bluuebunny/crossref_metadata_2025 huggingface dataset:
Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: A possible advantage of DNA over RNA as genomic material
Authors: Shibata Takehiko , Nishinaka Taro , Mikawa Tsutomu , Aihara Hideki , Kurumizaka Hitoshi , Yokoyama Shigeyuki , Ito Yutaka
Published: July 2002

Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes. A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA/Rad51-family of proteins. In this reaction, a RecA/Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology. Studies of the three-dimensional structures of single-stranded DNA bound either to
Escherichia coli
RecA or
Saccharomyces cerevisiae
Rad51 have revealed a novel extended DNA structure. This structure contains a hydrophobic interaction between the 2′ methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers. This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and single-stranded DNA during the formation of heteroduplex joints. The pivotal role of the 2′ methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes. Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival. The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA, enabling gene segment rearrangements without disturbing the coding frame (i.e., protein-segment shuffling). These properties may give an extensive evolutionary advantage to DNA.
DNA repair of a single UV photoproduct in a designed nucleosome
Authors: Kosmoski Joseph V. , Ackerman Eric J. , Smerdon Michael J.
Published: July 2002

Eukaryotic DNA repair enzymes must interact with the architectural hierarchy of chromatin. The challenge of finding damaged DNA complexed with histone proteins in nucleosomes is complicated by the need to maintain local chromatin structures involved in regulating other DNA processing events. The heterogeneity of lesions induced by DNA-damaging agents has led us to design homogeneously damaged substrates to directly compare repair of naked DNA with that of nucleosomes. Here we report that nucleotide excision repair in
Xenopus
nuclear extracts can effectively repair a single UV radiation photoproduct located 5 bases from the dyad center of a positioned nucleosome, although the nucleosome is repaired at about half the rate at which the naked DNA fragment is. Extract repair within the nucleosome is >50-fold more rapid than either enzymatic photoreversal or endonuclease cleavage of the lesion
in vitro
. Furthermore, nucleosome formation occurs (after repair) only on damaged naked DNA (165-bp fragments) during a 1-h incubation in these extracts, even in the presence of a large excess of undamaged DNA. This is an example of selective nucleosome assembly by
Xenopus
nuclear extracts on a short linear DNA fragment containing a DNA lesion.
Notch1 confers a resistance to glucocorticoid-induced apoptosis on developing thymocytes by down-regulating SRG3 expression
Authors: Choi Young I. , Jeon Sung H. , Jang Jiho , Han Sunmi , Kim Joong K. , Chung Heekyoung , Lee Han W. , Chung Hee-Y. , Park Sang D. , Seong Rho H.
Published: July 2002

We previously have reported that SRG3 is required for glucocorticoid (GC)-induced apoptosis in the S49.1 thymoma cell line. Activation of Notch1 was shown to induce GC resistance in thymocytes. However, the specific downstream target of Notch1 that confers GC resistance on thymocytes is currently unknown. We found that the expression level of SRG3 was critical in determining GC sensitivity in developing thymocytes. The expression of SRG3 also was down-regulated by the activated form of Notch1 (NotchIC). The promoter activity of the SRG3 gene also was down-regulated by NotchIC. Expression of transgenic SRG3 resulted in the restoration of GC sensitivity in thymocytes expressing transgenic Notch1. These results suggest that
SRG3
is the downstream target of Notch1 in regulating GC sensitivity of thymocytes.
Lateral transfer at the gene and subgenic levels in the evolution of eukaryotic enolase
Authors: Keeling Patrick J. , Palmer Jeffrey D.
Published: July 2002

Enolase genes from land plants and apicomplexa (intracellular parasites, including the malarial parasite,
Plasmodium
) share two short insertions. This observation has led to the suggestion that the apicomplexan enolase is the product of a lateral transfer event involving the algal endosymbiont from which the apicomplexan plastid is derived. We have examined enolases from a wide variety of algae, as well as ciliates (close relatives of apicomplexa), to determine whether lateral transfer can account for the origin of the apicomplexan enolase. We find that lateral gene transfer, likely occurring intracellularly between endosymbiont and host nucleus, does account for the evolution of cryptomonad and chlorarachniophyte algal enolases but fails to explain the apicomplexan enolase. This failure is because the phylogenetic distribution of the insertions—which we find in apicomplexa, ciliates, land plants, and charophyte green algae—directly conflicts with the phylogeny of the gene itself. Protein insertions have traditionally been treated as reliable markers of evolutionary events; however, these enolase insertions do not seem to reflect accurately the evolutionary history of the molecule. The lack of congruence between insertions and phylogeny could be because of the parallel loss of both insertions in two or more lineages, or what is more likely, because the insertions were transmitted between distantly related genes by lateral transfer and fine-scale recombination, resulting in a mosaic gene. This latter process would be difficult to detect without such insertions to act as markers, and such mosaic genes could blur the “tree of life” beyond the extent to which whole-gene lateral transfer is already known to confound evolutionary reconstruction.
Independent and combined analyses of sequences from all three genomic compartments converge on the root of flowering plant phylogeny
Authors: Barkman Todd J. , Chenery Gordon , McNeal Joel R. , Lyons-Weiler James , Ellisens Wayne J. , Moore Gerry , Wolfe Andrea D. , dePamphilis Claude W.
Published: July 2002

