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At least 847 records · Page 47Linked to original sources
PubMed1996

RNA-mediated transformation in Aspergillus nidulans recovers gene functions lost by deletion or by point mutations.

This work presents the results on two different approaches of RNA-mediated transformation in A. nidulans: a) the receptor strain was an argB2 (III) mutant deficient in arginine (OTCase deficient), and b) the receptor was an A. nidulans mutant defective in nitrate reductase synthesis due to a deletion in the niaD gene (VIII). The analyses of the arg+ and the nia+ retrotransformants allowed an insight on the fate and inheritance of the newly acquired characteristics. The occurrence and the study of Gene Inactivation Mechanism (RIP-like) inactivating the expression of extra copies of genes ectopically scattered over the receptor genome, was a byproduct of this research. Retrotransformants were also used as RNA-donor for a second turn of retrotransformation of the argB and niaD receptor strains. Genetic analyses of the new retrotransformants proved that the retrotransformation ability is kept by the re-extracted RNA when used in a second round of transformation process. This is the best genetic evidence that the newly acquired genetic characteristics were cDNA inserted, precisely transcribed and expressed. These are the first in vivo evidences of genetic information transference mediated by homologous RNA in lower eukaryotes.

Aspergillus nidulans↗
PubMed2006

Expression of vasa (vas)-related genes in germ cells and specific interference with gene functions by double-stranded RNA in the monogenean, Neobenedenia girellae.

Neobenedenia girellae, a monogenean, is an important pathogen in marine cultured fish such as yellowtail and amberjack. An effective control method is required but none has yet been established. Aiming to establish a new control method by interfering with the gametogenesis of N. girellae, we focused on vasa (vas)-related genes that are expressed exclusively in the germline granules in Drosophila, Caenorhabditis elegans and other animals. Three vas-related genes (N. girellae vasa-like gene, Ngvlg1, Ngvlg2 and Ngvlg3) were isolated by PCR and Ngvlg1 and Ngvlg2 were shown to be expressed only in germ cells. We demonstrated that introduction of double-stranded Ngvlg1 or Ngvlg2 RNA by soaking resulted in partial or complete loss of germ cells. Moreover, the hatching rate of eggs from animals showing partial loss of germ cells decreased significantly. These results suggest that Ngvlg1 and Ngvlg2 are essential genes for germ cell quantity and quality. The possibility that a new control method can be developed by controlling gametogenesis of N. girellae was proven, because sterilised N. girellae could be produced.

Amino Acid Sequence↗
PubMed1996

Salmonella typhimurium cobalamin (vitamin B12) biosynthetic genes: functional studies in S. typhimurium and Escherichia coli.

In order to study the Salmonella typhimurium cobalamin biosynthetic pathway, the S. typhimurium cob operon was isolated and cloned into Escherichia coli. This approach has given the new host of the cob operon the ability to make cobalamins de novo, an ability that had probably been lost by this organism. In total, 20 genes of the S. typhimurium cob operon have been transferred into E. coli, and the resulting recombinant strains have been shown to produce up to 100 times more corrin than the parent S. typhimurium strain. These measurements have been performed with a quantitative cobalamin microbiological assay which is detailed in this work. As with S. typhimurium, cobalamin synthesis is only observed in the E. coli cobalamin-producing strains when they are grown under anaerobic conditions. Derivatives of the cobalamin-producing E. coli strains were constructed in which genes of the cob operon were inactivated. These strains, together with S. typhimurium cob mutants, have permitted the determination of the genes necessary for cobalamin production and classification of cbiD and cbiG as cobl genes. When grown in the absence of endogenous cobalt, the oxidized forms of precorrin-2 and precorrin-3, factor II and factor III, respectively, were found to accumulate in the cytosol of the corrin-producing E. coli. Together with the finding that S. typhimurium cbiL mutants are not complemented with the homologous Pseudomonas denitrificans gene, these results lend further credence to the theory that cobalt is required at an early stage in the biosynthesis of cobalamins in S. typhimurium.

Amino Acid Sequence↗
PubMed2025

Exploring diagnostic m6A regulators in primary open-angle glaucoma: insight from gene signature and possible mechanisms by which key genes function.

