Indian Journal of Experimental Biology Vol. 45, May 2007, pp. 413-418

GC- rich heterochromatin in silver stained nucleolar organizer regions (NORs) fluoresces with Chromomycin A3 (CMA3) staining in three species of teleostean fishes (Pisces)

Jayanta Kumar Das & Anisur Rahman Khuda-Bukhsh* Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741 235, India Received 14 December 2005; revised 23 January 2007

In a bid to ascertain the molecular architecture of the silver positive regions (NORs) in chromosomes of three species of fish, namely, Hemibagrus menoda (Hamilton), Sperata seenghala (Sykes) (Fam: Bagridae) and Mastacembelus armatus

(Lacepède) (Fam: Mastacembelidae), an additional staining methodology using a fluorochrome dye (Chromomycin A3) was deployed along with the AgNO3 technique. The nucleolar organizing regions (NORs) were located terminally at the shorter arms (Tp) of one pair of submetacentric chromosomes (No.3) in H. menoda (2n=58), at the longer arms (Tq) of one pair of submetacentric chromosomes (No.5) in S. seenghala (2n=50) and at the shorter arm (Tp) of one pair of homologous submetacentric chromosomes (No.6) in M. armatus (2n=48). Staining with Chromomycin A3 produced bright fluorescing zones in GC- rich heterochromatin of Ag-positive NORs. The results indicate a more general trend of existence of an overlapping region between NOR and GC- rich fluorescing zones, the active sites of rRNA genes (rDNA) in this primitive group of vertebrates although exceptions to this situation has been reported in a couple of extant fish species earlier. More data utilizing such combined methodologies are warranted to understand the structural organization of fish chromosomes more precisely.

Keywords: Ag-NOR, Chromomycin A3 banding, G-C rich region, Hemibagrus menoda, Karyotype, Mastacembelus armatus, Sperata seenghala

The menoda catfish (Hemibagrus menoda) (Order: Bagridae appear to have been cytogenetically Siluriformes, Family: Bagridae) are found plenty in analyzed2-5 by their Giemsa karyotypes, Ag-NOR, rivers, bottom of ponds of soft wet clay of tropical Asia CMA3 and 3 species of family Mastacembelidae (the Ganges, Brahmaputra, Mahanadi, Godavari rivers appear to have been cytogenetically analyzed by their of India, drainages, rivers of Bangladesh, Nepal and Giemsa karyotypes only in India2 or elsewhere so far. Myanmar) and the other giant river catfish (Sperata Cytogenetical analysis of fish is often painstaking seenghala) (Order: Siluriformes, Family: Bagridae) are because most of the fishes possess a large number of mostly found in rivers, canals, beels, ditches, small chromosomes. Therefore, only a small freshwater fields of tropical Asia (39°N-8°N; percentage (about 12%) of karyological data in fish Afghanistan, Pakistan, India, Nepal and Bangladesh). have been known out of the ever-increasing list of The zig-zag eels (Mastacembelus armatus) (Order: 28,900 species so far recorded taxonomically1,2,6, 7. Synbranchiformes, Family: Mastacembelidae) are The localization of nucleolar organizer regions usually abounded in fresh water or brackish water (22°- (NORs) is considered as an important tool in certain 28°C, pH range 6.5-7.5) streams, still water ponds, studies on evolution and cytotaxonomy8. In fishes, the coastal marshes, dry zone tanks, rivers of tropical Asia1 number and location of the Ag-NORs represent one of (from Pakistan to Vietnam and Indonesia, 38°N-1°N). the few chromosomal markers useful in drawing Cytogenetic studies in the family Bagridae and meaningful conclusion on some cases of phylogenetic Mastacembelidae are scarce. Only 8 species of family enigma in a few groups of fishes studied till 9, 10-12 ______date . *Correspondent author In higher eukaryotes, ribosomal RNA (rRNA) genes Phone: 033-25828750 extn. 315 are organised as two distinct multigene families. One (Off), 033-25828768 (Res) E-mail: [email protected], class is represented by the major 45S rDNA, which [email protected] consists of a transcriptional unit that codes for the 18S, 414 INDIAN J EXP BIOL, MAY 2007

