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The single-crystal structure of a DNA Holliday junction assembled from four unique sequences shows a structure that conforms to the general features of models derived from similar constructs in solution. The structure is a compact stacked-X form junction with two sets of stacked B-DNA-type arms that coaxially stack to form semicontinuous duplexes interrupted only by the crossing of the junction. These semicontinuous helices are related by a right-handed rotation angle of 56.5 degrees, which is nearly identical to the 60 degree angle in the solution model but differs from the more shallow value of approximately 40 degrees for previous crystal structures of symmetric junctions that self-assemble from single identical inverted-repeat sequences. This supports the model in which the unique set of intramolecular interactions at the trinucleotide core of the crossing strands, which are not present in the current asymmetric junction, affects both the stability and geometry of the symmetric junctions. An unexpected result, however, is that a highly wobbled A.T base pair, which is ascribed here to a rare enol tautomer form of the thymine, was observed at the end of a CCCC/GGGG sequence within the stacked B-DNA arms of this 1.9 A resolution structure. We suggest that the junction itself is not responsible for this unusual conformation but served as a vehicle for the study of this CG-rich sequence as a B-DNA duplex, mimicking the form that would be present in a replication complex. The existence of this unusual base lends credence to and defines a sequence context for the "rare tautomer hypothesis" as a mechanism for inducing transition mutations during DNA replication.

A new method to detect mutations based on the serial analysis of gene expression (SAGE) technique, ligation-mediated (LM) PCR, and recombinant nuclease CEL I named LM-SAGE assay is reported in the present study. Mismatched DNA heteroduplexes formed from wild-type and mutant DNA are fragmented with CEL I nuclease at the mutant site to produce a double-strand fragment with an overhanging base at the 3'-end. The fragment is ligated to a linker, and digested with MmeI and then ligated to another linker. PCR is performed to amplify the ligation products, and NlaIII is used to release 17-bp tags containing mutation sites followed by purification, concatemerization, cloning, and sequencing. The locations of mutations can be identified from the homology analysis of tags. This new LM-SAGE assay can detect both known and unknown mutations with a sensitivity of 1:50 (mutant:wild-type DNA ratio) in 2.4 x 10(6) copies starting DNA sample. Our results show that this method could be used as a potentially high-throughput assay for mutation detection, particularly for the discovery of unknown mutations in genomic DNA.

Using optical microscopy, we have studied the phase behavior of mixtures of 12- to 22-bp-long nanoDNA oligomers. The mixtures are chosen such that only a fraction of the sample is composed of mutually complementary sequences, and hence the solutions are effectively mixtures of single-stranded and double-stranded (duplex) oligomers. When the concentrations are large enough, such mixtures phase-separate via the nucleation of duplex-rich liquid crystalline domains from an isotropic background rich in single strands. We find that the phase separation is approximately complete, thus corresponding to a spontaneous purification of duplexes from the single-strand oligos. We interpret this behavior as the combined result of the energy gain from the end-to-end stacking of duplexes and of depletion-type attractive interactions favoring the segregation of the more rigid duplexes from the flexible single strands. This form of spontaneous partitioning of complementary nDNA offers a route to purification of short duplex oligomers and, if in the presence of ligation, could provide a mode of positive feedback for the preferential synthesis of longer complementary oligomers, a mechanism of possible relevance in prebiotic environments.

Most metazoan genes are alternatively spliced, and a large number of alternatively spliced isoforms are likely to be functionally significant and expressed at specific stages of pathogenesis or differentiation. Splicing changes usually only affect a small portion of a gene, and these changes may cause significant mRNA degradation. After RT-PCR, minor variants can form heteroduplexes with the major variants. Affinity purification of these heteroduplexes using immobilized Thermus aquaticus single-stranded DNA-binding protein allows purification of alternative splice forms in a 1:1 ratio, which makes it easy to sequence the rare form. This chapter provides a detailed protocol of the technique I have developed to identify spliced isoforms called enrichment of alternatively spliced isoforms or EASI.

Uniform bimetallic nanowires, tunable in size, have been grown on artificial DNA templates via a two-step metallization process. Alkyne-modified cytosines were incorporated into 900-base-pair polymerase-chain-reaction fragments. The alkyne modifications serve as addressable metal-binding sites after conversion to a sugar triazole derivative via click chemistry. Reaction of the Tollens reagent with these sugar-coated DNA duplexes generates Ag0 metallization centers around the sugar modification sites of the DNA. After a subsequent enhancement step using gold, nanowires < or = 10 nm in diameter with a homogeneous surface profile were obtained. Furthermore, the advantage of this two-step procedure lies in the high selectivity of the process, due to the exact spatial control of modified DNA base incorporation and hence the confinement of metallization centers at addressable sites. Besides experiments on a membrane as a proof for the selectivity of the method, atomic force microscopy (AFM) studies of the wires produced on Si-SiO2 surfaces are discussed. Furthermore, we demonstrate time-dependent metallization experiments, monitored by AFM.

Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acids in complex samples according to strandness, conformation and length. Under the non-denaturing conditions of the first electrophoretic step, single-stranded DNA, double-stranded DNA and RNA.DNA hybrids of similar length migrate at different rates. The second electrophoretic step is performed under denaturing conditions (7 mol l(-1) urea, 55 degrees C) so that all the molecules are single-stranded and separate according to length only. 2D-SDE is useful for revealing important characteristics of complex nucleic acid samples in manipulations such as amplification, renaturation, cDNA synthesis and microarray hybridization. It can also be used to identify mispaired, nicked or damaged fragments in double-stranded DNA. The protocol takes approximately 2 h and requires only basic skills, equipment and reagents.

Efficient global scanning of single nucleotide variations in DNA sequences between related, complex DNA samples remains a challenge. In the present article we present an approach to this problem. We have used immobilized thymidine DNA glycosylases to capture and enrich DNA fragments containing internal mismatched base pairs and separate these fragments as a pool from perfectly base-paired fragments as another pool. Enrichments of up to several hundredfold were obtained with one cycle of treatment, and all of the four groups of single nucleotide mismatches were fully covered by combining use of two thymine DNA glycosylases generated here. We have used a heterohybrid-orientating strategy for selective amplification of duplexes with one strand derived from each of two input DNA samples, which can also be used for selective amplification of duplexes with both strands derived from one of two input samples when desired. By combining these methods, the single nucleotide variations either between two DNA pools or within one DNA pool can be obtained in one process. This approach has been applied to the total cDNA from a human cell line and has several potential applications in mapping genetic variations, particularly global scanning of cDNA single nucleotide variations or polymorphisms, and finally high-throughput mapping of complex genetic traits.

Fluorescence-assisted mismatch analysis (FAMA) methodology uses bifluorescent double-stranded DNA substrates to maximize the reliability and sensitivity of mutation-scanning procedures that rely on cleavage of mismatches using chemical. This unit presents a nested PCR procedure to fluorescently label each DNA strand, followed by chemical cleavage to detect the point mutations. Fluorescent end labeling with strand-specific fluorophores, electrophoretic separation of cleavage products in an automated Perkin-Elmer ABI 373 or 377 DNA sequencer and analysis using the Perkin-Elmer GENESCAN software expands sensitivity by highlighting weak signals through superimposition of strand-specific fluorescence electropherograms for different samples.

Mutation screening by the chemical-cleavage method is based on the fact that mismatched cytosine (C) and thymidine (T) are more reactive with the compounds hydroxylamine and osmium tetroxide, respectively, than are Watson and Crick-paired cytosine and thymidine bases. In this protocol, an excess of unlabeled target DNA is hybridized with labeled wild-type DNA probe and heteroduplexes are formed. One aliquot is treated with hydroxylamine, which reacts with mismatched C bases. Another aliquot is treated with osmium tetroxide, which reacts with mismatched T bases. The reactions are mixed with piperidine; the strands are then cleaved at the sites where hydroxylamine and osmium tetroxide react. Cleaved fragments are then electrophoresed and sized on polyacrylamide gels, identifying the point of cleavage (and hence the position of the mutation). Then only a small portion of the mutant gene needs to be sequenced to define a single change between two DNA sequences.

Deficiencies in DNA mismatch repair (MMR) have been found in hereditary colon cancers (hereditary non-polyposis colon cancer, HNPCC) as well as in sporadic cancers, illustrating the importance of MMR in maintaining genomic integrity. We have examined the interactions of specific mismatch repair proteins in human nuclear extracts. Western blot and co-immunoprecipitation studies indicate two complexes as follows: one consisting of hMSH2, hMSH6, hMLH1, and hPMS2 and the other consisting of hMSH2, hMSH6, hMLH1, and hPMS1. These interactions occur without the addition of ATP. Furthermore, the protein complexes specifically bind to mismatched DNA and not to a similar homoduplex oligonucleotide. The protein complex-DNA interactions occur primarily through hMSH6, although hMSH2 can also become cross-linked to the mismatched substrate when not participating in the MMR protein complex. In the presence of ATP the binding of hMSH6 to mismatched DNA is decreased. In addition, hMLH1, hPMS2, and hPMS1 no longer interact with each other or with the hMutSalpha complex (hMSH2 and hMSH6). However, the ability of hMLH1 to co-immunoprecipitate mismatched DNA increases in the presence of ATP. This interaction is dependent on the presence of the mismatch and does not appear to involve a direct binding of hMLH1 to the DNA.

