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Staining, and in Situ Digestion with Restriction Endonucleases

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Staining, and in Situ Digestion with Restriction Endonucleases Heredity66 (1991) 403—409 Received 23 August 1990 Genetical Society of Great Britain An analysis of coho salmon chromatin by means of C-banding, AG- and fluorochrome staining, and in situ digestion with restriction endonucleases R. LOZANO, C. RUIZ REJON* & M. RUIZ REJON* Departamento de Biologia Animal, Ecologia y Genética. E. /ngenierIa T. AgrIcola, Campus Universitario de Almeria, 04120 AlmerIa and *Facu/tad de Ciencias, 18071 Granada, Universidad de Granada, Spain Thechromosome complement of the coho salmon (Oncorhynchus kisutch) has been analysed by means of C-banding, silver and fluorochrome staining, and in situ digestion with restriction endo- nucleases. C-banding shows heterochromatic regions in the centromeres of most chromosomes but not in the telomeric areas. The fifteenth metacentric chromosome pair contains a large block of constitutive heterochromatin, which occupies almost all of one chromosome arm. This region is also the site where the ribosomal cistrons are located and it reacts positively to CMA3/DA fluorochrome staining. The NORs are subject to chromosome polymorphism, which might be explicable in terms of an amplification of ribosomal cistrons. The digestion banding patterns produced by four types of restriction endonucleases on the euchromatic and heterochromatic regions are described. Two kinds of highly repetitive DNAs can be distinguished and the role of restriction endonucleases as a valuable tool in chromosome characterization studies, as well as in the analysis of the structure and organization of fish chromatin, are also discussed. Keywords:C-banding,coho salmon, fluorochrome staining, restriction endonuclease banding. (Oncorhynchus kisutch), as well as applying conven- Introduction tional banding techniques, we have analysed the Theuse of restriction endonucleases (REs) is becom- mitotic chromosomes using DNA base-pair-specific ing common not only in molecular biology but also as fluorochromes and in situ digestion with restriction an important tool in molecular cytogenetics. Chromo- endonucleases. Three new observations resulted from some banding patterns induced by restriction endo- the application of these latter techniques to coho nuclease disgestion have led to the identification of salmon chromosomes: (i) extensive chromosome poly- animal satellite DNA regions (Miller et al., 1983; morphism affecting the NORs, which we attribute to an Bianchi et a!., 1985; Mezzanotte, 1986), although amplification phenomenon of the ribosomal cistrons; certain factors affecting enzyme activity still remain (ii) certain peculiarities in the NOR-associated hetero- unclear. chromatin compared to the remaining heterochromatin Salmonids constitute one of the most extensively and euchromatin; and (iii) cytogenetical differences analysed fish groups in cytogenetic terms mainly due to between centromeric and telomeric heterochromatin. their ancestral polyploid origin. Nevertheless, with Our results show that REs are useful in the charac- salmon belonging to the genus Oncorhynchus, chromo- terization of fish chromatin, particularly in fish species some differentiation methods have only been used to of polyploid origin. locate the NOR and the CMA regions (Phillips et a!., 1986). Thus, to characterize more accurately the Materialsand methods chromatin of one of these species, the coho salmon Thirty-sixspecimens of coho salmon, imported as eggs at eye-stage from the USA (Oregon Aquafood) and Correspondence: Dr Rafael Lozano Ruiz, Departamento de reared in a Spanish fresh-water fish farm (Marcultura, BiologIa Animal, Ecologia y Genética, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain. SA, Lugo), were employed in this study. 403 404 R. LOZANO ETAL. Some of the slides were destained with Carnoy fixative Cell collections and chromosome preparations for a few minutes and sequentially stained with fluoro- Livingspecimens were intraperitoneally injected with achromes or silver nitrate. yeast solution and 48 h later were coichicined accord- ing to the method of Lozano et at. (1988). The kidneys were removed and cell suspensions prepared by Results standard fixation. Other metaphase cells were obtained from lymphocyte cultures following the protocol of Giemsa karyotype Lloyd & Thorgaard (1988) with some modifications The coho salmon diploid complement is made up of 60 (Sanchez et at., 1990). Chromosome preparations were chromosomes (Fig. 1); 42 of them (pairs 1—21) are made according to the steps described by Lozano et at. medium and large metacentric or submetacentric (1988) and one slide per individual was stained with 10 chromosomes and 18 (pairs 22—30) are small telo- per cent Giemsa in phosphate buffer, pH 6.8, for centric or subtelocentric ones, although pairs 22 and karyotyping. 