An electron microscopic method for the mapping of proteins attached to nucleic acids.

An electron microscopic method for demonstrating the presence of and mapping the positions of proteins specifically bound to nucleic acids is described. The nucleic acid-protein complex is treated with dinitrofluorobenzene under conditions such that dinitrophenyl (DNP) groups are attached to nucleophilic groups on the protein, with only a low level of random attachment to the nuclei acid. This product is treated with rabbit anti-DNP IgG. The position of the protein-(DNP)n(IgG)m complex on the nucleic acid strand can be observed by electron microscopy by protein free spreading methods and, in many cases, by cytochrome-c spreading. If necessary for visualization by the latter method, the size of the labeled region can be increased by treatment with goat anti-rabbit IgG. High efficiency of electron microscopic labeling is achieved. Examples studied are: the adenovirus-2 DNA terminal protein, a protein covalently bound to SV40 DNA, DNA polymerase I bound to DNA, E. coli RNA polymerase bound to T7 DNA, and proteins UV crosslinked to avian sarcoma virus RNA.     

Nucleic acid capture assay,a new method for direct quantitation of nucleic acids

Technologies allowing direct detection of specific RNA/DNA sequences occasionally serve as an alternative to amplification methods for gene expression studies. In these direct methods the hybridization of probes takes place in complex mixtures, thus specificity and sensitivity still limit the use of current technologies. To address these challenges, we developed a new technique called the nucleic acid capture assay, involving a direct multi-capture system. This approach combines a 3′-ethylene glycol scaffolding with the incorporation of 2′-methoxy deoxyribonucleotides in the capture sequences. In our design, all nucleotides other than those complementary to the target mRNA have been replaced by an inert linker, resulting in significant reductions in non-specific binding. We also provide a versatile method to detect the presence of captured targets by using specific labeled probes with alkaline phosphatase-conjugated anti-label antibodies. This direct, flexible and reliable technique for gene expression analysis is well suited for high-throughput screening and has potential for DNA microarray applications.     

Fluorometric assay of secondary amino acids

A method is described for the conversion of secondary amino acids to primary amines which can be assayed with fluorescamine (I). Secondary amino acids undergo oxidative decar?ylation when reacted with halogenating agents. The resulting imines are hydrolyzed to primary amines, which are subsequently allowed to react with fluorescamine (I) to yield fluorescent pyrrolinones (II). This reaction sequence provides an efficient fluorometric assay for secondary amino acids. Thus, the fluorescamine procedure is now applicable to the full array of natural amino acids.     

Hydroxamate assay quantitation of ligands covalently bound to affinity chromatography gels

Affinity chromatography which utilizes specific biological interactions in the purification or analysis of a variety of biochemical systems is an exceedingly useful method. The technique was initially limited to the use of immunoadsorbants (1) and since has been broadened in scope largely by the efforts of Cuatrecases, Anfinsen and colleagues (2,3). In principle, a specific ligand interacting with a particular substance, usually a macromolecule, is covalently bound to an insoluble support. Substances with no affinity for the ligand will pass unretarded through a column of the bound support; whereas, the interacting materials will be retarded. The methods for preparing a variety of affinity-chromatographic systems are now readily available (2,3). However, a quantitative measure of the covalently bound ligand in many instances depends on the use of radioactively labelled ligands, access to an automatic amino acid analyser, or the subtraction of the amount of ligand recovered in washings after the binding reaction. The latter method is often inaccurate and the former methods may not be practical for all laboratories.We have employed successfully the hydroxamate assay (4,5) for measuring biotin linked through an amide bond to the ω amino group of a 3,3′-diaminodipropylamine substituted agarose gel. This method with individual modifications should be of general use in measuring ligands bound to solid supports through amide, ester, or thioester bonds.     

The characterisation of liposomes with covalently attached proteins

The problem of characterising liposomes with covalently attached proteins has been analysed theoretically in terms of a normal weight distribution of liposome diameters. The polydispersity of protein conjugation is considered in terms of the width (standard deviation) of the liposome size distribution. It is shown that the weight-average number of proteins per liposome is a convenient parameter to use to define the protein content of proteoliposomes. Two types of proteoliposome have been prepared (small unilamellar vesicles and reverse phase evaporation vesicles) in which wheat germ agglutinin is covalently coupled to the liposomal surface. The liposomes cover a range of weight average diameter from 65 to 240 nm and of polydispersity (weight to number average diameter (dw/dn) from 2.6 to 11.4. The liposomes have been characterised by chemical analysis and photon correlation spectroscopy and the results are discussed in terms of the theoretical consequences of an equivalent normal weight distribution of diameters.     

