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PubMed1994

Incorporation of adenovirus into a ligand-based DNA carrier system results in retention of original receptor specificity and enhances targeted gene expression.

Adenovirus type 5 was modified by coupling an asialoglycoprotein-polylysine conjugate to the virus by reactions that activate carbohydrate residues. Wild-type virus modified in this manner had greatly decreased infectivity toward normally susceptible HeLa S3 (asialoglycoprotein receptor (-)) and SK Hep1 (asialoglycoprotein receptor (-)) cells leaving 91 and 86% viable, respectively, after 48 h. However, with Huh 7 (asialoglycoprotein receptor (+)) cells, modified virus retained its infectivity leaving only 19% of cells viable under identical conditions. Modified virus was complexed to DNA in the form of a plasmid, pSVHBV surf, containing the gene for hepatitis B surface antigen as a marker of gene expression. Huh 7, receptor (+), cells treated with modified wild type, and modified replication-defective d1312 virus complexed to DNA raised antigen levels by approximately 13- and 30-fold, respectively, compared with asialoglycoprotein-polylysine DNA complex alone. Competition with a large excess of an asialoglycoprotein blocked the enhancement by more than 95%. Using a beta-galactosidase marker gene, the number of cells transfected by modified virus was found to be 200-fold higher than complex alone. Yet, specificity was retained exclusively for asialoglycoprotein receptor-bearing cells. These data indicate that adenovirus can be chemically modified by coupling ligands resulting in targeted gene expression dictated specifically by receptor recognition of the attached ligand.

Adenoviruses, Human↗
PubMed2000

Language, discourse and the chronotope: applying literary theory to the narratives in health care.

Language, discourse and the chronotope: applying literary theory to the narratives in health care One of the most significant developments in nursing has been the recognition of the power of the narrative approach. Narrative knowing has evolved from both the patient and the nurse experience. In the case of the individual, it can be told through case history (clinical) and life story (theory-building). The life story of the individual as a patient represents him or her to the nurse and is in turn responded to through appropriate care. Alternatively, the life story of the individual as research participant represents him or her to the researcher and is in turn responded to through appropriate interpretation. The value of the narrative is that the 'otherness' of the teller/writer can be regarded by the listener/reader. There resides a major tension, however, whether in theory building or the practice setting: meaning What this paper argues, is that the emphasis placed on the subject to provide an authentic story of their life is misguided, whether in the care setting or in interpretive research. My contention is that we need to develop a less naive understanding of the complexities of the language with which the individual speaks. This can be achieved by adopting post-structural analyses of the spoken or written word, from both the originator's and recipient's experiences. By reconceptualizing such narratives as 'autobiographies' it is possible to borrow from the field of literary criticism a tool known as chronotopic analysis to perform this task.

Attitude of Health Personnel↗
PubMed2004

Origin of the classical complement pathway: Lamprey orthologue of mammalian C1q acts as a lectin.

The lectin complement pathway in innate immunity is closely related to the classical complement pathway in adaptive immunity, with respect to the structures and functions of their components. Both pathways are initiated by complexes consisting of collagenous proteins and serine proteases of the mannose-binding lectin (MBL)-associated serine protease (MASP)/C1r/C1s family. It has been speculated that the classical pathway emerged after the lectin pathway, and that the activation mechanism of the latter was partially conserved. The classical and lectin pathways can be traced back to at least cartilaginous fish and ascidian (urochordata), respectively. To elucidate the evolution of the complement system, we isolated and characterized a GlcNAc-binding lectin from sera of lamprey (agnathans), the most primitive vertebrate that lacks the classical pathway. Lamprey GlcNAc-binding lectin was an oligomer consisting of 24-kDa subunits. cDNA and phylogenetic analyses revealed that the lamprey GlcNAc-binding lectin is an orthologue of mammalian C1q, a collagenous subcomponent of the first component involved in binding to immunoglobulins in the classical pathway. Lamprey C1q copurified with MASP-A, a serine protease of the MASP/C1r/C1s family, which exhibited proteolytic activity against lamprey C3. Surface plasmon resonance analysis showed that lamprey C1q specifically bound to GlcNAc, but not various other carbohydrates tested. These results suggest that C1q may have emerged as a lectin and may have functioned as an initial recognition molecule of the complement system in innate immunity before the establishment of adaptive immunity such as immunoglobulins in the cartilaginous fish.

