In the attempt to conclude investigation of the action of restriction endonucleases on eukaryote chromosomes, we carried out a series of experiments digesting in situ human metaphase chromosomes with AluI/TaqI followed by Giemsa staining. We focused on the centromeric regions of chromosomes1, 2 and 16 and noted that those areas appeared as intensely stained blocks after AluI digestion, but were dramatically reduced in size or completely destroyed after subsequent TaqI treatment. These results permitted us to draw some conclusions on the highly repetitive DNA composition of these regions, in terms of alphoid and classical satellite DNAs

G Pichiri

M Nieddu

R Mezzanotte

S Manconi

European Journal of Histochemistry

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281Restriction endonucleases (Res) are molecu-lar tools capable of localising in situ repet-itive sequences by specific attack, followedby removal/non-removal of DNA blocks from cer-tain regions of eukaryote chromosomes(Mezzanotte et al., 1983; Gosalvéz et al., 1997).Although most questions relative to the molecularmechanism(s) producing the cytological effectsobserved after RE digestion in situ have been clar-ified, some doubts remain. For instance, still opento discussion is the cause of the dramatic increasein hybridisation labelling areas when fluorescencein situ hybridisation (FISH) is carried out onhuman chromosomes using alphoid DNAs asprobes in preparations predigested with eitherAluI or TaqI (Nieddu et al., 1999). In this connec-tion, it has been hypothesized that this increase influorescence may be due to i) the reorganisationproduced in centromeric heterochromatin by REaction which, in turn, would render accessiblesequences impervious to hybridisation in standardconditions or ii) the presence in paracentromericheterochromatin of certain human chromosomes(1, 9, 16) of alphoid sequences, in addition to clas-sical satellite DNAs (Nieddu et al., 1999). Thiscontrasts with the belief that the former is foundonly in centromeres and the latter only in paracen-tromeric areas (Tyler-Smith &Willard, 1993).Thelast point to be clarified, ever-present when deal-ing with this subject, is what role, if any, specificproteins associated with specific DNA sequences,for instance specific centromeric proteic compo-nents (Tanaka et al., 2005), play in determiningthese results.We report and discuss the results ofa series of experiments designed to answer thesequestions.Materials and MethodsHuman metaphase chromosomes were obtainedfrom peripheral blood lymphocyte cultures by stan-dard procedure. Preparations were air-dried for©2006, European Journal of HistochemistryIn the attempt to conclude investigation of the action ofrestriction endonucleases on eukaryote chromosomes, wecarried out a series of experiments digesting in situ humanmetaphase chromosomes with AluI/TaqI followed byGiemsa staining. We focused on the centromeric regions ofchromosomes1, 2 and 16 and noted that those areasappeared as intensely stained blocks after AluI digestion,but were dramatically reduced in size or completelydestroyed after subsequent TaqI treatment. These resultspermitted us to draw some conclusions on the highly repet-itive DNA composition of these regions, in terms of alphoidand classical satellite DNAs.Key words: Restriction enzymes- DNA - repetitive DNAs.Correspondence: Roberto Mezzanotte,Sezione di Biologia e Genetica, Dipartimento di Scienze eTecnologie Biomediche, Cittadella Universitaria 09042Monserrato (CA) ItalyTel. +39.0706754122.Fax +39.0706754100.E-mail: mezzanotte@unica.itPaper accepted on September 26, 2006European Journal of Histochemistry2006; vol. 50 issue 4 (October-December):281-284Some remarks on the use of TaqI to detect highly repetitive DNAsequences in human chromosomesM. Nieddu, G. Pichiri, S. Manconi, R. MezzanotteSezione di Biologia e Genetica, Dipartimento di Scienze e Tecnologie Biomediche, CittadellaUniversitaria, Monserrato (CA), ItalyORIGINAL PAPER28224 