scholarly journals Ornithine Decarboxylase Knockout in Tapesia yallundae Abolishes Infection Plaque Formation In Vitro but Does Not Reduce Virulence Toward Wheat

2001 ◽  
Vol 14 (11) ◽  
pp. 1303-1311 ◽  
Author(s):  
E. Mueller ◽  
A. Bailey ◽  
A. Corran ◽  
A. J. Michael ◽  
P. Bowyer
Keyword(s):  
Ornithine Decarboxylase ◽  
Genomic Library ◽  
Normal Growth ◽  
Gene Replacement ◽  
Tapesia Yallundae ◽  
Suitable Target ◽  
Targeted Gene Replacement ◽  
Polymerase Chain ◽  
In Vitro Induction

A knockout strain of Tapesia yallundae lacking the single ornithine decarboxylase (ODC) allele has been created by targeted gene replacement. A central region of the ODC gene was isolated by polymerase chain reaction with degenerate oligonucleotides and used to probe a lambda genomic library. The gene was sequenced, and the encoded ODC protein sequence was shown to be similar to those from other fungi. The functionality of the T. yallundae ODC was confirmed by complementation of an Aspergillus nidulans mutant (puA) strain devoid of ODC activity, restoring growth in the absence of exogenous polyamines. Transformation-mediated gene replacement was used to create strains that were auxotrophic for putrescine and lack ODC coding sequences. ODC knockout strains were unable to differentiate infection structures after in vitro induction and showed an abnormal hyphal branching phenotype. Pathogenicity studies on these mutants showed that, surprisingly, they are not reduced in virulence compared with nondisrupted transformants. This suggests that the strains carrying an ODC disruption can obtain sufficient polyamines from the host plant for normal growth and differentiation and, therefore, that fungal ODC may not be a suitable target for fungicides.

scholarly journals Characterisation of a novel endophyte NRPS gene and its role in endophyte-grass symbioses

2007 ◽  
Vol 13 ◽  
pp. 491-493
Author(s):  
H. Harzer ◽  
R.D. Johnson ◽  
S. Rasmussen ◽  
C.R. Voisey ◽  
L.J. Johnson
Keyword(s):  
Fungal Endophytes ◽  
Gene Clusters ◽  
Gene Replacement ◽  
In Planta ◽  
Dna Library ◽  
Peptide Synthetase ◽  
Genomic Dna Library ◽  
Targeted Gene Replacement ◽  
Fungal Secondary Metabolite

Symbiotic grass associations with fungal endophytes (genera Neotyphodium and Epichloë) display enhanced fitness as well as prolonged field persistence over their endophyte free equivalents. Perennial ryegrass, an important agronomic grass, is typically associated with the N. lolii endophyte. The endophyte lives within the intercellular spaces without inducing any symptoms in the plant. The aim of this study is to elucidate the biosynthetic function of fungal secondary metabolite gene clusters. Non-ribosomal peptide synthetase genes (NRPSs) of unknown function were targeted, as these genes are commonly associated with the production of bioactive peptides some of which are ecologically important. Some novel endophyte NRPS genes have been identified using a degenerate PCR screen; one of these, NRPS5 will be discussed here. Clones were obtained by screening a fosmid Epichloë festucae genomic DNA library and we are currently determining gene function by using targeted gene replacement followed by an assessment in vitro and in planta using metabolomics and appropriate bioassay screens. Keywords: endophyte, NRPS, secondary metabolism

Construction of conjugal transfer system of Streptomyces cinnamonensis and effect of PCR-mediated nsdA gene disruption on its secondary metabolism

2008 ◽  
Vol 5 (1) ◽  
pp. 73-79
Author(s):  
Chen Fen ◽  
Xiong Wei ◽  
Min Yong ◽  
Fan Yu-Qing ◽  
Liang Yun-Xiang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Disruption ◽  
Gene Replacement ◽  
Transfer System ◽  
Null Mutant ◽  
Conjugation System ◽  
Streptomyces Cinnamonensis ◽  
Intergeneric Transfer ◽  
Targeted Gene Replacement

AbstractIntergeneric transfer of plasmid vectors pSET152 and pHL212 from donor Escherichia coli ET12567/pUZ8002 and S17-1 to Streptomyces cinnamonensis was demonstrated and optimized. Assisted by this conjugation system, nsdA gene disruption was achieved through PCR-targeted gene replacement. One AprRKanS exconjugant BIB309 was then isolated and confirmed to be the nsdA null mutant. Compared with the starting strain, monensin production by the nsdA− mutant BIB309 increased 270% in vitro.

