco leaf segments were transformed and selected, subsequent excision of the modified
Ac
produced marker-free transgenic tobacco plants without sexual crosses or seed production. In addition, the chimeric
ipt
gene could be visually used as a selectable marker for transformation of hybrid aspen (
Populus sieboldii
×
Populus grandidentata
). The chimeric
ipt
gene, therefore, is an attractive alternative to the most widely used selectable marker genes. The MAT vector system provides a promising way to shorten breeding time for genetically engineered crops. This method could be particularly valuable for fruit and forest trees, for which long generation times are a more significant barrier to breeding and genetic analysis.
Regular alcohol consumption mimics cardiac preconditioning by protecting against ischemia–reperfusion injury
Authors: Miyamae Masami , Diamond Ivan , Weiner Michael W. , Camacho S. Albert , Figueredo Vincent M.
Published: July 2002

Epidemiologic studies indicate that long-term alcohol consumption decreases the incidence of coronary disease and may improve outcome after myocardial infarction. Attenuation of ischemia–reperfusion injury after myocardial infarction improves survival. This study investigates the possibility that alcohol consumption can improve survival after myocardial infarction by reducing ischemia–reperfusion injury. Hearts were isolated from guinea pigs after drinking ethanol for 3–12 weeks and subjected to global ischemia and reperfusion. Hearts from animals drinking ethanol showed improved functional recovery and decreased myocyte damage when compared with controls. Adenosine A
1
receptor blockade abolished the protection provided by ethanol consumption. These findings indicate that long-term alcohol consumption reduces myocardial ischemia–reperfusion injury and that adenosine A
1
receptors are required for this protective effect of ethanol. This cardioprotective effect of long-term alcohol consumption mimics preconditioning and may, in part, account for the beneficial effect of moderate drinking on cardiac health.
Initial frequency of alleles for resistance to
Bacillus thuringiensis
toxins in field populations of
Heliothis virescens
Authors: Gould F. , Anderson A. , Jones A. , Sumerford D. , Heckel D. G. , Lopez J. , Micinski S. , Leonard R. , Laster M.
Published: July 2002

The risk of rapid pest adaptation to an insecticide is highly dependent on the initial frequency of resistance alleles in field populations. Because we have lacked empirical estimates of these frequencies, population–genetic models of resistance evolution have relied on a wide range of theoretical estimates. The recent commercialization of genetically engineered cotton that constitutively produces an insecticidal protein derived from the biocontrol agent,
Bacillus thuringiensis
(Bt) has raised concern that we lack data needed to quantify the risk of insect pests such as
Heliothis virescens
rapidly adapting to this ecologically valuable class of toxins. By individually mating over 2,000 male
H. virescens
moths collected in four states to females of a Bt toxin-resistant laboratory strain, and screening F
1
and F
2
offspring for tolerance of the toxic protein, we were able to directly estimate the field frequency of alleles for resistance as 1.5 × 10
−3
. This high initial frequency underscores the need for caution in deploying transgenic cotton to control insect pests. Our single-pair mating technique greatly increases the efficiency of detecting recessive resistance alleles. Because alleles that decrease target site sensitivity to Bt toxins and other insecticides are often recessive, this technique could be useful in estimating resistance allele frequencies in other insects exposed to transgenic insecticidal crops or conventional insecticides.
The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors
Authors: Marvin J. S. , Corcoran E. E. , Hattangadi N. A. , Zhang J. V. , Gere S. A. , Hellinga H. W.
Published: July 2002

Rational protein design is an emerging approach for testing general theories of structure and function. The ability to manipulate function rationally also offers the possibility of creating new proteins of biotechnological value. Here we use the design approach to test the current understanding of the structural principles of allosteric interactions in proteins and demonstrate how a simple allosteric system can form the basis for the construction of a generic biosensor molecular engineering system. We have identified regions in
Escherichia coli
maltose-binding protein that are predicted to be allosterically linked to its maltose-binding site. Environmentally sensitive fluorophores were covalently attached to unique thiols introduced by cysteine mutations at specific sites within these regions. The fluorescence of such conjugates changes cooperatively with respect to maltose binding, as predicted. Spatial separation of the binding site and reporter groups allows the intrinsic properties of each to be manipulated independently. Provided allosteric linkage is maintained, ligand binding can therefore be altered without affecting transduction of the binding event by fluorescence. To demonstrate applicability to biosensor technology, we have introduced a series of point mutations in the maltose-binding site that lower the affinity of the protein for its ligand. These mutant proteins have been combined in a composite biosensor capable of measuring substrate concentration within 5% accuracy over a concentration range spanning five orders of magnitude.
Association-induced folding of globular proteins
Authors: Uversky Vladimir N. , Segel Daniel J. , Doniach Sebastian , Fink Anthony L.
Published: July 2002

It has generally been assumed that the aggregation of partially folded intermediates during protein refolding results in the termination of further protein folding. We show here, however, that under some conditions the association of partially folded intermediates can induce additional structure leading to soluble aggregates with many native-like properties. The amount of secondary structure in a monomeric, partially folded intermediate of staphylococcal nuclease was found to double on formation of soluble aggregates at high protein or salt concentrations. In addition, more globularity, as determined from Kratky plots of small-angle x-ray scattering data, was also noted in the associated states.
Ligand-independent activation of platelet-derived growth factor receptor is a necessary intermediate in lysophosphatidic, acid-stimulated mitogenic activity in L cells
Authors: Herrlich Andreas , Daub Henrik , Knebel Axel , Herrlich Peter , Ullrich Axel , Schultz Günter , Gudermann Thomas
Published: July 2002