Plant phylogenetic estimates are most likely to be reliable when congruent evidence is obtained independently from the mitochondrial, plastid, and nuclear genomes with all methods of analysis. Here, results are presented from separate and combined genomic analyses of new and previously published data, including six and nine genes (8,911 bp and 12,010 bp, respectively) for different subsets of taxa that suggestAmborella+ Nymphaeales (water lilies) are the first-branching angiosperm lineage. Before and after tree-independent noise reduction, most individual genomic compartments and methods of analysis estimated theAmborella+ Nymphaeales basal topology with high support. Previous phylogenetic estimates placingAmborellaalone as the first extant angiosperm branch may have been misled because of a series of specific problems with paralogy, suboptimal outgroups, long-branch taxa, and method dependence. Ancestral character state reconstructions differ between the two topologies and affect inferences about the features of early angiosperms.
High refractive index substrates for fluorescence microscopy of biological interfaces with high
z
contrast
Authors: Ajo-Franklin Caroline M. , Kam Lance , Boxer Steven G.
Published: July 2002

Total internal reflection fluorescence microscopy is widely used to confine the excitation of a complex fluorescent sample very close to the material on which it is supported. By working with high refractive index solid supports, it is possible to confine even further the evanescent field, and by varying the angle of incidence, to obtain quantitative information on the distance of the fluorescent object from the surface. We report the fabrication of hybrid surfaces consisting of nm layers of SiO
2
on lithium niobate (LiNbO
3
,
n
= 2.3). Supported lipid bilayer membranes can be assembled and patterned on these hybrid surfaces as on conventional glass. By varying the angle of incidence of the excitation light, we are able to obtain fluorescent contrast between 40-nm fluorescent beads tethered to a supported bilayer and fluorescently labeled protein printed on the surface, which differ in vertical position by only tens of nm. Preliminary experiments that test theoretical models for the fluorescence-collection factor near a high refractive index surface are presented, and this factor is incorporated into a semiquantitative model used to predict the contrast of the 40-nm bead/protein system. These results demonstrate that it should be possible to profile the vertical location of fluorophores on the nm distance scale in real time, opening the possibility of many experiments at the interface between supported membranes and living cells. Improvements in materials and optical techniques are outlined.
Extension of the
Drosophila
lifespan by overexpression of a protein repair methyltransferase
Authors: Chavous David A. , Jackson F. Rob , O'Connor Clare M.
Published: July 2002

Atypical protein isoaspartyl residues arise spontaneously
during the aging process from the deamidation of protein asparaginyl
residues and the isomerization of protein aspartyl residues.
These abnormal residues are modified in cells by a strongly
conserved protein carboxyl methyltransferase (PCMT) as a first step in
a repair pathway. Because a decline in cellular repair mechanisms is
hypothesized to contribute to senescence, we determined whether
increased PCMT activity was correlated with enhanced longevity. Two
ubiquitous promoters were used with the binary GAL4-UAS system to drive
PCMT overexpression in
Drosophila melanogaster.
Flies
expressing PCMT activity under the regulation of either the
hsp70
or
actin5C
promoter had enzyme activities
that were 3- or 7-fold higher, respectively, than control flies at
29°C. Correlated with the observed increases in PCMT activities, such
flies lived on average 32–39% longer than control flies. Lifespan
extension was not observed at 25°C with either
hsp70
- or
actin5C
-driven expression, indicating a
temperature-dependent effect on longevity. We conclude that protein
repair is an important factor in the determination of lifespan under
certain environmental conditions. PCMT activity may become limiting
under mild stress conditions that accelerate rates of protein damage.
Direct binding of a soluble natural killer cell inhibitory receptor to a soluble human leukocyte antigen-Cw4 class I major histocompatibility complex molecule.
Authors: Fan Q R , Garboczi D N , Winter C C , Wagtmann N , Long E O , Wiley D C
Published: July 2002

Natural killer (NK) cells expressing specific p58 NK receptors are inhibited from lysing target cells that express human leukocyte antigen (HLA)-C class I major histocompatibility complex molecules. To investigate the interaction between p58 NK receptors and HLA-Cw4, the extracellular domain of the p58 NK receptor specific for HLA-Cw4 was overexpressed in Escherichia coli and refolded from purified inclusion bodies. The refolded NK receptor is a monomer in solution. It interacts specifically with HLA-Cw4, blocking the binding of a p58-Ig fusion protein to HLA-Cw4-expressing cells, but does not block the binding of a p58-Ig fusion protein specific for HLA-Cw3 to HLA-Cw3-expressing cells. The bacterially expressed extracellular domain of HLA-Cw4 heavy chain and beta2-microglobulin were refolded in the presence of a HLA-Cw4-specific peptide. Direct binding between the soluble p58 NK receptor and the soluble HLA-Cw4-peptide complex was observed by native gel electrophoresis. Titration binding assays show that soluble monomeric receptor forms a 1:1 complex with HLA-Cw4, independent of the presence of Zn2+. The formation of complexes between soluble, recombinant molecules indicates that HLA-Cw4 is sufficient for specific ligation by the NK receptor and that neither glycoprotein requires carbohydrate for the interaction.
Exon shuffling and the origin of the mitochondrial targeting function in plant cytochrome c1 precursor.
Authors: Long M , de Souza S J , Rosenberg C , Gilbert W
Published: July 2002

