scholarly journals Generation of barcoded plasmid libraries for massively parallel analysis of chromatin position effects

2019 ◽  
Vol 23 (2) ◽  
pp. 203-211
Author(s):  
M. O. Lebedev ◽  
L. A. Yarinich ◽  
A. V. Ivankin ◽  
A. V. Pindyurin
Keyword(s):  
High Throughput ◽  
Molecular Mechanisms ◽  
Position Effect ◽  
Critical Parameters ◽  
Position Effect Variegation ◽  
High Throughput Analysis ◽  
Simple Method ◽  
Position Effects ◽  
Gibson Assembly ◽  
Wide Range

The discovery of the position effect variegation phenomenon and the subsequent comprehensive analysis of its molecular mechanisms led to understanding that the local chromatin composition has a dramatic effect on gene activity. To study this effect in a high-throughput mode and at the genome-wide level, the Thousands of Reporters Integrated in Parallel (TRIP) approach based on the usage of barcoded reporter gene constructs was recently developed. Here we describe the construction and quality checks of high-diversity barcoded plasmid libraries supposed to be used for high-throughput analysis of chromatin position effects in Drosophila cells. First, we highlight the critical parameters that should be considered in the generation of barcoded plasmid libraries and introduce a simple method to assess the diversity of random sequences (barcodes) of synthetic oligonucleotides using PCR amplification followed by Sanger sequencing. Second, we compare the conventional restriction-ligation method with the Gibson assembly approach for cloning barcodes into the same plasmid vector. Third, we provide optimized parameters for the construction of barcoded plasmid libraries, such as the vector : insert ratio in the Gibson assembly reaction and the voltage used for electroporation of bacterial cells with ligation products. We also compare different approaches to check the quality of barcoded plasmid libraries. Finally, we briefly describe alternative approaches that can be used for the generation of such libraries. Importantly, all improvements and modifications of the techniques described here can be applied to a wide range of experiments involving barcoded plasmid libraries.

pitkinD, a Novel Gain-of-Function Enhancer of Position-Effect Variegation, Affects Chromatin Regulation During Oogenesis and Early Embryogenesis in Drosophila

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1227-1244 ◽  
Author(s):  
Steffi Kuhfittig ◽  
János Szabad ◽  
Gunnar Schotta ◽  
Jan Hoffmann ◽  
Endre Máthé ◽  
...  
Keyword(s):  
Germ Line ◽  
Position Effect ◽  
Early Embryogenesis ◽  
Position Effect Variegation ◽  
Chromatin Regulation ◽  
Gain Of Function ◽  
Position Effects ◽  
Dominant Female ◽  
Effect Variegation ◽  
Female Germ Line

Abstract The vast majority of the >100 modifier genes of position-effect variegation (PEV) in Drosophila have been identified genetically as haplo-insufficient loci. Here, we describe pitkinDominant (ptnD), a gain-of-function enhancer mutation of PEV. Its exceptionally strong enhancer effect is evident as elevated spreading of heterochromatin-induced gene silencing along euchromatic regions in variegating rearrangements. The ptnD mutation causes ectopic binding of the SU(VAR)3-9 heterochromatin protein at many euchromatic sites and, unlike other modifiers of PEV, it also affects stable position effects. Specifically, it induces silencing of white+ transgenes inserted at a wide variety of euchromatic sites. ptnD is associated with dominant female sterility. +/+ embryos produced by ptnD/+ females mated with wild-type males die at the end of embryogenesis, whereas the ptnD/+ sibling embryos arrest development at cleavage cycle 1-3, due to a combined effect of maternally provided mutant product and an early zygotic lethal effect of ptnD. This is the earliest zygotic effect of a mutation so far reported in Drosophila. Germ-line mosaics show that ptn+ function is required for normal development in the female germ line. These results, together with effects on PEV and white+ transgenes, are consistent with the hypothesis that the ptn gene plays an important role in chromatin regulation during development of the female germ line and in early embryogenesis.

The heterochromatin-associated protein HP-1 is an essential protein in Drosophila with dosage-dependent effects on position-effect variegation.

