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At least 343 records · Page 19Linked to original sources
PubMed2005

Compound heterozygosity (G71R/R140H) in the lecithin:cholesterol acyltransferase (LCAT) gene results in an intermediate phenotype between LCAT-deficiency and fish-eye disease.

The esterification of free cholesterol (FC) in plasma, catalyzed by the enzyme lecithin:cholesterol acyltransferase (LCAT; EC 2.3.1.43), is a key process in lipoprotein metabolism. The resulting cholesteryl esters (CE) represent the main core lipids of low (LDL) and high density lipoproteins (HDL). Primary (familial) LCAT-deficiency (FLD) is a rare autosomal recessive genetic disease caused by the complete or near absence of LCAT activity. In fish-eye disease (FED), residual LCAT activity is still detectable. Here, we describe a 32-year-old patient with corneal opacity, very low LCAT activity, reduced amounts of CE (low HDL-cholesterol level), and elevated triglyceride (TG) values. The lipoprotein pattern was abnormal with regard to lipoprotein composition and concentration, but distinct lipoprotein classes were still present. Despite of typical features of glomerular proteinuria, creatinine clearance was normal. DNA sequencing and restiction fragment analyses revealed two separate mutations in the patient's LCAT gene: a previously described G to A transition in exon 4 converting Arg140 to His, inherited from his mother, and a novel G to C transversion in exon 2 converting Gly71 to Arg, inherited from his father, indicating that M.P. was a compound heterozygote. Determination of enzyme activities of recombinant LCAT proteins obtained upon transfection of COS-7 cells with plasmids containing G71R-LCAT or wild-type LCAT cDNA revealed very low alpha- and absence of beta-LCAT activity for the G71R mutant. The identification of the novel G71R LCAT mutation supports the proposed molecular model for the enzyme implying that the "lid" domain at residues 50-74 is involved in enzyme:substrate interaction. Our data are in line with the hypothesis that a key event in the etiology of FLD is the loss of distinct lipoprotein fractions.

Adult↗
PubMed2004

p24 proteins, intracellular trafficking, and behavior: Drosophila melanogaster provides insights and opportunities.

The p24 transmembrane proteins, also known as EMP24/GP25 (endomembrane protein precursor of 24kD (Schimmoller et al., 1995)) proteins, are components of coat protein (COP)-coated vesicles and are present in species as diverse as fungi, plants, flies, worms, and mammals, indicating that they have important conserved functions. Genetic, molecular, and biochemical characterization of these proteins and the loci that encode them has provided insights into their potential cellular roles, including postulated functions in vesicle cargo protein selection and sorting, COPI and COPII vesicle formation and budding, and quality control of proteins that mature through the secretory pathway. Recently, the first mutations in a Drosophila melanogaster p24 gene have been isolated and characterized. These alleles produce an interesting behavioral phenotype in females, affecting their ability to oviposit. This identification and mutant characterization of a p24 locus in Drosophila will pave the way for a better understanding of cell-type-specific functions and interactions among p24 proteins.

Alleles↗
PubMed2005

Identification of low frequency knockout mutants in Dictyostelium discoideum created by single or double homologous recombination.

Generation and characterization of knockout clones is a widely used approach to evaluate the specific function of a gene product in Dictyostelium discoideum. The mutant clones are generally obtained by double homologous recombination in the target gene. A frequent limitation to obtaining mutants is the low frequency of homologous recombination. Here we present an easy method to identify rare mutants, based on PCR analysis of pools of clones. This method also allows the isolation of functional knockout mutants created by a single homologous recombination event, which can be more frequent than a double recombination event.

Animals↗
PubMed2006

Identification of a constitutively active mutant of JAK3 by retroviral expression screening.

To identify transforming genes in acute myeloid leukemia (AML) we here constructed a retroviral cDNA expression library from an AML patient, and then used this library to infect a mouse cell line 32Dcl3-mCAT. cDNA inserts of the cell clones which proliferated in the presence of granulocyte colony-stimulating factor were derived from JAK3 encoding a JAK3 mutant with a valine-to-alanine substitution at codon 674 and two additional amino acid substitutions. The transforming activity of JAK3(V674A) was confirmed by its introduction into 32Dcl3-mCAT. Sequencing of the original JAK3 cDNA derived from the patient, however, failed to detect the V674A mutation.

