A system for addressing in the construction of macromolecular assemblies can be based on the biospecificity of DNA (cytosine-5) methyltransferases and the capacity of these enzymes to form abortive covalent complexes at targeted 5-f luorocytosine residues in DNA. Using this system, macromolecular assemblies have been created using two representative methyltransferases: M⅐HhaI and M⅐MspI. When 5-f luorocytosine (F) is placed at the targeted cytosine in each recognition sequence in a synthetic oligodeoxynucleotide (GFGC for M⅐HhaI or FCGG for M⅐MspI), we show that the first recognition sequence becomes an address for M⅐HhaI, while the second sequence becomes an address for M⅐MspI. A chimeric enzyme containing a dodecapeptide antigen linked to the C terminus of M⅐HhaI retained its recognition specificity. That specificity served to address the linked peptide to the GFGC recognition site in DNA. With this assembly system components can be placed in a preselected order on the DNA helix. Axial spacing for adjacent addresses can be guided by the observed kinetic footprint of each methyltransferase. Axial rotation of the addressable protein can be guided by the screw axis of the DNA helix. The system has significant potential in the general construction of macromolecular assemblies. We anticipate that these assemblies will be useful in the construction of regular protein arrays for structural analysis, in the construction of protein-DNA systems as models of chromatin and the synaptonemal complex, and in the construction of macromolecular devices.Macromolecular assembly is easily approached with DNA. Branching through the formation of Watson-Crick paired duplexes in the shape of a Y or an X is now well known (1-4), and the feasibility of assembling 2-dimensional quadrilaterals and 3-dimensional cubes on which more extended structures can be based has been demonstrated (5, 6). However, the stable, site-directed attachment of labile enzymes and proteins to a DNA scaffold presents a formidable challenge in macromolecular fabrication. Candidate procedures in which the Watson-Crick base-pairing homology or triple-helix basepairing homology of an oligodeoxynucleotide is used to direct a tethered moiety to a preselected site in DNA (3, 4, 7-9) involve extremes of pH or temperature that can destroy the native structure of these proteins. Attachment systems based on antibodies directed against DNA are likely to lack specificity. On the other hand, antibodies to a hapten could be used to decorate a matrix depending on the pattern of haptens laid down during synthesis. The disadvantage here is that all hapten moieties are equivalent, and thus selective addressing would not be possible unless a series of haptens and antibodies directed to them could be developed. While a system of distinct haptens and antibodies is possible (3), it would be necessary to develop a set of hapten-phosphoramidites and the corresponding series of bifunctional antibodies to utilize this approach. Moreover, the use of noncovalent linkages sacrifice...

G4-DNA is a four-stranded structure that is formed by guanine-rich sequences. We report here the purification and characterization of a novel G4-DNA binding protein from Tetrahymena thermophila, designated TGP2. TGP2 was found to preferentially bind to G4-DNA oligonucleotides with adjacent single-stranded domains containing phosphorylated 5' ends and the sequence element, 5'-ACTG-3'. The amino acid sequence of TGP2 has high similarity to dihydrolipoamide dehydrogenase (DLDH) from a variety of species, and TGP2 was shown to have DLDH activity. Purified DLDH from porcine heart and bovine intestinal mucosa were shown to bind specifically to G4-DNA oligonucleotides. On the basis of these results we conclude that TGP2 is DLDH in T. thermophila and suggest that the G4-DNA binding capability of TGP2/DLDH may be biologically relevant.

The atomic force microscope (AFM)1 is capable of imaging and manipulating nucleic acids in solution and in air29' 13 We are developing methods for random and site-specific labeling of individual DNA molecules to facilitate manipulation of fragments excised in the AFM and for localization of specific DNA domains, such as protein binding sites and origins of replication. One successful method was to incorporate biotinylated nucleotides at random internal locations or specifically at the ends of linearized DNA molecules in vitro. Following complex formation with Snm diameter streptavidin-gold conjugates, chromatographic purification and passive adsorption of the complexes to mica, the biotinylated domains were easily localized in the AFM by virtue of the distinctive size and shape of the

Telomere length is dynamic in many organisms. Genetic screens that identify mutants with altered telomere lengths are essential if we are to understand how telomere length is regulated in vivo. In Tetrahymena thermophila, telomeres become long at 30°, and growth rate slows. A slow-growing culture with long telomeres is often overgrown by a variant cell type with short telomeres and a rapid-doubling rate. Here we show that this variant cell type with short telomeres is in fact a mutant with a genetic defect in telomere length regulation. One of these telomere growth inhibited forever (tgi) mutants was heterozygous for a telomerase RNA mutation, and this mutant telomerase RNA caused telomere shortening when overexpressed in wild-type cells. Several other tgi mutants were also likely to be heterozygous at their mutant loci, since they reverted to wild type when selective pressure for short telomeres was removed. These results illustrate that telomere length can regulate growth rate in Tetrahymena and that this phenomenon can be exploited to identify genes involved in telomere length regulation.

The application of atomic force microscopy (AFM) to biological investigation is attractive for a number of reasons. Foremost among these is the ability of the AFM to image samples, even living cells, under near native conditions and at resolution equal to, or exceeding, that possible by the best light microscopes. Moreover, the ability of the AFM to manipulate samples it images provides a novel and far reaching application of this technology.We have been studying a number of biological samples by AFM. These include conventional and non-conventional nucleic acid structures, ribosomes, neural cells and synapses, cellular organelles (chloroplasts and nuclei), among others. Each of these projects has its own set of associated difficulties and each reveals information about the uses and limits of the AFM in biology. Fig. 1 shows AFM images of various biological samples. In the case of nucleic acids, which have been extensively studied in a number of labs by AFM the problems of signal/noise sample deposition have been overcome in air and organic solvents.