The Hrp (type III protein secretion) system is essential for the plant parasitic ability of Pseudomonas syringae and most Gram-negative bacterial plant pathogens. AvrB and AvrPto are two P. syringae proteins that have biological activity when produced via heterologous gene expression inside plant cells or when produced by Hrp+ bacteria. Avr-like proteins, presumably injected by the Hrp system on bacterial contact with plant cells, appear to underlie pathogenic interactions, but none has been observed outside of the bacterial cytoplasm, and identifying novel genes encoding them is tedious and uncertain without a phenotype in culture. Here we describe a cloned Hrp secretion system that functions heterologously in Escherichia coli to secrete AvrB and AvrPto in culture and to promote AvrB and AvrPto biological activity in inoculated plants. The hrp gene cluster, carried on cosmid pCPP2156, was cloned from Erwinia chrysanthemi, a pathogen that differs from P. syringae in being host promiscuous. E. coli DH5α carrying pCPP2156, but not related Hrp-deficient cosmids, elicited a hypersensitive response in Nicotiana clevelandii only when also expressing avrB in trans. The use of pAVRB-FLAG2 and pAVRPTO-FLAG, which produce Avr proteins with a C-terminal FLAG-epitope fusion...
SR protein kinases (SRPKs) and their substrates, the SR family of serine/arginine-rich pre-mRNA splicing factors, appear to be key regulators of alternative splicing. Although SR proteins have been well characterized through biochemical experiments in metazoans, their functions in vivo are unclear. Because of the strict splice site consensus and near absence of alternative splicing in Saccharomyces cerevisiae, it had been thought that budding yeast would lack an SRPK and its substrates. Here, we present structural, biochemical, and cell-biological evidence that directly demonstrates an SR protein kinase, Sky1p, as well as a number of SRPK substrates in S. cerevisiae. One of these substrates is Npl3p, an SR-like protein involved in mRNA export. This finding raises the provocative possibility that Sky1p, and by extension metazoan SRPKs, regulates mRNA export or the nucleocytoplasmic shuttling of RS domain proteins. The unexpected discovery of an SR protein kinase in budding yeast provides a foundation for genetic dissection of the biological functions of SR proteins and their kinases.
Single-nucleotide polymorphisms (SNPs) are the most common type of genetic variation in man. Genes containing one or more SNPs can give rise to two or more allelic forms of mRNAs. These mRNA variants may possess different biological functions as a result of differences in primary or higher order structures that interact with other cellular components. Here we report the observation of marked differences in mRNA secondary structure associated with SNPs in the coding regions of two human mRNAs: alanyl tRNA synthetase and replication protein A, 70-kDa subunit (RPA70). Enzymatic probing of SNP-containing allelic fragments of the mRNAs revealed pronounced allelic differences in cleavage pattern at sites 14 or 18 nt away from the SNP, suggesting that a single-nucleotide variation can give rise to different mRNA folds. By using phosphorothioate oligodeoxyribonucleotides complementary to the region of different allelic structures in the RPA70 mRNA, but not extending to the SNP itself, we find that the SNP exerts an allele-specific effect on the accessibility of its flanking site in the endogenous human RPA70 mRNA. This further supports the allele-specific structural features identified by enzymatic probing. These results demonstrate the contribution of common genetic variation to structural diversity of mRNA and suggest a broader role than previously thought for the effects of SNPs on mRNA structure and...
A genetic annealing model for the universal ancestor of all extant life is presented; the name of the model derives from its resemblance to physical annealing. The scenario pictured starts when “genetic temperatures” were very high, cellular entities (progenotes) were very simple, and information processing systems were inaccurate. Initially, both mutation rate and lateral gene transfer levels were elevated. The latter was pandemic and pervasive to the extent that it, not vertical inheritance, defined the evolutionary dynamic. As increasingly complex and precise biological structures and processes evolved, both the mutation rate and the scope and level of lateral gene transfer, i.e., evolutionary temperature, dropped, and the evolutionary dynamic gradually became that characteristic of modern cells. The various subsystems of the cell “crystallized,” i.e., became refractory to lateral gene transfer, at different stages of “cooling,” with the translation apparatus probably crystallizing first. Organismal lineages, and so organisms as we know them, did not exist at these early stages. The universal phylogenetic tree, therefore, is not an organismal tree at its base but gradually becomes one as its peripheral branchings emerge. The universal ancestor is not a discrete entity. It is...
