bluuebunny/crossref_metadata_2025 huggingface dataset:
Heteronuclear NMR investigations of dynamic regions of intact
Escherichia coli
ribosomes
Authors: Christodoulou John , Larsson Göran , Fucini Paola , Connell Sean R. , Pertinhez Thelma A. , Hanson Charlotte L. , Redfield Christina , Nierhaus Knud H. , Robinson Carol V. , Schleucher Jürgen , Dobson Christopher M.
Published: July 2004

15
N-
1
H NMR spectroscopy has been used to probe the dynamic properties of uniformly
15
N labeled
Escherichia coli
ribosomes. Despite the high molecular weight of the complex (≈2.3 MDa), [
1
H-
15
N] heteronuclear single-quantum correlation spectra contain ≈100 well resolved resonances, the majority of which arise from two of the four C-terminal domains of the stalk proteins, L7/L12. Heteronuclear pulse-field gradient NMR experiments show that the resonances arise from species with a translational diffusion constant consistent with that of the intact ribosome. Longitudinal relaxation time (
T
1
) and
T
1ρ
15
N-spin relaxation measurements show that the observable domains tumble anisotropically, with an apparent rotational correlation time significantly longer than that expected for a free L7/L12 domain but much shorter than expected for a protein rigidly incorporated within the ribosomal particle. The relaxation data allow the ribosomally bound C-terminal domains to be oriented relative to the rotational diffusion tensor. Binding of elongation factor G to the ribosome results in the disappearance of the resonances of the L7/L12 domains, indicating a dramatic reduction in their mobility. This result is in agreement with cryoelectron microscopy studies showing that the ribosomal stalk assumes a single rigid orientation upon elongation factor G binding. As well as providing information about the dynamical properties of L7/L12, these results demonstrate the utility of heteronuclear NMR in the study of mobile regions of large biological complexes and form the basis for further NMR studies of functional ribosomal complexes in the context of protein synthesis.
Genetic organization of the
psbAD
region in phages infecting marine
Synechococcus
strains
Authors: Millard Andrew , Clokie Martha R. J. , Shub David A. , Mann Nicholas H.
Published: July 2004

The discovery of the genes
psbA
and
psbD
, encoding the D1 and D2 core components of the photosynthetic reaction center PSII (photosystem II), in the genome of the bacteriophage S-PM2 (a cyanomyovirus) that infects marine cyanobacteria begs the question as to how these genes were acquired. In an attempt to answer this question, it was established that the occurrence of the genes is widespread among marine cyanomyovirus isolates and may even extend to podoviruses. The phage
psbA
genes fall into a clade that includes the
psbA
genes from their potential
Synechococcus
and
Prochlorococcus
hosts, and thus, this phylogenetic analysis provides evidence to support the idea of the acquisition of these genes by horizontal gene transfer from their cyanobacterial hosts. However, the phage
psbA
genes form distinct subclades within this lineage, which suggests that their acquisition was not very recent. The
psbA
genes of two phages contain identical 212-bp insertions that exhibit all of the canonical structural features of a group I self-splicing intron. The different patterns of genetic organization of the
psbAD
region are consistent with the idea that the
psbA
and
psbD
genes were acquired more than once by cyanomyoviruses and that their horizontal transfer between phages via a common phage gene pool, as part of mobile genetic modules, may be a continuing process. In addition, genes were discovered encoding a high-light inducible protein and a putative key enzyme of dark metabolism, transaldolase, extending the areas of host-cell metabolism that may be affected by phage infection.
Conformation and orientation of a protein folding intermediate trapped by adsorption
Authors: Engel Maarten F. M. , Visser Antonie J. W. G. , van Mierlo Carlo P. M.
Published: July 2004

Although adsorption-induced conformational changes of proteins play an essential role during protein adsorption on interfaces, detailed information about these changes is lacking. To further the current understanding of protein adsorption, in this study, the orientation, conformation, and local stability of bovine α-lactalbumin (BLA) adsorbed on polystyrene nanospheres is characterized at the residue level by hydrogen/deuterium exchange and 2D NMR spectroscopy. Most of the adsorbed BLA molecules have conformational properties similar to BLA molecules in the acid-induced molten globule state (A state). A folding intermediate of BLA is thus induced and trapped by adsorption of the protein on the hydrophobic interface. Several residues, clustered on one side of the adsorbed folding intermediate of BLA, have altered amide proton exchange protection factors compared to those of the A state of BLA. This side preferentially interacts with the interface and includes residues in helix C, the calcium binding site, and part of the β–domain. Local unfolding of this interacting part of the adsorbed protein seems to initiate the adsorption-induced unfolding of BLA. Adsorption-induced protein unfolding apparently resembles more the mechanical unfolding of a protein than the global unfolding of a protein as induced by denaturant, pH, or pressure. 2D macromolecular crowding prevented the minority of adsorbed BLA molecules, which arrived late at the interface, to unfold to the A state. Protein adsorption is a novel and challenging approach to probe features of the free energy landscapes accessible to unfolding proteins.
Phylogeny and diversification of the largest avian radiation
Authors: Barker F. Keith , Cibois Alice , Schikler Peter , Feinstein Julie , Cracraft Joel
Published: July 2004