GB virus C/hepatitis G virus (GBV-C/HGV) is a positive-sense RNA virus belonging to the Flaviviridae family identified recently. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect GBV-C/HGV RNA using nested primers designed to amplify 245 bp of the 5'-untranslated region (UTR). GBV-C/HGV RNA was detected in 20.7% of 101 HCV-RNA positive and 6.8% of 44 HCV-RNA negative specimens. Sequencing of the PCR products demonstrated they had between 84.3 and 100% nucleotide identity. Most of the diversity corresponded to two variable regions identified within the 5'-UTR. Phylogenetic analysis indicated that GBV-C/HGV subtypes present in Australia belonged to group 2 and were closest in evolutionary terms to isolates from the USA and Europe. All isolates were analysed using single-strand conformation polymorphism (SSCP) and heteroduplex mobility analysis (HMA) on 8% non-denaturing polyacrylamide gels. SSCP of the isolates identified a number of distinct conformation polymorphisms that corresponded with sequence-determined genetic diversity. HMA was developed to assess the amount of genetic diversity between isolates without the need for sequencing. The average difference between the predicted divergence of two isolates calculated from the mobility of the heteroduplex and the actual value (based on nucleotide sequence) was 2.3% in this sample of isolates, where the mean sequence divergence was 8.52%.

A specialized form of ion-pair reversed-phase high-performance liquid chromatography is gaining widespread application in mutation detection for single nucleotide polymorphisms (SNP). The technique relies on temperature-modulated heteroduplex analysis (TMHA) by chromatographic separation of partially denatured DNA heteroduplexes from homoduplexes. Here, we demonstrate that fluorescent labeling is compatible with mutation analysis by this form of DNA chromatography and offers advantages over the use of unlabeled DNA fragments. Uniform labeling of wild-type and mutant alleles for TMHA yields peak patterns identical to unlabeled fragments. However, fluorescent labels increase retention times but do not influence resolution of heteroduplexes from homoduplexes. They increase sensitivity and decrease the amount of DNA required for analysis; e.g., in the case presented here, one allele can be detected in the presence of a 500-fold excess of another allele. Furthermore, allele-specific wild-type probes, fluorescently labeled on one strand only, make it possible to selectively monitor specific homoduplexes and wild-type/mutant heteroduplexes. This, in combination with an internal homoduplex standard, greatly reduces the complexity of fluorescence chromatograms compared with chromatograms recorded in the UV. These simplified chromatograms, in which only the internal homoduplex standard and the labeled heteroduplex are detected in the presence of a mutation, greatly facilitate the detection and identification of mutant alleles., (Copyright 1999 Academic Press.)

Capillary affinity gel electrophoresis was applied to sequence-specific and base composition-specific recognition of oligodeoxynucleotides, utilizing the formation of heteroduplexes between a nucleic acid analogue immobilized into the capillary gel and soluble oligodeoxynucleotides with different sequences. Capillary affinity gel electrophoresis using capillaries filled with a conjugated gel of polyacrylamide and a synthetic nucleic acid analogue [poly(9-vinyladenine)] was effective for the selective separation of hexathymidylic acid from a mixture of four homopolymers of A6, C6, G6, and T6 and for the complete resolution of five heteropolymers of hexadeoxynucleotides (TAAAAA, TTAAAA, TTTAAA, TTTTAA, TTTTTA). We also demonstrated that capillary affinity gel electrophoresis was useful for the selective and the sensitive sequence-specific recognition of sequence isomers of DNA (TTTTAA, TTTTAAT, TTTATA, and TTTTAA).

A 37-year old AIDS patient presented with foreign body sensation. Microsporidia were detected in smears from a conjunctival swab and urine sediment stained with calcofluor and a modified trichrome blue stain and by indirect fluorescent-antibody staining with murine polyclonal antiserum raised against Encephalitozoon hellem. This antiserum cross-reacted with other Encephalitozoon species, so PCR was performed to amplify the microsporidian ribosomal DNA (rDNA) with pan-Encephalitozoon primers. The PCR DNA products from the urine and conjunctival clinical specimens, along with the tissue culture-derived microsporidian controls, were assayed by Southern analysis with oligonucleotide probes specific for Encephalitozoon cuniculi, E. hellem, and Encephalitozoon (Septata) intestinalis. The PCR product amplified from the urine specimen hybridized with the E. hellem probe only, while insufficient DNA was amplified from the conjunctiva specimen for detection by Southern analysis. For corroboration of the PCR-Southern analysis results, aliquots of the urine and conjunctiva specimens were seeded onto RK-13 cell monolayers. The rDNA extracts of the cultured microsporidia were amplified by PCR with pan-Encephalitozoon primers, and the PCR DNA products were subjected to digestion with restriction endonuclease FokI. The amplified rDNA of both the urine and conjunctiva isolates generated digestion patterns that were identified to the E. hellem PCR rDNA digestion pattern. In addition, double-stranded heteroduplex mobility shift analysis with these PCR products indicated that the urine and conjunctiva isolates were identical to each other and to E. hellem. The patient was treated with albendazole and topical fumagillin and responded rapidly, with no recurrence of ophthalmologic signs. The results of this study demonstrate that PCR-Southern analysis provides a basis for distinguishing E. cuniculi, E. hellem, and E. intestinalis in clinical specimens.

