Satellite DNA and heterochromatin of the flour beetle Tribolium confusum

The chromosomes of Tribolium confusum have conspicuous bulks of pericentromeric constitutive heterochromatin. The amount of heterochromatin measured by C-banding in metaphase chromosomes is estimated to be 40–45%. It is composed of an A + T rich DNA according to the distamycin A/diamidinophenylindol staining of chromosomes. Restriction analysis of isolated T. confusum genomic DNA shows that this species has a satellite DNA that constitutes about 40% of the genome. Cloning and sequencing experiments reveal a monomer length of 158 base pairs and a copy number of 5.77 × 105 per haploid genome. Its sequence is A + T rich (73%), with direct and inverted repeats, one of them with a possibility of forming stable cruciform structure. The abundance, monomer length, and the mutation rate are similar to those found in other satellite families from different species of Tenebrionidae, but no sequence homology has been found among them. No retarded mobility of satellite DNA, characteristic for molecules with sequence-induced curvature, has been detected by electrophoresis on nondenaturing polyacrylamide gels. In situ digestions with restriction enzymes and in situ hybridization show that this satellite DNA is located in pericentromeric positions of all chromosomes coinciding with C-bands.Key words: tandem repeats, DNA sequence, bent DNA, inverted repeats, Coleoptera.

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Organization and distribution of a Sau3A tandem repeated DNA sequence in Picea (Pinaceae) species

Genome ◽

10.1139/g98-054 ◽

1998 ◽

Vol 41 (4) ◽

pp. 560-565 ◽

Cited By ~ 12

Author(s):

Garth R Brown ◽

Craig H Newton ◽

John E Carlson

Keyword(s):

In Situ Hybridization ◽

Dna Sequence ◽

Picea Glauca ◽

Tandem Repeats ◽

Picea Sitchensis ◽

Repeated Dna ◽

Metaphase Chromosomes ◽

Genes Encoding ◽

Repeated Dna Sequence

Repeated DNA families contribute to the large genomes of coniferous trees but are poorly characterized. We report the analysis of a 142 bp tandem repeated DNA sequence identified by the restriction enzyme Sau3A and found in approximately 20 000 copies in Picea glauca. Southern hybridization indicated that the repeated DNA family is specific to the genus, was amplified early in its evolution, and has undergone little structural alteration over evolutionary time. Fluorescence in situ hybridization localized arrays of the Sau3A repeating element to the centromeric regions of different subsets of the metaphase chromosomes of P. glauca and the closely related Picea sitchensis, suggesting that mechanisms leading to the intragenomic movement of arrays may be more active than those leading to mutation of the repeating elements themselves. Unambiguous identification of P. glauca and P. sitchensis chromosomes was made possible by co-localizing the Sau3A tandem repeats and the genes encoding the 5S and 18S-5.8S-26S ribosomal RNAs.Key words: Picea, repeated DNA, in situ hybridization, centromere.

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Mouse satellite DNA, centromere structure, and sister chromatid pairing.

The Journal of Cell Biology ◽

10.1083/jcb.103.4.1145 ◽

1986 ◽

Vol 103 (4) ◽

pp. 1145-1151 ◽

Cited By ~ 40

Author(s):

L M Lica ◽

S Narayanswami ◽

B A Hamkalo

Keyword(s):

Electron Microscopy ◽

Sister Chromatid ◽

Satellite Dna ◽

Marker Chromosome ◽

Restriction Enzymes ◽

Centromeric Heterochromatin ◽

Mitotic Chromosomes ◽

Metaphase Chromosomes ◽

Sister Chromatids ◽

Mouse Satellite Dna

The experiments described were directed toward understanding relationships between mouse satellite DNA, sister chromatid pairing, and centromere function. Electron microscopy of a large mouse L929 marker chromosome shows that each of its multiple constrictions is coincident with a site of sister chromatid contact and the presence of mouse satellite DNA. However, only one of these sites, the central one, possesses kinetochores. This observation suggests either that satellite DNA alone is not sufficient for kinetochore formation or that when one kinetochore forms, other potential sites are suppressed. In the second set of experiments, we show that highly extended chromosomes from Hoechst 33258-treated cells (Hilwig, I., and A. Gropp, 1973, Exp. Cell Res., 81:474-477) lack kinetochores. Kinetochores are not seen in Miller spreads of these chromosomes, and at least one kinetochore antigen is not associated with these chromosomes when they were subjected to immunofluorescent analysis using anti-kinetochore scleroderma serum. These data suggest that kinetochore formation at centromeric heterochromatin may require a higher order chromatin structure which is altered by Hoechst binding. Finally, when metaphase chromosomes are subjected to digestion by restriction enzymes that degrade the bulk of mouse satellite DNA, contact between sister chromatids appears to be disrupted. Electron microscopy of digested chromosomes shows that there is a significant loss of heterochromatin between the sister chromatids at paired sites. In addition, fluorescence microscopy using anti-kinetochore serum reveals a greater inter-kinetochore distance than in controls or chromosomes digested with enzymes that spare satellite. We conclude that the presence of mouse satellite DNA in these regions is necessary for maintenance of contact between the sister chromatids of mouse mitotic chromosomes.

