Two human genes isolated by a novel method encode DNA-binding proteins containing a common region of homology

Two cDNAs encoding new DNA-binding proteins (Dbps) have been cloned using a human placenta lambda gt11 recombinant cDNA library and DNA fragments as probes. Hybrid proteins expressed by the lambda gt11 cDNA library were blotted onto nitrocellulose filters, and incubated with three different radio-labeled DNA probes containing the human epidermal growth factor (EGF) receptor enhancer or the human c-erbB-2 promoter. Two kinds of clones, named dbpA and dbpB, showed high affinities for the DNA probes. The comparison of the nucleotide and the deduced amino acid (aa) sequences between these two cDNAs indicated that 100 of 109 aa located in the central region of these two Dbps were identical. The dbpA and dbpB-coded proteins also had an affinity for other cDNA probes such as the human c-ski gene, but not for poly(dI-dC).poly(dI-dC), suggesting that the sequence(s) recognized by the dbpA and dbpB-coded proteins may occur frequently, or that these proteins bind to DNA non-specifically in a different manner from that of histones. A simple method, described in this paper, can be used to isolate cDNA clones encoding Dbps. Strategies used for the detection of sequence-specific and non-specific Dbps are discussed.     

Multilocus DNA fingerprints in the plant Cynoglossum officinale L. and their use in the estimation of selfing

We have developed a nonradioactive oligonucleotide multilocus DNA fingerprinting method for Cynoglossum officinale . Of the 19 probes tested, six probes yielded banding patterns for all restriction enzymes used. All but one of the informative probes are repeats with a four-base motif. Approximately 60% of the loci appeared to be polymorphic. The sensitivity of the nonradioactive method was equal to that of the radioactive method. In addition, a new simple calculation method is presented to estimate selfing rates and approximate 95% confidence limits from the DNA fingerprint profiles avoiding 'between-gel' comparisons. The selfing rates differed significantly (as determined from 95% confidence intervals) between naturally pollinated individuals of C. officinale within the experimental population. The estimates ranged from 0 to 70% selfing.     

Gene isolation with human T lymphocyte probes. Isolation of a gene that expresses an epitope recognized by T cells specific for Mycobacterium bovis BCG and pathogenic mycobacteria

We have used human CD4+ T lymphocyte clones as primary probes to identify and isolate lambda gt11 rDNA clones that express epitopes recognized by T cells. The method that we describe here permits a direct survey of T cell epitope coding sequences in genomic DNA or cDNA libraries. A lambda gt11 library of Mycobacterium leprae DNA was screened with M. leprae-reactive human T cell clones as probes, allowing the isolation of a M. leprae DNA clone encoding the unidentified Ag. This DNA clone differs in restriction maps from those previously identified by antibody probes and encodes an epitope that is unique to vaccine strains of Mycobacterium bovis bacillus Calmette-Guérin and pathogenic mycobacteria. This method is generally applicable and should expedite the study of Ag and epitopes important to the T cell response in infections and in autoimmune diseases.     

Nonradioactive labeling with chemically modified cytosine tails by the polymerase chain reaction.

We developed a modified nonradioactive method for the detection of DNA. This method makes use of the polymerase chain reaction for preparation of probes; that is, a DNA fragment inserted in the polylinker region of an M13 or pUC vector is amplified with primers that have a modified cytosine tail at the 5' terminus (C-tailed primers). By this method, large amounts of labeled probes can be obtained easily. After hybridization, modified cytosine tails can be detected immunologically. DNA labeled by this method could be used in plaque hybridization. We could detect 0.05 pg of dot-blotted labeled DNA in 30 min with an enzyme-catalyzed chemiluminescence reaction.     

