scholarly journals Development of Genetic Modification Tools for Hanseniasporauvarum

2021 ◽  
Vol 22 (4) ◽  
pp. 1943
Author(s):  
Jennifer Badura ◽  
Niël van Wyk ◽  
Silvia Brezina ◽  
Isak S. Pretorius ◽  
Doris Rauhut ◽  
...  
Keyword(s):  
Genetic Modification ◽  
Genetic Manipulation ◽  
Molecular Genetic ◽  
Marker Gene ◽  
Reading Frame ◽  
Acetate Ester ◽  
Spoilage Yeasts ◽  
Ester Formation ◽  
Phenotypic Characterisation ◽  
Sequencing Studies

Apiculate yeasts belonging to the genus Hanseniaspora are commonly isolated from viticultural settings and often dominate the initial stages of grape must fermentations. Although considered spoilage yeasts, they are now increasingly becoming the focus of research, with several whole-genome sequencing studies published in recent years. However, tools for their molecular genetic manipulation are still lacking. Here, we report the development of a tool for the genetic modification of Hanseniaspora uvarum. This was employed for the disruption of the HuATF1 gene, which encodes a putative alcohol acetyltransferase involved in acetate ester formation. We generated a synthetic marker gene consisting of the HuTEF1 promoter controlling a hygromycin resistance open reading frame (ORF). This new marker gene was used in disruption cassettes containing long-flanking (1000 bp) homology regions to the target locus. By increasing the antibiotic concentration, transformants were obtained in which both alleles of the putative HuATF1 gene were deleted in a diploid H. uvarum strain. Phenotypic characterisation including fermentation in Müller-Thurgau must showed that the null mutant produced significantly less acetate ester, particularly ethyl acetate. This study marks the first steps in the development of gene modification tools and paves the road for functional gene analyses of this yeast.

scholarly journals Changes in Membrane Fluid State and Heat Shock Response Cause Attenuation of Virulence

2010 ◽  
Vol 192 (7) ◽  
pp. 1999-2005 ◽  
Author(s):  
Amalia Porta ◽  
Annamaria Eletto ◽  
Zsolt Török ◽  
Silvia Franceschelli ◽  
Attila Glatz ◽  
...  
Keyword(s):  
Heat Shock ◽  
Genetic Modification ◽  
Heat Shock Response ◽  
Mammalian Cells ◽  
Histoplasma Capsulatum ◽  
Genetic Manipulation ◽  
Microbial Pathogens ◽  
Fluid State ◽  
Shock Response ◽  
Serovar Typhimurium

ABSTRACT So far attenuation of pathogens has been mainly obtained by chemical or heat treatment of microbial pathogens. Recently, live attenuated strains have been produced by genetic modification. We have previously demonstrated that in several prokaryotes as well as in yeasts and mammalian cells the heat shock response is controlled by the membrane physical state (MPS). We have also shown that in Salmonella enterica serovar Typhimurium LT2 (Salmonella Typhimurium) overexpression of a Δ12-desaturase gene alters the MPS, inducing a sharp impairment of transcription of major heat shock genes and failure of the pathogen to grow inside macrophage (MΦ) (A. Porta et al., J. Bacteriol. 192:1988-1998, 2010). Here, we show that overexpression of a homologous Δ9-desaturase sequence in the highly virulent G217B strain of the human fungal pathogen Histoplasma capsulatum causes loss of its ability to survive and persist within murine MΦ along with the impairment of the heat shock response. When the attenuated strain of H. capsulatum was injected in a mouse model of infection, it did not cause disease. Further, treated mice were protected when challenged with the virulent fungal parental strain. Attenuation of virulence in MΦ of two evolutionarily distant pathogens was obtained by genetic modification of the MPS, suggesting that this is a new method that may be used to produce attenuation or loss of virulence in both other intracellular prokaryotic and eukaryotic pathogens. This new procedure to generate attenuated forms of pathogens may be used eventually to produce a novel class of vaccines based on the genetic manipulation of a pathogen's membrane fluid state and stress response.

scholarly journals Analysis of Codon Usage Patterns in Giardia duodenalis Based on Transcriptome Data from GiardiaDB

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1169
Author(s):  
Xin Li ◽  
Xiaocen Wang ◽  
Pengtao Gong ◽  
Nan Zhang ◽  
Xichen Zhang ◽  
...  
Keyword(s):  
Codon Usage ◽  
Genetic Manipulation ◽  
Molecular Genetic ◽  
Gc Content ◽  
Giardia Duodenalis ◽  
Codon Usage Pattern ◽  
Protein Size ◽  
New Genes ◽  
Optimal Codons ◽  
Usage Patterns

