Morphological and biochemical effects of endonucleases on isolated mammalian chromosomes in vitro

Abstract

Endonuclease digestion of isolated and unfixed mammalian metaphase chromosomes in vitro was examined as a means to study the higher-order regional organization of chromosomes related to banding patterns and the mechanisms of endonuclease-induced banding. Isolated mouse LM cell chromosomes, digested with the restriction enzymes AluI, HaeIII, EcoRI, BstNI, AvaII, or Sau96I, demonstrated reproducible G- and/or C-banding at the cytological level depending on the enzyme and digestion conditions. At the molecular level, specific DNA alterations were induced that correlated with the banding patterns produced. The results indicate that: (1) chromatin extraction is intimately involved in the mechanism of endonuclease-induced chromosome banding. (2) The extracted DNA fragments are variable in size, ranging from 200 bp to more than 4 kb in length. (3) For HaeIII, there appears to be variation in the rate of restriction site cleavage in G- and R-bands; HaeIII sites appear to be more rapidly cleaved in R-bands than in G-bands. (4) AluI and HaeIII ultimately produce banding patterns that reflect regional differences in the distribution of restriction sites along the chromosome. (5) BstNI restriction sites in the satellite DNA of constitutive heterochromatin are not cleaved intrachromosomally, probably reflecting an inaccessibility of the BstNI sites to enzyme due to the condensed nature of this chromatin or specific DNA-protein interactions. This implies that some enzymes may induce banding related to regional differences in the accessibility of restriction sites along the chromosome. (6) Several specific nonhistone protein differences were noted in the extracted and residual chromatin following an AluI digestion.(ABSTRACT TRUNCATED AT 250 WORDS)

Improving beetle karyotype analysis: restriction endonuclease banding of Tenebrio molitor chromosomes

“…In mouse Avail (GGCC) and BstNI (CCGG) reduce the staining of most of the centromeres (Kaelbling et aL, 1984), although in this case, the results of BstNI appears contradictory to those reported by Burkholder (1989). In general, four base pair cutters are more efficient in removing large amounts of DNA, while five or six base pair cutters usually do not produce longitudinal differentiation or give G bands (Mezzanotte et a!., 1985;Lopez-Fernandez et a!., 1989).…”

“…Recently, restriction endonucleases (RE) have been used to induce chromosome banding by in situ digestion of fixed chromatin in a large variety of organisms (Bianchi and Bianchi, 1987;Lopez-Fernandez et al, 1989), and these simple techniques are very useful for detection of specific sequences of DNA, such as for example satellite DNAs (Mezzanotte et al, 1986) or regions which remains cryptic with other banding techniques (Gosálvez et a!., 1987;1989). Miller et a!.…”

Prothymosin α, a mammalian c-myc-regulated acidic nuclear protein, provokes the decondensation of human chromosomes in vitro

“…ProT · Human metaphase chromosomes were prepared from peripheral blood lymphocytes and HeLa cells as described elsewhere (Burkholder, 1989). Briefly, lymphocytes were separated with Lymphoprep (Nygaard, Oslo) and the lymphocyte rich suspension was incubated at 37°C for 3 d in RPMI 1640 medium supplemented with 20 % fetal bovine serum (FBS) containing penicillin (50 U/ml), streptomycin (50 Ìg/ml) and L-Gln (0.29 mg/ml).…”

Autosomal, sex and B chromosomes in Eyprepocnemis plorans (Orthoptera) viewed with restriction endonuclease in situ digestion

“…The extraction of DNA from fixed chromosomes by REs can be affected by a series of factors such as fixation of the material (Burkholder, 1989), cleavage frequency (Miller et al, 1983;1984;Bianchi eta!., 1985;Mezzanotte, 1986) or structure of the RE and chromatin (Gosálvez et a!., 1989). Even so we believe that under homogeneous experimental conditions REs may be used to study processes that determine pathways of evolution of a given karyotype through the sensitivity of each chromosome region to a given RE.…”