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Banding of rye (Secale cereale) chromosomes using the restriction enzymes AluI, DraI, HpaII, and MspI

Genome ◽

10.1139/g93-131 ◽

1993 ◽

Vol 36 (5) ◽

pp. 998-1002 ◽

Cited By ~ 1

Author(s):

T. Stößer ◽

T. Günther ◽

C. U. Hesemann

Keyword(s):

Secale Cereale ◽

Restriction Enzymes ◽

Chromosome Band ◽

Banding Technique ◽

Recognition Sequence ◽

Metaphase Chromosomes ◽

Banding Patterns ◽

Secale Cereale L ◽

Characteristic Banding

Mitotic metaphase chromosomes of the rye inbred line L 301, which belongs to the Sortiment of the University of Hohenheim, were treated in situ with the restriction enzymes AluI (recognition sequence: 5′-AC/GT-3′), DraI (recognition sequence: 5′-TTT/AAA-3′), and the isoschizomeres HpaII and MspI (recognition sequence: 5′-C/CGG-3′) and stained with Giemsa. The chromosomes indicated similar banding patterns in comparison with the conventional Giemsa-C-banding. However, we have found in rye chromosomes after restrictase treatment that the telomeric bands were reduced in extension. In a lower degree the centromeric bands of individual chromosomes could be absent in dependence of the used restriction enzymes. The number of the intercalary bands were also reduced. Nevertheless, the tested restriction enzymes produced characteristic banding patterns of the rye genome. This uncomplicated banding technique is suited for a very quick banding method of karyotype analysis especially to obtain a first survey of the band patterns on the rye chromosomes.Key words: Secale cereale L., chromosome band pattern, in situ digestion, restriction endonuclease, restriction banding.

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Comparison of methods for the detection of in situ restriction enzyme – nick translation using fluorochromes and confocal microscopy

Genome ◽

10.1139/g95-136 ◽

1995 ◽

Vol 38 (5) ◽

pp. 1032-1036 ◽

Cited By ~ 3

Author(s):

L. Stuppia ◽

C. Cinti ◽

S. Santi ◽

R. Peila ◽

N. M. Maraldi ◽

...

Keyword(s):

Confocal Microscopy ◽

Restriction Enzyme ◽

Restriction Enzymes ◽

Chromosome Morphology ◽

Nick Translation ◽

Metaphase Chromosomes ◽

Direct Labelling ◽

Series Of Experiments ◽

The Cost

A series of experiments was carried out to determine the most efficient methods for detecting incorporated nucleotides in the "in situ" restriction enzyme – nick translation technique. Different methods were tested on fixed human metaphase chromosomes using confocal microscopy for the demonstration of the patterns produced. Of the various techniques tested, that using DIG-dUTP in conjunction with FITC-labelled anti-DIG appears to show the greatest sensitivity and specificity. The use of biotinylated nucleotides with FITC-avidin gives rather less sensitivity, while direct labelling with fluorescein-dUTP produces results more rapidly with better chromosome morphology but at the cost of reduced sensitivity. Resorufin-labelled dUTP was unusable, because of the low level of fluorescence and its very rapid fading. The successful fluorescence methods are more sensitive and faster than using horseradish peroxidase or alkaline phosphatase for detection.Key words: restriction enzymes, nick translation, chromosomes, fluorochromes, confocal microscopy.

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In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

The Journal of Cell Biology ◽

10.1083/jcb.95.2.609 ◽

1982 ◽

Vol 95 (2) ◽

pp. 609-618 ◽

Cited By ~ 85

Author(s):

NJ Hutchison ◽

PR Langer-Safer ◽

DC Ward ◽

BA Hamkalo

Keyword(s):

In Situ Hybridization ◽

Electron Microscope ◽

Satellite Dna ◽

Light Microscope ◽

Test System ◽

Microscope Level ◽

Metaphase Chromosomes ◽

Light Microscope Level ◽

Mouse Satellite Dna

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.

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Genome-Wide Distribution of Novel Ta-3A1 Mini-Satellite Repeats and Its Use for Chromosome Identification in Wheat and Related Species

Agronomy ◽

10.3390/agronomy9020060 ◽

2019 ◽

Vol 9 (2) ◽

pp. 60 ◽

Cited By ~ 3

Author(s):

Tao Lang ◽

Guangrong Li ◽

Zhihui Yu ◽

Jiwei Ma ◽

Qiheng Chen ◽

...