Since most of the examples of "exon shuffling" are between vertebrate genes, the view is often expressed that exon shuffling is limited to the evolutionarily recent lineage of vertebrates. Although exon shuffling in plants has been inferred from the analysis of intron phases of plant genes [Long, M., Rosenberg, C. & Gilbert, W. (1995) Proc. Natl. Acad. Sci. USA 92, 12495-12499] and from the comparison of two functionally unknown sunflower genes [Domon, C. & Steinmetz, A. (1994) Mol. Gen. Genet. 244, 312-317], clear cases of exon shuffling in plant genes remain to be uncovered. Here, we report an example of exon shuffling in two important nucleus-encoded plant genes: cytosolic glyceraldehyde-3-phosphate dehydrogenase (cytosolic GAPDH or GapC) and cytochrome c1 precursor. The intron-exon structures of the shuffled region indicate that the shuffling event took place at the DNA sequence level. In this case, we can establish a donor-recipient relationship for the exon shuffling. Three amino terminal exons of GapC have been donated to cytochrome c1, where, in a new protein environment, they serve as a source of the mitochondrial targeting function. This finding throws light upon an old important but unsolved question in gene evolution: the origin of presequences or transit peptides that generally exist in nucleus-encoded organelle genes.
The miniaturized nuclear genome of eukaryotic endosymbiont contains genes that overlap, genes that are cotranscribed, and the smallest known spliceosomal introns.
Authors: Gilson P R , McFadden G I
Published: July 2002

Chlorarachniophyte algae contain a complex, multi-membraned chloroplast derived from the endosymbiosis of a eukaryotic alga. The vestigial nucleus of the endosymbiont, called the nucleomorph, contains only three small linear chromosomes with a haploid genome size of 380 kb and is the smallest known eukaryotic genome. Nucleotide sequence data from a subtelomeric fragment of chromosome III were analyzed as a preliminary investigation of the coding capacity of this vestigial genome. Several housekeeping genes including U6 small nuclear RNA (snRNA), ribosomal proteins S4 and S13, a core protein of the spliceosome [small nuclear ribonucleoprotein (snRNP) E], and a cip-like protease (clpP) were identified. Expression of these genes was confirmed by combinations of Northern blot analysis, in situ hybridization, immunocytochemistry, and cDNA analysis. The protein-encoding genes are typically eukaryotic in overall structure and their messenger RNAs are polyadenylylated. A novel feature is the abundance of 18-, 19-, or 20-nucleotide introns; the smallest spliceosomal introns known. Two of the genes, U6 and S13, overlap while another two genes, snRNP E and clpP, are cotranscribed in a single mRNA. The overall gene organization is extraordinarily compact, making the nucleomorph a unique model for eukaryotic genomics.
Photochemical energy conversion in a helical oligoproline assembly.
Authors: McCafferty D G , Friesen D A , Danielson E , Wall C G , Saderholm M J , Erickson B W , Meyer T J
Published: July 2002

A general method is described for constructing a helical oligoproline assembly having a spatially ordered array of functional sites protruding from a proline-II helix. Three different redox-active carboxylic acids were coupled to the side chain of cis-4-amino-L-proline. These redox modules were incorporated through solid-phase peptide synthesis into a 13-residue helical oligoproline assembly bearing in linear array a phenothiazine electron donor, a tris(bipyridine)ruthenium(II) chromophore, and an anthraquinone electron acceptor. Upon transient 460-nm irradiation in acetonitrile, this peptide triad formed with 53% efficiency an excited state containing a phenothiazine radical cation and an anthraquinone radical anion. This light-induced redox-separated state had a lifetime of 175 ns and stored 1.65 eV of energy.
Antimycobacterial activities of antisense oligodeoxynucleotide phosphorothioates in drug-resistant strains.
Authors: Rapaport E , Levina A , Metelev V , Zamecnik P C
Published: July 2002

Strains of Mycobacterium smegmatis, a mycobacterium which shares genetic sequences, grows more rapidly, and is nonpathogenic in man as compared with Mycobacterium tuberculosis, were utilized for the initial development of new antimycobacterial therapy. Drug-resistant strains of M. smegmatis which are known to arise in a manner identical to the emergence of drug-resistant strains of M. tuberculosis were isolated and utilized as models for the antimycobacterial activities of modified and unmodified oligodeoxynucleotide phosphorothioates in broth cultures. Under normal conditions, oligodeoxynucleotide phosphorothioates do not enter mycobacteria, and several strategies were successfully utilized to afford entry of oligonucleotides into the mycobacterial cells. One involved the presence of very low levels of ethambutol, which enables the entry of oligonucleotides into mycobacteria because of its induced alterations in the cell wall, and another involved the utilization of oligonucleotides covalently attached to a D-cycloserine molecule, whereby entry into the mycobacterial cell is achieved by a receptor-mediated process. Another low molecular weight, covalently attached ligand that enabled the entry and subsequent antimycobacterial activities of oligodeoxynucleotide phosphorothioates in the absence of a cell wall modifying reagent was biotin. Significant sequence-specific growth inhibition of wild-type, as well as of drug-resistant, M. smegmatis was obtained by modified oligonucleotides complementary in sequence to a specific region of the mycobacterium aspartokinase (ask) gene when utilized in combinations with ethambutol (as compared to ethambutol alone) or as D-cycloserine or biotin covalent adducts without the presence of any other cytotoxic or cytostatic agent.
Mammalian orthologues of a yeast regulator of nonsense transcript stability.
Authors: Perlick H A , Medghalchi S M , Spencer F A , Kendzior R J , Dietz H C
Published: July 2002

