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Microarray technology has the unparalleled potential to simultaneously determine the dynamics and/or activities of most, if not all, of the microbial populations in complex environments such as soils and sediments. Researchers have developed several types of arrays that characterize the microbial populations in these samples based on their phylogenetic relatedness or functional genomic content. Several recent studies have used these microarrays to investigate ecological issues; however, most have only analyzed a limited number of samples with relatively few experiments utilizing the full high-throughput potential of microarray analysis. This is due in part to the unique analytical challenges that these samples present with regard to sensitivity, specificity, quantitation, and data analysis. This review discusses specific applications of microarrays to microbial ecology research along with some of the latest studies addressing the difficulties encountered during analysis of complex microbial communities within environmental samples. With continued development, microarray technology may ultimately achieve its potential for comprehensive, high-throughput characterization of microbial populations in near real time.

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Solid-phase synthesis is greatly dependent on the solid phase. We are interested in the development of a "pellicular" type of solid support where a more mobile polymer is grafted to rigid plastics. Compared to low cross-linked microporous beads that dominate the field, this approach allows great flexibility of design, as plastics are available as sheets, films, or threads, or can be molded into any shape, as required. Many different polymers or copolymers can be grafted onto any particular shape to give a wide choice of options in the physicochemical characteristics of the actual solid support. As an example of such a solid support, we report on polystyrene-grafted polypropylene in a particular shape that we have called "Lanterns." Its synthesis characteristics are compared to the commonly available low cross-linked polystyrene resins. As well, the handling advantages of these types of supports in multiple synthesis are highlighted.

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The utility of electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) for the characterisation of ligand-oligonucleotide adducts is demonstrated with adducts formed between the oligonucleotide 5'-CACGTG-3' and both a platinating agent, cis-diamminedichloroplatinum(II) (cisplatin), and an alkylating ligand, n-bromohexylphenanthridinium bromide (phenC6Br). We have demonstrated previously that negative ion MS/MS spectra of alkylated oligonucleotides show a highly specific fragmentation pathway that enables the site of binding of the ligand to be readily identified. In comparison, the positive ion ESI-MS/MS spectra reported here also show a single major fragmentation pathway, but the dominant ion is the protonated ligand-base adduct. MS/MS of this ion confirms the site on binding of the ligand to the guanine base. MS/MS spectra of cisplatin adducts show much less specific fragmentation than alkylated adducts, particularly in the negative ion mode. This suggests that the ESI-MS/MS spectra of ligand-DNA adducts are strongly influenced by the extent to which the ligand weakens the glycosidic bond in the residue to which it is bound. For platinating agents, which do not labilise the glycosidic bond, additional experiments involving MS/MS of source-generated product ions were required to enable isomeric adducts to be distinguished.

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The solid phase synthesis of a set of peptide aldehydes derived from the NS5A/NS5B junction of hepatitis C virus (HCV) viral polyprotein is demonstrated using an oxazolidine linker and the Multipin method. Deletion of the P6 and P5 residues results in a dramatic loss of inhibitory activity.

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Hedamycin, a member of the pluramycin class of antitumour antibiotics, consists of a planar anthrapyrantrione chromophore to which is attached two aminosugar rings at one end and a bisepoxide-containing sidechain at the other end. Binding to double-stranded DNA is known to involve both reversible and non-reversible modes of interaction. As a part of studies directed towards elucidating the structural basis for the observed 5'-pyGT-3' sequence selectivity of hedamycin, we conducted one-dimensional NMR titration experiments at low temperature using the hexadeoxyribonucleotide duplexes d(CACGTG)(2) and d(CGTACG)(2). Spectral changes which occurred during these titrations are consistent with hedamycin initially forming a reversible complex in slow exchange on the NMR timescale and binding through intercalation of the chromophore. Monitoring of this reversible complex over a period of hours revealed a second type of spectral change which corresponds with formation of a non-reversible complex.

