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At least 955 records · Page 53Linked to original sources
PubMed2007

Identification of genes displaying differential expression in the nuc-2 mutant strain of the mold Neurospora crassa grown under phosphate starvation.

Subtractive hybridization was used to isolate transcripts up-regulated in the nuc-2 mutant strain of Neurospora crassa grown under phosphate starvation. Following differential screening, 66 cDNA clones of the total enriched were screened in a second round by reverse Northern hybridization. The 17 cDNA candidates displaying visual positive differential expression were sequenced, and functional grouping identified putative proteins possibly involved in diverse cellular processes as, for example, protein synthesis, signal transduction mechanisms, and transport facilitation. Four of them, confirmed by both virtual and Northern blot analyses, revealed genes involved in the initiation of mRNA translation that are significantly up-regulated in the nuc-2 mutant strain, which may be relevant to a further understanding of the molecular events involved in the phosphorus sensing in N. crassa.

Ankyrin Repeat↗
PubMed2004

Activation of the anticancer prodrugs cyclophosphamide and ifosfamide: identification of cytochrome P450 2B enzymes and site-specific mutants with improved enzyme kinetics.

Cyclophosphamide (CPA) and ifosfamide (IFA) are oxazaphosphorine anticancer prodrugs metabolized by two alternative cytochrome P450 (P450) pathways, drug activation by 4-hydroxylation and drug inactivation by N-dechloroethylation, which generates the neurotoxic and nephrotoxic byproduct chloroacetaldehyde. CPA and IFA metabolism catalyzed by P450s 2B1, 2B4, 2B5, and seven site-specific 2B1 mutants was studied in a reconstituted Escherichia coli expression system to identify residues that contribute to the unique activities and substrate specificities of these enzymes. The catalytic efficiency of CPA 4-hydroxylation by rat P450 2B1 was 10- to 35-fold higher than that of rabbit P450 2B4 or 2B5. With IFA, approximately 50% of metabolism proceeded via N-dechloroethylation for 2B1 and 2B4, whereas CPA N-dechloroethylation corresponded to only approximately 3% of total metabolism (2B1) or was absent (2B4, 2B5). Improved catalytic efficiency of CPA and IFA 4-hydroxylation was obtained upon substitution of 2B1 Ile-114 by Val, and replacement of Val-363 by Leu or Ile selectively suppressed CPA N-dechloroethylation >or=90%. P450 2B1-V367A, containing the Ala replacement found in 2B5, exhibited only approximately 10% of wild-type 2B1 activity for both substrates. Canine P450 2B11, which has Val-114, Leu-363, and Val-367, was therefore predicted to be a regioselective CPA 4-hydroxylase with high catalytic efficiency. Indeed, P450 2B11 was 7- to 8-fold more active as a CPA and IFA 4-hydroxylase than 2B1, exhibited a highly desirable low K(m) (80-160 microM), and catalyzed no CPA N-dechloroethylation. These findings provide insight into the role of specific P450 2B residues in oxazaphosphorine metabolism and pave the way for gene therapeutic applications using P450 enzymes with improved catalytic activity toward these anticancer prodrug substrates.

Animals↗
PubMed1995

Spontaneous deletion mutants of the Lactococcus lactis temperate bacteriophage BK5-T and localization of the BK5-T attP site.

Spontaneous deletion mutants of the temperate lactococcal bacteriophage BK5-T were obtained when the phage was grown vegetatively on the indicator strain Lactococcus lactis subsp. cremoris H2. One deletion mutant was unable to form stable lysogens, and analysis of this mutant led to the identification of the BK5-T attP site and the integrase gene (int). The core sequences of the BK5-T attP and host attB regions are conserved in a number of lactococcal phages and L. lactis strains.

Amino Acid Sequence↗
PubMed2002

Molecular analyses of the natural transformation machinery and identification of pilus structures in the extremely thermophilic bacterium Thermus thermophilus strain HB27.

