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At least 721 records · Page 40Linked to original sources
PubMed2005

Biochemical and genetic analyses of integrase-interacting proteins lens epithelium-derived growth factor (LEDGF)/p75 and hepatoma-derived growth factor related protein 2 (HRP2) in preintegration complex function and HIV-1 replication.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) functions in cells within the context of high molecular weight preintegration complexes (PICs). Lens epithelium-derived growth factor (LEDGF) transcriptional coactivator/p75 and hepatoma-derived growth factor related protein 2 (HRP2) tightly bind to HIV-1 IN and stimulate its integration activity in vitro. Here, we show that each recombinant host cell factor efficiently reconstitutes the in vitro activity of HIV-1 PICs disrupted for functional integration by pre-treatment with high concentrations of salt. Mutational analysis reveals that both the IN-binding and DNA-binding activities of LEDGF/p75 contribute to functional PIC reconstitution. We also investigate a role(s) for these proteins in HIV-1 infection by using short-interfering RNA. HIV-1 infection was essentially unaffected in HeLa-P4 cells depleted for LEDGF/p75, HRP2, or both proteins. We conclude that cells knocked-out for LEDGF/p75 and/or HRP2 will be useful genetic tools to address the roles of these host cell factors in HIV-1 replication.

Cell Line↗
PubMed2004

bullwinkle is required for epithelial morphogenesis during Drosophila oogenesis.

Many organs, such as the liver, neural tube, and lung, form by the precise remodeling of flat epithelial sheets into tubes. Here we investigate epithelial tubulogenesis in Drosophila melanogaster by examining the development of the dorsal respiratory appendages of the eggshell. We employ a culture system that permits confocal analysis of stage 10-14 egg chambers. Time-lapse imaging of GFP-Moesin-expressing egg chambers reveals three phases of morphogenesis: tube formation, anterior extension, and paddle maturation. The dorsal-appendage-forming cells, previously thought to represent a single cell fate, consist of two subpopulations, those forming the tube roof and those forming the tube floor. These two cell types exhibit distinct morphological and molecular features. Roof-forming cells constrict apically and express high levels of Broad protein. Floor cells lack Broad, express the rhomboid-lacZ marker, and form the floor by directed cell elongation. We examine the morphogenetic phenotype of the bullwinkle (bwk) mutant and identify defects in both roof and floor formation. Dorsal appendage formation is an excellent system in which cell biological, molecular, and genetic tools facilitate the study of epithelial morphogenesis.

Animals↗
PubMed2000

Biophysical and structural characterization of proton-translocating NADH-dehydrogenase (complex I) from the strictly aerobic yeast Yarrowia lipolytica.

Mitochondrial proton-translocating NADH-dehydrogenase (complex I) is one of the largest and most complicated membrane bound protein complexes. Despite its central role in eukaryotic oxidative phosphorylation and its involvement in a broad range of human disorders, little is known about its structure and function. Therefore, we have started to use the powerful genetic tools available for the strictly aerobic yeast Yarrowia lipolytica to study this respiratory chain enzyme. To establish Y. lipolytica as a model system for complex I, we purified and characterized the multisubunit enzyme from Y lipolytica and sequenced the nuclear genes coding for the seven central subunits of its peripheral part. Complex I from Y lipolytica is quite stable and could be isolated in a highly pure and monodisperse state. One binuclear and four tetranuclear iron-sulfur clusters, including N5, which was previously known only from mammalian mitochondria, were detected by EPR spectroscopy. Initial structural analysis by single particle electron microscopy in negative stain and ice shows complex I from Y. lipolytica as an L-shaped particle that does not exhibit a thin stalk between the peripheral and the membrane parts that has been observed in other systems.

Amino Acid Sequence↗
PubMed2001

Efficient large scale purification of his-tagged proton translocating NADH:ubiquinone oxidoreductase (complex I) from the strictly aerobic yeast Yarrowia lipolytica.