PURPOSE: The purpose of this study was to interrogate the potential role of N6-methyladenosine (m6A) regulators in the process of trabecular meshwork (TM) tissue damage in patients with primary open-angle glaucoma (POAG). METHODS: Firstly, the expression profile of m6A regulators in TM tissues of POAG patients was comprehensively analyzed by bioinformatics analysis; Plasmid transfection and siRNA gene interference were used to enhance or weaken the expression levels of YTHDC2 in human trabecular meshwork cells (HTMCs); Cell migration ability was detected by transwell chamber assay; Immunofluorescence staining assay was used to evaluate the expression of extracellular matrix (ECM) related proteins. RESULTS: Through the analysis of GSE27276 database, 5 m6A regulators with different expression in POAG were screened out. The results of random forest model showed that these 5 m6A regulators exhibited diagnostic potential and were characteristic genes of POAG. All POAG samples could be effectively divided into two groups based on the expression levels of these 5 hub m6A regulators. Immune cell infiltration analysis indicated that the levels of activated CD8+ T cells and regulatory T cells were different in the two subtypes. HTMC oxidative stress cell model and TGF-β2 stimulation cell model were further constructed to verify the expression of the aforementioned hub m6A regulators, and it was found that YTHDC2 mRNA showed the same expression trend in both models. The silencing of YTHDC2 enhanced the migration ability of HTMCs and increased the synthesis ability of ECM. However, when YTHDC2ΔYTH, which lacks the YTH domain, is overexpressed in HTMCs, there is no significant change in the ECM synthesis ability. CONCLUSIONS: The differentially expressed m6A regulators in TM tissues may serve as potential diagnostic biomarkers for POAG. And, in HTMCs, the expression level of YTHDC2 mRNA was changed under oxidative stress or TGF-β2 intervention, and then exerted its regulation on cell migration and ECM synthesis capability through m6A modification, which may be an important part of the disease process of POAG.

Humans↗
PubMed1998

The Y chromosomal fertility factor Threads in Drosophila hydei harbors a functional gene encoding an axonemal dynein beta heavy chain protein.

To understand the contradiction between megabase-sized lampbrush loops and putative protein encoding genes both associated with the loci of Y chromosomal fertility genes of Drosophila on the molecular level, we used PCR-mediated cloning to identify and isolate the cDNA sequence of the Y chromosomal Drosophila hydei gene DhDhc7(Y). Alignment of the sequences of the putative protein DhDhc7(Y) and the outer arm dynein beta heavy chain protein DYH2 of Tripneustes gratilla shows homology over the entire length of the protein chains. Therefore the proteins can be assumed to fulfill orthologous functions within the sperm tail axonemes of both species. Functional dynein beta heavy chain molecules, however, are necessary for the assembly and attachment of outer dynein arms within the sperm tail axoneme. Localization of DhDhc7(Y) to the fertility factor Threads, comprising at least 5.1 Mb of transcriptionally active repetitive DNA, results from an infertile Threads- mutant where large clusters of Threads specifically transcribed satellites and parts of DhDhc7(Y) encoding sequences are missing simultaneously. Consequently, the complete lack of the outer dynein arms in Threads- males most probably causes sperm immotility and hence infertility of the fly. Moreover, preliminary sequence analysis and several other features support the hypothesis that DhDhc7(Y) on the lampbrush loops Threads in D. hydei and Dhc-Yh3 on the lampbrush loops kl-5 in Drosophila melanogaster on the heterochromatic Y chromosome of both species might indeed code for orthologous dynein beta heavy chain proteins.

Amino Acid Sequence↗
PubMed1997

Advancement through mitosis requires rae1 gene function in fission yeast.

Growth of the rae1-1 mutant of Schizosaccharomyces pombe at restrictive temperature results in accumulation of poly(A)+ RNA in the nucleus and a cell cycle arrest at the G2/M boundary. We demonstrate here that rae1 function is required for a process other than mRNA export which is essential for advancement through mitosis. Cells lacking rae1 function arrest with elevated Cdc2p kinase levels at a step before the formation of a mitotic spindle and without separation of the spindle pole bodies. Rae1p was localized to the nuclear periphery, consistent with a role in nucleocytoplasmic trafficking, which could include protein import. We propose a model where rae1 functions in cell cycle progression through trafficking of proteins required for mitosis.

CDC2 Protein Kinase↗
PubMed1988

Requirement of the Escherichia coli dnaA gene function for integrative suppression of dnaA mutations by plasmid R 100-1.