Growth factor-derived mitogenic signals from the cell surface are transmitted to the nucleus via receptor tyrosine kinases (RTKs), the adaptor proteins Shc and Grb2, and a Ras-dependent protein kinase cascade that activates the extracellular signal regulated kinase (ERK) subfamily of mitogen-activated protein kinases. ERKs also are activated by hormones that stimulate G protein-coupled receptors (GPCRs). We report here that, in agreement with previous data, the epidermal growth factor receptor (EGFR) is a signaling intermediate in ERK activation by GPCRs. Of import, we show that cross-talk between two classes of surface receptors, RTKs and GPCRs, is a general feature. Lysophosphatidic acid not only induces ligand-independent tyrosine autophosphorylation of EGFR but also of platelet-derived growth factor β receptor (PDGF-β-R) as shown by detection of tyrosine phosphorylation and by the use of specific inhibitors of RTKs. The cross-talk appears to be cell type-specific: In L cells that lack EGFR, lysophosphatidic acid-induced Shc and ERK activation is prevented completely by specific inhibition of PDGFR, whereas in COS-7 cells expressing only EGFR, the pathway via EGFR is chosen. In Rat-1 cells, however, that express both EGFR and PDGFR, the EGFR pathway dominates.
Recombinant modified vaccinia virus Ankara–simian immunodeficiency virusgag polelicits cytotoxic T lymphocytes in rhesus monkeys detected by a major histocompatibility complex class I/peptide tetramer
Authors: Seth Aruna , Ourmanov Ilnour , Kuroda Marcelo J. , Schmitz Jörn E. , Carroll Miles W. , Wyatt Linda S. , Moss Bernard , Forman Meryl A. , Hirsch Vanessa M. , Letvin Norman L.
Published: July 2002

The utility of modified vaccinia virus Ankara (MVA) as a vector for eliciting AIDS virus-specific cytotoxic T lymphocytes (CTL) was explored in the simian immunodeficiency virus (SIV)/rhesus monkey model. After two intramuscular immunizations with recombinant MVA-SIVSMgag pol, the monkeys developed a Gag epitope-specific CTL response readily detected in peripheral blood lymphocytes by using a functional killing assay. Moreover, those immunizations also elicited a population of CD8+ T lymphocytes in the peripheral blood that bound a specific major histocompatibility complex class I/peptide tetramer. These Gag epitope-specific CD8+ T lymphocytes also were demonstrated by using both functional and tetramer-binding assays in lymph nodes of the immunized monkeys. These observations suggest that MVA may prove a useful vector for an HIV-1 vaccine. They also suggest that tetramer staining may be a useful technology for monitoring CTL generation in vaccine trials in nonhuman primates and in humans.
PDZ-domain-mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor
Authors: Hock Björn , Böhme Beatrix , Karn Thomas , Yamamoto Takaharu , Kaibuchi Kozo , Holtrich Uwe , Holland Sacha , Pawson Tony , Rübsamen-Waigmann Helga , Strebhardt Klaus
Published: July 2002

Eph-related receptor tyrosine kinases (RTKs) have been implicated in intercellular communication during embryonic development. To elucidate their signal transduction pathways, we applied the yeast two-hybrid system. We could demonstrate that the carboxyl termini of the Eph-related RTKs EphA7, EphB2, EphB3, EphB5, and EphB6 interact with the PDZ domain of the ras-binding protein AF6. A mutational analysis revealed that six C-terminal residues of the receptors are involved in binding to the PDZ domain of AF6 in a sequence-specific fashion. Moreover, this PDZ domain also interacts with C-terminal sequences derived from other transmembrane receptors such as neurexins and the Notch ligand Jagged. In contrast to the association of EphB3 to the PDZ domain of AF6, the interaction with full-length AF6 clearly depends on the kinase activity of EphB3, suggesting a regulated mechanism for the PDZ-domain-mediated interaction. These data gave rise to the idea that the binding of AF6 to EphB3 occurs in a cooperative fashion because of synergistic effects involving different epitopes of both proteins. Moreover, in NIH 3T3 and NG108 cells endogenous AF6 is phosphorylated specifically by EphB3 and EphB2 in a ligand-dependent fashion. Our observations add the PDZ domain to the group of conserved protein modules such as Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains that regulate signal transduction through their ability to mediate the interaction with RTKs.
Mass spectrometric evidence for the enzymatic mechanism of the depolymerization of heparin-like glycosaminoglycans by heparinase II
Authors: Rhomberg Andrew J. , Shriver Zachary , Biemann Klaus , Sasisekharan Ram
Published: July 2002

