Chromosome Isolation Procedures

We have sequenced, assembled, and characterized a set of complexity-reduced genomic clones derived from a chromosome 18D-specific library from hexaploid oat ( Avena sativa L.). Sequences from 314 clones were assembled into 99 contigs of identical or nearly identical sequence. The Censor tool was used to identify similarity to known and characterized repeat sequences in RepBase. Eight repeat classes were scattered throughout 50 contigs, with most repeats belonging to seven transposon and retrotransposon classes. After accounting for known repeats, additional matches to orthologous genes from other species were identified in 24 regions of 22 contigs, and an additional 47 regions matched genomic sequences from oat and other related species. These results provide information about the types and density of transposable elements in the oat genome, as well as the potential for identifying unique or chromosome-specific sequence elements in oat. Overall, these results predict a low success rate in identifying chromosome-specific coding regions in oat through chromosome isolation and genome complexity reduction.

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The (6;9) chromosome translocation, associated with a specific subtype of acute nonlymphocytic leukemia, leads to aberrant transcription of a target gene on 9q34

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.4016-4026.1990 ◽

1990 ◽

Vol 10 (8) ◽

pp. 4016-4026

Author(s):

M von Lindern ◽

A Poustka ◽

H Lerach ◽

G Grosveld

Keyword(s):

Long Range ◽

Cdna Cloning ◽

Target Gene ◽

Chromosomal Translocation ◽

Chromosome Translocation ◽

Chromosome 9 ◽

Chromosome 6 ◽

Acute Nonlymphocytic Leukemia ◽

Chromosome Walking ◽

Chromosome Isolation

The specific (6;9)(p23;q34) chromosomal translocation is associated with a defined subtype of acute nonlymphocytic leukemia (ANLL). The 9q34 breakpoint is located at the telomeric side of the c-abl gene. Through a combination of chromosome jumping, long-range mapping, and chromosome walking, the chromosome 9 breakpoints of several t(6;9) ANLL patients were localized within a defined region of 8 kilobases (kb), 360 kb telomeric of c-abl. Subsequent cDNA cloning revealed that this region represented an intron in the middle of a gene, called Cain (can), encoding a 7.5-kb transcript. Disruption of the can gene by the translocation resulted in the expression of a new 5.5-kb can mRNA from the 6p- chromosome. Isolation of chromosome 6 sequences showed that breakpoints on 6p23 also clustered within a limited stretch of DNA. These data strongly suggest a direct involvement of the translocation in the leukemic process of t(6;9) ANLL.

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Plasmid chromosome isolation: An improved batch procedure for large plasmids

Plasmid ◽

10.1016/0147-619x(81)90011-1 ◽

1981 ◽

Vol 5 (3) ◽

pp. 366-370 ◽

Cited By ~ 1

Author(s):

J.W. Bornhoeft ◽

M. Stodolsky

Keyword(s):

Chromosome Isolation ◽

Large Plasmids

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Low angle x-ray diffraction studies of HeLa metaphase chromosomes: effects of histone phosphorylation and chromosome isolation procedure.

The Journal of Cell Biology ◽

10.1083/jcb.96.4.1132 ◽

1983 ◽

Vol 96 (4) ◽

pp. 1132-1137 ◽

Cited By ~ 19

Author(s):

J R Paulson ◽

J P Langmore

Keyword(s):

Diffraction Pattern ◽

Isolation Procedure ◽

X Ray Diffraction ◽

Metaphase Chromosomes ◽

Interphase Nuclei ◽

X Ray ◽

Low Concentrations ◽

Chromosome Isolation ◽

Chromatin Fibers ◽

High Concentrations

To test whether gross changes in chromatin structure occur during the cell cycle, we compared HeLa mitotic metaphase chromosomes and interphase nuclei by low angle x-ray diffraction. Interphase nuclei and metaphase chromosomes differ only in the 30-40-nm packing reflection, but not in the higher angle part of the x-ray diffraction pattern. Our interpretation of these results is that the transition to metaphase affects only the packing of chromatin fibers and not, to the resolution of our method, the internal structure of nucleosomes or the pattern of nucleosome packing within chromatin fibers. In particular, phosphorylation of histones H1 and H3 at mitosis does not affect chromatin fiber structure, since the same x-ray results are obtained whether or not histone dephosphorylation is prevented by isolating metaphase chromosomes in the presence of 5,5'-dithiobis(2-nitrobenzoate) or low concentrations of p-chloromercuriphenylsulfonate (ClHgPhSO3). We also compared metaphase chromosomes isolated by several different published procedures, and found that the isolation procedure can significantly affect the x-ray diffraction pattern. High concentrations of ClHgPhSO3 can also profoundly affect the pattern.

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Cell Culture for Chromosome Isolation

Flow Cytogenetics ◽

10.1016/b978-0-12-296110-6.50008-7 ◽

1989 ◽

pp. 35-41 ◽

Cited By ~ 3

Author(s):

CHARLOTTE LOZES

Keyword(s):

Cell Culture ◽

Chromosome Isolation

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New Application of the Comet Assay

Journal of Histochemistry & Cytochemistry ◽

10.1369/0022155411410884 ◽

2011 ◽

Vol 59 (7) ◽

pp. 655-660 ◽

Cited By ~ 16

Author(s):

Elva I. Cortés-Gutiérrez ◽

Martha I. Dávila-Rodríguez ◽

José Luís Fernández ◽

Carmen López-Fernández ◽

Altea Gosálbez ◽

...

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Single Cells ◽

White Blood Cells ◽

Human Biomonitoring ◽

Cell Types ◽

Dna Damage And Repair ◽

Dna Migration ◽

Sensitive Tool ◽

Chromosome Isolation

The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains.

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