Single-stranded Fragments Research Articles

Gene probes and their use in microbiology

A gene probe (nucleic acid probe) is a single-stranded nucleic acid fragment that interacts with a complementary sequence of a target nucleic acid. The test is based upon the principles of nucleic acid hybridization reactions. Different assay formats (dot-blot, colony, whole-cell hybridizations) can be applied. Gene probes can be used for the rapid and specific identification of microorganisms. The phylogenetic identification and in situ detection of uncultured bacteria will be discussed.

In Situ Polymerase Chain Reaction Detection of Viral DNA, Single-Copy Genes, and Gene Rearrangements in Cell Suspensions and Cytospins

The study of low-copy viral or genomic DNA sequences by in situ hybridization (ISH) is often limited by sensitivity. Using the polymerase chain reaction (PCR) for the amplification of target DNA sequences in fixed cells [in situ PCR] (ISPCR) before ISH, we have been able to greatly improve the sensitivity of ISH. Viral DNA present in low copy number, single-copy genes, as well as immunoglobulin gene rearrangements (VH3 family genes), were successfully amplified in cells in suspension or on glass slides (cytospins). Single primer pairs were used in the in situ amplification step and 35S- or digoxigenin-11-dUTP-labeled region specific oligonucleotide probes were used for detection of amplificants by ISH. Artifacts, presumably resulting from leakage of in situ amplificants out of cells, may be a significant problem in selected instances. By optimal fixation and permeabilization of cells, limiting PCR cycle number, amplification of long DNA sequences, and/or incorporation of biotinylated dNTPs to produce bulkier amplificants together with washing of cells after ISPCR, diffusion artifacts were significantly reduced. Probe hybridization to single-stranded long PCR fragments or messenger RNA were excluded as a source for false-positive ISPCR results. The techniques reported dramatically increase the sensitivity of ISH in the detection of low-copy viral infection as well as in the study of gene rearrangements, and provide unique opportunities to study chromosomal translocations and point mutations at the cellular level.

The nuclear 42-kDa phosphoprotein preferentially binds promoter-containing single-stranded DNA

The DNA-binding activity of a 42-kDa phosphoprotein from salivary gland cells and cultured epithelial cells of Chironomus tentans have been analyzed by the Southwestern technique. Both the salivary gland and the epithelial cell 42-kDa polypeptides were found to be single-stranded DNA-binding proteins. They bind to single-stranded promoter-containing restriction fragments including sequences from −204 to +74 from the ecdysterone controlled I-18C gene as well as sequences including the joint histone H2A H2B promoters in a sequence selective manner. By contrast, the 42-kDa polypeptides show no significant binding to intragenic restriction fragments from +71 to +351 from the I-18C gene. Previous and present data taken together suggest that the 42-kDa protein has a general role in the regulation of protein coding genes.

Presence of mitochondrial D-loop DNA in scrapie-infected brain preparations enriched for the prion protein

The prion preparation has, in recent years, been the focal point of scrapie research. The inability to identify agent-specific nucleic acids in this sample has led to the formulation of the infectious protein or prion hypothesis. In this study, we analyzed three different prion protein-enriched preparations and found all to contain significant amounts of mitochondrial nucleic acid. Southern blot analyses indicated that they are enriched for a specific component of the mitochondrial genome, the single-stranded displacement loop fragment. Our results suggest that if mitochondrial nucleic acids are involved in scrapie infection, it is the displacement loop fragment that is specifically responsible.

Escherichia coli RecQ protein is a DNA helicase.

The Escherichia coli recQ gene, a member of the RecF recombination gene family, was set in an overexpression plasmid, and its product was purified to near-homogeneity. The purified RecQ protein exhibited a DNA-dependent ATPase and a helicase activity. Without DNA, no ATPase activity was detected. The capacity as ATPase cofactor varied with the type of DNA in the following order: circular single strand greater than linear single strand much greater than circular or linear duplex. As a helicase, RecQ protein displaced an annealed 71-base or 143-base single-stranded fragment from circular or linear phage M13 DNA, and the direction of unwinding seemed to be 3'----5' with respect to the DNA single strand to which the enzyme supposedly bound. Furthermore, the protein could unwind 143-base-pair blunt-ended duplex DNA at a higher enzyme concentration. It is concluded that RecQ protein is a previously unreported helicase, which might possibly serve to generate single-stranded tails for a strand transfer reaction in the process of recombination.

