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.

scholarly journals Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

1983 ◽  
Vol 3 (11) ◽  
pp. 2089-2098 ◽  
Author(s):  
J Zieg ◽  
C E Clayton ◽  
F Ardeshir ◽  
E Giulotto ◽  
E A Swyryd ◽  
...  
Keyword(s):  
Cell Lines ◽  
Syrian Hamster ◽  
Resistant Cell ◽  
Single Step ◽  
Similar Degree ◽  
Fluctuation Analysis ◽  
Aspartate Transcarbamylase ◽  
Resistant Lines ◽  
Cad Gene

Eleven independent lines of Syrian hamster cells were selected by using very low levels of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbamylase. The protocol employed insured that each resistant cell arose during one of the last divisions before selection was applied. Cells of each mutant line contained an amplification of the structural gene for CAD, a trifunctional protein which includes aspartate transcarbamylase and two other enzymes of UMP biosynthesis. Strikingly, despite the minimal selection employed, the degree of amplification of the CAD gene was 6 to 10 times the normal diploid number in all 11 cases. In situ hybridization indicated that the amplified CAD genes were almost always present at a single chromosomal site in each line. Therefore, one of the two alleles was amplified 11- to 19-fold. The rates at which cells became resistant to PALA, determined by fluctuation analysis, were 100 times less dependent on drug concentration than were the frequencies of resistant cells in steady-state populations. The relatively shallow dependence of this rate upon PALA concentration is consistent with our independent observation that most events gave rise to a similar degree of amplification. In six of six cell lines examined, the levels of CAD mRNA and aspartate transcarbamylase activity were elevated two- to fourfold. These lines were resistant to PALA concentrations 20- to 80-fold higher than the ones used for selection. The organization of amplified DNA was examined by hybridizing Southern blots with cloned DNA fragments containing amplified sequences, previously isolated from two cell lines resistant to high levels of PALA. A contiguous region of DNA approximately 44 kilobases long which included the CAD gene was amplified in five of five single-step mutants examined. Outside this region, these mutants shared amplified sequences with only one of the two highly resistant lines.

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.

Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate

1983 ◽  
Vol 3 (11) ◽  
pp. 2089-2098
Author(s):  
J Zieg ◽  
C E Clayton ◽  
F Ardeshir ◽  
E Giulotto ◽  
E A Swyryd ◽  
...  
Keyword(s):  
Cell Lines ◽  
Syrian Hamster ◽  
Resistant Cell ◽  
Single Step ◽  
Similar Degree ◽  
Fluctuation Analysis ◽  
Aspartate Transcarbamylase ◽  
Resistant Lines ◽  
Cad Gene

Eleven independent lines of Syrian hamster cells were selected by using very low levels of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbamylase. The protocol employed insured that each resistant cell arose during one of the last divisions before selection was applied. Cells of each mutant line contained an amplification of the structural gene for CAD, a trifunctional protein which includes aspartate transcarbamylase and two other enzymes of UMP biosynthesis. Strikingly, despite the minimal selection employed, the degree of amplification of the CAD gene was 6 to 10 times the normal diploid number in all 11 cases. In situ hybridization indicated that the amplified CAD genes were almost always present at a single chromosomal site in each line. Therefore, one of the two alleles was amplified 11- to 19-fold. The rates at which cells became resistant to PALA, determined by fluctuation analysis, were 100 times less dependent on drug concentration than were the frequencies of resistant cells in steady-state populations. The relatively shallow dependence of this rate upon PALA concentration is consistent with our independent observation that most events gave rise to a similar degree of amplification. In six of six cell lines examined, the levels of CAD mRNA and aspartate transcarbamylase activity were elevated two- to fourfold. These lines were resistant to PALA concentrations 20- to 80-fold higher than the ones used for selection. The organization of amplified DNA was examined by hybridizing Southern blots with cloned DNA fragments containing amplified sequences, previously isolated from two cell lines resistant to high levels of PALA. A contiguous region of DNA approximately 44 kilobases long which included the CAD gene was amplified in five of five single-step mutants examined. Outside this region, these mutants shared amplified sequences with only one of the two highly resistant lines.