Proton translocating NADH:ubiquinone oxidoreductase (complex I) is the largest membrane bound multiprotein complex of the respiratory chain and the only one for which no molecular structure is available so far. Thus, information on the mechanism of this central enzyme of aerobic energy metabolism is still very limited. As a new approach to analyze complex I, we have recently established the strictly aerobic yeast Yarrowia lipolytica as a model system that offers a complete set of convenient genetic tools and contains a complex I that is stable after isolation. For crystallization of complex I and to obtain its molecular structure it is a prerequisite to prepare large amounts of highly pure enzyme. Here we present the construction of his-tagged complex I that for the first time allows efficient affinity purification. Our protocol recovers almost 40% of complex I present in Yarrowia mitochondrial membranes. Overall, 40-80 mg highly pure and homogeneous complex I can be obtained from 10 l of an overnight Y. lipolytica culture. After reconstitution into asolectin proteoliposomes, the purified enzyme exhibits full NADH:ubiquinone oxidoreductase activity, is fully sensitive to inhibition by quinone analogue inhibitors and capable of generating a proton-motive force.

Chromatography, Affinity↗
PubMed2002

Loss of the tailless gene affects forebrain development and emotional behavior.

We are studying the role of the evolutionarily conserved tlx gene in forebrain development in mice. Tlx is expressed in the ventricular zone that gives rise to neurons and glia of the forebrain. We have shown by mutating the tlx gene in mice, that in the absence of this transcription factor, mutant animals survive, but suffer specific anatomical defects in the limbic system. Because of these developmentally induced structural changes, mice with a mutation in the tlx gene can function, but exhibit extreme behavioral pathology. Mice show heightened aggressiveness, excitability, and poor cognition. In this article, we present a summary of our findings on the cellular and behavioral changes in the forebrain of mutant animals. We show that absence of the tlx gene leads to abnormal proliferation and differentiation of progenitor cells (PCs) in the forebrain from embryonic day 9 (E9). These abnormalities lead to hypoplasia of superficial cortical layers and subsets of GABAergic interneurons in the neocortex. We examined the behavior of mutant animals in three tests for anxiety/fear: the open field, the elevated plus maze, and fear conditioning. Mutant animals are less anxious and less fearful when assessed in the elevated plus and open-field paradigm. In addition, mutant animals do not condition to either the tone or the context in the fear-conditioning paradigm. These animals, therefore, provide a genetic tool to delineate structure/function relationships in defined regions of the brain and decipher how their disruption leads to behavioral abnormalities.

Aggression↗
PubMed2003

Future perspectives on malignant melanoma.

Emphasis for the treatment of melanoma should be shifted more to prevention and early diagnosis, because early melanoma may potentially be cured in most cases. Clinical trials are important to establish more effective adjuvant modalities against melanoma. Multifaceted aspects of micrometastasis, including differentiation of different clones with respect to the primary tumor, acquisition of adhesion molecules, and host interaction with the microscopic tumor, will shed new light on the biology and mechanism of early metastasis. New molecular and genetic tools may be used to dissect the mechanisms of lymphatic and hematogenous routes of metastasis. If such mechanisms can be understood, potential therapeutic maneuvers can be developed to prevent the process of micrometastasis.

Forecasting↗
PubMed2001

Clostridium botulinum and its neurotoxins: a metabolic and cellular perspective.

Clostridium botulinum comprises a diverse assemblage of clostridia that have the common property of producing a distinctive protein neurotoxin (BoNT) of similar pharmacological activity and extraordinary potency. BoNTs are produced in culture as molecular complexes consisting of BoNT, hemagglutinin (HA) and associated subcomponent proteins, nontoxic nonhemagglutinin (NTNH), and RNA. The genes encoding the protein components reside as a cluster on the chromosome, on bacteriophages, or on plasmids depending on the C. botulinum serotype. A gene BotR coding for a regulatory protein has been detected in toxin gene clusters from certain strains, as well as ORFs coding for uncharacterized components. The gene encoding TeNT is located on a large plasmid, and expression of the structural gene is controlled by the regulatory gene, TetR, located immediately upstream of the TeNT structural gene. TeNT is not known to be assembled into a protein/nucleic acid complex in culture. Cellular synthesis of BoNT and TeNT have been demonstrated to be positively regulated by the homologous proteins, BotR/A and TetR. Evidence suggests that negative regulatory factors and general control cascades such as those involved in nitrogen regulation and carbon catabolite repression also regulate synthesis of BoNTs. Neurotoxigenic clostridia have attracted considerable attention from scientists and clinicians during the past decade, and many excellent reviews are available on various aspects of these organisms and their neurotoxins. However, certain areas have not been well-studied, including metabolic regulation of toxin formation and genetic tools to study neurotoxigenic clostridia. These topics are the focus of this review.

Animals↗
PubMed2005

Genes and susceptibility to leishmaniasis.