5.8S and 28S rRNA and an intergenic spacer (IGS). nitrate by following the single-step method of Howell Multiple tandem copies of this array correspond to the and Black27 for Ag-NOR locations; and for nucleolar organizer regions (NORs). The other class, localization of rDNA, the CMA3 technique as codes for the minor 5S rRNA13. suggested by Schweizer28 was followed. A total of 60- Nucleolar organizer regions (NORs), formed by 70 metaphase spreads each of three species were major 45S rDNA can be visualized by using analyzed to detect and confirm the location and 14 chromomycin A3 or AgNO3 staining techniques and number of Ag-NOR as well as CMA3 stained NORs. only specific chromosomes (nucleolar chromosomes) The filter module having excitation filter BP 450- are involved in the formation of nucleoli in fish. 490nm and suppression or barrier filter LP 515nm Although the sites of synthesis of some nucleoli are with a dichroic mirror 510nm of Leica DMLS identified in prophase or metaphase chromosomes as fluorescent microscope was used to study CMA3 secondary chromosome constriction(s) in fish, these stained metaphase chromosome preparations. The sites of nucleolar synthesis are less defined regions and well spread preparations were photographed (using vary considerably in their distribution in different Kodak 400ASA film) with a manual camera (Pentax 15-20 groups of fish . Chemically, the nucleolar organizer K1000) at magnification of 1000 ×. regions have been reported to contain GC-rich DNA in many vertebrates, including fish21, but exceptions do Results occur. Similarly, not every secondary constriction of The Giemsa-stained karyotype of diploid chromosome will necessarily be on a NOR-bearing metaphase complements in H. menoda contained 29 chromosome; and not all sites of rDNA necessarily pairs of chromosomes (NF=100) comprising 11 pairs of metacentric (nos. 4, 6, 7, 12, 16-18, 23, 24, 27, 29), appear as a secondary constriction. Chromomycin A3 staining has been used for determination and 10 pairs of sub-metacentric (nos. 1-3, 5, 8, 9, 11, 13, confirmation of the number and localization of NORs, 14, 21), and 8 pairs of acrocentric (nos. 10, 15, 19, 20, as well as for pinpointing GC-rich sites of 22, 25, 26, 28) chromosomes, (Fig. 1a); in S. transcriptionally active rRNA genes for synthesizing seenghala contained 25 pairs of chromosomes 18S or 28S rRNA in several fish species4, 5, 22- 24. (NF=74) comprising 5 pairs of metacentric (nos. 2, 6, Therefore, the present work has been undertaken to 9, 14, 24), 7 pairs of sub-metacentric (nos. 1, 4, 5, 7, determine the number, localization and pattern of 10, 12, 13), 3 pairs of sub-telocentric (nos. 3, 11, 18) NORs and to analyze the relationship, if any, between and 10 pairs of acrocentric (nos. 8, 15-17, 19-23, 25) the active sites of GC- rich heterochromatin of rRNA chromosomes (Fig.1b) and in M. armatus contained genes (rDNA) and the silver-positive NORs in 24 pairs of chromosomes (NF=62) comprising 5 pairs Hemibagrus menoda, Sperata seenghal and of metacentric (nos.7-10, 13), 2 pairs of sub- Mastacembelus armatus by using of both, AgNO and metacentric (nos. 1, 6), 1 pair of sub-telocentric (nos. 3 12) and 16 pairs of telocentric (nos. 2-5, 11, 14-24) chromomycin A3 staining techniques. To our knowledge, such study has not been done in any of chromosomes (Fig. 1c). these species or in any other congeneric species The NORs were observed on the terminal position belonging to the three genera earlier in India or of the small arm (Tp) of one pair of sub-metacentric elsewhere. chromosomes in H. menoda (no. 3 in the Giemsa karyotype and the silver-stained NOR pair shown as Materials and Methods inset below the Giemsa karyotype in Fig.1a), of the Ten live specimens of H. menoda, eight specimens long arm (Tq) of one pair of sub-metacentric of S. seenghala, and nine live specimens of M. chromosomes (no.5 in the Giemsa karyotype and the armatus, collected locally, were intramuscularly silver-stained NOR pair shown as inset below the injected with 0.03% colchicine @ 1ml/100g body Giemsa karyotype in Fig.1b) in S. seenghala and of weight and kept for 2.5 hr prior to sacrifice. The the small arm (Tp) of one pair of sub-metacentric somatic chromosomes were prepared from their chromosomes (no.6 in the Giemsa karyotype and the kidney cells by the flame-drying technique described silver-stained NOR pair shown as inset below the elsewhere25 and their nomenclature adopted by Giemsa karyotype in Fig.1c) in M. armatus. following the method of Levan et al26. The silver preparations of metaphase complements Some of the slides were routinely stained with of H. menoda (Fig.2a), S. seenghala (Fig.2c) and M. Giemsa while some others were stained with silver armatus (Fig.2e) corresponded well to the greater DAS & KHUDA-BUKHSH: GC-RICH HETEROCHROMATION IN NORs IN FISHES 415