All eukaryotes that have been studied to date possess the ability to detect and degrade transcripts that contain a premature signal for the termination of translation. This process of nonsense-mediated RNA decay has been most comprehensively studied in the yeast Saccharomyces cerevisiae where at least three trans-acting factors (Upf1p through Upf3P) are required. We have cloned cDNAs encoding human and murine homologues of Upf1p, termed rent1 (regulator of nonsense transcripts). Rent1 is the first identified mammalian protein that contains all of the putative functional elements in Upf1p including zinc finger-like and NTPase domains, as well as all motifs common to members of helicase superfamily I. Moreover, expression of a chimeric protein, N and C termini of Upf1p, complements the Upf1p-deficient phenotype in yeast. Thus, despite apparent differences between yeast and mammalian nonsense-mediated RNA decay, these data suggest that the two pathways use functionally related machinery.
Distamycin A modulates the sequence specificity of DNA alkylation by duocarmycin A
Authors: Sugiyama Hiroshi , Lian Chenyang , Isomura Mariko , Saito Isao , Wang Andrew H.-J.
Published: July 2002

Duocarmycin A (Duo) normally alkylates adenine N3 at the 3′ end of
A+T-rich sequences in DNA. The efficient adenine alkylation by Duo is
achieved by its monomeric binding to the DNA minor groove. The addition
of another minor groove binder, distamycin A (Dist), dramatically
modulates the site of DNA alkylation by Duo, and the alkylation
switches preferentially to G residues in G+C-rich sequences. HPLC
product analysis using oligonucleotides revealed a highly efficient
G–N3 alkylation via the cooperative binding of a heterodimer between
Duo and Dist to the minor groove. The three-dimensional structure of
the ternary alkylated complex of
Duo/Dist/d(CAGGTGGT)·d(ACCACCTG) has been determined by
nuclear Overhauser effect (NOE)-restrained refinement using 750 MHz
two-dimensional NOE spectroscopy data. The refined NMR structure fully
explains the sequence requirement of such modulated alkylations. This
is the first demonstration of Duo DNA alkylation through cooperative
binding with another structurally different natural product, and it
suggests a promising new way to alter or modify the DNA alkylation
selectivity in a predictable manner.
Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon.
Authors: Tellier R , Bukh J , Emerson S U , Purcell R H
Published: July 2002

The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription-PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones.
Mutated mitogen-activated protein kinase: A tumor rejection antigen of mouse sarcoma
Authors: Ikeda Hiroaki , Ohta Nobuyoshi , Furukawa Keiko , Miyazaki Hiroshi , Wang Lijie , Furukawa Koichi , Kuribayashi Kagemasa , Old Lloyd J. , Shiku Hiroshi
Published: July 2002

The molecular basis of the polymorphic tumor rejection antigens of chemically induced sarcomas of inbred mice remains a mystery, despite the discovery of these antigens over 40 years ago and their critical importance to the foundation of tumor immunology. In an analysis of a panel of BALB/c 3-methylcholanthrene-induced tumors, we identified one tumor, CMS5, that elicited a strong cytotoxic T cell response with exquisite specificity for CMS5. A stable cloned line of T cells with this specificity (C18) was used to screen a CMS5 cDNA expression library. The gene encoding the C18-defined antigen was identified as a mutated form of a mouse mitogen-activated protein kinase, ERK2, and a peptide incorporating the resulting amino acid substitution (lysine to glutamine) was efficiently recognized by C18. Vaccination with this peptide elicited specific resistance to CMS5 challenge. Extensive efforts to isolate antigen-loss variants of CMS5 were unsuccessful, suggesting that the mutated mitogen-activated protein kinase is essential for maintenance of the malignant phenotype.
Preferential interaction of the
his
pause RNA hairpin with RNA polymerase β subunit residues 904–950 correlates with strong transcriptional pausing
Authors: Wang Daguang , Severinov Konstantin , Landick Robert
Published: July 2002

RNA secondary structures (hairpins) that form as the nascent RNA emerges from RNA polymerase are important components of many signals that regulate transcription, including some pause sites, all ρ-independent terminators, and some antiterminators. At the
his
leader pause site, a 5-bp-stem, 8-nt-loop pause RNA hairpin forms 11 nt from the RNA 3′ end and stabilizes a transcription complex conformation slow to react with NTP substrate. This stabilization appears to depend at least in part on an interaction with RNA polymerase. We tested for RNA hairpin interaction with the paused polymerase by crosslinking 5-iodoUMP positioned specifically in the hairpin loop. In the paused conformation, strong and unusual crosslinking of the pause hairpin to β904–950 replaced crosslinking to β′ and to other parts of β that occurred in nonpaused complexes prior to hairpin formation. These changes in nascent RNA interactions may inhibit reactive alignment of the RNA 3′ end in the paused complex and be related to events at ρ-independent terminators.
On the reaction mechanism of
l
-lactate oxidase: Quantitative structure-activity analysis of the reaction with
para
-substituted
l
-mandelates
Authors: Yorita Kazuko , Janko Karl , Aki Kenji , Ghisla Sandro , Palfey Bruce A. , Massey Vincent
Published: July 2002

