Susan A. Gerbi

Cytological localization of DNA complementary to ribosomal RNA in polytene chromosomes of Diptera

Molecular hybridization of radioactive ribosomal RNA with the DNA of cytological preparations is used to study the distribution of the ribosomal cistrons within the polytene chromosomes of three species of Diptera. It is shown that in Drosophila hydei the ribosomal cistrons are located within the nucleolus. In Rhynchosciara hollaenderi and Sciara coprophila, DNA coding for ribosomal RNA is present both in the nucleolus organizer regions of the chromosomes and in micronucleoli scattered throughout the nucleus. The DNA puffs in the salivary chromosomes of these sciarid flies do not contain detectable quantities of ribosomal cistrons. — RNA prepared by in vitro transcription has also been used to localize the ribosomal cistrons in Rhynchosciara. — Modifications in the technique of cytological hybridization are discussed.

The nucleolus: a site of ribonucleoprotein maturation

The nucleolus is the site of ribosomal RNA synthesis, processing and ribosome maturation. Various small ribonucleoproteins also undergo maturation in the nucleolus, involving RNA modification and RNA-protein assembly. Such steps and other activities of small ribonucleoproteins also take place in Cajal (coiled) bodies. Events of ribosome biogenesis are found solely in the nucleolus, which is the final destination of small nucleolar RNAs after their traffic through Cajal bodies. However, nucleoli are just a stopping point in the intricate cellular traffic for small nuclear RNAs and other ribonucleoproteins.

Chromosomal ARS1 Has a Single Leading Strand Start Site

Initiation sites for DNA synthesis in the chromosomal autonomously replicating sequence (ARS)1 of Saccharomyces cerevisiae were detected at the nucleotide level. The transition from discontinuous to continuous synthesis defines the origin of bidirectional replication (OBR), which mapped adjacent to the origin recognition complex binding site. To ascertain which sites represented starts for leading or lagging strands, we characterized DNA replication from ARS1 in a cdc9 (DNA ligase I) mutant, defective for joining Okazaki fragments. Leading strand synthesis in ARS1 initiated at only a single site, the OBR. Thus, replication in S. cerevisiae is not initiated stochastically by choosing one out of multiple possible sites but, rather, is a highly regulated process with one precise start point.

Conserved Boxes C and D are essential nucleolar localization elements of U14 and U8 snoRNAs

Sequences necessary for nucleolar targeting were identified in Box C/D small nucleolar RNAs (snoRNAs) by fluorescence microscopy. Nucleolar preparations were examined after injecting fluorescein‐labelled wild‐type and mutated U14 or U8 snoRNA into Xenopus oocyte nuclei. Regions in U14 snoRNA that are complementary to 18S rRNA and necessary for rRNA processing and methylation are not required for nucleolar localization. Truncated U14 molecules containing Boxes C and D with or without the terminal stem localized efficiently. Nucleolar localization was abolished upon mutating just one or two nucleotides within Boxes C and D. Moreover, the spatial position of Boxes C or D in the molecule is essential. Mutations in Box C/D of U8 snoRNA also impaired nucleolar localization, suggesting the general importance of Boxes C and D as nucleolar localization sequences for Box C/D snoRNAs. U14 snoRNA is shown to be required for 18S rRNA production in vertebrates.

Unusual Chromosome Movements in Sciarid Flies

Sciarid flies are Diptera (two-winged flies) of the suborder Nematocera; the family Sciaridae are commonly known as fungus gnats, since they eat mushrooms and other fungi. Morphological characteristics of Sciara species useful for taxonomic purposes were first tabulated by Johannsen (1909, 1912). More recently, the family of Sciara has been renamed Bradysia (Steffan 1966), but since almost none of the researchers who work with this family have switched over to the new name, I will use the original name of Sciara in this review.