Heparin-like glycosaminoglycans, acidic complex polysaccharides present on cell surfaces and in the extracellular matrix, regulate important physiological processes such as anticoagulation and angiogenesis. Heparin-like glycosaminoglycan degrading enzymes or heparinases are powerful tools that have enabled the elucidation of important biological properties of heparin-like glycosaminoglycans
in vitro
and
in vivo
. With an overall goal of developing an approach to sequence heparin-like glycosaminoglycans using the heparinases, we recently have elaborated a mass spectrometry methodology to elucidate the mechanism of depolymerization of heparin-like glycosaminoglycans by heparinase I. In this study, we investigate the mechanism of depolymerization of heparin-like glycosaminoglycans by heparinase II, which possesses the broadest known substrate specificity of the heparinases. We show here that heparinase II cleaves heparin-like glycosaminoglycans endolytically in a nonrandom manner. In addition, we show that heparinase II has two distinct active sites and provide evidence that one of the active sites is heparinase I-like, cleaving at hexosamine–sulfated iduronate linkages, whereas the other is presumably heparinase III-like, cleaving at hexosamine–glucuronate linkages. Elucidation of the mechanism of depolymerization of heparin-like glycosaminoglycans by the heparinases and mutant heparinases could pave the way to the development of much needed methods to sequence heparin-like glycosaminoglycans.
rRNA complementarity within mRNAs: A possible basis for mRNA-ribosome interactions and translational control
Authors: Tranque Pedro , Hu Michael C.-Y. , Edelman Gerald M. , Mauro Vincent P.
Published: July 2002

Our recent demonstration that many eukaryotic mRNAs contain sequences complementary to rRNA led to the hypothesis that these sequences might mediate specific interactions between mRNAs and ribosomes and thereby affect translation. In the present experiments, the ability of complementary sequences to bind to rRNA was investigated by using photochemical cross-linking. RNA probes with perfect complementarity to 18S or 28S rRNA were shown to cross-link specifically to the corresponding rRNA within intact ribosomal subunits. Similar results were obtained by using probes based on natural mRNA sequences with varying degrees of complementarity to the 18S rRNA. RNase H cleavage localized four such probes to complementary regions of the 18S rRNA. The effects of complementarity on translation were assessed by using the mRNA encoding ribosomal protein S15. This mRNA contains a sequence within its coding region that is complementary to the 18S rRNA at 20 of 22 nucleotides. RNA from an S15-luciferase fusion construct was translated in a cell-free lysate and compared with the translation of four related constructs that were mutated to decrease complementarity to the 18S rRNA. These mutations did not alter the amino acid sequence or the codon bias. A correlation between complementarity and translation was observed; constructs with less complementarity increased the amount of translation up to 54%. These findings raised the possibility that direct base-pairing of particular mRNAs to rRNAs within ribosomes may function as a mechanism of translational control.
Cloning and characterization of a gene from
Escherichia coli
encoding a transketolase-like enzyme that catalyzes the synthesis of
d
-1-deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis
Authors: Lois Luisa Maria , Campos Narciso , Putra Surya Rosa , Danielsen Knut , Rohmer Michel , Boronat Albert
Published: July 2002

For many years it was accepted that isopentenyl diphosphate, the common precursor of all isoprenoids, was synthesized through the well known acetate/mevalonate pathway. However, recent studies have shown that some bacteria, including
Escherichia coli
, use a mevalonate-independent pathway for the synthesis of isopentenyl diphosphate. The occurrence of this alternative pathway has also been reported in green algae and higher plants. The first reaction of this pathway consists of the condensation of (hydroxyethyl)thiamin derived from pyruvate with the C1 aldehyde group of
d
-glyceraldehyde 3-phosphate to yield
d
-1-deoxyxylulose 5-phosphate. In
E. coli
,
d
-1-deoxyxylulose 5-phosphate is also a precursor for the biosynthesis of thiamin and pyridoxol. Here we report the molecular cloning and characterization of a gene from
E. coli
, designated
dxs,
that encodes
d
-1-deoxyxylulose-5-phosphate synthase. The
dxs
gene was identified as part of an operon that also contains
ispA
, the gene that encodes farnesyl-diphosphate synthase.
d
-1-Deoxyxylulose-5-phosphate synthase belongs to a family of transketolase-like proteins that are highly conserved in evolution.
A cofactor, TIP30, specifically enhances HIV-1 Tatactivated transcription
Authors: Xiao Hua , Tao Yong , Greenblatt Jack , Roeder Robert G.
Published: July 2002

Replication of HIV-1 requires the viral Tat protein, which increases the extent of transcription elongation by RNA polymerase II after activation at the single viral long terminal repeat (LTR) promoter. This effect of Tat on transcription requires Tat interactions with a 5′ region (TAR) in nascent transcripts as well as Tat-specific cofactors. The present study identifies a cellular protein, TIP30, that interacts with Tat and with an SRB-containing RNA polymerase II complex both
in vivo
and
in vitro
. Coexpression of TIP30 specifically enhances transactivation by Tat in transfected cells, and immunodepletion of TIP30 from nuclear extracts abolishes Tat-activated transcription without affecting Tat-independent transcription. These results implicate TIP30 as a specific coactivator that may enhance formation of a Tat–RNA polymerase II holoenzyme complex.
Impaired fetal T cell development and perinatal lethality in mice lacking the cAMP response element binding protein
Authors: Rudolph Dorothea , Tafuri Anna , Gass Peter , Hämmerling Günter J. , Arnold Bernd , Schütz Günther
Published: July 2002