The order Passeriformes (“perching birds”) comprises extant species diversity comparable to that of living mammals. For over a decade, a single phylogenetic hypothesis based on DNA–DNA hybridization has provided the primary framework for numerous comparative analyses of passerine ecological and behavioral evolution and for tests of the causal factors accounting for rapid radiations within the group. We report here a strongly supported phylogenetic tree based on two single-copy nuclear gene sequences for the most complete sampling of passerine families to date. This tree is incongruent with that derived from DNA–DNA hybridization, with half of the nodes from the latter in conflict and over a third of the conflicts significant as assessed under maximum likelihood. Our historical framework suggests multiple waves of passerine dispersal from Australasia into Eurasia, Africa, and the New World, commencing as early as the Eocene, essentially reversing the classical scenario of oscine biogeography. The revised history implied by these data will require reassessment of comparative analyses of passerine diversification and adaptation.
Genome sequence of the enterobacterial phytopathogen
Erwinia carotovora
subsp.
atroseptica
and characterization of virulence factors
Authors: Bell K. S. , Sebaihia M. , Pritchard L. , Holden M. T. G. , Hyman L. J. , Holeva M. C. , Thomson N. R. , Bentley S. D. , Churcher L. J. C. , Mungall K. , Atkin R. , Bason N. , Brooks K. , Chillingworth T. , Clark K. , Doggett J. , Fraser A. , Hance Z. , Hauser H. , Jagels K. , Moule S. , Norbertczak H. , Ormond D. , Price C. , Quail M. A. , Sanders M. , Walker D. , Whitehead S. , Salmond G. P. C. , Birch P. R. J. , Parkhill J. , Toth I. K.
Published: July 2004

The bacterial family Enterobacteriaceae is notable for its well studied human pathogens, including
Salmonella
,
Yersinia
,
Shigella
, and
Escherichia
spp. However, it also contains several plant pathogens. We report the genome sequence of a plant pathogenic enterobacterium,
Erwinia carotovora
subsp.
atroseptica
(
Eca
) strain SCRI1043, the causative agent of soft rot and blackleg potato diseases. Approximately 33% of
Eca
genes are not shared with sequenced enterobacterial human pathogens, including some predicted to facilitate unexpected metabolic traits, such as nitrogen fixation and opine catabolism. This proportion of genes also contains an overrepresentation of pathogenicity determinants, including possible horizontally acquired gene clusters for putative type IV secretion and polyketide phytotoxin synthesis. To investigate whether these gene clusters play a role in the disease process, an arrayed set of insertional mutants was generated, and mutations were identified. Plant bioassays showed that these mutants were significantly reduced in virulence, demonstrating both the presence of novel pathogenicity determinants in
Eca
, and the impact of functional genomics in expanding our understanding of phytopathogenicity in the Enterobacteriaceae.
Recapitulation of B cell differentiation in the central nervous system of patients with multiple sclerosis
Authors: Corcione Anna , Casazza Simona , Ferretti Elisa , Giunti Debora , Zappia Emanuela , Pistorio Angela , Gambini Claudio , Mancardi Giovanni Luigi , Uccelli Antonio , Pistoia Vito
Published: July 2004

Clonally expanded populations of B cells carrying somatic mutations of Ig variable (V) region genes have been detected in the CNS of subjects with multiple sclerosis (MS), suggesting that a process of B cell affinity maturation with ensuing production of potentially pathogenic autoantibodies may occur inside the CNS. Here, we have characterized the B cell subsets present in the cerebrospinal fluid (CSF) of MS patients and of individuals with other inflammatory neurological disorders by flow cytometry. CD19+CD38high+CD77+, Ki67+, Bcl-2–centroblasts, i.e., a B cell subset found exclusively in secondary lymphoid organs, were detected in the CSF but not in paired peripheral blood from both patient groups. CD27+IgD–memory B cells, i.e., cells with hyper-mutated IgV genes, were significantly increased in the CSF vs. paired peripheral blood and displayed up-regulation of the CD80 and CD86 costimulatory molecules and of CC chemokine receptor (CCR) 1, CCR2, and CCR4 in both patient groups. Lymphotoxin-α, CXC ligand (CXCL) 12, and CXCL13, key mediators of lymphoid neogenesis, were present in the CSF from patients with MS and other inflammatory neurological disorders and were expressed in MS brain tissue, with selective localization in the outer layer of the capillary vessel wall. In conclusion, this study suggests that a compartmentalized B cell response occurs within the CNS during an ongoing inflammatory reaction, through a recapitulation of all stages of B cell differentiation observed in secondary lymphoid organs. The presence of lymphotoxin-α, CXCL12, and CXCL13 in the CNS may provide favorable microenvironmental conditions for these events.
Late viral interference induced by transdominant Gag of an endogenous retrovirus
Authors: Mura Manuela , Murcia Pablo , Caporale Marco , Spencer Thomas E. , Nagashima Kunio , Rein Alan , Palmarini Massimo
Published: July 2004

The sheep genome harbors ≈20 copies of endogenous retroviruses (enJSRVs) closely related to the exogenous and oncogenic Jaagsiekte sheep retrovirus (JSRV). One of the enJSRV loci, enJS56A1, has a defect for viral exit. We report a previously uncharacterized mechanism of retroviral interference. The defect possessed by enJS56A1 is determined by its Gag protein and is transdominant over the exogenous JSRV. By electron microscopy, cells transfected by enJS56A1, with or without JSRV, show agglomerates of tightly packed intracellular particles most abundant in the perinuclear area. The defect in exit and ability to interfere with JSRV exit could be largely attributed to the presence of tryptophan, rather than arginine, at position 21 of enJS56A1 Gag; C98 and V102 also contribute to these properties. We found that enJS56A1 or similar loci containing W21, C98, and V102 are expressed in sheep endometrium. enJS56A1 is a previously unrecognized example of a naturally occurring endogenous retrovirus expressing a dominant negative Gag acting at a late step of the viral replication cycle. Understanding the late blockade exerted by enJS56A1 could unravel fundamental aspects of retroviral biology and help to devise new antiretroviral strategies.
Cooperative action of coherent groups in broadly heterogeneous populations of interacting chemical oscillators
Authors: Mikhailov A. S. , Zanette D. H. , Zhai Y. M. , Kiss I. Z. , Hudson J. L.
Published: July 2004

