bluuebunny/crossref_metadata_2025 huggingface dataset:
Hierarchical formation of helical supramolecular polymers via stacking of hydrogen-bonded pairs in water
Authors: Brunsveld L. , Vekemans J. A. J. M. , Hirschberg J. H. K. K. , Sijbesma R. P. , Meijer E. W.
Published: July 2002

Bifunctional ureido-
s
-triazines provided with penta(ethylene oxide) side chains are able to self assemble in water, leading to helical columns via cooperative stacking of the hydrogen-bonded pairs (DADA array). Monofunctional ureido-
s
-triazines do not form such helical architectures. The presence of a linker, covalently connecting the two ureido-
s
-triazine units, is essential as it generates a high local concentration of aromatic units, favorable for stacking interactions. This hydrophobic stacking of the aromatic units occurs at concentrations as low as 5⋅10
−6
M and can be visualized by using fluorescence spectroscopy. The stacking generates a hydrophobic microenvironment that allows intermolecular hydrogen bonding to occur at higher concentrations because the hydrogen bonds are shielded from competitive hydrogen bonding with water. This hierarchical process results in the formation of a helical self-assembled polymer in water at concentrations above 10
−4
M. Chiral side chains attached to the ureido-
s
-triazine units bias the helicity of these columns as concluded from CD spectroscopy and “Sergeants and Soldiers” experiments.
Overexpression of 5-methylcytosine DNA glycosylase in human embryonic kidney cells EcR293 demethylates the promoter of a hormone-regulated reporter gene
Authors: Zhu Bing , Benjamin Don , Zheng Yong , Angliker Herbert , Thiry Stéphane , Siegmann Michel , Jost Jean-Pierre
Published: July 2002

We have shown that the DNA demethylation complex isolated from
chicken embryos has a G⋅T mismatch DNA glycosylase that also
possesses 5-methylcytosine DNA glycosylase (5-MCDG) activity. Herein we
show that human embryonic kidney cells stably transfected with 5-MCDG
cDNA linked to a cytomegalovirus promoter overexpress 5-MCDG. A 15- to
20-fold overexpression of 5-MCDG results in the specific demethylation
of a stably integrated ecdysone-retinoic acid responsive
enhancer-promoter linked to a β-galactosidase reporter gene.
Demethylation occurs in the absence of the ligand ponasterone A (an
analogue of ecdysone). The state of methylation of the transgene was
investigated by Southern blot analysis and by the bisulfite genomic
sequencing reaction. Demethylation occurs downstream of the hormone
response elements. No genome-wide demethylation was observed. The
expression of an inactive mutant of 5-MCDG or the empty vector does not
elicit any demethylation of the promoter-enhancer of the reporter gene.
An increase in 5-MCDG activity does not influence the activity of DNA
methyltransferase(s) when tested
in vitro
with a
hemimethylated substrate. There is no change in the transgene copy
number during selection of the clones with antibiotics.
Immunoprecipitation combined with Western blot analysis showed that an
antibody directed against 5-MCDG precipitates a complex containing the
retinoid X receptor α. The association between retinoid receptor and
5-MCDG is not ligand dependent. These results suggest that a complex of
the hormone receptor with 5-MCDG may target demethylation of the
transgene in this system.
Genome-wide screening for trait conferring genes using DNA microarrays
Authors: Gill R. T. , Wildt S. , Yang Y. T. , Ziesman S. , Stephanopoulos G.
Published: July 2002

We report a DNA microarray-based method for genome-wide monitoring of competitively grown transformants to identify genes whose overexpression confers a specific cellular phenotype. Whereas transcriptional profiling identifies differentially expressed genes that are correlated with particular aspects of the cellular phenotype, this functional genomics approach determines genes that result in a specific physiology. This parallel gene-trait mapping method consists of transforming a strain with a genomic library, enriching the cell population in transformants containing the trait conferring gene(s), and finally using DNA microarrays to simultaneously isolate and identify the enriched gene inserts. Various methods of enrichment can be used; here, genes conferring low-level antibiotic resistance were identified by growth in selective media. We demonstrated the method by transforming
Escherichia coli
cells with a genomic
E. coli
library and selecting for transformants exhibiting a growth advantage in the presence of the anti-microbial agent Pine-Sol. Genes conferring Pine-Sol tolerance (19 genes) or sensitivity (27 genes) were identified by hybridizing, on DNA microarrays containing 1,160
E. coli
gene probes, extra-chromosomal DNA isolated from transformed cells grown in the presence of various levels of Pine-Sol. Results were further validated by plating and sequencing of individual colonies, and also by assessing the Pine-Sol resistance of cells transformed with enriched plasmid library or individual resistance genes identified by the microarrays. Applications of this method beyond antibiotic resistance include identification of genes resulting in resistance to chemotherapeutic agents, genes yielding resistance to toxic products (recombinant proteins, chemical feedstocks) in industrial fermentations, genes providing enhanced growth in cell culture or high cell density fermentations, genes facilitating growth on unconventional substrates, and others.
A potent dimeric peptide antagonist of interleukin-5 that binds two interleukin-5 receptor α chains
Authors: England Bruce P. , Balasubramanian Palaniappan , Uings Iain , Bethell Sue , Chen Min-Jia , Schatz Peter J. , Yin Qun , Chen Yao-Fen , Whitehorn Erik A. , Tsavaler Alexander , Martens Christine L. , Barrett Ronald W. , McKinnon Murray
Published: July 2002