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Cisplatin analogues were synthesised that consisted of platinum(II) diamine complexes tethered via a polymethylene chain ( n = 3, 5, 8 and 10) to a phenanthridinium cation. Both chloro and iodo leaving groups were examined. DNA adduct formation was quantitatively analysed using a linear amplification system with the plasmid pGEM-3Zf(+). This system utilised Taq DNA polymerase to extend from an oligonucleotide primer to the damage site. This damage site inhibited the extension of the DNA polymerase. The products were electrophoresed on a DNA sequencing gel enabling adduct formation to be determined at base pair resolution. The damage intensity at each site was determined by densitometry. The platinum phenanthridinium complexes were shown to damage DNA at shorter incubation times than cisplatin. To produce similar levels of damage, an 18 h incubation was required for cisplatin compared to 30 min for the n = 3 platinum phenanthridinium complexes; this indicates that the intercalating chromophore causes a large increase in the rate of platination. A reaction mechanism involving direct displacement of the chloride by the N-7 of guanine may account for the rate increase. These results indicate that further development of these compounds could lead to more effective cancer chemotherapeutic agents.

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Hepatitis delta virus (HDV) is a small single-stranded RNA satellite of hepatitis B virus. Although it is a human pathogen, it shares a number of features with a subset of the small plant satellite RNA viruses, including self-cleaving sequences in the genomic and antigenomic sequences of the viral RNA. The self-cleaving sequence is critical to viral replication and is thought to function as a ribozyme in vivo to process the products of rolling-circle replication to unit-length molecules. A divalent cation is required for cleavage and while a structural role is implicated for metal ions, a more direct role for a metal ion in catalysis has not yet been proven. A minimal natural ribozyme sequence with proficient in vitro self-cleavage activity is about 85 nucleotides long and adopts a secondary structure with four paired regions (P1-P4). The two pairings that define the 5' and 3' boundaries of the ribozyme, P1 and P2, form an atypical pseudoknot arrangement. This secondary structure places a number of constraints on the possible tertiary folding of the sequence, which together with chemical probing, photo-cross-linking, mutagenesis and computer-assisted modeling provides clues to the three-dimensional structure. The data are consistent with a model in which the cleavage site, located at the 5' end of P1, is in close proximity to three single-stranded regions, consisting of a hairpin loop at the end of P3 and two sequences joining P1 to P4 and P4 to P2. While the natural forms of the HDV ribozymes appear to be prone to misfolding, biochemical and mutagenesis studies from a number of laboratories has allowed the production of trans-acting ribozymes and smaller more active cis-acting ribozymes, both of which will aid in further mechanistic and structural studies of this RNA.

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Previous studies involving fetal decapitation or hypophysectomy, and the treatment of neonates with hormones or antibodies, have suggested that changes in pituitary hormone status during the perinatal period may influence later body composition. In the present study, rats were treated for the first 21 days of life with twice daily subcutaneous injections of saline, recombinant bovine growth hormone (bGH) or pituitary ovine prolactin (oPRL). The bGH and oPRL were administered at doses of 0.2 or 0.4 microgram/g bodyweight/day. One-third of the rats in each treatment group were slaughtered at each of days 21, 60 and 120 of life and measurements made of: length and weight of the body; weights of bones and muscle groups in the hindlimb; weights of four fat depots (120-day group only); and the content of nitrogen (N) and fat in the carcass. bGH, but not oPRL, treatment increased weight of the femur and humerus (across ages) but neither treatment had marked effects on weights of muscle groups, carcass weight or carcass N content at any age. Both bGH and oPRL treatment significantly reduced weight of the subcutaneous scapular fat depot and reduced carcass fat content, but only in animals aged 120 days (i.e. 99 days after the cessation of treatment). It is concluded that treatment of rats with bGH and oPRL during the immediate postnatal period specifically retards the ability of animals to deposit body fat in later life by mechanisms which differ from those involved in the classical lipolytic/antilipogenic effects of bGH.