Leishmania are digenetic protozoa which inhabit two highly specific hosts, the sandfly where they grow as motile, flagellated promastigotes in the gut, and the mammalian macrophage where they grow intracellularly as non-flagellated amastigotes. Leishmaniasis is the outcome of an evolutionary 'arms race' between the host's immune system and the parasite's evasion mechanisms which ensure survival and transmission in the population. The spectrum of disease manifestations and severity reflects the interaction between the genome of the host and that of the parasite, and the pathology is caused by a combination of host and parasite molecules. This chapter examines the genetic basis of host susceptibility to disease in humans and animal models. It describes the genetic tools used to map and identify susceptibility genes, and the lessons learned from murine and human cutaneous leishmaniasis.

Animals↗
PubMed2004

Experimental studies on sexual reproduction in diatoms.

The diatoms are the most speciose group of algae, having global ecological significance in the carbon and silicon cycles. They are almost unique among algae in being diplontic, and sexual reproduction is an obligate stage in the life cycle of most diatom species. It is unclear which are the principal factors that have fostered the evolutionary success of diatoms, but the unique life cycle (which is correlated with a curious wall structure and cell division mechanism) and size-dependent control of sexuality must have played an important part. Progress in understanding life cycle dynamics and their interrelationships with population biology and evolution will depend on how successfully sex can be initiated and manipulated experimentally, and our review provides a foundation for such work. Relevant data are scattered in time and come mostly from non-English publications, producing a false impression of diatoms as recalcitrant with respect to sexualization. Recent advances dependent on experimental cultures include the discovery of widespread heterothallism (including some complex types of behavior) in pennate diatoms, sexual diversity among clones of centric diatoms, more flexible size restitution strategies in centric diatoms than had been suspected, and use of reproductive isolation as a criterion in diatom taxonomy. We identify unsolved problems in the life history of diatoms, including aspects of sexualization, cell-cell recognition, sexual reproduction, and the development of the special expanding cell (the auxospore), which is crucial to morphogenesis in this group. Some of these problems are being addressed using modern molecular genetic tools, and progress will be facilitated when whole-genome sequences are published (e.g., for Thalassiosira pseudonana). Problems of culture maintenance and methods for manipulating the life cycle are discussed.

Cell Communication↗
PubMed2005

Ectopic targeting of substrates to the ubiquitin pathway.

Explanation of the physiological function of a cellular protein often requires targeted removal of that protein to reveal the associated biochemical and phenotypic alterations. A variety of technologies such as gene targeting and RNAi have been developed to abrogate the biosynthesis of the protein of interest. Recently, targeted protein degradation by harnessing the cellular ubiquitin-proteolytic machinery has emerged as a novel reverse genetic tool for loss-of-function studies. Targeted proteolysis operates at the posttranslational level to directly accelerate the turnover rate of the target protein and opens up new avenues for the dissection of complicated protein functions associated with posttranslational events, which are unattainable by a simple blocking of the biosynthesis of the target protein.

Base Sequence↗
PubMed1997

Cocaine reward and locomotor activity in C57BL/6J and 129/SvJ inbred mice and their F1 cross.

Large individual differences exist among mice in their behavioral responses to drugs of abuse, and many of these differences have a substantial genetic basis. The creation of new animal models using recombinant DNA technology has provided new genetic tools for assessing the role of specific candidate genes in drug response. This study presents a characterization of cocaine activation and reward in the two strains used most commonly for production of knockout mice, C57BL/6J and 129/SvJ, and their outcrossed F1 offspring. Using conditioned place preference, the study demonstrates that there are large strain differences in spontaneous locomotor activity and in the rewarding effects of cocaine. The 129/SvJ strain is hypoactive and is very sensitive to the locomotor activating effects of cocaine but does not develop cocaine-conditioned place preference under conditions that yield significant place preference in C57BL/6J mice. These phenotypes are not inherited in a simple additive manner, but rather the F1 generation resembles the C57BL/6J progenitor strain for a number of the behaviors examined.

Animals↗
PubMed2000

Regulation of primary carbon metabolism in Kluyveromyces lactis.

