Joseph G. Gall

Chromosomal localization of mouse satellite DNA.

Hybridization of radioactive nucleic acids with the DNA of cytological preparations shows that the sequences of mouse satellite DNA are located in the centromeric heterochromatin of the mouse chromosomes. Other types of heterochromatin in the cytological preparations do not contain satellite DNA.

A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena

Abstract The extrachromosomal genes coding for ribosomal RNA (rDNA) in the ciliated protozoan Tetrahymena thermophila were studied with respect to sequences occurring at their termini. The linear rDNA molecules had previously been shown to be palindromic (Karrer and Gall, 1976; Engberg et al. , 1976). Within the terminal rDNA fragment produced by restriction endonuclease digestion, a tandomly repeated hexanucleotide sequence 5′ (C-C-C-C-A-A) n 3′ was found, where n is between 20 and 70. This fragment was heterogeneous in length as judged by gel electrophoresis. The repeating sequence was preferentially synthesized when rDNA was used as the template by Escherichia coli DNA polymerase I. Initiation occurred at specific single-strand discontinuities, probably one-nucleotide gaps, found every few repeats on the C-C-C-C-A-A strand. At least one discontinuity is present on the G-G-G-G-T-T strand. Experiments with T4 DNA polymerase suggested that there are no free cohesive ends on the rDNA of the kind found in bacteriophage λ DNA. The orientation of the strands carrying the repeating hexanucleotide sequence was determined, and a model for the termini of the rDNA based on these findings is presented.

Repetitive DNA sequences in Drosophila

The satellite DNAs of Drosophila melanogaster and D. virilis have been examined by isopycnic centrifugation, thermal denaturation, and in situ molecular hybridization. The satellites melt over a narrow temperature range, reassociate rapidly after denaturation, and separate into strands of differing buoyant density in alkaline CsCl. In D. virilis and D. melanogaster the satellites constitute respectively 41% and 8% of the DNA isolated from diploid tissue. The satellites make up only a minute fraction of the DNA isolated from polytene tissue. Complementary RNA synthesized in vitro from the largest satellite of D. virilis hybridized to the centromeric heterochromatin of mitotic chromosomes, although binding to the Y chromosome was low. The same cRNA hybridized primarily to the α-heterochromatin in the chromocenter of salivary gland nuclei. The level of hybridization in diploid and polytene nuclei was similar, despite the great difference in total DNA content. The centrifugation and hybridization data imply that the α-heterochromatin either does not replicate or replicates only slightly during polytenization. Similar but less extensive data are presented for D. melanogaster. — In D. melanogaster cRNA synthesized from total DNA hybridized to the entire chromocenter (α- and β-heterochromatin) and less intensely to many bands on the chromosome arms. The X chromosome was more heavily labeled than the autosomes. In D. virilis the X chromosome showed a similar preferential binding of cRNA copied from main peak sequences.—It is concluded that the majority of repetitive sequences in D. virilis and D. melanogaster are located in the α- and β-heterochromatin. Repetitive sequences constitute only a small percentage of the euchromatin, but they are widely distributed in the chromosomes. During polytenization the α-heterochromatin probably does not replicate, but some or all of the repetitive sequences in the β-heterochromatin and the euchromatin do replicate.

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.

Satellite DNA sequences in Drosophila virilis

Abstract The DNA of Drosophila virilis contains three highly repetitive density satellites that constitute approximately half the genome. Complementary RNA was synthesized enzymatically from the separated strands of the purified satellite DNAs. By sequencing the transcripts we have shown that the satellites are repeating heptanucleotides related to each other by simple base-pair changes. The sequences are: I, poly d(A-C-A-A-A-C-T): poly d(A-G-T-T-T-G-T); II, poly d(A-T-A-A-A-C-T): poly d(A-G-T-T-T-A-T); III, poly d(A-C-A-A-A-T-T): poly d(A-A-T-T-T-G-T). Thus, despite different physical properties (buoyant density and T m ), the three satellite DNAs of this species constitute a family of related sequences. The major satellite in another species, D. americana americana , was shown to be identical to satellite I of D. virilis . The evolution of satellite DNAs in Drosophila requires a mechanism that maintains homogeneity of repeated sequences over many thousands of generations while permitting the origin of new satellites with single base-pair changes in each repeating unit.

