scholarly journals Mutations in twinstar, a Drosophila gene encoding a cofilin/ADF homologue, result in defects in centrosome migration and cytokinesis.

1995 ◽  
Vol 131 (5) ◽  
pp. 1243-1259 ◽  
Author(s):  
K C Gunsalus ◽  
S Bonaccorsi ◽  
E Williams ◽  
F Verni ◽  
M Gatti ◽  
...  
Keyword(s):  
Essential Gene ◽  
Male Meiosis ◽  
P Element ◽  
Actin Dynamics ◽  
Northern Analysis ◽  
Contractile Ring ◽  
Protein Activity ◽  
Gene Encoding

We describe the phenotypic and molecular characterization of twinstar (tsr), an essential gene in Drosophila melanogaster. Two P-element induced alleles of tsr (tsr1 and tsr2) result in late larval or pupal lethality. Cytological examination of actively dividing tissues in these mutants reveals defects in cytokinesis in both mitotic (larval neuroblast) and meiotic (larval testis) cells. In addition, mutant spermatocytes show defects in aster migration and separation during prophase/prometaphase of both meiotic divisions. We have cloned the gene affected by these mutations and shown that it codes for a 17-kD protein in the cofilin/ADF family of small actin severing proteins. A cDNA for this gene has previously been described by Edwards et al. (1994). Northern analysis shows that the tsr gene is expressed throughout development, and that the tsr1 and tsr2 alleles are hypomorphs that accumulate decreased levels of tsr mRNA. These findings prompted us to examine actin behavior during male meiosis to visualize the effects of decreased twinstar protein activity on actin dynamics in vivo. Strikingly, both mutants exhibit abnormal accumulations of F-actin. Large actin aggregates are seen in association with centrosomes in mature primary spermatocytes. Later, during ana/telophase of both meiotic divisions, aberrantly large and misshaped structures appear at the site of contractile ring formation and fail to disassemble at the end of telophase, in contrast with wild-type. We discuss these results in terms of possible roles of the actin-based cytoskeleton in centrosome movement and in cytokinesis.

scholarly journals τ91, an Essential Subunit of Yeast Transcription Factor IIIC, Cooperates with τ138 in DNA Binding

1998 ◽  
Vol 18 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Rosalía Arrebola ◽  
Nathalie Manaud ◽  
Sophie Rozenfeld ◽  
Marie-Claude Marsolier ◽  
Olivier Lefebvre ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Essential Gene ◽  
Open Reading Frames ◽  
Class Iii ◽  
Gene Encoding ◽  
Promoter Recognition

ABSTRACT Transcription factor IIIC (TFIIIC) (or τ) is a large multisubunit and multifunctional factor required for transcription of all class III genes in Saccharomyces cerevisiae. It is responsible for promoter recognition and TFIIIB assembly. We report here the cloning and characterization of TFC6, an essential gene encoding the 91-kDa polypeptide, τ91, present in affinity-purified TFIIIC. τ91 has a predicted molecular mass of 74 kDa. It harbors a central cluster of His and Cys residues and has basic and acidic amino acid regions, but it shows no specific similarity to known proteins or predicted open reading frames. The TFIIIC subunit status of τ91 was established by the following biochemical and genetic evidence. Antibodies to τ91 bound TFIIIC-DNA complexes in gel shift assays; in vivo, a B block-deficient U6 RNA gene (SNR6) harboring GAL4 binding sites was reactivated by fusing the GAL4 DNA binding domain to τ91; and a point mutation in TFC6 (τ91-E330K) was found to suppress the thermosensitive phenotype of a tfc3-G349Emutant affected in the B block binding subunit (τ138). The suppressor mutation alleviated the DNA binding and transcription defects of mutant TFIIIC in vitro. These results indicated that τ91 cooperates with τ138 for DNA binding. Recombinant τ91 by itself did not interact with a tRNA gene, although it showed a strong affinity for single-stranded DNA.