CREB, the cAMP response element binding protein, is a key transcriptional regulator of a large number of genes containing a CRE consensus sequence in their upstream regulatory regions. Mice with a hypomorphic allele of CREB that leads to a loss of the CREBα and Δ isoforms and to an overexpression of the CREBβ isoform are viable. Herein we report the generation of CREB null mice, which have all functional isoforms (CREBα, β, and Δ) inactivated. In contrast to the CREBαΔ mice, CREB null mice are smaller than their littermates and die immediately after birth from respiratory distress. In brain, a strong reduction in the corpus callosum and the anterior commissures is observed. Furthermore, CREB null mice have an impaired fetal T cell development of the αβ lineage, which is not affected in CREBαΔ mice on embryonic day 18.5. Overall thymic cellularity in CREB null mice is severely reduced affecting all developmental stages of the αβ T cell lineage. In contrast γδ T cell differentiation is normal in CREB mutant mice.
Ubiquitination of RNA polymerase II large subunit signaled by phosphorylation of carboxyl-terminal domain
Authors: Mitsui Akira , Sharp Phillip A.
Published: July 2002

A sensitive assay using biotinylated ubiquitin revealed extensive ubiquitination of the large subunit of RNA polymerase II during incubations of transcription reactions
in vitro
. Phosphorylation of the repetitive carboxyl-terminal domain of the large subunit was a signal for ubiquitination. Specific inhibitors of cyclin-dependent kinase (cdk)-type kinases suppress the ubiquitination reaction. These kinases are components of transcription factors and have been shown to phosphorylate the carboxyl-terminal domain. In both regulation of transcription and DNA repair, phosphorylation of the repetitive carboxyl-terminal domain by kinases might signal degradation of the polymerase.
Structure of the soluble methane monooxygenase regulatory protein B
Authors: Walters Kylie J. , Gassner George T. , Lippard Stephen J. , Wagner Gerhard
Published: July 2002

The soluble methane monooxygenase (sMMO; EC
1.14.13.25
) from the pseudothermophile
Methylococcus capsulatus
(Bath) is a three-component enzyme system that catalyzes the selective oxidation of methane to methanol. We have used NMR spectroscopy to produce a highly refined structure of MMOB, the 16-kDa regulatory protein of this system. This structure has a unique and intricate fold containing seven β-strands forming two β-sheets oriented perpendicular to each other and bridged by three α-helices. The rate and efficiency of the methane hydroxylation by sMMO depend on dynamic binding interactions of the hydroxylase with the reductase and regulatory protein components during catalysis. We have monitored by NMR the binding of MMOB to the hydroxylase in the presence and absence of the reductase. The results of these studies provide structural insight into how the regulatory protein interacts with the hydroxylase.
Post-priming actions of ATP on Ca
2+
-dependent exocytosis in pancreatic beta cells
Authors: Takahashi Noriko , Kadowaki Takashi , Yazaki Yoshio , Ellis-Davies Graham C. R. , Miyashita Yasushi , Kasai Haruo
Published: July 2002

The role of cytosolic ATP in exocytosis was investigated by using amperometric measurement of insulin exocytosis in pancreatic beta cells, which were stimulated with photolysis of caged Ca
2+
compounds. Insulin exocytosis occurred with two rates. We found that ATP hastened and augmented the exocytosis via selective enhancement of the exocytosis with the faster rate. A nonhydrolysable analog of ATP, adenosine 5′-
O
-(3-thiotriphosphate), which blocks ATPase, was even more effective than ATP, indicating that the phosphorylation event occurred downstream of ATP-dependent vesicle transportation and priming. The action of ATP was eliminated by a competitive antagonist of cAMP, and by an inhibitor of adenylate cyclase. These data characterize an ATP sensing mechanism for the Ca
2+
-dependent exocytosis involving adenylate-cyclase, cAMP-dependent protein kinase, and, possibly, the fusion machinery itself. Thus, the fast exocytotic machinery requires both phosphorylation and Ca
2+
for the final triggering and likely constitutes a distal ATP sensor for insulin exocytosis that acts in concert with ATP-sensitive K
+
channels.
A coding RNA sequence acts as a replication signal in cardioviruses
Authors: Lobert Pierre-Emmanuel , Escriou Nicolas , Ruelle Jean , Michiels Thomas
Published: July 2002

Theiler’s virus and Mengo virus are representatives of the
Cardiovirus
genus within the picornavirus family. Their genome is an 8-kilobase long positive strand RNA molecule. This RNA molecule plays three roles in infected cells: It serves as a messenger RNA, acts as a template for genome replication, and is encapsidated to form progeny virions. We observed that a cis-acting signal required for replication of Theiler’s virus was contained within a 130-nt stretch of the region encoding the capsid protein VP2. This RNA sequence does not influence internal ribosome entry site-mediated translation initiation and thus likely acts directly as a signal for the replication complex. We found a similar signal in the VP2-coding sequence of Mengo virus, and both signals could be functionally exchanged. Within the replication element, a 9-nt sequence that is highly conserved among cardioviruses was shown to be essential for replication. This conserved sequence was contained in mostly unpaired regions of the RNA secondary structure predicted for the replication elements of the various cardioviruses. Interestingly, a similar replication element has been reported to occur in the distantly related human rhinovirus type 14, suggesting that such elements could be conserved throughout the picornavirus family. However, the different location of the replication elements in rhinovirus and cardioviruses, and the fact that they were not functionally exchangeable, is raising intriguing questions about the evolution of such signals in picornaviruses.
Direct visualization of the
elt-2
gut-specific GATA factor binding to a target promoter inside the living
Caenorhabditis elegans
embryo
Authors: Fukushige Tetsunari , Hendzel Michael J. , Bazett-Jones David P. , McGhee James D.
Published: July 2002

