Comparison of complex DNA mixtures with generic oligonucleotide microchips.

The reproducibility of melting curves for repeated hybridizations of target DNA with generic oligonucleotide microchips is shown experimentally to depend on the character of matching between fragments of target DNA and immobilized oligonucleotides. The reproducibility of melting curves is higher for the perfect match duplexes and decreases as the number of mismatched pairs within duplexes increases. This effect was applied to the comparative analysis of complex DNA mixtures. We developed a scheme in which we can identify and discriminate between the probe oligonucleotides responsible for the distinctions between target DNA mixtures. A scheme is illustrated by comparing DNA mixtures corresponding to V-D-J genes connected with populations of mRNAs CDR3 TCR Vb (T-cell receptor beta complementarity determining region 3) from the thymus and pancreas of NOD mice. Our results demonstrate that generic microchips can be applied efficiently to the analysis of DNA mixtures.

Detection of single nucleotide polymorphisms of the human mu opioid receptor gene by hybridization or single nucleotide extension on custom oligonucleotide gelpad microchips: potential in studies of addiction.

The human mu opioid receptor (MOR) plays a central role in mediating the effects of opioids, both endogenous and exogenous. Epidemiological studies have shown that addiction in general, and especially opiate addiction, has a heritable component. Clinical and laboratory studies suggest that the MOR gene may contribute to the heritable component of vulnerability to develop opiate addiction. Naturally occurring single nucleotide polymorphisms (SNPs) have been identified in the MOR gene by conventional methods. Two coding region SNPs, the A118G and C17T substitutions, occur at high allelic frequencies (10.5% and 6.6%, respectively, in our previous studies). These common SNPs cause amino acid changes in the receptor, and may have implications for differences in individual responses to opioids, as well as decreased or increased vulnerability to opiate addiction. The A118G substitution encodes a variant receptor with binding and signal transduction differences in response to beta-endorphin in cellular assays. Recent innovations in microchip technology offer new potential methods for SNP detection. We report here on the development of two separate approaches using custom oligonucleotide gelpad microarrays for detection of these two common SNPs of the MOR gene in human DNA samples. First, PCR-amplified genomic DNA samples were used to produce target sequences, which were labeled with fluorescent dye and hybridized to custom microchips. Oligonucleotides on these reusable microchips were designed to query nucleotide substitutions at positions 17 and 118 of the MOR gene. Thirty-six human DNA samples were assayed both on these custom microchips and by conventional automated gel sequencing, with highly concordant identification of both heterozygous and homozygous substitutions. A second approach was developed for the C17T SNP utilizing single nucleotide extension on custom microchips. These custom gelpad microchips have potential for the rapid and inexpensive detection of specific SNPs for genetic and genomic studies.

Fabrication of microarray of gel-immobilized compounds on a chip by copolymerization.

The manufacturing of microchips containing oligonucleotides and proteins immobilized within gel pads, ranging in size from 10 x 10 to 100 x 100 microns, is described. The microchips are produced by photo- or persulfate-induced copolymerization of unsaturated derivatives of biomolecules with acrylamide-bisacrylamide mixture. Oligonucleotides containing 5'-allyl or 5'-butenediol units were synthesized using standard phosphoramidite chemistry. Acryloyl residues were attached to a protein by a two-step procedure. Photopolymerization was induced by illumination of the monomer solution containing initiator with UV light through the mask. The mask was applied directly over the monomer solution or projected through a microscope. Alternatively, copolymerization was carried out in drops of aqueous solution of monomers containing ammonium persulfate. Drops with different allyl-oligonucleotides were distributed on a glass slide, and the polymerization was induced by diffusion of N,N,N',N'-tetramethylethylenediamine (TEMED) from a hexane solution that covered the aqueous drops.

Manual manufacturing of oligonucleotide, DNA, and protein microchips.

