scholarly journals Co-amplification of rRNA genes with CAD genes in N-(phosphonacetyl)-L-aspartate-resistant Syrian hamster cells.

1983 ◽  
Vol 3 (11) ◽  
pp. 2066-2075 ◽  
Author(s):  
G M Wahl ◽  
L Vitto ◽  
J Rubnitz
Keyword(s):  
In Situ Hybridization ◽  
Syrian Hamster ◽  
Blot Hybridization ◽  
Nucleolus Organizer ◽  
Rrna Genes ◽  
28S Rrna ◽  
Metaphase Chromosomes ◽  
Rdna Genes ◽  
Resistant Mutants

The amplified CAD genes in N-(phosphonacetyl)-L-aspartate (PALA)-resistant Syrian hamster cells are located in an expanded chromosomal region emanating from the site of the wild-type gene at the tip of the short arm of chromosome B-9. The terminus of B-9 in PALA-sensitive cells contains a cluster of rRNA genes (i.e., a nucleolus organizer, rDNA). We have used a molecular clone containing sequences complementary to Syrian hamster 28S rRNA to investigate whether rDNA is coamplified with CAD genes in the PALA-resistant mutants. In situ hybridization of this probe to metaphase chromosomes demonstrates that rDNA and CAD genes do coamplify in two independently isolated PALA-resistant mutants. The tight linkage of CAD and rDNA genes was demonstrated by their coordinate translocation from B-9 to the end of the long arm of chromosome C-11 in one mutant. Blot hybridization studies substantiate the in situ hybridization results. Both types of analysis indicate that only one or two rDNA genes, on the average, are coamplified per CAD gene. The data are consistent with the model that unequal exchanges between rDNA genes mediate the amplification of CAD genes in the Syrian hamster mutants that were analyzed.

Co-amplification of rRNA genes with CAD genes in N-(phosphonacetyl)-L-aspartate-resistant Syrian hamster cells

1983 ◽  
Vol 3 (11) ◽  
pp. 2066-2075
Author(s):  
G M Wahl ◽  
L Vitto ◽  
J Rubnitz
Keyword(s):  
In Situ Hybridization ◽  
Syrian Hamster ◽  
Blot Hybridization ◽  
Nucleolus Organizer ◽  
Rrna Genes ◽  
28S Rrna ◽  
Metaphase Chromosomes ◽  
Rdna Genes ◽  
Resistant Mutants

The amplified CAD genes in N-(phosphonacetyl)-L-aspartate (PALA)-resistant Syrian hamster cells are located in an expanded chromosomal region emanating from the site of the wild-type gene at the tip of the short arm of chromosome B-9. The terminus of B-9 in PALA-sensitive cells contains a cluster of rRNA genes (i.e., a nucleolus organizer, rDNA). We have used a molecular clone containing sequences complementary to Syrian hamster 28S rRNA to investigate whether rDNA is coamplified with CAD genes in the PALA-resistant mutants. In situ hybridization of this probe to metaphase chromosomes demonstrates that rDNA and CAD genes do coamplify in two independently isolated PALA-resistant mutants. The tight linkage of CAD and rDNA genes was demonstrated by their coordinate translocation from B-9 to the end of the long arm of chromosome C-11 in one mutant. Blot hybridization studies substantiate the in situ hybridization results. Both types of analysis indicate that only one or two rDNA genes, on the average, are coamplified per CAD gene. The data are consistent with the model that unequal exchanges between rDNA genes mediate the amplification of CAD genes in the Syrian hamster mutants that were analyzed.

Karyotyping Melandrium album, a dioecious plant with heteromorphic sex chromosomes

Genome ◽  
1990 ◽  
Vol 33 (4) ◽  
pp. 556-562 ◽  
Author(s):  
D. D. Ciupercescu ◽  
J. Veuskens ◽  
A. Mouras ◽  
D. Ye ◽  
M. Briquet ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Sex Chromosomes ◽  
Nucleolus Organizer ◽  
Rrna Genes ◽  
Hybridization Technique ◽  
Metaphase Chromosomes ◽  
Nucleolus Organizer Regions ◽  
Melandrium Album ◽  
Group B

Mitotic metaphase chromosomes of Melandrium album obtained from root protoplasts were studied. Morphologically, the chromosomes were either metacentrics or submetacentrics. They were classified into three distinct groups: group A comprising six pairs of autosomal metacentrics, group B comprising five pairs of autosomal submetacentrics, and the sex chromosomes: X and Y. The X chromosome is a metacentric (r = 1.44), which accounts for more than 14% of the genome. The Y chromosome is a metacentric with, virtually, equal arms (r = 1.09) and accounts for 21% of the genome, being the largest of the complement. The Y:X ratio was 1.4. Ethidium bromide, caffeine, and vinblastine were used to obtain a better resolution and higher frequency of satellited chromosomes 7q and 9p. The proposed karyotype of M. album is 2n = 24, XX, s(7q;9p) for female and 2n = 24, XY, s(7q;9p) for male plants. Nucleolus organizer regions (NORs) were present at the telomeric sites of three chromosome pairs: 7q, 9p, and 10p. The NORs were polymorphic, particularly between the nonhomologous chromosomes. The in situ hybridization technique localized the rRNA genes on four chromosome pairs: 5p, 7q, 9p, and 10p. The discrepancy between the NORs and the hybridization signals was probably due to the fact that NORs were restricted only to transcriptionally active rRNA genes. It was concluded that for a complete description and characterization of rRNA genes, both NOR detection and in situ hybridization techniques, as complementary methods, should be employed.Key words: Melandrium album, karyotype, satellites, idiogram, nucleolus organizer regions, in situ hybridization.

Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization

1982 ◽  
Vol 2 (3) ◽  
pp. 308-319
Author(s):  
G M Wahl ◽  
L Vitto ◽  
R A Padgett ◽  
G R Stark
Keyword(s):  
In Situ Hybridization ◽  
Syrian Hamster ◽  
Specific Radioactivity ◽  
Large Chromosome ◽  
Hybridization Technique ◽  
Wild Type ◽  
Aspartate Transcarbamylase ◽  
Metaphase Chromosomes ◽  
Cad Gene

Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional protein CAD, overproduce this protein as a result of amplification of the CAD gene. We have used a sensitive in situ hybridization technique to localize CAD genomes in spreads of metaphase chromosomes from several independent PALA-resistant lines and from wild-type PALA-sensitive cells. The amplified genes were always found within chromosomes, usually in an expanded region of the short arm of chromosome B9. In wild-type cells, the CAD gene was also on the short arm of chromosome B9. In one mutant line, 90 to 100 CAD genes were found within an expanded B9 chromosome and 10 to 15 more were near the distal end of one arm of several different chromosomes. Another line contained most the genes in a telomeric chromosome or large chromosome fragment. The amplified genes were in chromosomal regions that were stained in a banded pattern by trypsin-Giemsa. A few double minute chromosomes were observed in a very small fraction of the total spreads examined. The it situ hybridizations were performed in the presence of 10% dextral sulfate 500, which increases the signal by as much as 100-fold. Using recombinant DNA plasmids nick-translated with [125I]dCTP to high specific radioactivity, 10 CAD genes in a single chromosomal region were revealed after 1 week of autoradiographic exposure, and the position of the unique gene could be seen after 1 month.

scholarly journals Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization.

1982 ◽  
Vol 2 (3) ◽  
pp. 308-319 ◽  
Author(s):  
G M Wahl ◽  
L Vitto ◽  
R A Padgett ◽  
G R Stark
Keyword(s):  
In Situ Hybridization ◽  
Syrian Hamster ◽  
Specific Radioactivity ◽  
Large Chromosome ◽  
Hybridization Technique ◽  
Wild Type ◽  
Aspartate Transcarbamylase ◽  
Metaphase Chromosomes ◽  
Cad Gene

Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional protein CAD, overproduce this protein as a result of amplification of the CAD gene. We have used a sensitive in situ hybridization technique to localize CAD genomes in spreads of metaphase chromosomes from several independent PALA-resistant lines and from wild-type PALA-sensitive cells. The amplified genes were always found within chromosomes, usually in an expanded region of the short arm of chromosome B9. In wild-type cells, the CAD gene was also on the short arm of chromosome B9. In one mutant line, 90 to 100 CAD genes were found within an expanded B9 chromosome and 10 to 15 more were near the distal end of one arm of several different chromosomes. Another line contained most the genes in a telomeric chromosome or large chromosome fragment. The amplified genes were in chromosomal regions that were stained in a banded pattern by trypsin-Giemsa. A few double minute chromosomes were observed in a very small fraction of the total spreads examined. The it situ hybridizations were performed in the presence of 10% dextral sulfate 500, which increases the signal by as much as 100-fold. Using recombinant DNA plasmids nick-translated with [125I]dCTP to high specific radioactivity, 10 CAD genes in a single chromosomal region were revealed after 1 week of autoradiographic exposure, and the position of the unique gene could be seen after 1 month.

Distribution of rDNA and 28S, 18S, and 5S rRNA in micronuclei containing a single chromosome

1990 ◽  
Vol 68 (6) ◽  
pp. 957-964 ◽  
Author(s):  
B. Labidi ◽  
F. Broders ◽  
J. L. Meyer ◽  
D. Hernandez-Verdun
Keyword(s):  
In Situ Hybridization ◽  
Northern Blotting ◽  
Ribosomal Genes ◽  
5S Rrna ◽  
Rrna Genes ◽  
28S Rrna ◽  
X Chromosomes ◽  
Single Chromosome ◽  
Cell Flow Cytometry

