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At least 667 records · Page 37Linked to original sources
PubMed1999

Seeking a regulatory roadmap for heart morphogenesis.

Despite descriptive studies spanning centuries, we are still lacking an integrated concept of how molecular patterning information is developed in the heart, and how this information guides morphogenesis. With an increasing number of regionally-expressed cardiac genes and transgenes being identified, along with new animal models of cardiac dysmorphogenesis and an exciting array of genetic tools for further dissection, the need for an integrated morphogenetic concept is acute. Models invoking a linear array of cardiac segments are difficult to reconcile with the observation that those segments are evident only on the outer curvature of the heart tube. Molecular and anatomical evidence supports the view that chamber specification is achieved by interpretation of dorso-ventral (inner curvature/outer curvature), as well as anterior-posterior patterning information in the primary heart tube. In this essay, I examine some of the issues influencing and perhaps confusing our view of cardiac morphogenesis and briefly discuss regulatory genes in the context of an evolving morphogenetic model.

Animals↗
PubMed2025

Studying Mitotic Phosphorylation in Drosophila.

Mitosis is largely controlled by the reversible phosphorylation of effector proteins. The addition or removal of phosphate groups alters the activities of these proteins, resulting in changes in chromosome structure, cytoskeletal dynamics, nuclear envelope integrity, and other transformations that must occur as a cell progresses through mitosis. Drosophila has been instrumental in the elucidation of the molecular mechanisms of mitosis, which are mostly conserved among animals. In this model system, sophisticated genetic tools can be used to study mitosis in different tissues during development in vivo. Drosophila cell culture affords complementary possibilities. In this chapter, we present a phosphoproteomic protocol using Drosophila cell culture to identify phosphorylation sites that depend on mitotic kinases and phosphatases. We also provide protocols to dissect the roles of the identified sites in the regulation of protein interactions and localization during mitosis, using Drosophila embryos. We emphasize the advantages of the selected methods compared to possible alternatives in Drosophila or in other systems.

Animals↗
PubMed2004

Regulation of BMP and activin signaling in Drosophila.

Cytokines of the TGF-beta superfamily act through an evolutionarily conserved signaling pathway to elicit a diverse range of biological responses in vertebrates as well as invertebrates. Drosophila has proved to be a powerful system to unravel the profound complexities underlying the regulation of this superficially simple signaling system for two reasons--the availability of sophisticated genetic tools and the restricted number of core signaling components compared to vertebrates. A BMP signaling pathway in Drosophila that regulates growth, differentiation and morphogenesis of the embryo and the larva has been extensively characterized. This work has provided major insights into how gradients of secreted proteins can be established and maintained in vivo, allowing a single ligand to induce multiple cell fates rather than function as an on-off switch. More recently, an activin signaling pathway has also been delineated that is required for growth and neuronal function during development. This review provides an overview of TGF-beta signaling in Drosophila with emphasis on the extensive modulation of signaling activity both within and outside the cell, that enables ligands to trigger specific and context-dependent effects.

Activins↗
PubMed1994

Transposon mutagenesis of coryneform bacteria.

The Corynebacterium glutamicum insertion sequence IS31831 was used to construct two artificial transposons: Tn31831 and miniTn31831. The transposition vectors were based on a gram-negative replication origin and do not replicate in coryneform bacteria. Strain Brevibacterium flavum MJ233C was mutagenized by miniTn31831 at an efficiency of 4.3 x 10(4) mutants per microgram DNA. Transposon insertions occurred at different locations on the chromosome and produced a variety of mutants. Auxotrophs could be recovered at a frequency of approximately 0.2%. Transposition of IS31831 derivatives led not only to simple insertion, but also to cointegrate formation (5%). No multiple insertions were observed. Chromosomal loci of B. flavum corresponding to auxotrophic and pigmentation mutants could be rescued in Escherichia coli, demonstrating that these transposable elements are useful genetic tools for studying the biology of coryneform bacteria.

Base Sequence↗
PubMed1996

Genomic organization of lactic acid bacteria.

