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...

The number of telomeric DNA repeats at chromosome ends is maintained around a mean value by a dynamic balance between elongation and shortening. In particular, proteins binding along the duplex part of telomeric DNA set the number of repeats by progressively limiting telomere growth. The paradigm of this counting mechanism is the Rap1 protein in Saccharomyces cerevisiae. We demonstrate here that a Rap1-independent mechanism regulates the number of yeast telomeric repeats (TG 1±3 ) and of vertebrate repeats (T 2 AG 3 ) when TEL1, a yeast ortholog of the human gene encoding the ATM kinase, is inactivated. In addition, we show that a T 2 AG 3 -only telomere can be formed and maintained in humanized yeast cells carrying a template mutation of the gene encoding the telomerase RNA, which leads to the synthesis of vertebrate instead of yeast repeats. Genetic and biochemical evidences indicate that this telomere is regulated in a Rap1-independent manner, both in TEL1 and in tel1D humanized yeast cells. Altogether, these ®ndings shed light on multiple repeat-counting mechanisms, which may share critical features between lower and higher eukaryotes.

Genetic modification of cells and animals is an invaluable tool

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Accumulation of stable RNA and production of guanosine polyphosphates (ppGpp and pppGpp) were studied during amino acid starvation in four species of halobacteria. In two of the four species, stable RNA was under stringent control, whereas one of the remaining two species was relaxed and the other gave an intermediate phenotype. The stringent reaction was reversed by anisomycin, an effect analogous to the chloroamphenicol-induced reversal of stringency in the eubacteria. During the stringent response, neither ppGpp nor pppGpp accumulation took place during starvation. In both growing and starved cells a very low basal level of the two polyphosphates appeared to be present. In the stringent species the intracellular concentration of GTP did not diminish but actually increased during the course of the stringent response. These data demonstrate that (i) wild-type halobacteria can have either the stringent or the relaxed phenotype (all wild-type eubacteria tested have been shown to be stringent); (ii) stringency in the halobacteria is dependent on the deaminoacylation of tRNA, as in the eubacteria; and (iii) in the halobacteria, ppGpp is not an effector of stringent control over stable-RNA synthesis.

A natural human minichromosome (MC1) derived from human chromosome 1 was shown to be linear and to have a size of 5.5 Mb. Human IL-2 cDNA and the neo gene were co-transfected into a MC1-containing human-CHO hybrid cell line. Integration of the foreign genes was directed to the pericentromeric region of MC1 by co-transfection of chromosome 1-specific satellite 2 DNA. A number of G418-resistant transfectants were obtained and expression of IL-2 was determined. FISH analysis demonstrated co-localization in the minichromosome of the IL-2 gene and of the satellite 2 DNA. An IL-2-producing clone was used in cell fusion experiments with IL-2-dependent murine CTLL cells to generate CTLL-human hybrids containing the modified minichromosome (MC1- IL2 ). The hybrids were able to grow in medium lacking IL-2 for 17 mean population doublings (MPD), indicating that expression of the cytokine was sufficient to relieve the IL-2 dependence of CTLL proliferation. Endogenous IL-2 production delayed the onset of apoptosis in the IL-2-dependent CTLL cells. Mitotic stability was shown to be 100% in the human-CHO hybrids and 97% per MPD in CTLL cells. These results demonstrate that a natural human minichromosome can be utilized as a cloning and expression vector for mammalian cells and that the MC1 minichromosome can be engineered to deliver IL-2 to two types of cells, fibroblasts and lymphocytes.

Artificial chromosomes have been claimed to be the ideal vector for gene therapy, but their use has been hampered by an inability to produce stable and well designed molecules. We have used a structurally defined minichromosome to clone the human cystic fybrosis transmembrane conductance regulator (CFTR) locus. To guarantee the presence of the proper regulatory elements, we used the 320 kb yeast artificial chromosome (YAC) 37AB12 with the intact CFTR gene and upstream sequences. The resulting minichromosome was analyzed for the presence of the entire CFTR gene and for its functional activity by molecular and functional methods. We have identified clones showing the presence of both the transcript and the CFTR protein. Moreover, in the same clones, a chloride secretory response to cAMP was detected. Mitotic and molecular stability after prolonged growth without selection demonstrated that the constructs were stable. This is the first example of a structurally known minichromosome made to contain an active therapeutic gene.

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