DNA methylation in stingless bees with low and high heterochromatin contents as assessed by restriction enzyme digestion and image analysis

Abstract

Background: The stingless bee genus Melipona has been divided into two groups, based on their heterochromatin content. Melipona quadrifasciata and Melipona rufiventris have low and high levels of heterochromatin, respectively. Since condensed chromatin may be rich in methylated DNA sequences, M. quadrifasciata and M. rufiventris nuclei may contain different amounts of methylated CpG. These differences could be assessed by comparing Feulgen-DNA values obtained by image analysis of cells treated with the restriction enzymes Msp I and Hpa II that distinguish between methylated and unmethylated DNA. Msp I and Hpa II cleave the sequence -CCGG-, but there is no cleavage by Hpa II if the cytosine of the central CG dinucleotide is methylated. Methods: Malpighian tubules of M. quadrifasciata and M. rufiventris were treated with Msp I and Hpa II prior to

Spatial distribution of AT- and GC-rich DNA within interphase cell nuclei of Triatoma infestans Klug

“…In general, after the restriction enzyme recognizes, cleaves and removes determined DNA sequences from fixed chromosomes or interphase chromatin, specific images are generated (Mezzanotte et al, 1983;Bianchi et al, 1986;Sentis et al, 1989;Gonsálvez et al, 1995;Mello et al, 2000). The richness in -CCGG-sequences excised by Msp I/Hpa II, and the presence of methylated CpG sequences not cut by Hpa II, can be evaluated by quantifying the response to the Feulgen reaction, specific for DNA, under the action of these enzymes, using video or microspectrophotometric image analysis (Mello et al, 2000(Mello et al, , 2009bMampumbu and Mello, 2006).…”

Entropy of Feulgen-stained 17-β-estradiol-transformed human breast epithelial cells as assessed by restriction enzymes and image analysis

“…Then, the preparations were incubated with 1.0 U/μl MspI and HpaII (New England Biolabs, Ipswich, MA, USA) restriction enzymes in appropriate buffers and then covered with coverslips (15,18,24). The incubations were accomplished in a moist chamber at 37˚C for 10 h, after which the slides were rinsed in distilled water and air dried (18,24). As controls, preparations were treated with Triton X-100 and the assay buffers recommended for each of the enzymes used (18).…”

“…The incubations were accomplished in a moist chamber at 37˚C for 10 h, after which the slides were rinsed in distilled water and air dried (18,24). As controls, preparations were treated with Triton X-100 and the assay buffers recommended for each of the enzymes used (18).…”

“…CpG methylation affecting extensive global genome sites can be investigated in chromosomes and in interphase chromatin in situ after their treatment with the restriction enzymes MspI and HpaII (14)(15)(16)(17)(18). Both enzymes cleave the sequence -CCGG-, but HpaII does not cleave it if the central cytosine of this sequence is methylated (19).…”

“…Both enzymes cleave the sequence -CCGG-, but HpaII does not cleave it if the central cytosine of this sequence is methylated (19). Image analysis of Feulgen-stained cells previously treated with MspI and HpaII has permitted investigation into whether extensive CpG methylation participates in chromatin higher order structures (17,18). This approach could be extended to evaluate the participation of global DNA methylation in nuclear phenotypes that have various chromatin higher order packing states as transformation and tumorigenesis progress.…”