Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization

Although little is known about the molecular organization of most genes within heterochromatin, the unusual properties of these chromosomal regions suggest that genes therein may be organized and expressed very differently from those in euchromatin. We report here the cloning, by P transposon tagging, of sequences associated with the expression of the light locus, an essential gene found in the heterochromatin of chromosome 2 of Drosophila melanogaster. We conclude that this DNA is either a segment of the light locus, or a closely linked, heterochromatic sequence affecting its expression. While other functional DNA sequences previously described in heterochromatin have been repetitive, light gene function may be associated, at least in part, with single-copy DNA. This conclusion is based upon analysis of DNA from mutations and reversions induced by P transposable elements. The cloned region is unusual in that this single-copy DNA is embedded within middle-repetitive sequences. The in situ hybridization experiments also show that, unlike most other sequences in heterochromatin, this light-associated DNA evidently replicates in polytene chromosomes, but its diffuse hybridization signal may suggest an unusual chromosomal organization.Key words: polytene chromosomes, P transposon, in situ hybridization, middle-repetitive DNA.

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Physical localization of single-copy sequences on pachytene chromosomes in maize (Zea mays L.) by chromosome in situ suppression hybridization

Genome ◽

10.1139/g00-085 ◽

2000 ◽

Vol 43 (6) ◽

pp. 1081-1083 ◽

Cited By ~ 13

Author(s):

Monther T Sadder ◽

Norbert Ponelies ◽

Ute Born ◽

Gerd Weber

Keyword(s):

Zea Mays ◽

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Dna Sequence ◽

Zea Mays L ◽

Single Copy ◽

Chromosome 9 ◽

Copy Dna ◽

Single Copy Dna

A new approach for locating single-copy DNA sequences on pachytene chromosomes of maize (Zea mays L.) was developed. A cosmid clone with homologous sequences to a molecular marker (umc105a) linked to a quantitative trait locus (QTL) for resistance against sugarcane borer (SCB) was physically mapped by fluorescence in situ hybridization (FISH) to the short arm of chromosome 9. The marker umc105a was genetically placed in the centromeric region. To suppress signals generated by maize repetitive DNA, competitive in situ suppression (CISS) hybridization was necessary to obtain specific signals from umc105a. A centromere specific DNA probe (CentC) was used in a double-labeling technique as a reference marker. Fluorescence signals generated by umc105a cosmid and CentC were specific and highly reproducible. Thus the single-copy DNA sequence of umc105a was physically localized on the short arm of chromosome 9 near the telomere. This is the first report of physical localization of single-copy DNA sequence by CISS hybridization to a maize pachytene chromosome.Key words: fluorescence in situ hybridization, maize, pachytene chromosome, single-copy sequence, CISS hybridization.

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Characterization of the tomato (Lycopersicon esculentum) genome using in vitro and in situ DNA reassociation

Genome ◽

10.1139/g98-025 ◽

1998 ◽

Vol 41 (3) ◽

pp. 346-356 ◽

Cited By ~ 36

Author(s):

Daniel G Peterson ◽

William R Pearson ◽

Stephen M Stack

Keyword(s):

In Situ Hybridization ◽

Lycopersicon Esculentum ◽

Constitutive Heterochromatin ◽

Single Copy ◽

Tomato Genome ◽

Dna Reassociation ◽

Copy Dna ◽

Single Copy Dna

A detailed in vitro study of the kinetics of DNA renaturation, i.e., a C0t analysis, can be used to determine the size of a genome, the relative proportions of single-copy and repetitive sequences, and the complexity of genome components. Despite the dual importance of tomato (Lycopersicon esculentum) as a model for basic plant research and as a crop plant, to the best of our knowledge a C0t analysis has never been published for this species. This is probably due to difficulties associated with isolating sufficient quantities of polyphenol-free nuclear DNA from tomato. Recently we developed a technique for isolating milligram quantities of purified DNA from tomato nuclei, and we used DNA isolated in this manner to prepare a C0t curve for the tomato genome. Analysis of the C0t data indicates that the tomato genome (1C) consists of approximately 0.86 pg of DNA. In agreement with earlier molecular studies, the C0t analysis suggests that most (~73%) of the tomato genome is composed of single-copy sequences. Since 77% of the DNA in tomato chromosomes is found in constitutive heterochromatin, many of the single-copy sequences must reside in heterochromatin, an unexpected arrangement, considering that the constitutive heterochromatin of most species is predominantly repetitive DNA. To determine the distribution of repetitive and single-copy DNA along tomato pachytene chromosomes, we used hydroxyapatite-purified C0t fractions as probes for fluorescence in situ hybridization (FISH). Our FISH results indicate that highly repetitive DNA hybridizes almost exclusively with heterochromatin. While single-copy DNA comprises most of the DNA in euchromatin, heterochromatin contains the majority of single-copy DNA sequences, an observation consistent with our C0t data and previous cytological studies.Key words: tomato, Lycopersicon esculentum, genome size, heterochromatin, euchromatin, DNA reassociation, fluorescence in situ hybridization, FISH, C0t.

