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 endonucleases. 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 according 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 positive CMA3 regions flanking the (a) duplicated NORs (c). Arrowheads indicate the NORs.

4 / Fig. 4 Somatic metaphases of two a coho salmon: one carries hetero- a a.? morphism for the position of the NORs on the fifteenth pair (a) and the other is (b) heterozygous for the NOR poly- i7 morphism (b). Details of the NOR- 10 pm bearing chromosomes (c). Arrowheads indicate the NOR-bearing chromo- (d) (c) somes and the bar represents 10 urn. differentkinds of heterochromatic regions (centro- Japanese coho salmon, but do not coincide with the meres, telomeres and NOR-associated) and on euchro- results of Ueda (1983), working also with Japanese matin are summarized. It is worthwhile emphasizing specimens (48 M + SM and 12 ST+ T, NF= 108). The that Alu I and Mbo I generate a well-defined banding reasons for these discrepancies may be due to the pattern in which the NOR-associated heterochromatin denomination of one of the smallest chromosome pairs and most of the telomeres remain undigested (Figs 5, (subtelocentric or submetacentric?) or else to chromo- 6a and b), while Hae III and Hinf I digest these areas some rearrangements which are frequent in the selectively (Fig. 6d). The cytogenetical behaviour of salmonid group. The possibility that the fish analysed Dde I falls between these two types of enzymes (Fig. by each author belonged to different chromosome 6c), and no longitudinal differentiation is induced by races must also be considered. Bgl II or Hind III. The variability in the NOR-bearing pair could be due to in-tandem duplication. If this is the case, the Discussion original NORs would be located on the short arms of a subtelocentric pair, just as Phillips et al. (1986) found. Ourconclusions concerning the karyotype of 0. A progressive amplification involving the ribosomal kisutch concur with those of Uyeno (1972), who cistrons would then originate a new chromosome describes the existence of a complement composed of morphology, i.e. a metacentric chromosome with dupli- 46 metacentric and submetacentric and 14 subtelo- cated NORs (several silver-precipitated bands) along centric and telocentric chromosomes (NF= 106) in one of the arms. We have detected homozygous and COHO SALMON CHROMATIN 407 ; — L.a : c t a- _&t - -s 0 a • r1 •; =Il tics ' -s (#3 -s —s -s —S M 0 tc Wb — —s '.3 '.3 -4

Fig.5 Karyotype of coho salmon chromosomes after digestion with Alu I restriction endonuclease. This karyotype belongs to a heterozygous individual for the fifteenth pair (underlined). heterozygous individuals for this new NOR-bearing associated with the nucleolar organizer has certain chromosome type, depending on whether this amplifi- specific characteristics compared to the rest of the cation affects either one or two of the original NOR DNA: it is GC-rich, as shown by C-banding and its chromosomes. In the same way, a different activation high affinity for CMA3 staining, and it is resistant to status in the functional duplicated ribosomal cistrons endonuclease digestion with Alu I and Mbo I while would be responsible for the previously mentioned being very sensitive to treatment with Dde I, Hae III heteromorphism. Nevertheless, inversions, transloca- and Hinf I. This differential behaviour demonstrates tions, mobile genetic elements, or a combination of any the characteristic nature of the organization and/or of these processes, cannot be ruled out as possible structure of the chromatin in this zone, regardless of its explanations for these phenomena in 0. kisutch, just as base sequence. Furthermore, in situ digestion with Dde Wiley et at. (1989) have proposed in diploid—tetraploid I provides some evidence of the peculiarities of the treefrogs. It is worth bearing in mind that it is quite heterochromatin located at the NORs. The NOR- common in certain fish species for a number of riboso- heterochromatin is sensitive to Dde I while the remain- ma! genes in the NORs to have increased and various ing heterochromatic areas