An alternative nonradioactive method for labeling DNA using biotin

An alternative nonradioactive labeling method and a highly sensitive technique for detecting specific DNA sequences are described. The labeling method requires the "Klenow" fragment of DNA polymerase I and random hexanucleotides (synthesized or naturally extracted) as a primer for the production of highly sensitive DNA probes. The system has three main steps: (i) labeling of DNA with biotinylated 11-dUTP; (ii) detection of biotinylated DNA by a one-step procedure with streptavidin-alkaline phosphatase complex; (iii) blocking of background with Tween 20. Twenty attograms (2 X 10(-17) g) of pBR322 plasmid DNA was detected by dot-blot hybridization. Upon Southern blot hybridization, 7.4 fg (7.4 X 10(-15) g) of pBR322 HindIII DNA was detected using the biotinylated pBR322 plasmid DNA probe; 40.8 ag and 7.4 fg of lambda HindIII DNA were detected with the biotinylated whole lambda DNA probe by dot and Southern blot hybridization, respectively. Specific bands were also detected with the biotinylated argininosuccinolyase probe upon Northern blotting of mouse poly(A+) RNA. Further applications for in situ hybridization are also described.     

Use of biotinylated DNA probes in screening cells obtained from cervical swabs for human papillomavirus DNA sequences

A nonradioactive DNA-detection procedure using biotinylated DNA probes in the screening of cells from cervical swabs for DNA sequences homologous to human papillomavirus (HPV) DNA was tested. This alternative DNA-detection method yielded results comparable to those obtained with radioisotope-labeled DNA probes in 32 cases tested. This procedure obviates the special precautions required for radioisotope materials. Accordingly, this technique can be made available to many laboratories, and conclusive evidence as to the relation of HPV infection to cervical cancer may thus be accumulated.     

DNA polymerase lambda (Pol lambda), a novel eukaryotic DNA polymerase with a potential role in meiosis

A new gene (POLL) encoding a novel DNA polymerase (Pol lambda) has been identified at mouse chromosome 19. Murine Pol lambda, consisting of 573 amino acid residues, has a 32% identity to Pol beta, involved in nuclear DNA repair in eukaryotic cells. It is interesting that Pol lambda contains all the critical residues involved in DNA binding, nucleotide binding and selection, and catalysis of DNA polymerization, that are conserved in Pol beta and other DNA polymerases belonging to family X. Murine Pol lambda, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity when assessed by in situ gel analysis. Pol lambda also conserves the critical residues of Pol beta required for its intrinsic deoxyribose phosphate lyase (dRPase) activity. The first 230 amino acid residues of Pol lambda, that have no counterpart in Pol beta, contain a BRCT domain, present in a variety of cell-cycle check-point control proteins responsive to DNA damage and proteins involved in DNA repair. Northern blotting, in situ hybridization analysis and immunostaining showed high levels of Pol lambda specifically expressed in testis, being developmentally regulated and mainly associated to pachytene spermatocytes. These first evidences, although indirect, suggest a potential role of Pol lambda in DNA repair synthesis associated with meiosis.     

Cloning, nucleotide sequence, and mutagenesis of a gene (irpA) involved in iron-deficient growth of the cyanobacterium Synechococcus sp. strain PCC7942.

We describe the cloning and sequencing of a gene from the cyanobacterium Synechococcus sp. strain PCC7942, designated irpA (iron-regulated protein A), that encodes for a protein involved in iron acquisition or storage. Polyclonal antibodies raised against proteins which accumulate during iron-deficient growth were used as probes to isolate immunopositive clones from a lambda gt11 genomic expression library. The clone, designated lambda gtAN26, carried a 1.7-kilobase (kb) chromosomal DNA insert and was detected by cross-reactivity with antibody against a 36-kilodalton protein. It was possible to map a 20-kb portion of the chromosome with various DNA probes from lambda gt11 and lambda EMBL-3 clones, and Southern blot analysis revealed that the irpA gene was present in a single copy and localized within a 1.7-kb PstI fragment. DNA sequencing revealed an open reading frame of 1,068 nucleotides capable of encoding 356 amino acids which yields a protein with a molecular weight of 38,584. The hydropathy profile of the polypeptide indicated a putative N-terminal signal sequence of 44 amino acid residues. IrpA is a cytoplasmic membrane protein as determined by biochemistry and electron microscopy immunocytochemistry. The upstream region of the irpA gene contained a consensus sequence similar to the aerobactin operator in Escherichia coli. This fact, plus a mutant with a mutation in irpA that is unable to grow under iron-deficient conditions, led us to suggest that irpA is regulated by iron and that the gene product is involved in iron acquisition or storage.     

