Genomic characterization and physical mapping of two fucosyltransferase genes in Medicago truncatula

Genome ◽  
2005 ◽  
Vol 48 (1) ◽  
pp. 168-176 ◽  
Author(s):  
Alexandra Castilho ◽  
Margarida Cunha ◽  
Ana Rita Afonso ◽  
Leonor Morais-Cecílio ◽  
Pedro S Fevereiro ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Medicago Truncatula ◽  
Physical Mapping ◽  
Dna Sequences ◽  
Genomic Organization ◽  
Rrna Genes ◽  
Fucosyltransferase Gene ◽  
Gene Coding ◽  
Genomic Characterization

Fucosyltransferases catalyse fucose transfer onto oligosaccharides. Two fucosylated structures have been identified in plants: the α1,4-fucosylated Lewis-a epitope and the α1,3-fucosylated core. Here we report the cloning, genomic characterization, and physical mapping of two genes encoding proteins similar to α1,4-fucosyltransferase (EC 2.4.1.65, MtFUT1) and α1,3-fucosyltransferase (EC 2.4.1.214, MtFUT2) in Medicago truncatula. Analysis of the genomic organization of the fucosyltransferase genes in M. truncatula, revealed the presence of two genomic variants of the MtFUT1 gene coding sequence, one containing a single intron and the other intronless, whereas in MtFUT2, the gene coding region is interrupted by four introns. Using for the first time fluorescence in situ hybridization (FISH) to physically map fucosyltransferase genes in plants, this study reveals a high genomic dispersion of these genes in Medicago. The MtFUT1 genes are mapped on chromosomes 4, 7, and 8, colocalizing on three of the five MtFUT2 loci. Chromosomes 1 and 5 carry the additional MtFUT2 loci. Moreover, the intensity of the FISH signals reveals marked differences in the number of gene copies per locus for both genes. Simultaneous mapping of rRNA genes on chromosome 5 shows that several MTFUT2 gene loci are inserted within the rDNA array. Insertions of coding DNA sequences into the rDNA repeats were never reported to date.Key words: core α1,3-fucosyltransferase gene, α1,4-fucosyltransferase gene, genomic organization, in situ hybridization, Medicago truncatula.

Nonisotopic in situ hybridization and plant genome mapping: the first 10 years

Genome ◽  
1994 ◽  
Vol 37 (5) ◽  
pp. 717-725 ◽  
Author(s):  
Jiming Jiang ◽  
Bikram S. Gill
Keyword(s):  
In Situ Hybridization ◽  
Physical Mapping ◽  
Dna Sequences ◽  
Genome Mapping ◽  
Molecular Cytogenetics ◽  
Gene Families ◽  
Single Copy ◽  
Plant Genome ◽  
Metaphase Chromosomes

Nonisotopic in situ hybridization (ISH) was introduced in plants in 1985. Since then the technique has been widely used in various areas of plant genome mapping. ISH has become a routine method for physical mapping of repetitive DNA sequences and multicopy gene families. ISH patterns on somatic metaphase chromosomes using tandemly repeated sequences provide excellent physical markers for chromosome identification. Detection of low or single copy sequences were also reported. Genomic in situ hybridization (GISH) was successfully used to analyze the chromosome structure and evolution of allopolyploid species. GISH also provides a powerful technique for monitoring chromatin introgession during interspecific hybridization. A sequential chromosome banding and ISH technique was developed. The sequential technique is very useful for more precise and efficient mapping as well as cytogenetic determination of genomic affinities of individual chromosomes in allopolyploid species. A critical review is made on the present resolution of the ISH technique and the future outlook of ISH research is discussed.Key words: in situ hybridization, physical mapping, genome mapping, molecular cytogenetics.

scholarly journals 5-Methylcytosine distribution and genome organization in triticale before and after treatment with 5-azacytidine

1999 ◽  
Vol 112 (23) ◽  
pp. 4397-4404 ◽  
Author(s):  
A. Castilho ◽  
N. Neves ◽  
M. Rufini-Castiglione ◽  
W. Viegas ◽  
J.S. Heslop-Harrison
Keyword(s):  
In Situ Hybridization ◽  
Restriction Fragment ◽  
Dna Sequences ◽  
Genomic Dna ◽  
Restriction Enzymes ◽  
Hybrid Plant ◽  
Rrna Genes ◽  
Next Generation ◽  
Root Tips