Functional characterization of the Haemophilus influenzae 4.5S RNA

1998 ◽  
Vol 44 (1) ◽  
pp. 91-94
Author(s):  
G Scott Jenkins ◽  
Mark S Chandler ◽  
Pamela S Fink
Keyword(s):  
Haemophilus Influenzae ◽  
Functional Characterization ◽  
Coli Strain ◽  
Northern Analysis ◽  
Gene Encoding ◽  
E Coli ◽  
Functional Homolog ◽  
Polymerase Chain ◽  
4.5S Rna

The putative 4.5S RNA of Haemophilus influenzae was identified in the genome by computer analysis, amplified by the polymerase chain reaction, and cloned. We have determined that this putative 4.5S RNA will complement an Escherichia coli strain conditionally defective in 4.5S RNA production. The predicted secondary structures of the molecules were quite similar, but Northern analysis showed that the H. influenzae RNA was slightly larger than the E. coli RNA. The H. influenzae gene encoding this RNA is the functional homolog of the ffs gene in E. coli. Key words: ffs gene, complementation studies, small RNA, prokaryotic genetics.

scholarly journals Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P.

1991 ◽  
Vol 11 (2) ◽  
pp. 721-730 ◽  
Author(s):  
J Y Lee ◽  
C E Rohlman ◽  
L A Molony ◽  
D R Engelke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Essential Gene ◽  
Rnase P ◽  
Single Copy ◽  
Temperature Sensitive ◽  
Coding Region ◽  
Gene Encoding ◽  
Processing Steps ◽  
Potential Precursor

RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein. We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.

scholarly journals Isolation and Characterization of FecA- and FeoB-Mediated Iron Acquisition Systems of the Spirochete Leptospira biflexa by Random Insertional Mutagenesis

2005 ◽  
Vol 187 (9) ◽  
pp. 3249-3254 ◽  
Author(s):  
Hélène Louvel ◽  
Isabelle Saint Girons ◽  
Mathieu Picardeau
Keyword(s):  
Insertional Mutagenesis ◽  
Iron Acquisition ◽  
Transposon Mutagenesis ◽  
Ecological Niches ◽  
Acid Biosynthesis ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Transposon Mutants

ABSTRACT The specific mechanisms by which Leptospira spp. acquire iron from their ecological niches are unknown. A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. In this study, we have developed a system for random transposon mutagenesis of Leptospira biflexa using a mariner transposon, Himar1. To demonstrate the validity of Himar1 in vivo transposon mutagenesis in L. biflexa, a screen of mutants for clones impaired in amino acid biosynthesis was first performed, enabling the identification of tryptophan and glutamate auxotrophs. To investigate iron transporters, 2,000 L. biflexa transposon mutants were screened onto media with and without hemin, thus allowing the identification of five hemin-requiring mutants, and the putative genes responsible for this phenotype were identified. Three mutants had distinct insertions in a gene encoding a protein which shares homology with the TonB-dependent receptor FecA, involved in ferric citrate transport. We also identified two mutants with a Himar1 insertion into a feoB-like gene, the product of which is required for ferrous iron uptake in many bacterial organisms. Interestingly, the growth inhibition exhibited by the fecA and feoB mutants was relieved by deferoxamine, suggesting the presence of a ferric hydroxamate transporter. These results confirm the importance of iron for the growth of Leptospira and its ability to use multiple iron sources.

scholarly journals Mutations in the hrp48 gene, which encodes a Drosophila heterogeneous nuclear ribonucleoprotein particle protein, cause lethality and developmental defects and affect P-element third-intron splicing in vivo.

1997 ◽  
Vol 17 (12) ◽  
pp. 7260-7267 ◽  
Author(s):  
L E Hammond ◽  
D Z Rudner ◽  
R Kanaar ◽  
D C Rio
Keyword(s):  
Direct Evidence ◽  
Essential Gene ◽  
P Element ◽  
Developmental Defects ◽  
Nuclear Rna ◽  
Hnrnp Protein ◽  
Specific Alternative ◽  
Nuclear Ribonucleoprotein

The Drosophila melanogaster hnRNP protein, hrp48, is an abundant heterogeneous nuclear RNA-associated protein. Previous biochemical studies have implicated hrp48 as a component of a ribonucleoprotein complex involved in the regulation of the tissue-specific alternative splicing of the P-element third intron (IVS3). We have taken a genetic approach to analyzing the role of hrp48. Mutations in the hrp48 gene were identified and characterized. hrp48 is an essential gene. Hypomorphic mutations which reduce the level of hrp48 protein display developmental defects, including reduced numbers of ommatidia in the eye and morphological bristle abnormalities. Using a P-element third-intron reporter transgene, we found that reduced levels of hrp48 partially relieve IVS3 splicing inhibition in somatic cells. This is the first direct evidence that hrp48 plays a functional role in IVS3 splicing inhibition.