Origin recognition complex binding to a metazoan replication origin

The initiation of DNA replication in eukaryotic cells at the onset of S phase requires the origin recognition complex (ORC) [1]. This six-subunit complex, first isolated in Saccharomyces cerevisiae [2], is evolutionarily conserved [1]. ORC participates in the formation of the prereplicative complex [3], which is necessary to establish replication competence. The ORC-DNA interaction is well established for autonomously replicating sequence (ARS) elements in yeast in which the ARS consensus sequence [4] (ACS) constitutes part of the ORC binding site [2, 5]. Little is known about the ORC-DNA interaction in metazoa. For the Drosophila chorion locus, it has been suggested that ORC binding is dispersed [6]. We have analyzed the amplification origin (ori) II/9A of the fly, Sciara coprophila. We identified a distinct 80-base pair (bp) ORC binding site and mapped the replication start site located adjacent to it. The binding of ORC to this 80-bp core region is ATP dependent and is necessary to establish further interaction with an additional 65-bp of DNA. This is the first time that both the ORC binding site and the replication start site have been identified in a metazoan amplification origin. Thus, our findings extend the paradigm from yeast ARS1 to multicellular eukaryotes, implicating ORC as a determinant of the position of replication initiation.

Ribosomal DNA of the fly Sciara coprophila has a very small and homogeneous repeat unit

SummaryIn this report we show by hybridization of restriction fragments and by Miller spreads that the unit repeat of the fly Sciara coprophila is only 8.4 kb which is the smallest known for a multicellular eukaryote. The 8.4 kb EcoR1 fragment containing a complete unit of Sciara rDNA was cloned in pBR322, and mapped by the method of Parker (1977) and also by double digestion. The coding regions for 28S, 18S, and 5.8S RNA were localized by the method of Berk and Sharp (1977). From these data we conclude that the nontranscribed spacer, external transcribed spacer, and internal transcribed spacer are all shorter than in other organisms, thereby giving rise to the shorter overall rDNA repeat unit of Sciara.At least 90% of the Sciara rDNA repeats are homogeneous, with a length of 8.4 kb, but a 700 bp ladder of minor bands can also be found in digestions of total genome DNA. This profile of major and minor bands is identical between the X and X′ chromosomes, as seen by a comparison of several genotypes.There are only 45 rRNA genes per X chromosome of Sciara (Gerbi and Crouse, 1976). These can easily be counted by low magnification Miller speads which show that virtually all gene copies are actively being transcribed in the stage of spermatogenesis examined. This is the first demonstration for any reiterated gene family where all copies are shown to be simultaneously active.

Localization and characterization of the ribosomal RNA cistrons in Sciara coprophila

Abstract By molecular hybridization it was determined that the cistrons coding for ribosomal RNA in the fungus gnat, Sciara coprophila , are present in multiple copies located for the most part at the proximal heterochromatin of the X chromosome. The X′ chromosome which contains the mutation Wavy and a paracentric inversion, is deficient in ribosomal RNA cistrons. The Sciarid polytene nucleus often contains small refractile bodies known as “micronucleoli” which are associated with the chromosomes. These micronucleoli probably represent parts of a highly ramified nucleolus formed at the base of the X chromosome. The micronucleoli do not seem to have their origin in the heterochromatic bands of the chromosomes to which they are adjacent. Whether or not the micronucleoli represent amplification of the ribosomal RNA cistrons is discussed. The DNA puffs found in the Sciara salivary gland chromosomes do not contain detectable quantities of ribosomal RNA cistrons when examined by cytological hybridization.

DNA replication and chromatin

The study of DNA replication in eukaryotic chromosomes has revealed a multitude of different regulatory levels. Nuclear and chromosomal location as well as chromatin structure may affect the activity of replication origins and their modulation during development.

The basic repeat unit of a Chironomus Balbiani ring gene

A clone derived from the Balbiani ring b (BRb) gene of Chironomus thummi has been used to study the internal organization of that gene. Much of the gene consists of approximately 80 copies of a ca. 300 bp repeat unit, which are tandemly organized. The BRb clone contains a major part of that unit (242 bp). Sequence analysis shows that approximately 60% of the unit corresponds to short, tandemly organized subsequences, which encode peptides 8 to 11 residues long. In turn, each subsequence consists of even shorter internal repeats, corresponding to a tripeptide (consensus Proline. Serine. Lysine.). The remainder of the ca. 300 bp unit probably does not have obvious repetitive substructure.