We report a method that allows accurate, absolute quantification of gene expression in a single reverse transcriptase (RT)-PCR reaction. This method makes use of novel high-performance liquid chromatography (HPLC) technology to resolve and quantify the products of competitive, mutant RNA PCRs. The HPLC technique allows rapid, high resolution of reaction products. On-line UV detection eliminates the need for radiolabel or other tracers. The HPLC technique also demonstrates that these competition reactions readily generate heteroduplex products. The ability of HPLC to resolve and quantify heteroduplex products is fundamental to the accuracy of the technique. Accurate measurements of gene expression have been obtained over four orders of magnitude and experiments employing predetermined quantities of specific native RNA input have demonstrated the ability of the system to provide absolute estimates of gene expression. Large size differences between native and mutant RNA inputs affected reverse transcriptase (RT) efficiency, but not PCR amplification efficiency. However, the magnitude of the RT efficiency effect can be estimated, is reproducible, and can therefore be adjusted by a calculated correction factor. The RT efficiency difference can been eliminated by reduction in the magnitude of the sequence difference between native and mutant RNA so that no correction factor is required. The application of the technique to quantification of expression of the alpha 1 subunit of sodium, potassium-ATPase in microdissected nephron segments is demonstrated.

We examine the association of transcription factor TFIIIA with RNA-DNA heteroduplexes containing sequences from the Xenopus borealis 5 S rRNA gene. Under conditions where TFIIIA selectively binds to 5 S rRNA or the internal control region of the 5 S rRNA gene, no specific association of TFIIIA with DNA-RNA heteroduplexes containing either strand of 5 S DNA could be detected. We discuss our results with respect to specific models of TFIIIA recognition of the internal control region and of DNA-RNA hybrids by zinc finger proteins.

Familial defective apolipoprotein B-100 (FDB) is a dominantly-inherited genetic disorder causing primary hypercholesterolemia and premature coronary heart disease. To date, only two mutations causing FDB have been identified. A rapid non-radioactive technique is described to detect both disease-related apolipoprotein B point mutations in polymerase chain reaction (PCR) products amplified from genomic DNA. Heteroduplex formation between different alleles from FDB heterozygotes was shown to be visible directly after electrophoresis of PCR products and staining in low cross-linking polyacrylamide gels. We found that the simplicity of the method, in addition to its potential to identify new mutations in the amplified PCR product, makes heteroduplex detection the preferred initial method of screening potential heterozygotes.

Objective: To identify HIV-1 envelope sequence subtypes in infected individuals from the Russian Federation and Belarus., Patients: A cohort of children infected after exposure to non-sterile needles during the 1988-1989 HIV-1 epidemic in southern Russia (n = 20) and HIV-1-seropositive individuals from Russia (n = 1) and Belarus (n = 7) infected via sexual transmission., Methods: DNA samples derived from peripheral blood mononuclear cells were analysed for their HIV-1 genotypes by the heteroduplex mobility assay (HMA). The 1.3 kilobase-pair env gene fragments encoding a portion of gp120 were amplified by nested polymerase chain reaction, cloned and sequenced. The env sequences derived from these patients were aligned and phylogenetic neighbour-joining and maximum parsimony-derived trees generated., Results: The env sequences derived from eight individuals infected in Russia and Belarus belong to subtype A (one), B (four), C (two), and D (one). Sequences derived from children, infected during parenteral manipulations in southern Russia, and one mother were closely related, but highly divergent, as a group, from all prototypic strains (genetic divergence, 17.2-22.9%). However, they clustered together with env sequences of the V1525 and LBV21-7 isolates from Gabon, recently described to be members of a new HIV-1 env subtype G., Conclusion: Extensive heterogeneity of HIV-1 subtypes was evident in the Russian Federation and Belarus. Our data also support the existence of an HIV-1 env genetic subtype G, and such isolates are now apparently present on both the African and European continents. These variants were identified through V3 peptide enzyme-linked immunosorbent assay screening and subsequent HMA analysis. The combination of these techniques represents a model for screening HIV variants within a large population.