resist this enzyme treatment. chromosomal rearrangements to have taken place It would also appear that the structure and/or organi- during their evolution (Foresti et a!., 1981). In the zation of the chromatin differs between the centro- salmonid group, as well as in other teleostean fishes of meric and telomeric chromosome areas of salmonids. tetraploid origin (Sola et al., 1986), a diploidization This idea has also been suggested by Cau et at. (1988) process involving the rDNA zones must have occurred in another fish species of the family Muraenidae. at a time when many genetic changes affecting the Several authors have found a close correlation NORs (Robertsonian and others) were taking place. between C-banding and restriction banding patterns The NORs of this vertebrate group are variable (see for example Cau et at., 1988; Lloyd & Thorgaard, chromosome regions (Mayr et at., 1988; Phillips & 1988; Schmid & de Almeida, 1988), although some of Kapuscinksi, 1987; Phillips et at., 1988). them consider that this correlation is not entirely exact Two types of heterochromatin can be distinguished (Lloyd & Thorgaard, 1988; Lopez-Fernandez et at., in coho salmon mitotic chromosomes on the basis of 1989). Our findings in 0. kisutch indicate notable their response to conventional banding treatments (C-, differences between the banding methods. Thus, while Ag- and fluorochrome banding) and in situ RE diges- C-banded mitotic chromosomes show heterochromatic tion: the NOR-associated heterochromatin and the regions located mainly at the centromeres and only a remaining heterochromatic zones (centromeres and few telomeric areas appear to be positively stained, the telomeres). At the chromosome level, repetitive DNA banding patterns generated both by Alu I and by Mbo I 408 R. Distinctions clear gaplocatedcloseto thecentromere(Fig.5). the mostcharacteristicchromosome pairsduetoa sity oftheirtelomericbands. Theseventeenthisoneof telomeres. Pairsnumber12 and13differintheinten- both arecarriersofintercalary bandsneartheir distinguishes theeighthpair fromtheninth,although presence ofapositivelystainedcentromericregion presence orabsenceofdigestedcentromeres.Thusthe interstitial andterminalundigestedbandsthe Figs 1and5),accordingtothesizepositionof identification ofdifferenthomologouspairs(compare majority ofthechromosomes,whichpermits produce aclearlongitudinaldifferentiationinthe CMA3/DA (b),Dde1(c)andHaeIII(d).Arrowheadsindicate theNOR-associatedheterochromaticregions.Barrepresents Fig. 10 sum.

6 Metaphasechromosomesof0.kisutchdigestedwithdifferentenzymes:Mbo1(a),AluIandsequentiallystained 0' * • t LOZANO ETAL. 'a.a. can alsobemade betweensomeofthe - • t a •A F. a •'.'' t.-4 'i" ''::' t: t, 4 2 p '*4 .. .4 I.., •1 SI fl /* I,I•la 'lb ,4& S. — as —. t • a' ¶7/

C 9' fish issusceptible tobandingbymeans ofcertain digestion (G-banding),the euchromatin ofthisgroup salmonid euchromatinbymeans ofproteolyticenzyme possible toobtainaclear bandingpatternonthe C-banding, couldindicate thatalthoughitisnot ing fromAluIorMbo digestion, butabsentafter to REdigestion. 30th whichshowsadistinctivetelomericarearesistant 27th withaclearheteropycnoticcentromere,andthe its lackofanyregionresistanttoAluIorMboI,the that occupiesalmostallofthelongarm,24thwith group, e.g.the23rd,withitslargeAluI-resistantregion pairs thatbelongtothesubtelocentricandtelocentric S. Moreover, theexistenceof intercalarybandsresult- — 4- em íA, 4% a S S. Ifl / 1 4. 1> COHO SALMON CHROMATIN 409 restriction endonucleases. The absence of G-banding MAYR, B., KALAT, M. AND RAB, P. 1988. Heterochromatins and in some fish species has been attributed to a deficiency band karyotypes in three species of salmonids. Theoret. in heavy isochores (homogeneous GC-rich chromo- Appl. Genet., 76, 45—5 3. some regions) and the lack of compartmentalization of MEDRANO, L., BERNARDI, 0., COUTURIER, J., DUTRILLAUX, B. AND their genomes (Medrano et BERNARD!, G. 1988. Chromosome banding and genome a!.,1988). Nevertheless, compartimentalization in fishes. Chromosoma, 96, RE treatments lead to reproducible banding of the 178—183. chromosomes which, when followed by C-, Ag- or