We present laboratory experiments on the effects of global coupling in a population of electrochemical oscillators with a multimodal frequency distribution. The experiments show that complex collective signals are generated by this system through spontaneous emergence and joint operation of coherently acting groups representing hierarchically organized resonant clusters. Numerical simulations support these experimental findings. Our results suggest that some forms of internal self-organization, characteristic for complex multiagent systems, are already possible in simple chemical systems.
Discovery and characterization of sialic acid O-acetylation in group BStreptococcus
Authors: Lewis Amanda L. , Nizet Victor , Varki Ajit
Published: July 2004

Group BStreptococcus(GBS) is the leading cause of human neonatal sepsis and meningitis. The GBS capsular polysaccharide is a major virulence factor and the active principle of vaccines in phase II trials. All GBS capsules have a terminal α2–3-linked sialic acid [N-acetylneuraminic acid (Neu5Ac)], which interferes with complement-mediated killing. We show here that some of the Neu5Ac residues of the GBS type III capsule are O-acetylated at carbon position 7, 8, or 9, a major modification evidently missed in previous studies. Data are consistent with initial O-acetylation at position 7, and subsequent migration of theO-acetyl ester at positions 8 and 9. O-acetylation was also present on several other GBS serotypes (Ia, Ib, II, V, and VI). Deletion of the CMP-Neu5Ac synthase geneneuA by precise, in-frame allelic replacement gave intracellular accumulation of O-acetylated Neu5Ac, whereas overexpression markedly decreased O-acetylation. Given the known GBS Neu5Ac biosynthesis pathway, these data indicate that O-acetylation occurs on free Neu5Ac, competing with the CMP-Neu5Ac synthase. O-acetylation often generates immunogenic epitopes on bacterial capsular polysaccharides and can modulate human alternate pathway complement activation. Thus, our discovery has important implications for GBS pathogenicity, immunogenicity, and vaccine design.
Crystal structure of the p14/MP1 scaffolding complex: How a twin couple attaches mitogen-activated protein kinase signaling to late endosomes
Authors: Kurzbauer Robert , Teis David , de Araujo Mariana E. G. , Maurer-Stroh Sebastian , Eisenhaber Frank , Bourenkov Gleb P. , Bartunik Hans D. , Hekman Mirko , Rapp Ulf R. , Huber Lukas A. , Clausen Tim
Published: July 2004

Signaling pathways in eukaryotic cells are often controlled by the formation of specific signaling complexes, which are coordinated by scaffold and adaptor proteins. Elucidating their molecular architecture is essential to understand the spatial and temporal regulation of cellular signaling. p14 and MP1 form a tight (
K
d
= 12.8 nM) endosomal adaptor/scaffold complex, which regulates mitogen-activated protein kinase (MAPK) signaling. Here, we present the 1.9-Å crystal structure of a biologically functional p14/MP1 complex. The overall topology of the individual MP1 and p14 proteins is almost identical, having a central five-stranded β-sheet sandwiched between a two-helix and a one-helix layer. Formation of the p14/MP1 heterodimer proceeds by β-sheet augmentation and yields a unique, almost symmetrical, complex with several potential protein-binding sites on its surface. Mutational analysis allowed identification of the p14 endosomal adaptor motif, which seems to orient the complex relative to the endosomal membrane. Two highly conserved and hydrophobic protein-binding sites are located on the opposite “cytoplasmic” face of the p14/MP1 heterodimer and might therefore function as docking sites for the target proteins extracellular regulated kinase (ERK) 1 and MAPK/ERK kinase 1. Furthermore, detailed sequence analyses revealed that MP1/p14, together with profilins, define a protein superfamily of small subcellular adaptor proteins, named ProflAP. Taken together, the presented work provides insight into the spatial regulation of MAPK signaling, illustrating how p14 and MP1 collaborate as an endosomal adaptor/scaffold complex.
Density functional theory study of adamantanediyl dications C10H142+andprotio-adamantyl dications C10H162+
Authors: Rasul Golam , Olah George A. , Prakash G. K. Surya
Published: July 2004

Structures of the isomeric adamantanediyl dications C10H142+andprotio-1- andprotio-2-adamantyl dications C10H162+were investigated by using the density functional theory (DFT) method at the B3LYP/6–31G** level. Four structures, 1 b–e, were found to be minima on the potential energy surface of C10H142+. The 1,3-adamantanediyl dication 1b with two bridgehead tertiary carbocationic centers was found to be the most stable structure. On the potential energy surface of C10H162+(protonated adamantly cation), five structures, 2 b–f, were found to be minima. Each of the structure contains a two-electron, three-center bond. The C—C protonated 1-adamantyl dication, 2f, was characterized as the most stable structure.13C NMR chemical shifts of the structures were also calculated by using gauge-including atomic orbital-density functional theory and gauge-including atomic orbital-self-consistent field methods.
Testosterone activates mitogen-activated protein kinase and the cAMP response element binding protein transcription factor in Sertoli cells
Authors: Fix Charity , Jordan Cynthia , Cano Patricia , Walker William H.
Published: July 2004

