Isolation and expression of a new mouse homeobox gene

Development ◽  
1988 ◽  
Vol 102 (2) ◽  
pp. 397-407 ◽  
Author(s):  
P.T. Sharpe ◽  
J.R. Miller ◽  
E.P. Evans ◽  
M.D. Burtenshaw ◽  
S.J. Gaunt
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Southern Blotting ◽  
Homeobox Gene ◽  
Single Copy ◽  
Northern Blotting ◽  
Metaphase Chromosomes ◽  
Rnase Protection ◽  
Sequence Encoding

A homeobox-containing clone has been isolated from an adult mouse kidney cDNA library and shown by DNA sequence analysis to be a new isolate, Hox-6.1. A genomic clone containing Hox-6.1 has been isolated and found to contain another putative homeobox sequence (Hox-6.2), within 7 kb of Hox-6.1. In situ hybridization of mouse metaphase chromosomes shows this Hox-6 locus to be located on chromosome 14 (14E2). Hox-6.1 has been studied in detail and the predicted protein sequence of the homeobox is 100% homologous to the Xenopus Xeb1 (formally AC1) homeobox and the human c8 homeobox (Carrasco et al. 1984; Boncinelli et al. 1985; Simeone et al. 1987). Southern blotting shows that the DNA sequence encoding Hox-6.1 is single copy. Expression of Hox-6.1 has been studied in adult tissues and embryos by RNase protection assays, Northern blotting analysis and in situ hybridization. RNase protection assays show that Hox-6.1 transcripts are present in embryos between days 9 1/2 and 13 1/2 of gestation and in extraembryonic tissues at day 9 1/2. Adult expression is detectable in kidney and testis but not in liver, spleen and brain. One major transcript is detectable on Northern blots of kidney and day-13 1/2 embryo RNA. In kidney, this transcript is 2.7 kb whereas in embryos the major transcript is smaller at 1.9 kb, a much fainter band being visible at 2.7 kb. Localized expression of Hox-6.1 is observed in the spinal cord and prevertebral column of day-12 1/2 embryos, and in the posterior mesoderm and ectoderm of day-8 1/4 embryos. An anterior boundary of expression is located just behind the hindbrain whereas the boundary in the mesoderm is located at the level of the 7th prevertebra.

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.

Organization and distribution of a Sau3A tandem repeated DNA sequence in Picea (Pinaceae) species

Genome ◽  
1998 ◽  
Vol 41 (4) ◽  
pp. 560-565 ◽  
Author(s):  
Garth R Brown ◽  
Craig H Newton ◽  
John E Carlson
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Picea Glauca ◽  
Tandem Repeats ◽  
Picea Sitchensis ◽  
Repeated Dna ◽  
Metaphase Chromosomes ◽  
Genes Encoding ◽  
Repeated Dna Sequence

Repeated DNA families contribute to the large genomes of coniferous trees but are poorly characterized. We report the analysis of a 142 bp tandem repeated DNA sequence identified by the restriction enzyme Sau3A and found in approximately 20 000 copies in Picea glauca. Southern hybridization indicated that the repeated DNA family is specific to the genus, was amplified early in its evolution, and has undergone little structural alteration over evolutionary time. Fluorescence in situ hybridization localized arrays of the Sau3A repeating element to the centromeric regions of different subsets of the metaphase chromosomes of P. glauca and the closely related Picea sitchensis, suggesting that mechanisms leading to the intragenomic movement of arrays may be more active than those leading to mutation of the repeating elements themselves. Unambiguous identification of P. glauca and P. sitchensis chromosomes was made possible by co-localizing the Sau3A tandem repeats and the genes encoding the 5S and 18S-5.8S-26S ribosomal RNAs.Key words: Picea, repeated DNA, in situ hybridization, centromere.

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.

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.

Physical localization of single-copy sequences on pachytene chromosomes in maize (Zea mays L.) by chromosome in situ suppression hybridization

Genome ◽  
2000 ◽  
Vol 43 (6) ◽  
pp. 1081-1083 ◽  
Author(s):  
Monther T Sadder ◽  
Norbert Ponelies ◽  
Ute Born ◽  
Gerd Weber
Keyword(s):  
Zea Mays ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Dna Sequence ◽  
Zea Mays L ◽  
Single Copy ◽  
Chromosome 9 ◽  
Copy Dna ◽  
Single Copy Dna