Current knowledge of the genomes of the lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus, and members of the genera Lactobacillus, Leuconostoc, Pediococcus and Carnobacterium, is reviewed. The genomes contain a chromosome within the size range of 1.8 to 3.4 Mbp. Plasmids are common in Lactococcus lactis (most strains carry 4-7 different plasmids), some of the lactobacilli and pediococci, but they are not frequently present in S. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus or the intestinal lactobacilli. Five IS elements have been found in L. lactis and most strains carry multiple copies of at least two of them; some strains also carry a 68-kbp conjugative transposon. IS elements have been found in the genera Lactobacillus and Leuconostoc, but not in S. thermophilus. Prophages are also a normal component of the L. lactis genome and lysogeny is common in the lactobacilli, however it appears to be rare in S. thermophilus. Physical and genetic maps for two L. lactis subsp. lactis strains, two L. lactis subsp. cremoris strains and S. thermophilus A054 have been constructed and each reveals the presence of six rrn operons clustered in less than 40% of the chromosome. The L. lactis subsp. cremoris MG1363 map contains 115 genetic loci and the S. thermophilus map has 35. The maps indicate significant plasticity in the L. lactis subsp. cremoris chromosome in the form of a number of inversions and translocations. The cause(s) of these rearrangements is (are) not known. A number of potentially powerful genetic tools designed to analyse the L. lactis genome have been constructed in recent years. These tools enable gene inactivation, gene replacement and gene recovery experiments to be readily carried out with this organism, and potentially with other lactic acid bacteria and Gram-positive bacteria. Integration vectors based on temperate phage attB sites and the random insertion of IS elements have also been developed for L. lactis and the intestinal lactobacilli. In addition, a L. lactis sex factor that mobilizes the chromosome in a manner reminiscent to that seen with Escherichia coli Hfr strains has been discovered and characterized. With the availability of this new technology, research into the genome of the lactic acid bacteria is poised to undertake a period of extremely rapid information accrual.

Bacteriophages↗
PubMed2005

DNA transposons in vertebrate functional genomics.

Genome sequences of many model organisms of developmental or agricultural importance are becoming available. The tremendous amount of sequence data is fuelling the next phases of challenging research: annotating all genes with functional information, and devising new ways for the experimental manipulation of vertebrate genomes. Transposable elements are known to be efficient carriers of foreign DNA into cells. Notably, members of the Tc1/mariner and the hAT transposon families retain their high transpositional activities in species other than their hosts. Indeed, several of these elements have been successfully used for transgenesis and insertional mutagenesis, expanding our abilities in genome manipulations in vertebrate model organisms. Transposon-based genetic tools can help scientists to understand mechanisms of embryonic development and pathogenesis, and will likely contribute to successful human gene therapy. We discuss the possibilities of transposon-based techniques in functional genomics, and review the latest results achieved by the most active DNA transposons in vertebrates. We put emphasis on the evolution and regulation of members of the best-characterized and most widely used Tc1/mariner family.

Animals↗
PubMed2003

Catabolism of hydroxyacids and biotechnological production of lactones by Yarrowia lipolytica.

The gamma- and delta-lactones of less than 12 carbons constitute a group of compounds of great interest to the flavour industry. It is possible to produce some of these lactones through biotechnology. For instance, gamma-decalactone can be obtained by biotransformation of methyl ricinoleate. Among the organisms used for this bioproduction, Yarrowia lipolytica is a yeast of choice. It is well adapted to growth on hydrophobic substrates, thanks to its efficient and numerous lipases, cytochrome P450, acyl-CoA oxidases and its ability to produce biosurfactants. Furthermore, genetic tools have been developed for its study. This review deals with the production of lactones by Y. lipolytica with special emphasis on the biotransformation of methyl ricinoleate to gamma-decalactone. When appropriate, information from the lipid metabolism of other yeast species is presented.

Biotechnology↗
PubMed1999

Characterization of IS1676 from Rhodococcus erythropolis SQ1.

