Further Studies on the Ribosomal RNA Cistrons of SCIARA COPROPHILA (Diptera)

Additional experiments with homologous as well as heterologous hybridization confirmed our previous finding in Sciara coprophila that XX females have nearly twice the number of ribosomal RNA cistrons as XO males. A comparison between two different X' chromosomes revealed that only the one carrying the irradiation-induced Wavy mutation has a deletion of 70% of its ribosomal RNA cistrons as compared to the standard X. The deletion is relatively stable, and the remaining ribosomal RNA cistrons donot appear to undergo disproportionate replication or magnification as in Drosophila. Homologous hybridization experiments revealed an unusually low reiteration of ribosomal RNA cistrons in this fly, 45 gene copies per X chromosome. The question is raised as to whether such a low number of cistrons may be related to the unusual nucleolar condition encountered in the Sciaridae.     

Localization of ribosomal DNA within the proximal X heterochromatin of Sciara coprophila (Diptera,Sciaridae)

By means of several reciprocal translocations in Sciara coprophila, each having a break-point in the proximal X heterochromatin, it has been possible in the salivary gland nucleus to bring about separation of specific regions of this heterochromatin and then, by means of in situ hybridization, to determine the relative number of ribosomal RNA cistrons in each. The three blocks of heterochromatin delineated by the translocation break-points have been designated H1, H2, and H3; H1 is the most proximal, lying immediately to the right of the X centromere, and H3 is the most distal, constituting the very end (right) of the chromosome. The distribution of ribosomal RNA cistrons is as follows: 10% are located in H1; 50% in H2; and 40% in H3. For the first time it has been possible to confirm by grain count our previous biochemical estimate of a 60% deletion of rRNA cistrons in the proximal heterochromatin of the X _W homologue. The grain count data also support the conclusion of our previously published cytological analysis, that the exchange points in the X heterochromatin are identical in translocations T29 and T32 (between H1 and H2), also in translocations T23 and T70 (between H2 and H3). The coincidence of break-points in the X heterochromatin is considered in relation to the chromomere make-up of the region. Also, the occurrence of ribosomal RNA cistrons in all three heterochromomeres is discussed in relation to the functional significance of chromomeres.     

An investigation of some problems concerning nucleolus organizers in salamanders

Observed differences in the sizes of lampbrush nucleolus organizers in Plethodon cinereus have been shown by in situ hybridization to reflect true molecular differences in the numbers of ribosomal cistrons located at these organizers. Likewise, from in situ hybridization experiments on lampbrush and spermatocyte chromosomes it has been shown that animals may be, and indeed usually are, heterozygous with respect to the numbers of ribosomal cistrons on each half of the nucleolus bivalent. Filter hybridizations carried out on 33 males from a New Jersey population and 20 males from a Connecticut population have shown a 7.5-fold range in the numbers of ribosomal cistrons per diploid cell in the New Jersey population, and a 2.5-fold range in the Connecticut population. In view of the general heterozygosity of nucleolus organizers in these animals, the actual range in nucleolus organizer sizes in the New Jersey population is estimated to be at least 15-fold.     

Ribosomal RNA Cistron Multiplicity and Nucleolar Organizers in Hexaploid Wheat

The ribosomal DNA (DNA complementary to ribosomal RNA) content of twelve different wheat genotypes has been studied. Some of these genotypes are aneuploids with deletions or additions of chromosomes bearing nucleolar organisers. The rDNA contents of these genotypes provide several examples of a clear departure from a correlation between the number of rRNA cistrons and the number of nucleolar organisers. Thus the number of rRNA cistrons per nucleolar organiser is not constant in wheat. Wheat DNA was found to have a mean buoyant density of approximately 1.702 g/cc for all genotypes studied and rRNA hybridized selectively to DNA of buoyant density approximately 1.710 g/cc. The thermal stabilities of all the rRNA/DNA hybrids were essentially identical.     

