Sensitive Detection of DNA and mRNA Sequences by In Situ Hybridization and Immunogold-Silver Staining

1995 ◽  
pp. 113-130
Author(s):  
Gerhard W. Hacker ◽  
Ingeborg Zehbe ◽  
Cornelia Hauser-Kronberger ◽  
Jiang Gu ◽  
Angelika Graf ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Silver Staining ◽  
Sensitive Detection

Sequential silver staining and in situ hybridization reveal a direct association between rDNA levels and the expression of homologous nucleolar organizing regions: a hypothesis for NOR structure and function

1998 ◽  
Vol 111 (10) ◽  
pp. 1433-1439
Author(s):  
F. Zurita ◽  
R. Jimenez ◽  
M. Burgos ◽  
R.D. de la Guardia
Keyword(s):  
Transcription Factors ◽  
In Situ Hybridization ◽  
Silver Staining ◽  
Organizer Region ◽  
Nucleolar Organizer ◽  
Interindividual Variation ◽  
Nucleolar Organizer Regions ◽  
Single Pair ◽  
Ribosomal Cistrons

We have developed a procedure for sequential silver staining and in situ hybridization to analyze the relationship between the amount of rDNA present in nucleolar organizer regions, as estimated by in situ hybridization, and their level of expression, as estimated by the silver signal. For simplicity we used cells from the insectivorous mole Talpa occidentalis, which have a single pair of nucleolar organizer regions in chromosome pair 3. The relative content of ribosomal cistrons was also related to the hierarchy of activation of the nucleolar organizer regions present in this chromosomal pair. Statistical analyses demonstrated that both the relative level of expression and the activation hierarchy depended mainly on the number of ribosomal cistrons in nucleolar organizer regions. We propose a functional two-step hypothesis, which is consistent with most known data concerning interchromosomal, intercellular and interindividual variation in a number of plant and animal species, including Talpa occidentalis. In step one, the first available transcription factors bind randomly to the ribosomal promoters, such that larger nucleolar organizer regions are more likely to recruit them. In the second step the remaining transcription factors are recruited in a cooperative way, thus completing activation of one nucleolar organizer region, before the next one becomes active.

Physical localization of active and inactive rRNA gene loci in Hordeum marinum spp. gussoneanum (4x) by in situ hybridization

Genome ◽  
1992 ◽  
Vol 35 (6) ◽  
pp. 1032-1036 ◽  
Author(s):  
Ib Linde-Laursen ◽  
Elly Ibsen ◽  
Roland Von Bothmer ◽  
Henriette Giese
Keyword(s):  
In Situ Hybridization ◽  
Silver Staining ◽  
Chromosome Pair ◽  
Extra Chromosome ◽  
Rrna Gene ◽  
Morphological Similarity ◽  
Identical Position ◽  
Additional Support ◽  
Two Populations

Two populations of the diploid and 10 populations of the tetraploid cytotype of Hordeum marinum ssp. gussoneanum were studied for the presence of chromosomal segments harbouring rDNA. Conventional cytological methods established the presence of only one satellited (SAT) chromosome pair in both cytotypes. This was supported by silver staining revealing two NORs and two standard-sized nucleoli. Two additional micronucleoli were observed in a few interphases of two tetraploid populations indicating the presence of an extra chromosome pair with very low nucleolus-forming activity. In situ hybridization with the wheat rDNA probe pTA71 identified intense signals at the nucleolar constrictions of the SAT chromosomes of both cytotypes and weaker signals in a chromosome pair of the tetraploid cytotype, morphologically similar to the SAT chromosomes but without visible nucleolar constrictions. This confirms the presence of rDNA in two chromosome pairs in the tetraploid cytotype. The morphological similarity between these two pairs and the SAT-chromosome pair of the diploid cytotype as well as an identical position of the signals in all three pairs give additional support to an autoploid origin of the tetraploid cytotype. The rDNA at the nucleolar constrictions consisted of two segments of condensed rDNA of different sizes connected by diffuse rDNA. The rDNA of the chromosome pair without nucleolar constrictions was condensed supporting that this conformation is connected with inactivity. The tripartite structure of the rDNA at the nucleolar constrictions corresponds to similar tripartite structures observed after silver staining and Giemsa C-banding.Key words: in situ hybridization, rDNA location, Hordeum marinum, autoploidy, inactive NORs.

scholarly journals Combined beta-galactosidase and immunogold/silver staining for immunohistochemistry and DNA in situ hybridization.