The nontoxic proteolytic C fragment of tetanus toxin (TTC peptide) has the same ability to bind nerve cells and be retrogradely transported through a synapse as the native toxin. We have investigated its potential use as an in vivo neurotropic carrier. In this work we show that a hybrid protein encoded by the lacZ–TTC gene fusion retains the biological functions of both proteins in vivo—i.e., retrograde transynaptic transport of the TTC fragment and β-galactosidase enzymatic activity. After intramuscular injection, enzymatic activity could be detected in motoneurons and connected neurons of the brainstem areas. This strategy could be used to deliver a biological activity to neurons from the periphery to the central nervous system. Such a hybrid protein could also be used to map synaptic connections between neural cells.
An endogenous circadian biological clock controls the
temporal aspects of life in most organisms, including rhythmic control
of genes involved in clock output pathways. In the fungus
Neurospora crassa, one pathway known to be under control
of the clock is asexual spore (conidia) development. To understand more
fully the processes that are regulated by the N. crassa
circadian clock, systematic screens were carried out for genes that
oscillate at the transcriptional level. Time-of-day-specific cDNA
libraries were generated and used in differential screens to identify
six new clock-controlled genes (ccgs). Transcripts
specific for each of the ccgs preferentially accumulate
during the late night to early morning, although they vary with respect
to steady-state mRNA levels and amplitude of the rhythm. Sequencing of
the ends of the new ccg cDNAs revealed that
ccg-12 is identical to N. crassa cmt
encoding copper metallothionein, providing the suggestion that
not all clock-regulated genes in N. crassa are
specifically involved in the development of conidia. This was supported
by finding that half of the new ccgs, including
cmt(ccg-12), are not transcriptionally
induced by developmental or light signals. These data suggest a major
role for the clock in the regulation of biological processes distinct
A methodology, fluorescence-intensity distribution analysis, has been developed for confocal microscopy studies in which the fluorescence intensity of a sample with a heterogeneous brightness profile is monitored. An adjustable formula, modeling the spatial brightness distribution, and the technique of generating functions for calculation of theoretical photon count number distributions serve as the two cornerstones of the methodology. The method permits the simultaneous determination of concentrations and specific brightness values of a number of individual fluorescent species in solution. Accordingly, we present an extremely sensitive tool to monitor the interaction of fluorescently labeled molecules or other microparticles with their respective biological counterparts that should find a wide application in life sciences, medicine, and drug discovery. Its potential is demonstrated by studying the hybridization of 5′-(6-carboxytetramethylrhodamine)-labeled and nonlabeled complementary oligonucleotides and the subsequent cleavage of the DNA hybrids by restriction enzymes.
Chemokines comprise a family of low-molecular-weight proteins that elicit a variety of biological responses including chemotaxis, intracellular Ca2+ mobilization, and activation of tyrosine kinase signaling cascades. A subset of chemokines, including regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-1α (MIP-1α), and MIP-1β, also suppress infection by HIV-1. All of these activities are contingent on interactions between chemokines and cognate seven-transmembrane spanning, G protein-coupled receptors. However, these activities are strongly inhibited by glycanase treatment of receptor-expressing cells, indicating an additional dependence on surface glycosaminoglycans (GAG). To further investigate this dependence, we examined whether soluble GAG could reconstitute the biological activities of RANTES on glycanase-treated cells. Complexes formed between RANTES and a number of soluble GAG failed to induce intracellular Ca2+ mobilization on either glycanase-treated or untreated peripheral blood mononuclear cells and were unable to stimulate chemotaxis. In contrast, the same complexes demonstrated suppressive activity against macrophage tropic HIV-1. Complexes composed of 125I-labeled RANTES demonstrated saturable binding to glycanase-treated peripheral blood mononuclear cells...
Identification of cytokine-inducible genes is imperative for determining the mechanisms of cytokine action. A cytokine-inducible gene, mrg1 [melanocyte-specific gene (msg1) related gene], was identified through mRNA differential display of interleukin (IL) 9-stimulated and unstimulated mouse helper T cells. In addition to IL-9, mrg1 can be induced by other cytokines and biological stimuli, including IL-1α, -2, -4, -6, and -11, granulocyte/macrophage colony-stimulating factor, interferon γ, platelet-derived growth factor, insulin, serum, and lipopolysaccharide in diverse cell types. The induction of mrg1 by these stimuli appears to be transient, with induction kinetics similar to other primary response genes, implicating its role in diverse biological processes. Deletion or point mutations of either the Box1 motif (binds Janus kinase 1) or the signal transducer and activator of transcription 3 binding site-containing region within the intracellular domain of the IL-9 receptor ligand binding subunit abolished or greatly reduced mrg1 induction by IL-9, suggesting that the Janus kinase/signal transducer and activator of transcription signaling pathway is required for mrg1 induction, at least in response to IL-9. Transfection of mrg1 cDNA into TS1...