23 are submetacentric. Two-thirds of the individuals show heterozygosity for the fifteenth pair, charac- terized by the presence of a metacentric chromosome Chromosomebanding methods and one clearly submetracentric one. The remaining Themethod of Schwarzacher eta!.(1980) was individuals were homozygotes with two identical used to detect constitutive heterochromatic regions. metacentric chromosomes in the fifteenth pair. The NF NORs were visualized following the technique of was 106 in all. the cells. Conventional Giemsa staining Howell & Black (1980). Chromomycin A3/distamycin does not permit the identification of the various A and DAPI/actinomycin D counterstaining methods chromosome pairs. (Schweizer, 1976) were performed in order to locate the GC- and AT-rich chromosome regions respectively. In situ digestion with various restriction endonucleases C-banding was carried out by adding 30 1u1 of the specific enzyme Allthe metacentric chromosomes carry hetero- solution (suspended in the appropriate buffer) to the chromatic centromeric blocks, which vary in size and chromosome preparations, covering them with 24 x 32 staining intensity. The fifteenth pair stands out, show- mm coverslips and incubating the slides for 16 h at ing a wide region of constitutive heterochromatin, 37CC in a moist chamber. The concentration of the which occupies almost all of one chromosome arm but endonucleases varied from 0.5 to 1.5 units dUl, not the centromere (Fig. 2). The size of this region depending on their activity. After incubation the slides differs in individuals heterozygous for the poly- were carefully washed with distilled water and stained morphism mentioned above. A few thin telomeric with 5 per cent Giemsa in phosphate buffer, pH 6.8. bands can be seen on some of the pairs, especially the Preparations of the same individuals were simulta- 27th (Table 1 and Fig. 2). neously treated with the buffer alone as a control. 9 '8' 10 jim Fig. I Giemsa karyotype of a coho salmon (2 n =60)homozygous for the fifteenth polymorphic pair. Bar represents 10 tim. COHO SALMON CHROMATIN 405 " ' - ___ ta' —s a a 1 aS) 0i •, p. ;,-, ,- r'. _) a -S —s P4 -s—p w —. a (p a a -J — -4 0 ... sj•) a M (OS' Fig. 2 C-banded karyotype of 0. kisutch, heterozygous for the fifteenth pair. Table 1Summarizedresults obtained by means of C-banding and in situ digestion with various restriction endonucleases on different kinds of euchromatin in 0. kisutch Heterochromatin Treatment Target Centromeres Telomeres NOR-associated Euchromatin Cbanding* — + — + — Alu 1/ AG/CT — +1- —t Mbo I /GATC + Hae III! GG/CC + + +11 + Hinf I G/ANTC Dde II: C/TNAG + — + ¶ + § Bgl II! A/GATCT 0 0 0 0 Hind 1111: A/AGCTT * + denotespresence and —absenceof positive C-bands. tThe majority of the centromeres and/or telomeres. + denotesdigested regions, —undigestedregions and 0 denotes homogeneous digestion of the whole karyotype. § Some intercalary undigested bands also appear. 11 Region differentially digested compared to the rest of the heterochromatin. somes in homozygous individuals (Figs 3c and 6b), NOR silver staining while heterozygotes carried fluorescent regions in one Thesilver-stained karyotype of coho salmon, homo- metacentric and one subtelocentric chromosome. zygous for the fifteenth pair, shows two metacentric Chromomycin staining of metaphases previously chromosomes with interstitially or subterminally treated with silver nitrate showed that these chromo- located ribosomal cistrons, depending on the indivi- somes were the same as those which showed NOR dual (Figs 3a and b and 4a). For this reason the NORs activity, and thus CMA regions flanking the secon- are heteromorphic in position and even in size. In dary constrictions were observed on these chromo- heterozygotes for the polymorphic pair, only one such somes (the less condensed the chromosome the more metacentric chromosome can be seen, together with a clearly visible the regions). Treatment with the DAPI/ subtelocentric one, with NOR activity located at the AMD combination did not produce any differential end of the short arm (Fig. 4b). The NOR-bearing staining of the mitotic chromosomes of 0. kisutch. chromosomes correspond to those with the large heterochromatic blocks, i.e. the fifteenth pair. Restrictionendonuclease banding Fourgroups of restriction endonucleases can be F/uorochromestaining described on the basis of the digestion banding pattern Particularlybrilliant interstitial CMA3/DA clusters that they produce on fixed chromosomes of coho were found in two medium-sized metacentric chromo- salmon. In Table 1 the effects of these enzymes on the 406 R. LOZANO ETAL. 9 4 Fig. 3 Mitotic metaphase of 0. kisutch shown by silver-nitrate staining (a) and details of the fifteenth, NOR-bearing, chromosome pair (b) showing several
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