Deoxyribonucleic acid–ribonucleic acid hybridization. Annealing and quantitative recovery of intact ribosomal ribonucleic acid molecules from hybrids

A simple and efficient method for hybridization and subsequent recovery of non-fragmented ribosomal RNA from the hybrid is described. The procedure involves annealing of immobilized denatured DNA bound on cellulose nitrate membrane filters to complementary RNA in 50% (v/v) formamide-0.33m-potassium chloride-10mm-tris-hydrochloric acid buffer, pH7.4, at 33 degrees for 3hr. Under these conditions no detectable changes in the sedimentation coefficients of the input RNA were detected. The RNA can subsequently be recovered quantitatively from the hybrid in intact form by incubating the filters in formamide or in 85% (v/v) dimethyl sulphoxide. The applicability of the method for the evaluation of the absolute size of ribosomal RNA cistrons in Escherichia coli DNA and for the determination of the size of messenger RNA molecules is discussed.     

Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. III. Ribosomal RNA cistrons of the nucleus and leucoplast

The colorless alga Polytoma obtusum has been found to possess leucoplasts, and two kinds of ribosomes with sedimentation values of 73S and 79S. The ribosomal RNA (rRNA) of the 73S but not the 79S ribosomes was shown to hybridize with the leucoplast DNA (rho - 1.682 g/ml). Nuclear DNA of Polytoma (rho = 1.711) showed specific hybridization with rRNA from the 79S ribosomes. Saturation hybridization indicated that only one copy of the rRNA cistrons was present per leucoplast genome, with an average buoyant density of rho = 1.700. On the other hand, about 750 copies of the cytoplasmic rRNA cistrons were present per nuclear genome with a density of rho = 1.709. Heterologous hybridization studies with Chlamydomonas reinhardtii rRNAs showed an estimated 80% homology between the two cytoplasmic rRNAs, but only a 50% homology between chloroplast and leucoplast rRNAs of the two species. We conclude that the leucoplasts of Polytoma derive from chloroplasts of a Chlamydomonas-like ancestor, but that the leucoplast rRNA cistrons have diverged in evolution more extensively than the cistrons for cytoplasmic rRNA.     

Cellular content of chloroplast DNA and chloroplast ribosomal RNA genes in Euglena gracilis during chloroplast development.

The cellular content of chloroplast DNA in Euglena gracilis has been quantitatively determined. DNA was extracted from Euglena cells at various stages of chloroplast development and renatured in the presence of trace amounts of 3H-labeled chloroplast DNA. From the kinetics of renaturation of the 3H-labeled chloroplast DNA, compared with the kinetics of renaturation of excess nonradioactive chloroplast DNA, the fraction of cellular DNA represented by chloroplast DNA was calculated. The content of chloroplast DNA was found to increase from 4.9 to 14.6% of cellular DNA during light-induced chloroplast development. Correcting for the change in DNA mass per cell, the number of copies of chloroplast DNA is found to vary from 1400 to 2900 per cell. During this developmental transition, the cellular content of the chloroplast ribosomal RNA genes varies from 1900 to 5200 copies per cell. The ratio of the number of copies of rRNA genes to chloroplast genomes per cell remains in the range of 1-2 throughout chloroplast development, ruling out selective amplification of chloroplast rRNA genes as a means of regulation of rRNA gene expression. Direct measurement of the number of rRNA cistrons per 9.2 X 10(7) dalton genome yields a value of 1 or 2.     

