Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

In Vitro Cellular & Developmental Biology - Plant (Impact Factor: 1.14). 01/2001; 37(1):1-5. DOI: 10.1007/s11627-001-0001-6

ABSTRACT Fluorescence in situ hybridization (FISH) is a powerful tool for visualizing the chromosomal location of targeted sequences and has been applied
in many areas, including karyotyping, breeding and characterization of genes introduced into the plant genome. A simple, routine
and sensitive FISH procedure was developed for localizing single copy genes in rice (Oryza sativa L.) metaphase chromosomes. We used digoxygenin-labeled endogenous or T-DNA sequences as small as 5.6 kb to probe corresponding
endogenous sequences or the T-DNA insert in denatured rice metaphase chromosomes prepared from root meristem tissue. The hybridized
probe sequence was labeled with cy3-conjugated anti-mouse IgG and visualized using fluorescence microscopy. Single copy and
multiple copy introduced T-DNA sequences, as well as endogenous sequences, were localized on the chromosomes. The FISH protocol
was effectively used to sereen the chromosomal location of introduced T-DNA and number of integration loci in rice.

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    ABSTRACT: It is now well established that the seed storage proteins of major crop species are encoded as families of closely related genes. Similarities and divergencies are being defined at the levels of protein structure, amino acid sequence, and nucleotide sequence, (see Brown, Ersland, and Hall, 1982, and Ersland et al., 1983 for reviews). Phaseolin, the major storage protein of the French bean (Phaseolus vulgaris L.) can be separated into a group of about ten closely related polypeptides by two-dimensional PAGE. Analysis of some 150 cultivars yielded only three different patterns, characterized as T (Tendergreen), S (Sanilac) and C (Contender) types (Brown et al., 1981a). The component polypeptides range in apparent molecular weight from 45 to 51 kd, and in isoelectric point from pH 5.6–5.8 (Fig. 1). The T, S and C patterns contained 5, 8 and 8 polypeptides respectively.
    Structure and Function of Plant Genomes, Edited by O. Ciferri and L. Dure, 01/1983: pages 123-142; Springer U.S.., ISBN: 978-1-4684-4538-1
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    ABSTRACT: The inheritance of phaseolin and globulin-2 (G2)/albumin polypeptides was investigated in crosses involving varieties which exhibited the three electrophoretic banding patterns of phaseolin found in French bean. 'Total' seed protein extracts of single seeds of the F1 and F2 generations from the crosses: 'Sanilac' × 'Contender', 'BBL 240' × 'Contender', and 'Sanilac' × 'BBL 240' were analyzed by two-dimensional electrophoresis. Segregation of the genes controlling phaseolin and G2/albumin polypeptides, and those controlling a further five groups of seed proteins (A, B, D, E, and F) were observed. No recombinant electrophoretic phenotypes were seen for phaseolin or G2/albumin polypeptides suggesting that the genes controlling each of these groups of polypeptides are closely linked and segregate like single Mendelian genes. The phaseolin genes and G2/albumin genes were not linked to each other. The group of genes controlling phaseolin polypeptides were linked to those controlling group B proteins, and those controlling G2/albumin polypeptides were linked to those controlling group F proteins.
    Theoretical and Applied Genetics 10/1981; 60(4):251-9. · 3.66 Impact Factor
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    ABSTRACT: 3' terminal fragments of BMV RNA as short as 153 bases in length serve as efficient templates in vitro for BMV-specific RNA polymerase. Template activity of such fragments or of native BMV RNA is abolished when cDNA fragments as short as 39 bases are hybridized to their 3' termini. Hybridization of cDNa fragments to regions of BMV RNA 200 or more bases distal to the 3' end has no discernible effect on initiation and little effect on elongation. We conclude that BMV RNA polymerase initiates binding with an RNA template through a mechanism mediated by the tRNA-like 3' end of BMV RNA, requiring at least some of the last 39, but no more than the last 153 bases.
    Plant Molecular Biology 01/1984; 3(1):37-44. · 3.52 Impact Factor


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