Hereditas 131: 221-225 (1999)

Ribosomal RNA gene loci and silver-stained nucleolar organizer regions associated with heterochromatin in Alaskan char Salvelinus malma and chum salmon Oncorhynchus keta MONICA ALONSO’, ATUSHI FUJIWARA’, ETSURO YAMAHA2, SHIZUO KIMURA2 and SY UITI ABE’,3

I Laboratory of Cytogenetics, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-081 0, Japan ’ Nanae Fish Culture Experimental Station, Faculty of Fisheries, Hokkaido University, Kameda 041 - 1105, Japan ’ Chromosome Research Unit, Faculty of Science, Hokkaido University, Sapporo 060-081 0, Japan

Alonso, M., Fujiwara, A., Yamaha, E., Kimura, S. and Ahe, S. 1999. Ribosomal RNA gene loci and silver-stained nucleolar organizer regions associated with heterochromatin in Alaskan char Suluelinus n d m u and chum salmon Oncorhynchus kc,fu.-Hereditus 131: 221 -225. Lund, Sweden. ISSN 0018-0661. Received July 7, 1999. Accepted December 3, 1999

Nucleolus-forming 5.8S, 18s and 28s ribosomal RNA gene (rDNA) loci were assigned by fluorescence in situ hybridization (FISH) to the distal half of the short arms of a large-sized submetacentric pair in the Alaskan char (Suluelinu.7 mulmu) and to the distal region of the long arms of a medium-sized submetacentric pair in the chum salmon (Oncorhynchus k m ) , respectively. In each species, heteromorphic FISH signals, spanning whole satellite region and secondary constriction, imply an intraspecific variation in the size of rDNA loci. Size variation of silver-stained nucleolar organizer regions (AgNORs) was also apparent between or within the assigned rDNA loci in each species, suggesting a possible inter- or intralocus inactivation of rDNAs. C-band positivity of assigned rDNA loci and AgNORs unequivocally showed their association with heterochromatin in these species.

Syuiti Ahe. Chroniosome Research Unit, Fuculty of Science, Hokkaido Unioersify, North 10, West 8, Kita-ku, Supporo 060-0810, Jupan. E-mail: sabe@ees.hokudai.ac.jp

The ribosomal RNA genes (rDNAs) are usually en- coded in a tandemly repeated unit that is present in a high copy number. This unit possesses a transcribed zone with highly conserved coding regions for the nucleolus-forming 5.8S, 1 8 , and 28s rDNAs. It has been generally accepted that nucleolar organizer re- gions (NORs) are the chromosomal sites containing multiple copies of these rDNAs, and that transcrip- tionally active NORs, bearing a complex of residual acidic protein associated with the fibrillar center of the nucleolus and nascent pre-RNA (JORDAN 1987), are stained with silver (AgNORs) (REEDER 1990). AgNORs are hence useful for investigating the ex- pression of rDNAs. Moreover, the species-specific number and location of rDNA/AgNORs can provide a useful karyotypic marker, particularly in fishes (PENDAS et al. 1994; FUJIWARA et al. 1998) and amphibians (KING et al. 1990) because of no estab- lished G- or Q-banding techniques.

Previously, we have detected a considerable inter- or intraspecific variation in the size and number of rDNA loci/AgNORs in four salmonid species, by means of fluorescence in situ hybridization (FISH)

and Ag-staining, and found their association with heterochromatin as revealed by C-banding (FUJI- WARA et al. 1998). In order to ascertain whether such variation and heterochromatin association of rDNA loci/AgNORs are common in salmonid fishes, we examined here the localization of 5.8S, 18s and 28s rDNAs and AgNORs in the embryonic chromosomes of Alaskan char (Salvelinus malma) and chum salmon (Oncorhynchus keta) using the same techniques.

MATERIALS AND METHODS

Chromosome preparation Gametes from one pair of the parents of Alaskan char and chum salmon were obtained from the Nanae Fish Culture Experimental Station, Hokkaido University. Metaphase chromosomes were prepared from 15 early embryos at the tail-bud stage (BAL- LARD 1973) of each species after artificial fertiliza- tion, as previously described (INOKUCHI et al. 1994). At least 30 metaphases from each embryo were evalu- ated for FISH and chromosome banding as described below.