To develop a transposable element-based system for mutagenesis in Rhodococcus, we used the sacB gene from Bacillus subtilis to isolate a novel transposable element, IS1676, from R. erythropolis SQ1. This 1693 bp insertion sequence is bounded by imperfect (10 out of 13 bp) inverted repeats and it creates 4 bp direct repeats upon insertion. Comparison of multiple insertion sites reveals a preference for the sequence 5'-(C/T)TA(A/G)-3' in the target site. IS1676 contains a single, large (1446 bp) open reading frame with coding potential for a protein of 482 amino acids. IS1676 may be similar to an ancestral transposable element that gave rise to repetitive sequences identified in clinical isolates of Mycobacterium kansasii. Derivatives of IS1676 should be useful for analysis of Rhodococcus strains, for which few other genetic tools are currently available.

Base Sequence↗
PubMed2004

A new Hansenula polymorpha HAP4 homologue which contains only the N-terminal conserved domain of the protein is fully functional in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the HAP transcriptional complex is involved in the fermentation-respiration shift. This complex is composed of four subunits. Three subunits are necessary for DNA-binding, whereas the Hap4p subunit, glucose-repressed, contains the transcriptional activation domain. Hap4p is the key regulator of the complex activity in response to carbon sources in S. cerevisiae. To date, no HAP4 homologue has been identified, except in Kluyveromyces lactis. Examination of these two HAP4 sequences led to the identification of two very short conserved peptides also identified in other yeasts. In the yeast Hansenula polymorpha, two possible HAP4 homologues have been found. Their deduced amino acid sequences are similar to the ScHap4p and KlHap4p proteins only in the N-terminal 16-amino-acid basic motif. Since molecular genetic tools exist and complete genome sequence is known for this yeast, we expressed one of these putative HpHap4 proteins in S. cerevisiae and showed that this protein is able to restore the growth defect of the S. cerevisiae hap4-deleted strain. A set of experiments was performed to confirm the functional homology of this new gene with ScHAP4. The discovery of a Hap4-regulatory protein in H. polymorpha with only the N-terminal conserved domain of the S. cerevisiae protein indicates that this domain may play a crucial role during evolution.

CCAAT-Binding Factor↗
PubMed2005

X-ray-induced deletion complexes in embryonic stem cells on mouse chromosome 15.

Chromosomal deletions have long been used as genetic tools in dissecting the functions of complex genomes, and new methodologies are still being developed to achieve the maximum coverage. In the mouse, where the chromosomal deletion coverage is far less extensive than that in Drosophila, substantial coverage of the genome with deletions is strongly desirable. This article reports the generation of three deletion complexes in the distal part of mouse Chromosome (Chr) 15. Chromosomal deletions were efficiently induced by X rays in embryonic stem (ES) cells around the Otoconin 90 (Oc 90), SRY-box-containing gene 10 (Sox 10), and carnitine palmitoyltransferase 1b (Cpt 1 b) loci. Deletions encompassing the Oc 90 and Sox 10 loci were transmitted to the offspring of the chimeric mice that were generated from deletion-bearing ES cells. Whereas deletion complexes encompassing the Sox 10 and the Cpt 1 b loci overlap each other, no overlap of the Oc 90 complex with the Sox 10 complex was found, possibly indicating the existence of a haploinsufficient gene located between Oc 90 and Sox 10. Deletion frequency and size induced by X rays depend on the selective locus, possibly reflecting the existence of haplolethal genes in the vicinity of these loci that yield fewer and smaller deletions. Deletions induced in ES cells by X rays vary in size and location of breakpoints, which makes them desirable for mapping and for functional genomics studies.

Animals↗
PubMed2005

Molecular biology of insect olfaction: recent progress and conceptual models.

Insects have an enormous impact on global public health as disease vectors and as agricultural enablers as well as pests and olfaction is an important sensory input to their behavior. As such it is of great value to understand the interplay of the molecular components of the olfactory system which, in addition to fostering a better understanding of insect neurobiology, may ultimately aid in devising novel intervention strategies to reduce disease transmission or crop damage. Since the first discovery of odorant receptors in vertebrates over a decade ago, much of our view on how the insect olfactory system might work has been derived from observations made in vertebrates and other invertebrates, such as lobsters or nematodes. Together with the advantages of a wide range of genetic tools, the identification of the first insect odorant receptors in Drosophila melanogaster in 1999 paved the way for rapid progress in unraveling the question of how olfactory signal transduction and processing occurs in the fruitfly. This review intends to summarize much of this progress and to point out some areas where advances can be expected in the near future.

