scholarly journals Measurement of transcribed human X-chromosomal DNA sequences transferred to rodent cells by chromosome-mediated gene transfer.

1982 ◽  
Vol 2 (1) ◽  
pp. 52-65 ◽  
Author(s):  
O W McBride ◽  
A S Olsen ◽  
G S Aulakh ◽  
R S Athwal
Keyword(s):  
Dna Sequences ◽  
Genetic Information ◽  
Hybrid Cell ◽  
Single Copy ◽  
Nucleic Acid Hybridization ◽  
Chromosomal Dna ◽  
Metaphase Chromosomes ◽  
Transfer Event ◽  
Donor Chromosome ◽  
Labeled Probe

Transfer of genetic information can be effected by incubation of cultured eucaryotic cells with isolated metaphase chromosomes. In most cases, a resulting transformed cell contains only a fragment of a donor chromosome. The amount of transferred donor DNA has been quantified in 11 independent mouse A9 transformants by nucleic acid hybridization analysis. Each transformant had been selected for hprt (hypoxanthine phosphoribosyltransferase; EC 2.4.2.8) transfer and contained part of the human X chromosome. A labeled probe of transcribed human X-chromosomal DNA was prepared by hybridization of nick-translated unique-sequence human DNA with whole cellular RNA from a human-mouse hybrid cell line, A9/HRBC2-A, containing a single human chromosome., X. The amount of human X-chromosomal DNA in the transformants was quantitated by comparing the hybridization of this probe with transformant and A9/HRBC2-A DNAs. Two unstable transformants which had a microscopically detectable donor chromosome fragment contained 15% of the human X-chromosomal single-copy DNA. Four other unstable transformants contained 4 to 7% of human X-chromosomal DNA sequences. The transferred DNA was below the level of detection in three other unstable and in all three stable transformants. We conclude that the initial transfer event can introduce a substantial amount of genetic information but only smaller amounts of DNA are stably incorporated by integration.

Measurement of transcribed human X-chromosomal DNA sequences transferred to rodent cells by chromosome-mediated gene transfer

1982 ◽  
Vol 2 (1) ◽  
pp. 52-65
Author(s):  
O W McBride ◽  
A S Olsen ◽  
G S Aulakh ◽  
R S Athwal
Keyword(s):  
Dna Sequences ◽  
Genetic Information ◽  
Hybrid Cell ◽  
Single Copy ◽  
Nucleic Acid Hybridization ◽  
Chromosomal Dna ◽  
Metaphase Chromosomes ◽  
Transfer Event ◽  
Donor Chromosome ◽  
Labeled Probe

Transfer of genetic information can be effected by incubation of cultured eucaryotic cells with isolated metaphase chromosomes. In most cases, a resulting transformed cell contains only a fragment of a donor chromosome. The amount of transferred donor DNA has been quantified in 11 independent mouse A9 transformants by nucleic acid hybridization analysis. Each transformant had been selected for hprt (hypoxanthine phosphoribosyltransferase; EC 2.4.2.8) transfer and contained part of the human X chromosome. A labeled probe of transcribed human X-chromosomal DNA was prepared by hybridization of nick-translated unique-sequence human DNA with whole cellular RNA from a human-mouse hybrid cell line, A9/HRBC2-A, containing a single human chromosome., X. The amount of human X-chromosomal DNA in the transformants was quantitated by comparing the hybridization of this probe with transformant and A9/HRBC2-A DNAs. Two unstable transformants which had a microscopically detectable donor chromosome fragment contained 15% of the human X-chromosomal single-copy DNA. Four other unstable transformants contained 4 to 7% of human X-chromosomal DNA sequences. The transferred DNA was below the level of detection in three other unstable and in all three stable transformants. We conclude that the initial transfer event can introduce a substantial amount of genetic information but only smaller amounts of DNA are stably incorporated by integration.