Two series of peptides that specifically bind to the extracellular domain of the α chain of the human interleukin-5 receptor (IL-5Rα), but share no primary sequence homology to IL-5, were identified from libraries of random recombinant peptides. Affinity maturation procedures generated a 19-aa peptide that binds to the IL-5 receptor α/β heterodimer complex with an affinity equal to that of IL-5 and is a potent and specific antagonist of IL-5 activity in a human eosinophil adhesion assay. The active form of the peptide is a disulfide-crosslinked dimer that forms spontaneously in solution. Gel filtration analysis, receptor-binding studies, and analytical ultracentrifugation reveal that the dimeric peptide binds simultaneously to two receptor α chains in solution. Furthermore, the dimer peptide, but not IL-5, can activate a chimeric receptor consisting of the IL-5Rα extracellular domain fused to the intracellular domain of the epidermal growth factor receptor, thus demonstrating that the peptide also promotes receptor dimerization in a cellular context. The functional antagonism produced by the bivalent interaction of the dimeric peptide with two IL-5R α chains represents a distinctive mechanism for the antagonism of cytokines that use heteromeric receptors.
T cell-specific FADD-deficient mice: FADD is required for early T cell development
Authors: Kabra Nisha H. , Kang Chulho , Hsing Lianne C. , Zhang Jianke , Winoto Astar
Published: July 2002

FADD/Mort1, initially identified as a Fas-associated death-domain containing protein, functions as an adapter molecule in apoptosis initiated by Fas, tumor necrosis factor receptor-I, DR3, and TRAIL-receptors. However, FADD likely participates in additional signaling cascades. FADD-null mutations in mice are embryonic-lethal, and analysis ofFADD−/−T cells fromRAG-1−/−reconstituted chimeras has suggested a role for FADD in proliferation of mature T cells. Here, we report the generation of T cell-specific FADD-deficient mice via a conditional genomic rescue approach. We find that FADD-deficiency leads to inhibition of T cell development at the CD4−CD8−stage and a reduction in the number of mature T cells. The FADD mutation does not affect apoptosis or the proximal signaling events of the pre-T cell receptor; introduction of a T cell receptor transgene fails to rescue the mutant phenotype. These data suggest that FADD, through either a death-domain containing receptor or a novel receptor-independent mechanism, is required for the proliferative phase of early T cell development.
Characterization of the novel E3 ubiquitin ligase encoded in exon 3 of herpes simplex virus-1-infected cell protein 0
Authors: Hagglund Ryan , Roizman Bernard
Published: July 2002

Infected cell protein 0 (ICP0) of herpes simplex virus-1 is a 775-aa residue multifunctional protein that acts as a promiscuous transactivator linked to the degradation of several proteins. ICP0 is the only protein known which encodes two physically separated E3 ubiquitin (Ub) ligase domains, one, designated herpes virus Ub ligase 1 (HUL-1) located in a domain encoded in exon 3 and one designated herpes virus Ub ligase 2 (HUL-2) associated with the really interesting new gene (RING) finger domain encoded by exon 2. We report the following: (
i
) ICP0 residues 543–680 are sufficient for HUL-1 E3 activity and necessary determinants are encoded between residues 616 and 680. (
ii
) In substrate independent
in vitro
ubiquitylation reactions, a chimeric protein containing the HUL-1 domain promotes the ubiquitylation of itself and the ubiquitin conjugating enzyme (E2) cdc34 and interacts with cdc34. (
iii
) The mechanism of HUL-1 E3 function does not involve formation of a thioester between the HUL-1 domain and Ub. (
iv
) Residues 621–625 are essential for
in vitro
HUL-1 E3 activity and interaction between the HUL-1 domain and cdc34, suggesting that this interaction is required for HUL-1 E3 function.
Structural genomics of enzymes involved in sterol/isoprenoid biosynthesis
Authors: Bonanno Jeffrey B. , Edo Carme , Eswar Narayanan , Pieper Ursula , Romanowski Michael J. , Ilyin Valentin , Gerchman Sue Ellen , Kycia Helen , Studier F. William , Sali Andrej , Burley Stephen K.
Published: July 2002

X-ray structures of two enzymes in the sterol/isoprenoid biosynthesis pathway have been determined in a structural genomics pilot study. Mevalonate-5-diphosphate decarboxylase (MDD) is a single-domain α/β protein that catalyzes the last of three sequential ATP-dependent reactions which convert mevalonate to isopentenyl diphosphate. Isopentenyl disphosphate isomerase (IDI) is an α/β metalloenzyme that catalyzes interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, which condense in the next step toward synthesis of sterols and a host of natural products. Homology modeling of related proteins and comparisons of the MDD and IDI structures with two other experimentally determined structures have shown that MDD is a member of the GHMP superfamily of small-molecule kinases and IDI is similar to the nudix hydrolases, which act on nucleotide diphosphatecontaining substrates. Structural models were produced for 379 proteins, encompassing a substantial fraction of both protein superfamilies. All three enzymes responsible for synthesis of isopentenyl diphosphate from mevalonate (mevalonate kinase, phosphomevalonate kinase, and MDD) share the same fold, catalyze phosphorylation of chemically similar substrates (MDD decarboxylation involves phosphorylation of mevalonate diphosphate), and seem to have evolved from a common ancestor. These structures and the structural models derived from them provide a framework for interpreting biochemical function and evolutionary relationships.
Adenomatous polyposis coli protein contains two nuclear export signals and shuttles between the nucleus and cytoplasm
Authors: Neufeld Kristi L. , Nix David A. , Bogerd Hal , Kang Yibin , Beckerle Mary C. , Cullen Bryan R. , White Raymond L.
Published: July 2002