In analyzing the transcriptional networks that regulate development, one ideally would like to determine whether a particular transcription factor binds directly to a candidate target promoter inside the living embryo. Properties of the
Caenorhabditis elegans elt-2
gene, which encodes a gut-specific GATA factor, have allowed us to develop such a method. We previously have shown, by means of ectopic expression studies, that
elt-2
regulates its own promoter. To test whether this autoregulation is direct, we fused green fluorescent protein (GFP) close to the C terminus of
elt-2
in a construct that contains the full
elt-2
promoter and the full
elt-2
zinc finger DNA binding domain; the construct is expressed correctly (i.e., only in the gut lineage) and is able to rescue the lethality of an
elt-2
null mutant. Multicopy transgenic arrays of this rescuing
elt-2
∷GFP construct were integrated into the genome and transgenic embryos were examined when the developing gut has 4–8 cells; the majority of these embryonic gut nuclei show two discrete intense foci of fluorescence. We interpret these fluorescent foci as the result of ELT-2∷GFP binding directly to its own promoter within nuclei of the developing gut lineage. Numerous control experiments, both genetic and biochemical, all support this conclusion and support the specificity of the binding. The approach should be applicable to studying other transcription factors binding target promoters, all within the living
C. elegans
embryo.
Race-specific HIV-1 disease-modifying effects associated with
CCR5
haplotypes
Authors: Gonzalez Enrique , Bamshad Mike , Sato Naoko , Mummidi Srinivas , Dhanda Rahul , Catano Gabriel , Cabrera Sonia , McBride Megan , Cao Xuan-Hien , Merrill Gerald , O’Connell Peter , Bowden Donald W. , Freedman Barry I. , Anderson Stephanie A. , Walter Elizabeth A. , Evans James S. , Stephan Kevin T. , Clark Robert A. , Tyagi Sanjay , Ahuja Seema S. , Dolan Matthew J. , Ahuja Sunil K.
Published: July 2002

Genetic variation in CC chemokine receptor 5 (
CCR5
), the major HIV-1 coreceptor, has been shown to influence HIV-1 transmission and disease progression. However, it is generally assumed that the same
CCR5
genotype (or haplotype) has similar phenotypic effects in different populations. To test this assumption, we used an evolutionary-based classification of
CCR5
haplotypes to determine their associated HIV-1 disease-modifying effects in a large well-characterized racially mixed cohort of HIV-1-seropositive individuals. We demonstrate that the spectrum of
CCR5
haplotypes associated with disease acceleration or retardation differs between African Americans and Caucasians. Also, we show that there is a strong interactive effect between
CCR5
haplotypes with different evolutionary histories. The striking population-specific phenotypic effects associated with
CCR5
haplotypes emphasize the importance of understanding the evolutionary context in which disease susceptibility genes are expressed.
Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the β1-adrenergic receptor
Authors: Tang Yuting , Hu Liaoyuan A. , Miller William E. , Ringstad Niels , Hall Randy A. , Pitcher Julie A. , DeCamilli Pietro , Lefkowitz Robert J.
Published: July 2002

Several G-protein coupled receptors, such as the β1-adrenergic receptor (β1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein–protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the β1-AR either as a glutathione
S
-transferase fusion protein in biochemical “pull-down” assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4/p8/p13 (also referred to as endophilin 1/2/3), a SH3 domain-containing protein family, as binding partners for the β1-AR.
In vitro
and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the β1-AR but not to that of the β2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of β1-ARs in HEK293 cells while having no effect on β2-ARs. Thus, our studies demonstrate a role of the SH3p4/p8/p13 protein family in β1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.
Ubiquitin-mediated degradation of active Src tyrosine kinase
Authors: Harris Kimya F. , Shoji Ikuo , Cooper Eric M. , Kumar Sushant , Oda Hideaki , Howley Peter M.
Published: July 2002

Src family tyrosine kinases are involved in modulating various signal transduction pathways leading to the induction of DNA synthesis and cytoskeletal reorganization in response to cell-cell or cell-matrix adhesion. The critical role of these kinases in regulating cellular signaling pathways requires that their activity be tightly controlled. Src family proteins are regulated through reversible phosphorylation and dephosphorylation events that alter the conformation of the kinase. We have found evidence that Src also is regulated by ubiquitination. Activated forms of Src are less stable than either wild-type or kinase-inactive Src mutants and can be stabilized by proteasome inhibitors. In addition, poly-ubiquitinated forms of active Src have been detected
in vivo
. Taken together, our results establish ubiquitin-mediated proteolysis as a previously unidentified mechanism for irreversibly attenuating the effects of active Src kinase.
Promoter-specific hypoacetylation of X-inactivated genes
Authors: Gilbert Sandra L. , Sharp Phillip A.
Published: July 2002

The histone H4 acetylation status of the active X (Xa) and inactive X (Xi) chromosomes was investigated at the level of individual genes. A moderate level of acetylation was observed along the lengths of genes on both the Xi and Xa, regardless of their X inactivation status. However, this moderate level of acetylation was modified specifically in promoter regions. Transcriptionally active genes showed elevated levels of acetylation at their promoters on both the Xi and Xa. In contrast, promoters of X-inactivated genes were markedly hypoacetylated, which coincided with the methylation of adjacent CG dinucleotides. This promoter-specific hypoacetylation may be a key component of an X inactivation machinery that operates at the level of individual genes.
Germ-line transmission of transgenes in
Xenopus laevis
Authors: Marsh-Armstrong Nicholas , Huang Haochu , Berry Deborah L. , Brown Donald D.
Published: July 2002