MEzzAN0TFE, R. 1986. The selective digestion of polytene and fluorochrome banding, makes this technique an mitotic chromosomes of Drosophila melanogaster by the important tool for the analysis of chromosome struc- Alu I and Hae III restriction endonucleases. Chromo- ture and organization, as well as chromosome evolu- soma,93,249—255. tion, in the salmonid group. MILLER, D. A., CHOI, Y. AND MILLER, 0. .i. 1983. Chromosome localization of highly repetitive human DNA's and ampli- fied ribosomal DNA with restriction enzymes. Science, 219, 395—397. Acknowledgements PHILLIPS, R. B. AND KAPUSCINKSI, A. R. D. 1987. A Robertsonian Theauthors thank Dr J. Santos, Dr C. SentIs and Dr J. polymorphism in pink salmon (Oncorhynchus gorbuscha) Fernández-Piqueras for their technical assistance and involving the nucleolar organizer region. Cytogenet. Cell their valuable comments. We are grateful to Dr L. Genet., 44, 148—152. Sanchez, Ms A. Viñas and Mr F. Nieto for their PHILLIPS, R. B., PLEYTE, K. A. AND HARTLEY, S. E. 1988. Stock- specific differences in the number and chromosome posi- important contribution to cell culture. We also thank tions of the nucleolar organizer regions in arctic char our colleague Dr J. Trout for his revision of the English (Salvelinusalpinus). Cytogenet. Cell Genet., 48,9—12. text. This work was funded by a CICYT Project No. PHILLIPS, R. B., ZAJICEK, K. D. AND UIIER, F. M. 1986. Chromo- PB87/0881. some banding in salmonid fishes: nucleolar organizer regions in Oncorhynchus. Can. 3. Genet. Cytol., 28, 502—510. References SANCHEZ, L., MARTINEZ, P., VHAS, A. AND BOUZA, C. 1990. Analysis of the structure and variability of nucleolar BIANCHI,M. S., BlANCH!, N. 0., PANTELIAS, G. E. AND WOLFFS, G. organizer regions of Salmo truttabyC- Ag and restriction 1985. The mechanism and pattern of banding induced by endonuclease banding. Cytogenet.Cell Genet., 54,6—9. restriction endonucleases in human chromosomes. SCHMID, M. G. AND DE ALMEIDA, c. 1988. Chromosome banding Chromosoma,91, 131—136. in Amphibia. XII. Restriction endonuclease banding. CAU, A., SALVADORI, S., DEIANA, A. M., BELLA, J. L. AND MEZZANOTFE, Chromosoma,96,283—290. n. 1988. The characterization of Muraenahelena L. SCHWARZACHER, T., AMBROS, P. AND SCHWEIZER, D. 1980. mitotic chromosomes: karyotype, C-banding, nucleolar Application of Giemsa banding to orchid karyotype organizer regions, and insitu digestionwith restriction analysis. P1. Syst. Evol., 134, 293—297. endonucleases. Cytogenet.Cell Genet., 47, 223—226. SCI-IWEIZER, D. 1976. Reverse fluorescent chromosome band- FORESTI, F., ALMEIDA TOLEDO, L. F. AND TOLEDO F°. S A. 1981. ing with chromomycin and DAPI. Chromosoma, 58, Polymorphic nature of nucleolus organizer regions in 307—324. fishes. Cytogenet. Cell Genet., 31, 137—144. SOLA, L, ARCANGELI. R. AND CATAUDELLA, s 1986. Nucleolus HOWELL, W. M. AND BLACK, D. A. 1980. Controlled silver-staining organizer chromosomes in a teleostean species of tetra- of nucleolus organizer regions with a protective colloidal ploid origin. Cyprinuscarpio. Cytogenet. Cell Genet., 42, developer: a 1-step method. Experientia,36,1014—1015. 183—186. LLOYD, M. A. AND THORGAARD, 6. H. 1988. Restriction endo- UEDA, T. 1983. Cytogenetical characters of 4 species in the nuclease banding of rainbow trout chromosomes. salmonid fishes. Bull Fac. Educ. Utsunomiya University, Chromosoma,96, 171—177. 34, 53—6 1. LOPEZ-FERNANDEZ, C., GOSALVEZ, J. AND MEZZANOTFE, R. 1989. uYENO, T. 1972. Chromosomes of offspring resulting from Heterochromatin heterogeneity in Oedipodagermanica crossing coho salmon and brook trout. Jpn J. Ichthyol., 19, 166—17 1. (Orthoptera)detected by in situ digestion with restriction endonucleases. Heredity,62,269—277. WILEY, J. E., LITFLE, M. L., ROMANO, M. A., BLOUNT, D. A. AND CLINE, LOZANO, R.,RUIZREJON, C. AND RUIZ REJON, M. 1988. A method G. R. 1989. Polymorphism in the location of the 18S and for increasing the number of mitoses available for cyto- 28S rRNA genes on the chromosomes of the genetic analysis in rainbow trout. StainTechnol., 63, diploid—tetraploid treefrogs Hyla chrysoscelis and H. 335—338. versicolor.Chromosoma, 97, 481—487.