Mutational inactivation of the adenomatous polyposis coli (APC)
tumor suppressor initiates most hereditary and sporadic colon
carcinomas. Although APC protein is located in both the cytoplasm and
the nucleus, the protein domains required to maintain a predominantly
cytoplasmic localization are unknown. Here, we demonstrate that nuclear
export of APC is mediated by two intrinsic, leucine-rich, nuclear
export signals (NESs) located near the amino terminus. Each NES was
able to induce the nuclear export of a fused carrier protein. Both APC
NESs were independently able to interact with the Crm1 nuclear export
factor and substitute for the HIV-1 Rev NES to mediate nuclear mRNA
export. Both APC NESs functioned within the context of APC sequence: an
amino-terminal APC peptide containing both NESs interacted with Crm1
and showed nuclear export in a heterokaryon nucleocytoplasmic shuttling
assay. Also, mutation of both APC NESs resulted in the nuclear
accumulation of the full-length, ∼320-kDa APC protein, further
establishing that the two intrinsic APC NESs are necessary for APC
protein nuclear export. Moreover,
endogenous
APC accumulated in the nucleus of cells treated with the Crm1-specific
nuclear export inhibitor leptomycin B. Together, these data indicate
that APC is a nucleocytoplasmic shuttle protein whose predominantly
cytoplasmic localization requires NES function and suggests that APC
may be important for signaling between the nuclear and cytoplasmic
compartments of epithelial cells.
Increasing DNA repair methyltransferase levels via bone marrow stem cell transduction rescues mice from the toxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea, a chemotherapeutic alkylating agent.
Authors: Maze R , Carney J P , Kelley M R , Glassner B J , Williams D A , Samson L
Published: July 2002

The chloroethylnitrosourea (CNU) alkylating agents are commonly used for cancer chemotherapy, but their usefulness is limited by severe bone marrow toxicity that causes the cumulative depletion of all hematopoietic lineages (pancytopenia). Bone marrow CNU sensitivity is probably due to the inefficient repair of CNU-induced DNA damage; relative to other tissues, bone marrow cells express extremely low levels of the O6-methylguanine DNA methyltransferase (MGMT) protein that repairs cytotoxic O6-chloroethylguanine DNA lesions. Using a simplified recombinant retroviral vector expressing the human MGMT gene under control of the phosphoglycerate kinase promoter (PGK-MGMT) we increased the capacity of murine bone marrow-derived cells to repair CNU-induced DNA damage. Stable reconstitution of mouse bone marrow with genetically modified, MGMT-expressing hematopoietic stem cells conferred considerable resistance to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a CNU commonly used for chemotherapy. Bone marrow harvested from mice transplanted with PGK-MGMT-transduced cells showed extensive in vitro BCNU resistance. Moreover, MGMT expression in mouse bone marrow conferred in vivo resistance to BCNU-induced pancytopenia and significantly reduced BCNU-induced mortality due to bone marrow hypoplasia. These data demonstrate that increased DNA alkylation repair in primitive hematopoietic stem cells confers multilineage protection from the myelosuppressive effects of BCNU and suggest a possible approach to protecting cancer patients from CNU chemotherapy-related toxicity.
Intercellular transfer of a glycosylphosphatidylinositol (GPI)-linked protein: release and uptake of CD4-GPI from recombinant adeno-associated virus-transduced HeLa cells.
Authors: Anderson S M , Yu G , Giattina M , Miller J L
Published: July 2002

A diverse group of GPI-anchored protein structures are ubiquitously expressed on the external cell membranes of eukaryotes. Whereas the physiological role for these structures is usually defined by their protein component, the precise biological significance of the glycolipid anchors remains vague. In the course of producing a HeLa cell line (JM88) that contained a recombinant adeno-associated virus genome expressing a GPI-anchored CD4-GPI fusion protein on the surface of the cells, we noted the transfer of CD4-GPI to native HeLa cells. Transfer occurred after direct cell contact or exposure to JM88 cell supernatants. The magnitude of contact-mediated CD4-GPI transfer correlated with temperature. Supernatant CD4-GPI also attached to human red blood cells and could be cleaved with phosphatidylinositol-specific phospholipase C. The attached CD4-GPI remained biologically active after transfer and permitted the formation of syncytium when coated HeLa cells were incubated with glycoprotein 160 expressing H9 cells. JM88 cells provide a model for the production, release, and reattachment of CD4-GPI and may furnish insight into a physiologic role of naturally occurring GPI-anchored proteins. This approach may also allow the production of other recombinant GPI-anchored proteins for laboratory and clinical investigation.
Covalent assembly of a soluble T cell receptor-peptide-major histocompatibility class I complex.
Authors: Grégoire C , Lin S Y , Mazza G , Rebai N , Luescher I F , Malissen B
Published: July 2002

