Localisation of DNA sequences on plant chromosomes using PRINS and C-PRINS

2001 ◽  
pp. 71-82 ◽  
Author(s):  
M. Kubaláková ◽  
J. Vrána ◽  
J. Číhalíková ◽  
M. A. Lysák ◽  
Jaroslav Doležel
Keyword(s):  
Dna Sequences ◽  
Plant Chromosomes

A combined PRINS-FISH technique for simultaneous localisation of DNA sequences on plant chromosomes

1998 ◽  
Vol 41 (2) ◽  
pp. 293-296 ◽  
Author(s):  
M. Kubalakova ◽  
M. Nouzova ◽  
M. Dolezelova ◽  
J. Macas ◽  
J. Dolezel
Keyword(s):  
Dna Sequences ◽  
Fish Technique ◽  
Plant Chromosomes

scholarly journals A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps

2021 ◽  
Author(s):  
Natalya Kudryavtseva ◽  
Aleksey Ermolaev ◽  
Gennady Karlov ◽  
Ilya Kirov ◽  
Masayoshi Shigyo ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Assembly ◽  
Genetic Maps ◽  
Plant Genome ◽  
Probe Design ◽  
Fish Technique ◽  
Plant Chromosomes ◽  
Dual Color ◽  
Chromosome Level

In situ imaging of molecular markers on a physical chromosome is an indispensable tool for refin-ing of genetic maps and validation genome assembly at the chromosomal level. Despite tremen-dous progress in genome sequencing the plant genome assembly at chromosome level still remain a challenge. Recently developed optical and Hi-C mapping aim to assist in genome assembly. For high-confidence in the genome assembly at chromosome level more independent approaches will be required. The present study aimed to refined an ultrasensitive Tyr-FISH technique and to de-velop a reliable and easy method for in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH technique to find out the reasons for the failures of using the method. It has been shown that successful visualization of marker/gene depends significantly on method of chromosome slide preparation, probe design and labelling, high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.7Kb with a frequency of 51.6%. Based on our results, we developed a reliable and simple protocol for dual-color Tyr-FISH visualization of short unique DNA sequences on plant chromosomes. New protocol allows more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.

Mapping of repeated DNA sequences in plant chromosomes by PRINS and C-PRINS

1997 ◽  
Vol 94 (6-7) ◽  
pp. 758-763 ◽  
Author(s):  
M. Kubaláková ◽  
J. Macas ◽  
J. Dolez˘el
Keyword(s):  
Dna Sequences ◽  
Repeated Dna ◽  
Repeated Dna Sequences ◽  
Plant Chromosomes

scholarly journals A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps

2021 ◽  
Vol 22 (11) ◽  
pp. 5860
Author(s):  
Natalya Kudryavtseva ◽  
Aleksey Ermolaev ◽  
Gennady Karlov ◽  
Ilya Kirov ◽  
Masayoshi Shigyo ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Assembly ◽  
Genetic Maps ◽  
Plant Genome ◽  
Probe Design ◽  
Simple Method ◽  
Plant Chromosomes ◽  
Dual Color ◽  
Chromosome Level

In situ imaging of molecular markers on a physical chromosome is an indispensable tool for refining genetic maps and validation genome assembly at the chromosomal level. Despite the tremendous progress in genome sequencing, the plant genome assembly at the chromosome level remains a challenge. Recently developed optical and Hi-C mapping are aimed at assistance in genome assembly. For high confidence in the genome assembly at chromosome level, more independent approaches are required. The present study is aimed at refining an ultrasensitive Tyr-FISH technique and developing a reliable and simple method of in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH to find out the reasons behind the flaws of this technique. The accurate visualization of markers/genes appeared to be significantly dependent on the means of chromosome slide preparation, probe design and labeling, and high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.6 Kb with a frequency of 51.6%. Based on our results, we developed a more reliable and simple protocol for dual-color Tyr-FISH visualization of unique short DNA sequences on plant chromosomes. This new protocol can allow for more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.