Keyword(s):

Satellite Dna ◽

Tandem Repeats ◽

Chromosomal Rearrangements ◽

Wide Distribution ◽

Fish Analysis ◽

Satellite Repeats ◽

Genome Wide ◽

Rapid Sequence ◽

Group 5

A large proportion of the genomes of grasses is comprised of tandem repeats (TRs), which include satellite DNA. A mini-satellite DNA sequence with a length of 44 bp, named Ta-3A1, was found to be highly accumulated in wheat genome, as revealed by a comprehensive sequence analysis. The physical distribution of Ta-3A1 in chromosomes 3A, 5A, 5B, 5D, and 7A of wheat was confirmed by nondenaturing fluorescence in situ hybridization (ND-FISH) after labeling the oligonucleotide probe. The analysis of monomer variants indicated that rapid sequence amplification of Ta-3A1 occurred first on chromosomes of linkage group 5, then groups 3 and 7. Comparative ND-FISH analysis suggested that rapid changes occurred in copy number and chromosomal locations of Ta-3A1 among the different species in the tribe Triticeae, which may have been associated with chromosomal rearrangements during speciation and polyploidization. The labeling and subsequent use of Ta-3A1 by ND-FISH may assist in the precise identification and documentation of novel wheat germplasm engineered by chromosome manipulation.

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Characterization of a tandemly repeated subtelomeric sequence with inverted telomere repeats in maize

Genome ◽

10.1139/g09-005 ◽

2009 ◽

Vol 52 (3) ◽

pp. 286-293 ◽

Cited By ~ 11

Author(s):

Jun Li ◽

Fei Yang ◽

Jia Zhu ◽

Shibin He ◽

Lijia Li

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Tandem Repeats ◽

Repeated Sequence ◽

Metaphase Chromosomes ◽

Single Chromosome ◽

Amplification Band ◽

Subtelomeric Sequence ◽

Subtelomeric Regions

In this study, two complementary telomere primers were applied to a single-primer PCR. A clear amplification band was obtained with one primer, while a smear pattern was seen with the other primer. Sequence analysis of the isolated clones from this specific amplification band revealed that a 412 bp clone designated as MTAS1 shared high homology with a reported subtelomeric sequence (382 bp) from maize ( Zea mays L.), which indicated that this clone was possibly present at subtelomeric regions. The clone MTAS1 displayed a novel structural feature flanked by the forward and inverted telomere repeats. Southern hybridization revealed a ladder of hybridization bands, suggesting that MTAS1 was a tandemly repeated sequence. Fluorescence in situ hybridization results showed that the strong MTAS1 signals were present at the ends of short arms of several long chromosomes, confirming that MTAS1 was a subtelomeric sequence and the high brightness of signals further indicated this cloned sequence was a highly and tandemly repetitive sequence in maize. Fluorescence in situ hybridization with telomeric DNA and MTAS1 as probes on metaphase chromosomes and extended genomic DNA fibers showed that hybridization signals of this clone located adjacent to or overlapped with signals of telomere tandem repeats distributed heterogeneously in subtelomeric regions of several chromosomes and even exhibited differences in two subtelomeres of a single chromosome.

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ITS-RFLP analysis, an efficient tool for differentiation of Bursaphelenchus species

Nematology ◽

10.1163/156854108/399182 ◽

2009 ◽

Vol 11 (5) ◽

pp. 649-668 ◽

Cited By ~ 50

Author(s):

Wolfgang Burgermeister ◽

Helen Braasch ◽

Kai Metge ◽

Jianfeng Gu ◽

Thomas Schröder ◽

...

Keyword(s):

Tandem Repeats ◽

Restriction Analysis ◽

Rflp Analysis ◽

Restriction Enzymes ◽

Its Sequence ◽

Additional Species ◽

New Information ◽

Specific Fragment ◽

Species Specific ◽

Different Origins

Abstract Restriction analysis of amplified ribosomal ITS sequences has provided species-specific fragment patterns for nematodes of several genera, including Bursaphelenchus. We used restriction enzymes RsaI, HaeIII, MspI, HinfI and AluI to produce ITS-RFLP reference profiles of 44 Bursaphelenchus species, including two intraspecific types in each of B. mucronatus and B. leoni. In addition, reference profiles of Aphelenchoides stammeri and Ruehmaphelenchus asiaticus were produced. Reference profiles of six species are shown here for the first time. Identical ITS-RFLP patterns were usually obtained from different isolates and from individual specimens of the same species. However, in the case of B. 'corneolus', B. lini, B. singaporensis and B. sexdentati, additional bands in the patterns of certain isolates or individual nematodes were observed which may be explained by ITS sequence microheterogeneity, i.e., the presence of ITS sequence variants within the number of rDNA tandem repeats. Since these 'extra' bands appeared only with one out of the five restriction enzymes employed, they did not seriously impair identification of species based on the overall reference patterns. ITS-RFLP analysis has proved valuable for differentiation of the pathogenic pine wood nematode, B. xylophilus, from related species. In many recent descriptions of new Bursaphelenchus species, ITS-RFLP profiles have been used as additional species identification criteria. Comparison of profiles from isolates of many different origins has provided new information on intraspecific types or genetically distinct provenances of several Bursaphelenchus species.

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