Permuteins of interleukin 1 beta--a simplified approach for the construction of permutated proteins having new termini.

A technique for the rapid and simple generation of permutated versions of the interleukin-1 beta (IL-1 beta) gene is described. In this method, the human IL-1 beta cDNA is twice amplified by the polymerase chain reaction (PCR) and the resulting DNA fragments are ligated in tandem. Between the two genes, the DNA sequence encodes a short four amino acid loop to link the native N- and C-terminal ends of the IL-1 beta protein. By using PCR amplification from this starting template, a new version of the IL-1 beta cDNA was obtained that encodes a permutated form of the IL-1 beta protein where the new N- and C-terminal amino acids correspond to residues 65 and 64 of the native IL-1 beta sequence, respectively. The name 'permutein' is proposed to describe proteins generated by this technology. The molecular profile (IL-1 receptor binding, biologic activity and solution properties) of the IL-1 permutein produced by this technology, permutein 65/64, is shown to be identical to that of native IL-1 beta. The approach should be useful to define further the structural features of this protein that are important for its function.     

Nuclear internalisation and DNA binding activities of interleukin-1, interleukin-1 receptor and interleukin-1/receptor complexes.

This paper presents evidence to suggest that interleukin-1 alpha as a complex with its receptor is able to express DNA binding activity. Both the interleukin-1/receptor complex and the interleukin-1 receptor appear to be able to bind to DNA, however interleukin-1 on its own showed no binding activity. Interleukin-1 was found to be internalised into the nuclei of all cells examined (EL4, MEL, HL-60, K562, THP-1 and Jurkat cells). The data suggest the possible modulation of genes by interaction of interleukin-1/receptor complexes with DNA structures.     

Artificial DNA splicing using directed ligation]

An approach to the directed genetic recombination in vitro has been devised, which allows for joining, in a predetermined chemical-enzymatic way, a series of DNA segments to give a precisely spliced polynucleotide sequence (DNA Splicing by Directed Ligation, SDL). The approach makes use of amplification, by several polymerase chain reactions (PCR), of the chosen DNA segments. The corresponding primers contain recognition sites of the class IIS restriction endonucleases, yielding protruding ends of unique primary structures. The protruding ends of the segments to be joined together are structurally predetermined to make them mutually complementary. Ligation of the mixture of the segments so synthesized gives the desired sequence in an unambiguous way. The suggested approach has been exemplified by the synthesis of a totally processed (intronless) gene encoding human mature interleukin-1 alpha.     

Insertion sequence-based cassette PCR: cultivation-independent isolation of γ-hexachlorocyclohexane-degrading genes from soil DNA

gamma-Hexachlorocyclohexane (gamma-HCH) is a highly chlorinated pesticide that has caused serious environmental problems. Based on the frequently observed association of insertion sequence IS6100 with lin genes for gamma-HCH degradation in several gamma-HCH-degrading bacterial strains isolated to date, DNA fragments flanked by two copies of IS6100 were amplified by nested polymerase chain reaction (PCR) technique using a DNA sample extracted from soil contaminated with HCH. Four distinct DNA fragments with sizes of 6.6, 2.6, 1.6, and 1.3 kb were obtained, three of which carried lin genes: the 6.6-kb fragment carried linD and linE as well as linR; the 2.6-kb fragment showed a truncated form of linF; and the 1.6-kb fragment carried linB. Our approach, named as insertion sequence (IS)-based cassette PCR, was successful in the isolation of the lin genes from HCH-contaminated soil without cultivation of host cells and is applicable for the culture-independent isolation of other functional genes bordered by other IS elements.     

Direct and efficient cloning of full-length genes from environmental DNA by RT-qPCR and modified TAIL-PCR

Environmental DNA (eDNA) is defined as the total DNA that can be isolated from environmental samples. In total, therefore, eDNA includes a vast functional genes, and various approaches have been developed to retrieve full-length functional genes from eDNA. The efficiency of PCR amplification of eDNA is limited, however, because in truth, the net content of actual target functional genes is rather low in eDNA. To address this technical challenge, we developed a fast and effective approach to cloning full-length functional genes from eDNA. Two important modifications were made to existing PCR-based methods: first, a real-time quantitative PCR step was added to assess the difficulty of obtaining full-length genes; second, we improved the thermal asymmetric interlaced PCR program to make it more effective for cloning the flanking regions of known fragments that are present at low abundance in eDNA. Using this approach, five novel full-length functional genes with very low identity to known genes were cloned from environmental samples. This approach has great potential for allowing discovery of functional genes from environmental sources and may be broadly applicable to molecular biology research.     

