An efficient protocol for chromosome isolation from sterlet (A. ruthenus) embryos and larvae

In the present study, the development of an efficient and feasible protocol for chromosome preparation from sterlet (A. ruthenus) embryos and larvae was carried out. In the established protocol, the mean efficiency of chromosome extraction ranged from 70 to 100%. The average number of recorded metaphases per slide was between 9 to 15. In general, the most satisfactory results were obtained for embryos at 6 dpf and larvae at the age of up to 7 dph. In the 24 dpf group, chromosome isolation was possible without immersion in spindle poison, however; in successive developmental stages, the minimal immersion time exceeded 1.5 hours, regardless of chorionation. Immersion for 14 hours did not compromise the efficacy of chromosome isolation. In the current study, successful chromosome isolation was determined mainly by hypotonization conditions. Younger developmental stages generally require the shortest hypotonization times, whereas older larvae require longer hypotonization times. The optimal hypotonization period is 5-15 minutes for embryos at 24 dpf, 40 minutes for embryos at 4dpf, and 50-60 minutes for fish at 6 dpf-7 dph. The only exception was the 24 hpf group where only blastula cells were used. An additional overnight fixation step significantly enhanced chromosome quality and supported chromosome counting especially in the 24 dpf group. The quality and quantity of chromosome slides were also significantly determined by tissue type, and the slides prepared from heads (gill cells) produced the best results.

Characterization of chromosome-specific genomic DNA from hexaploid oat

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.

New Application of the Comet Assay

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.

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

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.

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

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.