Heterogeneity and persistence length in human ocular mucins.

Atomic force microscopy (AFM) has been used to investigate the heterogeneity and flexibility of human ocular mucins and their subunits. We have paid particular attention, in terms of theory and experiment, to the problem of inducing the polymers to assume equilibrium conformations at a surface. Mucins deposited from a buffer containing Ni(2+) ions adopt extended conformations on mica akin to those observed for DNA under similar conditions. The heterogeneity of the intracellular native mucins is evident from a histogram of contour lengths, reflecting, in part, the diversity of mucin gene products expressed. Reduction of the native mucin with dithiothreitol, thereby breaking the S==S bonds between cysteine residues, causes a marked reduction in polymer length. These results reflect the modes of transport and assembly of newly synthesized mucins in vivo. By modifying the worm-like chain model for applicability to two dimensions, we have confirmed that under the conditions employed mucin adsorbs to mica in an equilibrated conformation. The determined persistence length of the native mucin, 36 nm, is consistent with that of an extended, flexible polymer; such characteristics will influence the properties of the gels formed in vivo.

Secondary binding sites for triplex-forming oligonucleotides containing bulges, loops, and mismatches in the third strand.

We have used DNase I footprinting to examine the binding of five different 17-mer oligonucleotides to a 53-base oligopurine tract containing four pyrimidine interruptions. Although all the expected triplexes formed with high affinity (K(d) approximately 10-50 nM), one oligonucleotide produced a footprint at a second site with about 20-fold lower affinity. We have explored the nature of this secondary binding site and suggest that it arises when each end of the third strand forms a 7-mer triplex with adjacent regions on the duplex, generating a contiguous 14-base triplex with a bulge in the center of the third strand oligonucleotide. This unusual binding mode was examined by use of oligonucleotides that were designed with the potential to form different length third-strand loops of various base composition. We find that triplexes containing single-base bulges are generally more stable than those with dinucleotide loops, though triplexes can be formed with loops of up to nine thymines, generating complexes with submicromolar dissociation constants. These structures are much more stable than those formed by adding two separate 7-mer oligonucleotides, which do not generate DNase I footprints, though a stable complex is generated when the two halves are covalently joined by a hexa(ethylene glycol) linker. MPE produces less clear footprints, presumably because this cleavage agent binds to triplex DNA, but confirms that the oligonucleotides can bind in unexpected places. These results suggest that extra care needs to be taken when designing long triplex-forming oligonucleotides so as to avoid triplex formation at shorter secondary sites.

Alternative geometries of DNA looping: an analysis using the SfiI endonuclease.

Many processes are governed by proteins that bind to separate sites in DNA and loop out the intervening DNA, but the geometries of the loops have seldom been determined. The SfiI endonuclease cleaves DNA after interacting with two recognition sites, and is a favourable system for the analysis of DNA looping. A gel-shift assay was used here to examine the binding of SfiI to a series of linear DNA molecules containing two SfiI sites separated by 109-170 base-pairs. The complexes in which SfiI trapped a loop by binding to two sites in the same DNA were separated from the complexes containing SfiI bound to separate DNA molecules. Step-wise changes in the inter-site spacing generated two forms of the looped complex with different electrophoretic mobilities. The yields of each looped complex and the complexes from intermolecular synapses all varied cyclically with the inter-site spacing, with similar periodicities ( approximately 10.5 base-pairs) but with different phases. One looped complex predominated whenever the DNA between the sites needed to be underwound in order to produce the correct helical orientation of the binding sites. The other looped complex predominated whenever the intervening DNA needed to be overwound. We conclude that the former has trapped a right-handed loop with a negative node and the latter a left-handed loop with a positive node.

DNA triple helix formation at target sites containing several pyrimidine interruptions: stabilization by protonated cytosine or 5-(1-propargylamino)dU.