We created a simulation based on experimental data from bacteriophage T7 that computes the developmental cycle of the wild-type phage and also of mutants that have an altered genome order. We used the simulation to compute the fitness of more than 105 mutants. We tested these computations by constructing and experimentally characterizing T7 mutants in which we repositioned gene 1, coding for T7 RNA polymerase. Computed protein synthesis rates for ectopic gene 1 strains were in moderate agreement with observed rates. Computed phage-doubling rates were close to observations for two of four strains, but significantly overestimated those of the other two. Computations indicate that the genome organization of wild-type T7 is nearly optimal for growth: only 2.8% of random genome permutations were computed to grow faster, the highest 31% faster, than wild type. Specific discrepancies between computations and observations suggest that a better understanding of the translation efficiency of individual mRNAs and the functions of qualitatively “nonessential” genes will be needed to improve the T7 simulation. In silico representations of biological systems can serve to assess and advance our understanding of the underlying biology. Iteration between computation...
Photosynthesis, biological nitrogen fixation, and carbon dioxide
assimilation are three fundamental biological processes catalyzed by
photosynthetic bacteria. In the present study, it is shown that mutant
strains of the nonsulfur purple photosynthetic bacteria
Rhodospirillum rubrum and Rhodobacter
sphaeroides, containing a blockage in the primary
CO2 assimilatory pathway, derepress the synthesis of
components of the nitrogen fixation enzyme complex and abrogate normal
control mechanisms. The absence of the Calvin–Benson–Bassham (CBB)
reductive pentose phosphate CO2 fixation pathway removes an
important route for the dissipation of excess reducing power. Thus, the
mutant strains develop alternative means to remove these reducing
equivalents, resulting in the synthesis of large amounts of nitrogenase
even in the presence of ammonia. This response is under the control of
a global two-component signal transduction system previously found to
regulate photosystem biosynthesis and the transcription of genes
required for CO2 fixation through the CBB pathway and
alternative routes. In addition, this two-component system directly
controls the ability of these bacteria to grow under nitrogen-fixing
conditions. These results indicate that there is a molecular link
between the CBB and nitrogen fixation process...
Thionein (T) has not been isolated previously from biological material. However, it is generated transiently in situ by removal of zinc from metallothionein under oxidoreductive conditions, particularly in the presence of selenium compounds. T very rapidly activates a group of enzymes in which zinc is bound at an inhibitory site. The reaction is selective, as is apparent from the fact that T does not remove zinc from the catalytic sites of zinc metalloenzymes. T instantaneously reverses the zinc inhibition with a stoichiometry commensurate with its known capacity to bind seven zinc atoms in the form of clusters in metallothionein. The zinc inhibition is much more pronounced than was previously reported, with dissociation constants in the low nanomolar range. Thus, T is an effective, endogenous chelating agent, suggesting the existence of a hitherto unknown and unrecognized biological regulatory system. T removes the metal from an inhibitory zinc-specific enzymatic site with a resultant marked increase of activity. The potential significance of this system is supported by the demonstration of its operations in enzymes involved in glycolysis and signal transduction.
Quantitative, chemically specific images of biological systems
would be invaluable in unraveling the bioinorganic chemistry of
biological tissues. Here we report the spatial distribution and
chemical forms of selenium in Astragalus bisulcatus
(two-grooved poison or milk vetch), a plant capable of
accumulating up to 0.65% of its shoot dry biomass as Se in its natural
habitat. By selectively tuning incident x-ray energies close to the Se
K-absorption edge, we have collected quantitative, 100-μm-resolution
images of the spatial distribution, concentration, and chemical form of
Se in intact root and shoot tissues. To our knowledge, this is the
first report of quantitative concentration-imaging of specific chemical
forms. Plants exposed to 5 μM selenate for 28 days contained
predominantly selenate in the mature leaf tissue at a concentration of
0.3–0.6 mM, whereas the young leaves and the roots contained
organoselenium almost exclusively, indicating that the ability to
biotransform selenate is either inducible or developmentally specific.
