Cytologic and molecular analysis of 46,XXq- cells to identify a DNA segment that might serve as a probe for a putative human X chromosome inactivation center

Tantravahi, Umadevi; Kirschner, Daniel A.; Beauregard, Laurent J.; Page, Laurie S.; Kunkel, Louis M.; Latt, Samuel A.

doi:10.1007/bf00289475

Cited by 31 publications

(14 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The samples were then boiled for 10 min, incubated at 65°C for 4-8 hr, mixed with prehybridization solution, and added to the filter bags containing the Southern blots. DNA hybridization to nitrocellulose blots (16) and washing were as described (21).…”

Section: Methodsmentioning

confidence: 99%

“…Initial mapping of single-copy inserts was accomplished by using DNA from Chinese hamster-human somatic cell hybrid cell lines containing various human chromosomes, which have been described by Bruns et al (22). These lines and their corresponding chromosomes are G35D3 (2p, 4,7,8,9,10,14,16,17,19,20,21,22); G35D5 (1,2,3,6,7,10,13,14,15,16,17,18,19,20,22); and G35E4 (4p-q-, 9,15,19). Probes hybridizing to cell lines G35D5 and G35E4, which contain chromosome 15 as their only common human chromosome, but not to G35D3 were tentatively assigned to human chromosome 15.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Isolation of molecular probes associated with the chromosome 15 instability in the Prader-Willi syndrome.

Donlon¹,

Lalande²,

Wyman³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

134

View full text Add to dashboard Cite

Flow cytometry and recombinant DNA techniques have been used to obtain reagents for a molecular analysis of the Prader-Willi syndrome (PWS). HindIll totaldigest libraries were prepared in X phage Charon 21A from flow-sorted inverted duplicated no. 15 human chromosomes and propagated on recombination-proficient (LE392) and recBC-, sbcB-(DB1257) bacteria. (2)(3)(4)(5)(6).The most common chromosome aberration in PWS patients is a deletion of band 15q11.2 (2); however, the size of these deletions is variable and presumably some are undetectable by using current cytogenetic techniques (see Fig. 1). Other aberrations at 15q11.2 occur in 2-5% of PWS patients (6-8), including translocations and duplications (both tandem and inverted), the latter termed inv dup(15) chromosomes.This multiplicity of chromosome rearrangements involving 15qll->13 in the PWS suggests that the proximal long arm of chromosome 15 contains DNA sequences, such as tandem or inverted repeats, which could predispose this segment to structural instability.To define this region of the human genome on a molecular level, several DNA segments from the proximal long arm of chromosome 15 were isolated from X phage Charon 21A libraries constructed from HindIII-digested DNA obtained from flow-sorted (9) inv dup(15) chromosomes. Because these inverted duplicated chromosomes contain two copies of all sequences within 15pter->15ql3, we were able to more easily isolate DNA segments from the proximal long arm of chromosome 15. Using different cloning and insert screening strategies we determined that many of these sequences are genetically unstable in a recombination-proficient bacterial strain. Electron micrographs of heteroduplexes showed that unstable clones contain inverted repeats, which suggests at least one possible mechanism for their instability. In total, we describe in this report ll different cloned DNA segments that map to 15qll-43; 4 of 8 tested are deleted in one of two PWS patients with a deletion of 15qll.2. These data suggest that there is molecular heterogeneity between 15qll deletions in different PWS patients and that the cytogenetic instability of band 15q11.2 might be explained in terms ofthe types ofDNA sequences it contains. MATERIALS AND METHODSCell Lines. The lymphoblastoid cell lines used were from a karyotypically normal (46,XY) individual (MD-li), two patients with the PWS and different-sized deletions in 15qll.2 (DON-5, and DON-10), and two individuals with inv dup(15) chromosomes of different size [ALD-6 (10) and ALD-24 (11)] (Fig. 1).Bacterial Strains. Strain DB1257 is related to strain DB1170 (12) and was constructed by P1 transduction of CES 200 (13) to kanamycin resistance using a lysate prepared on NK 5857 (Trp::TnS SupF 58). The partial genotype of CES 200 is recBC-, sbcB-. Strain LE392 has been described (14).Construction of the inv dup(15) Library. Charon 21A libraries were created from 50 ng of HindIII-digested DNA from 6 million flow-sorted, inv dup(15) chromosomes, isolated from cell line ALD-24. Chromosomes were...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Isolation of molecular probes associated with the chromosome 15 instability in the Prader-Willi syndrome.