Thermus thermophilus HB27, an extremely thermophilic bacterium, exhibits high competence for natural transformation. To identify genes of the natural transformation machinery of T. thermophilus HB27, we performed homology searches in the partially completed T. thermophilus genomic sequence for conserved competence genes. These analyses resulted in the detection of 28 open reading frames (ORFs) exhibiting significant similarities to known competence proteins of gram-negative and gram-positive bacteria. Disruption of 15 selected potential competence genes led to the identification of 8 noncompetent mutants and one transformation-deficient mutant with a 100-fold reduced transformation frequency. One competence protein is similar to DprA of Haemophilus influenzae, seven are similar to type IV pilus proteins of Pseudomonas aeruginosa or Neisseria gonorrhoeae (PilM, PilN, PilO, PilQ, PilF, PilC, PilD), and another deduced protein (PilW) is similar to a protein of unknown function in Deinococcus radiodurans R1. Analysis of the piliation phenotype of T. thermophilus HB27 revealed the presence of single pilus structures on the surface of the wild-type cells, whereas the noncompetent pil mutants of Thermus, with the exception of the pilF mutant, were devoid of pilus structures. These results suggest that pili and natural transformation in T. thermophilus HB27 are functionally linked.

Amino Acid Sequence↗
PubMed1972

Murein synthesis and identification of cell wall precursors of temperature-sensitive lysis mutants of Escherichia coli.

A group of temperature-sensitive lysis mutants of Escherichia coli K-12 was studied. Mutants impaired in the synthesis of uridine diphosphate-N-acetylmuramyl (UDP-MurNAc)-pentapeptide or in the synthesis of murein amino acids were found. Their rate of murein synthesis at the restrictive temperature was decreased. A large number of mutants did not differ from the parent strain with respect to the rate of murein synthesis and the precursor pattern. The behavior of these mutants is discussed. It was impossible to accumulate UDP-MurNAc-pentapeptide in E. coli by the antibiotics penicillin and vancomycin. The hypothesis is put forward that the amount of this murein precursor is regulated by feedback inhibition.

Alanine↗
PubMed1994

Identification of FtsW and characterization of a new ftsW division mutant of Escherichia coli.

The product of the ftsW gene has been identified as a polypeptide that, like the related RodA protein, shows anomalous mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FtsW is produced at low levels that can be increased by altering the translation initiation region of the mRNA. Overproduction of FtsW strongly inhibits cell growth. A new mutant allele, ftsW201, causes a temperature-dependent block in the initiation stage of cell division which is similar to the division block in ftsZ mutants. The block in initiation of division in the ftsW201 allele is shown to be independent of FtsZ or the FtsZ inhibitor, SulA. In addition, the ftsW201 mutant is hypersensitive to overproduction of the division initiation protein FtsZ at the permissive temperature. Our results suggest a role for FtsW in an early stage of division which may involve an interaction with FtsZ.

Alleles↗
PubMed1996

Cloning and characterization of two Serratia marcescens genes involved in core lipopolysaccharide biosynthesis.

Bacteriocin 28b from Serratia marcescens binds to Escherichia coli outer membrane proteins OmpA and OmpF and to lipopolysaccharide (LPS) core (J. Enfedaque, S. Ferrer, J. F. Guasch, J. Tomás, and M. Requé, Can. J. Microbiol. 42:19-26, 1996). A cosmid-based genomic library of S. marcescens was introduced into E. coli NM554, and clones were screened for bacteriocin 28b resistance phenotype. One clone conferring resistance to bacteriocin 28b and showing an altered LPS core mobility in polyacrylamide gel electrophoresis was found. Southern blot experiments using DNA fragments containing E. coli rfa genes as probes suggested that the recombinant cosmid contained S. marcescens genes involved in LPS core biosynthesis. Subcloning, isolation of subclones and Tn5tac1 insertion mutants, and sequencing allowed identification of two apparently cotranscribed genes. The deduced amino acid sequence from the upstream gene showed 80% amino acid identity to the KdtA protein from E. coli, suggesting that this gene codes for the 3-deoxy-manno-octulosonic acid transferase of S. marcescens. The downstream gene (kdtX) codes for a protein showing 20% amino acid identity to the Haemophilus influenzae kdtB gene product. The S. marcescens KdtX protein is unrelated to the KdtB protein of E. coli K-12. Expression of the kdtX gene from S. marcescens in E. coli confers resistance to bacteriocin 28b.