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Sensitive Multicolor Fluorescence In Situ Hybridization Using Catalyzed Reporter Deposition (CARD) Amplification

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215549704501013 ◽

1997 ◽

Vol 45 (10) ◽

pp. 1439-1446 ◽

Cited By ~ 43

Author(s):

Ernst J.M. Speel ◽

Frans C.S. Ramaekers ◽

Anton H.N. Hopman

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Peroxidase Activity ◽

Single Copy ◽

Metaphase Chromosomes ◽

Detection Systems ◽

Copy Dna ◽

Catalyzed Reporter Deposition ◽

Single Copy Dna

We describe the simultaneous localization of DNA sequences in cell and chromosome preparations by means of differently fluorochrome-labeled (AMCA, FITC, TRITC) tyramides using the catalyzed reporter deposition (CARD) procedure. For this purpose, repeated as well as single-copy DNA probes were labeled with biotin, digoxigenin, and FITC, hybridized, and visualized with three different cytochemical detection systems based on horseradish peroxidase conjugates. These were sequentially applied to interphase nuclei and metaphase chromosomes at low concentrations to prevent crossreaction and nonspecific background. In situ localized peroxidase activity was visualized by the deposition of fluorochrome-labeled tyramide molecules. To allow specific deposition of a second and a third tyramide conjugate for multiple-target fluorescence in situ hybridization (FISH), remaining peroxidase activity was always completely inactivated by a mild acid treatment before application of the next peroxidase conjugate. The CARD reactions were optimized for maximal signal-to-noise ratio and discrete localization by tuning reaction time, H2O2, and tyramide concentrations. For both repeated and single-copy DNA targets, high FISH signal intensities were obtained, providing improvement of sensitivity over conventional indirect detection systems. In addition, the fluorescence CARD detection system proved to be highly efficient and easy to implement in multiple-labeling studies, such as reported here for FISH.

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Flow cytometry assay for the detection of single-copy DNA in human lymphocytes

Nucleic Acids Research ◽

10.1093/nar/gkaa515 ◽

2020 ◽

Vol 48 (15) ◽

pp. e86-e86

Author(s):

Naoki Uno ◽

Norihito Kaku ◽

Yoshitomo Morinaga ◽

Hiroo Hasegawa ◽

Katsunori Yanagihara

Keyword(s):

Flow Cytometry ◽

In Situ Hybridization ◽

Dna Sequences ◽

Human Lymphocytes ◽

Single Copy ◽

Target Sequence ◽

Flow Cytometry Assay ◽

Flow Cytometric ◽

Copy Dna

Abstract Specific nucleic acid sequences can be detected in individual cells by in situ hybridization. However, when very few copies of a target sequence are present per cell, its signal is undetectable by flow cytometry. Although various approaches have been developed to increase fluorescence signals for in situ hybridization, flow cytometric detection of specific genomic DNA sequences has not been established. Here, we present a flow cytometry assay for detection of single-copy genomic sequences in human lymphocytes using in situ PCR with universal energy transfer-labelled primers.

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Isolation of 37 single-copy DNA probes from human chromosome 6 and physical mapping of 11 probes by in situ hybridization

Genomics ◽

10.1016/0888-7543(91)90262-d ◽

1991 ◽

Vol 9 (2) ◽

pp. 338-343 ◽

Cited By ~ 3

Author(s):

Mladen Golubić ◽

Marie-Genevieve Mattei ◽

Nguyen van Cong ◽

Felipe Figueroa ◽

Jan Klien

Keyword(s):

In Situ Hybridization ◽

Human Chromosome ◽

Physical Mapping ◽

Single Copy ◽

Dna Probes ◽

Chromosome 6 ◽

Human Chromosome 6 ◽

Copy Dna ◽

Single Copy Dna

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Fluorescence in situ hybridization on plant extended chromatin DNA fibers for single-copy and repetitive DNA sequences

Plant Cell Reports ◽

10.1007/s00299-011-1086-y ◽

2011 ◽

Vol 30 (9) ◽

pp. 1779-1786 ◽

Cited By ~ 5

Author(s):

Kun Yang ◽

Hecui Zhang ◽

Richard Converse ◽

Yong Wang ◽

Xiaoying Rong ◽

...

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Repetitive Dna ◽

Dna Sequences ◽

Single Copy ◽

Repetitive Dna Sequences

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Electron microscopy of Achlya deoxyribonucleic acid sequence organization

Molecular and Cellular Biology ◽

10.1128/mcb.1.2.136-143.1981 ◽

1981 ◽

Vol 1 (2) ◽

pp. 136-143

Author(s):

M Pellegrini ◽

W E Timberlake ◽

R B Goldberg

Keyword(s):

Dna Sequences ◽

Deoxyribonucleic Acid ◽

Microscopic Analysis ◽

Single Copy ◽

Electron Microscopic ◽

Sequence Organization ◽

Double Stranded Dna ◽

Copy Dna ◽

Gene 32 ◽

Single Copy Dna

Electron microscopic analysis of reassociated deoxyribonucleic acid (DNA) from the aquatic fungus Achlya bisexualis revealed details of the sequence arrangement of the inverted repeats and both the highly and moderately repetitive sequence clusters. We used the gene 32 protein-ethidium bromide technique for visualizing the DNA molecules, a procedure which provides excellent contrast between single- and double-stranded DNA regions. Long (greater than 6-kilobase) DNA fragments were isolated after reannealing to two different repetitive C0t values, and the renatured structures were then visualized in an electron microscope. Our results showed that the inverted repeat sequences were short (0.5 kilobase, number-average) and separated by nonhomologous DNA of various lengths. These pairs of sequences were not clustered within the genome. Both highly repetitive and moderately repetitive DNA sequences were organized as tandem arrays of precisely paired, regularly repeating units. No permuted clusters of repeating sequences were observed, nor was there evidence of interspersion of repetitive with single-copy DNA sequences in the Achlya genome.

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