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

Genome ◽  
1990 ◽  
Vol 33 (3) ◽  
pp. 405-415 ◽  
Author(s):  
Robert H. Devlin ◽  
David G. Holm ◽  
Karen R. Morin ◽  
Barry M. Honda

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.

Genome ◽  
2000 ◽  
Vol 43 (6) ◽  
pp. 1081-1083 ◽  
Author(s):  
Monther T Sadder ◽  
Norbert Ponelies ◽  
Ute Born ◽  
Gerd Weber

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.


Genetics ◽  
1980 ◽  
Vol 95 (3) ◽  
pp. 673-691
Author(s):  
J Lynn Zimmerman ◽  
David L Fouts ◽  
Jerry E Manning

ABSTRACT The amount, by mass, of poly(A+) mRNA present in the polyribosomes of third-instar larvae of Drosophila melanogaster, and the relative contribution of the poly(A+) mRNA to the sequence complexity of total polysomal RNA, has been determined, Selective removal of poly(A+) mRNA from total polysoma1 RNA by use of either oligo-dT-cellulose, or poly (U)-sepharose affinity chromatography, revealed that only 0.15% of the mass of the polysomal RNA was present as poly(A+) mRNA. The present study shows that this RNA hybridized at saturation with 3.3% of the single-copy DNA in the Drosophila genome. After correction for asymmetric transcription and reactability of the DNA, 7.4% of the single-copy DNA in the Drosophila genome is represented in larval poly(A+) mRNA. This corresponds to 6.73 × 1O6 nucleotides of mRNA coding sequences, or approximately 5,384 diverse RNA sequences of average size 1,250 nucleotides. However, total polysomal RNA hybridizes at saturation to 10.9% of the single-copy DNA sequences. After correcting this value for asymmetric transcripti0n and tracer DNA reactability, 24% of the single-copy DNA in Drosophila is represented in total polysomal RNA. This corresponds to 2.18 × 107 nucleotides of RNA coding sequences or 17,440 diverse RNA molecules of size 1,250 nucleotides. This value is 3.2 times greater than that observed for poly(A+) mRNA, and indicates that ≃69% of the polysomal RNA sequence complexity is contributed by n0nadenylated RNA. Furthermore, if the number of different structural genes represented in total polysomal RNA is ≃1.7 × 104, then the number of genes expressed in thirdinstar larvae exceeds the number of chromomeres in Drosophila by about a factor of three. This numerology indicates that the number of chromomeres observed in polytene chromosomes does not reflect the number of structural gene sequences in the Drosophila genome.


Genome ◽  
1998 ◽  
Vol 41 (3) ◽  
pp. 346-356 ◽  
Author(s):  
Daniel G Peterson ◽  
William R Pearson ◽  
Stephen M Stack

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.


1997 ◽  
Vol 45 (10) ◽  
pp. 1439-1446 ◽  
Author(s):  
Ernst J.M. Speel ◽  
Frans C.S. Ramaekers ◽  
Anton H.N. Hopman

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.


2020 ◽  
Vol 48 (15) ◽  
pp. e86-e86
Author(s):  
Naoki Uno ◽  
Norihito Kaku ◽  
Yoshitomo Morinaga ◽  
Hiroo Hasegawa ◽  
Katsunori Yanagihara

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.


Genomics ◽  
1991 ◽  
Vol 9 (2) ◽  
pp. 338-343 ◽  
Author(s):  
Mladen Golubić ◽  
Marie-Genevieve Mattei ◽  
Nguyen van Cong ◽  
Felipe Figueroa ◽  
Jan Klien

2011 ◽  
Vol 30 (9) ◽  
pp. 1779-1786 ◽  
Author(s):  
Kun Yang ◽  
Hecui Zhang ◽  
Richard Converse ◽  
Yong Wang ◽  
Xiaoying Rong ◽  
...  

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