To assist in the preparation for the testing of vaccines against human immunodeficiency virus (HIV) we, as part of the World Health Organization Network for HIV Isolation and Characterization (WHO-NHIC), evaluated the genotypic variation of HIV-1 in cohorts from Brazil, Rwanda, Thailand, and Uganda. Here we report the results from a pilot study of 65 HIV-1-infected individuals. In all cases in which viral envelope gene fragments could be amplified by polymerase chain reaction, subtypes could be assigned using a heteroduplex mobility assay (HMA)1 by comparison with HIV-1 strains representing six HIV-1 envelope subtypes. All subtype classifications matched those found by envelope gene sequencing. Phylogenetic relationships were further clarified by heteroduplex formation between samples within each subtype. A relatively homogeneous subtype E virus population predominated over subtype B viruses in the sample set from Thailand. Viruses from the other countries were also limited to one or two subtypes but were more divergent within each subtype. All samples from Rwanda (13/13) and some from Uganda (3/16) were of subtype A; all Brazilian samples were of subtype B, except for one belonging to subtype C; most samples from Uganda (13/16) clustered with the subtype D. Analysis by HMA is therefore applicable for screening of HIV-1 genotypes in countries under consideration for large-scale vaccine trials. It should be generally useful when samples containing at least one variable genetic locus need to be rapidly classified by genotype and/or analyzed for epidemiological clustering.

Uncrosslinked polyacrylamide forms gels in the concentration range of 15-40% acrylamide. Electrophoresis in these gels of a commercially available 350 bp heteroduplex DNA preparation separates it from the homoduplex DNA of the same size. The separation is qualitatively equivalent to that previously achieved in a commercial proprietary gel ("Mutation Detection Gel" of AT-Biochem), or in an equivalent 14% T, 0.15% C (N,N'-methylenebisacrylamide) gel, but the mechanical stability of mutation detection electrophoresis (MDE) gels or 0.15% C gels is better than that of uncrosslinked polyacrylamide gels. The separation in any of these three gel media can be carried out in short gel tubes within a few hours of electrophoresis time. In both uncrosslinked polyacrylamide and MDE gel media, the Ferguson plots [log(mobility) vs. gel concentration] and the plots of effective molecular radius vs. gel concentration ("T-plots") of both the heteroduplex and homoduplex DNA indicate an augmented size but similar flexibility upon passage through the gel than exhibited by the components of a DNA standard ladder. Homoduplex and heteroduplex DNA correspondingly exhibit a parallelism of their Ferguson curves in transverse MDE pore gradient gel electrophoresis, suggesting a surface net charge difference, possibly due to a conformational reorientation too subtle to be detected by a shift in the slope of the Ferguson plot, as has been observed once previously with a "kinked" DNA species. The gel fiber radius or length per unit volume of uncrosslinked polyacrylamide and MDE gels do not differ significantly within confidence limits, which are wide compared to unconventionally crosslinked gels, presumably because of their greater swelling.

We have investigated DNA interactions between homologs and between sister chromatids during meiosis in S. cerevisiae. We have detected a DNA species containing information from both parental chromosomes at a specific hotspot for meiotic recombination and double strand breaks (DSBs). These joint molecules are a prominent feature of meiotic prophase. They appear to be a major intermediate stage in DSB-promoted recombination, because they occur with appropriate timing and require known recombination functions. Other possibilities cannot be completely dismissed, however. Most or all joint molecules contain two full-length nonrecombinant strands from each parental duplex and thus do not consist of single Holliday junctions. Joint molecules form between sister chromatids at approximately 10% the interhomolog level. Also, joint molecule formation is aberrant in a mutant defective in the HOP1 gene, which encodes a meiotic chromosome structure component. General models for discrimination between homologs and sisters during meiosis are discussed.

Murine monoclonal antibodies (MAbs) specific for DNA-RNA hybrids were successfully produced with two different heteropolymers as antigens, cDNA-mRNA and phi X174 DNA-RNA heteroduplexes. The former was simpler to prepare. Both had shown similar immunogenicities. Two different immunoglobulin M MAbs were isolated. The 20D3 MAb, generated with the phi X174 DNA-RNA hybrid, showed association constants of 1.05 x 10(12), 2.12 x 10(10), and 1.68 x 10(7) for the antigens phi X174 DNA-RNA, cDNA-mRNA, and poly(rA)-poly(dT), respectively. The 6B5 MAb, obtained with the cDNA-mRNA hybrid, showed association constants of 1.59 x 10(5), 5 x 10(12), and 7.1 x 10(8) for the above-described antigens, respectively. With the 20D3 MAb, an immunoassay was developed for the detection of Listeria DNA-RNA hybrids. In brief, a biotinylated rRNA gene probe specific for the genus Listeria was hybridized with rRNA in the solution phase. The hybrids thus formed were then captured in microtiter plate wells precoated with the purified 20D3 MAb, and the probe-target hybrids were detected with a streptavidin-alkaline phosphatase conjugate. This assay was shown to be specific for the genus Listeria and highly sensitive, allowing the detection of as little as 2.5 pg of target rRNA.