Use of fluorescence in situ hybridization to study the arrangement of DNA sequences in normal and disease-related chromosomes

1995 ◽  
Vol 53 ◽  
pp. 748-749
Author(s):  
Barbara J. F. Trask ◽  
Hillary Massa ◽  
Cynthia Friedman ◽  
Richard Esposito ◽  
Ger van den Engh ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Dna Sequences ◽  
Single Copy ◽  
Two Dimensions ◽  
Genetic Maps ◽  
Epifluorescence Microscopy ◽  
Metaphase Chromosomes ◽  
Interphase Nuclei

The sites of specific DNA sequences can be fluorescently tagged by fluorescence in situ hybridization (FISH). Different sequences can be labeled with different fluorochromes so that their arrangement can be studied using epifluorescence microscopy. The distances between points on the same or different chromosomes can be determined easily in a large number of interphase nuclei or metaphase chromosomes. A variety of probe types, ranging from single-copy sequences to highly repeated sequences can be employed. Our work has focussed on the analysis of hybridization patterns in two dimensions using conventional fluorescence microscopy.We have used FISH to study various aspects of genome organization that are difficult to study using other techniques. Examples of these applications will be presented.FISH is now the method of choice for determining the chromosomal location of DNA sequences. DNA sequences can be positioned in the genome with <1:1000 accuracy (to a 3-Mbp region within a 3000-Mbp genome). Through FISH, the cytogenetic, physical and genetic maps of chromosomes can be linked.

Histochemical aspects of nucleic acid detection

1995 ◽  
Vol 53 ◽  
pp. 746-747
Author(s):  
B. A. Hamkalo ◽  
Elizabeth R. Unger
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
High Resolution ◽  
Dna Sequences ◽  
Single Copy ◽  
Nucleic Acid Detection ◽  
Metaphase Chromosomes ◽  
Potential Applications ◽  
Nucleic Acid Sequences

This symposium brings together several approaches for the detection of specific nucleic acid sequences that have potential applications at the histochemical level.Trask et al. report on the use of fluorescence in situ hybridization (FISH) techniques to study the arrangement of DNA sequences in normal and diseaserelated chromosomes. The sites of specific DNA sequences can be fluorescently tagged. Different sequences can be labeled with different fluorochromes so that their arrangement can be studied using fluorescence microscopy. The distances between points on the same or different chromosomes can be determined in a large number of interphase nuclei or metaphase chromosomes. A variety of probe types, ranging from single-copy sequences to highly repeated sequences can be employed.Hamkalo and co-workers have used non-radioactive methods at the EM level for the detection of nucleic acid sequences by in situ hybridization. Analysis of metaphase chromosomes by electron microscopy allows for high resolution mapping of chromosomes. A variety of labelling procedures have been employed to illustrate the utility of high resolution nucleic acid sequence mapping in these preparations.

Correlating the Genetic and Physical Map of Barley Chromosome 3H Revealed Limitations of the FISH-Based Mapping of Nearby Single-Copy Probes Caused by the Dynamic Structure of Metaphase Chromosomes

2017 ◽  
Vol 152 (2) ◽  
pp. 90-96 ◽  
Author(s):  
Fernanda O. Bustamante ◽  
Lala Aliyeva-Schnorr ◽  
Jörg Fuchs ◽  
Sebastian Beier ◽  
Andreas Houben
Keyword(s):  
Dna Sequences ◽  
Physical Map ◽  
Dynamic Structure ◽  
Single Copy ◽  
Physical Distance ◽  
Genetic Distances ◽  
Genetic Maps ◽  
Metaphase Chromosomes ◽  
Physical Maps

Genetic maps are based on the recombination frequency of molecular markers which often show different positions in comparison to the corresponding physical maps. To decipher the position and order of DNA sequences genetically mapped to terminal and interstitial regions of barley (Hordeum vulgare) chromosome 3H, fluorescence in situ hybridization (FISH) on mitotic metaphase chromosomes was performed with 16 genomic single-copy probes derived from fingerprinted BAC contigs. Long genetic distances at subterminal regions translated into short physical distances, confirming that recombination events occur more often at distal regions of chromosome 3H. Nonoverlapping FISH signals were frequently obtained for probes with a physical distance of at least 30-60 kb. Only 8% of the analyzed chromosomes showed a symmetric order of FISH signals on both sister chromatids. Due to the dynamic packing of metaphase chromatin, the order of 2 adjacent single-copy signals along the chromosome arms outside the (peri)centromeric region can only reliably be determined if the cytological distance is approximately 3%, corresponding to 21.6 Mb.