Polyphotobiotin--a new reagent for introducing biotin into nucleic acids]

A new reagent, polyphotobiotin (PPhB), for labelling DNA probes has been obtained using low molecule oligoethylenimine (M 600 Da). Photoreactive group of PPhB is 4-azido-salicylic acid. The procedure is simple and quick, the reaction time being about 20 min. PPhB-labelled DNA has the same efficiency in the hybridisation analysis as DNA labelled with Bio-4-dUTP via PCR.     

Conversion of covalently mercurated nucleic acids to tritiated and halogenated derivatives.

Mercurated nucleic acids are converted to the corresponding tritiated, brominated, and iodinated derivatives by treatment with sodium borotritiide, N-bromosuccinimide, and elemental iodine, respectively. All three reactions occur under mild conditions in neutral aqueous solutions. Mercury-halogen conversions are essentially quantitative at both the mono- and polynucleotide levels. Tritiation reactions also proceed efficiently with mononucleotides, although polymers undergo incomplete demercuration. In spite of the latter limitation , these reactions provide novel and efficient synthetic routes to radiolabeled nucleic acid derivatives.     

Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins

Background

The post-translational modification pathway referred to as pupylation marks proteins for proteasomal degradation in Mycobacterium tuberculosis and other actinobacteria by covalently attaching the small protein Pup (prokaryotic ubiquitin-like protein) to target lysine residues. In contrast to the functionally analogous eukaryotic ubiquitin, Pup is intrinsically disordered in its free form. Its unfolded state allows Pup to adopt different structures upon interaction with different binding partners like the Pup ligase PafA and the proteasomal ATPase Mpa. While the disordered behavior of free Pup has been well characterized, it remained unknown whether Pup adopts a distinct structure when attached to a substrate.

Results

Using a combination of NMR experiments and biochemical analysis we demonstrate that Pup remains unstructured when ligated to two well-established pupylation substrates targeted for proteasomal degradation in Mycobacterium tuberculosis, malonyl transacylase (FabD) and ketopantoyl hydroxylmethyltransferase (PanB). Isotopically labeled Pup was linked to FabD and PanB by in vitro pupylation to generate homogeneously pupylated substrates suitable for NMR analysis. The single target lysine of PanB was identified by a combination of mass spectroscopy and mutational analysis. Chemical shift comparison between Pup in its free form and ligated to substrate reveals intrinsic disorder of Pup in the conjugate.

Conclusion

When linked to the proteasomal substrates FabD and PanB, Pup is unstructured and retains the ability to interact with its different binding partners. This suggests that it is not the conformation of Pup attached to these two substrates which determines their delivery to the proteasome, but the availability of the degradation complex and the depupylase.

     

Sequence-targeted photochemical modifications of nucleic acids by complementary oligonucleotides covalently linked to porphyrins

Porphyrins linked to oligonucleotides produce various types of photodamage on a complementary target DNA. The observed reactions include oxidation of guanine bases and cross-linking reactions of the oligonucleotide to its target sequence. Guanines located close to the porphyrin macrocycle were the most altered as compared to more remote guanines on the target sequence. No specific reaction was observed when the complexes were dissociated at temperatures above the melting temperature of the oligonucleotide-target hybrid. Both cross-linking and oxidation reactions accounted for ca. 60% modification of the target chains in the complex. Our results show that oligonucleotides covalently linked to porphyrins are efficient systems for inducing irreversible sequence-specific photodamage on a target DNA.     

Fluorometric quantification of RNA and DNA in solutions containing both nucleic acids

A fluorescence-based method for quantitative determination of RNA and DNA in probes containing both nucleic acids has been developed. The total concentration of nucleic acids is determined using SYBR Green II dye under conditions providing independent binding of the fluorophore with DNA and RNA. The concentration of DNA is specifically measured using the Hoechst 33258 dye and the RNA concentration is calculated from these data. The procedure allows for accurate determination of DNA concentration in the range 10-1000 ng/ml in the presence of 200-fold excess of RNA and determination of RNA concentrations in the range 10-1000 ng/ml in the presence of large excess of DNA. An absence of the treatment of mixed samples with RNase-free DNase I provides rapid, reproducible, and accurate RNA quantification.     

Sequence-targeted photosensitized reactions in nucleic acids by oligo-alpha-deoxynucleotides and oligo-beta-deoxynucleotides covalently linked to proflavin

Proflavin was covalently linked to the 3'-end or to the 5'-end of an octadeoxythymidylate. This oligonucleotide was synthesized with either the natural beta-anomer of thymidine or its synthetic alpha-anomer. A polymethylene chain was used to link one of the amino groups of proflavin to a terminal thiophosphate group of the oligonucleotide. A 27-mer oligodeoxynucleotide containing an octadeoxyadenylate sequence was used as a target for the proflavin-substituted octadeoxythymidylates. Upon irradiation with visible light, photo-cross-linking reactions induced the formation of branched species that migrated more slowly than the 27-mer on denaturing polyacrylamide gels. Piperidine treatment of the photo-cross-linked species induced strand breaks in the 27-mer. In addition, proflavin induced photosensitized reactions at guanine residues in the 27-mer sequence which were converted to strand breaks following piperidine treatment. Triple-helix formation by the oligothymidylates with their complementary oligodeoxyadenylate sequence at high salt concentration led to photo-cross-linking and cleavage reactions on both sides of the target sequence. These results show that it is possible to target photosensitized reactions to specific sequences on nucleic acids. This opens new possibilities for site-directed mutagenesis and the development of photoactive anti-messenger oligodeoxynucleotides.     