The rate constants for reduction of the flavoenzyme,
l
-lactate oxidase, and a mutant (in which alanine 95 is
replaced by glycine), by a series of
para
-substituted
mandelates, in both the 2-
1
H- and 2-
2
H- forms,
have been measured by rapid reaction spectrophotometry. In all cases,
significant isotope effects (
1
H/
2
H =
3–7) on the rate constants of flavin reduction were found, indicating
that flavin reduction is a direct measure of α-C-H bond breakage. The
rate constants show only a small influence of the electronic
characteristics of the substituents, but show a good correlation when
combined with some substituent volume parameters. A surprisingly good
correlation is found with the molecular mass of the substrate. The
results are compatible with any mechanism in which there is little
development of charge in the transition state. This could be a transfer
of hydride to the flavin N(5) position or a synchronous mechanism in
which the α-C-H is formally abstracted as a H
+
while the
resulting charge is simultaneously neutralized by another event.
Metabolism of an insect diuretic hormone by Malpighian tubules studied by liquid chromatography coupled with electrospray ionization mass spectrometry
Authors: Li Hong , Wang Houle , Schegg Kathleen M. , Schooley David A.
Published: July 2002

The larger of two diuretic hormones of the tobacco hornworm,
Manduca sexta
, (Mas-DH) is a peptide of 41 residues. It is one of a family of seven currently known insect diuretic hormones that are similar to the corticotropin-releasing factor–urotensin–sauvagine family of peptides. We investigated the possible inactivation of Mas-DH by incubating it
in vitro
with larval Malpighian tubules (Mt), the target organ of the hormone. The medium was analyzed, and degradation products were identified, using on-line microbore reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry (RPLC-ESI-MS). This sensitive technique allows identification of metabolites of Mas-DH (present at an initial level of ≈1 μM). An accurate
M
r
value for a metabolite is usually sufficient for unambiguous identification. Mas-DH is cleaved by Mt proteases initially at L
29
–R
30
and R
30
–A
31
under our assay conditions; some Mas-DH is also oxidized, apparently at M
2
and M
11
. The proteolysis can be inhibited by 5 mM EDTA, suggesting that divalent metals are needed for peptide cleavage. The oxidation of the hormone can be inhibited by catalase or 1 mM methionine, indicating that H
2
O
2
or related reactive oxygen species are responsible for the oxidative degradation observed. RPLC-ESI-MS is shown here to be an elegant and efficient method for studying peptide hormone metabolism resulting from unknown proteases and pathways.
HIV Rev-dependent binding of SF2/ASF to the Rev response element: Possible role in Rev-mediated inhibition of HIV RNA splicing
Authors: Powell Douglas M. , Amaral M. Catherine , Wu Jane Y. , Maniatis Tom , Greene Warner C.
Published: July 2002

Production of the structural and enzymatic proteins of type 1 human immunodeficiency virus (HIV-1) is controlled by the
rev
regulatory gene product. The 116-amino acid Rev protein acts by binding to the Rev response element (RRE), a complex RNA stem–loop structure located within the
env
gene of HIV. Rev exerts a series of posttranscriptional effects, including the inhibition of viral RNA splicing, the activation of nuclear export of incompletely spliced viral RNAs, and the enhancement of translation of RRE-containing RNAs. Our studies now demonstrate that at least one member of the SR family of splicing factors, SF2/ASF, specifically binds to a subregion of the RRE
in vitro
in a Rev-dependent manner. Furthermore, expression of high levels of SF2/ASF inhibits Rev function and impairs HIV replication
in vivo
. Both the
in vitro
binding of SF2/ASF to the Rev/RRE complex and the
in vivo
inhibition of Rev action by SF2/ASF are abrogated by mutation of the N-terminal RNA recognition motif but are not affected by mutation of the C-terminal arginine–serine-rich domain. These findings suggest that Rev inhibition of HIV splicing likely involves recruitment of the essential splicing factor SF2/ASF to the Rev/RRE complex. However, these inhibitory effects of Rev on viral RNA splicing are apparently overcome by augmenting the intracellular levels of SF2/ASF expression.
A post-tetanic time window for the reinforcement of long-term potentiation by appetitive and aversive stimuli
Authors: Seidenbecher Thomas , Reymann Klaus G. , Balschun Detlef
Published: July 2002

Current theories on the encoding and storage of information in the brain commonly suppose that a short-term memory is converted into a lasting one; thus, it becomes consolidated over time. Within a finite period after training, such a short-term memory can be reinforced by behavioral and humoral stimuli. We have found that, long-term potentiation (LTP), a likely candidate for a memory-encoding mechanism at the cellular level, displays similar features. LTP in the dentate gyrus of freely moving rats was reinforced after its induction by appetitive and aversive stimuli. The efficacy of these stimuli terminates about 1 h after tetanization, which may reflect the time constants of the mechanisms underlying the consolidation that takes place. The reinforcement by appetitive and aversive stimulation was blocked by the β-adrenergic antagonist propranolol, implicating norepinephrine in the underlying cellular processes.
Regulation of the expression of the tissue transglutaminase gene by DNA methylation
Authors: Lu Shan , Davies Peter J. A.
Published: July 2002