scholarly journals Characterization of the dnaG Locus inMycobacterium smegmatis Reveals Linkage of DNA Replication and Cell Division

1998 ◽  
Vol 180 (1) ◽  
pp. 65-72 ◽  
Author(s):  
Amy G. Klann ◽  
Aimee E. Belanger ◽  
Angelica Abanes-De Mello ◽  
Janice Y. Lee ◽  
Graham F. Hatfull
Keyword(s):  
Cell Division ◽  
Dna Replication ◽  
Mycobacterium Smegmatis ◽  
Genomic Library ◽  
Essential Gene ◽  
Coupled Processes ◽  
Temperature Sensitive ◽  
Macromolecular Synthesis ◽  
Gene Encoding

ABSTRACT We have isolated a UV-induced temperature-sensitive mutant ofMycobacterium smegmatis that fails to grow at 42°C and exhibits a filamentous phenotype following incubation at the nonpermissive temperature, reminiscent of a defect in cell division. Complementation of this mutant with an M. smegmatis genomic library and subsequent subcloning reveal that the defect lies within the M. smegmatis dnaG gene encoding DNA primase. Sequence analysis of the mutant dnaG allele reveals a substitution of proline for alanine at position 496. Thus, dnaG is an essential gene in M. smegmatis, and DNA replication and cell division are coupled processes in this species. Characterization of the sequences flanking the M. smegmatis dnaG gene shows that it is not part of the highly conserved macromolecular synthesis operon present in other eubacterial species but is part of an operon with a dgt gene encoding dGTPase. The organization of this operon is conserved in Mycobacterium tuberculosis andMycobacterium leprae, suggesting that regulation of DNA replication, transcription, and translation may be coordinated differently in the mycobacteria than in other bacteria.

scholarly journals The SR protein B52/SRp55 is essential for Drosophila development.

1994 ◽  
Vol 14 (11) ◽  
pp. 7499-7506 ◽  
Author(s):  
H Z Ring ◽  
J T Lis
Keyword(s):  
Germ Line ◽  
Essential Gene ◽  
Critical Role ◽  
Protein Family ◽  
P Element ◽  
Null Mutant ◽  
Sr Protein ◽  
Drosophila Development

B52, also called SRp55, is a 52-kDa member of the Drosophila SR protein family of general splicing factors. Escherichia coli-produced B52 is capable of both activating splicing and affecting the alternative splice site choice in human in vitro splicing reactions. Here we report the isolation of a B52 null mutant generated by remobilizing a P element residing near the B52 gene. The resulting deletion, B52(28), is confined to the B52 gene and its neighbor the Hrb87F gene. Second-instar larvae homozygous for the deletion are deficient in both B52 mRNA and protein. The B52 null mutant is lethal at the first- and second-instar larval stages. Germ line transformation of Drosophila flies with B52 genomic DNA rescues this lethality. Thus, B52 is an essential gene and has a critical role in Drosophila development. Larvae deficient in B52 are still capable of splicing the five endogenous pre-mRNAs tested here, including both constitutively and alternatively spliced genes. Therefore, B52 is not required for all splicing in vivo. This is the first in vivo deficiency analysis of a member of the SR protein family.

scholarly journals Identification and Characterization of the Bacillus thuringiensis phaZ Gene, Encoding New Intracellular Poly-3-Hydroxybutyrate Depolymerase

2006 ◽  
Vol 188 (21) ◽  
pp. 7592-7599 ◽  
Author(s):  
Chi-Ling Tseng ◽  
Hui-Ju Chen ◽  
Gwo-Chyuan Shaw
Keyword(s):  
Bacillus Thuringiensis ◽  
Wild Type ◽  
Gene Encoding ◽  
Significant Similarity ◽  
Phb Depolymerase ◽  
New Type ◽  
Identification And Characterization