Self-assembly of proteins and their nucleic acids

We have developed an artificial protein scaffold, herewith called a protein vector, which allows linking of an in-vitro synthesised protein to the nucleic acid which encodes it through the process of self-assembly. This protein vector enables the direct physical linkage between a functional protein and its genetic code. The principle is demonstrated using a streptavidin-based protein vector (SAPV) as both a nucleic acid binding pocket and a protein display system. We have shown that functional proteins or protein domains can be produced in vitro and physically linked to their DNA in a single enzymatic reaction. Such self-assembled protein-DNA complexes can be used for protein cloning, the cloning of protein affinity reagents or for the production of proteins which self-assemble on a variety of solid supports. Self-assembly can be utilised for making libraries of protein-DNA complexes or for labelling the protein part of such a complex to a high specific activity by labelling the nucleic acid associated with the protein. In summary, self-assembly offers an opportunity to quickly generate cheap protein affinity reagents, which can also be efficiently labelled, for use in traditional affinity assays or for protein arrays instead of conventional antibodies.     

Characterization and quantitation of fatty acids covalently bound to erythrocyte membrane proteins: anion transporter contains 1 mol of fatty acid thiol ester

Human erythrocyte membranes which had been thoroughly extracted with organic solvents contained 20 nmol of fatty acids/mg dry wt. The major fatty acids were palmitic and stearic with their monoethenoic derivatives as minor constituents. No other fatty acids were detected. When solvent-extracted membranes were digested with Pronase about 90% of the original content of fatty acids was retained in the insoluble residue. Fatty acids were linked to membrane proteins through alkali-labile bonds of which 30% were of a thiol ester and the remainder of an O-ester type. This conclusion is based on differential liberation of fatty acids by hydroxylamine at pH 7.0 and pH 11.0. Two extracts of membranes enriched in peripheral proteins (bands 1, 2, 5 and 2.1, 4.1, 4.2, 6) were prepared and extracted with organic solvents but each contained about six times less fatty acids than the parent solvent-extracted membranes. Glycophorin A contains little if any covalently bound fatty acids. Anion transporter (band 3) contains about 1 mol of thiol ester of fatty acid. This accounts for about half of the thiol ester-linked fatty acids in the parent solvent-extracted membranes. Most of the O-ester-linked fatty acids are linked to an undisclosed membrane protein.     

How to stain nucleic acids and proteins in Miller spreads

The spreading technique proposed by Miller and Beatty in 1969 allowed for the first time the visualization at transmission electron microscopy of nucleic acids and chromatin in an isolated and distended conformation. This approach is beneficial since it can reveal many aspects of chromatin organization and function that otherwise can only be indirectly inferred by biochemical methods. The final step of staining chromatin spreads is critical because it can strongly influence the interpretation of the results. We evaluated different staining techniques, and almost all provided a good result. Specifically, well-contrasted micrographs were obtained when staining with H3PW12O40 (phosphotungstic acid, PTA), as originally proposed by Miller and Beatty, and with two alternatives proposed here: uranyl acetate or terbium citrate. Quite a good contrast of the spread DNA could also be achieved using osmium ammine; while no or little contrast of nucleic acids was observed by staining with KMnO₄ (potassium permanganate) and H3PMo12O40 (phosphomolybdic acid, PMA) respectively.Key words: Chromatin spread, transmission electron microscopy, staining techniques     

Sequence-targeted chemical modifications of nucleic acids by complementary oligonucleotides covalently linked to porphyrins.

Oligo-heptathymidylates covalently linked to porphyrins bind to complementary sequences and can induce local damages on the target molecule. In dark reactions, iron porphyrin derivatives exhibited various chemical reactivities resulting in base oxidation, crosslinking and chain scission reactions. Reactions induced by reductants, such as ascorbic acid, dithiothreitol or mercapto-propionic acid, led to very localised reactions. A single base was the target for more than 50% of the damages. Oxidising agents such as H2O2 and its alkyl derivatives induced reactions that extended to a wider range of altered bases. The specificity of the chemical modifications observed in these systems is discussed from a mechanistic point of view.