The phenotype of Escherichia coli dnaA missense and nonsense mutations was integratively suppressed by plasmid R100-1. The suppressed strains, however, could not survive when the dnaA function was totally inactivated. This was demonstrated by the inability of replacing the dnaA allele in the suppressed strain by a dnaA::Tn10 insertion using phage P1-mediated transduction. When the intact dnaA+ allele was additionally supplied by a specialized transducing phage, lambda imm21 dnaA+, which integrated at the att lambda site on the E. coli chromosome, then the dnaA::Tn10 insertion, together with a delta oriC deletion, were able to be introduced into the suppressed strain. Thus, the mechanisms of dnaA function for oriC and for the replication origin of R100-1 may not be quite the same.

Bacterial Proteins↗
PubMed1971

Control of gene function in bacteriophage Tr. 3. Preventing the shutoff of early enzyme synthesis.

Synthesis of early T4 protein, which is normally shut off at 10 min after infection, continues until lysis when host cells have been preinfected with T3 sam(+). In host cells preinfected with T3 sam(-), synthesis of early enzymes is shut off as normal. Thus, S-adenosylmethionine is required for the turnoff of early T4 functions (at least when host cells have been preinfected with T3).

Aminohydrolases↗
PubMed1981

Ir gene function in an I-A subregion mutant B6.C-H-2bm12.

The B6.C-H2bm12 (bm 12) strain has a mutation in the I-A subregion of the murine H-2 complex and is characterized by a loss of serologically detected Ia antigens and a strong graft rejection and mixed lymphocyte response between parent and mutant. It was presumed that the mutation affected the Ia-1 gene and to determine the relationship of Ia antigens and Ir genes, the immune responses of mutant and parent were compared. The immune responses to poly(L-Tyr,LGlu)-poly(DLAla)--poly(LLys), poly(Phe,Glu)-poly(DLAla)--poly(LLys), and poly(His,Glu)-poly(DLAla)--poly(LLys) in parent and mutant were same, indicating the Ia-1 and the Ir genes for these antigens are not identical. By contrast, although C57BL/6 gave a good response, the mutant strain was unable to generate cytotoxic T lymphocytes to the male-specific H-Y antigen--a response under I-A subregion Ir gene control, which now must be considered to be the Ia-1 gene. In addition, complementary Ir genes in the H-2b haplotype for the H-Y immune response could be detected when the bm12 mutant was used.

Animals↗
PubMed1994

Multicopy tRNA genes functionally suppress mutations in yeast eIF-2 alpha kinase GCN2: evidence for separate pathways coupling GCN4 expression to unchanged tRNA.

GCN2 is a protein kinase that stimulates translation of GCN4 mRNA in amino acid-starved cells by phosphorylating the alpha subunit of translation initiation factor 2 (eIL-2). We isolated multicopy plasmids that overcome the defective derepression of GCN4 and its target genes caused by the leaky mutation gcn2-507. One class of plasmids contained tRNA(His) genes and conferred efficient suppression only when cells were starved for histidine; these plasmids suppressed a gcn2 deletion much less efficiently than they suppressed gcn2-507. This finding indicates that the reduction in GCN4 expression caused by gcn2-507 can be overcome by elevating tRNA(His) expression under conditions in which the excess tRNA cannot be fully aminoacylated. The second class of suppressor plasmids all carried the same gene encoding a mutant form of tRNA(Val) (AAC) with an A-to-G transition at the 3' encoded nucleotide, a mutation shown previously to reduce aminoacylation of tRNA(Val) in vitro. In contrast to the wild-type tRNA(His) genes, the mutant tRNA(Val) gene efficiently suppressed a gcn2 deletion, and this suppression was independent of the phosphorylation site on eIF-2 alpha (Ser-51). Overexpression of the mutant tRNA(Val) did, however, stimulate GCN4 expression at the translational level. We propose that the multicopy mutant tRNA(Val) construct leads to an accumulation of uncharged tRNA(Val) that derepresses GCN4 translation through a pathway that does not involve GCN2 or eIF-2 alpha phosphorylation. This GCN2-independent pathway was also stimulated to a lesser extent by the multicopy tRNA(His) constructs in histidine-deprived cells. Because the mutant tRNA(Val) exacerbated the slow-growth phenotype associated with eIF-2 alpha hyperphosphorylation by an activated GCN2c kinase, we suggest that the GCN2-independent derepression mechanism involves down-regulation of eIF-2 activity.