Heteroduplex detection by hydrolink gel electrophoresis was performed to screen for small mutations in 12 Lesch-Nyhan syndrome families with characterised molecular pathology which included nine point mutations, two small deletions, and a 1-bp insertion. This modified protocol for heteroduplex detection by hydrolink gel electrophoresis detected all 12 of these mutations and was utilised to rapidly determine the carrier status of females from affected families. On the basis of these results this approach appears to be a rapid and reliable screening method for point mutations in addition to small length mutations and for carrier detection in Lesch-Nyhan syndrome.

Immunopurification and characterization of dysfunctional factor VIII-like molecules in CRM-positive and CRM-reduced hemophilia A permit correlation of structural changes with molecular defects. The technique described here is sufficiently sensitive to characterize the molecular mass and enzymatic fragments of the factor VIII chains in patients with as little VIII: Ag as 0.05 units/ml. Specific abnormalities have been identified in 5 of the first 24 samples tested. In each case, the mutation responsible for factor VIII dysfunction has been determined by sequencing a part of the abnormal gene. Mutations have been identified that abolish critical thrombin cleavage sites or which generate new N-glycosylation sites. The technique provides a useful approach to the study factor VIII structure-function relationships, and it has the potential to clarify further the molecular basis of factor VIII procoagulant activity.

Structural isoforms of the insulin receptor that occur in various tissues have been postulated to be involved in certain actions of insulin in target cells. To determine whether these insulin-receptor subtypes are caused by alterations in the receptor primary structure, we used RNA heteroduplex mapping and amplification of cDNA to detect variation in the coding region of insulin-receptor mRNA from 5 rat tissues. A complete series of overlapping antisense [32P]RNA probes was prepared from plasmids containing segments of a full-length rat insulin-receptor cDNA, and probes were hybridized individually in solution with polyadenylated RNA from rat brain, kidney, liver, skeletal muscle, and spleen. After ribonuclease digestion, probe fragments were analyzed by denaturing gel electrophoresis. Tissue-specific cleavage of the mRNA:RNA probe heteroduplex, attributable to sequence mismatch, was detected only for a single probe covering the distal alpha-subunit, as expected for the known alternative splicing of rat insulin-receptor mRNA in this region. No evidence for additional heterogeneity of the receptor mRNA coding region was observed in the 5 tissues studied either by RNA heteroduplex mapping or, in some areas, by regional amplification of insulin-receptor cDNA. Cell-free translation of size-fractionated polyadenylated RNA was used to further demonstrate that each of the major insulin-receptor mRNA size classes in rat liver contained both forms of the alternatively spliced mRNA transcripts and produced two insulin-proreceptor polypeptides. These results suggest that heterogeneity of the insulin-receptor mRNA coding region affecting the receptor primary structure is limited to the distal alpha-subunit near the subunit cleavage site.(ABSTRACT TRUNCATED AT 250 WORDS)

A method is presented that allows simultaneous analysis of the effects of all possible point mutations within a specific mutation window of at least 50 base pairs on a DNA fragment that codes for a selectable function. It relies on the detection of mismatched base pairs with hydroxylamine and osmium tetroxide. A mutant plasmid library of randomly distributed point mutations within the lacZ' gene of Escherichia coli was selected for functional alpha-complementation by growth on lactose. The DNA fragments of the selected and unselected library were each heat denatured and again renatured, thereby generating a randomly distributed set of all possible mismatches within the mutagenesis window. Cytidine-containing mismatches were then detected with hydroxylamine, and thymidine-containing mismatches were detected with osmium tetroxide. When this procedure was performed for both DNA strands, all mismatches could be detected. A comparison of the results of the unselected and selected library leads to an estimation of the effects of each detectable mutation on alpha-complementation in vivo. This method, called "mutant profiling," should be applicable to all selectable genetic elements.

We have characterized three temperate bacteriophages of pneumococcus (HB-3, HB-623, and HB-746). Although all the phages belong to the same family, the polypeptide composition of the virions and the DNA restriction endonuclease analysis of their DNAs revealed differences among the three phages. The genomes of these bacteriophages have been isolated as DNA-protein complexes. The protein is specifically associated with the two 5' termini of the DNA as shown by experiments carried out with exonucleases. The protein bound to the DNA in the three phages studied, iodinated in vitro with 125I, has a molecular weight of 23,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of the complexes with chaotropic agents suggested that the protein is covalently bound to the 5' termini of the DNA. Comparative pulsed-field gel electrophoresis analysis and Southern hybridization of the SmaI restriction fragments of DNAs from one lysogenic bacteria and its parental strain revealed that the prophage genome was integrated in the host chromosome.