The androgen testosterone is essential for the Sertoli cell to support the maturation of male germ cells and the production of spermatozoa (spermatogenesis). In the classical view of androgen action, binding of androgen to the intracellular androgen receptor (AR) produces a conformational change in AR such that the receptor–steroid complex has high affinity for specific DNA regulatory elements and is able to stimulate gene transcription. Here, we demonstrate that testosterone can act by means of an alternative, rapid, and sustainable mechanism in Sertoli cells that is independent of AR–DNA interactions. Specifically, the addition of physiological levels of testosterone to Sertoli cells stimulates the mitogen-activated protein kinase signaling pathway and causes phosphorylation of the cAMP response element binding protein transcription factor on serine 133, a modification known to be required for Sertoli cells to support spermatogenesis. Androgen-mediated activation of mitogen-activated protein kinase and cAMP response element binding protein occurs within 1 min, extends for at least 12 h and requires AR. Furthermore, androgen induces endogenous cAMP response element binding protein-mediated transcription in Sertoli cells. These newly identified mechanisms of androgen action in Sertoli cells suggest new targets for developing male contraceptive agents.
Reovirus oncolysis: The Ras/RalGEF/p38 pathway dictates host cell permissiveness to reovirus infection
Authors: Norman Kara L. , Hirasawa Kensuke , Yang An-Dao , Shields Michael A. , Lee Patrick W. K.
Published: July 2004

Reovirus is a benign human virus that was recently found to have oncolytic properties and is currently in clinical trials as a potential cancer therapy. We have previously demonstrated that activation of Ras signaling, a common event in cancer, renders cells susceptible to reovirus oncolysis. In this study, we investigate which elements downstream of Ras are important in reovirus infection. By using a panel of NIH 3T3 cells transformed with activated Ras mutated in the effector-binding domain, we found that only the RasV12G37 mutant, which was unable to signal to Raf or phosphatidylinositol 3-kinase but retained signaling capability to guanine nucleotide-exchange factors (GEFs) for the small G protein, Ral (known as RalGEFs), was permissive to reovirus. Expression of the activated mutant of the RalGEF, Rlf, also allowed reovirus replication. Specific inhibition of the Ral pathway by using dominant-negative RalA rendered normally permissive H-Ras cells (cells expressing activated Ras) resistant to reovirus. To further identify elements downstream of RalGEF that promote reovirus infection, we used chemical inhibitors of the downstream signaling elements p38 and JNK. We found that reovirus infection was blocked in the presence of the p38 inhibitor but not the JNK inhibitor. Together, these results implicate a Ras/RalGEF/p38 pathway in the regulation of reovirus replication and oncolysis.
A role for heme in Alzheimer's disease: Heme binds amyloid β and has altered metabolism
Authors: Atamna Hani , Frey William H.
Published: July 2004

Heme is a common factor linking several metabolic perturbations in Alzheimer's disease (AD), including iron metabolism, mitochondrial complex IV, heme oxygenase, and bilirubin. Therefore, we determined whether heme metabolism was altered in temporal lobes obtained at autopsy from AD patients and age-matched nondemented subjects. AD brain demonstrated 2.5-fold more heme-
b
(
P
< 0.01) and 26% less heme-
a
(
P
= 0.16) compared with controls, resulting in a highly significant 2.9-fold decrease in heme-
a
/heme-
b
ratio (
P
< 0.001). Moreover, the strong Pearson correlation between heme-
a
and heme-
b
measured in control individuals (
r
2
= 0.66,
P
< 0.002,
n
= 11) was abolished in AD subjects (
r
2
= 0.076,
P
= 0.39,
n
= 12). The level of ferrochelatase (which makes heme-
b
in the mitochondrial matrix) in AD subjects was 4.2 times (
P
< 0.04) that in nondemented controls, suggesting up-regulated heme synthesis. To look for a possible connection between these observations and established mechanisms in AD pathology, we examined possible interactions between amyloid β (Aβ) and heme. Aβ
(1–40)
and Aβ
(1–42)
induced a redshift of 15–20 nm in the spectrum of heme-
b
and heme-
a
, suggesting that heme binds Aβ, likely to one or more of the histidine residues. Lastly, in a tissue culture model, we found that clioquinol, a metal chelator in clinical trials for AD therapy, decreased intracellular heme. In light of these observations, we have proposed a model of AD pathobiology in which intracellular Aβ complexes with free heme, thereby decreasing its bioavailability (e.g., heme-
a
) and resulting in functional heme deficiency. The model integrates disparate observations, including Aβ, mitochondrial dysfunction, cholesterol, and the proposed efficacy of clioquinol.
Employment patterns and economic independence of women in intimate relationships
Authors: Klement Carmen , Rudolph Brigitte
Published: July 2004