222 M . Alonso et al. Hereditas 131 (1999)

FISH

FISH procedures were essentially the same as de- scribed previously (FUJIWARA et al. 1998). In brief, two clones, pHr21Ab and pHr14E3, containing 5.8S, 18s and 28s rDNAs of human rDNA obtained from Japanese Cancer Research Resources Bank, Tokyo, and a rainbow trout 5.8s rDNA clone (FUJIWARA et al. 1998) were used simultaneously as the mixed probe after labeling with biotin-1 6-dUTP by nick translation (Boehringer Mannheim). Formamide-de- natured chromosome slides were hybridized with 150 ng of rDNA probe per slide. After posthybridization washing, chromosome slides were subjected to detec- tion with avidin-FITC (Boehringer Mannheim) and counterstained with propidium iodide (PI, Sigma). Hybridization signals were observed and pho- tographed under a Nikon fluorescence microscope using a Nikon B-2A filter, with Fujifilm Provia ASA 400 color reversal film.

Chromosome handing

Either freshly prepared or FISH-treated chromosome slides were used for chromosome banding as previ- ously (FUJIWARA et al. 1998). The one-step silver staining (HOWELL and BLACK 1980) and C-banding (SUMNER 1982) were performed as described (FUJI- WARA et al. 1998).

RESULTS

Chromosome numbers of the Alaskan char and chum salmon were 2n = 82 and 2n = 74, respectively. Kary- otype of Alaskan char comprised 16 meta- (M) or submetacentric (SM), 4 subtelocentric (ST), and 62 acrocentric (A) chromosomes as previously described in Japanese char (ABE and MURAMOTO 1974). A large-sized SM pair in this species has a prominent secondary constriction in the distal part of the short arms (Fig. la), which can be seen in nearly 100 % of metaphases scanned. Chum salmon karyotype con- tained 32 M or SM and 42 A, in agreement with our previous report (INOKUCHI et al. 1994). A secondary constriction in the distal portion of the long arms of a medium-sized SM pair (Fig. lb) is rarely seen in metaphases examined.

Representative profiles of FISH with the mixed rDNA probe in two salmonid species examined are shown in Fig. 2. FISH signals occupied distal half of the short arms of a large-sized SM pair, including the secondary constriction and satellite region, in the Alaskan char (Fig. 2a). In the chum salmon, rDNA loci were assigned to the distal half of the long arms of a medium-sized SM pair, spanning the secondary constriction and satellite region (Fig. 2b). Although

no other chromosome sites had FISH signals besides these regions, the assigned rDNA loci were appar- ently heteromorphic in most of metaphases examined in each species (Fig. 2a and b).

Silver-staining revealed polymorphic AgNORs oc- cupied a limited region within the rDNA loci in both species, resulting in a varying size of AgNORs be- tween the loci (Fig. l a and b). Such a polymorphism was more distinct in the Alaskan char than the chum salmon. The region bearing rDNA loci and AgNORs was unequivocally stained with C-banding in both species (Fig. l a and b), in agreement with our previ- ous findings (FUJIWARA et al. 1998).

DISCUSSION

The present FISH study unequivocally assigned the 5.8S, 18s and 28s (major) rDNA loci on a single chromosome pair in both the Alaskan char and chum salmon. To our knowledge, six salmonid species, i.e., rainbow trout (Oncorhynchus mykiss), masu salmon (0. masou), Atlantic salmon (Salmo salar), brown trout ( S . trutru), brook trout (Saluelinus jontinalis), and Japanese huchen (Hucho perryi), have so far been studied for localization of rDNA loci with FISH (PENDAS et al. 1993a, b; PENDAS et al. 1994; MORAN et al. 1996; FUJIWARA et al. 1997, 1998). Among these, three have the major rDNA loci on one chro- mosome pair, although non-nucleolus-forming 5 s (minor) rDNA loci tended to scatter on two or more (up to eight) chromosome pairs in five of the six species (PENDAS et al. 1994; MORAN et al. 1996; FUJIWARA et al. 1998). Including the Alaskan char and chum salmon, five out of eight salmonids exam- ined with FISH have the major rDNA loci on a single chromosome pair, among which the rainbow trout and Atlantic salmon showed tandemly aligned minor and major rDNA loci, with or without extra minor rDNA loci on other chromosomes (PENDAS et al. 1994; MORAN et al. 1996; FUJIWARA et al. 1998). These findings may indicate a different evolutionary trait of two classes of rDNA loci in salmonid fishes (FUJIWARA et al. 1998), although localization of the minor rDNA loci remained to be investigated in the present two salmonid species.