The ornithine decarboxylase (ODC)-deficient Chinese hamster ovary (CHO) cell line C55.7 has normal amounts of ODC mRNA with very low amounts of immunologically detectable ODC protein, suggesting a structural mutation; however, 5-azacytidine treatment leads to phenotypical reversion (Steglich, C., and Scheffler, I. E. (1985) Somat. Cell Mol. Genet. 11, 11-23). We have demonstrated by chemical cleavage a single base mismatch in DNA heteroduplexes composed of wild-type and mutant cDNA strands. DNA sequencing showed that the mutant phenotype results from an aspartate-glycine substitution at amino acid 381 of the protein. When 5-azacytidine-revertant cell lines were selected for resistance to alpha-difluoromethylornithine, the resulting amplified ODC gene was structurally indistinguishable from the wild type gene. These results suggested the existence of a single active ODC locus in CHO cells. Using the methylation-sensitive restriction endonucleases AvaI and HpaII, we found evidence for two differentially methylated alleles in wild type, ODC-deficient and alpha-difluoromethylornithine-resistant cells. One of the alleles appeared completely inactivated by hypermethylation but could be reactivated by demethylation in spontaneous or 5-azacytidine-induced revertants.

Amino acid residues of the carboxyl-terminal region of kanamycin nucleotidyltransferase were modified using segment-directed mutagenesis. Six different mutant enzymes with single amino acid replacements were selected out of 59 clones by DNA sequence analyses. The mutant enzymes were purified and it was found that the thermostability of one mutant enzyme was identical to the wild type, whereas the other five were less thermostable at varying degrees. The data suggested that changes in the enzyme thermostability depend not only on the position but also on the species of amino acid residue replaced.

Heteroduplex DNA molecules were generated in which one of the two strands contained a 7-base-pair (double-stranded) stem and 3-base-pair (single-stranded) loop. The heteroduplexes and their corresponding homoduplex parental molecules, each of approximately 260 base pairs, were used for nucleosomes reconstitution. Protection from restriction endonuclease digestion was used to probe the structure of the resulting dinucleosomes. Although 50% or more of the potential cleavage sites in the homoduplex DNAs and in the linear portion of the heteroduplex DNAs were inaccessible to nuclease digestion, no protection of the stem-loop structures was observed. The results imply that a stem-loop structure preferentially occupies the spacer region between nucleosomes, but if it is part of a core particle the stem-loop is always pointed "outward" in such a way as to be accessible to nuclease digestion.

The human p53 gene was cloned and characterized by using a battery of p53 DNA clones. A series of human cDNA clones of various sizes and relative localizations to the mRNA molecule were isolated by using the human p53-H14 (2.35-kilobase) cDNA probe which we previously cloned. One such isolate, clone p53-H7 (2.65 kilobases), spans the entire human mature p53 mRNA molecule. Construction of the human cDNA clones in the pSP65 RNA transcription vector facilitated the generation of p53 transcripts by the SP6 bacteriophage RNA polymerase. The p53-specific RNA transcripts obtained without further processing were translated into p53 proteins in a cell-free system. By using this rapid in vitro transcription-translation assay, we found that whereas clone p53-H7 (2.65 kilobases) coded for a mature-sized p53 protein, a shorter cDNA clone, p53-H13 (1.8 kilobases), dictated the synthesis of a smaller-sized p53 protein (45 kilodaltons). The p53 proteins synthesized in vitro immunoprecipitated efficiently with human-specific anti-p53 antibodies. Genomic analysis of human DNA revealed the presence of a single p53 gene residing within two EcoRI fragments. Heteroduplex analysis between the full-length cDNA clone p53-H7 and the cloned p53 gene indicated the presence of seven major exons.

Some properties of bacteriophage phi T isolated from the vaccine strain Bordetella pertussis Tohama phase I and propagated in Bordetella parapertussis 504 cells are presented. Phage phi T belongs to the IV group in accordance with Tikhonenko classification. The diameter of head and length of noncontractile tail sheath are 49.5 +/- 0.5 and 145 +/- 7 nm, respectively. Diameter of the tail sheath is 3.2 +/- 0.6 nm. Molecular mass of the phage DNA is 37 +/- 3 kb. Population of phi T phage is polymorphous and consists of particles the genomes or which vary from each other by the "insert" located 6.8 +/- 0.6 kb from the end of molecule. The blot hybridization has demonstrated that the bacteriophage genome is not inserted into the chromosome of the lysogenic strain. Autonomous location of the phage genome in the host cell is suggested. The temperature and hydrogen ions concentration effects on bacteriophage phi T stability were studied. The conditions for phage suspension storage are described.