High resolution molecular cytogenetic techniques in plants: Pachytene- and fibre-FISH

2012 ◽  
Vol 60 (2) ◽  
pp. 157-165
Author(s):  
G. Linc ◽  
M. Molnár-Láng
Keyword(s):  
Dna Sequences ◽  
Physical Map ◽  
Single Copy ◽  
Molecular Cytogenetic ◽  
Plant Chromosomes ◽  
Powerful Research Tool ◽  
Increased Sensitivity ◽  
Molecular Cytogenetic Techniques ◽  
Copy Dna ◽  
Fibre Fish

Fluorescence in situ hybridization (FISH) is the most versatile and accurate molecular cytogenetic technique for determining euchromatic-heterochromatic boundaries and the locations of repetitive and single-copy DNA sequences and of chromosome-specific BAC clones on chromosomes. The combination of cytogenetic and genetic methods yields a highresolution physical map. FISH allows direct mapping of specific DNA sequences inside the cell (interphase nuclei), along meiotic pachytene chromosomes and isolated chromatin (DNA fibres). The increased sensitivity of the technique and its ability to detect gene locations provide a powerful research tool for genetic and pre-breeding studies. FISH-based physical mapping plays an important role and is increasingly used for studies at the cytological level on the chromatin organization that controls gene expression and regulation. The present minireview describes some of the benefits of alternative FISH-based techniques and their application for studying plant chromosomes and genomes.

Transcription factor/DNA interactions visualized by electron spectroscopic imaging

1992 ◽  
Vol 50 (1) ◽  
pp. 450-451
Author(s):  
David P. Bazett-Jones ◽  
Mark L. Brown
Keyword(s):  
Transcription Factor ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Phosphorus Content ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
Dna Backbone ◽  
Two Parameters ◽  
High Level

A multisubunit RNA polymerase enzyme is ultimately responsible for transcription initiation and elongation of RNA, but recognition of the proper start site by the enzyme is regulated by general, temporal and gene-specific trans-factors interacting at promoter and enhancer DNA sequences. To understand the molecular mechanisms which precisely regulate the transcription initiation event, it is crucial to elucidate the structure of the transcription factor/DNA complexes involved. Electron spectroscopic imaging (ESI) provides the opportunity to visualize individual DNA molecules. Enhancement of DNA contrast with ESI is accomplished by imaging with electrons that have interacted with inner shell electrons of phosphorus in the DNA backbone. Phosphorus detection at this intermediately high level of resolution (≈lnm) permits selective imaging of the DNA, to determine whether the protein factors compact, bend or wrap the DNA. Simultaneously, mass analysis and phosphorus content can be measured quantitatively, using adjacent DNA or tobacco mosaic virus (TMV) as mass and phosphorus standards. These two parameters provide stoichiometric information relating the ratios of protein:DNA content.

DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 496-497
Author(s):  
Barbara Trask ◽  
Susan Allen ◽  
Anne Bergmann ◽  
Mari Christensen ◽  
Anne Fertitta ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Dual Band ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

Three-Dimensional Structure of Cloned T3 Connector Protein at 1.6nm Resolution

1990 ◽  
Vol 48 (1) ◽  
pp. 282-283
Author(s):  
José L. Carrascosa ◽  
José M. Valpuesta ◽  
Hisao Fujisawa
Keyword(s):  
Dna Sequences ◽  
Expression Vector ◽  
Three Dimensional ◽  
Deae Cellulose ◽  
Dna Packaging ◽  
Dimensional Structure ◽  
Two Dimensional ◽  
Freezing And Thawing ◽  
Three Dimensional Structure ◽  
Dimensional Reconstruction

The head to tail connector of bacteriophages plays a fundamental role in the assembly of viral heads and DNA packaging. In spite of the absence of sequence homology, the structure of connectors from different viruses (T4, Ø29, T3, P22, etc) share common morphological features, that are most clearly revealed in their three-dimensional structure. We have studied the three-dimensional reconstruction of the connector protein from phage T3 (gp 8) from tilted view of two dimensional crystals obtained from this protein after cloning and purification.DNA sequences including gene 8 from phage T3 were cloned, into Bam Hl-Eco Rl sites down stream of lambda promotor PL, in the expression vector pNT45 under the control of cI857. E R204 (pNT89) cells were incubated at 42°C for 2h, harvested and resuspended in 20 mM Tris HC1 (pH 7.4), 7mM 2 mercaptoethanol, ImM EDTA. The cells were lysed by freezing and thawing in the presence of lysozyme (lmg/ml) and ligthly sonicated. The low speed supernatant was precipitated by ammonium sulfate (60% saturated) and dissolved in the original buffer to be subjected to gel nitration through Sepharose 6B, followed by phosphocellulose colum (Pll) and DEAE cellulose colum (DE52). Purified gp8 appeared at 0.3M NaCl and formed crystals when its concentration increased above 1.5 mg/ml.

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