Size polymorphism between the assigned rDNA loci is apparent in both the Alaskan char and chum salmon (Fig. 2a and b), as has been observed in other salmonid species (PENDAS et al. 1993a, b; PENDAS et al. 1994; MORAN et al. 1996; FUJIWARA et al. 1998). However, it is unknown which parent has homomor- phic or heteromorphic rDNA loci resulting in the observed polymorphism because only one pair of the parents was used in the present study without know- ing their karyotypes. The observed size polymor-

Hereditas 131 (1999) rDNA loci in salmonid jishes 223

phism may be relevant to the enlargement of rDNA loci in these species. Similar enlarged rDNA loci have also been observed in other salmonid species (PEN- DAS et al. 1993a, b; MORAN et al. 1996; FUJIWARA et al. 1998). Since the salmonid rDNA loci so far exam- ined coincide with C-banding positive region as seen in the present two species, implication of heterochro- matin in the accumulation of rDNA loci through unequal crossing-over or sister chromatid exchanges involving repeated sequences and adjacent loci has been stressed previously (PENDAS et al. 1993b; FUJI-

WARA et al. 1998). Such an accumulation might have been different between or among rDNA loci during salmonid evolution. This may be favored by the observation of large interindividual differences in the number of rDNA units in salmonid fishes (SCHMIDTKE et al. 1976).

Polymorphic AgNORs in the present salmonid spe- cies, distinctly in the Alaskan char (Fig. l a and b), are thought to be related to transcriptional regulation rather than structural alteration of the major rDNAs (SOLA et a]. 1984; LOPEZ et al. 1989; PENDAS et al.

Fig. l a and b. Conventionally stained partial karyotype (top) of Alaskan char (Saluelinus malma) (a) and chum salmon (Oncorhynchus keta) (b). Arrows indicate the secondary constriction on a large-sized submetacentric pair in Alaskan char and on a medium-sized submetacentric pair in chum salmon. AgNORs and C-bands, with apparent size variations, on the corresponding chromosome pairs from different metaphases are indicated by arrowheads (bottom) in each species.

224 M. Alvnso et al. Hereditas 131 (1999)

Fig. 2a and b. Representative profile of fluorescence in situ hybridization (FISH), showing chromosomal location of 5.8S, 18s and 28s rDNAs (arrows) in Alaskan char (a) and chum salmon (b).

1993b; FUJIWARA et al. 1998). This is favored by the findings that a majority of potentially active numer- ous dispersed major rDNA loci in the brown trout and brook trout become transcriptionally silence, re- sulting in a frequent AgNOR polymorphism (PEN- DAS et al. 1993b; FUJIWARA et al. 1998). AgNOR expression in a limited region within o r between the major rDNA loci in the salmonid species studied could be explained by the intra- or interlocus inacti- vation to be controlled by different mechanisms, as have been discussed previously (FUJIWARA et al. 1998). Nevertheless, it remains to be ascertained why and to what extent the untranscribed rDNA loci are conserved in the genome of salmonid fishes, and whether the inactive NORs become active when a structural change occurs in the rDNA loci.

In conclusion, the present findings on the Alaskan char and chum salmon further advocate the limited gene activation among the abundant heterochromatic major rDNA loci in salmonid fishes. Although molec- ular composition of heterochromatic rDNA loci and the underlying mechanism(s) for their inactivation remain to be elucidated, mapping of rDNA loci by FISH should be pursued to analyze salmonid genome organization.