ABSTRACTA gene that codes for a novel intracellular poly-3-hydroxybutyrate (PHB) depolymerase has now been identified in the genome ofBacillus thuringiensissubsp.israelensisATCC 35646. This gene, previously annotated as a hypothetical 3-oxoadipate enol-lactonase (PcaD) gene and now designatedphaZ, encodes a protein that shows no significant similarity with any known PHB depolymerase. Purified His-tagged PhaZ could efficiently degrade trypsin-activated native PHB granules as well as artificial amorphous PHB granules and release 3-hydroxybutyrate monomer as a hydrolytic product, but it could not hydrolyze denatured semicrystalline PHB. In contrast, purified His-tagged PcaD ofPseudomonas putidawas unable to degrade trypsin-activated native PHB granules and artificial amorphous PHB granules. TheB. thuringiensisPhaZ was inactive againstp-nitrophenylpalmitate, tributyrin, and triolein. Sonication supernatants of the wild-typeB. thuringiensiscells exhibited a PHB-hydrolyzing activity in vitro, whereas those prepared from aphaZmutant lost this activity. ThephaZmutant showed a higher PHB content than the wild type at late stationary phase of growth in a nutrient-rich medium, indicating that this PhaZ can function as a PHB depolymerase in vivo. PhaZ contains a lipase box-like sequence (G-W-S102-M-G) but lacks a signal peptide. A purified His-tagged S102A variant had lost the PHB-hydrolyzing activity. Taken together, these results indicate thatB. thuringiensisharbors a new type of intracellular PHB depolymerase.

scholarly journals Characterization of novel mutations at the Schizosaccharomyces pombe cdc2 regulatory phosphorylation site, tyrosine 15.

1996 ◽  
Vol 7 (10) ◽  
pp. 1573-1586 ◽  
Author(s):  
K L Gould ◽  
A Feoktistova
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Phosphorylation Site ◽  
Loss Of Function ◽  
Contractile Ring ◽  
Mutant Proteins ◽  
Regulatory Phosphorylation ◽  
Binding Loop

The cdc2 protein kinase family is regulated negatively by phosphorylation in the glycine ATP-binding loop at a conserved tyrosine residue, Y15, alone or in combination with T14 phosphorylation. In Schizosaccharomyces pombe and other systems, substitution of these residues with structurally similar but nonphosphorylatable amino acids has generated proteins (Y15F or T14AY15F) that behave as constitutively tyrosine-dephosphorylated proteins or threonine and tyrosine-dephosphorylated proteins. Here we report the characteristics of three additional mutants at Y15--Y15E, Y15S, and Y15T--in S. pombe cdc2p. All three mutant proteins are active in in vitro kinase assays, but are unable to functionally complement cdc2 loss-of-function mutations in vivo. Additionally, all three mutants are dominant negatives. A more detailed analysis of the Y15T mutant indicates that it can initiate chromosome condensation and F-actin contractile ring formation, but is unable to drive the reorganization of microtubules into a mitotic spindle.

Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE): Biochemical Features and Therapeutic Approaches

2007 ◽  
Vol 27 (1-3) ◽  
pp. 151-163 ◽  
Author(s):  
M. C. Lara ◽  
M. L. Valentino ◽  
J. Torres-Torronteras ◽  
M. Hirano ◽  
R. Martí
Keyword(s):  
Molecular Genetic ◽  
In Vivo Studies ◽  
Gene Encoding ◽  
Mitochondrial Neurogastrointestinal Encephalomyopathy ◽  
Mtdna Replication ◽  
Biochemical Features

Over the last 15 years, important research has expanded our knowledge of the clinical, molecular genetic, and biochemical features of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). The characterization of mitochondrial involvement in this disorder and the seminal determination of its genetic cause, have opened new possibilities for more detailed and deeper studies on the pathomechanisms in this progressive and fatal disease. It has been established that MNGIE is caused by mutations in the gene encoding thymidine phosphorylase (TP), which lead to absolute or nearly complete loss of its catalytic activity, producing systemic accumulations of its substrates, thymidine (dThd) and deoxyuridine (dUrd). Findings obtained from in vitro and in vivo studies indicate that the biochemical imbalances specifically impair mitochondrial DNA (mtDNA) replication, repair, or both leading to mitochondrial dysfunction. We have proposed that therapy for MNGIE should be aimed at reducing the concentrations of these toxic nucleosides to normal or nearly normal levels. The first treatment, allogeneic stem-cell transplantation (alloSCT) reported in 2006, produced a nearly full biochemical correction of the dThd and dUrd imbalances in blood. Clinical follow-up of this and other patients receiving alloSCT is necessary to determine whether this and other therapies based on a permanent restoration of TP will be effective treatment for MNGIE.

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