h and subsequently digested in situ with 30 Uof AluI (Invitrogen) for 16 – 18 h a 37°C accord-ing to Mezzanotte et al. (1983). Slides werestained with 4% Giemsa (Sigma) in deionisedwater.After microscopic observation, slides weredestained in methylic alcol: acetic acid 3:1.Subsequently, the same slides were digested insitu with 30 U of TaqI ( Invitrogen) and treated aspreviously described. Before digestion in situ withRE , chromosomes were pretreated on some slideswith proteinasi K (50 ng/mL) (Sigma) in Tris HCl20 mM pH 7.4, Ca Cl 2 2 mM for 2-3 min at roomtemperature. Chromosomes were then dehydratedwith an alcohol series (60%, 80%, 100%). Forreasons of brevity, we focused our attention onchromosomes 1, 2 and 16, although our observa-tions with Giemsa suggest that results relative tothese chromosomes may be extended to otherswhose heterochromatic areas possess similar char-acteristics.Results and DiscussionAluI digestion of human chromosomes followedby simple Giemsa staining produces the typical C-like bands previously described (Mezzanotte et al.,1983). Double digestion in situ using AluI followedby TaqI shows a conspicuous gap produced in theconstitutive hetechromatin regions of a number ofchromosomes, including the paracentromeric areaof chromosomes 1 and 16,while some dot-like cen-tromeric structures are still present (Figure 1 a, b).In other words, the thick, AluI-induced band, simi-lar to the classical C-band (Sumner, 1972), isreduced to a small dot plus a clearcut paracen-tromeric gap after subsequent TaqI digestion.Moreover, the thin AuI C-like band present in chro-mosome 2 is completely removed, and a sort ofsmall gap is evident in the centromeric area of thesame chromosome when TaqI follows AluI diges-tion. When proteinase K treatment is carried outon fixed, standard chromosomes, a type of G-likebanding appears (Sumner et al., 1971). In chro-M. Nieddu et al.Figure 1. Human metaphase chromosomes stained with Giemsa after digestion in situ with AluI (a) and subsequent digestion with TaqI(b). Note heterochromatic centromeric blocks on chromosomes 1, 2 and 16 (see arrows), which are partially (chromosomes 1 and 16)or totally digested (chromosome 2) after TaqI treatment.283Original Papermosomes pretreated with proteinase K, RE diges-tion does not reveal changes, unlike what weobserved in chromosomes digested only with Res.In this connection, it is important to stress that weused proteinase K at a concentration which permit-ted us to visualise a chromosome structure similar,even though often more swollen, to that observedin standard fixed chromosomes. This increase inproteinase K concentration produced dramaticmorphological alteration of chromosomes which,as a consequence, could not be used in subsequentexperiments. It is known that two main classes ofhighly repetitive DNA sequences are located in theconstitutive heterochromatin of human chromo-somes, representing about 13% of total genomichuman DNA (Tyler-Smith & Willard, 1993): i)alphoid DNAs, variants of a basic repetitivesequence unit composed of 170 bp and ii) three so-called classical satellite DNAs, isolated by CsClgradients and indicated as satellites I, II and III(Prosser et al., 1986). The centromeric hete-rochromatic area of human chromosomes 1 and16 contains specific alphoid DNAs and massivequantities of classical satellite II DNA (Tagarro,1993), but only small amounts of classical satelliteIII DNA in chromosome 1 (Nieddu et al., 2003).On the other hand, TaqI cuts classical satellite IIvery efficiently (Frommer et al., 1982)), showinglimited activity on alphoid DNA ( Nieddu et al.,1999).These considerations would account for thegap observed in the paracentromeric areas of chro-mosomes 1 and 16, as well as for the Giemsa-pos-itive spot found in the centromeric region of thesame chromosome after AluI/TaqI double diges-tion, due to the fact that the AluI C-like band,except for its centromeric component, is destroyed.As already shown by the persistence