All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3' terminus.

Abstract

In hypotrichous ciliates, all of the macronuclear DNA is in the form of low molecular weight molecules with an average size of -2200 base pairs. Total macronuclear DNA from four hypotrichs has been shown to have inverted terminal repeats by direct sequence analysis. In Oxytricha nova, Oxytricha sp., and Stylonychia pustulata, this terminal sequence may be written as 5'-C4A4C4A4C4 ... 3'-G4T4G4T4G4T4G4T4G4 ...In Euplotes aediculatus, the sequence is similar but differs in the lengths of the duplex region (28 base pairs) and of the putative 3' extension (14 base pairs). Also in Euplotes, a second common sequence of 5 base pairs (A:T:G-A-A) occurs internal to the terminal repeat and a 17-base-pair heterogeneous region:The length of the terminal repeat sequence for 0. nova was confirmed in cloned macronuclear DNA molecules.Ciliated protozoa of the order Hypotrichida have two types of nuclei-a small transcriptionally inactive (germinal) micronucleus and a large DNA-rich macronucleus, which contains the entire functional vegetative genome. Whereas the DNA of the micronucleus is large (>>150 X 10' daltons) and is contained in chromosomes, the transcriptionally active macronuclear genome is achromosomal; the DNA exists as a heterogeneous distribution of molecules ranging in size from 400 to ==20,000 base pairs (bp) with an average of 2200 bp (1, 2).The macronucleus is formed de novo from a diploid micronucleus after sexual conjugation. In brief, formation of the macronucleus involves the polytenization of micronuclear chromosomes, followed by fragmentation of polytene chromosomes at interbands and degradation of --95% of the DNA. The degradation phase eliminates all detectable repetitive DNA and most of the unique sequence DNA. What remains are small DNA molecules, which are extensively replicated to form the mature macronuclear genome (for review, see ref.3).This subset of the micronuclear DNA sequences that is retained in the macronucleus contains all of the genetic information (except for mitochondrial) for vegetative growth.The macronucleus contains --24,000 different DNA sequences, present at an average of 1000 copies per macronucleus. Each of these gene-sized molecules is a separate functional genetic unit. Denaturation of macronuclear DNA followed by a short period of reassociation leads to formation of singlestranded circles in more than 80% ofthe molecules as observed by electron microscopy. The single-stranded DNA is held in a circular configuration by a short duplex region. Therefore, all or nearly all of the gene-sized molecules of the macronucleus have an inverted terminal repeat (4). The sequence of this terminal repeat was shown to be the same in many ofthe molecules (5), although the sequence reported for the terminal repeat was not correct. We report here the correct sequence and show that the sequence forming the inverted terminal repeat is almost the same in four different hypotrich species. To establish the structure of the termini of native DNA molecules, we determined both the 3'-and 5'-en...

Regulatory structures of gene expression, DNA-replication and DNA-rearrangement in macronuclear genes of Stylonychia lemnae, a hypotrichous ciliate

“…Each DNA molecule is thought to represent a transcription and replication unit and consists of a single intronless coding region flanked by short (74 bp to 1000 bp) AT-rich (75%-80%) noncoding sequences terminating in telomeric inverted repeats of C4A4. The coding regions show, like those of some other ciliates, an altered genetic code using the "universal" stop codons TAA and TAG to specify glutamine [8,10,18,19,22,25,36,40].…”

G-Wires: Self-Assembly of a Telomeric Oligonucleotide, d(GGGGTTGGGG), into Large Superstructures

“…Inmostcases, telomeric DNA consists of short repetitive sequences containing a guanine-rich strand running 5' to 3' toward the end of the chromosome and a complementary cytosine-rich strand (Greider, 1991;Zakian et al, 1990). At the very end of the chromosome, the guanine strand forms a 3' overhang of approximately two sequence repeats (Henderson & Blackburn, 1989;Klobutcher et al, 1981;Pluta et al, 1982). In many instances, telomeric repeat sequences from diverse species are very similar in composition.…”

A Protein from Tetrahymena thermophila That Specifically Binds Parallel-Stranded G4-DNA

“…The G-strand extends approximately 12-16 nucleotides beyond the end of G/C duplex, forming a 3' overhang in organisms where this has been studied (Klobutcher et al, 1981;Pluta et al, 1982;Henderson & Blackburn, 1989). Synthetic oligonucleotides containing telomeric G-strand sequences are able to form unusual structures mediated by cyclic hydrogen-bonded arrays of guanines (G-quartets) [reviewed by Sundquist (1991), Williamson et al (1989), Sundquist and Klug (1989), Kang et al (1992), and Smith and Feigon (1992)l.…”