Amino Acid Sequence↗
PubMed2004

The long and short flavodoxins: I. The role of the differentiating loop in apoflavodoxin structure and FMN binding.

Flavodoxins are well known one-domain alpha/beta electron-transfer proteins that, according to the presence or absence of a approximately 20-residue loop splitting the fifth beta-strand of the central beta-sheet, have been classified in two groups: long and short-chain flavodoxins, respectively. Although the flavodoxins have been extensively used as models to study electron transfer, ligand binding, protein stability and folding issues, the role of the loop has not been investigated. We have constructed two shortened versions of the long-chain Anabaena flavodoxin in which the split beta-strand has been spliced to remove the original loop. The two variants have been carefully analyzed using various spectroscopic and hydrodynamic criteria, and one of them is clearly well folded, indicating that the long loop is a peripheral element of the structure of long flavodoxins. However, the removal of the loop (which is not in contact with the cofactor in the native structure) markedly decreases the affinity of the apoflavodoxin-FMN complex. This seems related to the fact that, in long flavodoxins, the adjacent tyrosine-bearing FMN binding loop (which is longer and thus more flexible than in short flavodoxins) is stabilized in its competent conformation by interactions with the excised loop. The modest role played by the long loop of long flavodoxins in the structure of these proteins (and in its conformational stability, see Lopez-Llano, J., Maldonado, S., Jain, S., Lostao, A., Godoy-Ruiz, R., Sanchez-Ruiz, Cortijo, M., Fernandez-Recio, J., and Sancho, J. (2004) J. Biol. Chem. 279, 47184-47191) opens the possibility that its conservation in so many species is related to a functional role yet to be discovered. In this respect, we discuss the possibility that the long loop is involved in the recognition of some flavodoxin partners. In addition, we report on a structural feature of flavodoxins that could indicate that the short flavodoxins derive from the long ones.

Anabaena↗
PubMed1996

Responses of mental health professionals to man-made trauma: the Israeli experience.

The reactions and responses of mental health professionals in the area of armed conflict is the focus of this paper. It examines the way the therapeutic community has dealt with the survivors of two catastrophes-the Holocaust and warfare. A parallel process of a gradual change of attitudes towards the survivors was observed: emotional detachment, lack of recognition in the early stages and, eventually, social acceptance and empathy. The origins of these attitudes will be discussed, and three explanations will be offered. Israel is a small, stress-ridden country that has known seven full-scale wars and countless hostilities during its 47 years of existence. Our national history over 2000 years has been beset with persecution, programs and deportations, culminating in the Nazi Holocaust. The establishment of the State of Israel brought with it the hope of a secure existence. Unfortunately, this has not been achieved, and Israel is a natural laboratory of war stress. The reactions and responses of mental health professionals in areas of armed conflict is the focus of this paper. Presented here will be this author's analysis of the way the Israeli society and the helping professions in Israel have dealt with two kinds of man-made catastrophic events: the Nazi Holocaust and seven Arab-Israeli wars. In these different events of human violence, a parallel process of a gradual change of attitude towards the survivors was observed. This remarkable parallel presents emotional detachment, lack of recognition and at times blaming the victims in the early stages and, eventually, social acceptance and empathy. The process of social change becomes complex when the agents of change are themselves members of the social entity undergoing the change. This paper shall demonstrate that therapists and mental health planners had considerable difficulties in transcending public attitudes toward survivors of the Holocaust and psychiatric casualties of the Israeli-Arab conflict. As a result, they were unable to treat properly those injured by trauma until certain social changes took place. This paper submits that the Israeli experience is not isolated and limited to our part of the globe. It represents a general, universal process, from which parallel processes in other countries and in other man-made trauma can be drawn.

Attitude of Health Personnel↗
PubMed1995

Methodological considerations in estimating speed of cognitive operations.