Adult
Xenopus
laevis
frogs made transgenic by restriction enzyme-mediated integration were bred to test the feasibility of establishing lines of frogs that express transgenes. All of the 19 animals raised to sexual maturity generated progeny that expressed the transgene(s). The patterns and levels of expression of green fluorescent protein transgenes driven by a viral promoter, rat promoter, and four
X. laevis
promoters were all unaffected by passage through the germ line. These results demonstrate the ease of establishing transgenic lines in
X. laevis
.
Structure of a cytochrome P450–redox partner electron-transfer complex
Authors: Sevrioukova Irina F. , Li Huiying , Zhang Hong , Peterson Julian A. , Poulos Thomas L.
Published: July 2002

The crystal structure of the complex between the heme- and FMN-binding domains of bacterial cytochrome P450BM-3, a prototype for the complex between eukaryotic microsomal P450s and P450 reductase, has been determined at 2.03 Å resolution. The flavodoxin-like flavin domain is positioned at the proximal face of the heme domain with the FMN 4.0 and 18.4 Å from the peptide that precedes the heme-binding loop and the heme iron, respectively. The heme-binding peptide represents the most efficient and coupled through-bond electron pathway to the heme iron. Substantial differences between the FMN-binding domains of P450BM-3 and microsomal P450 reductase, observed around the flavin-binding sites, are responsible for different redox properties of the FMN, which, in turn, control electron flow to the P450.
Evidence for a physical interaction between presenilin and Notch
Authors: Ray William J. , Yao Min , Nowotny Petra , Mumm Jeff , Zhang Wanjiang , Wu Jane Y. , Kopan Raphael , Goate Alison M.
Published: July 2002

Genetic analyses in
Caenorhabditis elegans
demonstrate that
sel-12
and
hop-1
, homologues of the Alzheimer’s disease-associated presenilin genes, modify signaling through LIN-12 and GLP-1, homologues of the Notch cell surface receptor. To gain insight into the biochemical basis of this genetic interaction, we tested the possibility that presenilin-1 (PS1) physically associates with the Notch1 receptor in mammalian cells. Notch1 and PS1 coimmunoprecipitated from transiently transfected human embryonic kidney 293 cell lysates in a detergent-sensitive manner, consistent with a noncovalent physical association between the two proteins. The interaction predominantly occurred early in the secretory pathway prior to Notch cleavage in the Golgi, because PS1 immunoprecipitation preferentially recovered the full-length Notch1 precursor. When PS1 was immunoprecipitated from 293 cells that had been metabolically labeled with [
35
S]methionine and [
35
S]cysteine, Notch1 was the primary protein detected in PS1 immunoprecipitates, suggesting that this interaction is specific. Furthermore, endogenous Notch and presenilin coimmunoprecipitated from cultured
Drosophila
cells, indicating that physical interaction can occur at physiological expression levels. These results suggest that the genetic relationship between presenilins and the Notch signaling pathway derives from a direct physical association between these proteins in the secretory pathway.
The Cys
4
zinc finger of bacteriophage T7 primase in sequence-specific single-stranded DNA recognition
Authors: Kusakabe Takahiro , Hine Anna V. , Hyberts Sven G. , Richardson Charles C.
Published: July 2002

Bacteriophage T7 DNA primase recognizes 5′-GTC-3′ in single-stranded DNA. The primase contains a single Cys
4
zinc-binding motif that is essential for recognition. Biochemical and mutagenic analyses suggest that the Cys
4
motif contacts cytosine of 5′-GTC-3′ and may also contribute to thymine recognition. Residues His
33
and Asp
31
are critical for these interactions. Biochemical analysis also reveals that T7 primase selectively binds CTP in the absence of DNA. We propose that bound CTP selects the remaining base G, of 5′-GTC-3′, by base pairing. Our deduced mechanism for recognition of ssDNA by Cys
4
motifs bears little resemblance to the recognition of trinucleotides of double-stranded DNA by Cys
2
His
2
zinc fingers.
Functions of the DNA damage response pathway target Ho endonuclease of yeast for degradation via the ubiquitin-26S proteasome system
Authors: Kaplun Ludmila , Ivantsiv Yelena , Kornitzer Daniel , Raveh Dina
Published: July 2002

Ho endonuclease of
Saccharomyces cerevisiae
is a homing endonuclease that makes a site-specific double-strand break in the
MAT
gene in late G
1
. Here we show that Ho is rapidly degraded via the ubiquitin-26S proteasome system through two ubiquitin-conjugating enzymes UBC2
Rad6
and UBC3
Cdc34
. UBC2
Rad6
is complexed with the ring finger DNA-binding protein Rad18, and we find that Ho is stabilized in
rad18
mutants. We show that the Ho degradation pathway involving UBC3
Cdc34
goes through the Skp1/Cdc53/F-box (SCF) ubiquitin ligase complex and identify a F-box protein, Yml088w, that is required for Ho degradation. Components of a defined pathway of the DNA damage response,
MEC1
,
RAD9
, and
CHK1
, are also necessary for Ho degradation, whereas functions of the
RAD24
epistasis group and the downstream effector
RAD53
have no role in degradation of Ho. Our results indicate a link between the endonuclease function of Ho and its destruction.
Mik1 levels accumulate in S phase and may mediate an intrinsic link between S phase and mitosis
Authors: Christensen Per U. , Bentley Nicola J. , Martinho Rui G. , Nielsen Olaf , Carr Antony M.
Published: July 2002