In the recent past, through advances in development of genetic tools, the budding yeast Kluyveromyces lactis has become a model system for studies on molecular physiology of so-called "Nonconventional Yeasts." The regulation of primary carbon metabolism in K. lactis differs markedly from Saccharomyces cerevisiae and reflects the dominance of respiration over fermentation typical for the majority of yeasts. The absence of aerobic ethanol formation in this class of yeasts represents a major advantage for the "cell factory" concept and large-scale production of heterologous proteins in K. lactis cells is being applied successfully. First insight into the molecular basis for the different regulatory strategies is beginning to emerge from comparative studies on S. cerevisiae and K. lactis. The absence of glucose repression of respiration, a high capacity of respiratory enzymes and a tight regulation of glucose uptake in K. lactis are key factors determining physiological differences to S. cerevisiae. A striking discrepancy exists between the conservation of regulatory factors and the lack of evidence for their functional significance in K. lactis. On the other hand, structurally conserved factors were identified in K. lactis in a new regulatory context. It seems that different physiological responses result from modified interactions of similar molecular modules.

Journal Article↗
PubMed1998

A genetic and multifactorial analysis of anxiety-related behaviours in Lewis and SHR intercrosses.

Lewis (LEW) and spontaneously hypertensive rats (SHR) have been shown to differ in a series of fear-related behaviours measured in different anxiety/emotionality tests. In the present study, we have investigated some of the genetic mechanisms underlying these differences. To this end, male and female rats from the two inbred strains were crossed to produce two parental (LEW and SHR), two F1 (LEW or SHR mother), and two F2 (LEW or SHR grandmother) groups. All rats were tested in the elevated plus-maze and in the open field, besides being characterised for systolic blood pressure (BP). LEW rats approached the open arms of the plus-maze and the central area of the open field less than SHRs. The two strains also differed in their BP (SHR > LEW). LEW/SHR differences were found to be due to direct effects of the genes, rather than to indirect maternal and grand-maternal effects. Central locomotion in the open field was shown to be the most heritable of all the traits considered herein. A factor analysis on the segregating F2 population produced three independent factors. The first one was associated to measures of anxiety from the elevated plus-maze, and the second to measures of locomotion in novel environments. Factor scores revealed that the parental strains differ in relation to the first but not to the second factor. This study demonstrates the usefulness of coupling genetic and multifactorial methods to investigate behavioural traits and it confirms LEW and SHR strains as an interesting genetic tool for the study of anxiety.

Analysis of Variance↗
PubMed2000

Isolation and characterization of Leishmania mutants defective in glycosomal protein import.

Kinetoplastid parasites contain a unique microbody organelle called the glycosome. Several important metabolic pathways found in the cytoplasm of higher eukaryotes are compartmentalized within the glycosome in these pathogens. This fundamental difference between the host and parasite has led to consideration of the glycosome as a potential chemotherapeutic target. The genetic basis of glycosome biogenesis is therefore of great interest. This report describes the isolation of multiple Leishmania mutant cell lines defective in glycosomal protein import, and the detailed characterization of three such lines. The mutants examined partially mislocalize a subset of glycosomal proteins to the cytosol yet retain wild-type numbers of glycosomes. One of the mutants has a mutation in the previously identified LdPEX2 (GIM1) gene. The other two mutants are demonstrated to contain cell-specific lesions in one or more genes distinct from PEX2. The identification of multiple genetically distinct mutants with defects in glycosome import provides an important genetic tool to facilitate the identification of genes involved in glycosome biogenesis.

Animals↗
PubMed2002

Construction of transcriptional and translational lacZ gene reporter plasmids for use in Streptococcus mutans.

Reporter genes have become standard genetic tools used to evaluate either the transcriptional or the translational activity associated with genes of interest, whose products cannot be easily assayed. The lacZ gene from Escherichia coli has been used very effectively to quantify such regulated activities in many different organisms. This report describes the construction of a pair of plasmids that may be used for either transcriptional or translational lacZ gene fusions in Streptococcus mutans. The translational E. coli beta-galactosidase gene (lacZ) fusion plasmid, pALH109, as well as the transcriptional lacZ gene fusion plasmid, pALH122, have been used successfully in S. mutans to measure the activity of various PTS genes. Both plasmids employ fusions with the E. coli lacZ gene that can be easily quantified using standard O-nitrophenyl-beta-D-galactopyranoside (ONPG) based enzyme assays or the more sensitive fluorometric assays using 4-methyl-umbelliferyl beta-D-galactopyranoside (MUG) as the enzyme substrate. Currently, there has been only one other report of the use of lacZ as a gene reporter in S. mutans. The plasmids described in this paper will provide new tools and techniques for the analysis of S. mutans gene regulation. In addition, we have compiled the complete nucleotide sequences of these gene reporter plasmids.

