Localization of the human CD59 gene by fluorescence in situ hybridization and pulsed-field gel electrophoresis

1993 ◽  
Vol 63 (3) ◽  
pp. 144-146 ◽  
Author(s):  
B. Heckl-Östreicher ◽  
S. Ragg ◽  
M. Drechsler ◽  
H. Scherthan ◽  
B. Royer-Pokora
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Gel Electrophoresis ◽  
Pulsed Field Gel Electrophoresis ◽  
Pulsed Field ◽  
Human Cd59

Somatic chromosome karyotype of tomato based on in situ hybridization of the TGRI satellite repeat

Genome ◽  
1989 ◽  
Vol 32 (6) ◽  
pp. 992-998 ◽  
Author(s):  
Nora L. V. Lapitan ◽  
Martin W. Ganal ◽  
Steven D. Tanksley
Keyword(s):  
Molecular Weight ◽  
In Situ Hybridization ◽  
Satellite Dna ◽  
Gel Electrophoresis ◽  
Morphological Characteristics ◽  
Pulsed Field Gel Electrophoresis ◽  
Mitotic Chromosomes ◽  
High Molecular Weight Dna ◽  
Pulsed Field

A karyotype of tomato mitotic chromosomes was constructed based on in situ hybridization to a 162-bp telomeric DNA repeat, TGRI. Variation in the spatial and quantitative distribution of this repeat creates distinct patterns for most of the chromosomes, which along with other morphological characteristics (i.e., length and arm length ratio), allow the identification of each of the 12 mitotic chromosomes of tomato. The structure and physical size of the TGRI clusters were further investigated by means of pulsed-field gel electrophoresis. Approximately 30 hybridizing fragments were observed in the range of 25 to 1000 kb when high molecular weight DNA was digested with BglII and probed with TGRI. The total molecular weight of these fragments is approximately 14 million bp, which is close to the estimated total length of TGRI in the genome (12.5 million bp) based on genomic reconstruction experiments. The results suggest that most of the TGRI clusters consist of single, uninterrupted blocks of satellite DNA. Assignment of somatic chromosomes, identified by TGRI hybridization to the previously established tomato linkage groups, was accomplished via in situ hybridization to mitotic spreads of primary trisomic lines. Using this information, we estimate the somatic length and DNA content of each of the tomato chromosomes and chromosome arms. Key words: Lycopersicon esculentum, somatic karyotype, in situ hybridization, satellite DNA, pulsed-field gel electrophoresis.

The photosynthetic endosymbiont in cryptomonad cells produces both chloroplast and cytoplasmic-type ribosomes

1994 ◽  
Vol 107 (2) ◽  
pp. 649-657
Author(s):  
G.I. McFadden ◽  
P.R. Gilson ◽  
S.E. Douglas
Keyword(s):  
In Situ Hybridization ◽  
Gel Electrophoresis ◽  
Small Volume ◽  
Pulsed Field Gel Electrophoresis ◽  
Rrna Gene ◽  
Pulsed Field ◽  
Host Cytoplasm ◽  
Cytoplasmic Type ◽  
Host Nucleus

Cryptomonad algae contain a photosynthetic, eukaryotic endosymbiont. The endosymbiont is much reduced but retains a small nucleus. DNA from this endosymbiont nucleus encodes rRNAs, and it is presumed that these rRNAs are incorporated into ribosomes. Surrounding the endosymbiont nucleus is a small volume of cytoplasm proposed to be the vestigial cytoplasm of the endosymbiont. If this compartment is indeed the endosymbiont's cytoplasm, it would be expected to contain ribosomes with components encoded by the endosymbiont nucleus. In this paper, we used in situ hybridization to localize rRNAs encoded by the endosymbiont nucleus of the cryptomonad alga, Cryptomonas phi. Transcripts of the endosymbiont rRNA gene were observed within the endosymbiont nucleus, and in the compartment thought to represent the endosymbiont's cytoplasm. These results indicate that the endosymbiont produces its own set of cytoplasmic translation machinery. We also localized transcripts of the host nucleus rRNA gene. These transcripts were found in the nucleolus of the host nucleus, and throughout the host cytoplasm, but never in the endosymbiont compartment. Our rRNA localizations indicate that the cryptomonad cell produces two different of sets of cytoplasmic-type ribosomes in two separate subcellular compartments. The results suggest that there is no exchange of rRNAs between these compartments. We also used the probe specific for the endosymbiont rRNA gene to identify chromosomes from the endosymbiont nucleus in pulsed field gel electrophoresis. Like other cryptomonads, the endosymbiont nucleus of Cryptomonas phi contains three small chromosomes.