Individuals infected with Human Immunodeficiency Virus (HIV) and having cognitive impairment have been described as having slow mentation. Data supporting this proposition come from a variety of sources, including Sternberg's (1966) item recognition memory task. The procedure nominally provides an index of speed of mental operations, independent from input/output demands. However, since the original use of this procedure in the 1960s, advances in cognitive psychology have revealed many of its limitations. The purpose of the present study was to examine the psychometric characteristics of this task. Each participant performed the Sternberg item recognition task twice, 6 mo apart. The stability of the estimate of the slope of regression equations and for zero intercept ranged from excellent (r = .87) to poor (r = .30), and the data from many individual subjects could not be reliably modelled using multiple linear regression techniques. These data, as well as those from previous research, demonstrate the limited practical use of this task in clinical samples. Furthermore, as cognitive psychological theory has advanced in the past 30 yr, the conceptual underpinnings of the procedure have essentially evaporated.

AIDS Dementia Complex↗
PubMedDate not supplied

Seven years' experience with Integra as a reconstructive tool.

The bilayered dermal substitute Integra (Integra Life Sciences Corp., Plainsboro, NJ) was developed and has been widely used as primary coverage for excised acute burns. Our take has been slightly different, finding it most useful in the management of complex soft-tissue loss and threatened extremities as the result of tendon, joint, or bone exposure. Often tasked to fill significant volume loss, we have become adept at stacked multiple-layer applications. Creative use of this material has resulted in unexpected successes with distal limb salvage; the technique takes its place beside adjacent tissue transfer, composite flaps, and vascular pedicle flaps in our burn reconstructive practice. A prospective registry (44 patients) has been kept during the past 7 years that catalogs wounds with complex soft-tissue loss treated with Integra grafts. Many of these patients were at risk of extremity loss because of exposed tendons, joints, or bone. Integra was applied after 1:1 meshing. With profound soft-tissue defects, multiple layers of Integra were serially applied 1 to 2 weeks apart for reconstitution of soft-tissue contours. Local Integra graft infections were managed by silicone unroofing followed by topical sulfamylon liquid dressings. Wounds addressed included fourth-degree burns, necrotizing fasciitis, pit-viper envenomations, and total abdominal wall avulsion in one patient after being run over by a bus. Patients generally were free of pain from their wounds during the maturation phase of the Integra neodermis. Restoration of tissue contour was significantly better when using multiple layers for deep defects. Second and third layers of Integra were successfully applied after an abbreviated first graft maturation period of 7 days. Epithelial autografts on multilayer Integra applications frequently "ghosted"; they would auto-digest to dispersed cells followed subsequently by the reappearance of a confluent epithelial layer. Final grafted skin morphology over palmar and plantar surfaces assumed the type and fingerprint pattern of the original tissues. Infections were readily visible. Early recognition kept them to easily treated circumscribed areas, which did not jeopardize the entire wound. Lengths of stay were long (range, 2-246 days) but not significantly greater than with traditional techniques. The specific reconstructive use of Integra permitted unexpected salvage of several threatened extremities by protecting exposed tendons, bones and joints. Long-term histologic examination revealed unexpected persistence of Integra collagen. Large volume loss wounds benefited from the ability to fill voids with multilayered applications.

Abdominal Injuries↗
PubMed2005

Peptide charge state determination for low-resolution tandem mass spectra.

Mass spectrometry is a particularly useful technology for the rapid and robust identification of peptides and proteins in complex mixtures. Peptide sequences can be identified by correlating their observed tandem mass spectra (MS/MS) with theoretical spectra of peptides from a sequence database. Unfortunately, to perform this search the charge of the peptide must be known, and current chargestate- determination algorithms only discriminate singlyfrom multiply-charged spectra: distinguishing +2 from +3, for example, is unreliable. Thus, search software is forced to search multiply-charged spectra multiple times. To minimize this inefficiency, we present a support vector machine (SVM) that quickly and reliably classifies multiplycharged spectra as having either a +2 or +3 precursor peptide ion. By classifying multiply-charged spectra, we obtain a 40% reduction in search time while maintaining an average of 99% of peptide and 99% of protein identifications originally obtained from these spectra.