Acute Disease↗
PubMed2005

Enhanced swarming of bacteria on agar plates containing the surfactant Tween 80.

A widely used method for quantifying swarming motility is the swarm plate assay. A significant increase in the motility halo size formed by Escherichia coli or Azospirillum brasilense was measured on Tween 80-containing agar relative to untreated agar. This improvement could benefit the identification of mutants in swarming motility.

Agar↗
PubMed2004

Identification of radiation-sensitive mutants in the Medaka, Oryzias latipes.

We screened populations of N-ethyl-N-nitrosourea (ENU)-mutagenized Medaka, (Oryzias latipes) for radiation-sensitive mutants to investigate the mechanism of genome stability induced by ionizing radiation in developing embryos. F3 embryos derived from male founders that were homozygous for induced the mutations were irradiated with gamma-rays at the organogenesis stage (48hpf) at a dose that did not cause malformation in wild-type embryos. We screened 2130 F2 pairs and identified three types of mutants with high incidence of radiation-induced curly tailed (ric) malformations using a low dose of irradiation. The homozygous strain from one of these mutants, ric1, which is highly fertile and easy to breed, was established and characterized related to gamma-irradiation response. The ric1 strain also showed higher incidence of malformation and lower hatchability compared to the wild-type CAB strain after gamma-irradiation at the morula and pre-early gastrula stages. We found that the decrease in hatching success after gamma-irradiation, depends on the maternal genotype at the ric1 locus. Terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling assays showed a high frequency of apoptosis in the ric1 embryos immediately after gamma-irradiation at the pre-early gastrula stage but apoptotic cells were not observed before midblastula transition (MBT). The neutral comet assay revealed that the ric1 mutant has a defect in the rapid repair of DNA double-strand breaks induced by gamma-rays. These results suggest that RIC1 is involved in the DNA double strand break repair in embryos from morula to organogenesis stages, and unrepaired DNA double strand breaks in ric1 trigger apoptosis after MBT. These results support the use of the ric1 strain for investigating various biological consequences of DNA double strand breaks in vivo and for sensitive monitoring of genotoxicity related to low dose radiation.

Animals↗
PubMed2005

Systemic signaling in the wound response.

In many plants, localized tissue damage elicits an array of systemic defense responses against herbivore attack. Progress in our understanding of the long-distance signaling events that control these responses has been aided by the identification of mutants that fail to mount systemic defenses in response to wounding. Grafting experiments conducted with various mutants of tomato indicate that systemic signaling requires both the biosynthesis of jasmonic acid at the site of wounding and the ability to perceive a jasmonate signal in remote tissues. These and other studies support the hypothesis that jasmonic acid regulates the production of, or acts as, a mobile wound signal. Following its synthesis in peroxisomes, further metabolism of jasmonic acid might enhance its stability, transport, or action in remote tissues. Recent studies in tomato suggest that the peptide signal systemin promotes long-distance defense responses by amplifying jasmonate production in vascular tissues.

Cyclopentanes↗
PubMed2004

Using the zebrafish model to study GATA transcription factors.

The zebrafish is an established animal model system that profits from the availability of strong experimental approaches in both genetics and embryology. As a vertebrate, zebrafish can be used to model many aspects of human development and disease. GATA transcription factors play important roles in the development of many organ systems, including those for hematopoietic, cardiovascular, reproductive, and gut-endoderm derived tissues. The six vertebrate GATA factors are highly conserved in zebrafish at the level of sequence, expression pattern, and function. The identification of mutants, establishment of transgenic GFP reporter fish, and the ease of performing loss- and gain-of-function experiments have all contributed new insight into our understanding of the regulation and function of GATA factors. We review recent advances toward this goal using the zebrafish system with a focus on hematopoiesis and cardiogenesis, and suggest how comparative genetics using the zebrafish genes might reveal core conserved properties, as well as changes in gene function that reflect different morphogenetic programs utilized by various vertebrate embryos.