(2n=54)31-33, (2n=50)34-36 and three species of Mastacembelus, namely, M. aculeatum (2n=48), M. pancalus (2n=48)37 and M. armatus (2n=48)38 have earlier been studied from India. The present findings of 2n=58 in H. menoda (with a chromosome formula of 22m+20Sm+16A; NF=100), 2n=50 in S. seenghala (10m+14sm+6st+20A, NF=74) and 2n=48 in M. armatus (10m+4sm+2st+32t, NF=62) confirm the karyotypes reported for these three species of fish earlier.

However, the localization, number and pattern of NORs in Hemibagrus menoda, Sperata seenghala and Mastacembelus armatus, have not been reported earlier either by Ag-NOR technique, or by the additional method of CMA3 staining in these species. Staining with GC-specific fluorochrome CMA3 produced positive signals at all Ag-NORs. The results indicated that active NORs fluoresced brightly with CMA3 that bound preferentially to GC- rich chromatin segments characteristically located on rDNA sites of these cold-blooded vertebrates. Therefore, this seems to be the first report on NORs as revealed from a combined method using both silver nitrate and CMA3 staining. Like a few other extant species of fish, an intimate association between the NOR-bearing chromosomes and GC-rich heterochromatin of active rRNA genes appeared to exist in these three species of fish as well. In three species of Indian bagrid, namely, Mystus tangera, Rita rita and Mystus gulio studied earlier4-5, the two species of bagrid under present study had also a similar pattern of NOR localization. However, to our knowledge, no other congeneric species of Mastacembelus belonging to the family Mastacembelidae has been studied through both the techniques, for which no specific comments on the pattern of localization of NORs in this family could be made. However, it is quite enigmatic that the Fig. 1⎯Giemsa stained karyotypes of diploid metaphase occurrence of GC-rich heterochromatin sequences complements of (a) H. menoda, (b) S. seenghala, (c) M. armatus, either adjacent to or interspersed among NORs has the silver-stained NOR pair of H. menoda, S. seenghala and M. also been reported to occur widely in many species armatus shown as inset below the Giemsa karyotypes of Figs a, b, 39, 11 and c respectively. Bar= 10 μm. belonging to diverse other families .

40 fluorescing regions of chromosomes prepared by In one of their earlier studies, Jankun et al. reported a positive correlation to exist between CMA3 CMA3 technique (Fig. 2b, 2d, 2f), indicating that the active transcribing zones of NOR-bearing stained sites and active rRNA genes (45S rRNA unit chromosomes actually represented the GC-rich active which consists of a transcriptional unit that codes for sites for rRNA genes. 18S, 5.8S, 28S rRNA, IGS and minor 5S rRNA) in some coregonid fish, suggesting its role in ribosomal Discussion gene activity. The same authors23 later verified by The somatic Giemsa karyotypes of Hemibagrus deploying a further improved method of menoda (2n=58)29 (2n=56)30, Sperata seenghala PRINS/CMA3 sequential staining and confirmed the 416 INDIAN J EXP BIOL, MAY 2007

Fig. 2⎯Photomicrographs of silver stained diploid metaphase complements of (a) H. menoda, (c) S. seenghala, (e) M. armatus, and CMA3 stained diploid metaphase complements of (b) H. menoda, (d) S. seenghala and (f) M. armatus; CMA3 fluorescent regions are indicated by arrows. Bar= 10 μm. karyotypic correspondence between rRNA genes and in fish and the improved techniques (FISH) used by NOR sites in some Eurasian coregonid fish, although others44-46 may be needed to verify the relationship. the correspondence between Ag-positive CMA3 Our preliminary results also highlight the need for a fluorescence signals and true NORs has been called wider scale study of such relationship. However, it into question in several recent papers41-43. seems more probable that the kind of association Therefore, from the data available so far, no noted in the present study through the combined definite conclusion can yet be drawn as the exact and staining procedures may be more typical in fish as a precise relationship between NORs and rRNA genes group. More work in this direction is necessary and DAS & KHUDA-BUKHSH: GC-RICH HETEROCHROMATION IN NORs IN FISHES 417

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