Giardia duodenalis, a flagellated parasitic protozoan, the most common cause of parasite-induced diarrheal diseases worldwide. Codon usage bias (CUB) is an important evolutionary character in most species. However, G. duodenalis CUB remains unclear. Thus, this study analyzes codon usage patterns to assess the restriction factors and obtain useful information in shaping G. duodenalis CUB. The neutrality analysis result indicates that G. duodenalis has a wide GC3 distribution, which significantly correlates with GC12. ENC-plot result—suggesting that most genes were close to the expected curve with only a few strayed away points. This indicates that mutational pressure and natural selection played an important role in the development of CUB. The Parity Rule 2 plot (PR2) result demonstrates that the usage of GC and AT was out of proportion. Interestingly, we identified 26 optimal codons in the G. duodenalis genome, ending with G or C. In addition, GC content, gene expression, and protein size also influence G. duodenalis CUB formation. This study systematically analyzes G. duodenalis codon usage pattern and clarifies the mechanisms of G. duodenalis CUB. These results will be very useful to identify new genes, molecular genetic manipulation, and study of G. duodenalis evolution.

scholarly journals The Plastid of Toxoplasma gondii Is Divided by Association with the Centrosomes

2000 ◽  
Vol 151 (7) ◽  
pp. 1423-1434 ◽  
Author(s):  
Boris Striepen ◽  
Michael J. Crawford ◽  
Michael K. Shaw ◽  
Lewis G. Tilney ◽  
Frank Seeber ◽  
...  
Keyword(s):  
Cell Division ◽  
Toxoplasma Gondii ◽  
Genetic Manipulation ◽  
Fluorescent Proteins ◽  
Recombinant Expression ◽  
Molecular Genetic ◽  
Time Lapse ◽  
Microtubule Organization ◽  
Apicomplexan Parasites ◽  
Daughter Cells

Apicomplexan parasites harbor a single nonphotosynthetic plastid, the apicoplast, which is essential for parasite survival. Exploiting Toxoplasma gondii as an accessible system for cell biological analysis and molecular genetic manipulation, we have studied how these parasites ensure that the plastid and its 35-kb circular genome are faithfully segregated during cell division. Parasite organelles were labeled by recombinant expression of fluorescent proteins targeted to the plastid and the nucleus, and time-lapse video microscopy was used to image labeled organelles throughout the cell cycle. Apicoplast division is tightly associated with nuclear and cell division and is characterized by an elongated, dumbbell-shaped intermediate. The plastid genome is divided early in this process, associating with the ends of the elongated organelle. A centrin-specific antibody demonstrates that the ends of dividing apicoplast are closely linked to the centrosomes. Treatment with dinitroaniline herbicides (which disrupt microtubule organization) leads to the formation of multiple spindles and large reticulate plastids studded with centrosomes. The mitotic spindle and the pellicle of the forming daughter cells appear to generate the force required for apicoplast division in Toxoplasma gondii. These observations are discussed in the context of autonomous and FtsZ-dependent division of plastids in plants and algae.

scholarly journals The Expanding Molecular Genetics Tool Kit in Chlamydia

2018 ◽  
Vol 200 (24) ◽  
Author(s):  
Raphael H. Valdivia ◽  
Robert J. Bastidas
Keyword(s):  
Genetic Engineering ◽  
Molecular Genetics ◽  
Genetic Manipulation ◽  
Molecular Genetic ◽  
Model System ◽  
New Method ◽  
Content Type ◽  
Host Interactions ◽  
Additional Tool ◽  
Genomic Regions

ABSTRACT Chlamydia has emerged as an important model system for the study of host pathogen interactions, in part due to a resurgence in the development of tools for its molecular genetic manipulation. An additional tool, published by Keb et al. (G. Keb, R. Hayman, and K. A. Fields, J. Bacteriol. 200:e00479-18, 2018, https://doi.org/10.1128/JB.00479-18), now allows for custom genetic engineering of genomic regions that were traditionally recalcitrant to genetic manipulation, such as genes within operons. This new method will be an essential instrument for the elucidation of Chlamydia-host interactions.

