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

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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LOCALIZATION OF SINGLE COPY DNA SEQUENCES ON G-BANDED HUMAN CHROMOSOMES BY IN SITU HYBRIDIZATION

Mobilization and Reassembly of Genetic Information ◽

10.1016/b978-0-12-633360-2.50076-0 ◽

1980 ◽

pp. 443

Author(s):

Mary E. Chandler ◽

Michael B. Mann ◽

Grady F. Saunders

Keyword(s):

In Situ Hybridization ◽

Dna Sequences ◽

Single Copy ◽

Human Chromosomes ◽

Copy Dna ◽

Single Copy Dna

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

Chromosoma ◽

10.1007/bf00327364 ◽

1981 ◽

Vol 83 (3) ◽

pp. 431-439 ◽

Cited By ~ 521

Author(s):

Mary E. Harper ◽

Grady F. Saunders

Keyword(s):

In Situ Hybridization ◽

Dna Sequences ◽

Single Copy ◽

Human Chromosomes ◽

Copy Dna ◽

Single Copy 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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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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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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Identifying a single-copy DNA sequence associated with the expression of a heterochromatic gene, the light locus of Drosophila melanogaster

Genome ◽

10.1139/g90-062 ◽

1990 ◽

Vol 33 (3) ◽

pp. 405-415 ◽

Cited By ~ 17

Author(s):

Robert H. Devlin ◽

David G. Holm ◽

Karen R. Morin ◽

Barry M. Honda

Keyword(s):

Drosophila Melanogaster ◽

In Situ Hybridization ◽

Dna Sequences ◽

Polytene Chromosomes ◽

Single Copy ◽

Molecular Organization ◽

Copy Dna ◽

Single Copy Dna ◽

P Transposon

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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Mapping of single-copy DNA sequences on human chromosomes by in situ hybridization with biotinylated probes: enhancement of detection sensitivity by intensified-fluorescence digital-imaging microscopy.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.86.2.582 ◽

1989 ◽

Vol 86 (2) ◽

pp. 582-586 ◽

Cited By ~ 79

Author(s):

E. Viegas-Pequignot ◽

B. Dutrillaux ◽

H. Magdelenat ◽

M. Coppey-Moisan

Keyword(s):

In Situ Hybridization ◽

Dna Sequences ◽

Digital Imaging ◽

Single Copy ◽

Detection Sensitivity ◽

Human Chromosomes ◽

Copy Dna ◽

Single Copy Dna ◽

Biotinylated Probes

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The pericentromeric heterochromatin of the grass Zingeria biebersteiniana (2n = 4) is composed of Zbcen1-type tandem repeats that are intermingled with accumulated dispersedly organized sequences

Genome ◽

10.1139/g01-092 ◽

2001 ◽

Vol 44 (6) ◽

pp. 955-961 ◽

Cited By ~ 19

Author(s):

Verity A Saunders ◽

Andreas Houben

Keyword(s):

In Situ Hybridization ◽

Tandem Repeat ◽

Tandem Repeats ◽

Repetitive Sequences ◽

Pericentromeric Region ◽

Pericentromeric Heterochromatin ◽

Repeat Family ◽

Dna Reassociation ◽

Copy Dna

DNA reassociation and hydroxyapatite chromatography were used to isolate high-copy DNA of the grass Zingeria biebersteiniana (2n = 4). In situ hybridization demonstrated that the DNA isolated was enriched for pericentromere-specific repetitive sequences. One abundant pericentromere-specific component is the differentially methylated tandem-repeat family Zbcen1. Other sequences isolated, Zb46 and Zb47A, are dispersed and display similarity to parts of the gypsy- and copia-like retrotransposable elements of other grasses. In situ hybridization with the copia-like sequence Zb47A resulted in dispersed labelling along the chromosome arms, with a significant signal accumulation in the pericentromeric region of all chromosomes. It is concluded that the pericentromeric heterochromatin of Z. biebersteiniana is composed of members of the Zbcen1 tandem repeat family and that these tandem arrays are intermingled with accumulated putative copia-like retrotransposon sequences. An observed Rabl interphase orientation suggests that the length of the chromosomes rather than the genome size is the determining factor of the Rabl phenomenon.Key Words: centromere, heterochromatin, tandemly repeated DNA, retrotransposon-like, DNA reassociation.

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An evaluation of a new series of fluorescent dUTPs for fluorescence in situ hybridization.

Journal of Histochemistry & Cytochemistry ◽

10.1177/44.5.8627009 ◽

1996 ◽

Vol 44 (5) ◽

pp. 525-529 ◽

Cited By ~ 12

Author(s):

J Wiegant ◽

N Verwoerd ◽

S Mascheretti ◽

M Bolk ◽

H J Tanke ◽

...

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Human Lymphocytes ◽

Single Copy ◽

Cyanine Dye ◽

Chromosome 1 ◽

Nick Translation ◽

Qualitative Comparison ◽

Copy Dna

Synthesis of fluorochrome-modified deoxyribonucleotides has been carried out mostly by linking the fluorochrome molecule to the C-5 position of dUTP via an allylamine spacer, similar to the modification of allylamine-dUTP with the haptens biotin and digoxigenin. Recently, a new series of fluorescent nucleotides has been prepared by using an alkynyl bridge between the uracil moiety and the fluorochrome. Here we report the qualitative and quantitative analysis of fluorescence in situ hybridization results obtained on interphase cells and chromosomes with a variety of highly repetitive and single-copy DNA probes that were modified by nick translation with such alkynyl dUTPs. A qualitative comparison was made of the alkynyl dUTPs conjugated to the fluorochromes fluorescein, the cyanine dye Cy3, tetramethylrhodamine, Lissamine and Texas Red. With the exception of tetramethylrhodamine, all fluorochromes performed satisfactorily. The cyanine dye Cy3 provided the highest sensitivity, i.e., cosmid and YAC probes could easily be visualized by conventional fluorescence microscopy. In a quantitative assay, different nick translation conditions were tested using a human chromosome 1 satellite III probe (pUC1.77) and alkynyl dUTPs labeled with fluorescein and Cy3. Using these two nucleotides, FISH signal intensities on interphase nuclei from human lymphocytes were quantitated by digital imaging microscopy. The strongest signals were obtained when during nick translation the ratio between dTTP and fluorescein-dUTP or Cy3-dUTP was 1:5.

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