Affinity chromatography of DNA fragments and P-modified oligonucleotides

The wide possibilities for use of affinity chromatography are demonstrated by two examples: (i) isolation of a single-stranded fragment of the tick-borne encephalitis virus DNA (302-mer) and an oligonucleotide (34 bases) from reaction mixtures and (ii) fractionation of mixtures of diastereoisomers of octathymidylates with modified internucleotide phosphates. All affinity sorbents are constructed by the covalent attachment of the oligonucleotides to solid supports and can be used repeatedly.

The basis of high resolution separation of small DNAs by asymmetric-voltage field inversion electrophoresis and its application to DNA sequencing gels

We have previously shown that asymmetric-voltage field inversion electrophoresis produces more uniform separation for fragments between 1 and 50 kilobases (kb) than other modes of pulsed field gel electrophoresis. We now report on the basis of this phenomenon. As in conventional electrophoresis, the pulsed field mobility of DNAs between 1 and 50 kb varies with voltage in a size dependent manner. The complex migration pattern obtained with asymmetric-voltage field inversion electrophoresis reflects the difference between the mobilities of each sized fragment under the conditions used for the forward and reverse fields. We have applied this technique to DNA sequencing gels and find improvement in resolution for single-stranded fragments in polyacrylamide gels.

Fine mapping of DNA single-stranded regions using base-specific chemical probes: study of an open complex formed between RNA polymerase and the lac UV5 promoter.

We have used diethyl pyrocarbonate (DEP), which carbethoxylates adenine bases, and dimethyl sulfate (DMS), which methylates guanine residues and single-stranded cytosines, to probe bases in open complexes between RNA polymerase and the lac UV5 promoter in vitro. We compared the kinetics of reactivity between bases in an open complex and those in a single-stranded 35-mer fragment corresponding to the lower template strand of lac UV5 in the region -25 to +10 relative to the transcription start site. We observed that cytosine and adenine residues in the 35-mer fragment reacted according to a second-order process with DMS and DEP, respectively, at sufficiently low concentrations of the reagents and that the degree of reactivity was base position independent. In an open complex in the absence of substrates, we observed reactivity with DEP in adenines from -12 to +4 as well as +21 on the template strand and methylation by dimethyl sulfate of cytosines -6, -4, -2, and -1. No hyperreactivity was observed on the nontemplate strand. The degree of reactivity of bases between -12 and +4 was position dependent, maximum reactivity being displayed by bases in the middle of the region. The reaction was first order within the range of reagent concentration investigated. It was confirmed that in the presence of ApA and UTP cytosine +5, as well as cytosines -6, -4, -2, and -1, in an open complex became reactive to DMS. With regard to DEP the extent of reactivity of the adenine at position +3 was increased markedly, adenine +4 was brought into the single-stranded region, and the overall reactivity of adenine -10 decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence-targeted Cleavage of single- and Double-stranded DNA by Oligothymidylates Covalently Linked to 1,10-Phenanthroline

The nuclease activity of 1,10-phenanthroline copper ion was targeted to a specific sequence by attachment of the ligand to the 5' or 3' end of octathymidylates. An acridine derivative was also attached to the other end of the oligothymidylate-phenanthroline conjugate. The duplex formed by the oligothymidylate with its complementary sequence was stabilized by intercalation of the acridine derivative. The reaction induced by 3-mercaptopropionic acid led to a very localized cleavage of a 27-nucleotide-long DNA fragment containing a (dA)8 sequence. At high NaCl concentration or in the presence of spermine, cleavage of the single-stranded 27-mer fragment occurred on both sides of the target sequence. This was ascribed to the formation of a triple helix involving two 1,10-phenanthroline-octathymidylate strands that adopt an antiparallel orientation with respect to each other. When a 27-mer duplex was used as a substrate, cleavage sites were observed on both strands. The location of the cleavage sites led us to conclude that the octathymidylate was bound to the (dA)8.(dT)8 sequence in a parallel orientation with respect to the (dA)8-containing strand. This result reflects the ability of thymine to form two hydrogen bonds with an adenine already engaged in a Watson-Crick base pair. This study shows that it is possible to design DNA-binding oligodeoxynucleotides that could selectively recognize and cleave polypurine-polypyrimidine sequences in double-stranded DNA.

Expression of mRNA of beta 1- and beta 2-adrenergic receptors in Xenopus oocytes results from structurally distinct receptor mRNAs.

