Molecular analysis of a NOR site polymorphism in brown trout (Salmo trutta): organization of rDNA intergenic spacers.

Using restriction endonuclease mapping, we have analyzed the organization of rDNA (DNA coding for ribosomal RNA (rRNA)) units in the salmonid fish Salmo trutta, as an initial step toward understand the molecular basis of a nucleolar organizer region (NOR) site polymorphism detected in this species. The size of the rDNA units ranged between 15 and 23 kb, with remarkable variation both within individuals and between populations. Three regions of internal tandem repetitiveness responsible for this length polymorphism were located to the intergenic spacers. NOR site polymorphic individuals showed a higher number of length classes, in some cases forming a complete 1 kb fragment ladder. The amount of rRNA genes was as much as 8-fold higher in polymorphic individuals compared with standard individuals. All individuals from the most polymorphic population showed a 14-kb insertion of unknown nature in a small proportion (below 25%) of the 28S rRNA genes.     

Genetic Analysis of the 5s RNA Genes in DROSOPHILA MELANOGASTER

The 5S RNA genes of Drosophila melanogaster in either an isogenic wild-type or a multiply inverted (SM1) chromosome 2 increase their multiplicity when opposite a deficiency for the 5S gene site. This is analogous to the compensation phenomenon previously described for the 18S and 28S ribosomal RNA genes of the X chromosome nucleolus organizer region. Molecular hybridization of 5S RNA to DNA containing various doses of the 56F1-9 region of chromosome 2 demonstrates that most, if not all, of the 5S genes reside in or near this region. Also, a deficiency missing approximately one-half of the wild-type number of 5S genes was isolated and genetically localized. This mutant has a phenotype like that of bobbed, a mutant known to be partially deficient in 18S and 28S ribosomal RNA genes. Finally, we report the existence of a chromosomal rearrangement which splits the second chromosome into two segments, each containing 5S DNA.     

The presence of 5 S RNA genes in two consecutive chromosome bands in Chironomus tentans

It has earlier been shown by in situ hybridization that the 5 S RNA genes are located in region 2A of chromosome II in Chironomus tentans [1]. In the present study the resolution in this chromosome region could be considerably improved by using the inversion 1 A/9C of chromosome II, which carries region 2A, not in the usual position close to one end of chromosome II, but in the middle of the chromosome. It was then revealed that electrophoretically isolated 5 S RNA hybridized in situ with two adjacent bands in the region 2A. It was also observed that in heterozygotes harbouring one normal chromosome II and one with the inversion 1 A/9C, there is a close physical contact between the bands containing the 5 S RNA genes and the nucleolar organizer region.     

The location of repeated DNA sequences in the chromosomes of Chironomus tentans

Polytene chromosomes of Chironomus tentans were hybridized in situ with in vivo labelled nuclear and chromosomal RNA. Nuclear RNA formed hybrids preferentially in five distinct regions considered to contain clustered, repeated DNA sequences. These are the two nucleolar organizer regions, Balbiani ring 1 and 2, and the 5 S RNA genes in region 2A of chromosome II, which together comprised almost 70% of the total number of grains over the complement. The remaining grains were diffusely distributed over the chromosomes. There was a significant difference in the distribution of grains when RNA from different chromosomes was used for hybridization. Chromosome I RNA hybridized preferentially with chromosome I, and chromosome II+III RNA preferentially with chromosome II+III. Some regions within the chromosomes hybridized significantly more chromosomal RNA than other regions. A considerable cross-hybridization of RNA from one particular type of chromosome with the other chromosomes was also found. It is concluded that repeated DNA sequences which hybridize with heterogeneous chromosomal RNA in C. tentans are widely dispersed in the genome. Some of these sequences have a delimited localization, others are dispersed, and some sequences which are transcribed in one particular chromosome are present also in the other chromosomes.     

The 5' end of U3 snRNA can be crosslinked in vivo to the external transcribed spacer of rat ribosomal RNA precursors

From previous work it was known that U3 RNA is hydrogen bonded to nucleolar 28 S to 35 S RNA and can be covalently crosslinked to RNA of greater than 28 S by irradiation in vivo with long-wave ultraviolet light in the presence of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT psoralen). Here we use a novel sandwich blot technique to identify these large nucleolar RNA species as rRNA precursors and to map the site(s) of crosslinking in vivo. The crosslink occurs between one or more residues near the 5' end of U3 RNA and a 380 nucleotide region of the rat rRNA external transcribed spacer (ETS1). We have sequenced this region of the rat ETS and we show that it includes an RNA-processing site analogous to those previously mapped to approximately 3.5 kb upstream from the 5' end of mouse and human 18 S rRNAs.     

Fidelity of ribosomal ribonucleic acid synthesis by nucleoli and nucleolar chromatin.

Isolated nucleoli, nucleolar chromatin, and nucleolar DNA were used as templates for DNA synthesis in appropriately supplemented systems in which RNA polymerases other than RNA polymerase I were blocked by alpha-amanitin. With the aid of nucleotide analysis, DNA-RNA hybridization, and homochromatography fingerprinting, it was found that isolated nucleoli and nucleolar chromatin serve primarily as templates for synthesis of rRNA. However, the products formed with purified nucleolar DNA as a template do not contain the specific rRNA oligonucleotides nor are they appreciably hybridized to the rDNA region on cesium chloride gradients. These results indicate that whole nucleoli and nucleolar chromatin contain control mechanisms that restrict readouts by RNA polymerase I of nucleolar DNA to rDNA.     