The macronuclear ribosomal DNA of Tetrahymena pyriformis is a palindrome

The macronuclear ribosomal DNA of Tetrahymena pyriformis (strain BVII, syngen 1) is extraohromosomal. The majority of the molecules are linear with measured mean molecular weights of 13.0 × 106 to 13.8 × 106 in various experiments. Several lines of evidence indicated that the double-stranded rDNA molecule is a palindrome in the terminology of Wilson & Thomas (1974). That is, taking into account the polarity of the polynucleotide strands, the sequence is the same read from either end of the molecule. The rDNA molecules had a partial denaturation map that was symmetrical about the center of the molecule. There were two sites susceptible to cleavage by the restriction enzyme EcoRI. Partial denaturation of the fragments indicated that these two sites were equidistant from the center of the rDNA molecule. There were two different sites susceptible to cleavage by the restriction enzyme Bam, which were also equidistant from the center of the rDNA molecule. The two polynucleotide strands of a palindromic DNA molecule are similar or identical and each is self-complementary. Thus each strand will rapidly renature with first-order kinetics following denaturation. Thermal denaturation of the rDNA of Tetrahymena revealed a complex melting curve with at least six transitions. When the temperature was reduced under conditions such that intermolecular reassociation was not expected to occur, the rDNA abruptly lost 35% of its hyperchromicity. When the products of this snapback renaturation were examined in the electron microscope, many of the molecules observed were double stranded, and had a contour length half the length of the native rDNA. Thermal denaturation of the snapback molecules yielded the same transitions as the original melt, but in different proportions, as predicted from the partial denaturation map. There was little or no mismatching in the self-complementary strands that could be detected by comparison of melting temperature (tm) between the melt and the remelt. Cruciform structures were generated by incubating the rDNA in vitro. Although the rDNA of the amicronucleate Tetrahymena strain GL is slightly different in size and buoyant density, and in its partial denaturation map from that of strain BVII, it too has a palindromic structure.

A single integrated gene for ribosomal RNA in a eucaryote, tetrahymena pyriformis

The macronucleus of the protozoan, Tetrahymena, is known to contain multiple rRNA genes which are not linked to the chromosomes. Here we present evidence that the germinal micronucleus of this organism contains a single gene for rRNA integrated into the chromosomal DNA. Unlike the extrachromosomal copies of the macronucleus, which are composed of a pair of reversely repeated sequences (a palindrome), the integrated copy of rDNA is nonrepetitive or half the size of the extrachromosomal rDNA. Furthermore, we have failed to detect such an integrated copy of rDNA in the macronucleus. The implications of these observations for the amplification and evolution of rDNA are discussed.

Transcripts from both strands of a satellite DNA occur on lampbrush chromosome loops of the newt notophthalmus

We have isolated genomic DNA clones from the newt Notophthalmus which contain one or more copies of a 222 bp tandemly repeated sequence (satellite 1). Satellite 1 occurs in the pericentromeric heterochromatin of all chromosomes and at the sphere loci of chromosomes 2 and 6. Lampbrush chromosome loops associated with the spheres contain transcripts of satellite 1, as shown by in situ hybridization. The matrix on these loops consists of multiple thin--thick transcription units, most but not all of which have the same polarity within a given loop. By in situ hybridization with single-stranded probes we show that transcription units of opposite morphological polarity contain transcripts of opposite strands of satellite 1. Satellite 1 occurs as the spacer between clusters of histone genes. We postulate that transcription initiates at a histone gene promoter, fails to terminate at the end of the gene and continues without interruption into the adjacent satellite DNA. In this way, long transcripts are produced which contain both histone and satellite 1 sequences. A general failure of termination signals might explain the unusually long transcripts of lampbrush chromosomes.

Gene Amplification in the Oocytes of Dytiscid Water Beetles

A conspicuous mass of extrachromosomal DNA (Giardinas body) is found in oogonia and oocytes of Dytiscid water beetles. Since in older oocytes this DNA is associated with numerous nucleoli, it seemed probable that the ovary might contain extra copies of the genes for ribosomal RNA (rRNA). This hypothesis has been confirmed by centrifugation and molecular hybridization studies. —In Dytiscus marginalis and Colymbetes fuscus a high density satellite DNA is found in somatic cells and in sperm. Hybridization experiments show that all of the rDNA, i.e., those sequences complementary to rRNA, are located in this satellite, although quantitatively they make up only a small fraction of the satellite. In both species the DNA isolated from ovariole tips is enriched with respect to the satellite. A parallel enrichment of the rDNA has been shown in ovariole tips of Colymbetes, but for technical reasons has not been examined in Dytiscus. —The following model is proposed. In somatic cells and sperm the rDNA is part of an extensive region of high density DNA in one or more chromosomes. In oogonia and oocytes the entire high density region is replicated extrachromosomally and appears cytologically as Giardinas body.

Chromosome fibers studied by a spreading technique

Chromosomes and interphase nuclei can be spread on the surface of water in a simplified Langmuir trough. Interphase nuclei of Triturus erythrocytes display fibers with a diameter of about 250–300 Å. Very similar fibers are seen in metaphase chromosomes of cultured human cells. Fibers from grasshopper spermatocyte chromosomes (prophase) are more variable in diameter, and many fibers thinner than 200 Å extend laterally from the chromosome. In the grasshopper spermatocyte, fibers align in parallel to form plates. It is suggested that the 250–300 Å fibers may represent an inactive state of the chromosome material, and that only the thinner fibers are involved in RNA synthesis. The 250–300 Å fibers may result from the folding or coiling of a thinner fiber having the approximate dimensions of the nucleohistone molecule.