We have investigated the role of DNA methylation in the regulation of the expression of the human tissue transglutaminase gene. Studies on the methylation of the transglutaminase promoter in normal and neoplastic human cells demonstrated that the promoter is methylated
in vivo
and hypomethylation of the promoter is correlated with constitutive gene expression. Demethylation of the promoter
in vivo
by treatment of the cells with 5-azacytidine increased transglutaminase expression and hypermethylation of the promoter
in vitro
suppressed its activity. These studies suggest that alternations in DNA methylation may be one of the mechanisms regulating the tissue-specific expression of the tissue transglutaminase gene.
The Shake-and-Bake structure determination of triclinic lysozyme
Authors: Deacon Ashley M. , Weeks Charles M. , Miller Russ , Ealick Steven E.
Published: July 2002

The crystal structure of triclinic lysozyme, comprised of 1,001 non-H protein atoms and ≈200 bound water molecules, has been determined
ab initio
(using native data alone) by the “Shake-and-Bake” method by using the computer program SnB. This is the largest structure determined so far by the SnB program. Initial experiments, using default SnB parameters derived from studies of smaller molecules, were unsuccessful. In fact, such experiments produced electron density maps dominated by a single large peak. This problem was overcome by considering the choice of protocol used during the parameter-shift phase refinement. When each phase was subjected to a single shift of ±157.5° during each SnB cycle, an unusually high percentage of random trials (≈22%) yielded correct solutions within 750 cycles. This success rate is higher than that typically observed, even for much smaller structures.
Two yeast nuclear pore complex proteins involved in mRNA export form a cytoplasmically oriented subcomplex
Authors: Hurwitz Michael E. , Strambio-de-Castillia Caterina , Blobel Günter
Published: July 2002

We sublocalized the yeast nucleoporin Nup82 to the cytoplasmic side of the nuclear pore complex (NPC) by immunoelectron microscopy. Moreover, by
in vitro
binding assays we showed that Nup82 interacts with the C-terminal region of Nup159, a yeast nucleoporin that previously was also localized to the cytoplasmic side of the NPC. Hence, the two nucleoporins, Nup82 and Nup159, form a cytoplasmically oriented subcomplex that is likely to be part of the fibers emanating from the cytoplasmic ring of the NPC. Overexpression of Rss1/Gle1, a putative nucleoporin and/or mRNA transport factor, was shown previously to partially rescue depletion of Nup159. We show here that overexpression of Rss1/Gle1 also partially rescued depletion of Nup82. Depletion of either Nup82, Nup159, or Rss1/Gle1 was shown previously to inhibit mRNA export. As was reported previously for depletion of Nup159 or of Rss1/Gle1, we show here that depletion of Nup82 has no detectable effect on classical nuclear localization sequence-mediated nuclear import. In summary, the nucleoporins Nup159 and Nup82 form a cytoplasmically oriented subcomplex of the NPC that is likely associated with Rss1/Gle1; this complex is essential for RNA export, but not for classical nuclear localization sequence-mediated nuclear protein import.
v-K-
ras
leads to preferential farnesylation of p21

ras

in FRTL-5 cells: Multiple interference with the isoprenoid pathway
Authors: Laezza Chiara , Di Marzo Vincenzo , Bifulco Maurizio
Published: July 2002

The isoprenoid pathway in FRTL-5 thyroid cells was found to be deeply altered on transformation with v-K-
ras
. A dramatic overall reduction of protein prenylation was found in v-K-
ras
-transformed cells in comparison with the parent FRTL-5 cells, as shown by labeling cells with [
3
H]mevalonic acid. This phenomenon was accompanied by a relative increase of p21

ras

farnesylation and by a decrease of the ratio between the amounts of geranylgeraniol and farnesol bound to prenylated proteins. Analysis of protein prenylation in FRTL-5 cells transformed by a temperature-sensitive mutant of the v-K-
ras
oncogene indicated that these variations represent an early and specific marker of active K
-ras
. Conversely, FRTL-5 cells transformed with Harvey-
ras
showed a pattern of [
3
H]-mevalonate (MVA)-labeled proteins similar to that of nontransformed cells. The K-
ras
oncogene activation also resulted in an overall decrease of [
3
H]-MVA incorporation into isopentenyl-tRNA together with an increase of unprocessed [
3
H]-MVA and no alteration in [
3
H]-MVA uptake. The effects of v-K-
ras
on protein prenylation could be mimicked in FRTL-5 cells by lowering the concentration of exogenous [
3
H]-MVA whereas increasing the [
3
H]-MVA concentration did not revert the alterations observed in transformed cells. Accordingly
,
v-K-
ras
expression was found to: (
i
) down-regulate mevalonate kinase; (
ii
) induce farnesyl-pyrophosphate synthase expression; and (
iii
) augment protein farnesyltransferase but not protein geranylgeranyl-transferase-I activity. Among these events, mevalonate kinase down-regulation appeared to be related strictly to differential protein prenylation. This study represents an example of how expression of the v-K-
ras
oncogene, through multiple interferences with the isoprenoid metabolic pathway, may result in the preferential farnesylation of the
ras
oncogene product p21

ras

.
Spontaneous liposome formation induced by grafted poly(ethylene oxide) layers: Theoretical prediction and experimental verification
Authors: Szleifer Igal , Gerasimov Oleg V. , Thompson David H.
Published: July 2002