of positive sig-nal in FISH experiments using specific alphoidDNA probes (Nieddu et al., 1999), this componentwould represent alphoid DNA and, above all,demonstrates that this DNA fraction is localisedexclusively in the centromere, excluding the possi-bility that it is scattered in the paracentromericarea of chromosomes such as 1 and 16, as previ-ously postulated (Buno, 1997). On the other hand,taking into account that human classical satelliteII is extensively digested by TaqI (Frommer et al.,1982), our data would indicate that the cen-tromere of chromosome 2, in addition to its specif-ic alphoid DNA, also contains classical satellite IIDNA that is not attacked by AluI, thus producing aC-like band, which is cleaved and removed by TaqI.In this case, it is interesting to note that in spite ofthe gap observed in the chromosome 2 centromereafterTaqI digestion, FISH carried out using specif-ic alphoid DNA as a probe shows positive hybridi-sation signal in this area. This observation allowsus to hypothesize that, among the factors respon-sible for the production of C-like bands, one shouldconsider above all the presence of classical satel-lite DNAs and not alphoid DNAs. Lastly, the factthat proteinase K pre-treatment does not affectour results would suggest that, at least in this case,the proteic component, associated with specificDNA fractions (Tanaka et al., 2005) does not playany role in determining the activity of these Res infixed eukaryote chromosomes.ReferencesBuno BI (1997). Quantification del Quimerismo hematopoyético enpacientes con leucemias sometidos a transplante alogénico demedula osea. Utilizaciòn de polimorfisms revelados mediante diges-tion in situ con endonucleasas de restriccion. Tesis doctoral,Universidad Autonoma de Madrid.Frommer M, Prosser J, Tkachuk D, Vincent PC. Simple repeatedsequences in human satellite DAN.Nucleic Acid Res 1982;10:547-63.Gosalvéz J, Lopez-Fernandéz C, Goyanes V, Mezzanotte R. (1997).Chromosome differentiation using nucleases: an overview. In:Chromosome Today. Ed. By N. Henriques-Gil, J.S. Parker and M.J.Puertas. Chapman and Hall, pp. 23-49.Mezzanotte R, Ferrucci L, Vanni R, Bianchi U. Selective digestion ofhuman metaphase chromosomes by AluI restriction endonuclese. J.Histochem. Cytochem 1983;31:553-6.Miller DA, Choi YC, Miller OJ (1983). Chromosome localization of ahighly repetitive human dans and amplified ribosomal DNA withrestriction enzymes. Science 1983;219:395-7.Nieddu M, Rossino R, Pichiri G, Rocchi M, Setzu MD,Mezzanotte R.The efficiency of in situ hybridization on human chromosomes withalphoid DNAs is enhanced by previous digestion with AluI andTaqI.Chrom Res 1999;7, 593-602.Nieddu M, Pichiri G, Diaz G, Mezzanotte R.The organization of clas-sical satellite DNAs in human chromosomes: an approach usingAluI and TaqI restriction endonucleases. Eur J Histochem2003;47:209-14.Prosser J, Frommer M, Paul C, Vincent J. Sequence relationships ofthree human satellite DNAs. J Mol Biol 1986; 187:145-55.Sumner AT, Evans HJ,Buckland R.A. New technique for distinguish-ing between human chromosomes. Nature New Biology 1971;232,31-2.Sumner AT. A simple technique for demonstrating centromeric hete-rochromatin. Exp. Cell Res 1972;75:304-6.Tagarro-Garcia I. Caracterizacion citogenetico-molecular del ADNsatelite umano de sequencia simple. Tesis doctoral. UniversidadAutonoma de Madrid. 1993.Tanaka Y,Tachiwana H,Yoda K,Masumoto H, Okazaki T, KurumizakaH et al. Human centromere protein B induces translational posi-tioning of nucleosomes on alpha-satellite sequences. J Biol2005;280:41609-618.Tyler-Smith C, Willard HF. Mammalian chromosome structure. CurrOpin Genet Dev 1993;3:390-7.284M. Nieddu et al.

Some remarks on the use of TaqI to detect highly repetitive DNA sequences in human chromosomes

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Some remarks on the use of TaqI to detect highly repetitive DNA sequences in human chromosomes

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