Novel method for identifying sequence-specific DNA-binding proteins.

We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.     

Detection of protein-DNA interaction with a DNA probe: distinction between single-strand and double-strand DNA-protein interaction

A simple, direct method for the detection of DNA-protein interaction was developed with electrochemical methods. Single-stranded DNA (ss-DNA) probes were prepared through the chemical bonding of an oligonucleotide to a polymer film bearing carboxylic acid groups, and double-stranded DNA (ds-DNA) probes were prepared through hybridization of the complementary sequence DNA on the ss-DNA probe. Impedance spectroscopy and differential pulse voltammetry (DPV) distinguished the interaction between the DNA probes with mouse Purbeta (mPurbeta), an ss-DNA binding protein, and with Escherichia coli MutH, a ds-DNA binding protein. Impedance spectra obtained before and after the interaction of DNA probes with these proteins clearly showed the sequence-specific ss-DNA preference of mPurbeta and the sequence-specific ds-DNA preference of MutH. The concentration dependence of proteins on the response of the DNA probes was also investigated, and the detection limits of MutH and mPurbeta were 25 and 3 microg/ml, respectively. To confirm the impedance results, the variation of the current oxidation peak of adenine of the DNA probe was monitored with DPV. The formation constants of the complexes formed between the probe DNA and the proteins were estimated based on the DPV results.     

Interactions between phage lambda replication proteins, lambda DNA and minicell membrane

Gentle methods for minicell lysis and lysate fractionation have been elaborated: lysis by T4 lysozyme without detergents, and fractionation by equilibrium sedimentation in a metrizamide density gradient, both at low ionic strength. In the lysates of phage-lambda-infected minicells the lambda DNA, trapped at a prereplicative step [Witkiewicz, H. and Taylor, K. (1979) Biochim. Biophys. Acta 564, 31-36], appeared in two peaks of different buoyant densities: as a membrane-bound and a free lambda DNA. The covalently-closed-circular form of lambda DNA appeared exclusively in the membrane fraction. The lambda-coded proteins, synthesized in lambda-infected minicells, appeared in two major fractions: as membrane-bound and as free proteins, and in one minor fraction, bound with free lambda DNA. Neither lambda protein engaged in the initiation of DNA replication was present in the fraction of free proteins: the P-gene product was membrane-associated, and the O-gene product formed a complex with free lambda DNA. The effect of high ionic strength (KCl) and of detergents (Triton X-100 and sarcosyl) on the binding of replication proteins with lambda DNA and with the membrane was studied. The non-ionic detergent, Triton X-100 caused displacement of a part of lambda DNA from the membrane to the free lambda DNA peak; both lambda replication proteins were bound with free lambda DNA. The binding of the O protein with lambda DNA was relatively stable, but was destroyed by the ionic detergent, sarcosyl.     

Immunoblotting of a Staphylococcus aureus DNA library as a tool for isolating potential antigenic proteins

A Staphylococcus aureus DNA library was created in the lambda vector EMBL4 and expressed in Escherichia coli strain Y1090. Plaques were screened using serum from a S. aureus-infected patient. A number of positive clones that expressed proteins recognised by serum antibodies were further analysed using Western blots of total lysates from cultures infected with the lambda clones. Differences were found between patient and control sera in both the specificity and titre of anti-S. aureus antibodies. Expressed staphylococcal proteins appeared to be stable and were easily distinguishable from E. coli proteins in the Western blot. This method may have applications in specifically selecting clones encoding antigens associated with infection and may be of potential use clinically.     

Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene.

Monoclonal antibodies were prepared against a fraction of nuclear proteins of Drosophila melanogaster identified as tightly binding to DNA. Four of these antibodies were directed against a 19-kilodalton nuclear protein; immunofluorescence staining of the polytene chromosomes localized the antigen to the alpha, beta, and intercalary heterochromatic regions. Screening of a lambda gt11 cDNA expression library with one of the monoclonal antibodies identified a recombinant DNA phage clone that produced a fusion protein immunologically similar to the heterochromatin-associated protein. Polyclonal sera directed against the bacterial lacZ fusion protein recognized the same nuclear protein on Western blots. A full-length cDNA clone was isolated from a lambda gt10 library, and its DNA sequence was obtained. Analysis of the open reading frame revealed an 18,101-dalton protein encoded by this cDNA. Two overlapping genomic DNA clones were isolated from a Charon 4 library of D. melanogaster with the cDNA clone, and a restriction map was obtained. In situ hybridization with these probes indicated that the gene maps to a single chromosome location at 29A on the 2L chromosome. This general strategy should be effective for cloning the genes and identifying the genetic loci of chromosomal proteins which cannot be readily assayed by other means.     

High-density functional display of proteins on bacteriophage lambda

We designed a bacteriophage lambda system to display peptides and proteins fused at the C terminus of the head protein gpD of phage lambda. DNA encoding the foreign peptide/protein was first inserted at the 3' end of a DNA segment encoding gpD under the control of the lac promoter in a plasmid vector (donor plasmid), which also carried lox P(wt) and lox P(511) recombination sequences. Cre-expressing cells were transformed with this plasmid and subsequently infected with a recipient lambda phage that carried a stuffer DNA segment flanked by lox P(wt) and lox P(511) sites. Recombination occurred in vivo at the lox sites and Amp(r) cointegrates were formed. The cointegrates produced recombinant phage that displayed foreign protein fused at the C terminus of gpD. The system was optimised by cloning DNA encoding different length fragments of HIV-1 p24 (amino acid residues 1-72, 1-156 and 1-231) and the display was compared with that obtained with M13 phage. The display on lambda phage was at least 100-fold higher than on M13 phage for all the fragments with no degradation of displayed products. The high-density display on lambda phage was superior to that on M13 phage and resulted in selective enrichment of epitope-bearing clones from gene-fragment libraries. Single-chain antibodies were displayed in functional form on phage lambda, strongly suggesting that correct disulphide bond formation takes place during display.This lambda phage display system, which avoids direct cloning into lambda DNA and in vitro packaging, achieved cloning efficiencies comparable to those obtained with any plasmid system. The high-density display of foreign proteins on bacteriophage lambda should be extremely useful in studying low-affinity protein-protein interactions more efficiently compared to the M13 phage-based system.     

Nonradioactive detection of nucleic acid by the universal probe system

A convenient and nonradioactive method for DNA hybridization tests termed the "Universal probe system" has been developed. This method is based on the principle of sandwich hybridization. This system consists of two single-stranded DNA probes (a primary probe and a biotin-labeled secondary probe). The primary probe is prepared from a chimeric phage-plasmid vector containing the complementary sequence to a target gene. The secondary probe has a sequence complementary to the vector portion of the primary probe and is labeled with biotin via the transamination reaction. An advantage of this method is that the single-stranded primary probe can be prepared with ease by using the chimeric phage-plasmid vector system, thereby avoiding tedious labeling of individually different probes. As the primary probe is not modified with biotin and other labels, it conserves the sequence to be hybridized with a target. Accordingly, the primary probe containing a relatively short hybridizing region (ca. 50 bp) can efficiently hybridize with the target. In fact, the universal probe is sensitive enough to detect a single-copy human gene on Southern blots.     

DNA microarray probe preparation by gel isolation nested PCR

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.