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2D NMR spectroscopic methods have been used to determine the structure of the adduct formed between the antitumor antibiotic hedamycin and the oligodeoxyribonucleotide duplex d(CACGTG)2. Evidence for both intercalation and alkylation in the adduct was observed, and a model for the binding interaction was constructed based on intermolecular NOEs and distance-restrained molecular dynamics. In our computationally refined model, the anthrapyrantrione chromophore of hedamycin is intercalated between the 5'-CG-3' bases with the two aminosugar groups placed in the minor groove and the six carbon bisepoxide side chain located in the major groove. The anglosamine sugar attached at C8 is oriented in the 3' direction relative to the intercalation site, while the N,N-dimethylvancosamine attached at C10 is oriented to the 5' side, with each aminosugar wedged between a guanine exocyclic amino group and one of the groove walls. The terminal epoxide carbon C18 is covalently bound to the N7 atom of the central guanine, as evidenced by lability of the C8 hydrogen of this purine upon reaction with hedamycin. Our binding model places the C10-attached N,N-dimethylvancosamine of hedamycin in van der Waals contact with the alkylated strand. A strong NOE contact verifies the close proximity of the terminal methyl group (C19) of the bisepoxide side chain to the methyl group of the thymine on the 3' side of the alkylated guanine. This, in conjunction with other data, suggests hydrophobic interactions between the bisepoxide chain and the floor of the major groove may contribute to sequence recognition. Furthermore, it is proposed that the 5'-CGT sequence selectivity of hedamycin arises, in part, from complementarity in shape between the chromophore substituents and the major and minor groove at the binding site.

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The sequence specificity of DNA damage of n-bromoalkylphenanthridinium bromides, with linker chain lengths (n) of 4,6,8 and 10 methylene groups, was investigated in the plasmid pUC8 and in intact human cells. A linear amplification assay was used to elucidate the DNA sequence specificity of the alkylating agents. In this assay Taq DNA polymerase extends from an oligonucleotide primer up to the damage site and the products run on a DNA sequencing gel to reveal the precise sites of DNA damage. For both the plasmid and cellular experiments, the compound that caused the most damage to DNA was the n=6 compound, followed by (in decreasing order) the n=4, n=8, and n=10 compounds. There were significant differences in the sequence specificity of DNA damage between n-bromoalkylphenanthridinium bromides of different linker chain length; (1) the main sites of damage were at guanines for the n=4,6 and 8 compounds but at guanines and adenines for the n=10 compound; (2) a consensus sequence of 5'-c(a/t)Ggg-3' was obtained for the n=4,6 and 8 compounds but 5'-c(a/c)(G/A)(g/a)-3' for the n=10 compound; (3) runs of consecutive Gs were the major site of damage for the n=4,6 and 8 compounds, but consecutive Gs or consecutive As for the n=10 compound; (4) for damage at single isolated guanines, the most damaged sequences were at 5'-Ga-3' for the n=4 compound but at 5'-Gt-3' for the n=6,8 and 10 compounds. The tandemly repeated alpha RI DNA sequence was the DNA target in intact human K562 cells. In intact human cells, the compounds produced damage with similar DNA sequence selectivity to that found in plasmid DNA. The n=4 and 6 compounds possess marginal anti-tumour activity and these compounds produced the most damage in intact human cells. The n=8 and 10 compounds do not demonstrate significant anti-tumour activity and these compounds resulted in the least damage in cells.

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A series of terephthalamide derivatives with the substituents R = NO2, NH2 and NHCOCH2NH2 on the central aryl ring have been synthesized, and their interaction with the DNA decamer d(GGTAATTACC)2 has been studied by 1H NMR. The amine and nitro (R = NH2, NO2) derivatives bind with micromolar affinities and exhibit NMR spectra characteristic of fast exchange on the chemical shift time scale. The glycine derivative (R = NHCOCH2NH2) binds more tightly and a number of its resonances are in intermediate to slow exchange on the chemical shift time scale. Estimates of binding affinities and bound chemical shifts of ligand and DNA resonances were made from an analysis of chemical shifts and linewidths in a series of spectra with ligand duplex mole ratios ranging from 0:1 to 2:1. The data unequivocally suggest that all three ligands bind in the minor groove of the DNA decamer and more specifically that the binding site is localized over the ATTA sequence. The ligand is able to exchange rapidly between two symmetry-related ATTA sites per decamer.