New selectable marker/auxotrophic host strain combinations for molecular genetic manipulation of Pichia pastoris

Gene ◽  
2001 ◽  
Vol 263 (1-2) ◽  
pp. 159-169 ◽  
Author(s):  
Geoffrey P. Lin Cereghino ◽  
Joan Lin Cereghino ◽  
Anthony Jay Sunga ◽  
Monique A. Johnson ◽  
May Lim ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Selectable Marker ◽  
Genetic Manipulation ◽  
Molecular Genetic ◽  
Host Strain

Molecular Genetic Manipulation of Mosquito Vectors

1995 ◽  
Vol 40 (1) ◽  
pp. 359-388 ◽  
Author(s):  
J Carlson ◽  
K Olson ◽  
S Higgs ◽  
B Beaty
Keyword(s):  
Genetic Manipulation ◽  
Molecular Genetic ◽  
Mosquito Vectors

scholarly journals Harnessing the Endogenous 2μ Plasmid of Saccharomyces cerevisiae for Pathway Construction

2021 ◽  
Vol 12 ◽  
Author(s):  
Jing Yang ◽  
Yujuan Tian ◽  
Huayi Liu ◽  
Yeyi Kan ◽  
Yi Zhou ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Manipulation ◽  
Marker Gene ◽  
Insertion Site ◽  
Selection Marker ◽  
Plasmid Copy ◽  
Genetic Manipulations ◽  
Copy Numbers ◽  
Improved Stability ◽  
Valuable Product

pRS episomal plasmids are widely used in Saccharomyces cerevisiae, owing to their easy genetic manipulations and high plasmid copy numbers (PCNs). Nevertheless, their broader application is hampered by the instability of the pRS plasmids. In this study, we designed an episomal plasmid based on the endogenous 2μ plasmid with both improved stability and increased PCN, naming it p2μM, a 2μ-modified plasmid. In the p2μM plasmid, an insertion site between the REP1 promoter and RAF1 promoter was identified, where the replication (ori) of Escherichia coli and a selection marker gene of S. cerevisiae were inserted. As a proof of concept, the tyrosol biosynthetic pathway was constructed in the p2μM plasmid and in a pRS plasmid (pRS423). As a result, the p2μM plasmid presented lower plasmid loss rate than that of pRS423. Furthermore, higher tyrosol titers were achieved in S. cerevisiae harboring p2μM plasmid carrying the tyrosol pathway-related genes. Our study provided an improved genetic manipulation tool in S. cerevisiae for metabolic engineering applications, which may be widely applied for valuable product biosynthesis in yeast.

scholarly journals Constitutional chromothripsis of the APC locus as a cause of genetic predisposition to colon cancer

2021 ◽  
pp. jmedgenet-2021-108147
Author(s):  
Florentine Scharf ◽  
Rafaela Magalhaes Leal Silva ◽  
Monika Morak ◽  
Alex Hastie ◽  
Julia M A Pickl ◽  
...  
Keyword(s):  
Molecular Genetic ◽  
Genomic Analysis ◽  
Random Order ◽  
Molecular Genetic Analysis ◽  
Chromosomal Microarray ◽  
Reading Frame ◽  
Monogenic Diseases ◽  
Long Read ◽  
Allele Specific ◽  
Tool Set

PurposeApproximately 20% of patients with clinical familial adenomatous polyposis (FAP) remain unsolved after molecular genetic analysis of the APC and other polyposis genes, suggesting additional pathomechanisms.MethodsWe applied multidimensional genomic analysis employing chromosomal microarray profiling, optical mapping, long-read genome and RNA sequencing combined with FISH and standard PCR of genomic and complementary DNA to decode a patient with an attenuated FAP that had remained unsolved by Sanger sequencing and multigene panel next-generation sequencing for years.ResultsWe identified a complex 3.9 Mb rearrangement involving 14 fragments from chromosome 5q22.1q22.3 of which three were lost, 1 reinserted into chromosome 5 and 10 inserted into chromosome 10q21.3 in a seemingly random order and orientation thus fulfilling the major criteria of chromothripsis. The rearrangement separates APC promoter 1B from the coding ORF (open reading frame) thus leading to allele-specific downregulation of APC mRNA. The rearrangement also involves three additional genes implicated in the APC–Axin–GSK3B–β-catenin signalling pathway.ConclusionsBased on comprehensive genomic analysis, we propose that constitutional chromothripsis dampening APC expression, possibly modified by additional APC–Axin–GSK3B–β-catenin pathway disruptions, underlies the patient’s clinical phenotype. The combinatorial approach we deployed provides a powerful tool set for deciphering unsolved familial polyposis and potentially other tumour syndromes and monogenic diseases.

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