Nonisotopic in situ hybridization and plant genome mapping: the first 10 years

Genome ◽  
1994 ◽  
Vol 37 (5) ◽  
pp. 717-725 ◽  
Author(s):  
Jiming Jiang ◽  
Bikram S. Gill
Keyword(s):  
In Situ Hybridization ◽  
Physical Mapping ◽  
Dna Sequences ◽  
Genome Mapping ◽  
Molecular Cytogenetics ◽  
Gene Families ◽  
Single Copy ◽  
Plant Genome ◽  
Metaphase Chromosomes

Nonisotopic in situ hybridization (ISH) was introduced in plants in 1985. Since then the technique has been widely used in various areas of plant genome mapping. ISH has become a routine method for physical mapping of repetitive DNA sequences and multicopy gene families. ISH patterns on somatic metaphase chromosomes using tandemly repeated sequences provide excellent physical markers for chromosome identification. Detection of low or single copy sequences were also reported. Genomic in situ hybridization (GISH) was successfully used to analyze the chromosome structure and evolution of allopolyploid species. GISH also provides a powerful technique for monitoring chromatin introgession during interspecific hybridization. A sequential chromosome banding and ISH technique was developed. The sequential technique is very useful for more precise and efficient mapping as well as cytogenetic determination of genomic affinities of individual chromosomes in allopolyploid species. A critical review is made on the present resolution of the ISH technique and the future outlook of ISH research is discussed.Key words: in situ hybridization, physical mapping, genome mapping, molecular cytogenetics.

scholarly journals New Thermosensitive Delivery Vector and Its Use To Enable Nisin-Controlled Gene Expression in Lactobacillus gasseri

2003 ◽  
Vol 69 (3) ◽  
pp. 1377-1382 ◽  
Author(s):  
T. Neu ◽  
B. Henrich
Keyword(s):  
Dna Sequences ◽  
Expression System ◽  
Single Copy ◽  
Temperature Sensitive ◽  
Chromosomal Dna ◽  
Erythromycin Resistance ◽  
Lactobacillus Curvatus ◽  
Lactobacillus Gasseri ◽  
Delivery Vector ◽  
Derivatives Of

ABSTRACT Derivatives of a cryptic plasmid from Lactobacillus curvatus showed temperature-sensitive replication in thermophilic lactobacilli. The thermosensitive replicon was used to construct the new delivery vector pTN1, which allows site-specific replacement of chromosomal DNA sequences. pTN1 carries an erythromycin resistance marker suitable for selection of single-copy integrants and replicates readily at 35°C, whereas replication is efficiently shut down at 42°C. To demonstrate the functionality of pTN1, the signal transduction genes (nisRK) of the nisin-controlled expression system were integrated downstream of the pepN gene into the chromosome of Lactobacillus gasseri. In the resulting strain, UKLbg1, expression of nisRK was likely driven by cotranscription with pepN and enabled nisin-dependent induction of a fusion of a reporter gene (pepI) to the nisA promoter. The induction rates were correlated with the amount of nisin used, and maximum pepI expression was achieved with nisin concentrations (above 25 ng/ml) at which growth of the bacteria was already inhibited.

scholarly journals Localization of Single- and Low-Copy Sequences on Tomato Synaptonemal Complex Spreads Using Fluorescence in Situ Hybridization (FISH)

Genetics ◽  
1999 ◽  
Vol 152 (1) ◽  
pp. 427-439 ◽  
Author(s):  
Daniel G Peterson ◽  
Nora L V Lapitan ◽  
Stephen M Stack
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Synaptonemal Complex ◽  
Dna Sequences ◽  
Single Copy ◽  
Differential Staining ◽  
Rflp Markers ◽  
Metaphase Chromosomes ◽  
Fish Mapping

Abstract Fluorescence in situ hybridization (FISH) is a powerful means by which single- and low-copy DNA sequences can be localized on chromosomes. Compared to the mitotic metaphase chromosomes that are normally used in FISH, synaptonemal complex (SC) spreads (hypotonically spread pachytene chromosomes) have several advantages. SC spreads (1) are comparatively free of debris that can interfere with probe penetration, (2) have relatively decondensed chromatin that is highly accessible to probes, and (3) are about ten times longer than their metaphase counterparts, which permits FISH mapping at higher resolution. To investigate the use of plant SC spreads as substrates for single-copy FISH, we probed spreads of tomato SCs with two single-copy sequences and one low-copy sequence (ca. 14 kb each) that are associated with restriction fragment length polymorphism (RFLP) markers on SC 11. Individual SCs were identified on the basis of relative length, arm ratio, and differential staining patterns after combined propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) staining. In this first report of single-copy FISH to SC spreads, the probe sequences were unambiguously mapped on the long arm of tomato SC 11. Coupled with data from earlier studies, we determined the distance in micrometers, the number of base pairs, and the rates of crossing over between these three FISH markers. We also observed that the order of two of the FISH markers is reversed in relation to their order on the molecular linkage map. SC-FISH mapping permits superimposition of markers from molecular linkage maps directly on pachytene chromosomes and thereby contributes to our understanding of the relationship between chromosome structure, gene activity, and recombination.