Animals↗
PubMed2003

The medaka fish Tol2 transposable element can undergo excision in human and mouse cells.

Tol2 is an active DNA-based transposable element identified in the medaka fish, Oryzias latipes. Originating from a vertebrate and belonging to the hAT ( hobo/ Activator/ Tam3) transposable element family, featuring a wide distribution among organisms, Tol2 would be expected to be active if introduced into mammals. We, therefore, examined if excision, one part of the transposition reaction, can occur in human and mouse culture cells. A Tol2 clone was introduced into cells and, after incubation, recovered. PCR and sequencing analysis provided evidence for precise and near precise excision in these cells. Tol2 can thus be expected to serve as a material for developing a gene transfer vector and other genetic tools applicable to mammals. It was also suggested that an intact Tol2 element could retain autonomy as a transposable element in mammalian cells.

3T3 Cells↗
PubMed2002

Gene transfer and cloning of flanking chromosomal regions using the medaka fish Tol2 transposable element.

For the ultimate purpose of developing genetic tools using the medaka fish Tol2 transposable element, we examined whether it can transfer a marker gene into the fish genome and also be applied for cloning of chromosomal regions adjacent to insertion points. An internal region of Tol2 was removed and replaced with the green fluorescent protein (GFP) gene and a bacterial plasmid replication origin. This modified Tol2 clone was microinjected into fertilized eggs together with messenger RNA for the Tol2 transposase. The GFP gene was found to be integrated into chromosomes and transmitted to subsequent generations. Restriction enzyme digestion of genomic DNA of a transformant fish, followed by ligation and introduction into bacteria, produced a plasmid containing the entire element and flanking chromosomal regions. Sequencing analysis of this clone demonstrated transposition of the element in the germline of the first generation. Thus, the basic requirements for a gene transfer vector and gene tagging system were fulfilled.

Journal Article↗
PubMed2003

Development of cloning vectors and transformation methods for Amycolatopsis.

The genus Amycolatopsis is of industrial importance, as its species are known to produce commercial antibiotics. It belongs to the family Pseudonocardiaceae and has an eventful taxonomic history. Initially strains were identified as Streptomyces, then later as Nocardia. However, based on biochemical, morphological and molecular features, the genus Amycolatopsis, containing seventeen species, was created. The development of molecular genetic techniques for this group has been slow. The scarcity of molecular genetic tools including stable plasmids, antibiotic resistance markers, transposons, reporter genes, cloning vectors, and high efficiency transformation protocols has made progress slow, but efforts in the past decade have led to the development of cloning vectors and transformation methods for these organisms. Some of the cloning vectors have broad host range (pRL series) whereas others have limited host range (pMEA300 and pMEA100). The cloning vector pMEA300 has been completely sequenced, while only the minimal replicon (pA- rep) has been sequenced from pRL plasmids. Direct transformation of mycelia and electroporation are the most widely applicable methods for transforming species of Amycolatopsis. Conjugational transfer from Escherichia coli has been reported only in the species A. japonicum, and gene disruption and replacements using homologous recombination are now possible in some strains.

Actinomycetales↗
PubMed2025

Engineered Lactiplantibacillus plantarum and Levilactobacillus brevis utilizing ribonucleoprotein-mediated editing for inactivation of hemolysin gene.