Base Sequence↗
PubMed2003

Characterization of an Escherichia coli O157:H7 marR mutant.

One mechanism for generation of multiple antibiotic resistance in enteric bacteria involves the global regulatory system marRAB. The operon, when induced, encodes for resistance to structurally and functionally unrelated antibiotics. Exposure of Escherichia coli O157:H7 to increasing levels of chloramphenicol (CHL) resulted in survival of mutants that were resistant to the inducing agent and to tetracycline (TET), nalidixic acid (NAL), and ciprofloxacin (CIP). A mutant (RU122) that lacks MarR, the transcriptional repressor of the multiple antibiotic resistance (mar) operon, served as a genetic tool to study the role of MarR in growth of E. coli O157:H7. No significant difference (P>0.05) was observed in growth curves of the wild-type or the mutant under the conditions examined (rich and minimal media, acidic conditions, and temperatures from 24 to 42 C). A preconditioned mutant (indRU122; cultured for 17 h in Luria-Bertani (LB) containing chloramphenicol and then examined) exhibited greater growth under all treatments tested compared to the mutant. marCRAB of E. coli O157:H7 was sequenced and compared to other known mar sequences to determine divergence from other bacteria (Salmonella, Klebsiella, and Enterobacter).

Base Sequence↗
PubMed2004

High efficiency transformation of Penicillium nalgiovense with integrative and autonomously replicating plasmids.

Penicillium nalgiovense is a filamentous fungus that is acquiring increasing biotechnological importance in the food industry due to its widespread use as starter culture for cured and fermented meat products. Strains of P. nalgiovense can be improved by genetic modification to remove the production of penicillin and other potentially hazardous secondary metabolites, to improve its capacity to control the growth of undesirable fungi and bacteria on the meat product, and other factors that contribute to the ripening of the product in order to get safer and better quality foods. Genetic manipulation of P. nalgiovense has been limited by the lack of molecular genetics tools that were available for this fungus, particularly for "self-cloning" avoiding the use of exogenous DNAs. In this article we describe a series of vectors, selectable markers and transformation methods that can be used for efficient transformation of P. nalgiovense, gene cloning and expression. A uridine auxotrophic P. nalgiovense mutant with an inactive pyrG gene has been isolated. The P. nalgiovense wild-type pyrG gene was cloned and sequenced, and vectors carrying the gene were shown to complement the pyrG mutant. Autonomously replicating plasmids carrying the AMA1 region from Aspergillus nidulans transformed P. nalgiovense very efficiently; these plasmids were shown to be maintained as stable extrachromosomal elements in P. nalgiovense and could be rescued in Escherichia coli. The mitotic stability of self-replicative AMA1 plasmids in P. nalgiovense was higher than that reported for Penicillium chrysogenum.

Biotechnology↗
PubMed1999

Experimental approaches to understanding virulence in toxoplasmosis.

Toxoplasma gondii is a widespread protozoan parasite that causes severe disease only in immunocompromised individuals. Equipped with excellent animal models and relatively advanced systems for genetics, T. gondii provides an excellent system for understanding pathogenesis. Resistance to toxoplasmosis is governed by rapid innate and adaptive immunity that is characterized by a Th1 type profile of cytokines. Despite this effective response, acute infections can cause considerable damage and the parasite effectively establishes a long-term chronic infection that predisposes the host to reactivation and provides a means of eventual transmission. This complex interaction is brought about by the differentiation of the parasite from a rapidly replicating, lytic form (known as the tachyzoite) to a slow-growing form (known as the bradyzoite) that gives rise to chronic infection. The population structure of T. gondii is remarkably clonal, consisting of just three predominant lineages that are geographically widespread and found in a variety of hosts including humans. Acute virulence is strongly associated with the type I genotype which exhibits an enhanced replication rate in vitro and higher tissue burdens in vivo relative to non-virulent lineages. The pathology associated with acute infection appears to be due to excessive production of acute inflammatory mediators, suggesting that disease is partly due to over-response of the host immune system. A combination of refined animal models and newly developed genetic tools for establishing the relative contribution of genes to pathogenesis will enable a comprehensive analysis of the molecular basis of virulence in toxoplasmosis.

Acute Disease↗