In European societies there are some differences in the way and extent of women's integration into the labour market due to different economic development, political ideas and cultural traditions. In this paper two countries are compared, namely Finland and Germany, which differ considerably concerning women's employment patterns, employment prospects and their life orientations. While most Finnish women are participants of the labour force in full-time jobs, the working pattern of German women is of rather discountinous nature, depending on their chosen intimate relationships in private life, implicating their role as unpaid carers. As there is no sufficient provision of public care services, many German women are dependent on a male breadwinner while leaving their jobs for family purposes. In contrast, Finnish mothers return to their full-time jobs after maternity leave as the welfare state provides enough public care services for children and the dependent elderly. Thus it appears that Finnish women have gained financial autonomy and economic independence, a pre-requisite for living in democratic, individualized intimate relationships with equal decision power. Even if, during the last decades, empirical evidence has revealed the transformation of the well known breadwinner model to a modernized version, or partially a shift to an ‘adult worker model’, in Germany there is still no evidence for a general trend to a fully individualized life pattern of women with children.
External labour market flexibility and social inequality
Authors: Giesecke Johannes , Groß Martin
Published: July 2004

In this paper we examine the impact of temporary work on two dimensions of social inequality: income and career mobility. Additionally, we are taking a comparative perspective on this subject by comparing Germany and the UK. To investigate the effects of temporary work we use data from the German Socio-Economic Panel and the British Household Panel Study on non-self-employed respondents. The results show that temporary work does influence the system of social inequality: we found wage penalties and an increased probability of severe negative effects on the working careers of temporarily employed persons in both countries (net of education, age, and a variety of other covariates). Thus we can conclude that temporary employment represents a substantial socio-economic risk for employees. Most importantly, this holds true for both the German and the British case, two quite distinct labour market regimes.
Persisting class inequality?
Authors: McGinnity Frances , Hillmert Steffen
Published: July 2004

In conditions of increasing economic uncertainty, a body of social stratification researchers claims that significant class inequality in life chances persists (Breen, 1997; Goldthorpe, 2000). Can we find evidence of this, and are developments similar across countries? Differences in education systems, welfare and labour market institutions may have implications for class risks and class theory. Britain and Germany differ significantly in these institutions. Selecting the risk of unemployment as an indicator of class inequality, we investigate how this risk varies by social class in different cohorts in the two countries. Using life course data, we find that in both countries class is a good predictor of the risk of unemployment, though there is less of a difference in class risks in Germany. We find evidence of persisting inequality in both Britain and Germany. We reflect on the interaction between social class and labour market institutions for labour market outcomes, and discuss the implications of our findings for the theory and measurement of class.
Novel Expression Pattern of Cytosolic Gln Synthetase in Nitrogen-Fixing Root Nodules of the Actinorhizal Host, Datisca glomerata
Authors: Berry Alison M. , Murphy Terence M. , Okubara Patricia A. , Jacobsen Karin R. , Swensen Susan M. , Pawlowski Katharina
Published: July 2004

Gln synthetase (GS) is the key enzyme of primary ammonia assimilation in nitrogen-fixing root nodules of legumes and actinorhizal (Frankia-nodulated) plants. In root nodules of Datisca glomerata (Datiscaceae), transcripts hybridizing to a conserved coding region of the abundant nodule isoform, DgGS1-1, are abundant in uninfected nodule cortical tissue, but expression was not detectable in the infected zone or in the nodule meristem. Similarly, the GS holoprotein is immunolocalized exclusively to the uninfected nodule tissue. Phylogenetic analysis of the full-length cDNA of DgGS1-1 indicates affinities with cytosolic GS genes from legumes, the actinorhizal species Alnus glutinosa, and nonnodulating species, Vitis vinifera and Hevea brasilensis. The D. glomerata nodule GS expression pattern is a new variant among reported root nodule symbioses and may reflect an unusual nitrogen transfer pathway from the Frankia nodule microsymbiont to the plant infected tissue, coupled to a distinctive nitrogen cycle in the uninfected cortical tissue. Arg, Gln, and Glu are the major amino acids present in D. glomerata nodules, but Arg was not detected at high levels in leaves or roots. Arg as a major nodule nitrogen storage form is not found in other root nodule types except in the phylogenetically related Coriaria. Catabolism of Arg through the urea cycle could generate free ammonium in the uninfected tissue where GS is expressed.
Microarray Analyses of Gene Expression during Adventitious Root Development in Pinus contorta
Authors: Brinker Monika , van Zyl Leonel , Liu Wenbin , Craig Deborah , Sederoff Ronald R. , Clapham David H. , von Arnold Sara
Published: July 2004

In order to investigate the gene expression pattern during adventitious root development, RNA of Pinus contorta hypocotyls, pulse-treated with the auxin indole-3-butyric acid and harvested at distinct developmental time points of root development, was hybridized to microarrays containing 2,178 cDNAs from Pinus taeda. Over the period of observation of root development, the transcript levels of 220 genes changed significantly. During the root initiation phase, genes involved in cell replication and cell wall weakening and a transcript encoding a PINHEAD/ZWILLE-like protein were up-regulated, while genes related to auxin transport, photosynthesis, and cell wall synthesis were down-regulated. In addition, there were changes in transcript abundance of genes related to water stress. During the root meristem formation phase the transcript abundances of genes involved in auxin transport, auxin responsive transcription, and cell wall synthesis, and of a gene encoding a B-box zinc finger-like protein, increased, while those encoding proteins involved in cell wall weakening decreased. Changes of transcript abundance of genes related to water stress during the root meristem formation and root formation phase indicate that the plant roots had become functional in water transport. Simultaneously, genes involved in auxin transport were up-regulated, while genes related to cell wall modification were down-regulated. Finally, during the root elongation phase down-regulation of transcripts encoding proteins involved in cell replication and stress occurred. Based on the observed changes in transcript abundances, we suggest hypotheses about the relative importance of various physiological processes during the auxin-induced development of roots in P. contorta.
Autophosphorylation and Subcellular Localization Dynamics of a Salt- and Water Deficit-Induced Calcium-Dependent Protein Kinase from Ice Plant
Authors: Chehab E. Wassim , Patharkar O. Rahul , Hegeman Adrian D. , Taybi Tahar , Cushman John C.
Published: July 2004