ACKNOWLEDGEMENTS

The authors are grateful to Professor emeritus Dr. M.C. Yoshida, Faculty of Science, Hokkaido University, for his keen interest on the present study. Supported partly by the Grant-in-Aid for Specially Promoted Research (2) from the Ministry of Education, Science, Sports and Culture, Japan. M.A. was awarded the Scholarships for Foreign Researcher from the same Ministry.

REFERENCES

Abe S and Muramoto J, (1974). Differential staining of chromosomes of two salmonoid species, Salvelinus leu- comaenis (Pallas) and Salvelinus matma (Walbaum). Proc. Japan Acad. 50: 507-511.

Ballard WM, (1973). Normal embryonic stages for salmonid fishes, based on Salmo gairdneri (Richardson) and Salvelinus fontinalis (Mitchell). J. Exp. Zool. 148: 7-26.

Fujiwara A, Abe S, Yamaha E, Yamazaki F and Yoshida MC, (1997). Uniparental chromosome elimination in the early embryogenesis of the inviable salmonid hybrids between maw salmon female and rainbow trout male. Chromosoma 106: 44-52.

Fujiwara A, Abe S, Yamaha E, Yamazaki F and Yoshida MC, (1 998). Chromosomal localization and heterochro- matin association of ribosomal RNA gene loci and silver-stained nucleolar organizer regions in salmonid fishes. Chromosome Res. 6: 463-471.

Howell WM and Black DA, (1 980). Controlled silver-stain- ing of nucleolus organizer regions with a protective colloidal developer: A 1 -step method. Experientia 36: 1014-1015.

Inokuchi T, Abe S, Yamaha E, Yamazaki F and Yoshida MC, (1994). BrdU replication banding studies on the chromosomes in early embryos of salmonid fishes. Hereditas 121: 255-265.

Jordan G, (1987). At the heart of the nucleolus. Nature 329: 489-499.

King M, Contreras N and Honeycut RL, (1990). Variation within and between nucleolar organizer regions in Aus- tralian hylid frogs (Anura) shown by 18s + 28s in situ hybridization. Genetica 80: 17-29.

Lopez JR, Alvarez MC, Thode G and Martinez G, (1989). Karyotype divergence in Symphodus melops and Sym- phodus roissali (Labridae, Perciforms): C-banded and Ag-NOR karyotypes. Genome 32: 35-39.

Morrin P, Martinez JL, Garcia-Vasquez E and Pendas AM, (1996). Sex chromosome linkage of 5s rDNA in rainbow

Hereditas 13 1 (1999) rDNA loci in sulmonidjshes 225

trout (Oncorhynchus mykiss). Cytogenet. Cell Genet. 75: 145-150.

Pendis AM, Moran P and Garcia-Vasquez E, (1993a). Ribosomal RNA genes are interspersed throughout a heterochromatic chromosome arm in Atlantic salmon. Cytogenet. Cell Genet 63: 128-130.

Pendas AM, Moran P and Garcia-Visquez E, (1993b). Multi-chromosomal location of ribosomal RNA genes and heterochromatin association in brown trout. Chro- mosome Res. 1: 63-67.

Pendas AM, Moran P, Freije JP and Garcia-Visquez E, (1 994). Chromosomal mapping and nucleotide sequence of two tandem repeats of Atlantic salmon 5s rDNA. Cytogenet. Cell Genet.67: 31 -36.

Reeder RH, (1990). rRNA synthesis in the nucleolus. Trends Genet.6: 390-395.

Schmidtke J, Zenzes MT, Weiler C, Bross K and Engel W, (1976). Gene action in fish of tetrdploid origin. IV. Ribosomal DNA amount in clupeoid and salmonoid fish. Biochem. Genet. 14: 293-297.

Sola L, Camerini B and Cataudella S, (1984). Cytogenetics of Atlantic eels: C- and G-banding, nucleolus organizer regions, and DNA content. Cytogenet. Cell Genet. 38: 206- 2 10.

Sumner AT, (1982). A simple technique for demonstrating centromeric heterochromatin. Exp. Cell Res. 75: 304- 306.