Investigating genes and their transcription products in nuclear compartments corresponding to one mammalian chromosome, the ribosomal genes 18S–28S and 5S were localized in PtK1 micronucleated cells and rRNA was characterized in sorted micronuclei containing single identified chromosomes. In situ hybridization revealed the presence of 18S–28S rRNA genes in two micronuclei per cell and 5S rRNA genes in four micronuclei per cell. Flow cytometry histograms of isolated micronuclei stained with Hoechst 33342 exhibited five peaks (a–e) in which peaks b and c, respectively, corresponded to chromosomes 4 and X. Restricted genomic DNA from sorted peak c micronuclei showed the presence of 28S gene sequences. Direct sorting of the micronuclei from each peak on nitrocellulose and their hybridization with the 18S–28S rDNA probe revealed that the rRNA genes were exclusively located in micronuclei containing X chromosomes. Northern blotting showed the presence of 18S–28S and 5S rRNAs in peak c micronuclei and their absence from peak b micronuclei. Consequently, these procedures allowed us to show the presence of ribosomal genes and the corresponding rRNA in micronuclei containing single X chromosomes, and the absence of rRNA from micronuclei that do not contain the ribosomal genes. In regards to the transcription of these genes, the micronuclei from peak c can be considered as functional interphase X chromosomes.Key words: micronuclei, rDNA, rRNA, single chromosome, in situ hybridization.

DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 496-497
Author(s):  
Barbara Trask ◽  
Susan Allen ◽  
Anne Bergmann ◽  
Mari Christensen ◽  
Anne Fertitta ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Dual Band ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

Karyotypes and Distribution of Tandem Repeat Sequences in Brassica nigra Determined by Fluorescence in situ Hybridization

2017 ◽  
Vol 152 (3) ◽  
pp. 158-165 ◽  
Author(s):  
Gui-xiang Wang ◽  
Qun-yan He ◽  
Jiri Macas ◽  
Petr Novák ◽  
Pavel Neumann ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Fluorescence Intensity ◽  
Tandem Repeat ◽  
Brassica Nigra ◽  
Metaphase Chromosomes ◽  
Repeat Sequences ◽  
Tandem Repeat Sequences ◽  
Black Mustard

Whole-genome shotgun reads were analyzed to determine the repeat sequence composition in the genome of black mustard, Brassica nigra (L.) Koch. The analysis showed that satellite DNA sequences are very abundant in the black mustard genome. The distribution pattern of 7 new tandem repeats (BnSAT13, BnSAT28, BnSAT68, BnSAT76, BnSAT114, BnSAT180, and BnSAT200) on black mustard chromosomes was visualized using fluorescence in situ hybridization (FISH). The FISH signals of BnSAT13 and BnSAT76 provided useful cytogenetic markers; their position and fluorescence intensity allowed for unambiguous identification of all 8 somatic metaphase chromosomes. A karyotype showing the location and fluorescence intensity of these tandem repeat sequences together with the position of rDNAs and centromeric retrotransposons of Brassica (CRB) was constructed. The establishment of the FISH-based karyotype in B. nigra provides valuable information that can be used in detailed analyses of B. nigra accessions and derived allopolyploid Brassica species containing the B genome.

Optimization of in situ hybridization to human metaphase chromosomes

1989 ◽  
Vol 182 (1) ◽  
pp. 25-31 ◽  
Author(s):  
Bodil Lomholt ◽  
Pernille Dissing Sørensen ◽  
Henrik Simonsen ◽  
Sune Frederiksen
Keyword(s):  
In Situ Hybridization ◽  
Metaphase Chromosomes

The use of double fluorescence in situ hybridization to physically map the positions of 5S rDNA genes in relation to the chromosomal location of 18S–5.8S–26S rDNA and a C genome specific DNA sequence in the genus Avena

Genome ◽  
1996 ◽  
Vol 39 (3) ◽  
pp. 535-542 ◽  
Author(s):  
Concha Linares ◽  
Juan González ◽  
Esther Ferrer ◽  
Araceli Fominaya
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Diploid Species ◽  
5S Rdna ◽  
Rdna Genes ◽  
26S Rdna ◽  
A Genome ◽  
Rdna Loci ◽  
C Genome

A physical map of the locations of the 5S rDNA genes and their relative positions with respect to 18S–5.8S–26S rDNA genes and a C genome specific repetitive DNA sequence was produced for the chromosomes of diploid, tetraploid, and hexaploid oat species using in situ hybridization. The A genome diploid species showed two pairs of rDNA loci and two pairs of 5S loci located on both arms of one pair of satellited chromosomes. The C genome diploid species showed two major pairs and one minor pair of rDNA loci. One pair of subtelocentric chromosomes carried rDNA and 5S loci physically separated on the long arm. The tetraploid species (AACC genomes) arising from these diploid ancestors showed two pairs of rDNA loci and three pairs of 5S loci. Two pairs of rDNA loci and 2 pairs of 5S loci were arranged as in the A genome diploid species. The third pair of 5S loci was located on one pair of A–C translocated chromosomes using simultaneous in situ hybridization with 5S rDNA genes and a C genome specific repetitive DNA sequence. The hexaploid species (AACCDD genomes) showed three pairs of rDNA loci and six pairs of 5S loci. One pair of 5S loci was located on each of two pairs of C–A/D translocated chromosomes. Comparative studies of the physical arrangement of rDNA and 5S loci in polyploid oats and the putative A and C genome progenitor species suggests that A genome diploid species could be the donor of both A and D genomes of polyploid oats. Key words : oats, 5S rDNA genes, 18S–5.8S–26S rDNA genes, C genome specific repetitive DNA sequence, in situ hybridization, genome evolution.

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