A salinity and dehydration stress-responsive calcium-dependent protein kinase (CDPK) was isolated from the common ice plant (Mesembryanthemum crystallinum; McCPK1). McCPK1 undergoes myristoylation, but not palmitoylation in vitro. Removal of the N-terminal myristate acceptor site partially reduced McCPK1 plasma membrane (PM) localization as determined by transient expression of green fluorescent protein fusions in microprojectile-bombarded cells. Removal of the N-terminal domain (amino acids 1–70) completely abolished PM localization, suggesting that myristoylation and possibly the N-terminal domain contribute to membrane association of the kinase. The recombinant, Escherichia coli-expressed, full-length McCPK1 protein was catalytically active in a calcium-dependent manner (K  0.5 = 0.15 μ  m). Autophosphorylation of recombinant McCPK1 was observed in vitro on at least two different Ser residues, with the location of two sites being mapped to Ser-62 and Ser-420. An Ala substitution at the Ser-62 or Ser-420 autophosphorylation site resulted in a slight increase in kinase activity relative to wild-type McCPK1 against a histone H1 substrate. In contrast, Ala substitutions at both sites resulted in a dramatic decrease in kinase activity relative to wild-type McCPK1 using histone H1 as substrate. McCPK1 undergoes a reversible change in subcellular localization from the PM to the nucleus, endoplasmic reticulum, and actin microfilaments of the cytoskeleton in response to reductions in humidity, as determined by transient expression of McCPK1-green fluorescent protein fusions in microprojectile-bombarded cells and confirmed by subcellular fractionation and western-blot analysis of 6× His-tagged McCPK1.
Isolation and Characterization of a TERMINAL FLOWER Homolog and Its Correlation with Juvenility in Citrus
Authors: Pillitteri Lynn Jo , Lovatt Carol J. , Walling Linda L.
Published: July 2004

TERMINAL FLOWER is a key regulator of floral timing in Arabidopsis and other herbaceous species. A homolog of this gene, CsTFL, was isolated from the hybrid perennial tree crop Washington navel orange (Citrus sinensis L. Osbeck). The deduced amino acid sequence of CsTFL was 65% identical to the Arabidopsis TFL1 protein. Wild-type Arabidopsis plants ectopically expressing CsTFL showed late-flowering phenotypes similar to those described for overexpression of Arabidopsis TFL1. In addition, the 35S:CsTFL transgene complemented the tfl1-2 mutant. The severity of the overexpression phenotypes correlated with the amount of CsTFL transcript that accumulated. Unlike many model systems that have been studied, C. sinensis maintains two distinguishable CsTFL alleles. CsTFL transcripts from either allele were not detected in adult vegetative tissues using reverse transcription-PCR, but CsTFL RNAs were detected in all floral organs. In addition, real-time PCR determined that juvenility in citrus was positively correlated with CsTFL transcript accumulation and negatively correlated with the floral-regulatory genes, LEAFY and APETALA1, RNA levels.
Down-Regulation of Metallothionein, a Reactive Oxygen Scavenger, by the Small GTPase OsRac1 in Rice
Authors: Wong Hann Ling , Sakamoto Tsuyoshi , Kawasaki Tsutomu , Umemura Kenji , Shimamoto Ko
Published: July 2004

Metallothioneins are small, ubiquitous Cys-rich proteins known to be involved in reactive oxygen species (ROS) scavenging and metal homeostasis. We found that the expression of a metallothionein gene (OsMT2b) was synergically down-regulated by OsRac1 and rice (Oryza sativa) blast-derived elicitors. Transgenic plants overexpressing OsMT2b showed increased susceptibility to bacterial blight and blast fungus. OsMT2b-overexpressing cells showed reduced elicitor-induced hydrogen peroxide production. In contrast, homozygous OsMT2b::Tos17-inserted mutant and OsMT2b-RNAi-silenced transgenic cells showed significantly higher elicitor-induced hydrogen peroxide production than the wild-type cells. In vitro assay showed that recombinant OsMT2b protein possessed superoxide- and hydroxyl radical-scavenging activities. Taken together, these results showed that OsMT2b is an ROS scavenger and its expression is down-regulated by OsRac1, thus potentiating ROS, which function as signals in resistance response. The results suggest that OsRac1 plays a dual role as an inducer of ROS production and a suppressor of ROS scavenging.
Phylogenetic Analyses inCornusSubstantiate Ancestry of Xylem Supercooling Freezing Behavior and Reveal Lineage of Desiccation Related Proteins
Authors: Karlson Dale T. , Xiang (Jenny) Qiu-Yun , Stirm Vicki E. , Shirazi A.M. , Ashworth Edward N.
Published: July 2004

