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Macronuclear DNA from the protozoan G. chattoni, a holotrichous ciliate, was analyzed. Most, if not all, of the macronuclear DNA is subchromosomal, ranging in size from above 100 kb down to 2.1 kb, with molecules in the lower molecular weight range being resolvable by gel electrophoresis into reproducible, specific, discrete size classes. A prominent class of linear 9.3 kb molecules consists of single free rRNA genes. Upon denaturation and partial renaturation, a high percentage of total macronuclear DNA was found as single-stranded circles. Sequence analysis showed that a minimum of 38 tandem repeats of the sequence CCCCAA is present in inverted orientation at each end of most or all Glaucoma macronuclear DNA molecules, including the rDNA. This sequence must therefore be recognized during site-specific fragmentation of chromosomes in macronuclear development.

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.

A tandemly repeated DNA hexanucleotide sequence, 5'C-C-C-C-A-A3', that occurs at the termini of extrachromosomal DNA molecules coding for rRNA (rDNA) in Tetrahymena macronuclei was examined to determine whether it is packaged as nucleosomes. This repeated DNA sequence comprises the terminal few hundred base pairs at each end of the linear rDNA molecules. Digestion of macronuclei with micrococcal nuclease showed that this DNA sequence is protected from digestion but is left, following digestion, as a single but broad size class of DNA fragments several hundred base pairs long, under conditions in which bulk macronuclear DNA and rDNA were digested to fragments that were multiples of approximately 200 base pairs in length. The repeated C-C-C-C-A-A was found protected as fragments longer than the bulk macronuclear DNA digestion products at all times during digestion. Together with putative associated protein(s), this protected DNA was soluble after lysis of micrococcal nuclease-digested macronuclei at low salt concentrations but was insoluble in 0.075--0.2 M KCl, regardless of the extend of digestion. The size and solubility properties of the repeated C-C-C-C-A-A DNA nucleoprotein complex after micrococcal nuclease digestion of macronuclei are clearly distinguishable from those of nucleosomes, and it is inferred that this DNA sequence in macronuclei is packaged in chromatin by proteins other than histones.

We analyzed the extent, reproducibility, and developmental control of genomic rearrangements in the somatic macronucleus of the ciliate Tetrahymena thermophila. To exclude differences caused by genetic polymorphisms, we constructed whole-genome homozygotes, and we compared the homozygous progeny derived from single macronuclear differentiation events. This strategy enabled us to identify a novel form of variable rearrangement and to confirm previous findings that rearranged sequences occur at a high frequency in the Tetrahymena genome. Rearrangements studied here were deletions of both unique and interchromosomally dispersed repetitive DNA sequences involving DNA rejoining of internal, nontelomeric regions of macronuclear DNAs. We showed that although rearrangements of some sequence classes are reproducible among independently developed macronuclei, other specific sequence classes are variably rearranged in macronuclear development. The variable somatic genomes so produced may be the source of phenotypically variant cell lines.

The reactivity of single-stranded thymidines with osmium tetraoxide was used to demonstrate the existence of a terminal overhang of the G-rich strand of telomeres from two distantly related eucaryotes, the ciliated protozoan Tetrahymena spp. and the acellular slime mold Didymium spp. Conservation of a G-strand overhang at the molecular terminus of telomeres is consistent with our suggestion that an unusual DNA structure formed by the G-strand overhang is important for telomere function (E. Henderson, C. C. Hardin, S. K. Wolk, I. Tinoco Jr., and E. H. Blackburn, Cell 51:899-908, 1987).

The metabolism of glutamine by isolated, respiring rat liver mitochondria produced ammonia and asparate as the main identified products. The dependence of ammonia and aspartate production on glutamine concentration was sigmoidal. Glutaminase activity was located in the inner membrane plus matrix subfraction of liver mitochondria. Rotenone inhibited glutaminase activity in whole mitochondria; addition of succinate did not remove the inhibition. Glutamine and glutamate at low concentrations were metabolized, to some extent independently, to form ammonia and aspartate. It was concluded that the metabolism of both glutamine and glutamate is integrated with the tricarboxylic acid cycle to produce the two precursors of urea, ammonia and aspartate.

Elizabeth H. Blackburn Department of Molecular Biology University of California Berkeley, California 94720 For decades it has been known that the ends of linear eucaryotic chromosomes are specialized structures (re- viewed in Blackburn and Szostak, Ann. Rev. Biochem. 53, 163-194, 1984). This fact was most evident when chro- mosomes were broken by mechanical rupture or X-irradia- tion Unlike true telomeres, the ends of broken chromo- somes are usually unstable; they fuse chromosomes end- to-end and manifest other abnormal reactions, One likely function of a telomere is to allow completion of the replication of the end of the linear chromosomal DNA molecule. All known template-dependent DNA polym- erases require a primer (RNA or DNA) to initiate 5’*3’ strand synthesis, which after chromatid replication would leave some unreplicated bases at each of its ends. Studies on linear viral DNAs show that viruses exploit various types of structures at their DNA termini to solve this problem, but the question of whether any of these viral models apply to cellular, chromosomal DNAs in eucaryotic nuclei re- mains Molecular Structure of Telomeres in Lower Eucaryotes Direct analysis of chromosomal ends has not been prac- ticable until recently, because telomeric sequences repre- sent a minute proportion of the enormously long chromo- somal DNAs. However, the nuclei of certain lower eucary otes contain relatively short linear DNAs (