We used stepwise photochemical cross-linking for specifically assembling soluble and covalent complexes made of a T-cell antigen receptor (TCR) and a class I molecule of the major histocompatibility complex (MHC) bound to an antigenic peptide. For that purpose, we have produced in myeloma cells a single-chain Fv construct of a TCR specific for a photoreactive H-2Kd-peptide complex. Photochemical cross-linking of this TCR single-chain Fv with a soluble form of the photoreactive H-2Kd-peptide ligand resulted in the formation of a ternary covalent complex. We have characterized the soluble ternary complex and showed that it reacted with antibodies specific for epitopes located either on the native TCR or on the Kd molecules. By preventing the fast dissociation kinetics observed with most T cell receptors, this approach provides a means of preparing soluble TCR-peptide-MHC complexes on large-scale levels.
Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix.
Authors: Sanderson I R , Ezzell R M , Kedinger M , Erlanger M , Xu Z X , Pringault E , Leon-Robine S , Louvard D , Walker W A
Published: July 2002

The differentiation of small intestinal epithelial cells may require stimulation by microenvironmental factors in vivo. In this study, the effects of mesenchymal and luminal elements in nonmalignant epithelia] cells isolated from the human fetus were studied in vitro. Enterocytes from the human fetus were cultured and microenvironmental factors were added in stages, each stage more closely approximating the microenvironment in vivo. Four stages were examined: epithelial cells derived on plastic from intestinal culture and grown as a cell clone, the same cells grown on connective tissue support, primary epithelial explants grown on fibroblasts with a laminin base, and primary epithelial explants grown on fibroblasts and laminin with n-butyrate added to the incubation medium. The epithelial cell clone dedifferentiated when grown on plastic; however, the cells expressed cytokeratins and villin as evidence of their epithelial cell origin. Human connective tissue matrix from Engelbreth-Holm-Swarm sarcoma cells (Matrigel) modulated their phenotype: alkaline phosphatase activity increased, microvilli developed on their apical surface, and the profile of insulin-like growth factor binding proteins resembled that secreted by differentiated enterocytes. Epithelial cells taken directly from the human fetus as primary cultures and grown as explants on fibroblasts and laminin expressed greater specific enzyme activities in brush border membrane fractions than the cell clone. These activities were enhanced by the luminal molecule sodium butyrate. Thus the sequential addition of connective tissue and luminal molecules to nonmalignant epithelia] cells in vitro induces a spectrum of changes in the epithelial cell phenotype toward full differentiation.
Cellular strategies for accommodating replication-hindering adducts in DNA: control by the SOS response in Escherichia coli.
Authors: Koffel-Schwartz N , Coin F , Veaute X , Fuchs R P
Published: July 2002

The replication of double-stranded plasmids containing a single adduct was analyzed in vivo by means of a sequence heterology that marks the two DNA strands. The single adduct was located within the sequence heterology, making it possible to distinguish trans-lesion synthesis (TLS) events from damage avoidance events in which replication did not proceed through the lesion. When the SOS system of the host bacteria is not induced, the C8-guanine adduct formed by the carcinogen N-2-acetylaminofluorene (AAF) yields less than 1% of TLS events, showing that replication does not readily proceed through the lesion. In contrast, the deacetylated adduct N-(deoxyguanosin-8-yl)-2-aminofluorene yields approximately 70% of TLS events under both SOS-induced and uninduced conditions. These results for TLS in vivo are in good agreement with the observation that AAF blocks DNA replication in vitro, whereas aminofluorene does so only weakly. Induction of the SOS response causes an increase in TLS events through the AAF adduct (approximately 13%). The increase in TLS is accompanied by a proportional increase in the frequency of AAF-induced frameshift mutations. However, the polymerase frameshift error rate per TLS event was essentially constant throughout the SOS response. In an SOS-induced delta umuD/C strain, both US events and mutagenesis are totally abolished even though there is no decrease in plasmid survival. Error-free replication evidently proceeds efficiently by means of the damage avoidance pathway. We conclude that SOS mutagenesis results from increased TLS rather than from an increased frameshift error rate of the polymerase.
Migration of vesicular stomatitis virus glycoprotein to the nucleus of infected cells.
Authors: Da Poian A T , Gomes A M , Oliveira R J , Silva J L
Published: July 2002

A new means of direct visualization of the early events of viral infection by selective fluorescence labeling of viral proteins coupled with digital imaging microscopy is reported. The early phases of viral infection have great importance for understanding viral replication and pathogenesis. Vesicular stomatitis virus, the best-studied rhabdovirus, is composed of an RNA genome of negative sense, five viral proteins, and membrane lipids derived from the host cell. The glycoprotein of vesicular stomatitis virus was labeled with fluorescein isothiocyanate, and the labeled virus was incubated with baby hamster kidney cells. After initiation of infection, the fluorescence of the labeled glycoprotein was first seen inside the cells in endocytic vesicles. The fluorescence progressively migrated to the nucleus of infected cells. After 1 h of infection, the virus glycoprotein was concentrated in the nucleus and could be recovered intact in a preparation of purified nuclei. These results suggest that uncoating of the viral RNA occurs close to the nuclear membrane, which would precede transcription of the leader RNA that enters the nucleus to shut off cellular RNA synthesis and DNA replication.
Induction of nephrogenic mesenchyme by osteogenic protein 1 (bone morphogenetic protein 7).
Authors: Vukicevic S , Kopp J B , Luyten F P , Sampath T K
Published: July 2002

