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.

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.

Recovery of a telomere-truncated chromosome via a compensating translocation in maize

Genome ◽  
2011 ◽  
Vol 54 (3) ◽  
pp. 184-195 ◽  
Author(s):  
Robert T. Gaeta ◽  
Tatiana V. Danilova ◽  
Changzeng Zhao ◽  
Rick E. Masonbrink ◽  
Morgan E. McCaw ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Transgene Expression ◽  
De Novo ◽  
Extra Chromosome ◽  
Single Gene ◽  
B Chromosomes ◽  
Chromosome 1 ◽  
Chromosome 6

Maize-engineered minichromosomes are easily recovered from telomere-truncated B chromosomes but are rarely recovered from A chromosomes. B chromosomes lack known genes, and their truncation products are tolerated and transmitted during meiosis. In contrast, deficiency gametes resulting from truncated A chromosomes prevent their transmission. We report here a de novo compensating translocation that permitted recovery of a large truncation of chromosome 1 in maize. The truncation (trunc-1) and translocation with chromosome 6 (super-6) occurred during telomere-mediated truncation experiments and were characterized using single-gene fluorescent in situ hybridization (FISH) probes. The truncation contained a transgene signal near the end of the broken chromosome and transmitted together with the compensating translocation as a heterozygote to approximately 41%–55% of progeny. Transmission as an addition chromosome occurred in ~15% of progeny. Neither chromosome transmitted through pollen. Transgene expression (Bar) cosegregated with trunc-1 transcriptionally and phenotypically. Meiosis in T1 plants revealed eight bivalents and one tetravalent chain composed of chromosome 1, trunc-1, chromosome 6, and super-6 in diplotene and diakinesis. Our data suggest that de novo compensating translocations allow recovery of truncated A chromosomes by compensating deficiency in female gametes and by affecting chromosome pairing and segregation. The truncated chromosome can be maintained as an extra chromosome or together with the super-6 as a heterozygote.

scholarly journals Investigation of an Acetate-Fed Denitrifying Microbial Community by Stable Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescent In Situ Hybridization-Microautoradiography

2005 ◽  
Vol 71 (12) ◽  
pp. 8683-8691 ◽  
Author(s):  
Maneesha P. Ginige ◽  
Jürg Keller ◽  
Linda L. Blackall
Keyword(s):  
In Situ Hybridization ◽  
Stable Isotope ◽  
Activated Sludge ◽  
Fluorescent In Situ Hybridization ◽  
Batch Reactor ◽  
External Source ◽  
Stable Isotope Probing ◽  
Rrna Gene ◽  
The Impact

ABSTRACT The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [13C]acetate was used in SIP to label the DNA of the denitrifiers. The [13C]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking up [14C]acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the wastewater industry to enhance denitrification.

Genetic and chromosomal localization of the 5S rDNA locus in sugar beet (Beta vulgaris L.)

Genome ◽  
1997 ◽  
Vol 40 (2) ◽  
pp. 171-175 ◽  
Author(s):  
J. Schondelmaier ◽  
T. Schmidt ◽  
C. Jung ◽  
J. S. Heslop-Harrison
Keyword(s):  
In Situ Hybridization ◽  
Linkage Group ◽  
Sugar Beet ◽  
Beta Vulgaris ◽  
5S Rdna ◽  
Rdna Locus ◽  
5S Rrna ◽  
Rrna Genes ◽  
Rrna Gene

A digoxigenin-labelled 5S rDNA probe containing the 5S rRNA gene and the adjacent intergenic spacer was used for in situ hybridization to metaphase and interphase chromosomes of a trisomic stock from sugar beet (Beta vulgaris L.). Three chromosomes of primary trisomic line IV (T. Butterfass. Z. Bot. 52: 46–77. 1964) revealed signals close to the centromeres. Polymorphisms of 5S rDNA repeats in a segregating population were used to map genetically the 5S rRNA genes within a cluster of markers in linkage group II of sugar beet. The concentration of genetic markers around the centromere presumably reflects the suppressed recombination frequency in centromeric regions. The correlation of physical and genetic data allowed the assignment of a linkage group to sugar beet chromosome IV according to line IV of the primary trisomics.Key words: Beta vulgaris, sugar beet, 5S rRNA, in situ hybridization, RFLPs, trisomics.

Heterogeneity between 16S ribosomal RNA gene copies borne by one Desulfitobacterium strain is caused by different 100-200 bp insertions in the 5´ region

2007 ◽  
Vol 53 (1) ◽  
pp. 116-128 ◽  
Author(s):  
Richard Villemur ◽  
Philippe Constant ◽  
Annie Gauthier ◽  
Martine Shareck ◽  
Réjean Beaudet
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Ribosomal Rna ◽  
16S Ribosomal Rna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Gene Copies