1990 ◽  
Vol 38 (3) ◽  
pp. 325-329 ◽  
Author(s):  
W van den Brink ◽  
C van der Loos ◽  
H Volkers ◽  
R Lauwen ◽  
F van den Berg ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Silver Staining ◽  
Staining Method ◽  
Double Staining ◽  
Reaction Products ◽  
Silver Enhancement ◽  
Silver Staining Method ◽  
Double Staining Method ◽  
Beta Galactosidase

A combination of beta-galactosidase enzyme and the immunogold/silver staining method was studied for evaluation of double-staining experiments. Applications are shown for immunohistochemical double staining using two monoclonal antibodies and for combined immunohistochemistry and DNA in situ hybridization in one tissue section. The following advantages for the present double-staining method were evaluated: superior sensitivity of the immunogold/silver staining method for at least one epitope, which also allows detection of biotinylated DNA probes. The structure of the indolyl precipitate after revelation of beta-galactosidase activity did not show a concealing effect during a sequential double-staining method, as compared with the visualization of peroxidase with diaminobenzidine. These factors, and the sharply contrasting colored reaction products of beta-galactosidase (blue-green) and the immunogold/silver staining method including silver enhancement (brown-black), allow clear distinction of mixed-stained cell constituents.

In situ hybridization technique using an immunogold silver staining system

1989 ◽  
Vol 21 (7) ◽  
pp. 425-428 ◽  
Author(s):  
P. Jackson ◽  
F. A. Lewis ◽  
M. Wells
Keyword(s):  
In Situ Hybridization ◽  
Silver Staining ◽  
Hybridization Technique

Nucleolar fragmentation in polytene trichogen cells of Lucilia cuprina and Chrysomya bezziana (Diptera: Calliphoridae)

Genome ◽  
1992 ◽  
Vol 35 (2) ◽  
pp. 283-293 ◽  
Author(s):  
D. G. Bedo
Keyword(s):  
In Situ Hybridization ◽  
Silver Staining ◽  
Secondary Constriction ◽  
Ribosomal Gene ◽  
Ribosomal Genes ◽  
Lucilia Cuprina ◽  
Gene Probe ◽  
Y Chromosomes ◽  
Chrysomya Bezziana

The location of genes coding for 18S and 28S ribosomal RNA in mitotic and polytene cells of Lucilia cuprina and Chrysomya bezziana was investigated using in situ hybridization of an 18 + 28S ribosomal gene probe and silver staining. In both species ribosomal genes were localized to secondary constriction regions in sex chromosome heterochromatin. In L. cuprina mitotic cells the probe hybridizes to a distal secondary constriction region in the short arms of the X and Y chromosomes. In C. bezziana mitotic chromosomes ribosomal genes were located in distal secondary constriction regions in the long arms of the X and Y chromosomes. In polytene trichogen cells of both species, hybridization results varied with the level of polyteny. Cells of low polyteny have a single hybridization site, but with greater polytenization, increasing numbers of extrachromosomal fragments strongly hybridize to the ribosomal gene probe. No hybridization occurs in structures representing the sex chromosomes or in the autosomes. These results indicate that fragmentation and dispersal of the nucleolus occurs during polytenization. Silver staining of both unsquashed and squashed polytene nuclei show identical behaviour of multiple, varied-sized nucleolar bodies, thus confirming the in situ hybridization results. Uridine incorporation studies in L. cuprina indicate that transcription occurs in extrachromosomal bodies similar to nucleolar fragments. Nucleolar fragmentation is more pronounced in L. cuprina males, particularly in those with the translocation T(Y;2)540. Chromosomally normal C. bezziana show nucleolar fragmentation levels similar to that in L. cuprina males. Ribosomal genes are disproportionately replicated in trichogen cells to a much greater extent than surrounding heterochromatin. Nucleolar fragmentation may be a gene amplification system, but it is not known to what degree, relative to diploid amounts, ribosomal genes replicate in trichogen cells.Key words: Lucilia cuprina, Chrysomya bezziana, polytene cells, ribosomal genes, nucleolar fragmentation, disproportionate replication, heterochromatin.

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