The definitive mammalian kidney forms as the result of reciprocal interactions between the ureteric bud epithelium and metanephric mesenchyme. As osteogenic protein 1 (OP-1/bone morphogenetic protein 7), a member of the TGF-beta superfamily of proteins, is expressed predominantly in the kidney, we examined its involvement during metanephric induction and kidney differentiation. We found that OP-1 mRNA is expressed in the ureteric bud epithelium before mesenchymal condensation and is subsequently seen in the condensing mesenchyme and during glomerulogenesis. Mouse kidney metanephric rudiments cultured without ureteric bud epithelium failed to undergo mesenchymal condensation and further epithelialization, while exogenously added recombinant OP-1 was able to substitute for ureteric bud epithelium in restoring the induction of metanephric mesenchyme. This OP-1-induced nephrogenic mesenchyme differentiation follows a developmental pattern similar to that observed in the presence of the spinal cord, a metanephric inducer. Blocking OP-1 activity using either neutralizing antibodies or antisense oligonucleotides in mouse embryonic day 11.5 mesenchyme, cultured in the presence of metanephric inducers or in intact embryonic day 11.5 kidney rudiment, greatly reduced metanephric differentiation. These results demonstrate that OP-1 is required for metanephric mesenchyme differentiation and plays a functional role during kidney development.
Alternatively spliced forms in the cytoplasmic domain of the human growth hormone (GH) receptor regulate its ability to generate a soluble GH-binding protein.
Authors: Dastot F , Sobrier M L , Duquesnoy P , Duriez B , Goossens M , Amselem S
Published: July 2002

The mechanism underlying the generation of soluble growth hormone binding protein (GHBP) probably differs among species. In rats and mice, it involves an alternatively spliced mRNA, whereas in rabbits, it involves limited proteolysis of the membrane-bound growth hormone receptor (GHR). In humans, this latter mechanism is favored, as no transcript coding for a soluble GHR has been detected so far. To test this hypothesis, we analyzed COS-7 cells transiently expressing the full-length human (h) GHR and observed specific GH-binding activity in the cell supernatants. Concomitantly, an alternatively spliced form in the cytoplasmic domain of GHR, hGHR-tr, was isolated from several human tissues. hGHR-tr is identical in sequence to hGHR, except for a 26-bp deletion leading to a stop codon at position 280, thereby truncating 97.5% of the intracellular domain of the receptor protein. When compared with hGHR, hGHR-tr showed a significantly increased capacity to generate a soluble GHBP. Interestingly, this alternative transcript is also expressed in liver from rabbits, mice, and rats, suggesting that, in these four species, proteolysis of the corresponding truncated transmembrane GHR is a common mechanism leading to GHBP generation. These findings support the hypothesis that GHBP may at least partly result from alternative splicing of the region encoding the intracellular domain and that the absence of a cytoplasmic domain may be involved in increased release of GHBP.
A single G-to-C change causes human centromere TGGAA repeats to fold back into hairpins.
Authors: Zhu L , Chou S H , Reid B R
Published: July 2002

Recently, we established that satellite III (TGGAA)n tandem repeats, which occur at the centromeres of human chromosomes, pair with themselves to form an unusual "self-complementary" antiparallel duplex containing (GGA)2 motifs in which two unpaired guanines from opposite strands intercalate between sheared G.A base pairs. In separate studies, we have also established that the GCA triplet does not form bimolecular (GCA)2 motifs but instead promotes the formation of hairpins containing a GCA-turn motif in which the loop contains a single cytidine closed by a sheared G.A pair. Since TGCAA is the most frequent variant of TGGAA found in satellite III repeats, we reasoned that the potential of this variant to form GCA-turn miniloop fold-back structures might be an important factor in modulating the local structure in natural (TGGAA)n repeats. We report here the NMR-derived solution structure of the heptadecadeoxynucleotide (G)TGGAATGCAATGGAA(C) in which a central TGCAA pentamer is flanked by two TGGAA pentamers. This 17-mer forms a rather unusual and very stable hairpin structure containing eight base pairs in the stem, only four of which are Watson-Crick pairs, and a loop consisting of a single cytidine residue. The stem contains a (GGA)2 motif with intercalative 14G/4G stacking between two sheared G.A base pairs; the loop end of the stem consists of a sheared 8G.10A closing pair with the cytosine base of the 9C loop stacked on 8G. The remarkable stability of this unusual hairpin structure (Tm = 63 degrees C) suggests that it probably plays an important role in modulating the folding of satellite III (TGGAA)n repeats at the centromere.
Retroviral transduction of a mutant methylguanine DNA methyltransferase gene into human CD34 cells confers resistance to
O
6
-benzylguanine plus 1,3-bis(2-chloroethyl)-1-nitrosourea
Authors: Reese Jane S. , Koç Omer N. , Lee Keun Myoung , Liu Lili , Allay James A. , Phillips Weldon P. , Gerson Stanton L.
Published: July 2002

Human CD34 cells express low levels of the DNA repair protein
O
6
-alkylguanine-DNA alkyltransferase (AGT)
and are sensitive to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Gene
transfer of the AGT gene, methylguanine DNA methyltransferase
(
MGMT
), results in only modest BCNU resistance.
Recently, an AGT inhibitor,
O
6
-benzylguanine
(BG), entered clinical trials. In preclinical studies, BG potentiated
the cytotoxic effect of BCNU in tumors but increased toxicity to normal
CD34 cells. We transferred a mutant
MGMT
containing a
glycine-to-alanine mutation at position 156, resulting in marked
resistance to BG, into Chinese hamster cells; the K562 cell line and
human CD34 cells used the retroviral backbone MFG. In each instance,
cells expressed increased AGT and were much more resistant to the
combination of BG and BCNU than the parental cells or cells transduced
with wild-type
MGMT
. Furthermore, the transduction
efficiency in human CD34 cells was in excess of 70%, and the
proportion of CD34 transduced cells resistant to the combination was
>30%. Thus, retroviral-mediated transduction of a mutant
MGMT
into CD34 cells appears to be an effective way to
induce selective resistance to a drug combination designed to overcome
a significant resistance mechanism to nitrosoureas in tumors.
Ultrafast signals in protein folding and the polypeptide contracted state
Authors: Sosnick T. R. , Shtilerman M. D. , Mayne L. , Englander S. W.
Published: July 2002