While the concentration of organoselenium in the majority of the root
tissue was much lower than that of the youngest leaves (0.2–0.3
compared with 3–4 mM), isolated areas on the extremities of the roots
contained concentrations of organoselenium an order of magnitude
greater than the rest of the root. These imaging results were
corroborated by spatially resolved x-ray absorption near-edge spectra
collected from selected 100 × 100 μm2 regions of
the same tissues.
Retinoic acid (RA) exerts diverse biological effects in the control of cell growth in embryogenesis and oncogenesis. These effects of RA are thought to be mediated by the nuclear retinoid receptors. Mannose-6-phosphate (M6P)/insulin-like growth factor-II (IGF-II) receptor is a multifunctional membrane glycoprotein that is known to bind both M6P and IGF-II and function primarily in the binding and trafficking of lysosomal enzymes, the activation of transforming growth factor-β, and the degradation of IGF-II. M6P/IGF-II receptor has recently been implicated in fetal development and carcinogenesis. Despite the functional similarities between RA and the M6P/IGF-II receptor, no direct biochemical link has been established. Here, we show that the M6P/IGF-II receptor also binds RA with high affinity at a site that is distinct from those for M6P and IGF-II, as identified by a photoaffinity labeling technique. We also show that the binding of RA to the M6P/IGF-II receptor enhances the primary functions of this receptor. The biological consequence of the interaction appears to be the suppression of cell proliferation and/or induction of apoptosis. These findings suggest that the M6P/IGF-II receptor mediates a RA response pathway that is important in cell growth regulation. This discovery of the interaction of RA with the M6P/IGF-II receptor may have important implications for our understanding of the roles of RA and the M6P/IGF-II receptor in development...
The phytochemical resveratrol, which is found in grapes and wine, has been reported to have a variety of anti-inflammatory, anti-platelet, and anti-carcinogenic effects. Based on its structural similarity to diethylstilbestrol, a synthetic estrogen, we examined whether resveratrol might be a phytoestrogen. At concentrations (≈3–10 μM) comparable to those required for its other biological effects, resveratrol inhibited the binding of labeled estradiol to the estrogen receptor and it activated transcription of estrogen-responsive reporter genes transfected into human breast cancer cells. This transcriptional activation was estrogen receptor-dependent, required an estrogen response element in the reporter gene, and was inhibited by specific estrogen antagonists. In some cell types (e.g., MCF-7 cells), resveratrol functioned as a superagonist (i.e., produced a greater maximal transcriptional response than estradiol) whereas in others it produced activation equal to or less than that of estradiol. Resveratrol also increased the expression of native estrogen-regulated genes, and it stimulated the proliferation of estrogen-dependent T47D breast cancer cells. We conclude that resveratrol is a phytoestrogen and that it exhibits variable degrees of estrogen receptor agonism in different test systems. The estrogenic actions of resveratrol broaden the spectrum of its biological actions and may be relevant to the reported cardiovascular benefits of drinking wine.
ADP-ribosylation factor (ARF) GTPases and their regulatory
proteins have been implicated in the control of diverse biological
functions. Two main classes of positive regulatory elements for ARF
have been discovered so far: the large Sec7/Gea and the small
cytohesin/ARNO families, respectively. These proteins harbor
guanine–nucleotide-exchange factor (GEF) activity exerted by the
common Sec7 domain. The availability of a specific inhibitor, the
fungal metabolite brefeldin A, has enabled documentation of the
involvement of the large GEFs in vesicle transport. However, because of
the lack of such tools, the biological roles of the small GEFs have
remained controversial. Here, we have selected a series of RNA aptamers
that specifically recognize the Sec7 domain of cytohesin 1. Some
aptamers inhibit guanine–nucleotide exchange on ARF1, thereby
preventing ARF activation in vitro. Among them, aptamer
M69 exhibited unexpected specificity for the small GEFs, because it
does not interact with or inhibit the GEF activity of the related
Gea2-Sec7 domain, a member of the class of large GEFs. The inhibitory
effect demonstrated in vitro clearly is observed as well
in vivo, based on the finding that M69 produces similar
results as a dominant-negative...
