Fluorescent in situ hybridization of a bacterial artificial chromosome

Genome ◽  
1995 ◽  
Vol 38 (4) ◽  
pp. 646-651 ◽  
Author(s):  
Robert E. Hanson ◽  
Michael S. Zwick ◽  
Sangdun Choi ◽  
M. Nurul Islam-Faridi ◽  
Rod A. Wing ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Bacterial Artificial Chromosome ◽  
Fluorescent In Situ Hybridization ◽  
Organizer Region ◽  
Repetitive Sequences ◽  
Nucleolar Organizer Region ◽  
Artificial Chromosome ◽  
Genome Species ◽  
A Genome

Fluorescent in situ hybridization (FISH) of a 130 kilobase cotton (Gossypium hirsutum L.) bacterial artificial chromosome (BAC) clone containing a high proportion of single-copy DNA produced a large pair of FISH signals on the distal end of the long arm of a pair of chromosomes of the D-genome species G. raimondii Ulbr. and produced a fainter pair of signals on a small submetacentric pair of chromosomes of the A-genome species G. herbaceum L. The signals were syntenic with a nucleolar organizer region in G. raimondii and G. herbaceum. Signal pairs were easily recognized in interphase and metaphase cells either with or without suppression of repetitive sequences with unlabeled G. hirsutum C0t-1 DNA. High quality FISH results were consistently obtained and image analysis was not required for viewing or photography. Results indicate that FISH of BAC clones is an excellent tool for the establishment of new molecular cytogenetic markers in plants and will likely prove instrumental in the development of useful physical maps for many economically important crop species.Key words: bacterial artificial chromosome, BAC, Gossypium, in situ hybridization, physical mapping.

scholarly journals Repetitive DNA sequences in Crocus vernus Hill (Iridaceae): The genomic organization and distribution of dispersed elements in the genus Crocus and its allies

Genome ◽  
2000 ◽  
Vol 43 (5) ◽  
pp. 902-909 ◽  
Author(s):  
S Frello ◽  
J S Heslop-Harrison
Keyword(s):  
In Situ Hybridization ◽  
Sequence Analysis ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Southern Hybridization ◽  
Genomic Organization ◽  
Organizer Region ◽  
Nucleolar Organizer Region ◽  
Taxonomic Structure

Eight clones of repetitive DNA were isolated from Crocus vernus Hill. The genomic organization of the clones was analyzed by in situ hybridization to C. vernus and Southern hybridization to a range of Crocus and other species. Seven clones were used for in situ hybridization. Sequence analysis showed that all eight clones were nonhomologous, and thus represented eight different sequence-families. In situ hybridization showed that six were dispersed in high copy numbers on all chromosomes of the C. vernus genome, whereas one was localized proximal to the secondary constriction, at the NOR (nucleolar organizer region) and was not further analyzed, as it was considered part of the 18S-25S rDNA repeat. Except for short palindromes, none of the sequences showed notable internal structures. Clone pCvKB4 showed homology to the reverse transcriptase gene of Ty1-copia-like retrotransposons; the others showed no homology to known sequences. When used as probes for Southern hybridization, four showed a ladder of 3-4 bands superimposed by irregular patterns, indicating organization in short tandem arrays. Each clone had a unique distribution among Crocus species (12-16 species analyzed with each clone) and six species of Iridaceae, Liliaceae, and Amaryllidaceae; all seven investigated sequences were Iridaceae specific and four were Crocus specific. The species distribution of these seven clones showed notable discrepancies with the taxonomic subdivision of the genus at the subgenus, section, and series levels. The results suggest that the phylogeny and taxonomic structure of the genus Crocus might need reconsideration. The analysis of repetitive DNA as a major and rapidly evolving part of the genome could contribute to the study of species relationships and evolution.Key words: phylogeny, evolution, in situ hybridization, sequence analysis, dispersed elements.