To test the significance of ultrafast protein folding signals (≪1 msec), we studied cytochrome
c
(Cyt c) and two Cyt
c
fragments with major C-terminal segments deleted. The fragments remain unfolded under all conditions and so could be used to define the unfolded baselines for protein fluorescence and circular dichroism (CD) as a function of denaturant concentration. When diluted from high to low denaturant in kinetic folding experiments, the fragments readjust to their new baseline values in a “burst phase” within the mixing dead time. The fragment burst phase reflects a contraction of the polypeptide from a more extended unfolded condition at high denaturant to a more contracted unfolded condition in the poorer, low denaturant solvent. Holo Cyt
c
exhibits fluorescence and CD burst phase signals that are essentially identical to the fragment signals over the whole range of final denaturant concentrations, evidently reflecting the same solvent-dependent, relatively nonspecific contraction and not the formation of a specific folding intermediate. The significance of fast folding signals in Cyt
c
and other proteins is discussed in relation to the hypothesis of an initial rate-limiting search-nucleation-collapse step in protein folding [Sosnick, T. R., Mayne, L. & Englander, S. W. (1996)
Proteins Struct. Funct. Genet.
24, 413–426].
Gene-based approach to human gene-phenotype correlations
Authors: Dryja Thaddeus P.
Published: July 2002

Elucidating the genetic basis of human phenotypes is a major goal of contemporary geneticists. Logically, two fundamental and contrasting approaches are available, one that begins with a phenotype and concludes with the identification of a responsible gene or genes; the other that begins with a gene and works toward identifying one or more phenotypes resulting from allelic variation of it. This paper provides a conceptual overview of phenotype-based vs. gene-based procedures with emphasis on gene-based methods. A key feature of a gene-based approach is that laboratory effort first is devoted to developing an assay for mutations in the gene under regard; the assay then is applied to the evaluation of large numbers of unrelated individuals with a variety of phenotypes that are deemed potentially resulting from alleles at the gene. No effort is directed toward chromosomally mapping the loci responsible for the phenotypes scanned. Example is made of my laboratory’s successful use of a gene-based approach to identify genes causing hereditary diseases of the retina such as retinitis pigmentosa. Reductions in the cost and improvements in the speed of scanning individuals for DNA sequence anomalies may make a gene-based approach an efficient alternative to phenotype-based approaches to correlating genes with phenotypes.
On the geometry of solutions of the quasi-geostrophic and Euler equations
Authors: Cordoba Diego
Published: July 2002

We study solutions of the two-dimensional quasi-geostrophic thermal active scalar equation involving simple hyperbolic saddles. There is a naturally associated notion of simple hyperbolic saddle breakdown. It is proved that such breakdown cannot occur in finite time. At large time, these solutions may grow at most at a quadruple-exponential rate. Analogous results hold for the incompressible three-dimensional Euler equation.
Construction of a high-affinity receptor site for dihydropyridine agonists and antagonists by single amino acid substitutions in a non-
l
-type Ca
2+
channel
Authors: Hockerman Gregory H. , Peterson Blaise Z. , Sharp Elizabeth , Tanada Timothy N. , Scheuer Todd , Catterall William A.
Published: July 2002

The activity of
l
-type Ca
2+
channels is increased by dihydropyridine (DHP) agonists and inhibited by DHP antagonists, which are widely used in the therapy of cardiovascular disease. These drugs bind to the pore-forming α
1
subunits of
l
-type Ca
2+
channels. To define the minimal requirements for DHP binding and action, we constructed a high-affinity DHP receptor site by substituting a total of nine amino acid residues from DHP-sensitive
l
-type α
1
subunits into the S5 and S6 transmembrane segments of domain III and the S6 transmembrane segment of domain IV of the DHP-insensitive P/Q-type α
1A
subunit. The resulting chimeric α
1A/DHPS
subunit bound DHP antagonists with high affinity in radioligand binding assays and was inhibited by DHP antagonists with high affinity in voltage clamp experiments. Substitution of these nine amino acid residues yielded 86% of the binding energy of the
l
-type α
1C
subunit and 92% of the binding energy of the
l
-type α
1S
subunit for the high-affinity DHP antagonist PN200–110. The activity of chimeric Ca
2+
channels containing α
1A/DHPS
was increased 3.5 ± 0.7-fold by the DHP agonist (−)Bay K8644. The effect of this agonist was stereoselective as in
l
-type Ca
2+
channels since (+) Bay K8644 inhibited the activity of α
1A/DHPS
. The results show conclusively that DHP agonists and antagonists bind to a single receptor site at which they have opposite effects on Ca
2+
channel activity. This site contains essential components from both domains III and IV, consistent with a domain interface model for binding and allosteric modulation of Ca
2+
channel activity by DHPs.
σ
54
, a vital protein for
Myxococcus xanthus
Authors: Keseler Ingrid M. , Kaiser Dale
Published: July 2002