DNA-Binding Proteins↗
PubMed2006

Double-stranded DNA breaks and gene functions in recombination and meiosis.

Meiotic prophase I is a long and complex phase. Homologous recombination is an important process that occurs between homologous chromosomes during meiotic prophase I. Formation of chiasmata, which hold homologous chromosomes together until the metaphase I to anaphase I transition, is critical for proper chromosome segregation. Recent studies have suggested that the SPO11 proteins have conserved functions in a number of organisms in generating sites of double-stranded DNA breaks (DSBs) that are thought to be the starting points of homologous recombination. Processing of these sites of DSBs requires the function of RecA homologs, such as RAD51, DMC1, and others, as suggested by mutant studies; thus the failure to repair these meiotic DSBs results in abnormal chromosomal alternations, leading to disrupted meiosis. Recent discoveries on the functions of these RecA homologs have improved the understanding of the mechanisms underlying meiotic homologous recombination.

Animals↗
PubMed2003

NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis.

Reactive oxygen species (ROS) have been proposed to function as second messengers in abscisic acid (ABA) signaling in guard cells. However, the question whether ROS production is indeed required for ABA signal transduction in vivo has not yet been addressed, and the molecular mechanisms mediating ROS production during ABA signaling remain unknown. Here, we report identification of two partially redundant Arabidopsis guard cell-expressed NADPH oxidase catalytic subunit genes, AtrbohD and AtrbohF, in which gene disruption impairs ABA signaling. atrbohD/F double mutations impair ABA-induced stomatal closing, ABA promotion of ROS production, ABA-induced cytosolic Ca(2+) increases and ABA- activation of plasma membrane Ca(2+)-permeable channels in guard cells. Exogenous H(2)O(2) rescues both Ca(2+) channel activation and stomatal closing in atrbohD/F. ABA inhibition of seed germination and root elongation are impaired in atrbohD/F, suggesting more general roles for ROS and NADPH oxidases in ABA signaling. These data provide direct molecular genetic and cell biological evidence that ROS are rate-limiting second messengers in ABA signaling, and that the AtrbohD and AtrbohF NADPH oxidases function in guard cell ABA signal transduction.

Abscisic Acid↗
PubMed1995

Transcriptional regulation of human CYP2C genes: functional comparison of CYP2C9 and CYP2C18 promoter regions.

The cytochrome P4502C subfamily comprises a group of constitutive microsomal hemoproteins which are expressed primarily in liver. In humans, this subfamily is responsible for metabolism of a variety of therapeutic drugs such as warfarin, mephenytoin, omeprazole, and antiinflammatory drugs. In the present study, we analyzed the promoter activity of the 5'-flanking region of two human CYP2C genes, CYP2C9 and CYP2C18. The ability of the 2.2-kb 5'-flanking region of the CYP2C9 gene to direct expression of a luciferase reporter gene in HepG2 cells was 25 times greater than that of the 1.3-kb 5'-flanking region of CYP2C18. Deletional analysis of CYP2C9 indicated that the minimal promoter was located between the translation start site and nucleotide -155, and an HPF-1 domain consensus sequence was identified in this region. Gel shift analysis demonstrated that nuclear proteins from HepG2 cells had a high binding affinity for a 20-bp oligonucleotide containing the HPF-1 site of CYP2C9. Antiserum to rat HNF-4 supershifted this DNA--protein complex, and an oligonucleotide derived from an HNF-4 motif present in the human apolipoprotein CIII promoter competed for the supershifted complex. Cotransfection with an HNF-4 expression plasmid increased transcriptional activity of the CYP2C9 minimal promoter (approximately 2-fold) in HepG2 cells and elevated activity more substantially in nonhepatic NIH3T3 cells (26-fold) and Cos 1 cells (9-fold).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Probing Drosophila gene function by antisense RNA.