Amino Acid Sequence↗
PubMed2006

Precise identification of a human immunodeficiency virus type 1 antigen processing mutant.

Human immunodeficiency virus type 1 (HIV-1) evokes a strong immune response, but the virus persists. Polymorphisms within known antigenic sites result in loss of immune recognition and can be positively selected. Amino acid variation outside known HLA class I restricted epitopes can also enable immune escape by interfering with the processing of the optimal peptide antigen. However, the lack of precise rules dictating epitope generation and the enormous genetic diversity of HIV make prediction of processing mutants very difficult. Polymorphism E169D in HIV-1 reverse transcriptase (RT) is significantly associated with HLA-B*0702 in HIV-1-infected individuals. This polymorphism does not map within a known HLA-B*0702 epitope; instead, it is located five residues downstream of a HLA-B*0702-restricted epitope SPAIFQSSM (SM9). Here we investigate the association between E169D and HLA-B*0702 for immune escape via the SM9 epitope. We show that this single amino acid variation prevents the immune recognition of the flanked SM9 epitope by cytotoxic T cells through lack of generation of the epitope, which is a result of aberrant proteasomal cleavage. The E169D polymorphism also maps within and abrogates the recognition of an HLA-A*03-restricted RT epitope MR9. This study highlights the potential for using known statistical associations as indicators for viral escape but also the complexity involved in interpreting the immunological consequences of amino acid changes in HIV sequences.

Amino Acid Sequence↗
PubMed2001

Identification of a system that allows a Rhizobium tropici dctA mutant to grow on succinate, but not on other C4-dicarboxylates.

A defined insertion mutant of a gene encoding a homolog of the rhizobial C4-dicarboxylate permease (dctA) was constructed in Rhizobium tropici strain CIAT899. This mutant (GA1) was unable to grow on fumarate or malate; however, in contrast with other rhizobial dctA mutants, it retained a limited ability to grow on succinate with ammonia as a nitrogen source. Our results suggest the presence of a novel succinate-specific transport system in R. tropici. Biochemical characterization indicated that this alternative transport system in GAI is active and dependent on an energized membrane. It was also induced by succinate and aspartate, and was repressed by glucose and glycerol. Bean plants inoculated with GA1 showed a reduced nitrogen-fixing ability, achieving only 29% of the acetylene reduction activity determined in CIAT899 strain nodules, 33 days after inoculation. Also, bean plants inoculated with GA1 had reduced shoot dry weight compared with plants inoculated with the wild-type strain.

Amino Acid Sequence↗
PubMed2000

Courtship in Drosophila.

Courtship is a complex behavior in Drosophila that recruits a wide range of genes for its realization, including those concerning sex determination, ion channels, and circadian rhythms. Results from different experimental approaches-behavioral and genetic comparisons between species, analysis of mutants and mosaics, and identification of specific sensory stimuli-sketch the outlines of a set of pleiotropic genes acting on a distributed system in the brain to produce the species-specific sequence of responses and actions.

Animals↗
PubMed2004

Macromolecular interactions and ion transport in cystic fibrosis.