High-density fluorescence in situ hybridization signal detection on barley (Hordeum vulgare L.) chromosomes with improved probe screening and reprobing procedures

Genome ◽  
2011 ◽  
Vol 54 (2) ◽  
pp. 151-159 ◽  
Author(s):  
Akio Kato
Keyword(s):  
In Situ Hybridization ◽  
Hordeum Vulgare ◽  
Fluorescence In Situ Hybridization ◽  
Gel Electrophoresis ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Hordeum Vulgare L ◽  
Pcr Products ◽  
Break Points

The barley ( Hordeum vulgare L.) genome was screened to identify sequences that could be used for fluorescence in situ hybridization (FISH). From 2000 transformed bacterium colonies carrying barley clones, 56 colonies were selected on the basis of the patterns that their PCR products produced when subjected to agarose gel electrophoresis. Among them, 42 (75%) exhibited fluorescent signals on barley chromosomes after in situ hybridization using the directly labeled PCR products. Sequencing revealed seven clones, pHv-365, pHv-177, pHv-1112, pHv-689, pHv-1476, pHv-1889, and pHv-1972, to be newly identified FISH-positive sequences. The remainder possess previously described sequences such as 5S, GAA microsatellite, centromere repeats, HVT01, and pHvMWG2315 (324 bp repeat). It is shown here that a combination of five probes, which produce strong signals on barley chromosomes, pHv-38 (5S), pHv-365, pHv-961 (HVT01), GAA, and TAG microsatellites, offer unequivocal recognition of each chromosome. The combination of three probes, i.e., pHv-1123 (barley 324 bp repeat), GAA, and TAG, decorated entire chromosomes with fine dotted signals and was useful for detecting the break points of aberrant chromosomes. The signals’ distributions of pHv-177, pHv-1112, and TAG were highly polymorphic. An improved reprobing procedure and its usefulness are also discussed.

scholarly journals Telomeric DNA in ALT Cells Is Characterized by Free Telomeric Circles and Heterogeneous t-Loops

2004 ◽  
Vol 24 (22) ◽  
pp. 9948-9957 ◽  
Author(s):  
Anthony J. Cesare ◽  
Jack D. Griffith
Keyword(s):  
Cell Lines ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Pulsed Field Gel Electrophoresis ◽  
Telomeric Dna ◽  
Pulsed Field ◽  
Telomere Elongation ◽  
Alternative Lengthening ◽  
Circle Formation

ABSTRACT A prerequisite for cellular immortalization in human cells is the elongation of telomeres through the upregulation of telomerase or by the alternative lengthening of telomeres (ALT) pathway. In this study, telomere structure in multiple ALT cell lines was examined by electron microscopy. Nuclei were isolated from GM847, GM847-Tert, and WI-38 VA13 ALT cells, psoralen photo-cross-linked in situ, and the telomere restriction fragments were purified by gel filtration chromatography. Examination of telomere-enriched fractions revealed frequent extrachromosomal circles, ranging from 0.7 to 56.8 kb. t-loops were also observed, with the loop portion ranging from 0.5 to 70.2 kb. The total length of the loop plus tail of the t-loops corresponded to the telomere restriction fragment length from the ALT cell lines as determined by pulsed-field gel electrophoresis. The presence of extrachromosomal circles containing telomeric DNA was confirmed by two-dimensional pulsed-field gel electrophoresis. These results show that extrachromosomal telomeric DNA circles are present in ALT nuclei and suggest a roll-and-spread mechanism of telomere elongation similar to that seen in previous observations of multiple yeast species. Results presented here also indicate that expression of telomerase in GM847 cells does not affect t-loop or extrachromosomal circle formation.

scholarly journals Genomic Analysis of Clostridium botulinum Group II by Pulsed-Field Gel Electrophoresis

1998 ◽  
Vol 64 (2) ◽  
pp. 703-708 ◽  
Author(s):  
Sebastian Hielm ◽  
Johanna Björkroth ◽  
Eija Hyytiä ◽  
Hannu Korkeala
Keyword(s):  
Gel Electrophoresis ◽  
Clostridium Botulinum ◽  
Genomic Analysis ◽  
Restriction Enzymes ◽  
Pulsed Field Gel Electrophoresis ◽  
Pulsed Field ◽  
Epidemiological Method ◽  
Genomic Analyses ◽  
Group Ii

ABSTRACT Pulsed-field gel electrophoresis (PFGE) was optimized for genomic analyses of Clostridium botulinum (nonproteolytic) group II. DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. A rapid (4-h) in situ DNA isolation method was also assessed and gave indistinguishable results. Genomic DNA from 21 strains of various geographical and temporal origins was digested with 15 rare-cutting restriction enzymes. Of these, ApaI, MluI,NruI, SmaI, and XhoI gave the most revealing PFGE patterns, enabling strain differentiation. Twenty strains yielded PFGE patterns containing 13 pulsotypes. From summation of MluI, SmaI, and XhoI restriction fragments, the genome size of C. botulinum group II was estimated to be 3.6 to 4.1 Mb (mean ± standard deviation = 3,890 ± 170 kb). The results substantiate that after problems due to DNases are overcome, PFGE analysis will be a reproducible and highly discriminating epidemiological method for studying C. botulinum group II at the molecular level.

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