Amino Acid Sequence↗
PubMed2000

Centrosome replication in somatic cells: the significance of G1 phase.

Proper cell division requires that the cell be able to form a bipolar spindle during mitosis. To achieve this, the centrosome must be replicated accurately during interphase. Our understanding of the mechanisms that allow centrosome doubling to be coordinated with other cell cycle progression processes is advancing at a rapid pace. Several different experimental systems have been developed that are allowing detailed studies of centrosome replication. For example, the identification of mutants in yeast that are unable to duplicate the SPB accurately during interphase has provided important insights concerning centrosome duplication. In addition, intact embryonic cells and extracts prepared from unfertilized eggs are powerful tools for investigating the molecular regulation of centrosome doubling during the cell cycle. Many of the observations from these embryonic systems are directly applicable to understanding centrosome doubling in somatic cells. Finally, transgenic mouse models and cultured mammalian cell systems have been developed for analyzing the regulation of centrosome doubling in cells with more complex cell cycles. As our knowledge of the cell cycle advances, particularly our understanding of the intricate series of events that must occur for somatic cells to traverse G1 phase, it should be possible to use the systems that have been developed to determine how the replication of the centrosome is coordinated with other cell cycle progression processes. The next few years should see rapid advances in our understanding of this critical cell biological process.

Animals↗
PubMed2000

Functional implications of protein isoprenylation in plants.

Plant protein isoprenylation has received considerable attention in the past decade. Since the initial discovery of isoprenylated plant proteins and their respective protein isoprenyltransferases, several research groups have endeavored to understand the physiological significance of this process in plants. Various experimental approaches, including inhibitor studies, systematic methods of protein identification, and mutant analyses in Arabidopsis thaliana, have enabled these groups to elucidate important roles for isoprenylated proteins in cell cycle control, signal transduction, cytoskeletal organization, and intracellular vesicle transport. This article reviews recent progress in understanding the functional implications of protein isoprenylation in plants.

Plant Physiological Phenomena↗
PubMed1997

Stage-specific expression of a selectable marker in Toxoplasma gondii permits selective inhibition of either tachyzoites or bradyzoites.

The establishment of culture conditions suitable for inducing differentiation of Toxoplasma gondii tachyzoites into parasites resembling the latent bradyzoite form has opened this important developmental transition to experimental analysis. In order to develop a genetic marker suitable for positive and negative selection during parasite differentiation. the T. gondii HXGPRT gene was placed under control of 5' flanking sequences derived from two bradyzoite-specific genes: BAG1 and LDH2. Random transgene integration at undefined genomic loci resulted in modest regulation (approximately 5-6-fold induction) above relatively high background levels (approximately 4% of wild-type controls). Integration of transgenes at a defined genomic position was achieved by targeting the uracil phosphoribosyl transferase (UPRT) locus using flanking homologous sequences and fluorouracil selection. This strategy was found to provide the added advantage of enhancing bradyzoite induction frequencies under conditions of pyrimidine starvation (low CO2). Constructs integrated in the direction of normal UPRT transcription exhibited moderate levels of inducibility, but transgenes integrated in the opposite direction were dramatically induced under differentiation conditions: 50-100-fold above the very low levels observed in tachyzoites (< 1% control). Positive selection (using mycophenolic acid) was shown to inhibit tachyzoites but not bradyzoites, while negative selection (using 8-azahypoxanthine) inhibited bradyzoites only. Stage-specific regulation of the HXGPRT selectable marker should permit genetic selections for the identification of mutants in the bradyzoite differentiation process.

Animals↗
PubMed2003

Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli.