DNA, chromosomes, and in situ hybridization

Genome ◽  
2003 ◽  
Vol 46 (6) ◽  
pp. 953-962 ◽  
Author(s):  
Trude Schwarzacher
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequences ◽  
Physical Map ◽  
Repetitive Sequences ◽  
Sequence Evolution ◽  
Molecular Sequence ◽  
Repeat Units ◽  
Satellite Repeats ◽  
A Genome

In situ hybridization is a powerful and unique technique that correlates molecular information of a DNA sequence with its physical location along chromosomes and genomes. It thus provides valuable information about physical map position of sequences and often is the only means to determine abundance and distribution of repetitive sequences making up the majority of most genomes. Repeated DNA sequences, composed of units of a few to a thousand base pairs in size, occur in blocks (tandem or satellite repeats) or are dispersed (including transposable elements) throughout the genome. They are often the most variable components of a genome, often being species and, occasionally, chromosome specific. Their variability arises through amplification, diversification and dispersion, as well as homogenization and loss; there is a remarkable correlation of molecular sequence features with chromosomal organization including the length of repeat units, their higher order structures, chromosomal locations, and dispersion mechanisms. Our understanding of the structure, function, organization, and evolution of genomes and their evolving repetitive components enabled many new cytogenetic applications to both medicine and agriculture, particularly in diagnosis and plant breeding.Key words: repetitive DNA, genome organization, sequence evolution, telomere, centromere.

Highly repetitive sequences in B chromosomes of Secale cereale revealed by fluorescence in situ hybridization

Genome ◽  
1994 ◽  
Vol 37 (4) ◽  
pp. 709-712 ◽  
Author(s):  
Angeles Cuadrado ◽  
Nicolas Jouve
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Secale Cereale ◽  
Fluorescent In Situ Hybridization ◽  
Repetitive Sequences ◽  
5S Rdna ◽  
B Chromosomes ◽  
Multigene Families ◽  
Hybridization Probes

An analysis of the presence and distribution of the rye and wheat repeated sequences in rye B chromosomes was carried out by fluorescent in situ hybridization. Probes used consisted of three highly repetitive sequences from rye (pSc119.2, pSc74, and pSc34) and the multigene families for the 25S–5.8S–18S and 5S rDNA from wheat (pTa71 and pTa794, respectively). pSc74 and pSc119.2 showed hybridization signals in the telomeric regions of rye B chromosomes. The remaining DNA clones did not hybridize to the B chromosomes.Key words: Secale cereale, rye, repetitive DNA, fluorescence in situ hybridization, B chromosomes.

scholarly journals Construction and characterization of a bacterial artificial chromosome (BAC) library for the A genome of wheat

Genome ◽  
1999 ◽  
Vol 42 (6) ◽  
pp. 1176-1182 ◽  
Author(s):  
D Lijavetzky ◽  
G Muzzi ◽  
T Wicker ◽  
B Keller ◽  
R Wing ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Repetitive Sequences ◽  
Bac Library ◽  
Artificial Chromosome ◽  
High Density ◽  
Triticum Monococcum ◽  
Average Insert Size ◽  
Insert Size ◽  
High Molecular Weight Dna ◽  
A Genome

A genomic bacterial artificial chromosome (BAC) library of the A genome of wheat has been constructed. Triticum monococcum accession DV92 was selected for this purpose because it is a cultivated diploid wheat and one of the parental lines used in the construction of a saturated genetic map. Leaves from this accession were used to isolate high-molecular-weight DNA from nuclei. This DNA was partially digested with restriction enzyme Hind III, subjected to double size selection, electroeluted and cloned into the pINDIGO451 BAC vector. The library consists of 276 480 clones with an average insert size of 115 kb. Excluding the 1.33% of empty clones and 0.14% of clones with chloroplast DNA, the coverage of this library is 5.6 genome equivalents. With this genome coverage the probability of having any DNA sequence represented in this library is higher than 99.6%. Clones were sorted in 720 384-well plates and blotted onto 15 high-density filters. High-density filters were screened with several single or low-copy clones and five positive BAC clones were selected for further analysis. Since most of the T. monococcum BAC ends included repetitive sequences, a modification was introduced into the classical end-isolation procedure to select low copy sequences for chromosome walking.Key words: bacterial artificial chromosome, BAC library, Triticum monococcum, wheat.

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