Poly(A)+-selected RNA prepared from cells or tissues that express a homogeneous population of either beta 1- or beta 2-adrenergic receptors was isolated and then microinjected into Xenopus laevis oocytes. Following microinjection, the expression of beta-adrenergic receptors was assessed by equilibrium radioligand binding analysis using the antagonist ligand [3H]dihydroalprenolol. The pharmacology of the newly- expressed beta-adrenergic receptors in oocyte membranes was the same as that of the original tissue used as a source of RNA. Hybridization of nick-translated cDNA of hamster beta 2-adrenergic receptor to poly(A)+-selected RNA from tissues containing beta 2-adrenergic receptors was to a mRNA species of 2.2 kilobases. In contrast, hybridization of the cDNA probe to poly(A)+-selected RNA from tissues containing beta 1-adrenergic receptors was to a mRNA species of 2.0 kilobases. A single-stranded fragment of hamster beta 2-adrenergic receptor cDNA corresponding to nucleotides 730-886 was isolated and uniformly radiolabeled. This region of the gene is predicted to encode for the entire second exofacial loop (L4-5), the entire fifth transmembrane-spanning region, and the first 5 amino acid residues of the third cytoplasmic loop (L5-6) of the beta 2-adrenergic receptor. Hybridization at 48 and 56 degrees C of poly(A)+-selected RNA prepared from sources that express either beta 1 or beta 2-adrenergic receptors to the antisense orientation strand of this region of the beta 2-adrenergic receptor cDNA was followed by S1 endonuclease digestion of nonhybridized sequences. At 48 degrees C, S1-resistant hybrids from both sources of RNA protected the probe from S1 endonuclease digestion. At 56 degrees C, however, only the RNA prepared from the source of beta 2-adrenergic receptors protected the probe from S1 endonuclease digestion. These results demonstrate that the mRNAs encoding for the structurally homologous beta 1- and beta 2-adrenergic receptors are distinct in the pharmacological specificity of their translation products and in their size and structure.

Site-directed protection of RNA during nuclease digestion

We report the use of oligodeoxynucleotides to block the nucleolytic hydrolysis of single-stranded regions of RNA. Using complementary oligomers, the hydrolysis of the CCA terminus of methionine initiator tRNA could be prevented. This method can be useful in the production of specific single-stranded fragments of RNA, which are necessary in recombinant RNA technology.

Effect of SSB protein on cleavage of single-stranded DNA by phi X gene A protein and A* protein.

Gene A protein of bacteriophage phi X174 plays a role as a site-specific endonuclease in the initiation and termination of phi X rolling circle DNA replication. To clarify the sequence requirements of this protein we have studied the cleavage of single-stranded restriction fragments from phi X and G4 viral DNAs using purified gene A protein. The results show that in both viral DNAs cleavage occurs at the origin and at one additional site which shows striking sequence homology with the origin region. During rolling circle replication the single-stranded viral DNA tail is covered with single-stranded DNA binding (SSB) protein. Therefore, we have also studied the effect of SSB on phi X gene A protein cleavage. In these conditions only single-stranded fragments containing the complete or almost complete origin region of 30 bases are cleaved, whereas cleavage at the additional sites of phi X or G4 viral DNAs does not occur. A model for termination of rolling circle replication which is based on these findings is presented. Finally, we present evidence that the second product of gene A, the A* protein, cleaves phi X viral DNA at the additional cleavage site in the presence of SSB, not only in vitro but also in vivo. The functional significance of this cleavage in vivo is discussed.

Presence of random single-strand gaps in mycobacteriophage 13 DNA

The genomic double-stranded DNA of mycobacteriophage 13, when denatured with alkali, heat, formamide or dimethylsulfoxide, breaks down to heterogeneous-sized single-strand (ss) fragments smaller than the expected intact unit genome length suggesting the presence of random ss interruptions on both the strands. The occurrence of the interruptions at random is also demonstrated by two-dimensional gel electrophoresis of the restriction fragments of 13 DNA. These interruptions have no adverse effect on the phage infectivity or DNA transfectivity. Studies with nuclease BAL 31 and end-labeling analysis confirm the presence of random interruptions. Detailed analysis using T4 DNA ligase, nuclease S1 and DNA polymerase I Klenow fragment revealed that the interruptions are in the form of small gaps rather than single phosphodiester bond breaks. The average length of the gap is about 10 nucleotides long and there are 13 to 14 such gaps per DNA molecule.