In Vitro Induction of Crystals of MT Protein by Vinblastine-Colcemid in Mouse Spermatids

1974 ◽  
Vol 32 ◽  
pp. 132-133
Author(s):  
John J. Wolosewick ◽  
John H. D. Bryan
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Nuclear Ring ◽  
Rough Er ◽  
Distal Direction ◽  
In Vitro Induction

Early in spermiogenesis the manchette is rapidly assembled in a distal direction from the nuclear-ring-densities. The association of vesicles of smooth endoplasmic reticulum (SER) and the manchette microtubules (MTS) has been reported. In the mouse, osmophilic densities at the distal ends of the manchette are the organizing centers (MTOCS), and are associated with the SER. Rapid MT assembly and the lack of rough ER suggests that there is an existing pool of MT protein. Colcemid potentiates the reaction of vinblastine with tubulin and was used in this investigation to detect this protein.

Characterization of the Original Christmas Disease Mutation (Cysteine 206 → Serine): From Clinical Recognition to Molecular Pathogenesis

1992 ◽  
Vol 67 (01) ◽  
pp. 063-065 ◽  
Author(s):  
Sherryl A M Taylor ◽  
Jacalyn Duffin ◽  
Cherie Cameron ◽  
Jerome Teitel ◽  
Bernadette Garvey ◽  
...  
Keyword(s):  
Nucleotide Substitution ◽  
Novel Mutation ◽  
Factor Ix ◽  
Causative Mutation ◽  
Coding Region ◽  
Body Of Knowledge ◽  
Polymerase Chain ◽  
Splice Junctions

SummaryChristmas disease was first reported as a distinct clinical entity in two manuscripts published in 1952 (1, 2). The eponym associated with this disorder, is the surname of the first patient examined in detail and reported by Biggs and colleagues in a paper describing the clinical and laboratory features of seven affected individuals (3). This patient has severe factor IX coagulant deficiency (less than 0.01 units/ml) and no detectable circulating factor IX antigen (less than 0.01 units/ml). Coding sequence and splice junctions of the factor IX gene from this patient have been amplified in vitro through the polymerase chain reaction (PCR). One nucleotide substitution was identified at nucleotide 30,070 where a guanine was replaced by a cytosine. This mutation alters the amino acid encoded at position 206 in the factor IX protein from cysteine to serine. The non conservative nature of this substitution, the absence of this change in more than 200 previously sequenced factor IX genes and the fact that the remainder of the coding region of this gene was normal, all provide strong circumstantial evidence in favour of this change being the causative mutation in this patient. The molecular characterization of this novel mutation in the index case of Christmas disease, contributes to the rapidly expanding body of knowledge pertaining to Christmas disease pathogenesis.

Triploidy in a Live-Born Extremely Low Birth Weight Twin: Clinical Aspects

2020 ◽  
Author(s):  
Liliya Vakrilova ◽  
Stanislava Hitrova-Nikolova ◽  
Irena Bradinova
Keyword(s):  
Left Lung ◽  
Extremely Low Birth Weight ◽  
Clinical Aspects ◽  
Severe Respiratory Distress ◽  
Vitro Fertilization ◽  
Gestational Week ◽  
Polymerase Chain ◽  
Fluorescent Polymerase Chain Reaction ◽  
Second Twin

AbstractTriploidy is a rare chromosomal aberration characterized by a karyotype with 69 chromosomes. Triploid fetuses usually are miscarried in early pregnancy. We present a case of a triploid twin and a genetically unaffected co-twin, conceived through in vitro fertilization. A discordant growth was registered at 20 weeks of gestation. Cesarean section was performed at 355/7 gestational week. The second twin was extremely growth restricted female (780 g) with oligohydramnios and severe respiratory distress, and died at 20 hours of age. The autopsy revealed unilobar left lung, bilobar right lung, and cysts of the terminal bronchioles. Quantitative fluorescent polymerase chain reaction detected triploidy compatible pattern. So, early intrauterine growth restriction may be a sign of triploidy, which must be proven by pre or postnatal genetic testing.

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