Algorithms↗
PubMed1989

Aging, selective attention, and feature integration.

This study used feature-integration theory as a means of determining the point in processing at which selective attention deficits originate. The theory posits an initial stage of processing in which features are registered in parallel and then a serial process in which features are conjoined to form complex stimuli. Performance of young and older adults on feature versus conjunction search is compared. Analyses of reaction times and error rates suggest that elderly adults in addition to young adults, can capitalize on the early parallel processing stage of visual information processing, and that age decrements in visual search arise as a result of the later, serial stage of processing. Analyses of a third, unconfounded, conjunction search condition reveal qualitatively similar modes of conjunction search in young and older adults. The contribution of age-related data limitations is found to be secondary to the contribution of age decrements in selective attention.

Adult↗
PubMed1986

The organization and development of the hippocampal mossy fiber system.

The anatomical organization and development of the hippocampal mossy fiber system has been reviewed with special reference to its organization in the common laboratory rat. The mossy fibers originate from the granule cells of the dentate granular layer and the few granule cells found scattered in the dentate molecular layer and hilus. Via a complex system of collaterals the mossy fibers terminate on several types of neurons in the hilus, e.g. the basket cells and the mossy cells. Upon leaving the hilus to pass into Ammon's horn, the mossy fibers converge to form a distinct band of fibers that terminates on the proximal part of the apical and basal dendrites of the pyramidal and basket cells of the regio inferior. In some mammalian species the mossy fibers may continue into the adjacent part of the regio superior. Despite differences in the number of granule cells and pyramidal cells at different septotemporal levels this organization is relatively uniform along the septotemporal extent of the hippocampus. During development the mossy fibers grow out in a sequential manner that matches the pattern of neurogenesis and the aggregation of the cells of origin. From the level at which they originate, the fibers diverge along the septotemporal axis in such a way that the oldest granule cells have the most extensive projections. The adult topographic organization, which is already apparent at the earliest developmental stages, is thus formed in a stepwise fashion. It is concluded that the organization of the hippocampal mossy fibers indicates that neuronal specificity should not be explained by cellular recognition alone, but rather as the cumulated product of the preceding sequence of developmental events that include neurogenesis, migration, aggregation and directed axonal outgrowth.

Animals↗
PubMed1987

Circuitry of the frontal association cortex and its relevance to dementia.

The prefrontal cortex reaches its peak size and complexity in the human brain where it occupies more than a quarter of the cerebral cortical surface. This paper reviews studies on that portion of the prefrontal cortex that is buried in and around the principal sulcus of macaque monkeys and corresponds to Brodmann's area 46 in man. Neuropsychological research as well as neurophysiology and 2-deoxyglucose metabolic mapping indicate that the principal sulcus is essential for regulation of motor behavior by internalized representations of visuo-spatial events. Conversely, the prefrontal cortex is unnecessary for behavior regulated by external stimuli, as is the case with many associative learning and recognition memory processes. Research over the past decade suggests that the principal sulcus accomplishes its regulatory functions by its interconnections with (1) the posterior parietal cortex which provides it with access to visuo-spatial data, (2) the parahippocampal gyrus and subiculum which allows information to be held 'on line' and deposited in long-term storage, and (3) motor centers such as the basal ganglia, deep layers of the superior colliculus and several premotor areas that control head, eye and hand movements. In addition, modulatory influences on prefrontal functions are exerted by (4) dopamine-, norepinephrine- and serotonin-containing fiber systems that originate in the brain stem and innervate the prefrontal cortex in a selective manner. Comparison between syndromes present in patients with a variety of diagnoses and human and nonhuman subjects with prefrontal injuries provides suggestive evidence that prefrontal dysfunction may underly the disordered thinking and abnormal social and affective responses found in many of these diseases. Accordingly, knowledge of the neural mechanisms underlying cognitive processing in nonhuman primates should be helpful in the analysis of the 'neurology' of many neurological and psychiatric illnesses.