Genetics ◽  
1992 ◽  
Vol 131 (2) ◽  
pp. 345-352 ◽  
Author(s):  
J C Eissenberg ◽  
G D Morris ◽  
G Reuter ◽  
T Hartnett
Keyword(s):  
Germ Line ◽  
Position Effect ◽  
Inducible Promoter ◽  
Specific Protein ◽  
P Element ◽  
Position Effect Variegation ◽  
Chromosomal Protein ◽  
Gene Encoding ◽  
Position Effects ◽  
Effect Variegation

Abstract Chromosome rearrangements which place euchromatic genes adjacent to a heterochromatic breakpoint frequently result in gene repression (position-effect variegation). This repression is thought to reflect the spreading of a heterochromatic structure into neighboring euchromatin. Two allelic dominant suppressors of position-effect variegation were found to contain mutations within the gene encoding the heterochromatin-specific chromosomal protein HP-1. The site of mutation for each allele is given: one converts Lys169 into a nonsense (ochre) codon, while the other is a frameshift after Ser10. In flies heterozygous for one of the mutant alleles (Su(var)2-504), a truncated HP-1 protein was detectable by Western blot analysis. An HP-1 minigene, consisting of HP-1 cDNA under the control of an Hsp70 heat-inducible promoter, was transduced into flies by P element-mediated germ line transformation. Heat-shock driven expression of this minigene results in elevated HP-1 protein level and enhancement of position-effect variegation. Levels of variegating gene expression thus appear to depend upon the level of expression of a heterochromatin-specific protein. The implications of these observations for mechanism of heterochromatic position effects and heterochromatin function are discussed.

scholarly journals Dung beetles as vertebrate samplers – a test of high throughput analysis of dung beetle iDNA

2021 ◽  
Author(s):  
Rosie Drinkwater ◽  
Elizabeth L. Clare ◽  
Arthur Y. C. Chung ◽  
Stephen J. Rossiter ◽  
Eleanor M. Slade
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Dung Beetle ◽  
Blood Feeding ◽  
Preliminary Evidence ◽  
Dung Beetles ◽  
High Throughput Analysis ◽  
Humid Forest ◽  
Terrestrial Vertebrates ◽  
Wide Range

AbstractThe application of environmental DNA (eDNA) sampling in biodiversity surveys has gained widespread acceptance, especially in aquatic systems where free eDNA can be readily collected by filtering water. In terrestrial systems, eDNA-based approaches for assaying vertebrate biodiversity have tended to rely on blood-feeding invertebrates, including leeches and mosquitoes (termed invertebrate-derived DNA or iDNA). However, a key limitation of using blood-feeding taxa as samplers is that they are difficult to trap, and, in the case of leeches, are highly restricted to humid forest ecosystems. Dung beetles (superfamily Scarabaeoidea) feed on the faecal matter of terrestrial vertebrates and offer several potential benefits over blood-feeding invertebrates as samplers of vertebrate DNA. Importantly, these beetles can be easily captured in large numbers using simple, inexpensive baited traps; are globally distributed; and also occur in a wide range of biomes, allowing mammal diversity to be compared across habitats. In this exploratory study, we test the potential utility of dung beetles as vertebrate samplers by sequencing the mammal DNA contained within their guts. First, using a controlled feeding experiment, we show that mammalian DNA can be retrieved from the guts of large dung beetles (Catharsius renaudpauliani) for up to 10 hours after feeding. Second, by combining high-throughput sequencing of a multi-species assemblage of dung beetles with PCR replicates, we show that multiple mammal taxa can be identified with high confidence. By providing preliminary evidence that dung beetles can be used as a source of mammal DNA, our study highlights the potential for this widespread group to be used in future biodiversity monitoring surveys.