We have developed a method called "gene gorging" to make precise mutations in the Escherichia coli genome at frequencies high enough (1-15%) to allow direct identification of mutants by PCR or other screen rather than by selection. Gene gorging begins by establishing a donor plasmid carrying the desired mutation in the target cell. This plasmid is linearized by in vivo expression of the meganuclease I-SceI, providing a DNA substrate for lambda Red mediated recombination. This results in efficient replacement of the wild type allele on the chromosome with the modified sequence. We demonstrate gene gorging by introducing amber stop codons into the genes xylA, melA, galK, fucI, citA, ybdO, and lacZ. To compliment this approach we developed an arabinose inducible amber suppressor tRNA. Controlled expression mediated by the suppressor was demonstrated for the lacZ and xylA amber mutants.

Anti-Bacterial Agents↗
PubMed2000

Multiple endocrine neoplasia type 1.

Combined clinical and laboratory investigations of MEN-1 have resulted in an increased understanding of this disorder, which may be inherited as an autosomal dominant condition. Defining the features of each disease manifestation in MEN-1 has improved patient management and treatment and has facilitated a screening protocol. Application of the techniques of molecular biology has enabled the identification of the gene causing MEN-1 and the detection of mutations in patients. The protein encoded by the MEN1 gene has been shown to be involved in the regulation of JunD-mediated transcription, but much still remains to be elucidated. Recent advances permit the identification of mutant MEN1 gene carriers who are at a high risk for this disorder and who require regular and biochemical screening to detect the development of endocrine tumors.

Adult↗
PubMed1996

Cell division: why daughters cannot be like their mothers.

A cell-fate determinant that segregates asymmetrically at cell division has been identified in budding yeast. Possible mechanisms for this asymmetric segregation are suggested by the identification of mutants in genes encoding cortically localized proteins.

Animals↗
PubMed2001

Genes controlling expression of defense responses in Arabidopsis--2001 status.

In the past two years, the focus of studies of the genes controlling expression of defense responses in Arabidopsis has shifted from the identification of mutants to gene isolation and the ordering of genes within branches of the signal transduction networks. It is now clear that gene-for-gene resistance can be mediated through at least three genetically distinguishable pathways. Additional genes affecting salicylic-acid-dependent signaling have been identified, and double-mutant analyses have begun to reveal the order in which they act. Genes required for jasmonic-acid-dependent signaling and for induced systemic resistance have also been identified.

Antifungal Agents↗
PubMed1998

Ovule and embryo development, apomixis and fertilization.

Genetic analyses, particularly in Arabidopsis, have led to the identification of mutants that define different steps of ovule ontogeny, pollen stigma interaction, pollen tube growth, and fertilization. Isolation of the genes defined by these mutations promises to lead to a molecular understanding of these processes. Mutants have also been obtained in which processes that are normally triggered by fertilization, such as endosperm formation and initiation of seed development, occur without fertilization. These mutants may illuminate apomixis, a process of seed development without fertilization extant in many plants.

Arabidopsis↗
PubMed2001

Early and multiple Ac transpositions in rice suitable for efficient insertional mutagenesis.

A GFP excision assay was developed to monitor the excision of Ac introduced into rice by Agrobacterium-mediated transformation. The presence of a strong double enhancer element of the CaMV 35S promoter adjacent to the Ac promoter induced very early excision, directly after transformation into the plant cell, exemplified by the absence of Ac in the T-DNA loci. Excision fingerprint analysis and characterization of transposition events from related regenerants revealed an inverse correlation between the number of excision events and transposed Ac copies, with single early excisions after transformation generating Ac amplification. New transpositions were generated at a frequency of 15-50% in different lines, yielding genotypes bearing multiple insertions, many of which were inherited in the progeny. The sequence of DNA flanking Ac in three representative lines provided a database of insertion tagged sites suitable for the identification of mutants of sequenced genes that can be examined for phenotypes in a reverse genetics strategy to elucidate gene function. Remarkably, two-thirds of Ac tagged sites showing homology to sequences in public databases were in predicted genes. A clear preference of transposon insertions in genes that are either predicted by protein coding capacity or by similarity to ESTs suggests that the efficiency of recovering knockout mutants of genes could be about three times higher than random. Linked Ac transposition, suitable for targeted tagging, was documented by segregation analysis of a crippled Ac element and by recovery of a set of six insertions in a contiguous sequence of 70 kb from chromosome 6 of rice.

Base Sequence↗