PMID- 8919917
OWN - NLM
STAT- MEDLINE
DCOM- 19970102
LR  - 20240109
IS  - 0960-7412 (Print)
IS  - 0960-7412 (Linking)
VI  - 9
IP  - 3
DP  - 1996 Mar
TI  - High-resolution physical mapping in Arabidopsis thaliana and tomato by 
      fluorescence in situ hybridization to extended DNA fibres.
PG  - 421-30
AB  - A technique to detect DNA sequences on extended DNA fibres (EDF) prepared from 
      interphase nuclei from tomato (Lycopersicon esculentum) and Arabidopsis thaliana 
      leaves by fluorescence in situ hybridization (FISH) is described. Three nuclear 
      lysis procedures have been tested for their ability to decondense chromatin and 
      to generate highly extended intact DNA fibres on microscopic slides. DNA probes 
      of various sizes have been used in FISH experiments to EDFs to establish the 
      resolution and sensitivity of the technique. The fluorescent signals of a 5S rDNA 
      probe hybridized to tomato EDFs revealed continuous strings of about 200 microns, 
      that corresponded to a molecular size of about 660 kb. In A. thaliana, a contig 
      of three cosmids spanning a genomic region with a total length of about 89 kb was 
      analysed. By means of multicolour hybridization the physical positions of the 
      cosmids were visualized as red and green fluorescence strings with overlapping 
      regions in yellow. Comparison of the length of the fluorescent signals with the 
      molecular data revealed a stretching degree of the DNA fibres at 3.27 kb 
      microns-1, which is close to the Watson-Crick DNA length estimate of 2.9 kb 
      microns-1. Other experiments on small size molecular probes with both lambda 
      clones (13.5-17 kb insert sizes) and plasmids (4.2 and 5 kb) in a contig of A. 
      thaliana, and the 5S rDNA region in tomato showed close agreement with molecular 
      data. The lower limit of the detection, which was established in a hybridization 
      experiment with two DNA probes from the 45S ribosomal gene on extended fibres of 
      tomato, was about 0.7 kb. Consistent patterns of alternating fluorescent red and 
      green spots were obtained reflecting the tandemly repeated arrangement of the 18S 
      and 25S ribosomal sequences. On the basis of the microscopic distance between 
      these hybridization spots the size of the ribosomal unit was estimated at 8.2 kb. 
      This implies a drastic improvement of high-resolution physical mapping of DNA 
      sequences by FISH on plant DNA.
FAU - Fransz, P F
AU  - Fransz PF
AD  - Department of Genetics, Wageningen Agricultural University, Netherlands.
FAU - Alonso-Blanco, C
AU  - Alonso-Blanco C
FAU - Liharska, T B
AU  - Liharska TB
FAU - Peeters, A J
AU  - Peeters AJ
FAU - Zabel, P
AU  - Zabel P
FAU - de Jong, J H
AU  - de Jong JH
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Plant J
JT  - The Plant journal : for cell and molecular biology
JID - 9207397
RN  - 0 (DNA Probes)
RN  - 0 (DNA, Plant)
SB  - IM
MH  - Arabidopsis/*genetics
MH  - Chromosome Mapping/*methods
MH  - Cloning, Molecular
MH  - Cosmids
MH  - DNA Probes
MH  - DNA, Plant/*genetics
MH  - Genes, Plant
MH  - In Situ Hybridization, Fluorescence
MH  - Solanum lycopersicum/genetics
MH  - Plasmids
MH  - Repetitive Sequences, Nucleic Acid
EDAT- 1996/03/01 00:00
MHDA- 1996/03/01 00:01
CRDT- 1996/03/01 00:00
PHST- 1996/03/01 00:00 [pubmed]
PHST- 1996/03/01 00:01 [medline]
PHST- 1996/03/01 00:00 [entrez]
AID - 10.1046/j.1365-313x.1996.09030421.x [doi]
PST - ppublish
SO  - Plant J. 1996 Mar;9(3):421-30. doi: 10.1046/j.1365-313x.1996.09030421.x.