A new approach for locating single-copy DNA sequences on pachytene chromosomes of maize (Zea mays L.) was developed. A cosmid clone with homologous sequences to a molecular marker (umc105a) linked to a quantitative trait locus (QTL) for resistance against sugarcane borer (SCB) was physically mapped by fluorescence in situ hybridization (FISH) to the short arm of chromosome 9. The marker umc105a was genetically placed in the centromeric region. To suppress signals generated by maize repetitive DNA, competitive in situ suppression (CISS) hybridization was necessary to obtain specific signals from umc105a. A centromere specific DNA probe (CentC) was used in a double-labeling technique as a reference marker. Fluorescence signals generated by umc105a cosmid and CentC were specific and highly reproducible. Thus the single-copy DNA sequence of umc105a was physically localized on the short arm of chromosome 9 near the telomere. This is the first report of physical localization of single-copy DNA sequence by CISS hybridization to a maize pachytene chromosome.Key words: fluorescence in situ hybridization, maize, pachytene chromosome, single-copy sequence, CISS hybridization.

scholarly journals Sensitive Multicolor Fluorescence In Situ Hybridization Using Catalyzed Reporter Deposition (CARD) Amplification

1997 ◽  
Vol 45 (10) ◽  
pp. 1439-1446 ◽  
Author(s):  
Ernst J.M. Speel ◽  
Frans C.S. Ramaekers ◽  
Anton H.N. Hopman
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Peroxidase Activity ◽  
Single Copy ◽  
Metaphase Chromosomes ◽  
Detection Systems ◽  
Copy Dna ◽  
Catalyzed Reporter Deposition ◽  
Single Copy Dna

We describe the simultaneous localization of DNA sequences in cell and chromosome preparations by means of differently fluorochrome-labeled (AMCA, FITC, TRITC) tyramides using the catalyzed reporter deposition (CARD) procedure. For this purpose, repeated as well as single-copy DNA probes were labeled with biotin, digoxigenin, and FITC, hybridized, and visualized with three different cytochemical detection systems based on horseradish peroxidase conjugates. These were sequentially applied to interphase nuclei and metaphase chromosomes at low concentrations to prevent crossreaction and nonspecific background. In situ localized peroxidase activity was visualized by the deposition of fluorochrome-labeled tyramide molecules. To allow specific deposition of a second and a third tyramide conjugate for multiple-target fluorescence in situ hybridization (FISH), remaining peroxidase activity was always completely inactivated by a mild acid treatment before application of the next peroxidase conjugate. The CARD reactions were optimized for maximal signal-to-noise ratio and discrete localization by tuning reaction time, H2O2, and tyramide concentrations. For both repeated and single-copy DNA targets, high FISH signal intensities were obtained, providing improvement of sensitivity over conventional indirect detection systems. In addition, the fluorescence CARD detection system proved to be highly efficient and easy to implement in multiple-labeling studies, such as reported here for FISH.

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.

Detection of a 2.6 kb single/low copy DNA sequence on chromosomes of wheat (Triticum aestivum) and rye (Secale cereale) by fluorescence in situ hybridization

Genome ◽  
1995 ◽  
Vol 38 (2) ◽  
pp. 246-249 ◽  
Author(s):  
Haishui Dong ◽  
James S. Quick
Keyword(s):  
Triticum Aestivum ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Dna Sequence ◽  
Secale Cereale ◽  
Genomic Library ◽  
Fluorescein Isothiocyanate ◽  
Single Copy ◽  
Copy Dna

A fluorescence in situ hybridization procedure was developed to detect a 2.6 kb single/low copy DNA sequence on chromosomes of wheat (Triticum aestivum) and rye (Secale cereale). The probe, pTtksuI26, was from the wheat genomic library generated at Kansas State University and was labeled with Bio-11-dUTP by nick translation. The signal was amplified by a protocol of avidin–FITC (fluorescein isothiocyanate), biotinylated goat anti-avidin antibody, avidin–FITC. Two rye chromosomes and several wheat chromosomes showed the hybridization sites. The multiple sites in wheat are probably the result of the homoeologous nature of the three genomes in wheat. The ability to detect the hybridization signal of a small single/low copy DNA sequence is a very important step towards the physical mapping of plant genomes. This procedure might also be useful for studying the genomic relationship among wheat relative species.Key words: wheat, Triticum aestivum, rye, Secale cereale, fluorescence in situ hybridization, single copy DNA mapping.

Fluorescence in situ hybridization on plant extended chromatin DNA fibers for single-copy and repetitive DNA sequences

2011 ◽  
Vol 30 (9) ◽  
pp. 1779-1786 ◽  
Author(s):  
Kun Yang ◽  
Hecui Zhang ◽  
Richard Converse ◽  
Yong Wang ◽  
Xiaoying Rong ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Single Copy ◽  
Repetitive Dna Sequences
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