松毛虫DpKettin基因片段的克隆与初步定位研究

对于ZW型鳞翅目昆虫, 雄性为ZZ, 雌性为ZW; 细胞内Z染色体数与常染色体组数之比在雌雄间存在差异, 雄性为2Z∶2A=1.0, 雌性为Z∶2A=0.5, 如果某物种一个基因(假如K基因)位于Z染色体上, 另一个基因(假如N基因)位于常染色体上, 则雄体中2K∶2N=1.0, 雌体中K∶2N=0.5。研究利用家蚕、棉铃虫等昆虫Kettin基因序列, 克隆了松毛虫的同源基因(DpKettin)片段, 并采用荧光定量PCR技术, 以松毛虫的ANT基因为参照基因, 检测松毛虫雌雄不同个体间DpKettin基因与腺苷酸转移酶基因(ANT)的拷贝数之比, 结果表明: 雄体DpKettin∶ANT=1.0, 雌体DpKettin∶ANT=0.5, 说明DpKettin基因位于松毛虫Z染色体上。     

The Nucleolus Organizer Region of Maize (ZEA MAYS L.): Tests for Ribosomal Gene Compensation or Magnification

Ribosomal gene compensation and magnification that might be detected on a whole-plant basis was not found in maize. Plants monosomic for chromosome 6 (the NOR chromosome) were compared with monosomic-8 and monosomic-10 plants, disomic sibs, and parental lines. Assuming no rDNA compensation, monosomic-6 plants showed approximately the decrease expected in rRNA cistron number. Monosomic-8 had a normal ribosomal gene number, while monosomic-10 showed a decrease; but further documentation is needed. Besides demonstrating the absence of gene compensation, the results document our previous conclusion that maize chromosome 6 carries DNA complementary to ribosomal RNA. Further documentation was provided from studies with trisomic chromosome 6 plants showing proportional increases in ribosomal gene number. Progeny of the monosomic plants crossed as males to a standard singlecross hybrid possessed expected ribosomal gene numbers suggesting the lack of ribosomal gene magnification.—The ragged (rgd) mutant of maize, suspected of being deficient in rRNA cistrons, had a normal number.     

Distribution of Ribosomal Ribonucleic Acid Cistrons Among Yeast Chromosomes

High-molecular-weight deoxyribonucleic acid (DNA) of Saccharomyces carls bergensis has been fractionated by sucrose density gradient centrifugation. The main DNA fraction has an average molecular weight of about 500 x 10(6). A major fraction of the DNA molecules containing sequences homologous to ribosomal ribonucleic acid (RNA) sediments as material of this molecular weight. The remainder sediments as material of a molecular weight of about 250 x 10(6). The latter fraction contains relatively more ribosomal RNA cistrons than the former. Studies on the buoyant density of high-molecular-weight DNA homologous to ribosomal RNA have led to the conclusion that the ribosomal RNA cistrons occur in groups attached to a relatively large amount of nonribosomal RNA and suggest that ribosomal RNA cistrons are distributed over a number of yeast chromosomes.     

Genetically Induced Mitotic Exchange in the Heterochromatin of DROSOPHILA MELANOGASTER

Multiple copies of the 18S and 28S ribosomal RNA cistrons are present in both the X and Y chromosomes of Drosophila melanogaster. Data are presented here that identify a locus, Rex, that causes exchange-like events between duplicated ribosomal complexes at the ends of an attached-XY chromosome. Rex: (1) is close to or in the basal heterochromatin of the X chromosome; (2) is semidominant and (its effect) is temperature sensitive; (3) acts maternally; and (4) affects behavior of paternally derived attached-XY chromosomes shortly after fertilization. Though, at this point, the existence of Rex is known only from its effects on behavior of a particular compound chromosome, it presents intriguing possibilities for understanding regulation of chromosome behavior and organization of the ribosomal cistrons.     

Cytophotometric study of nuclear proteins during gametogenesis in Ascaris lumbricoides.