Two paradigms exist for maintaining order during cell-cycle progression: intrinsic controls, where passage through one part of the cell cycle directly affects the ability to execute another, and checkpoint controls, where external pathways impose order in response to aberrant structures. By studying the mitotic inhibitor Mik1, we have identified evidence for an intrinsic link between unperturbed S phase and mitosis. We propose a model in which S/M linkage can be generated by the production and stabilization of Mik1 protein during S phase. The production of Mik1 during unperturbed S phase is independent of the Rad3- and Cds1-dependent checkpoint controls. In response to perturbed S phase, Rad3-Cds1 checkpoint controls are required to maintain high levels of Mik1, probably indirectly by extending the S phase period, where Mik1 is stable. In addition, we find that Mik1 protein can be moderately induced in response to irradiation of G
2
cells in a Chk1-dependent manner.
Design of polyzinc finger peptides with structured linkers
Authors: Moore Michael , Choo Yen , Klug Aaron
Published: July 2002

Zinc finger domains are perhaps the most versatile of all known DNA
binding domains. By fusing up to six zinc finger modules, which
normally recognize up to 18 bp of DNA, designer transcription factors
can be produced to target unique sequences within large genomes.
However, not all continuous DNA sequences make good zinc finger binding
sites. To avoid having to target unfavorable DNA sequences, we designed
multizinc finger peptides with linkers capable of spanning long
stretches of nonbound DNA. Two three-finger domains were fused by using
either transcription factor IIIA for the
Xenopus
5S RNA
gene (TFIIIA) finger 4 or a non-sequence-specific zinc finger as a
“structured” linker. Our gel-shift results demonstrate that these
peptides are able to bind with picomolar affinities to target sequences
containing 0–10 bp of nonbound DNA. Furthermore, these peptides
display greater sequence selectivity and bind with higher affinity than
similar six-finger peptides containing long, flexible linkers. These
peptides are likely to be of use in understanding the behavior of
polydactyl proteins in nature and in the targeting of human, animal, or
plant genomes for numerous applications. We also suggest that in
certain polydactyl peptides an individual finger can “flip” out
of the major groove to allow its neighbors to bind shorter, nontarget
DNA sequences.
Ash1p is a site-specific DNA-binding protein that actively represses transcription
Authors: Maxon Mary E. , Herskowitz Ira
Published: July 2002

ASH1
encodes a protein that is localized
specifically to the daughter cell nucleus, where it has been proposed
to repress transcription of the
HO
gene. Using Ash1p
purified from baculovirus-infected insect cells, we have shown that
Ash1p binds specific DNA sequences in the
HO
promoter.
DNase I protection analyses showed that Ash1p recognizes a consensus
sequence, YTGAT. Mutation of this consensus abolishes Ash1p DNA binding
in vitro
. We have shown that Ash1p requires an intact
zinc-binding domain in its C terminus for repression of
HO in
vivo
and that this domain may be involved in DNA binding. A
heterologous DNA-binding domain fused to an N-terminal segment of Ash1p
functions as an active repressor of transcription. Our studies indicate
that Ash1p is a DNA-binding protein of the GATA family with a separable
transcriptional repression domain.
John Rae: The Lost Letters
Authors: James R. Warren
Published: July 2002

Charles Whitney Mixter started a minor controversy when he published an article, “A Forerunner of Böhm-Bawerk,” in the Quarterly Journal of Economics in 1897, in which he suggested that John Rae had anticipated some aspects of Böhm-Bawerk's theory of capital. At this time, not much was known in academic circles about Rae's life in Canada except for the remark in the preface to his 1834 book, Statement of Some New Principles on the Subject of Political Economy …: “I exchanged the literary leisure of Europe for the solitude and labors of the Canadian backwoods.”
Cell Size Control and a Cell-intrinsic Maturation Program in Proliferating Oligodendrocyte Precursor Cells
Authors: Gao Fen-Biao , Raff Martin
Published: July 2002

We have used clonal analysis and time-lapse video recording to study the proliferative behavior of purified oligodendrocyte precursor cells isolated from the perinatal rat optic nerve growing in serum-free cultures. First, we show that the cell cycle time of precursor cells decreases with increasing concentrations of PDGF, the main mitogen for these cells, suggesting that PDGF levels may regulate the cell cycle time during development. Second, we show that precursor cells isolated from embryonic day 18 (E18) nerves differ from precursor cells isolated from postnatal day 7 (P7) or P14 nerves in a number of ways: they have a simpler morphology, and they divide faster and longer before they stop dividing and differentiate into postmitotic oligodendrocytes. Third, we show that purified E18 precursor cells proliferating in culture progressively change their properties to resemble postnatal cells, suggesting that progressive maturation is an intrinsic property of the precursors. Finally, we show that precursor cells, especially mature ones, sometimes divide unequally, such that one daughter cell is larger than the other; in each of these cases the larger daughter cell divides well before the smaller one, suggesting that the precursor cells, just like single-celled eucaryotes, have to reach a threshold size before they can divide. These and other findings raise the possibility that such stochastic unequal divisions, rather than the stochastic events occurring in G1 proposed by “transition probability” models, may explain the random variability of cell cycle times seen within clonal cell lines in culture.
Increased Apoptosis Arising from Increased Expression of the Alzheimer's Disease–associated Presenilin-2 Mutation (N141I)
Authors: Janicki Susan , Monteiro Mervyn J.
Published: July 2002