The response of woody plant tissues to freezing temperature has evolved into two distinct behaviors: an avoidance strategy, in which intracellular water supercools, and a freeze-tolerance strategy, where cells tolerate the loss of water to extracellular ice. Although both strategies involve extracellular ice formation, supercooling cells are thought to resist freeze-induced dehydration. Dehydrin proteins, which accumulate during cold acclimation in numerous herbaceous and woody plants, have been speculated to provide, among other things, protection from desiccative extracellular ice formation. Here we use Cornus as a model system to provide the first phylogenetic characterization of xylem freezing behavior and dehydrin-like proteins. Our data suggest that both freezing behavior and the accumulation of dehydrin-like proteins in Cornus are lineage related; supercooling and nonaccumulation of dehydrin-like proteins are ancestral within the genus. The nonsupercooling strategy evolved within the blue- or white-fruited subgroup where representative species exhibit high levels of freeze tolerance. Within the blue- or white-fruited lineage, a single origin of dehydrin-like proteins was documented and displayed a trend for size increase in molecular mass. Phylogenetic analyses revealed that an early divergent group of red-fruited supercooling dogwoods lack a similar protein. Dehydrin-like proteins were limited to neither nonsupercooling species nor to those that possess extreme freeze tolerance.
The Putative Arabidopsis Homolog of Yeast Vps52p Is Required for Pollen Tube Elongation, Localizes to Golgi, and Might Be Involved in Vesicle Trafficking
Authors: Lobstein Eglantine , Guyon Anouchka , Férault Madina , Twell David , Pelletier Georges , Bonhomme Sandrine
Published: July 2004

The screening of the Versailles collection of Arabidopsis T-DNA transformants allowed us to identify several male gametophytic mutants, including poky pollen tube (pok). The pok mutant, which could only be isolated as a hemizygous line, exhibits very short pollen tubes, explaining the male-specific transmission defect observed in this line. We show that the POK gene is duplicated in the Arabidopsis genome and that the predicted POK protein sequence is highly conserved from lower to higher eukaryotes. The putative POK homolog in yeast (Saccharomyces cerevisiae), referred to as Vps52p/SAC2, has been shown to be located at the late Golgi and to function in a complex with other proteins, Vps53p, Vps54p, and Vps51p. This complex is involved in retrograde trafficking of vesicles between the early endosomal compartment and the trans-Golgi network. We present the expression patterns of the POK gene and its duplicate P2 in Arabidopsis, and of the putative Arabidopsis homologs of VPS53 and VPS54 of yeast. We show that a POK::GFP fusion protein localizes to Golgi in plant cells, supporting the possibility of a conserved function for Vps52p and POK proteins. These results, together with the expression pattern of the POK::GUS fusion and the lack of plants homozygous for the pok mutation, suggest a more general role for POK in polar growth beyond the pollen tube elongation process.
Tomato Phospholipid Hydroperoxide Glutathione Peroxidase Inhibits Cell Death Induced by Bax and Oxidative Stresses in Yeast and Plants
Authors: Chen Shaorong , Vaghchhipawala Zarir , Li Wei , Asard Han , Dickman Martin B.
Published: July 2004

Using a conditional life or death screen in yeast, we have isolated a tomato (Lycopersicon esculentum) gene encoding a phospholipid hydroperoxide glutathione peroxidase (LePHGPx). The protein displayed reduced glutathione-dependent phospholipid hydroperoxide peroxidase activity, but differs from counterpart mammalian enzymes that instead contain an active seleno-Cys. LePHGPx functioned as a cytoprotector in yeast (Saccharomyces cerevisiae), preventing Bax, hydrogen peroxide, and heat stress induced cell death, while also delaying yeast senescence. When tobacco (Nicotiana tabacum) leaves were exposed to lethal levels of salt and heat stress, features associated with mammalian apoptosis were observed. Importantly, transient expression of LePHGPx protected tobacco leaves from salt and heat stress and suppressed the apoptotic-like features. As has been reported, conditional expression of Bax was lethal in tobacco, resulting in tissue collapse and membrane permeability to Evans blue. When LePHGPx was coexpressed with Bax, little cell death and no vital staining were observed. Moreover, stable expression of LePHGPx in tobacco conferred protection against the fungal phytopathogen Botrytis cinerea. Taken together, our data indicated that LePHGPx can protect plant tissue from a variety of stresses. Moreover, functional screens in yeast are a viable tool for the identification of plant genes that regulate cell death.
Computational Identification and Characterization of Novel Genes from Legumes
Authors: Graham Michelle A. , Silverstein Kevin A.T. , Cannon Steven B. , VandenBosch Kathryn A.
Published: July 2004

The Fabaceae, the third largest family of plants and the source of many crops, has been the target of many genomic studies. Currently, only the grasses surpass the legumes for the number of publicly available expressed sequence tags (ESTs). The quantity of sequences from diverse plants enables the use of computational approaches to identify novel genes in specific taxa. We used BLAST algorithms to compare unigene sets from Medicago truncatula, Lotus japonicus, and soybean (Glycine max and Glycine soja) to nonlegume unigene sets, to GenBank's nonredundant and EST databases, and to the genomic sequences of rice (Oryza sativa) and Arabidopsis. As a working definition, putatively legume-specific genes had no sequence homology, below a specified threshold, to publicly available sequences of nonlegumes. Using this approach, 2,525 legume-specific EST contigs were identified, of which less than three percent had clear homology to previously characterized legume genes. As a first step toward predicting function, related sequences were clustered to build motifs that could be searched against protein databases. Three families of interest were more deeply characterized: F-box related proteins, Pro-rich proteins, and Cys cluster proteins (CCPs). Of particular interest were the >300 CCPs, primarily from nodules or seeds, with predicted similarity to defensins. Motif searching also identified several previously unknown CCP-like open reading frames in Arabidopsis. Evolutionary analyses of the genomic sequences of several CCPs in M. truncatula suggest that this family has evolved by local duplications and divergent selection.
Expansins Abundant in Secondary Xylem Belong to Subgroup A of the α-Expansin Gene Family
Authors: Gray-Mitsumune Madoka , Mellerowicz Ewa J. , Abe Hisashi , Schrader Jarmo , Winzéll Anders , Sterky Fredrik , Blomqvist Kristina , McQueen-Mason Simon , Teeri Tuula T. , Sundberg Björn
Published: July 2004