The ribosomal RNA gene cluster in aneuploid chickens: evidence for increased gene dosage and regulation of gene expression

In the chicken, the nucleolus organizer regions, or sites of the genes encoding 18S, 5.8S, and 28S ribosomal RNA (rRNA), map to one pair of microchromosomes that can be identified by silver nitrate cytochemistry. This nucleolar organizer chromosome also contains the major histocompatibility complex. Chickens aneuploid for this chromosome have been identified and reproduced for over seven generations. Crossing two trisomic parents results in the production of viable disomic, trisomic, and tetrasomic progeny, showing two, three, and four nucleoli and nucleolar organizers per cell, respectively. A molecular analysis of rRNA genes was undertaken to establish the gene copy numbers in the aneuploid genotypes, and to determine if elevated numbers of rRNA genes are stably maintained and inherited over multiple generations. Gene copy numbers were determined using hybridization analysis of erythrocyte DNA obtained from individuals comprising a family which segregated disomic, trisomic, and tetrasomic genotypes. The values obtained were 290, 420, and 570 rDNA repeats per cell for disomic, trisomic, and tetrasomic animals, respectively. These results provide molecular confirmation of the two aneuploid states and show that elevated gene copy numbers have been maintained over multiple generations. Fibroblasts derived from disomic and tetrasomic embryos were found to grow at similar rates in culture, and mature rRNA levels in chicken embryo fibroblasts from disomic, trisomic and tetrasomic embryos were also found to have similar levels of mature rRNA. Therefore, despite the increase in rDNA content, the level of rRNA is regulated to diploid amounts in aneuploid fibroblasts.     

Isolation of Euglena gracilis chloroplast 5S ribosomal RNA and mapping the 5S rRNA gene on chloroplast DNA.

Ribosomal RNA (5S) from Euglena gracilis chloroplasts was isolated by preparative electrophoresis, labeled in vitro with 125I, and hybridized to restriction nuclease fragments from chloroplast DNA or cloned chloroplast DNA segments. Euglena chloroplast 5S rRNA is encoded in the chloroplast genome. The coding region of 5S rRNA has been positioned within the 5.6 kilobase pair (kbp) repeat which also codes for 16S and 23S rRNA. There are three 5S rRNA genes on the 130-kbp genome. The order of RNAs within a single repeat is 16S-23S-5S. The organization and size of the Euglena chloroplast ribosomal repeat is very similar to the ribosomal RNA operons of Escherichia coli.     

Quantitative analysis of rat liver nucleolar and nucleoplasmic ribosomal ribonucleic acids.

rRNA from detergent-purified nuclei was fractionated quantitatively, by two independent methods, into nucleolar and nucleoplasmic RNA fractions. The two RNA fractions were analysed by urea/agar-gel electrophoresis and the amount of pre-rRNA (precursor of rRNA) and rRNA components was determined. The rRNA constitutes 35% of total nuclear RNA, of which two-thirds are in nucleolar RNA and one-third in nucleoplasmic RNA. The identified pre-rRNA components (45 S, 41 S, 39 S, 36 S, 32 S and 21 S) are confined to the nucleolus and constitute about 70% of its rRNA. The remaining 30% are represented by 28 S and 18 S rRNA, in a molar ratio of 1.4. The bulk of rRNA in nucleoplasmic RNA is represented by 28 S and 18 S rRNA in a molar ratio close to 1.0. Part of the mature rRNA species in nucleoplasmic RNA originate from ribosomes attached to the outer nuclear membrane, which resist detergent treatment. The absolute amount of nuclear pre-rRNA and rRNA components was evaluated. The amount of 32 S and 21 S pre-rRNA (2.9 x 10(4) and 2.5 x 10(4) molecules per nucleus respectively) is 2-3-fold higher than that of 45 S, 41 S and 36 S pre-rRNA.     

Localization of 5S RNA and rRNA genes in the Norway rat

The genes coding for the two classes of ribosomal RNA molecules, 5S RNA and 18+28S RNA, have been localized in the Norway rat (Rattus norvegicus). The 18+28S RNA cistrons are found on three chromosomes, at secondary constrictions on the short arms of chromosomes 3 and 12 and at the telomere of the short arm of chromosome 11. These sites were confirmed using the silver staining technique for nucleolar organizer regions. Two sites were found for the 5S RNA genes; one is closely linked to the 18+28S gene site on chromosome 12. The second site is at or near the telomere of the long arm of chromosome 19.     

Hybridization properties of nucleolar ribonucleic acid.

Rat liver nuclei were fractionated into chromatin and nucleolar fractions. Chromatin DNA, which does not form hybrids with rRNA, was, nevertheless, able to hybridize with 32P-labelled total nucleolar RNA. The optimal temperature for this hybridization was 55 degrees C when the reaction was carried out in 2 X SSC (0.3 MnaCl + 0.3 M-sodium citrate). The hybrids formed were specific, as judged by analysis of thermal elution profiles. The low Tm (73 degreesC) observed could be explained by the low amount of DNA in the filters. The lenth of the hybridized sequences was extimated as 54 mucleotide pairs. Contamination to nucleolar RNA by nucleoplasmic RNA was ruled out by showing the former was able to form more hybrids than the latter. Competition experiments showed that hybridization of nucleolar RNA, although not competed with by rRNA, suffered pronounced competition from total microsomal RNA, even though the levels of competition obtained did not equal thsoe with cold nucleolar RNA as competitor.