Cystic fibrosis (CF) is a genetic disease caused by autosomal recessive mutations of the CF transmembrane regulator, CFTR. CFTR functions in the plasma membrane of epithelial cells lining the lung, pancreas, liver, intestines, sweat duct, and the epididymis. The primary problem in CF is that mutations in CFTR affect its ability to be made, processed, and trafficked to the plasma membrane and/or its function as a Cl(-) channel and conductance regulator. Many proteins and processes normally interact with normal CFTR throughout its life cycle and mutant CFTR during the disease process. Understanding the function of these proteins and processes is expected to provide a clearer understanding of how normal CFTR is involved in salt movement and how mutant CFTR is handled by the cell and leads to the pathophysiology of CF. Recently, efforts to find therapies that correct defective CFTR have been intensifying. To facilitate our understanding of normal and mutant CFTR and the identification of new drug targets for developing novel therapies, a panel of experts was convened by the National Heart, Lung, and Blood Institute to explore the critical questions, challenges, and current opportunities to highlight new areas of research that would facilitate a integrated understanding of the processes and proteins that impact CFTR. The meeting highlighted the multiple pathways and interacting proteins involved in CFTR folding and biosynthesis, processing, and trafficking. A number of critical areas for future study were identified. Although these therapies are promising, a big question remains as to whether simply correcting defective CFTR will lead to significant improvement in patient health or whether the symptoms manifested in CF will require therapies in addition to those that target defective CFTR specifically.

Cell Membrane↗
PubMed1995

Identification, molecular characterization, and cellular studies of an apolipoprotein E mutant (E1) in three unrelated families with hyperlipidemia.

Remnants of triglyceride-rich lipoproteins accumulate in plasma of subjects with type III hyperlipoproteinemia (HLP) due to defective clearance by hepatic receptors. Although most subjects with type III HLP are homozygous for apolipoprotein (apo) E2 (arg158-->cys, R158C), a variant that binds defectively to cell surface receptors, some individuals with type III HLP have rare mutations of apo E. We identified six subjects from three families with type III HLP who had either an apo E3/1 or E4/1 phenotype by isoelectric focusing. Using DNA restriction isotyping with HhaI, all six subjects were determined to have only one apo E allele encoding cys158 and the other encoding arg158. Subsequently, digestion of polymerase chain reaction-amplified portions of exon 4 of the apo E gene with endonucleases HaeIII, TaqI, and Sau3AI demonstrated a second DNA variant that encoded a single amino acid substitution (gly127-->asp, G127D) due to a guanosine-to-adenosine nucleotide change resulting in the apo E1 isoform (G127D, R158C), which had arisen from a parent apo E2 allele. This mutation was confirmed with direct DNA sequencing. Incubation of very low density lipoprotein (VLDL) isolated from hyperlipidemic apo E1 subjects with J774 macrophages resulted in a 7- to 12-fold increase in cellular cholesterol ester compared with VLDL from apo E2/2 subjects. Although heterozygosity for apo E1 alone did not impair the interaction of VLDL with cellular receptors in vitro, its presence in subjects with type III HLP suggests that apo E1, perhaps in combination with secondary factors, may be causative for the dyslipidemia.

Adolescent↗
PubMed2003

Na,K-ATPase is essential for embryonic heart development in the zebrafish.

Na,K-ATPase is an essential gene maintaining electrochemical gradients across the plasma membrane. Although previous studies have intensively focused on the role of Na,K-ATPase in regulating cardiac function in the adults, little is known about the requirement for Na,K-ATPase during embryonic heart development. Here, we report the identification of a zebrafish mutant, heart and mind, which exhibits multiple cardiac defects, including the primitive heart tube extension abnormality, aberrant cardiomyocyte differentiation, and reduced heart rate and contractility. Molecular cloning reveals that the heart and mind lesion resides in the alpha1B1 isoform of Na,K-ATPase. Blocking Na,K-ATPase alpha1B1 activity by pharmacological means or by morpholino antisense oligonucleotides phenocopies the patterning and functional defects of heart and mind mutant hearts, suggesting crucial roles for Na,K-ATPase alpha1B1 in embryonic zebrafish hearts. In addition to alpha1B1, the Na,K-ATPase alpha2 isoform is required for embryonic cardiac patterning. Although the alpha1B1 and alpha2 isoforms share high degrees of similarities in their coding sequences, they have distinct roles in patterning zebrafish hearts. The phenotypes of heart and mind mutants can be rescued by supplementing alpha1B1, but not alpha2, mRNA to the mutant embryos, demonstrating that alpha1B1 and alpha2 are not functionally equivalent. Furthermore, instead of interfering with primitive heart tube formation or cardiac chamber differentiation, blocking the translation of Na,K-ATPase alpha2 isoform leads to cardiac laterality defects.