Reduction in the number of nucleoli/nucleus and increase in their size were usually observed in rat liver after partial hepatectomy. These changes of nucleoli were greatest 16–18 h after the operation, when RNA biosynthesis in the nucleoli is reported to be highest. Approx. 50% of the nuclei had one enlarged nucleolus at this time but after the increase in nuclear DNA synthesis less than 15% of the nuclei had one nucleolus, as in normal liver. Before the next peak of nuclear DNA synthesis, nucleolar changes appeared again, though less conspicuously.The enlarged nucleoli of regenerating liver were separated from smaller ones by discontinuous sucrose gradient centrifugation and the contents of nucleic acid and ribosomal cistrons in different-sized nucleoli were measured. The large nucleoli in regenerating liver were found to have increased DNA content, whereas smaller ones had the normal content. The total number of ribosomal cistrons in the enlarged nucleoli from regenerating liver was also increased roughly in proportion to the DNA content. No significant difference was found between the percentages of ribosomal cistrons in whole nuclear DNAs from regenerating and normal liver. Small but reproducible [³H]TdR incorporation into nucleolar DNA was observed and this was similar in normal liver and regenerating liver 12 h after partial hepatectomy. Therefore, the nucleolar changes in regenerating liver were not accompanied by any particular DNA synthesis in the nucleolus itself. These results suggest that in the nuclei of regenerating liver nucleolar chromatins may be redistributed and assembled into large nucleoli, rather than that any amplification of ribosomal cistrons occurs.     

Isolation and Characterization of Bacterial Ribosomal RNA Cistrons

The DNA sequences which code for ribosomal DNA have been isolated and purified. The technique used has general application for RNA:DNA hybridization studies and enables the isolation of any gene for which sufficient gene product can be obtained. Experiments with isolated ribosomal RNA cistrons demonstrated that (a) the majority of the ribosomal cistrons are similar to one another; (b) the cistrons which are similar to one another are virtually identical to one another; (c) ribosomal cistrons of different bacterial species are closely related to one another.     

Magnification of the Ribosomal Genes in Female DROSOPHILA MELANOGASTER

The genetically induced increase in the number of 18S + 28S ribosomal genes known as magnification has been reported to occur in male Drosophila but has not previously been observed in females. We now report that bobbed magnified (bbm) is recovered in progeny of female Drosophila carrying three different X bobbed (Xbb) chromosomes and the helper XYbb chromosome, which is a derivative of the Ybb- chromosome. Using different combinations of bb or bb+ X and Y chromosomes, we show that magnification in females requires both a deficiency in ribosomal genes and the presence of a Y chromosome: X/X females that are rDNA-deficient but do not carry a Y chromosome do not produce bbm; similarly, X/X/Y females that carry a Y chromosome but are not rDNA-deficient do not produce bbm. Bobbed magnified is only recovered from rDNA-deficient X/XY, X/X/Y or XX/Y females. We have also found that females carrying a ring Xbb chromosome together with the XYbb- chromosome do not produce bbm, indicating that ring X chromosomes are inhibited to magnify in females as in males. We postulate that the requirement for a Y chromosome is due to sequences on the Y chromosome that regulate or encode factor(s) required for magnification, or alternatively, affect pairing of the ribosomal genes.--These studies demonstrate that magnification is not limited to males but also occurs in females. Magnification in females is induced by rDNA-deficient conditions and the presence of a Y chromosome, and probably occurs by a mechanism similar to that in males.     

A family of repetitive nucleotide sequences (Sau 3A family) in the genome of 2 forms of the sockeye salmon Oncorhynchus nerka

A family of DNA sequences (Sau3A repeat sequences) has been revealed in genomes of two forms of O. nerka. A DNA fragment belonging to this "family" has been cloned in bacterial plasmid. Its copy number (in genomes of normal (570 +/- 33) and dwarf (66 +/- 12) forms) has been counted. The structural organization of sequences of Sau3A family in genomes of males and females of normal form is not the same. The organization of Sau3A sequences is the same in genomes of dwarf males and dwarf females of normal form, but the quantity of Sau3A sequences copies is smaller. It is supposed that the DNA sequences of Sau3A family bear the sex determination of dwarf form of O. nerka. The fragment of rRNA gene of nerka has been cloned and the number of rRNA gene copies in genomes of normal (200 +/- 62) and dwarf (1690 +/- 24) forms has been defined.     