Mechanisms for the construction and developmental control of heterochromatin formation and imprinted chromosome domains

Development ◽  
1990 ◽  
Vol 108 (Supplement) ◽  
pp. 35-45 ◽  
Author(s):  
Kenneth D. Tartof ◽  
Marilyn Bremer
Keyword(s):  
Position Effect ◽  
Model System ◽  
Chromosome Inactivation ◽  
Position Effect Variegation ◽  
Position Effects ◽  
Developmental Control ◽  
Heterochromatin Formation ◽  
Material Basis ◽  
Chromosome Domains ◽  
Effect Variegation

The study of variegating position effects in Drosophila provides a model system to explore the mechanism and material basis for the construction and developmental control of heterochromatin domains and the imprinted genomic structures that they may create. The results of our experiments in this regard have implications for a diverse assortment of long-range chromosome phenomena related to gene and chromosome inactivation. Specifically, as a consequence of our studies on position effect variegation, we propose a simple mechanism of X-chromosome inactivation, suggest a purpose for genomic imprinting, and postulate a general means for regulating the time in development at which certain genes become heterochromatically repressed.

scholarly journals High throughput sequencing unravels tomato-pathogen interactions towards a sustainable plant breeding

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Maria Doroteia Campos ◽  
Maria do Rosário Félix ◽  
Mariana Patanita ◽  
Patrick Materatski ◽  
Carla Varanda
Keyword(s):  
Plant Breeding ◽  
High Throughput ◽  
Plant Pathogen ◽  
Molecular Mechanisms ◽  
High Throughput Sequencing ◽  
Molecular Networks ◽  
Sequencing Data ◽  
Sources Of Resistance ◽  
Tomato Plants ◽  
Wide Range

AbstractTomato (Solanum lycopersicum) is one of the most economically important vegetables throughout the world. It is one of the best studied cultivated dicotyledonous plants, often used as a model system for plant research into classical genetics, cytogenetics, molecular genetics, and molecular biology. Tomato plants are affected by different pathogens such as viruses, viroids, fungi, oomycetes, bacteria, and nematodes, that reduce yield and affect product quality. The study of tomato as a plant-pathogen system helps to accelerate the discovery and understanding of the molecular mechanisms underlying disease resistance and offers the opportunity of improving the yield and quality of their edible products. The use of functional genomics has contributed to this purpose through both traditional and recently developed techniques, that allow the identification of plant key functional genes in susceptible and resistant responses, and the understanding of the molecular basis of compatible interactions during pathogen attack. Next-generation sequencing technologies (NGS), which produce massive quantities of sequencing data, have greatly accelerated research in biological sciences and offer great opportunities to better understand the molecular networks of plant–pathogen interactions. In this review, we summarize important research that used high-throughput RNA-seq technology to obtain transcriptome changes in tomato plants in response to a wide range of pathogens such as viruses, fungi, bacteria, oomycetes, and nematodes. These findings will facilitate genetic engineering efforts to incorporate new sources of resistance in tomato for protection against pathogens and are of major importance for sustainable plant-disease management, namely the ones relying on the plant’s innate immune mechanisms in view of plant breeding.

scholarly journals The role of heterochromatin in the expression of a heterochromatic gene, the rolled locus of Drosophila melanogaster.

Genetics ◽  
1993 ◽  
Vol 134 (1) ◽  
pp. 277-292 ◽  
Author(s):  
D F Eberl ◽  
B J Duyf ◽  
A J Hilliker
Keyword(s):  
Drosophila Melanogaster ◽  
Chromosomal Rearrangements ◽  
Position Effect ◽  
Centromeric Heterochromatin ◽  
Position Effect Variegation ◽  
Position Effects ◽  
Major Function ◽  
Effect Variegation ◽  
Heterochromatic Genes

Abstract Constitutive heterochromatic regions of chromosomes are those that remain condensed through most or all of the cell cycle. In Drosophila melanogaster, the constitutive heterochromatic regions, located around the centromere, contain a number of gene loci, but at a much lower density than euchromatin. In the autosomal heterochromatin, the gene loci appear to be unique sequence genes interspersed among blocks of highly repeated sequences. Euchromatic genes do not function well when brought into the vicinity of heterochromatin (position-effect variegation). We test the possibility that the blocks of centromeric heterochromatin provide an environment essential for heterochromatic gene function. To assay directly the functional requirement of autosomal heterochromatic genes to reside in heterochromatin, the rolled (rl) gene, which is normally located deep in chromosome 2R heterochromatin, was relocated within small blocks of heterochromatin to a variety of euchromatic positions by successive series of chromosomal rearrangements. The function of the rl gene is severely affected in rearrangements in which the rl gene is isolated in a small block of heterochromatin, and these position effects can be reverted by rearrangements which bring the rl gene closer to any large block of autosomal or X chromosome heterochromatin. There is some evidence that five other 2R heterochromatic genes are also affected among these rearrangements. These findings demonstrate that the heterochromatic genes, in contrast to euchromatic genes whose function is inhibited by relocation to heterochromatin, require proximity to heterochromatin to function properly, and they argue strongly that a major function of the highly repeated satellite DNA, which comprises most of the heterochromatin, is to provide this heterochromatic environment.