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The sequence specificity of the pluramycin antibiotics hedamycin and DC92-B, was established in intact human cells using a linear amplification system. In this system an oligonucleotide primer is extended by Taq DNA polymerase up to a damage site. The products are run on a DNA sequencing gel and the damage can be determined to the exact base pair. The human repetitive alpha RI DNA was used as the target DNA sequence for these experiments. It was found that G residues were the main site of adduct formation, for both hedamycin and DC92-B. The sequences 5'-TGT and 5'-CGT were the most intense sites of DNA damage. A comparison of the DNA damage intensity in intact cells and purified DNA revealed that the sequence position of adduct formation was very similar in the two environments. However, a densitometric comparison of the damage intensity in the two environments revealed significant differences. Two regions were found (120 and 130 bp in length) where the damage intensity was relatively lower in intact cells compared to purified DNA. But at the boundaries of these sequences, there were regions (approx. 50-60 bp long) that were relatively more damaged in intact cells compared to purified DNA. One explanation of this phenomenon is the presence of a protecting nucleosome core on each of the 120/130 bp regions and flanking nucleosome linker regions of 50-60 bp. This postulated sequence phasing of the nucleosomes corresponds almost exactly with the major nucleosome phasing found in African green monkey cells. Also the centromere protein B binding site is found in the border region between the nucleosome core and linker DNA regions. Hedamycin and DC92-B produced nearly identical results in this human cell system.

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The sequence selectivity of DNA alkylation by the recently isolated pluramycin antitumour antibiotic DC92-B has been investigated using two methods: a piperidine-induced strand-breaking procedure and a Taq DNA polymerase/linear amplification method. These techniques reveal that guanines are the most reactive sites for alkylation and that the level of adduct formation at these sites is clearly sequence dependent. The highest levels of alkylation occurred at isolated guanines located in 5'-CGT sequences and also at the 5'-G in some 5'-CGG sequences. Isolated guanines in 5'-TGT sequences were also quite reactive. We have also re-examined, in parallel, the sequence selectivity of binding of the structurally-related compound hedamycin: the first known example of a bis(epoxide)-containing, DNA-alkylating pluramycin. Our studies included a more extensive sequence analysis of hedamycin binding than that previously reported and we are able, therefore, to define more precisely the sequence preference. Despite significant differences in the stereochemistry and substitution of their bis(epoxide) sidechains, hedamycin and DC92-B exhibited very similar sequence selectivities in our assays.

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Covalent binding of the antitumour antibiotic hedamycin to the self-complementary hexadeoxyribonucleotide 5'-CACGTG-3' has been investigated by electrospray ionization mass spectrometry (ESI-MS). Ions due to double-stranded forms of the free 5'-CACGTG-3' and the hedamycin-5'- CACGTG-3' adduct have been observed in ESI mass spectra and their identity has been confirmed by resolution of individual charge states in ESI-MS spectra. Clear evidence that specific base-paired associations are being observed in ESI-MS is provided by the results of a titration experiment involving alkylated and non-alkylated complementary strands. This work demonstrates the potential of this powerful new tool for studying ligand-DNA binding.

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Electrospray ionization mass spectrometry (ESI-MS) has been used to examine the covalent binding of the anti-tumour agents cisplatin and hedamycin with self-complementary oligonucleotides 5'-TACGTA-3', 5'-CACGTG-3', 5'-AGGCCT-3' or 5'-CGTACG-3' as models for binding to cellular DNA. The observation of duplex forms of oligonucleotide adducts of these compounds in the gas phase has been found to correlate with the stability of the adducts in solution. Hedamycin, which both intercalates into and alkylates DNA, enhances the stability of duplex ions in ESI mass spectra. In contrast, the binding of cisplatin is known to destabilise duplexes in solution and only weak double-stranded peaks are observed in the ESI spectra of cisplatin-oligonucleotide adducts. Results of titration experiments with the hedamycin-5'-CACGTG-3' adduct and complementary and non-complementary oligo-nucleotides provide strong evidence that the observed duplex ions are the result of specific base-paired associations in the gas phase, rather than non-specific interactions. Finally, estimates of the extent of cisplatin binding to different sequences based on ESI mass spectra of crude reaction mixtures are found to correlate well with data obtained by reversed-phase high-performance liquid chromatography. This work demonstrates the considerable potential of ESI-MS as a tool for characterization of the interactions of antitumour agents with DNA.