A simple procedure for manufacturing microchips containing various gel-immobilized compounds is described. A gel photopolymerization technique is introduced to produce micromatrices of polyacrylamide gel pads (25 x 25 x 20 microm and larger) separated by a hydrophobic glass surface. A pin device for the manual application of a compound in solution onto the activated polyacrylamide gel pad for immobilization is described. Oligonucleotide, DNA, and protein microchips have been produced by this method and tested by hybridization and immunoanalysis monitored with a fluorescence microscope. The effect of the lengths of the immobilized oligonucleotides and the hybridized RNA and DNA on hybridization of the oligonucleotide microchips was evaluated. This method can also be used for manufacturing microchips containing a variety of other compounds.

Laminin E8 alveolarization site: heparin sensitivity, cell surface receptors, and role in cell spreading.

Cell adhesion to amino acids 2179-2198 (SN-peptide) of the laminin-1 alpha1-chain is required for lung alveolar formation in vitro (M. L. Matter and G. W. Laurie. J. Cell Biol. 124: 1083-1090, 1994). The nature of the SN-peptide receptor(s) was probed with neutralizing anti-integrin monoclonal antibodies (MAb), cells lacking integrin subunits, soluble heparin, and SN-peptide columns. Cell adhesion and spreading studies confirmed the specificity of SN-peptide and revealed adhesion to be unaffected by inclusion of anti-beta1-, anti-alpha(2-6)- or anti-alpha(V)beta5-integrin MAb. Cells lacking beta1- or alpha6-integrin subunits were fully adherent. Adhesion was heparin, but not chondroitin sulfate or heparinase, sensitive, much as is alpha-dystroglycan-laminin-1 binding. Heparin eluted approximately 155- and 180-kDa cell-surface proteins from SN-peptide columns. An additional approximately 91-kDa protein was eluted by EDTA. All were unrecognized by anti-beta1-integrin MAb. SN-peptide therefore interacts with three cell-surface proteins for which the identity remains to be determined.

Distribution of high mobility group proteins 1/2, E and 14/17 and linker histones H1 and H5 on transcribed and non-transcribed regions of chicken erythrocyte chromatin.

Quantitative analysis of distribution of chromosomal proteins on single copy DNA sequences has been further developed. Our approach consists of DNA-protein crosslinking within whole cells or isolated nuclei, specific immunoaffinity isolation of crosslinked complexes via protein and identification of crosslinked DNA by hybridisation with single-stranded DNA probes. The present study shows that transcribed chromatin of chicken embryonic erythrocyte beta globin gene is characterized by about 1.5-2.5-fold higher density of HMG 14/17 and 2-fold lower density of H1 and H5 as compared with non-transcribed chromatin of ovalbumin and lysozyme genes, whereas HMG 1/2, E proteins were equally distributed between DNA of both transcribed and non-transcribed genes. The depletion of H1/H5 in beta globin sequences was verified by the 'protein image' hybridisation technique (1). The DNase I hypersensitive site located 5' upstream from beta globin gene is deficient in all the proteins assayed, what implies a drastic disruption in the nucleosomal array. Minor quantitative changes of protein pattern suggest transient local perturbation of the chromatin on transcription.

An oligonucleotide hybridization approach to DNA sequencing.

We have proposed a DNA sequencing method based on hybridization of a DNA fragment to be sequenced with the complete set of fixed-length oligonucleotides (e.g., 4(8) = 65,536 possible 8-mers) immobilized individually as dots of a 2-D matrix [(1989) Dokl. Akad. Nauk SSSR 303, 1508-1511]. It was shown that the list of hybridizing octanucleotides is sufficient for the computer-assisted reconstruction of the structures for 80% of random-sequence fragments up to 200 bases long, based on the analysis of the octanucleotide overlapping. Here a refinement of the method and some experimental data are presented. We have performed hybridizations with oligonucleotides immobilized on a glass plate, and obtained their dissociation curves down to heptanucleotides. Other approaches, e.g., an additional hybridization of short oligonucleotides which continuously extend duplexes formed between the fragment and immobilized oligonucleotides, should considerably increase either the probability of unambiguous reconstruction, or the length of reconstructed sequences, or decrease the size of immobilized oligonucleotides.

Interaction of high mobility group proteins HMG 1 and HMG 2 with nucleosomes studied by gel electrophoresis.