scholarly journals Nitrocellulose redox permanganometry: a simple method for reductive capacity assessment

2020 ◽  
Author(s):  
J Homolak ◽  
I Kodvanj ◽  
A Babic Perhoc ◽  
D Virag ◽  
A Knezovic ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Detailed Analysis ◽  
Manganese Dioxide ◽  
High Throughput ◽  
Biological Samples ◽  
Capacity Assessment ◽  
Robust Method ◽  
Nitrocellulose Membrane ◽  
High Throughput Analysis ◽  
Simple Method

AbstractWe propose a rapid, simple and robust method for measurement of reductive capacity of liquid and solid biological samples based on potassium permanganate reduction followed by trapping of manganese dioxide precipitate on a nitrocellulose membrane. Moreover, we discuss how nitrocellulose redox permanganometry (NRP) can be used for high-throughput analysis of biological samples and present HistoNRP, its modification used for detailed analysis of reductive capacity spatial distribution in tissue with preserved anatomical relations.

scholarly journals High-throughput analysis of endogenous fruit glycosyl hydrolases using a novel chromogenic hydrogel substrate assay

2017 ◽  
Vol 9 (8) ◽  
pp. 1242-1247 ◽  
Author(s):  
Julia Schückel ◽  
Stjepan Krešimir Kračun ◽  
Thomas Frederik Lausen ◽  
William George Tycho Willats ◽  
Bodil Jørgensen
Keyword(s):  
High Throughput ◽  
Enzyme Activities ◽  
Glycosyl Hydrolases ◽  
Fruiting Bodies ◽  
High Throughput Analysis ◽  
Efficient Manner ◽  
Throughput Analysis ◽  
Wide Range

A broad range of enzyme activities can be found in a wide range of different fruits and fruiting bodies and we demonstrate how many samples and diverse activities can be handled in a high-throughput and efficient manner.

scholarly journals A transposing forked-duplication with position effect variegation in Drosophila

1980 ◽  
Vol 36 (1) ◽  
pp. 41-56 ◽  
Author(s):  
P. T. Shukla ◽  
C. Auerbach
Keyword(s):  
Position Effect ◽  
Good Evidence ◽  
Position Effect Variegation ◽  
Interesting Aspect ◽  
Position Effects ◽  
X Ray ◽  
Effect Variegation ◽  
Insertion Sites ◽  
The Individual ◽  
Chromosome Ii

SUMMARYIn the course of an X-ray experiment, the normal allele of forked was transposed to the second chromosome, where it acts as a suppressor of forked. In this position, which is near the centromere, the duplication (Dp-f+) is subject to a variegated position effect. This was studied in its dependence on the hetero-euchromatin balance; the results agree with and extend those found for other position effects. In addition, we found regional preferences for variegation in the individual flies. The most interesting aspect of Dp-f+ is its tendency to transpose either to the homologous second chromosome or to Chromosome IV. In the latter position, Dp-f+ acts as a dominant near-lethal, so that the apparent selectivity of insertion sites is at least in part due to deleterious effects at insertion sites other than its original one. In a new, and presumably, centromere-far position of Dp-f+ on Chromosome II the variegated position effect disappeared and transposition was reduced in frequency or wholly abolished. The frequency of losses of Dp-f+ approximately equalled that of transpositions. Since there is good evidence that transpositions occurred pre-meiotically, the apparent losses of Dp-f+ may have been due to meiotic segregation separating the loss from the new insertion.

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