The largest biological fractionations of stable carbon isotopes
observed in nature occur during production of methane by methanogenic
archaea. These fractionations result in substantial (as much as
≈70‰) shifts in δ13C relative to the initial
substrate. We now report that a stable carbon isotopic fractionation of
comparable magnitude (up to 70‰) occurs during oxidation of methyl
halides by methylotrophic bacteria. We have demonstrated biological
fractionation with whole cells of three methylotrophs (strain IMB-1,
strain CC495, and strain MB2) and, to a lesser extent, with the
purified cobalamin-dependent methyltransferase enzyme obtained from
strain CC495. Thus, the genetic similarities recently reported between
methylotrophs, and methanogens with respect to their pathways for
C1-unit metabolism are also reflected in the carbon
isotopic fractionations achieved by these organisms. We found that only
part of the observed fractionation of carbon isotopes could be
accounted for by the activity of the corrinoid methyltransferase
enzyme, suggesting fractionation by enzymes further along the
degradation pathway. These observations are of potential biogeochemical
significance in the application of stable carbon isotope ratios to
constrain the tropospheric budgets for the ozone-depleting
The plant-derived steroid, digoxin, a specific inhibitor of Na,K-ATPase, has been used for centuries in the treatment of heart disease. Recent studies demonstrate the presence of a digoxin analog, ouabain, in mammalian tissue, but its biological role has not been elucidated. Here, we show in renal epithelial cells that ouabain, in doses causing only partial Na,K-ATPase inhibition, acts as a biological inducer of regular, low-frequency intracellular calcium ([Ca2+]i) oscillations that elicit activation of the transcription factor, NF-κB. Partial inhibition of Na,K-ATPase using low extracellular K+ and depolarization of cells did not have these effects. Incubation of cells in Ca2+-free media, inhibition of voltage-gated calcium channels, inositol triphosphate receptor antagonism, and redistribution of actin to a thick layer adjacent to the plasma membrane abolished [Ca2+]i oscillations, indicating that they were caused by a concerted action of inositol triphosphate receptors and capacitative calcium entry via plasma membrane channels. Blockade of ouabain-induced [Ca2+]i oscillations prevented activation of NF-κB. The results demonstrate a new mechanism for steroid signaling via plasma membrane receptors and underline a novel role for the steroid hormone...
Four-stranded guanine tetraplexes in RNA have been identified to be involved in crucial biological functions, such as dimerization of retroviral RNA, translational repression, and mRNA turnover. However, the structural basis for these biological processes is still largely unknown. Here we report the RNA tetraplex structure (UGGGGU)4 at ultra-high resolution (0.61 Å). The space group is P4212, and cell constants are a = b = 36.16 Å and c = 74.09 Å. The structure was solved by the multiple-wavelength anomalous dispersion method using a set of three-wavelength data of the isomorphous bromo derivative brUGGGGU and refined to 0.61-Å resolution. Each of the four strands in the asymmetric unit forms a parallel tetraplex with symmetry-related molecules. The tetraplex molecules stack on one another in opposite polarity (head-to-head or tail-to-tail) to form a pseudocontinuous column. All of the 5′-end uridines rotate around the backbone of G2, swing out, and form unique octaplexes with the neighboring G tetraplexes, whereas the 3′-end uridines are stacked-in and form uridine tetrads. All of the bases are anti, and the riboses are in the mixed C2′- and C3′-puckering mode. Strontium ions are observed in every other guanine tetrad plane...
Endogenous circadian clocks are robust regulators of physiology and behavior. Synchronization or entrainment of biological clocks to environmental time is adaptive and important for physiological homeostasis and for the proper timing of species-specific behaviors. We studied subjects in the laboratory for up to 55 days each to determine the ability to entrain the human clock to a weak circadian synchronizing stimulus [scheduled activity–rest cycle in very dim (≈1.5 lux in the angle of gaze) light–dark cycle] at three ≈24-h periods: 23.5, 24.0, and 24.6 h. These studies allowed us to test two competing hypotheses as to whether the period of the human circadian pacemaker is near to or much longer than 24 h. We report here that imposition of a sleep–wake schedule with exposure to the equivalent of candlelight during wakefulness and darkness during sleep is usually sufficient to maintain circadian entrainment to the 24-h day but not to a 23.5- or 24.6-h day. Our results demonstrate functionally that, in normally entrained sighted adults, the average intrinsic circadian period of the human biological clock is very close to 24 h. Either exposure to very dim light and/or the scheduled sleep–wake cycle itself can entrain this near-24-h intrinsic period of the human circadian pacemaker to the 24-h day.