DNase I footprinting has been used to study the formation of parallel triplexes at oligopurine target sequences which are interrupted by pyrimidines at regular intervals. TA interruptions are targeted with third strand oligonucleotides containing guanine, generating G x TA triplets, while CG base pairs are targeted with thymine, forming T x CG triplets. We have attempted to optimize the stability of these complexes by varying the base composition and sequence arrangement of the target sites, and by replacing the third strand thymines with the positively charged analogue 5-(1-propargylamino)dU (U(P)). For the target sequence (AAAT)(5)AA, in which pyrimidines are positioned at every fourth residue, triplex formation with TG-containing oligonucleotides is only detected in the presence of a triplex-binding ligand, though stable triplexes were detected at the target site (AAAAAT)(3)AAAA. Triplex stability at targets containing pyrimidines at every fourth residue is increased by introducing guanines into the duplex repeat unit using the targets (AGAT)(5)AA and (ATGA)(5)AA. In contrast, placing C(+) x GC triplets on the 5'-side of G x TA, using the target (AGTA)(5)TT, produces complexes of lower stability. We have attempted further to increase the stability of these complexes by using the positively charged thymine base analogue U(P), and have shown that (TU(P)TG)(5)TT forms a more stable complex with target (AAAT)(5)AA than the unmodified third strand, generating a footprint in the absence of a triplex-binding ligand. Triplex formation at (AGTA)(5)AA is improved by using the modified oligonucleotide (TCGU(P))(5)TT, generating a complex in which the charged triplets C(+) x GC and U(P) x AT alternate with uncharged triplets. In contrast, placing U(P) x AT triplets adjacent to C(+) x GC, using the third strand oligonucleotide (U(P)CGT)(5)TT, reduces triplex formation, while the third strand with both substitutions, (U(P)CGU(P))(5)TT, produces a complex with intermediate stability. It appears that, although adjacent U(P) x AT triplets form stable triplexes, placing U(P) x AT adjacent to C(+) x GC is unfavorable. Similar results were obtained with fragments containing CG inversions within the oligopurine tract, though triplexes at (AAAAAC)(3)AA were only detected in the presence of a triplex-binding ligand. Placing C(+) x GC on the 5'-side of T x CG triplets also reduces triplex formation, while a 3'-C(+) x GC produces complexes with increased stability.

Towards mixed sequence recognition by triple helix formation.

The formation of intermolecular DNA triple helices offers the possibility of designing compounds with extensive sequence recognition properties which may be useful as antigene agents or tools in molecular biology. One major limitation of this approach is that these structures are generally restricted to homo-purine. homopyrimidine target sites. This review describes the strategies that have been employed to overcome this drawback and outlines the potential for triplex formation at mixed sequence DNA targets.

Health surveillance using an occupational medical database.

This pilot study sought associations between liver function tests (LFTs) and membership in homogeneous exposure groups (HEGs) at a target plant as pre-clinical indications of possible future occupational health problems. A large company database yielded linear models for each of six LFTs (total bilirubin, alkaline phosphatase, gammaglutamyl transferase, lactate dehydrogenase, aspartate transaminase, and alanine transaminase) in terms of sex, body mass index, age, race (white/non-white), alcohol and cigarette consumption, and production/non-production (P/NP) job, permitting control for these in analyses of LFTs vs HEGs at the plant. These analyses, with HEG substituted for P/NP in the large group model, resulted in loosely "suspect" associations significant at P < 0.10. Collapsed HEG variable (containing "suspects" separately and all other non-significant HEG levels pooled) yielded "confirmed suspects" at P < 0.05 in the analysis of an independent LFT set taken at the plant approximately one year later.

Triple helix formation at (AT)n adjacent to an oligopurine tract.