The binding of isolated high mobility group proteins HMG (1 + 2) with nucleosomes was studied using gel electrophoresis. The interaction of HMG (1 + 2) with mononucleosomes could be detected as a new discrete electrophoretic band with a decreased mobility only after cross-linking of HMG (1 + 2)-nucleosome complex by formaldehyde. Approximately two molecules of the large HMG proteins were bound per nucleosomal particle of a DNA length of approximately 185 base pairs, lacking histones H1 and H5. Using the same techniques, no binding was observed with core particles of a DNA length of approximately 145 base pairs.

Primary organization of nucleosomes. Interaction of non-histone high mobility group proteins 14 and 17 with nucleosomes, as revealed by DNA-protein crosslinking and immunoaffinity isolation.

The binding sites for histones and high mobility group proteins (HMG) 14 and 17 have been located on DNA in the nucleosomal cores and H1/H5-containing nucleosomes. The nucleosomes were specifically associated with two molecules of the non-histone proteins HMG 14 and/or HMG 17 when followed by DNA-protein crosslinking and immunoaffinity isolation of the crosslinked HMG-DNA complexes. HMGs 14 and 17 were shown to be crosslinked in a similar manner to each core DNA strand at four sites: to both 3' and 5' DNA ends and also at distances of about 25 and 125 nucleotides from the 5' termini of the DNA. These sites are designated as HMG(143), (0), (25) and (125). The site HMG(125) is located at the place where no significant histone-DNA crosslinking was observed. The HMG(125) and HMG(25) sites lie opposite one another on the complementary DNA strands across the minor DNA groove and are placed, similarly to histones, on the inner side of the DNA superhelix in the nucleosome. The crosslinking of HMG 17 to the 3' ends of the DNA is much weaker than that of HMG 14. These data indicate that each of two molecules of HMG 14 and/or HMG 17 is bound to the double-stranded core DNA at two discrete sites: to the 3' and 5' ends of the DNA and at a distance of 20 to 25 base-pairs from each DNA terminus inside the nucleosome on a histone-free DNA region. Binding of HMG 14 or 17 does not induce any detectable rearrangement of histones on DNA and both HMGs seem to choose the same sites for attachment in nucleosomal cores and H1/H5-containing nucleosomes.

Primary organization of nucleosomal core particles is invariable in repressed and active nuclei from animal, plant and yeast cells.

A refined map for the linear arrangement of histones along DNA in nucleosomal core particles has been determined by DNA-protein crosslinking. On one strand of 145-bp core DNA, histones are aligned in the following order: (5') H2B25,35-H455,65-H375,85,95/H488-H2B105,11 5-H2A118-H3135,145/H2A145 (3') (the subscripts give approximate distance in nucleotides of the main histone contacts from the 5'-end). Hence, the histone tetramer (H3,H4)2 and two dimers (H2A-H2B) are arranged on double-stranded core DNA in a symmetrical and rather autonomous way: H2A/H3-(H2A-H2B)-(H3,H4)2-(H2B-H2A)-H3/H2A. The primary organization was found to be very similar in core particles isolated from repressed nuclei of sea urchin sperm and chicken erythrocytes, from active in replication and transcription nuclei of Drosophila embryos and yeast and from somatic cells of lily. These data show that (i) the core structure is highly conserved in evolution and (ii) the overall inactivation of chromatin does not affect the arrangement of histones along DNA and thus does not seem to be regulated on this level of the core structure.

Primary organization of nucleosome core particle of chromatin: sequence of histone arrangement along DNA.

A high-resolution map for the arrangement of histones along DNA in the nucleosome core particle has been determined by a sequencing procedure based on crosslinking histones to the 5'-terminal DNA fragments produced by scission of one DNA strand at the point of crosslinking. The position of histones on DNA has been identified by measuring the length of crosslinked DNA fragments. The results demonstrate that each of the histones is arranged within several adjacent or dispersed DNA segments of a little less than 10 nucleotides in length. Histone-free intervals are located between these segments at the regular distances of about (10)n nucleotides from the 5' end of the DNA and are likely to face one side of the DNA helix. Histones appear to be arranged in a similar manner on both DNA strands and do not form "locks" around DNA. A linearized model of the core particle is proposed.