Relative ribosomal RNA cistron multiplicity in oocytes and postembryonic stages of the eutelic nematodePanagrellus silusiae

Summary The number of ribosomal RNA cistrons has been measured in the total DNA extracted from L2 juvenile and adult stages of the free-living nematodePanagrellus silusiae. Saturation hybridization studies with homologous rRNA indicate that both stages have about 275 ribosomal genes per haploid equivalent. Using homologous¹²⁵I-labelled rRNA for in situ hybridization, the mean number of silver grains per DNA content for oocyte, hypodermis and gut nuclei was similar. The mean DNA contents of maturing oocyte, hypodermis and gut nuclei are about 20C, 2C, and 10C respectively. We conclude that rDNA amplification alone is insufficient to account for the variation in DNA content of oocytes and that postembryonic development in this eutelic organism occurs without a significant differential increase in the number of ribosomal cistrons per worm.Supported by the National Research Council of Canada     

Evidence for male XO sex-chromosome system in Pentodon bidens punctatum (Coleoptera Scarabaeoidea: Scarabaeidae) with X-linked 18S-28S rDNA clusters

In scarab beetle species of the genus Pentodon, the lack of analysis of sex chromosomes in females along with the poor characterization of sex chromosomes in the males, prevented all previous investigations from conclusively stating sex determination system. In this study, somatic chromosomes from females and spermatogonial chromosomes from males of Pentodon bidens punctatum (Coleoptera: Scarabaeoidea: Scarabaeidae) from Sicily have been analyzed using non-differential Giemsa staining. Two modal numbers of chromosomes were obtained: 2n = 20 and 19 in females and males, respectively. This finding along with other karyological characteristics such as the occurrence of one unpaired, heterotypic chromosome at metaphase-I and two types of metaphase-II spreads in spermatocytes demonstrate that a XO male/XX female sex determining mechanism - quite unusual among Scarabaeoidea - operates in the species investigated here. Spermatocyte chromosomes have also been examined after a number of banding techniques and fluorescent in situ hybridization with ribosomal sequences as a probe (rDNA FISH). The results obtained showed that silver and CMA(3) staining were inadequate to localize the chromosome sites of nucleolus organizer regions (NORs) due to the over-all stainability of both constitutive heterochromatin and heterochromatin associated to the NORs. This suggests that heterochromatic DNA of P. b. punctatum is peculiar as compared with other types of heterochromatin studied so far in other invertebrate taxa. By rDNA FISH major ribosomal genes were mapped on the X chromosome.     

Regulation of ribosomal RNA cistron number in a strain of Neurospora crassa with a duplication of the nucleolus organizer region

Some progeny from a cross of the translocation mutant T(VL→IVL)AR33 with wild-type Neurospora crassa are double nucleolus organizer (DNO) strains, usually displaying two distinct nucleolus organizer regions. The DNO strain is sterile but displays the same growth response as normal laboratory strains of Neurospora. We used DNA-DNA hybridization techniques to quantify the number of rRNA cistrons in the DNO mutant and its vegetative progeny. Comparisons of the rate of hybridization of genomic DNA from the parental AR33 strain and from the DNO strain showed that hybridization was more rapid for the DNO strain than for the parental strain. Successive vegetative progeny of the DNO strain displayed hybridization rates intermediate to those of the original DNO strain and the parental single nucleolus strain, indicating that the number of rRNA cistrons had decreased during vegetative propagation. Estimates of rRNA cistron number obtained from comparisons of the amount of single copy DNA and rDNA hybridized to genomic DNO and AR33 DNA at saturation indicate that the parental AR33 strain contains 225 copies of the rRNA repeat unit, while the DNO strain has approx. 440 copies. The number of rRNA cistrons decreases gradually in the successive vegetative progeny, approximating the parental haploid value by the eleventh vegetative transfer.