Certain single base substitutions causing genetic diseases or resulting in polymorphisms linked to mutant alleles, alter a restriction enzyme cleavage site and can therefore be detected in total genomic DNA using DNA blots. Many base substitutions do not lead to an altered restriction site, but these can be detected using synthetic oligonucleotides as hybridization probes if the DNA sequence surrounding the base substitution is known. In the case of beta-thalassaemia, where 22 different single base mutations have been identified, a large number of probes would be required for diagnosis. An approach which was used to detect mutations in viral DNA involves the S1 nuclease treatment of heteroduplexes formed between wild-type and mutant DNA. Although certain single base mismatches are cleaved by S1 nuclease (ref. 11 and T. Shenk, personal communication), many other mismatches examined by this procedure are not cleaved (B. Seed, personal communication; R.M.M., unpublished data). Heteroduplexes between mutant and wild-type subgenomic fragments of double-stranded reovirus RNA migrate slower than the corresponding homoduplexes in polyacrylamide gels containing 7 M urea, but it is not known whether this method is applicable to DNA heteroduplexes containing single base mismatches. Here we describe a procedure that involves the electrophoretic separation of DNA heteroduplexes in a well-characterized gel system. We show that four different human beta-thalassaemia alleles with known single base mutations can be detected with as little as 5 micrograms of total genomic DNA. The method should be useful in the localization and diagnosis of mutations associated with genetic diseases.

Double-stranded segments (c-ds) have been studied in the poly(A)+ cytoplasmic rat liver RNA. Duplexes about 40 base pairs long have been shown to be of intermolecular character and originate from the interaction between ss-RNA and complementary regions of the poly(A)-containing RNA molecules. Shorter ds-sequences are, mainly, of intramolecular nature. Double-stranded sequences of different length differ also in their oligonucleotide composition, according to fingerprint analysis data. Under the action of cortisone, only several kinds of double-stranded sequences have been demonstrated to increase in the population of cytoplasmic poly(A)+RNA. The function of ds-regions in the hormonal regulation of gene expression is suggested.

NMR studies were carried out on various equimolar mixtures consisting of a combination of oligomers: d(ACGGCT) (I). d(pACGGCT) (Ia), d(TGCAGT) (II), d(AGCCGTACTGCA) (III), d(TGCAGTACGGCT) (IV). It is shown that I + II + III (MI) and Ia + II + III (M2) form stable duplexes with nicks in the centre of the respective double helices. A close analysis of the NOESY experiments of M1 and M2 revealed that these fragments form B-DNA type duplex structures. A comparison of the chemical-shift data of the nicked duplexes with those of the intact duplex of III + IV (M3) demonstrated that only small local distortions occur when a nick is introduced. The chemical-shift profiles of M1 and M3 were used to obtain the thermodynamic data for the duplex/coil transitions. The profiles of M1 were analysed by means of a new thermodynamic model (TRIDUP). From the calculated thermodynamic data of M1 and M3 it is concluded that the melting behaviour of M1 occurs cooperatively. A ligation experiment demonstrated that the relatively small substrate (M2) was almost completely joined after an overnight incubation at 14 degrees C.

Understanding the nature of DNA sequence differences among individuals is important to the understanding of fundamental questions in biology. To analyze such differences in complex genomes new approaches must be developed. We report two new techniques which aid in this effort. First, we have developed a modification of the Phenol Emulsion Reassociation Technique (PERT) that allows hybridization of long (20 kb and longer) single copy heteroduplex DNA fragments from human genomic DNAs. Secondly, by using a differential methylase protection technique we have shown that double methylase resistant heteroduplex DNA molecules can be size fractionated away from reannealed single methylase resistant homoduplex DNA molecules. These methods will be useful in obtaining DNA from chromosomal subregions linked to the inheritance of a specific trait or condition as described in the preceding paper and could also be used to create a map of the chromosomal subregion which includes the gene for the trait.

We propose, and test using a Monte-Carlo analysis (a computer-based numerical analysis using a random number generator), a novel and efficient method to obtain sets of DNA markers linked to any inherited genetic locus. The method consists of a targeted search that is based on the common inheritance among members of an outbred pedigree, of discrete chromosome lengths, which we call inheritance units, to obtain DNA markers linked to the locus. In cases where two individuals inherit the same trait through two different lines of descent from a common ancestor, the set of inheritance units in each of the two genomes includes an inheritance unit that is identical in both individuals for a substantial distance on both sides of the DNA sequence which confers the trait. The power of the technique derives from the genetic selection that reduces the size and number of the inheritance units as the generational distance between the two individuals being compared increases.