Animals↗
PubMed2005

Endoplasmic reticulum-associated protein quality control and degradation: screen for ERAD mutants after ethylmethane sulfonate mutagenesis.

Proteins destined for secretion in eukaryotic cells enter the endoplasmic reticulum (ER) in an unfolded state and are properly folded in this organelle and sent to their final destination. Misfolded or orphan proteins are retained in the ER by a quality control system, retrotranslocated into the cytosol and degraded. Soluble and membrane proteins were found to require a basic machinery for elimination. It is composed of (1) the E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligase) enzymes, which polyubiquitinate the substrate proteins during retrotranslocation; (2) the trimeric AAA-ATPase complex Cdc48-Ufd1-Npl4p, which liberates the polyubiquitinated proteins from the ER; and (3) the 26S proteasome, finally degrading the misfolded proteins. Additional components for degradation of soluble or membrane proteins may vary depending on the nature of malfolded proteins. It is therefore of utmost importance to gain insight into the different components of the ER protein quality control and degradation system required for the elimination of the substrate variety. Protein quality control of the ER and subsequent degradation are evolutionarily highly conserved from yeast to human. The yeast Saccharomyces cerevisiae is therefore an elegant model organism for a search of new components of the ER quality control and degradation machinery, because it is easily amenable to genetic and molecular biological experimentation. In this chapter, a genetic approach is presented, which leads to the isolation of mutants and to the identification of proteins involved in protein quality control and ER-associated degradation (ERAD). The method resides in ethylmethane sulfonate (EMS) mutagenesis of a yeast strain followed by screening for stabilization of soluble ERAD substrates, two mutated and consequently malfolded vacuolar enzymes, carboxypeptidase yscY (CPY*) and proteinase yscA (PrA*). Both malfolded proteins are retained in the ER lumen and become substrates of the ERAD machinery.

Endoplasmic Reticulum↗
PubMed2005

Endoplasmic reticulum-associated protein quality control and degradation: genome-wide screen for ERAD components.

In this chapter, a genetic approach is presented that leads to the isolation of mutants and to the identification of proteins involved in protein quality control and endoplasmic reticulum-associated degradation (ERAD). The method makes use of a genomic screen of a yeast deletion library (EUROSCARF). Transformation of each of the approx 5000 strains deleted in one nonvital gene each with a CPY* chimera containing CPY* C-terminally fused to a transmembrane domain and the cytosolic Leu2 protein (3-isopropylmalate dehydrogenase) constitutes the basic screening procedure. Because of a Leu2p deficiency in all deletion strains, cells can grow only when the CTL* chimera is present. As the CPY* module of CTL* will be recognized in ERAD-proficient cells, CTL* will be degraded and the strain is unable to grow. Therefore the absence of genes necessary for ER quality control and ERAD will allow cell growth and indicate the necessity of the respective gene for these processes.

3-Isopropylmalate Dehydrogenase↗
PubMed2006

Mutations in the inter-SH2 domain of the regulatory subunit of phosphoinositide 3-kinase: effects on catalytic subunit binding and holoenzyme function.