Lactiplantibacillus plantarum and Levilactobacillus brevis are widely used probiotics with significant potential as chassis organisms for probiotic engineering. However, their bioengineering remains underdeveloped compared to that of other probiotic bacteria due to the limited availability of genetic tools. Although CRISPR-Cas systems have shown promise for genome editing in Lactobacillus species, strain- or site-specific targeting challenges must be overcome to enhance their broader applicability. This study aimed to develop a novel editing system with reduced dependency on plasmids and antibiotics in L. plantarum WCFS1, L. plantarum SPC 72 - 1 and L. brevis SPC-SNU 70 - 2 using a Cas9-gRNA ribonucleoprotein (RNP) complex. Although the hlyIII gene has been annotated as a hemolysin-related gene in several Lactobacillus genomes, no functional hemolytic activity has been definitively demonstrated to date. In this study, hlyIII was selected as a target to evaluate genome editing efficiency and to assess its potential relevance to strain safety. To construct ΔhlyIII strains, the RNP complex targeting hlyIII was separately transformed with recombinase RecE/T and double-stranded donor DNA. As a result, ΔhlyIII mutants were obtained under optimized electroporation conditions. Sequencing analysis revealed a 50 bp deletion and the introduction of a stop codon in hlyIII across all mutant strains. The hemolytic activity test showed a reduction in free hemoglobin levels in the ΔhlyIII strains compared to the wild type: 27.0%, 74.3%, and 5.0% in L. plantarum WCFS1, L. plantarum SPC 72 - 1, and L. brevis SPC-SNU 70 - 2, respectively. These results suggest strain-dependent differences in hemolytic activity and indicate that inactivation of hlyIII may contribute to reduced hemolysis, although further validation is needed to clarify its functional role. In conclusion, the hlyIII gene was successfully edited in L. plantarum and L. brevis using Cas9-gRNA ribonucleoprotein-mediated editing, demonstrating the feasibility of this genome editing platform for application in probiotic strains.

Gene Editing↗
PubMed2006

RNAi in embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells that can be isolated and grown in vitro from the inner cell mass of blastocysts. Their potential to differentiate into any cell of the body makes them a promising starting material for cell therapy. Much progress has been made in recent years to develop ES cell differentiation protocols employing cocktails of certain growth factors or by using cell-type-restricted promoters driving the expression of selection markers or fluorescent proteins. However, little is known about the molecular details underlying the earliest processes of mammalian development. Genetic tools that provide novel insight into these processes would be very helpful to gain a better molecular understanding and to design better differentiation protocols. Recently, RNAi has emerged as a powerful technology to perform loss-of-function studies in mammalian cells. This technology should be ideal to identify and study genes required for ES cell self-renewal and differentiation. Here, we review the recent advances and challenges of RNAi research in ES cells and we provide a perspective on possible applications to enhance our understanding of ES cell self-renewal and early differentiation.

Animals↗
PubMed1989

Genetic control of sexually dimorphic axon morphology in Drosophila sensory neurons.

The mechanism by which orderly axonal projections are formed during development remains an important and largely unsolved problem in neurobiology. It may be possible to examine the control of axon growth in Drosophila and take advantage of genetic tools to better understand the phenomenon. We show here that some gustatory axons in Drosophila are sexually dimorphic and that genes involved in sex determination control the anatomy of these axons. Both males and females possess gustatory receptors on their legs but males possess more of these receptors than females. More significantly, the axons of the male receptors usually cross the midline and they never do so in females, indicating a central zone of bilateral input in the male but not in the female nervous system. In chromosomal females, expressing a tra or Sxl mutation, the gustatory system is transformed toward the male phenotype. Mutant XX adults resemble normal males externally, because they have more gustatory receptors, and internally, because their axons cross the midline. Gynandromorphs show that the sex of the sensory neuron, and apparently not the central nervous system, controls the growth of the axons. We conclude that the anatomical site of control for this dimorphism is the gustatory neurons.

Animals↗
PubMed1994

Cloning, sequencing and expression of stress genes from the ethanol-producing bacterium Zymomonas mobilis: the groESL operon.

Zymomonas mobilis is unique among bacteria in its ability to produce high levels of ethanol (EtOH) during fermentation. Elevated EtOH concentration, like elevated temperature, is a microbial stress and a universal inducer of stress proteins. For Z. mobilis, exposure to high levels of EtOH represents a natural stress. By using a simple strategy which combines the genetic tools of Escherichia coli and Bacillus subtilis, we have cloned genes encoding two of the most abundant stress proteins in Z. mobilis, GroES and GroEL. Both genes were expressed at high levels in E. coli. Despite the unique environment (EtOH concentrations of above 10%) in which the translated products from these genes function in Z. mobilis, the amino-acid sequences encoded were remarkably similar to their homologues from bacteria which are not known to accumulate EtOH. Two small regions were observed, however, which appear more similar to Saccharomyces cerevisiae hsp60 (groEL homologue) than to E. coli or the concensus. These may be related to EtOH tolerance.

Amino Acid Sequence↗