Strains of Desulfitobacterium hafniense, such as strains PCP-1, DP7, TCE1, and TCP-A, have unusual long 16S ribosomal RNA (rRNA) genes due to an insertion of approximately 100 bp in the 5' region. In this report, we analyzed the 16S rRNA genes of different Desulfitobacterium strains to determine if such an insertion is a common feature of desulfitobacteria. We amplified this region by polymerase chain reaction (PCR) from eight Desulfitobacterium strains (D. hafniense strains PCP-1, DP7, TCP-A, TCE1, and DCB-2; D. dehalogenans; D. chlororespirans; and Desulfitobacterium sp. PCE1) and resolved each PCR product by denaturing gradient gel electrophoresis (DGGE). All strains had from two to seven DGGE- migrating bands, suggesting heterogeneity in their 16S rRNA gene copies. For each strain, the 5' region of the 16S rRNA genes was amplified and a clone library was derived. Clones corresponding to most PCR–DGGE migration bands were isolated. Sequencing of representative clones revealed that the heterogeneity was generated by insertions of 100–200 bp. An insertion was found in at least one copy of the 16S rRNA gene in all examined strains. In total, we found eight different types of insertions (INS1–INS8) that varied from 123 to 193 nt in length. Two-dimensional structural analyses of transcribed sequences predicted that all insertions would form an energetically stable loop. Reverse transcriptase – PCR experiments revealed that most of the observed insertions in the Desulfitobacterium strains were excised from the mature 16S rRNA transcripts. Insertions were not commonly found in bacterial 16S rRNA genes, and having a different insertion in several 16S rRNA gene copies borne by a single bacterial species was rarely observed. The function of these insertions is not known, but their occurrence can have an important impact in deriving 16S rRNA oligonucleotidic fluorescence in situ hybridization probes, as these insertions can be excised from 16S rRNA transcripts.Key words: Desulfitobacterium, 16S ribosomal RNA genes, heterogeneity, gene insertions, fluorescence in situ hybridization.

Lupin kernel fiber consumption modifies fecal microbiota in healthy men as determined by rRNA gene fluorescent in situ hybridization

2006 ◽  
Vol 45 (6) ◽  
pp. 335-341 ◽  
Author(s):  
Stuart C. Smith ◽  
Rachel Choy ◽  
Stuart K. Johnson ◽  
Ramon S. Hall ◽  
Alida C.M. Wildeboer-Veloo ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Fecal Microbiota ◽  
Rrna Gene ◽  
Healthy Men ◽  
Fiber Consumption

Characterization of Thinopyrum distichum chromosomes using double fluorescence in situ hybridization, RFLP analysis of 5S and 26S rRNA, and C-banding of parents and addition lines

Genome ◽  
1997 ◽  
Vol 40 (5) ◽  
pp. 689-696 ◽  
Author(s):  
A Fominaya ◽  
S. Molnar ◽  
G. Fedak ◽  
K. C. Armstrong ◽  
N.-S. Kim ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Chromosome Pair ◽  
Triticum Turgidum ◽  
5S Rrna ◽  
Polymorphism Analysis ◽  
Addition Lines ◽  
C Banding ◽  
26S Rrna

Diagnostic markers for eight Thinopyrum distichum addition chromosomes in Triticum turgidum were established using C-banding, in situ hybridization, and restriction fragment length polymorphism analysis. The C-band karyotype conclusively identified individual Th. distichum chromosomes and distinguished them from chromosomes of T. turgidum. Also, TaqI and BamHI restriction fragments containing 5S and 18S–5.8S–26S rRNA sequences were identified as positive markers specific to Th. distichum chromosomes. Simultaneous fluorescence in situ hybridization showed both 5S and 18S–5.8S–26S ribosomal RNA genes to be located on chromosome IV. Thinopyrum distichum chromosome VII carried only a 18S–5.8S–26S rRNA locus and chromosome pair II carried only a 5S rRNA locus. The arrangement of these loci on Th. distichum chromosome IV was different from that on wheat chromosome pair 1B. Two other unidentified Th. distichum chromosome pairs also carried 5S rRNA loci. The homoeologous relationship between Th. distichum chromosomes IV and VII and chromosomes of other members of the Triticeae was discussed by comparing results obtained using these physical and molecular markers.Key words: Triticum turgidum, homoeologous relationship, Triticeae, addition lines, NOR.

Chromosomal localization of two novel repetitive sequences isolated from the Chenopodium quinoa Willd. genome

Genome ◽  
2011 ◽  
Vol 54 (9) ◽  
pp. 710-717 ◽  
Author(s):  
B. Kolano ◽  
B.W. Gardunia ◽  
M. Michalska ◽  
A. Bonifacio ◽  
D. Fairbanks ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Repetitive Sequences ◽  
Chenopodium Quinoa ◽  
5S Rdna ◽  
Rrna Gene ◽  
Fish Analysis ◽  
Rdna Loci

The chromosomal organization of two novel repetitive DNA sequences isolated from the Chenopodium quinoa Willd. genome was analyzed across the genomes of selected Chenopodium species. Fluorescence in situ hybridization (FISH) analysis with the repetitive DNA clone 18–24J in the closely related allotetraploids C. quinoa and Chenopodium berlandieri Moq. (2n = 4x = 36) evidenced hybridization signals that were mainly present on 18 chromosomes; however, in the allohexaploid Chenopodium album L. (2n = 6x = 54), cross-hybridization was observed on all of the chromosomes. In situ hybridization with rRNA gene probes indicated that during the evolution of polyploidy, the chenopods lost some of their rDNA loci. Reprobing with rDNA indicated that in the subgenome labeled with 18–24J, one 35S rRNA locus and at least half of the 5S rDNA loci were present. A second analyzed sequence, 12–13P, localized exclusively in pericentromeric regions of each chromosome of C. quinoa and related species. The intensity of the FISH signals differed considerably among chromosomes. The pattern observed on C. quinoa chromosomes after FISH with 12–13P was very similar to GISH results, suggesting that the 12–13P sequence constitutes a major part of the repetitive DNA of C. quinoa.

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