The
rpoN
gene encoding the transcription factor σ
54
in
Myxococcus xanthus
has been cloned using a heterologous
rpoN
probe. The sequence of the cross-hybridizing DNA confirmed the existence of an ORF 1518 bp long that encodes a well conserved member of the σ
54
family of σ factors. Low- as well as high-stringency hybridizations detected only a single
rpoN
gene in the
M. xanthus
chromosome. In other bacteria, σ
54
is an alternative σ, and null mutants are viable. However, all attempts to construct a strain containing a null mutation in the
M. xanthus rpoN
have been unsuccessful. Partial diploids of
rpoN
+
/
rpoN
null are viable. Recombination experiments with such partial diploids showed the impossibility of constructing, either by segregation or by transduction, a viable null haploid under any of a wide range of growth conditions. The product of the
rpoN
gene, σ
54
, therefore appears to be essential for growth in
M. xanthus
.
Low dimensionality of supraspinally induced force fields
Authors: d’Avella A. , Bizzi E.
Published: July 2002

Recent experiments using electrical and N-methyl-
d
-aspartate microstimulation of the spinal cord gray matter and cutaneous stimulation of the hindlimb of spinalized frogs have provided evidence for a modular organization of the frog’s spinal cord circuitry. A “module” is a functional unit in the spinal cord circuitry that generates a specific motor output by imposing a specific pattern of muscle activation. The output of a module can be characterized as a force field: the collection of the isometric forces generated at the ankle over different locations in the leg’s workspace. Different modules can be combined independently so that their force fields linearly sum. The goal of this study was to ascertain whether the force fields generated by the activation of supraspinal structures could result from combinations of a small number of modules. We recorded a set of force fields generated by the electrical stimulation of the vestibular nerve in seven frogs, and we performed a principal component analysis to study the dimensionality of this set. We found that 94% of the total variation of the data is explained by the first five principal components, a result that indicates that the dimensionality of the set of fields evoked by vestibular stimulation is low. This result is compatible with the hypothesis that vestibular fields are generated by combinations of a small number of spinal modules.
Mechanism of DNA segregation in prokaryotes: Replicon pairing by
parC
of plasmid R1
Authors: Jensen Rasmus Bugge , Lurz Rudi , Gerdes Kenn
Published: July 2002

Prokaryotic chromosomes and plasmids encode partitioning systems that are required for DNA segregation at cell division. The systems are thought to be functionally analogous to eukaryotic centromeres and to play a general role in DNA segregation. The
parA
system of plasmid R1 encodes two proteins ParM and ParR, and a cis-acting centromere-like site denoted
parC.
The ParR protein binds to
parC in vivo
and
in vitro
. The ParM protein is an ATPase that interacts with ParR specifically bound to
parC
. Using electron microscopy, we show here that
parC
mediates efficient pairing of plasmid molecules. The pairing requires binding of ParR to
parC
and is stimulated by the ParM ATPase. The ParM mediated stimulation of plasmid pairing is dependent on ATP hydrolysis by ParM. Using a ligation kinetics assay, we find that ParR stimulates ligation of
parC
-containing DNA fragments. The rate-of-ligation was increased by wild type ParM protein but not by mutant ParM protein deficient in the ATPase activity. Thus, two independent assays show that
parC
mediates pairing of plasmid molecules
in vitro
. These results are consistent with the proposal that replicon pairing is part of the mechanism of DNA segregation in prokaryotes.
Differential rescue of visceral and cardiac defects in
Drosophila
by vertebrate
tinman
-related genes
Authors: Park Maijon , Lewis Carol , Turbay David , Chung Amy , Chen Jau-Nian , Evans Sylvia , Breitbart Roger E. , Fishman Mark C. , Izumo Seigo , Bodmer Rolf
Published: July 2002

tinman
, a mesodermal NK2-type homeobox gene, is absolutely required for the subdivision of the early
Drosophila
mesoderm and for the formation of the heart as well as the visceral muscle primordia. Several vertebrate relatives of
tinman
, many of which are predominately expressed in the very early cardiac progenitors (and pharyngeal endoderm), also seem to promote heart development. Here, we show that most of these vertebrate
tinman
-related genes can readily substitute for
Drosophila tinman
function in promoting visceral mesoderm-specific marker gene expression, but much less in promoting cardiac-specific gene expression indicative of heart development. In addition, another mesodermal NK2-type gene from
Drosophila
,
bagpipe
, which is normally only needed for visceral mesoderm but not heart development, cannot substitute for
tinman
at all. These data indicate that the functional equivalence of the
tinman
-related subclass of NK2-type genes (in activating markers of visceral mesoderm development in
Drosophila
) is specific to this subclass and distinct from other homeobox genes. Despite the apparent overall conservation of heart development between vertebrates and invertebrates, the differential rescue of visceral mesoderm versus heart development suggests that some of the molecular mechanisms of organ formation may have diverged during evolution.
UVB-induced association of tumor necrosis factor (TNF) receptor 1/TNF receptor-associated factor-2 mediates activation of Rel proteins
Authors: Tobin Derek , van Hogerlinden Max , Toftgård Rune
Published: July 2002