Spontaneous liposome formation is predicted in binary mixtures of fluid phase phospholipids and poly(n)ethylene oxide (PEO)-bearing lipids by using single chain mean field theory. The range of stability of the spontaneous liposomes is determined as a function of percentage of PEO-conjugated lipids and polymer molecular weight. These predictions were tested by using cast films of 1,2-diacyl-
sn
-glycerophosphocholines (e.g., egg
l
-α-lecithin, 1,2-dimyristoyl-
sn
-glycero-3-phosphocholine, 1,2-dipalmitoyl-
sn
-glycero-3-phosphocholine, and 1,2-distearoyl-
sn
-glycero-3-phosphocholine) and 1,2-dipalmitoyl-
sn
-glycerophosphatidylethanolamine-PEO conjugates (i.e., 1,2-dipalmitoyl-
sn
-glycero-3-phosphoethanolamine-
N
-[methoxypoly(ethylene glycol)2000]carboxamide and 1,2-dipalmitoyl-
sn
-glycero-3-phosphoethanolamine-
N
-[methoxypoly(ethylene oxide)5000]carboxamide) that were hydrated above their gel-liquid crystal phase transition temperatures. Particle sizes of the resulting dispersions, analyzed by quasielastic light scattering, solute retention,
31
P NMR, and freeze-fracture electron microscopy measurements, confirmed the single chain mean field predictions. These data indicate that thermodynamically stable, unilamellar liposomes are formed spontaneously by simple hydration of fluid phase phospholipid bilayer films containing low molar ratios of PEO-based amphiphiles. They further suggest that the equilibrium size and colloidal properties of fluid phase, PEO-modified liposomes can be predicted by using this theoretical approach. The implication of these results on the design and processing of sterically stabilized liposomes used in drug delivery applications also is described.
Putative pheromone receptors related to the Ca
2+
-sensing receptor in
Fugu
Authors: Naito Takayuki , Saito Yutaka , Yamamoto Jun , Nozaki Yuko , Tomura Keiko , Hazama Masaaki , Nakanishi Shigetada , Brenner Sydney
Published: July 2002

By a combination of PCR with degenerate primers and low stringency probing, we have isolated a large family of genes related to the Ca
2+
-sensing receptor from the genome of
Fugu rubripes
. One of the genes (type I) is the
Fugu
homologue of the Ca
2+
-sensing receptor. The remaining genes can be divided into five classes (type II–VI) on the bases of gene structure. In several types, the genes occur in clusters as tandem arrays. These genes appear to be the homologues of the vomeronasal pheromone receptors recently described in rodents. The
Fugu
genes are expressed in the tissues of the nose, suggesting that they may have a similar physiological role.
Predicting amino acid sequences of the antibody human V
H
chains from its first several residues
Authors: Galitsky Boris A. , Gelfand Israel M. , Kister Alexander E.
Published: July 2002

A new method for classification of Ig sequences is suggested. The defining characteristic of a class is presence of particular residues at several class-determining positions. Sequences within a class follow the same amino acid pattern, i.e., residues at identical positions are, in an overwhelming majority of sequences of that class, identical or chemically related. Thus, once the class of a sequence is determined, one can predict the residue(s) at almost any position in the sequence. In this paper, results of analysis of 1,172 human heavy chains are presented. It was shown that a sequence can be assigned to one of six classes depending on which residues are found at its positions 1, 3, 5, 6, 7, 9, 10, 12, and 13. It is important to note that it is possible to achieve same six-class classification of the human heavy chains on the basis of a different set of positions found not at the beginning but near the end of the sequence (around position 80). For every class, an amino acid pattern of an entire sequence (complementarity determining regions excepting) has been determined. Our approach allowed us to reconstruct the incomplete human heavy chains in which residues at certain positions at the beginning or end of the chain are known. We developed a software tool for analysis, classification, and prediction of residues in sequences of the Ig family.
Protochlorophyllide oxidoreductase B-catalyzed protochlorophyllide photoreduction
in vitro
: Insight into the mechanism of chlorophyll formation in light-adapted plants
Authors: Lebedev Nikolai , Timko Michael P.
Published: July 2002

The mechanism of the protochlorophyllide (PChlide) photoreduction reaction operating in light-adapted plants and catalyzed by NADPH:protochlorophyllide oxidoreductase B (PORb) has been analyzed by low-temperature fluorescence spectroscopy by using purified barley PORb overexpressed heterologously in
Escherichia coli
as a fusion protein with the maltose-binding protein. We show that the PORb-catalyzed PChlide reduction reaction consists of two steps, one photochemical and the other nonphotochemical. The initial photochemical reaction follows a single quantum mechanism and leads to the formation of an unstable intermediate with mixed pigment electronic structure and an EPR spectrum that suggests the presence of a free electron. The second step involves the spontaneous conversion of the unstable intermediate into chlorophyllide as defined by its spectroscopic characteristics and migration on an HPLC column. Both steps of the reaction can be performed at subzero temperatures in frozen samples, suggesting that they do not include major changes in enzyme conformation or pigment rearrangement within the active site. The rate of the reaction at room temperature depends linearly on enzyme and substrate (PChlide) concentration, and the kinetic parameters are consistent with one molecule of substrate bound per active monomer in solution. The PORb-catalyzed reaction
in vitro
is spectroscopically similar to that identified in leaves of light-adapted plants, suggesting that the same reaction sequence observed operates
in planta
.
A multiplasmid approach to preparing large libraries of polyketides
Authors: Xue Qun , Ashley Gary , Hutchinson C. Richard , Santi Daniel V.
Published: July 2002