Active maize genes are unmodified and flanked by diverse classes of modified, highly repetitive DNA

Genome ◽  
1994 ◽  
Vol 37 (4) ◽  
pp. 565-576 ◽  
Author(s):  
Jeffrey L. Bennetzen ◽  
Kathrin Schrick ◽  
Patricia S. Springer ◽  
Willis E. Brown ◽  
Phillip SanMiguel
Keyword(s):  
Repetitive Dna ◽  
Dna Sequences ◽  
Copy Number ◽  
Repetitive Sequences ◽  
Restriction Enzymes ◽  
Single Copy ◽  
Chromosomal Dna ◽  
Coding Regions ◽  
Highly Repetitive Dna ◽  
Copy Dna

We have characterized the copy number, organization, and genomic modification of DNA sequences within and flanking several maize genes. We found that highly repetitive DNA sequences were tightly linked to most of these genes. The highly repetitive sequences were not found within the coding regions but could be found within 6 kb either 3′ or 5′ to the structural genes. These highly repetitive regions were each composed of unique combinations of different short repetitive sequences. Highly repetitive DNA blocks were not interrupted by any detected single copy DNA. The 13 classes of highly repetitive DNA identified were found to vary little between diverse Zea isolates. The level of DNA methylation in and near these genes was determined by scoring the digestibility of 63 recognition/cleavage sites with restriction enzymes that were sensitive to 5-methylation of cytosines in the sequences 5′-CG-3′ and 5′-CNG-3′. All but four of these sites were digestible in chromosomal DNA. The four undigested sites were localized to extragenic DNA within or near highly repetitive DNA, while the other 59 sites were in low copy number DNAs. Pulsed field gel analysis indicated that the majority of cytosine modified tracts range from 20 to 200 kb in size. Single copy sequences hybridized to the unmodified domains, while highly repetitive sequences hybridized to the modified regions. Middle repetitive sequences were found in both domains.Key words: genome organization, interspersed repetitive DNA, DNA modification.

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.

Mapping by Sequencing the Pneumocystis Genome Using the Ordering DNA Sequences V3 Tool

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1299-1313
Author(s):  
Zheng Xu ◽  
Britton Lance ◽  
Claudia Vargas ◽  
Budak Arpinar ◽  
Suchendra Bhandarkar ◽  
...  
Keyword(s):  
Physical Mapping ◽  
Dna Sequences ◽  
Pneumocystis Carinii ◽  
Single Copy ◽  
Fungal Genome ◽  
Mapping Data ◽  
Genome Database ◽  
Rdna Genes ◽  
Genome Map ◽  
Mapping By Sequencing

Abstract A bioinformatics tool called ODS3 has been created for mapping by sequencing. The tool allows the creation of integrated genomic maps from genetic, physical mapping, and sequencing data and permits an integrated genome map to be stored, retrieved, viewed, and queried in a stand-alone capacity, in a client/server relationship with the Fungal Genome Database (FGDB), and as a web-browsing tool for the FGDB. In that ODS3 is programmed in Java, the tool promotes platform independence and supports export of integrated genome-mapping data in the extensible markup language (XML) for data interchange with other genome information systems. The tool ODS3 is used to create an initial integrated genome map of the AIDS-related fungal pathogen, Pneumocystis carinii. Contig dynamics would indicate that this physical map is ∼50% complete with ∼200 contigs. A total of 10 putative multigene families were found. Two of these putative families were previously characterized in P. carinii, namely the major surface glycoproteins (MSGs) and HSP70 proteins; three of these putative families (not previously characterized in P. carinii) were found to be similar to families encoding the HSP60 in Schizosaccharomyces pombe, the heat-shock Ψ protein in S. pombe, and the RNA synthetase family (i.e., MES1) in Saccharomyces cerevisiae. Physical mapping data are consistent with the 16S, 5.8S, and 26S rDNA genes being single copy in P. carinii. No other fungus outside this genus is known to have the rDNA genes in single copy.

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