Animals↗
PubMed1985

The molecular origin of DNA-drug specificity in netropsin and distamycin.

X-ray analysis of the complex of netropsin with the B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-BrC-G-C-G reveals that the antitumor antibiotic binds within the minor groove by displacing the water molecules of the spine of hydration. Netropsin amide NH furnish hydrogen bonds to bridge DNA adenine N-3 and thymine O-2 atoms occurring on adjacent base pairs and opposite helix strands, exactly as with the spine of hydration. The narrowness of the groove forces the netropsin molecule to sit symmetrically in the center, with its two pyrrole rings slightly non-coplanar so that each ring is parallel to the walls of its respective region of the groove. Drug binding neither unwinds nor elongates the double helix, but it does force open the minor groove by 0.5-2.0 A, and it bends back the helix axis by 8 degrees across the region of attachment. The netropsin molecule has an intrinsic twist that favors insertion into the minor groove of B-DNA, and it is given a small additional twist upon binding. The base specificity that makes netropsin bind preferentially to runs of four or more A X T base pairs is provided not by hydrogen bonding but by close van der Waals contacts between adenine C-2 hydrogens and CH groups on the pyrrole rings of the drug molecule. Substitution of one or more pyrroles by imidazole could permit recognition of G X C base pairs as well, and it could lead to a class of synthetic "lexitropsins," capable of reading any desired short sequence of DNA base pairs.

Base Sequence↗
PubMed2004

Synthetic creatinine receptor: imprinting of a Lewis acidic zinc(II)cyclen binding site to shape its molecular recognition selectivity.

Molecularly imprinted polymers (MIPs) from polymerizable Lewis acidic zinc(II)cyclen complexes and ethylene glycol dimethyl acrylate have been prepared. For the imprinting process the template molecule creatinine is reversibly coordinated to the zinc atom. The high strength of this interaction allows analyte binding to the MIP from aqueous solution with high affinity. Its pH dependence is used for controlled guest release with nearly quantitative analyte recovery rate. The binding capacity and selectivity profile of the MIP remains constant through several pH controlled binding and release cycles. MIPs missing a suitable metal binding site showed no significant affinity for thymine or creatinine. Flavin adsorbs nonspecifically to all polymers. The imprinting process reverses the binding selectivity of zinc(II)cyclen for creatinine and thymine from 1:34 in homogeneous solution to 3.5:1 in the MIP. Scatchard plot analysis of creatinine binding isotherms reveals uniform binding of the imprint, with fits indicating a one-site model; however, similar analysis for thymine indicate high and low affinity sites. This corresponds to unrestricted coordination sites freely accessible for thymine, e.g., at the polymer surface, and misshaped imprinted sites, which still can accommodate thymine. More than 50% of all binding sites exclusively bind creatinine and are not accessible to thymine. The binding properties of a copolymer of polymerizable zinc(II)cyclen and ethylene glycol dimethyl acrylate missing the creatinine template, which match the binding selectivity of the complex in solution, confirm that the origin of altered selectivities is the imprinting process. With binding ability at physiological pH, the MIPs are applicable for tasks in medicinal diagnostics or biotechnology. Imprinted zinc(II)cyclen complexes provide, like a metalloenzyme binding motif, high binding affinity by reversible coordination while the surrounding macromolecule determines binding selectivity.

Cations, Divalent↗
PubMed1997

A role for the cyclin-dependent kinase inhibitor p21 in the G1 cell cycle arrest mediated by the type I interferons.