Class IA phosphoinositide 3-kinases (PI3Ks) represent a group of heterodimeric lipid kinases with important functions in cellular signal transduction. The regulatory p85 subunit constitutively binds to the catalytic p110 subunit and mediates the recruitment of the heterodimer to various membrane-localized proteins upon activation by a vast array of stimuli. The functional characterization of protein domains that mediate p85 function has been hampered by a lack of structural data. Therefore, we investigated a 35-aa region in the inter-SH2 domain of p85, reported to be necessary for binding of p110, by site-directed mutagenesis and evaluated the importance of individual amino acids for PI3K heterodimer formation. This approach led to the identification of an 11-aa region required for p110 binding in vitro and mesoderm induction during early Xenopus development in vivo. Further analyses revealed two pairs of hydrophobic amino acids within this region, which are particularly important for high-affinity intersubunit interaction. Thus, our data provide further insight into the molecular mechanisms of PI3K intersubunit interaction and led to the identification of new p85 mutant proteins with varying degrees of dominant-negative effects that will be helpful for future PI3K-related research.

Amino Acid Sequence↗
PubMed2006

Kunjin virus replicons: an RNA-based, non-cytopathic viral vector system for protein production, vaccine and gene therapy applications.

The application of viral vectors for gene expression and delivery is rapidly evolving, with several entering clinical trials. However, a number of issues, including safety, gene expression levels, cell selectivity and antivector immunity, are driving the search for new vector systems. A number of replicon-based vectors derived from positive-strand RNA viruses have recently been developed, and this paper reviews the current knowledge on the first flavivirus replicon system, which is based on the Australian flavivirus Kunjin (KUN). Like most replicon systems, KUN replicons can be delivered as DNA, RNA or virus-like particles, they replicate their RNA in the cytoplasm and direct prolonged high-level gene expression. However, unlike most alphavirus replicon systems, KUN replicons are non-cytopathic, with transfected cells able to divide, allowing the establishment of cell lines stably expressing replicon RNA and heterologous genes. As vaccine vectors KUN replicons can induce potent, long-lived, protective, immunogen-specific CD8+ T cell immunity, a feature potentially related to extended production of antigen and double-stranded RNA-induced 'danger signals'. The identification of KUN replicon mutants that induce increased levels of IFN-alpha/beta has also spawned investigation of KUN replicons for use in cancer gene therapy. The unique characteristics of KUN replicons may thus make them suitable for specific protein production, vaccine and gene therapy applications.

Animals↗
PubMed2000

MEN1 gene mutation analysis in Italian patients with multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN 1) is a familial syndrome characterized by parathyroid, enteropancreatic and pituitary tumors. The gene responsible for this syndrome is localized at chromosomal 11q13 region and DNA markers from this region cosegregate with the disease. The recent identification of the MEN1 gene, encoding for a protein termed menin of 610 amino acids, allowed mutational screening to be performed both in affected families and sporadic cases. To date many different heterozygous mutations, spreading across all the encoding sequence, have been identified in MEN 1 patients with no apparent mutational hot spots or genotype-phenotype correlation. To analyze the genetic alterations of the MEN1 gene occurring in Italian patients we performed mutational screening by Denaturant Gradient Gel Electrophoresis followed by sequencing of exons 2-10 of the MEN1 gene in 27 Italian MEN 1 families and in five sporadic cases. We identified 17 different heterozygous mutations in 60% of analyzed cases. Twelve of these mutations are novel. Two mutations each occurred twice in unrelated families but no evidence of genotype-phenotype correlation can be established for these families. The extension of genetic diagnosis to asymptomatic family members allowed the identification of 10 MEN1 mutant gene carriers, one newly described and nine previously detected by linkage analysis with DNA markers from the 11q13 region. Our findings add new information to the diversity of mutations occurring in the MEN1 gene and confirm that the mutational screening of MEN 1 is a useful approach to detect individuals at higher risk of developing MEN 1-associated tumors.

DNA Mutational Analysis↗