Exposure of mammalian skin to UV light results in induced gene transcription, playing a role in inflammation, immunosuppression, and tumor promotion. One important group of transcription factors induced by UV radiation is composed of members of the Rel/NF-κB family, which are known to play a major role in the transcriptional activation of many genes encoding inflammatory cytokines, adhesion molecules, and viral proteins. However, the upstream events in the transduction of the UVB signal to Rel protein activity are, as yet, unknown. Here, we provide biochemical evidence that exposure of keratinocytes to UVB causes rapid association of tumor necrosis factor (TNF) receptor 1 with its downstream partner TRAF-2. The functional relevance of this association is demonstrated by experiments showing that expression of a dominant negative TNF receptor 1 or TRAF-2 protein inhibits UVB-induced Rel-dependent transcription. Inclusion of a neutralizing antibody toward TNFα has no effect on UVB activation of a Rel-responsive reporter gene. Therefore, UVB-induced activation of Rel proteins via TNF receptor 1, independent of ligand activation, is a key component in the UV response in keratinocytes.
Promoter opening via a DNA fork junction binding activity
Authors: Guo Yuli , Gralla Jay D.
Published: July 2002

The rate-limiting step in transcriptional initiation typically is opening the promoter DNA to expose the template strand. Opening is tightly regulated, but how it occurs is not known. These experiments identify an activity, recognition of specific DNA fork junctions, and suggest that it is critical to bacterial promoter opening. This activity is both sequence and structure specific; it recognizes the bases that constitute the upstream double-stranded/single-stranded boundary of the open complex. Promoter mutations known to reduce opening rates lead to comparable reductions in fork junction binding affinity. The activity acts to establish the upstream boundary of melted DNA and works in conjunction with two single-stranded DNA binding activities that recognize separately the two melted strands. The junction binding activity is contained within the sigma factor component of the holoenzyme. The activity occurs in both a typical prokaryotic transcription system and in a eukaryotic-like bacterial system that responds to enhancers and needs ATP. Thus DNA opening catalyzed by fork junction binding may occur in a variety of systems in which DNA must be opened to be copied.
In vivo
genomic footprinting analysis reveals that the complex
Bradyrhizobium japonicum fixRnifA
promoter region is differently occupied by two distinct RNA polymerase holoenzymes
Authors: Barrios Humberto , Grande Ricardo , Olvera Leticia , Morett Enrique
Published: July 2002

The
Bradyrhizobium japonicum fixRnifA
operon is transcribed from two promoters:
fixR
p1, a −24/−12 promoter recognized by the σ
54
-holoenzyme form of the RNA polymerase, and
fixR
p2, a −35/−10 promoter that is transcribed by a second, unidentified, form of RNA polymerase holoenzyme. The
fixR
p1 promoter is autoregulated during microaerobiosis by NifA, whereas
fixR
p2 is also activated, but by a different regulatory protein. The main transcription start sites for these promoters are just two nucleotides apart, such that the conserved −12 and −10 regions of
fixR
p1 and
fixR
p2, respectively, must overlap each other, whereas the −24 and −35 regions lie one DNA helical turn apart. Using
in vivo
genomic dimethyl sulfate and KMnO
4
footprinting, we showed that the promoter region is differentially protected, depending upon which holoenzyme is bound. Mutagenesis analyses indicated that positions from −12 to −14 are critical for the activity of both promoters, whereas mutations at −10 and −11 affected mainly
fixR
p2 expression. When the sequence of the putative −35 region of
fixR
p2 was modified to match the putative consensus, expression from this promoter was increased 3-fold and the reactivity toward KMnO
4
, but not the transcriptional start site, moved two nucleotides further upstream, indicating that the altered promoter forms a different open complex. Additionally, we detected NifA-dependent methylation protection of two atypical NifA binding sites and protection of guanine −75. The latter residue is located in a region critical for
fixR
p2 promoter activation. The results present direct physical evidence of the complexity of the organization, regulation, and function of the
fixRnifA
promoter region.
Intermolecular cleavage by UmuD-like mutagenesis proteins
Authors: McDonald John P. , Frank Ekaterina G. , Levine Arthur S. , Woodgate Roger
Published: July 2002

The activity of a number of proteins is regulated by self-processing reactions. Elegant examples are the cleavage of the prokaryotic LexA and λCI transcriptional repressors and the UmuD-like mutagenesis proteins. Various studies support the hypothesis that LexA and λCI cleavage reactions are predominantly intramolecular in nature. The recently described crystal structure of the
Escherichia coli
UmuD′ protein (the posttranslational cleavage product of the UmuD protein) suggests, however, that the region of the protein corresponding to the cleavage site is at least 50 Å away from the catalytic active site. We considered the possibility, therefore, that the UmuD-like proteins might undergo self-processing that, in contrast to LexA and λCI, occurs via an intermolecular rather than intramolecular reaction. To test this hypothesis, we introduced into
E. coli
compatible plasmids with mutations at either the cleavage or the catalytic site of three UmuD-like proteins. Cleavage of these proteins only occurs in the presence of both plasmids, indicating that the reaction is indeed intermolecular in nature. Furthermore, this intermolecular reaction is completely dependent upon the multifunctional RecA protein and leads to the restoration of cellular mutagenesis in nonmutable
E. coli
strains. Intermolecular cleavage of a biotinylated UmuD active site mutant was also observed
in vitro
in the presence of the wild-type UmuD′ protein, indicating that in addition to the intact UmuD protein, the normal cleavage product (UmuD′) can also act as a classical enzyme.