Type I interferons (IFN), such as IFN-alpha, are potent antiproliferative and antitumor agents. IFN-tau, originally identified as a pregnancy recognition hormone, is a type I IFN that is related to IFN-alpha. We examine here the mechanism of the antiproliferative effects of IFN-alpha and IFN-tau in terms of their effects on intracellular events that regulate the cell cycle. Both IFN inhibited proliferation of the human Burkitt lymphoma cell line, Daudi, causing accumulation of cells in the G1 phase of the cell cycle. IFN-alpha was more effective than IFN-tau in this regard. Both IFN were found to inhibit the kinase activity of the cyclin-dependent kinase cdk2 in a manner that correlated with their relative abilities to cause cells to accumulate in the G1 phase of the cell cycle. Further, IFN treatment did not affect the expression of cdk2 protein, suggesting that the IFN modulated cdk2 activity through a cdk inhibitor. Consistent with this conclusion, both IFN induced the expression of the cyclin-dependent kinase inhibitor protein p21. The levels of p21 induced also correlated with the relative abilities of the IFN to inhibit cdk2 activity and to arrest cell growth in the G1 phase of the cell cycle. Moreover, following IFN treatment, increased levels of p21 were found complexed with cdk2, consistent with its role in the inhibition of cdk2 activity. These data suggest that p21-mediated inhibition of cdk2 activity plays an important role in the antiproliferative activity of type I IFN. The findings highlight interesting similarities between these cytokines and the products of tumor suppressor genes, such as p53, and may indicate a mechanism for the antitumor effects of the type I IFN.

Antineoplastic Agents↗
PubMed2006

Developmental regulation of prostacyclin synthase and prostacyclin receptors in the ovine uterus and conceptus during the peri-implantation period.

This study documents the expression of prostacyclin (PGI2) synthase (PTGIS) and PGI2 receptors in the trophoblast and uterus of the ewe at the time of maternal recognition of pregnancy (i.e. days 7, 9, 12, 14 and 17). The membrane receptor for PGI2 (PTGIR) and the nuclear receptors, i.e. peroxisome proliferator-activated receptors (PPAR) and their heterodimer partners the retinoid X receptors (RXR), were analysed. In the endometrium, PTGIS transcript and protein were expressed at day 9 of pregnancy and levels declined from days 12 to 17. Immunohistochemistry and in situ hybridization indicated that PTGIS was mainly located in the luminal epithelium of the endometrium. Endometrial PTGIR, PPARA, PPARG and RXRG expression was regulated during the peri-implantation period whereas PPARD, RXRA and RXRB were consistently expressed. In the trophoblast, PTGIS transcript levels rose as development progressed and peaked at day 17. PTGIR and PPARA transcripts peaked before day 12 and then declined and became nearly undetectable by day 17, whereas PPARD and PPARG transcript levels rose steadily from days 12 to 17. Because the PPARs and the RXRs display different expression profiles, we suggest that different heterodimers may form and support distinct functions as development proceeds. Our results also underline the importance of PTGIS and PPARD in the trophoblast and PTGIR in the uterus, suggesting that PGI2 is of both uterine and trophoblastic origin and is involved in a complex signalling pathway at around the time of implantation in the ewe.

Animals↗
PubMed1992

The nonequivalence of behavioral and mathematical equivalence.

Sidman and his colleagues derived behavioral tests for stimulus equivalence from the axiom in logic and mathematics that defines a relation of equivalence. The analogy has generated abundant research in which match-to-sample methods have been used almost exclusively to study interesting and complex stimulus control phenomena. It has also stimulated considerable discussion regarding interpretation of the analogy and speculation as to its validity and generality. This article reexamines the Sidman stimulus equivalence analogy in the context of a broader consideration of the mathematical axiom than was included in the original presentation of the analogy and some of the data that have accumulated in the interim. We propose that (a) mathematical and behavioral examples of equivalence relations differ substantially, (b) terminology is being used in ways that can lead to erroneous conclusions about the nature of the stimulus control that develops in stimulus equivalence experiments, and (c) complete analyses of equivalence and other types of stimulus-stimulus relations require more than a simple invocation of the analogy. Implications of our analysis for resolving current issues and prompting new research are discussed.

Animals↗
PubMed1994

Molecular evolution of SRP cycle components: functional implications.

Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein that targets a subset of nascent presecretory proteins to the endoplasmic reticulum membrane. We have considered the SRP cycle from the perspective of molecular evolution, using recently determined sequences of genes or cDNAs encoding homologs of SRP (7SL) RNA, the Srp54 protein (Srp54p), and the alpha subunit of the SRP receptor (SR alpha) from a broad spectrum of organisms, together with the remaining five polypeptides of mammalian SRP. Our analysis provides insight into the significance of structural variation in SRP RNA and identifies novel conserved motifs in protein components of this pathway. The lack of congruence between an established phylogenetic tree and size variation in 7SL homologs implies the occurrence of several independent events that eliminated more than half the sequence content of this RNA during bacterial evolution. The apparently non-essential structures are domain I, a tRNA-like element that is constant in archaea, varies in size among eucaryotes, and is generally missing in bacteria, and domain III, a tightly base-paired hairpin that is present in all eucaryotic and archeal SRP RNAs but is invariably absent in bacteria. Based on both structural and functional considerations, we propose that the conserved core of SRP consists minimally of the 54 kDa signal sequence-binding protein complexed with the loosely base-paired domain IV helix of SRP RNA, and is also likely to contain a homolog of the Srp68 protein. Comparative sequence analysis of the methionine-rich M domains from a diverse array of Srp54p homologs reveals an extended region of amino acid identity that resembles a recently identified RNA recognition motif. Multiple sequence alignment of the G domains of Srp54p and SR alpha homologs indicates that these two polypeptides exhibit significant similarity even outside the four GTPase consensus motifs, including a block of nine contiguous amino acids in a location analogous to the binding site of the guanine nucleotide dissociation stimulator (GDS) for E. coli EF-Tu. The conservation of this sequence, in combination with the results of earlier genetic and biochemical studies of the SRP cycle, leads us to hypothesize that a component of the Srp68/72p heterodimer serves as the GDS for both Srp54p and SR alpha. Using an iterative alignment procedure, we demonstrate similarity between Srp68p and sequence motifs conserved among GDS proteins for small Ras-related GTPases. The conservation of SRP cycle components in organisms from all three major branches of the phylogenetic tree suggests that this pathway for protein export is of ancient evolutionary origin.

Amino Acid Sequence↗
PubMed1998

Recruitment of the RNA polymerase II holoenzyme and its implications in gene regulation.

In yeast cells, interaction between a DNA-bound protein and a single component of the RNA polymerase II (poIII) holoenzyme is sufficient to recruit the latter to a promoter and thereby activate gene transcription. Here we review results which have suggested such a simple mechanism for how genes can be turned on. The series of experiments which eventually led to this model was originally instigated by studying gene expression in a yeast strain which carries a point mutation in Gal11, a component of the poIII holoenzyme. In cells containing this mutant protein termed Gall11P, a derivative of the transcriptional activator Gal4 devoid of any classical activating region is turned into a strong activator. This activating function acquired by an otherwise silent DNA-binding protein is solely due to a novel and fortuitous interaction between Gal11P and a fragment of the Gal4 dimerization region generated by the P mutation. The simplest explanation for these results is that tethering Gal11 to DNA recruits the poIII holoenzyme and, consequently, activates gene transcription. Transcription factors that are believed not to be integral part of the poIII holoenzyme but are nevertheless required for this instance of gene activation, e.g. the TATA-binding TFIID complex, may bind DNA cooperatively with the holoenzyme when recruited to a promoter, thus forming a complete poIII preinitiation complex. One prediction of this model is that recruitment of the entire poIII transcription complex and consequent gene activation can be achieved by tethering different components to DNA. Indeed, fusion of a DNA-binding domain to a variety of poIII holoenzyme components and TFIID subunits leads to activation of genes bearing the recognition site for the DNA-binding protein. These results imply that accessory factors, which are required to remove or modify nucleosomes do not need to be directly contacted by activators, but can rather be engaged in the activation process when the poIII complex is recruited to DNA. In fact, recruitment of the poIII holoenzyme suffices to remodel nucleosomes at the PHO5 promoter and presumably at many other promoters. Other events in the process of gene expression following recruitment of the transcription complex, e.g. initiation, promoter clearance, elongation and termination, could unravel as a consequence of the recruitment step and the formation of an active preinitiation complex on DNA. This view does not exclude the possibility that classical activators also act directly on chromatin remodeling and post-recruitment steps to regulate gene expression.

Animals↗