Differentiation of xylem cells in dicotyledonous plants involves expansion of the radial primary cell walls and intrusive tip growth of cambial derivative cells prior to the deposition of a thick secondary wall essential for xylem function. Expansins are cell wall-residing proteins that have an ability to plasticize the cellulose-hemicellulose network of primary walls. We found expansin activity in proteins extracted from the cambial region of mature stems in a model tree species hybrid aspen (Populus tremula × Populus tremuloides Michx). We identified three α-expansin genes (PttEXP1, PttEXP2, and PttEXP8) and one β-expansin gene (PttEXPB1) in a cambial region expressed sequence tag library, among which PttEXP1 was most abundantly represented. Northern-blot analyses in aspen vegetative organs and tissues showed that PttEXP1 was specifically expressed in mature stems exhibiting secondary growth, where it was present in the cambium and in the radial expansion zone. By contrast, PttEXP2 was mostly expressed in developing leaves. In situ reverse transcription-PCR provided evidence for accumulation of mRNA of PttEXP1 along with ribosomal rRNA at the tips of intrusively growing xylem fibers, suggesting that PttEXP1 protein has a role in intrusive tip growth. An examination of tension wood and leaf cDNA libraries identified another expansin, PttEXP5, very similar to PttEXP1, as the major expansin in developing tension wood, while PttEXP3 was the major expansin expressed in developing leaves. Comparative analysis of expansins expressed in woody stems in aspen, Arabidopsis, and pine showed that the most abundantly expressed expansins share sequence similarities, belonging to the subfamily A of α-expansins and having two conserved motifs at the beginning and end of the mature protein, RIPVG and KNFRV, respectively. This conservation suggests that these genes may share a specialized, not yet identified function.
Contrasting Modes of Diversification in the Aux/IAA and ARF Gene Families
Authors: Remington David L. , Vision Todd J. , Guilfoyle Thomas J. , Reed Jason W.
Published: July 2004

The complete genomic sequence for Arabidopsis provides the opportunity to combine phylogenetic and genomic approaches to study the evolution of gene families in plants. The Aux/IAA and ARF gene families, consisting of 29 and 23 loci in Arabidopsis, respectively, encode proteins that interact to mediate auxin responses and regulate various aspects of plant morphological development. We developed scenarios for the genomic proliferation of the Aux/IAA and ARF families by combining phylogenetic analysis with information on the relationship between each locus and the previously identified duplicated genomic segments in Arabidopsis. This analysis shows that both gene families date back at least to the origin of land plants and that the major Aux/IAA and ARF lineages originated before the monocot-eudicot divergence. We found that the extant Aux/IAA loci arose primarily through segmental duplication events, in sharp contrast to the ARF family and to the general pattern of gene family proliferation in Arabidopsis. Possible explanations for the unusual mode of Aux/IAA duplication include evolutionary constraints imposed by complex interactions among proteins and pathways, or the presence of long-distance cis-regulatory sequences. The antiquity of the two gene families and the unusual mode of Aux/IAA diversification have a number of potential implications for understanding both the functional and evolutionary roles of these genes.
Gene Duplication in the Carotenoid Biosynthetic Pathway Preceded Evolution of the Grasses
Authors: Gallagher Cynthia E. , Matthews Paul D. , Li Faqiang , Wurtzel Eleanore T.
Published: July 2004

Despite ongoing research on carotenoid biosynthesis in model organisms, there is a paucity of information on pathway regulation operating in the grasses (Poaceae), which include plants of world-wide agronomic importance. As a result, efforts to either breed for or metabolically engineer improvements in carotenoid content or composition in cereal crops have led to unexpected results. In comparison to maize (Zea mays), rice (Oryza sativa) accumulates no endosperm carotenoids, despite having a functional pathway in chloroplasts. To better understand why these two related grasses differ in endosperm carotenoid content, we began to characterize genes encoding phytoene synthase (PSY), since this nuclear-encoded enzyme appeared to catalyze a rate-controlling step in the plastid-localized biosynthetic pathway. The enzyme had been previously associated with the maize Y1 locus thought to be the only functional gene controlling PSY accumulation, though function of the Y1 gene product had never been demonstrated. We show that both maize and rice possess and express products from duplicate PSY genes, PSY1 (Y1) and PSY2; PSY1 transcript accumulation correlates with carotenoid-containing endosperm. Using a heterologous bacterial system, we demonstrate enzyme function of PSY1 and PSY2 that are largely conserved in sequence except for N- and C-terminal domains. By database mining and use of ortholog-specific universal PCR primers, we found that the PSY duplication is prevalent in at least eight subfamilies of the Poaceae, suggesting that this duplication event preceded evolution of the Poaceae. These findings will impact study of grass phylogeny and breeding of enhanced carotenoid content in an entire taxonomic group of plant crops critical for global food security.