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.

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.

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.

scholarly journals Combination of Direct Viable Count and Fluorescent In Situ Hybridization (DVC-FISH) as a Potential Method for Identifying Viable Vibrio parahaemolyticus in Oysters and Mussels

Foods ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1502
Author(s):  
Jorge García-Hernández ◽  
Manuel Hernández ◽  
Yolanda Moreno
Keyword(s):  
In Situ Hybridization ◽  
Vibrio Parahaemolyticus ◽  
Fluorescent In Situ Hybridization ◽  
Potential Method ◽  
Viable Count ◽  
Hybridization Technique ◽  
Fish Technique ◽  
Viable Cells

Vibrio parahaemolyticus is a human food-borne pathogen with the ability to enter the food chain. It is able to acquire a viable, non-cultivable state (VBNC), which is not detected by traditional methods. The combination of the direct viable count method and a fluorescent in situ hybridization technique (DVC-FISH) makes it possible to detect microorganisms that can present VBNC forms in complex samples The optimization of the in vitro DVC-FISH technique for V. parahaemolyticus was carried out. The selected antibiotic was ciprofloxacin at a concentration of 0.75 μg/mL with an incubation time in DVC broth of 5 h. The DVC-FISH technique and the traditional plate culture were applied to detect and quantify the viable cells of the affected pathogen in artificially contaminated food matrices at different temperatures. The results obtained showed that low temperatures produced an important logarithmic decrease of V. parahaemolyticus, while at 22 °C, it proliferated rapidly. The DVC-FISH technique proved to be a useful tool for the detection and quantification of V. parahaemolyticus in the two seafood matrices of oysters and mussels. This is the first study in which this technique has been developed to detect viable cells for this microorganism.

Microbial ecology of sulfate-reducing bacteria in wastewater biofilms analyzed by microelectrodes and fish (fluorescent in situ hybridization) technique

1999 ◽  
Vol 39 (7) ◽  
pp. 41-47 ◽  
Author(s):  
Satoshi Okabe ◽  
Hisashi Satoh ◽  
Tsukasa Itoh ◽  
Yoshimasa Watanabe
Keyword(s):  
In Situ Hybridization ◽  
Sulfate Reduction ◽  
Sulfate Reducing Bacteria ◽  
Hybridization Technique ◽  
Concentration Profiles ◽  
Reduction Zone ◽  
Sulfate Reducing ◽  
Reducing Bacteria ◽  
Culture Dependent

The vertical distribution of sulfate-reducing bacteria (SRB) in microaerophilic wastewater biofilms grown on fully submerged rotating disk reactors (RDR) was determined by the conventional culture-dependent MPN method and in situ hybridization of fluorescently-labelled 16S rRNA-targeted oligonucleotide probes for SRB in parallel. Chemical concentration profiles within the biofilm were also measured using microelectrodes for O2, S2-, NO3- and pH. In situ hybridization revealed that the SRB probe-stained cells were distributed throughout the biofilm even in the oxic surface zone in all states from single scattered cells to clustered cells. The higher fluorescence intensity and abundance of SRB probe-stained cells were found in the middle part of the biofilm. This result corresponded well with O2 and H2S concentration profiles measured by microelectrodes, showing sulfate reduction was restricted to a narrow anaerobic zone located about 500 μm below the biofilm surface. Results of the MPN and potential sulfate reducing activity (culture-dependent approaches) indicated a similar distribution of cultivable SRB in the biofilm. The majority of the general SRB probe-stained cells were hybridized with SRB 660 probe, suggesting that one important member of the SRB in the wastewater biofilm could be the genus Desulfobulbus. An addition of nitrate forced the sulfate reduction zone deeper in the biofilm and reduced the specific sulfate reduction rate as well. The sulfate reduction zone was consequently separated from O2 and NO3- respiration zones. Anaerobic H2S oxidation with NO3- was also induced by addition of nitrate to the medium.

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