A three-plasmid system for heterologous expression of 6-deoxyerythronolide B synthase (DEBS) has been developed to facilitate combinatorial biosynthesis of polyketides made by type I modular polyketide synthases (PKSs). The
eryA
PKS genes encoding the three DEBS subunits were individually cloned into three compatible
Streptomyces
vectors carrying mutually selectable antibiotic resistance markers. A strain of
Streptomyces lividans
transformed with all three plasmids produced 6-deoxyerythronolide B at a level similar to that of a strain transformed with a single plasmid containing all three genes. The utility of this system in combinatorial biosynthesis was demonstrated through production of a library of modified polyketide macrolactones by using versions of each plasmid constructed to contain defined mutations. Combinations of these vector sets were introduced into
S. lividans
, resulting in strains producing a wide range of 6-deoxyerythronolide B analogs. This method can be extended to any modular PKS and has the potential to produce thousands of novel natural products, including ones derived from further modification of the PKS products by tailoring enzymes.
Nonlinear indicial response of complex nonstationary oscillations as pulmonary hypertension responding to step hypoxia
Authors: Huang Wei , Shen Zheng , Huang Norden E. , Fung Yuan Cheng
Published: July 2002

This paper is devoted to the quantization of the degree of nonlinearity of the relationship between two biological variables when one of the variables is a complex nonstationary oscillatory signal. An example of the situation is the indicial responses of pulmonary blood pressure (P) to step changes of oxygen tension (ΔpO
2
) in the breathing gas. For a step change of ΔpO
2
beginning at time
t
1
, the pulmonary blood pressure is a nonlinear function of time and ΔpO
2
, which can be written as P(
t
-
t
1
| ΔpO
2
). An effective method does not exist to examine the nonlinear function P(
t
-
t
1
| ΔpO
2
). A systematic approach is proposed here. The definitions of mean trends and oscillations about the means are the keys. With these keys a practical method of calculation is devised. We fit the mean trends of blood pressure with analytic functions of time, whose nonlinearity with respect to the oxygen level is clarified here. The associated oscillations about the mean can be transformed into Hilbert spectrum. An integration of the square of the Hilbert spectrum over frequency yields a measure of oscillatory energy, which is also a function of time, whose mean trends can be expressed by analytic functions. The degree of nonlinearity of the oscillatory energy with respect to the oxygen level also is clarified here. Theoretical extension of the experimental nonlinear indicial functions to arbitrary history of hypoxia is proposed. Application of the results to tissue remodeling and tissue engineering of blood vessels is discussed.
G protein βγ subunit-dependent Rac-guanine nucleotide exchange activity of Ras-GRF1/CDC25
Mm
Authors: Kiyono Mari , Satoh Takaya , Kaziro Yoshito
Published: July 2002

Ras-GRF1 has been implicated as a Ras-specific guanine nucleotide exchange factor (GEF), which mediates calcium- and muscarinic receptor-triggered signals in the brain. Although a Dbl homology domain known as a motif conserved among GEFs that target Rho family GTP-binding proteins exists in Ras-GRF1, GEF activity toward Rho family proteins has not been observed. Here we show that Ras-GRF1 exhibits Rac1-specific GEF activity when recovered from cells overexpressing G protein βγ subunits (G
β
γ
). Substitution of conserved amino acids within the Dbl homology domain abolished this activity. Activation of the Rac pathway in the cell was further evidenced by synergistic activation of the stress kinase JNK1 by Ras-GRF1 and G
β
γ
, which is sensitive to inhibitory action of dominant-negative Rac1(17N). In addition, association of Ras-GRF1 with Rac1(17N) was demonstrated by coimmunoprecipitation. Evidence for the involvement of tyrosine kinase(s) in G
β
γ
-mediated induction of Rac1-specific GEF activity was provided by the use of specific inhibitors. These results suggest a role of Ras-GRF1 for regulating Rac-dependent as well as Ras-dependent signaling pathways, particularly in the brain functions.
Patterns of Telecommuting Engagement and Frequency: A Cluster Analysis of Telecenter Users
Authors: Mokhtarian Patricia L. , Meenakshisundaram Ravikumar
Published: July 2002

Cluster analysis of sign-in log data for 115 users of California telecenters was conducted to identify patterns of telecommuting engagement and frequency over a six-month window. Three engagement clusters were identified: Persisters, Decliners, and Dabblers. Four frequency clusters were identified, classified as Low, Medium, High, and Erratic. Nearly half of the Persisters belonged to the Low Frequency cluster, highlighting the need to count not just telecommuters, but telecommuting occasions. Variables significantly associated with cluster membership were identified. Consistent with other research, management-related issues seem to play a substantial role in affecting both the engagement in, and frequency of, telecommuting.