Mutations in the genes for presenilin 1 and 2 (PS-1 and PS-2) have been linked to development of early-onset Alzheimer's disease (AD). As neither the normal function of either presenilin is known nor why mutations cause disease, we examined the properties of wild-type, truncated, and mutant PS-2 upon expression in HeLa cells. Although HeLa cells are strongly predisposed to continued mitosis, expression of PS-2 induced programmed cell death (apoptosis). Direct evidence for apoptosis was obtained by double staining for terminal deoxynucleotide transferase nick end labeling (TUNEL) and PS-2 expression and by following green fluorescent protein–tagged PS-2 over time. Deletion analysis indicates that as little as 166 NH2-terminal residues of PS-2 are sufficient for endoplasmic reticulum (ER) localization and apoptosis. Moreover, the AD- associated PS-2 missense mutation (N141I) more efficiently induced cell death compared to wild-type PS-2 despite lower mutant protein accumulation. Expression of the presenilins in several other cell lines and transgenic mice has been accompanied by rapid protein cleavage without the induction of cell death. In contrast, PS-2 expressed in HeLa cells was not cleaved, and cell death occurred. We hypothesize that full-length but not cleaved PS-2 may be important in the regulation or induction of apoptosis.
Complementary Roles for Receptor Clustering and Conformational Change in the Adhesive and Signaling Functions of Integrin αIIbβ3
Authors: Hato Takaaki , Pampori Nisar , Shattil Sanford J.
Published: July 2002

Integrin αIIbβ3 mediates platelet aggregation and “outside-in” signaling. It is regulated by changes in receptor conformation and affinity and/or by lateral diffusion and receptor clustering. To document the relative contributions of conformation and clustering to αIIbβ3 function, αIIb was fused at its cytoplasmic tail to one or two FKBP12 repeats (FKBP). These modified αIIb subunits were expressed with β3 in CHO cells, and the heterodimers could be clustered into morphologically detectable oligomers upon addition of AP1510, a membrane-permeable, bivalent FKBP ligand. Integrin clustering by AP1510 caused binding of fibrinogen and a multivalent (but not monovalent) fibrinogen-mimetic antibody. However, ligand binding due to clustering was only 25–50% of that observed when αIIbβ3 affinity was increased by an activating antibody or an activating mutation. The effects of integrin clustering and affinity modulation were additive, and clustering promoted irreversible ligand binding. Clustering of αIIbβ3 also promoted cell adhesion to fibrinogen or von Willebrand factor, but not as effectively as affinity modulation. However, clustering was sufficient to trigger fibrinogen-independent tyrosine phosphorylation of pp72Syk and fibrinogen-dependent phosphorylation of pp125FAK, even in non-adherent cells. Thus, receptor clustering and affinity modulation play complementary roles in αIIbβ3 function. Affinity modulation is the predominant regulator of ligand binding and cell adhesion, but clustering increases these responses further and triggers protein tyrosine phosphorylation, even in the absence of affinity modulation. Both affinity modulation and clustering may be needed for optimal function of αIIbβ3 in platelets.
The Nonreceptor Protein Tyrosine Phosphatase PTP1B Binds to the Cytoplasmic Domain of N-Cadherin and Regulates the Cadherin–Actin Linkage
Authors: Balsamo Janne , Arregui Carlos , Leung TinChung , Lilien Jack
Published: July 2002

Cadherin-mediated adhesion depends on the association of its cytoplasmic domain with the actin-containing cytoskeleton. This interaction is mediated by a group of cytoplasmic proteins: α-and β- or γ- catenin. Phosphorylation of β-catenin on tyrosine residues plays a role in controlling this association and, therefore, cadherin function. Previous work from our laboratory suggested that a nonreceptor protein tyrosine phosphatase, bound to the cytoplasmic domain of N-cadherin, is responsible for removing tyrosine-bound phosphate residues from β-catenin, thus maintaining the cadherin–actin connection (Balsamo et al., 1996). Here we report the molecular cloning of the cadherin-associated tyrosine phosphatase and identify it as PTP1B. To definitively establish a causal relationship between the function of cadherin-bound PTP1B and cadherin-mediated adhesion, we tested the effect of expressing a catalytically inactive form of PTP1B in L cells constitutively expressing N-cadherin. We find that expression of the catalytically inactive PTP1B results in reduced cadherin-mediated adhesion. Furthermore, cadherin is uncoupled from its association with actin, and β-catenin shows increased phosphorylation on tyrosine residues when compared with parental cells or cells transfected with the wild-type PTP1B. Both the transfected wild-type and the mutant PTP1B are found associated with N-cadherin, and recombinant mutant PTP1B binds to N-cadherin in vitro, indicating that the catalytically inactive form acts as a dominant negative, displacing endogenous PTP1B, and rendering cadherin nonfunctional. Our results demonstrate a role for PTP1B in regulating cadherin-mediated cell adhesion.