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The phylogenetic diversity of a well-known pink filament community associated with the 84 to 88 degrees C outflow from Octopus Spring, Yellowstone National Park, was examined. Three phylogenetic types ("phylotypes"), designated EM 3, EM 17, and EM 19, were identified by cloning and sequencing the small subunit rRNA genes (16S rDNA) obtained by PCR amplification of mixed-population DNA. All three phylotypes diverge deeply within the phylogenetic domain Bacteria sensu Woese (C. R. Woese, O. Kandler, and M. L. Wheelis, Proc. Natl. Acad. Sci. USA 87:4576-4579, 1990). No members of the Archaea or Eucarya were detected. EM 3 comprises a unique lineage within the Thermotogales group, and EM 17 and EM 19 are affiliated with the Aquificales. A total of 35 clones were examined, of which the majority (26 clones) were of a single sequence type (EM 17) closely related to Aquifex pyrophilus. In situ hybridization with clone-specific probes attributes the majority sequence, EM 17, to the pink filaments.

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Bidirectional transcription footprinting has been used to probe the platination of DNA by cisplatin, and to examine the modulation of these interactions by (a) cyclisation of the non-reactive amino group by either ethyl or propyl groups, and (b) the further addition of a pendant intercalator (9-amino acridine) linked by either phenylethyl or phenylpentyl groups. Intrastrand crosslinking was detected for all derivatives at all 5'-GG and 5'-AG sequences on the template strand, but the same sites did not result in transcriptional blockages when on the non-template strand. There was little effect of cyclysation of the amino groups, but the further addition of an intercalator resulted in three responses: a time-dependent increase of the blocked transcript by one and three nucleotides; a reduction of the sequence selectivity of platination; a decrease of apparent interstrand crosslinking for these derivatives with a pendant intercalator tethered to the amino moiety of cisplatin.

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The sequence specificity of DNA damage caused by cis-diamminedichloroplatinum(II) (cisplatin) and four analogues in human (HeLa) cells was studied using Taq DNA polymerase and a linear amplification system. The primer extension is inhibited by the drug-DNA adducts, and hence the sites of these lesions can be analyzed on DNA sequencing gels. The repetitive alphoid DNA was used as the target DNA in human cells. A comparison was made between adduct formation in human cells and in purified DNA. The sequence-specific position and relative intensity of damage was similar in both systems for cisplatin, dichloro(ethylenediammine)platinum(II) (PtenCl2), and N-[3-N-(ethylenediamino)propyl]acridine-4-carboxamidedichloropl atinum(II) (4AcC3PtenCl2). However, no DNA damage could be detected in cells for trans-diamminedichloroplatinum(II) (transPt) or N-[3-N-(ethylenediamino)propyl]acridine-2-carboxamide-dichloroplat inum(II) (2AcC3PtenCl2) despite the ability of these latter analogues to damage purified DNA. Cisplatin, PtenCl2, and 4AcC3PtenCl2, which significantly damaged DNA inside cells, also show antitumor activity in mouse models. However, transPt and 2AcC3PtenCl2, which did not detectably damage DNA inside cells, did not show such antitumor activity. This correlation between intracellular DNA damaging ability and in vivo antitumor activity indicates the potential use of the human cells/Taq DNA polymerase/linear amplification technique as a convenient method for screening new cisplatin analogues for useful chemotherapeutic activity.

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A series of dimers of the monofunctional platinum species [Pt(dien)Cl]+, linked by a variety of flexible (polymethylene) and more rigid chains, was prepared and evaluated for DNA interactions and cytotoxic activity. The polymethylene-linked dimers were prepared by acylation of N(1),N(3)-bistrityldiethylenetriamine with alpha, omega-dicarboxylic acid chlorides, followed by reduction with diborane. Platination of these ligands was achieved with K2PtI4 prepared in situ, followed by anion exchange. Solutions of the bis(Pt(dien)Cl)2+ complexes were stable, and shown to be pure by 195Pt NMR, but solid products could not be isolated. All of the bis(Pt(dien)Cl)2+ complexes unwound closed circular supercoiled DNA more efficiently than the monomer, and were more efficient than the difunctional platinum complex cisplatin at cross-linking linearized plasmid DNA, as measured on non-denaturing agarose gels. None of the bis(Pt(dien)Cl)2+ complexes were as cytotoxic as cisplatin in both the wild-type and platinum-resistant P388 murine leukaemia cell lines. The more rigid analogues were equitoxic in both sensitive and cisplatin-resistant cells, but none showed in vitro activity against the P388 tumour.

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