Separation of single-stranded oligonucleotides on polyvinyl alcohol gel column

Separation mechanisms for single-stranded oligodeoxyribo-or oligoribonucleic acid fragments were explored on an Asahipak polyvinyl alcohol gel column (GS-320) by use of sequential isomers of such molecules. Substrates having different base numbers were found to be separated by size-exclusion chromatography while those having the same numbers with different base sequences were isolated by use of the reversed-phase mode. By using those dual modes, a limit for the separation of the samples was found to arise because one mode shifted the peaks of the substrates in the sense opposite to the shift resulting from the other mode and it was found that when substrates had less than nine bases, the solutes eluted separately.

Repair of psoralen and acetylaminofluorene DNA adducts by ABC excinuclease

Escherichia coli UvrA, UvrB and UvrC proteins acting in concert remove the major ultraviolet light-induced photoproduct, the pyrimidine dimer, from DNA in the form of a 12 to 13-nucleotide long single-stranded fragment. In vivo data indicate that the UvrABC enzyme is also capable of removing other nucleotide diadducts as well as certain nucleotide monoadducts from DNA and initiating the repair process that leads to removal of interstrand crosslinks caused by some bifunctional chemical agents. We have determined the action mechanism of the enzyme on nucleotide monoadducts produced by 4′-hydroxymethyl-4,5′,8-trimethylpsoralen and N-acetoxy- N-2-acetylaminofluorene. In both cases we find that the enzyme hydrolyzes the eighth phosphodiester bond 5′ and the fifth phosphodiester bond 3′ to the modified base. This cutting pattern is similar to that observed with diadduct substrate, the only difference being that while the enzyme incises the fourth or fifth phosphodiester bond 3′ to the pyrimidine dimer it always hydrolyzes the fifth bond relative to monoadducts. Our results also suggest that ABC excinuclease cuts the same two phosphodiester bonds on both sides of a T whether that T has a psoralen monoadduct or is involved in psoralen-mediated interstrand crosslink.

RecBC enzyme nicking at chi sites during DNA unwinding: Location and orientation-dependence of the cutting

Homologous recombination by the E. coli RecBC pathway occurs at elevated frequency near Chi sites. We reported previously that Chi induces RecBC enzyme to cleave one DNA strand—that containing the Chi sequence 5′G-C-T-G-G-T-G-G3′. We report here that the Chi-dependent cleavage occurs four, five, or six nucleotides to the 3′ side of the Chi octamer and produces nicks with 3′-OH and 5′-PO 4 groups. Chi-dependent cleavage occurs if RecBC enzyme approaches the Chi sequence from the right, but not if it approaches only from the left, during unwinding of the duplex DNA substrate. A single RecBC enzyme molecule appears to cleave the DNA and to release part of it as a single-stranded fragment. These and previous results indicate that Chi-dependent cleavage is concomitant with DNA unwinding by RecBC enzyme and provide an enzymatic basis for the orientation-dependence of Chi recombinational hotspot activity. These observations demonstrate a key step of a proposed model of recombination in which RecBC enzyme produces a potentially invasive single-stranded DNA tail extending from Chi to its left. We discuss the relation between the action of Chi sites and that of special sites enhancing eukaryotic recombination.

Breakage of single-stranded DNA by eukaryotic type 1 topoisomerase occurs only at regions with the potential for base-pairing

Eukaryotic type 1 DNA topoisomerases break single-stranded DNA at specific sites. A preferred site for rat liver topoisomerase breakage in single-stranded φX174 DNA was located within a region of the DNA with the potential for duplex formation. To investigate the relationship between sites of breakage in duplex and single-stranded DNA, a restriction fragment containing sequences from the transcriptional regulatory and enhancer region of the simian virus 40 genome was used as a substrate for topoisomerase. While different patterns of breakage in the native and denatured forms of the DNA were observed, some sites of breakage were common to both forms. The break sites in the denatured DNA were a subset of the break sites in the duplex DNA and were located in regions which had the potential for intrastrand base-pairing due to distal complementary sequences. A series of single-stranded fragments were generated with the distal complementary sequences deleted and these fragments were used as substrates for topoisomerase breakage. The lack of detectable breakage at a site when the complementary sequence was deleted, suggests that topoisomerase acts at duplex regions in the single-stranded DNA and that it is not active on regions of single-stranded DNA that are not base-paired.