We have used DNase I footprinting to investigate the recognition of (AT) n tracts in duplex DNA using GT-containing oligonucleotides designed to form alternating G.TA and T.AT triplets. Previous studies have shown that the formation of these complexes is facilitated by anchoring the triplex with a block of adjacent T.AT triplets, i.e. using T11(TG)6to recognize the target A11(AT)6. (AT)6T11. In the present study we have examined how the stability of these complexes is affected by the length of either the T.AT tract or the region of alternating G.TA and T.AT triplets, using oligonucleotides of type T x (TG) y to recognize the sequence A11(AT)11. We find that successful triplex formation at (AT)n (n = 3, 6 or 11) can be achieved with a stabilizing tail of 11xT.AT triplets. The affinity of the third strand increases with the length of the (GT) n tract, suggesting that the alternating G.TA and T.AT triplets are making a positive contribution to stability. These complexes are stabilized by the presence of manganese or a triplex-specific binding ligand. Shorter oligo-nucleotides, such as T7(TG)5, bind less tightly and require the addition of a triplex-binding ligand. T4(GT)5showed no binding under any conditions. Oligo-nucleotides forming a 3'-terminal T.AT are marginally more stable that those with a terminal G.TA. The stability of these complexes was further increased by replacing two of the T.AT triplets in the T n tail region with two C+.GC triplets.

DNA triple helix formation at oligopurine sites containing multiple contiguous pyrimidines.

We have used DNase I footprinting to assess the formation of triple helices at 15mer oligopurine target sites which are interrupted by several (up to four) adjacent central pyrimidine residues. Third strand oligonucleotides were designed to generate complexes containing central (X.TA)nor (X.CG)n triplets (X = each base in turn) surrounded by C+.GC and T.AT triplets. It has previously been shown that G.TA and T.CG are the most stable triplets for recognition of single TA and CG interruptions. We show that these triplets are the most useful for recognizing consecutive pyrimidine interruptions and find that addition of each pyrimidine residue leads to a 30-fold decrease in third strand affinity. The addition of 10 microM naphthylquinoline triplex-binding ligand stabilizes each complex so that all the oligonucleotides produce footprints at similar concentrations (0.3 microM). Targets containing two pyrimidines are only bound by oligonucleotides generating (G.TA)2 and (T.CG)2 with a further 30-fold decrease in affinity. (G.TA)2 is slightly more stable than (T.CG)2. In the presence of the triplex-binding ligand the order of stability is (G.TA)2 > (C.TA)2 > (T.TA)2 > (A.TA)2 and (T.CG)2 > (C.CG)2 > (G.CG)2 = (A.CG)2. No oligonucleotide footprints are generated at target sites containing three consecutive pyrimidines, though addition of 10 microM triplex-binding ligand produces stable complexes with oligonucleotides generating (G.TA)3, (T.CG)3 and (C.CG)3, with a further 30-fold reduction in affinity. No footprints are generated at targets containing four Ts, though the ligand induces a weak interaction with the oligonucleotide generating (T.CG)4.

Incidence of respiratory cancer among workers exposed to chloromethyl-ethers.

A factory in France had used technical-grade chloromethyl-methyl-ether in the manufacture of anion exchange resins since 1958. Technical-grade chloromethyl-methyl-ether contains a human lung carcinogen, bis-chloromethyl-ether. The purpose of this cohort study was to determine if workers at the factory whose jobs had involved potential exposure to technical chloromethyl-methyl-ether had a higher incidence of lung cancer than coworkers, or others without potential exposure. Lung cancer occurred at a higher rate among potentially exposed workers than among nonexposed workers at the same plant (rate ratio (RR) = 5.0, 95% confidence interval (CI) 2.0-12.3), or among an external reference population (RR = 7.6, 95% CI 4.3-13.5). The average age at diagnosis for exposed cases was 10.5 years lower than for nonexposed cases. The predominantly small-cell cancers of the exposed were mostly oat-cell. There was a positive dose-response relation. The mean time from first exposure to diagnosis was 13 years (95% CI 8-18). Cumulative dose and induction time were not associated. The rate for the exposed workers peaked between 7 and 13 years after the start-up of the chloromethylation process, and was still above background in 1986, the end of the study period.