Triticale (2n=6x=42) is a hybrid plant including rye (R) and wheat (A and B) genomes. Using genomic in situ hybridization with rye DNA as a probe, we found the chromosomes of the R genome were not intermixed with the wheat chromosomes in 85% of nuclei. After treatment of seedlings with low doses of the drug 5-azacytidine (5-AC), leading to hypomethylation of the DNA, the chromosomes became intermixed in 60% of nuclei; the next generation showed intermediate organization. These results correlate with previous data showing that expression of R-genome rRNA genes, normally suppressed, is activated by 5-AC treatment and remains partially activated in the next generation. The distribution of 5-methylcytosine (5-mC) was studied using an antibody to 5-mC. Methylation was detected along the lengths of all chromosomes; there were some chromosome regions with enhanced and reduced methylation, but these were not located at consistent positions, nor were there differences between R and wheat genome chromosomes. After 5-AC treatment, lower levels of methylation were detected. After 5-AC treatment, in situ hybridization with rye genomic DNA sometimes showed micronuclei of rye origin and multiple translocations between wheat and rye chromosomes. Genomic DNA was analysed using methylation-sensitive restriction enzymes and, as probes, two rDNA sequences, two tandemly organised DNA sequences from rye (pSc200 and pSc250), and copia and the gypsy group retrotransposon fragments from rye and wheat. DNA extracted immediately after 5-AC treatment was cut more by methylation-sensitive restriction enzymes than DNA from untreated seedlings. Each probe gave a characteristic restriction fragment pattern, but rye- and wheat-origin probes behaved similarly, indicating that hypomethylation was induced in both genomes. In DNA samples from leaves taken 13–41 days after treatment, RFLP (Restriction Fragment Length Polymorphism) patterns were indistinguishable from controls and 5-AC treatments with all probes. Surprising differences in hybridization patterns were seen between DNA from root tips and leaves with the copia-fragment probes.

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.

Physical mapping of ribosomal DNA sites in Festuca arundinacea and related species by in situ hybridization

Genome ◽  
1997 ◽  
Vol 40 (3) ◽  
pp. 406-410 ◽  
Author(s):  
H. M. Thomas ◽  
J. A. Harper ◽  
M. R. Meredith ◽  
W. G. Morgan ◽  
I. P. King
Keyword(s):  
In Situ Hybridization ◽  
Ribosomal Dna ◽  
Physical Mapping ◽  
Festuca Arundinacea ◽  
Rrna Genes ◽  
Structural Rearrangements ◽  
Phylogenetic Studies ◽  
Rdna Sites ◽  
5S Rrna Genes

The positions of the 18S–5.8S–26S and 5S rRNA genes have been physically mapped on the chromosomes of diploid, tetraploid, and hexaploid Festuca species by in situ hybridization. The number and position of the rDNA sites in the species were compared. The results confirm some of the earlier phylogenetic studies of these species but suggest that some structural rearrangements have occurred and that sites have been lost during polyploidization. Keywords: Festuca, in situ hybridization, phylogeny, physical mapping, rDNA.

Physical mapping of plant DNA sequences by simultaneous in situ hybridization of two differently labelled fluorescent probes

Genome ◽  
1991 ◽  
Vol 34 (3) ◽  
pp. 329-333 ◽  
Author(s):  
I. J. Leitch ◽  
A. R. Leitch ◽  
J. S. Heslop-Harrison
Keyword(s):  
In Situ Hybridization ◽  
Physical Mapping ◽  
Dna Sequences ◽  
Secale Cereale ◽  
Repeat Sequence ◽  
Tandem Repeat Sequence ◽  
Plant Dna ◽  
Multiple Labelling ◽  
Ribosomal Dna Sequence

Two haptens, biotin and digoxigenin, were used to label two highly repetitive plant DNA sequences: pTa71 (a clone containing a ribosomal DNA sequence from wheat, Triticum aestivum) and pSc119.2 (a clone containing a 120-bp tandem repeat sequence from rye, Secale cereale). These probes were simultaneously localized by in situ hybridization on chromosome spreads of rye, Secale cereale cv. Petkus Spring. The ability to localize two sequences simultaneously will be of major importance for physically ordering DNA sequences along plant chromosomes.Key words: physical mapping, DNA–DNA in situ hybridization, Secale, fluorescent mapping, multiple labelling.

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