Replication origin of the Bacillus subtilis chromosome determined by hybridization of the first-replicating DNA with cloned fragments from the replication origin region of the chromosome

The replication origin ( ori) on the Bacillus subtilis genome was determined by the hybridization between the first-replicating DNA region and the cloned fragments from the ori region. The first-replicating DNA region was labeled specifically by [ 3H]thymidine in the presence of an inhibitor for DNA polymerase during a synchronous initiation of the chromosomal replication by germinating spores starved for thymine, and isolated by a sucrose density gradient centrifugation. Most of the labeled DNA molecules are small in size (up to 1000 bases long). The 45-kb ori region was cloned first in a λ Charon vector and then subcloned in pBR vectors. Restriction fragments from these cloned DNAs were purified by electrophoresis in agarose gels. Only one region within the 45-kb ori region shows strong hybridization with the first-replicating DNA. Restriction fragments from this region were cloned in a phage M13 vector and separated into complementary strands. Hybridization of the labeled DNA with these cloned single-stranded fragments revealed that one site of the ori is located in each strand and they are some 2-kb apart from each other. Replication starts from these sites and proceeds inwards to pass each other.

Characterization of rapidly labeled detergent-soluble DNA in murine splenocytes

Freshly prepared spleen cells from concanavalin A stimulated mice incorporate [3H]thymidine into DNA which can be recovered in detergent-soluble (NP40) and detergent-insoluble forms. The presence of detergent-soluble forms occurs despite the fact that the cells are lysed at 4 degrees C in the presence or absence of 25 mM ethylenediaminetetraacetic acid. After a 2-h pulse with [3H]thymidine, the detergent-soluble fraction contains about 1-3% of the total cellular DNA but 25% of the total labeled high molecular weight material. Since the specific activity of the extensively purified DNA from the detergent-soluble fraction is considerably higher than that of chromosomal DNA, it meets the criteria for being metabolically active. We propose the name "MADS" DNA for metabolically active detergent-soluble DNA. MADS DNA has a density of 1.699 g/mL on cesium chloride gradients and a slightly higher G + C content than chromosomal DNA as determined by high-pressure liquid chromatography. Electrophoresis using native or denaturing agarose gels resolves MADS DNA into discreet sizes between 200 and 4500 base pairs. Nuclease S-1 treatment of native MADS DNA does not alter the size distribution as resolved by means of gel electrophoresis under denaturing conditions. Therefore, MADS DNA is not a collection of single-stranded Okazaki fragments. Southern blot analysis reveals that mitochondrial DNA is a minor component of higher molecular weights above the bulk of the DNA visualized either by staining with ethidium bromide or by incorporation of [3H]thymidine. Inhibitors of ribonucleotide reductase or DNA polymerase alpha inhibit incorporation of [3H]thymidine into MADS DNA, and hence chromosomal DNA synthesis is required for MADS DNA production. Since Southern blot analysis also reveals homology of larger fragments with the 32P-labeled 200 base pair fragment, the presence of repetitive sequences is suggested.

Purification and properties of the recA protein of Proteus mirabilis. Comparison with Escherichia coli recA protein; specificity of interaction with single strand binding protein.

The product of the cloned recA+ gene of Proteus mirabilis substitutes for a defective recA protein in Escherichia coli recA- mutants and restores recombination, repair, and prophage induction functions to near normal levels (Eitner, G., Adler, B., Lanzov, V. A., and Hofemeister, J. (1982) Mol. Gen. Genet. 185, 481-486). In this paper, we report the purification to near homogeneity of the P. mirabilis recA protein (recApm). The polypeptide has a molecular weight similar to that of E. coli recA protein (recAec) and shows partial identity with recAec when reacted against antibodies specific for the E. coli recA protein. recApm catalyzes the hydrolysis of ATP in the presence of single-stranded but not double-stranded DNA. We have compared the recombination-like activities of recApm with those of recAec and found them to be similar. In the presence of ATP and Mg2+, stoichiometric amounts of recApm promote the complete reciprocal exchange of strands between gapped circular and linear duplex DNA molecules. The enzyme also efficiently promotes the formation of D-loops from circular duplex DNA and homologous single-stranded fragments. However, although recApm and recAec share the above physical and functional similarities, they differ in their ability to interact with the E. coli single strand binding protein to catalyze the transfer of one DNA strand from a linear duplex to a single-stranded circle.