The conventional technique for assigning a particular genetic function to a cloned transcription unit has relied on the rescue of the mutant phenotype by germ line transformation. An alternative approach is to mimic a mutant phenotype by the use of antisense RNA injections to produce phenocopies. This approach has been successfully used to identify genes involved in early pattern forming processes in the Drosophila embryo. At the time when antisense RNA is injected, the embryo develops as a syncytium composed of about 5000 nuclei which share a common cytoplasm. The gene interactions required to establish the body plan occur before cellularization at the blastoderm stage. Thus the nuclei and their exported transcripts are accessible to the injected antisense RNA. The antisense RNA interferes with the endogenous RNA by an as yet unidentified mechanism. The extent of interference is only partial and produces phenocopies with characteristics of weak mutant alleles. In our lab and others, this approach has been successfully used to identify several genes required for normal Drosophila pattern formation.

Animals↗
PubMed2002

Using Drosophila melanogaster to uncover human disease gene function and potential drug target proteins.

The fruit fly Drosophila melanogaster is a powerful genetic model organism, which has been instrumental in the determination of essential developmental and neurological pathways conserved from invertebrates to humans. With the completion of both the human and Drosophila genomes, the revelation that we are more similar to this simple organism than previously suspected was realised. 75% of human genetic disease genes have clear homologues in the fly. By utilising an array of genetic tools available to disrupt or misexpress these proteins, it is now feasible to perform large-scale genetic screens in Drosophila to identify other members of a particular human genetic pathway. This review outlines some of the reasons Drosophila is a useful tool for the discovery of therapeutic targets, covers some of the tools available to manipulate this organism and discusses specific examples of how to use Drosophila as a genetic test tube for revealing proteins which act in a common pathway.

Animals↗
PubMed2000

A SAF binding site in the promoter region of human gamma-fibrinogen gene functions as an IL-6 response element.

Expression of fibrinogen is highly induced during inflammation, and such abnormal expression of this protein is considered as a major cardiovascular risk factor. IL-6 is one of the main mediators of abnormal expression of fibrinogen leading to the pathogenic conditions. Transient transfection and EMSA were performed to investigate the molecular mechanism of IL-6-induced gamma-fibrinogen gene expression in hepatic cells. Using progressively deleted 5' fragments of the gamma-fibrinogen promoter coupled to chloramphenicol acetyltransferase gene, an IL-6 responsive element located between positions -273 and -259 was identified. Mutation of this element abrogates IL-6 responsiveness of the gamma-fibrinogen promoter. Interaction of this promoter with a zinc finger transcription factor, serum amyloid A activating factor (SAF)-1, was demonstrated by EMSA. Furthermore, overexpression of wild-type SAF-1 in transfected liver cells can increase transcription of the gamma-fibrinogen promoter. These data show that transcription factor SAF-1 is involved in the regulation of IL-6-mediated induction of the human gamma-fibrinogen gene in liver cells.

Binding Sites↗
PubMed1991

Loss of a tumor suppressor gene function is correlated with downregulation of chondrocyte-specific collagen expression in Syrian hamster embryo cells.

We previously described the isolation of closely related, preneoplastic Syrian hamster cell lines that have retained (supB+) or lost (supB-) the ability to suppress the anchorage-independent growth and tumorigenicity of a sarcoma cell line (BP6T) in cell hybrids. In this report, we have used differential cDNA screening to clone several genes that are expressed in supB+ cells and downregulated in supB- cells. The nontumorigenic supB+ and supB- variants are advantageous for differential cDNA cloning because multiple independent cell lines differing in their tumor suppressor activity have been isolated. Differentially expressed cDNAs were isolated and placed into one of four groups based on DNA cross-hybridization. Representative cDNAs from Groups I and II, which were expressed at relatively high levels in two independently derived supB+ cell lines (DES4 and 10W) and downregulated in the supB- and tumor cell lines, were sequenced. The DNA and predicted amino acid sequences of these genes were found to be highly homologous to the chondrocyte-specific collagens type II and type IX. In contrast to the chondrocyte-specific collagens, another collagen isoform, collagen type I, was expressed at similar levels in both supB+ and supB- cells. These results suggest that carcinogen-induced immortalization selected for chondrocyte-like cell lines from the mixed embryo cell population. As these cells progressed toward tumorigenicity, the ability to express the chondrocyte differentiation markers was lost concomitantly with the ability to suppress the tumorigenicity of the BP6T sarcoma cell line. These results are consistent with the hypothesis that the supB+ tumor suppressor gene is involved in the regulation of differentiation. The identification of genes regulated by this suppressor gene may aid in its isolation.

Animals↗