The Research About Chromosome Banding Technology On Oncorhynchus Mykiss

Metaphase chromosome specimens of Oncorhynchus mykiss injected with PHA and colchicin,were prepared from short-term culture of kidney cells with air-drying technique.The number of the chromosome ranged from 56 to 64,but the modal number of the chromosome was 60,which was in proportion to 77.27%.It diploid chromosome number is 2n = 60.The karyotype is composed of 20m,10sm,16t chromosomes,NF = 104.Diploid nucleus DNA content was measured from the somatic cell of Oncorhynchus mykiss using flow cytometer (Beckman cellLab Quant,made in USA) and the DNA content(2.3pg.N^-1) of erythrocytes of chick(Gallus sp) as standard.The Oncorhynchus mykiss DNA content is 3.56pg.N^-1,which is correspond with the result measured chromosome number as ploidy.According to the present results and date reported,Oncorhynchus mykiss is possibly an tetraploid(4n).Using the methods of Sumner(1972) with slightly modified,the C-bands were showed in all chromosomes of Oncorhynchus mykiss.Some variable bands were also observed.It is well known that C-bands are equated with constitutive heterochromatin.Although many functions of the heterochromatin have been proposed,it should be admitted that the heterochromatin holds very few"active"genes and the majority are made up of many"silent"genes or"nonfunctioning"fragments of DNA.Most of the macrochromosomes of Oncorhynchus mykiss show a prominent centromeric C-banding.The GTG banding(G-banding) was carried out by the standard trypsin method with slight modification,which works well for protochordate because a good number of reproducible Gbands are consistently obtained from the kidney cells of late blastulae and early gastrulae of Oncorhynchus mykiss.The trypsin treatment for G-banding is an important factor in obtaining well-banded chromosomes,while the slide aging is not.After G-banding,87%of the chromosome surface is positively stained.It is generally accepted that chromosome banding patterns appear closely related to differences in DNA base composition.The nucleolar organizer regions are the chromosomal sites of the genes for ribosomal RNA (rRNA),Which are readily stained by the Ag-staining.The chromosomes of Oncorhynchus mykiss were thus stained according to the method of Howell and Black(1980) that was slightly modified.The posotion of nucleolar organizer regions were situated on the Large arm region of no.1 and 13 pair chromosomes and the total number of NORs was 4.A modified Brdu-Hoechst-Giemsa Method is described for the demonstration of easily reproducible DNA-replication bands in the chromosomes of Oncorhynchus mykiss.It involves culturing the cell in medium RPMI-1640 supplemented with 20%fetal calf serum at a temperature of 16℃,treating living cells with a final concentration of 10 ug/ml Brdu for 16～18h as well as with 2 ug/ml AMD along with 0.05ug/ml colchicine for 2h before harvest. Using this method,the DNA-Replication Bands in the chromosomes of Oncorhynchus mykiss is observed with excellent results.We used silver nitrate staining,CMA₃ and FISH to study the chromosomal localization of both the major ribosomal genes and the nucleolar organizer regions as well as that of the minor ribosomal genes(5S rDNA) in Oncorhynchus mykiss,all the metaphases examined showed the presence of multiple NOR-bearing sites,while the gene for 5S rRNA proved to be localized on chromosome pairs.Furthermore,one of the two 5S rDNA was shown to be co-localized with the major ribosomal cluster(18 S rDNA).Ribosomal RNA is by far the most abundant product of transcription and accounts for between 80 and 90%of the total mass of cellular RNA,both in prokaryotes and in eukaryotes.In addition to the major class,which consists of the genes for 18S,28S and 5.8S rRNA,the genes for the ribosomal RNA of the eukaryotes are represented by a class of genes that code for 5S RNA(5S rDNA),a component of the major subunit of ribosomes.In the majority of higher eukaryotes,the genes for 5S rRNA are separated in the genome from the major cluster and are present as tandem repetitions,separated by nontranscribed spacer regions,with a"head-to-tail"arrangement.