Donlon¹,

Lalande²,

Wyman³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

134

View full text Add to dashboard Cite

show abstract

“…DNA samples from three negative controls (normal blood, placenta, and a normal lymphoblastoid line) and one positive control (IMR-32) were run on each gel, and each blot was also hybridized with the control probe H2-26 (22). The copy number of every probe in each sample was estimated by scanning densitometry readings (16), which were normalized for experimental variation against the standard probe H2-26. The results (Figs.…”

Section: Resultsmentioning

confidence: 99%

Differential amplification, assembly, and relocation of multiple DNA sequences in human neuroblastomas and neuroblastoma cell lines.

Shiloh¹,

Shipley²,

Brodeur³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

DNA amplification, manifested by homogeneously staining regions in chromosomes and by extrachromosomal, double minute bodies, is characteristic of many neuroblastoma cell lines. Sequences recruited from a specific domain on the short arm of chromosome 2 (2p) are amplified in advanced-stage primary neuroblastomas, whereas sequences from distinctly different regions of 2p are amplified in the neuroblastoma cell line IMR-32. Five different DNA segments, which include the oncogene N-myc, three other fragments derived from the homogeneously staining region of the neuroblastoma cell line IMR-32, and a fifth fragment, derived from the neuroblastoma cell line NB-9, showed differential and variable amplification in 24 advanced-stage neuroblastoma tumors out of 112 tested specimens. AU five fragments were mapped within the chromosomal region 2p23-2p25 by three different approaches. However, eight other fragments cloned from the homogeneously staining region of IMR-32 cells, which were not amplified in the tumor tissues examined, were mapped to two more proximal domains of 2p, thousands of kilobases apart from each other and from the chromosomal domain that is amplified in the tumors. These results establish the amplification, to different degrees, of a variable-sized segment of one domain near the terminus of 2p in advanced neuroblastomas. These tumors might ultimately be distinguished according to the pattern of amplification of DNA segments within this domain. The data presented also indicate the existence of a new and complex amplification mechanism in at least one neuroblastoma cell line (IMR-32), which involves not only relocation of DNA from specific genomic domains but also the formation of novel units by splicing together very distant DNA segments.

show abstract

“…Accurate measurement of DNA was determined by a fluorometric assay using the dye 4', 6-diamidino-2-phenylindole (DAPI) (12). Gel electrophoresis and Southern hybridization analysis (13) were as previously described (3,14). cDNA construction and plasmid screening HeLa cytoplasmic poly A+ RNA was a gift of Dr. R. Singer, University of Mass., Amherst.…”

Section: Enzyme Reactionsmentioning

confidence: 99%

“…Poly A+ RNA from Hela cells was fractionated on 1% agarose, 6% formaldehyde gels according to the procedures of Lehrach et al (22) and Goldberg (23). RNA from the gels was transferred to nitrocellulose filters in 10 X SSC, in a manner similar to that of a modified DNA blot (13) procedure (14). Hybridization to immobilized RNA, with DNA fragments labeled via T4 DNA polymerase, was carried out in 50% formamide at 420C according to the procedure of Wahl et al (24).…”

Section: Rna Isolation and Hybridizationmentioning

confidence: 99%

Identification and isolation of transcribed human X chromosome DNA sequences

et al. 1983

Self Cite

View full text Add to dashboard Cite

A human X chromosome specific phage library has been used as a source of X-specific genomic DNA clones which hybridize with cellular RNA. Random cDNA clones were mapped for X chromosome sequence localization and 8 were identified as hybridizing to X chromosome Hind III fragments. All eight also hybridized with autosomal Hind III fragments. The X chromosome genomic sequences corresponding to two of these cDNA clones were isolated from a phage library constructed with the Hind III endonuclease digest products of X enriched DNA. One genomic DNA segment, localized to the short arm of the X, shared sequence homology with at least one region of the human Y chromosome. The methodology developed represents a rapid means to obtain a specific genomic DNA clone from a single chromosome when multiple different genomic loci homologous to an expressed DNA sequence exist.INTRODUCTION:

show abstract

Cytologic and molecular analysis of 46,XXq- cells to identify a DNA segment that might serve as a probe for a putative human X chromosome inactivation center

Cited by 31 publications

References 38 publications

Isolation of molecular probes associated with the chromosome 15 instability in the Prader-Willi syndrome.

Isolation of molecular probes associated with the chromosome 15 instability in the Prader-Willi syndrome.

Differential amplification, assembly, and relocation of multiple DNA sequences in human neuroblastomas and neuroblastoma cell lines.

Identification and isolation of transcribed human X chromosome DNA sequences

Contact Info

Product

Resources

About