Exon concatenation to increase the efficiency of mutation screening by DGGE

For genes that have a substantial number of exons and long intronic sequences, mutation screening by denaturing gradient gel electrophoresis (DGGE) requires the amplification of each exon from genomic DNA by PCR. This results in a high number of fragments to be analyzed by DGGE so that the analysis of large sample sets becomes labor intensive and time consuming. To address this problem, we have developed a new strategy for mutation analysis, lexon-DGGE, which combines the joining of different exons by PCR (also known as lexons) with a highly sensitive technique such as DGGE to screen for mutations. The lexon technique is based on the concatenation of several exons, adjacent or not, from genomic DNA into a single DNA fragment so that this approach could simultaneously be used to check the mutational status of several small genes. To show the feasibility of the approach, we have used the lexon-DGGE technique to analyze all coding exons, intron-exon junctions, noncoding exon 1, and part of the noncoding region of exon 11 of the TP53 gene. The validity and performance of the technique were confirmed by using negative and positive controls for each of the DNAfragments analyzed.     

A PCR-based amplification method retaining the quantitative difference between two complex genomes

With the increasing emergence of genome-wide analysis technologies (including comparative genomic hybridization (CGH), expression profiling on microarrays, differential display (DD), subtractive hybridization, and representational difference analysis (RDA)), there is frequently a need to amplify entire genomes or cDNAs by PCR to obtain enough material for comparisons among target and control samples. A major problem with PCR is that amplification occurs in a nonlinear manner and reproducibility is influenced by stray impurities. As a result, when two complex DNA populations are amplified separately, the quantitative relationship between two genes after amplification is generally not the same as their relation before amplification. Here we describe balanced PCR, a procedure that faithfully retains the difference among corresponding amplified genes by using a simple principle. Two distinct genomic DNA samples are tagged with oligonucleotides containing both a common and a unique DNA sequence. The genomic DNA samples are pooled and amplified in a single PCR tube using the common DNA tag. By mixing the two genomes, PCR loses the ability to discriminate among the different alleles and the influence of impurities is eliminated. The PCR-amplified pooled samples can be separated using the DNA tag unique to each individual genomic DNA sample. The principle of this method has been validated with synthetic DNA, genomic DNA, and cDNA applied on microarrays. By removing the bias of PCR, this method allows a balanced amplification of allelic fragments from two complex DNAs even after three sequential rounds of PCR. This balanced PCR approach should allow genetic analysis in minute laser-microdissected tissues, paraffin-embedded archived material, or single cells.     

New inducers revealed by the promoter sequence analysis of two interferon-activated human genes

In order to investigate the molecular basis of the regulation of interferon-inducible genes, we isolated the promoter region of two such genes coding for the (2'-5')oligo(adenylate) synthetase and a 56-kDa protein (IFI-56K). The regions surrounding the cap site were sequenced and compared with the sequences of vertebrate and viral DNA present in the Genbank data bank. Small DNA segments were found in both genes which are homologous to part of the promoter region of other genes, such as those of interferon-beta, tumor necrosis factor beta, interleukin-2 and its receptor. Since these homologies were found located in functionally important regions of these genes, we tested whether their inducers also enhance the (2'-5')oligo(adenylate) synthetase and IFI-56K gene expression. We found that poly(rI).poly(rC) and interleukin-1, activators of the interferon-beta gene and of T lymphocytes respectively, are both able to enhance IFI-56K mRNA accumulation in all cell lines tested. Cycloheximide even superinduces this gene when added together with poly(rI).poly(rC) and interleukin-1 (but not when added with interferon). We showed that these inductions are direct and not mediated by interferon produced by cells in response to poly(rI).poly(rC) or interleukin-1. The promoter sequence analyses have thus led to the discovery of unexpected inducers, i.e. an interferon inducer such as poly(rI).poly(rC) is also able to directly induce a gene that is under the control of interferon.     

Reducing the complexity of the transforming Epstein-Barr virus genome to 64 kilobase pairs.

Transformation-competent, replication-defective Epstein-Barr virus (EBV) recombinants which are deleted for 18 kbp of DNA encoding the largest EBNA intron and for 58 kbp of DNA between the EBNA1 and LMP1 genes were constructed. These recombinants were made by transfecting three overlapping cosmid-cloned EBV DNA fragments into cells infected with a lytic replication-competent but transformation-defective EBV (P3HR-1 strain) and were identified by clonal transformation of primary B lymphocytes into lymphoblastoid cell lines. One-third of the lymphoblastoid cell lines were infected with recombinants which had both deletions and carried the EBNA2 and EBNA3 genes from the transfected EBV DNA and therefore are composed mostly or entirely from the transfected EBV DNA fragments. The deleted DNA is absent from cells infected with most of these recombinants, as demonstrated by Southern blot and sensitive PCR analyses for eight different sites within the deleted regions. Cell growth and EBNA, LMP, and BZLF1 gene expression in lymphoblastoid cell lines infected with these recombinants are similar to those in cells infected with wild-type EBV recombinants. Together with previous data, these experiments reduce the complexity of the EBV DNA necessary for transformation of primary B lymphocytes to 64 kbp. The approach should be useful for molecular genetic analyses of transforming EBV genes or for the insertion of heterologous fragments into transforming EBV genomes.     

Re-usable DNA template for the polymerase chain reaction.

DNA covalently bound to an uncharged nylon membrane was used for consecutive amplifications of several different genes by PCR. Successful PCR amplifications were obtained for membrane-bound genomic and plasmid DNA. Membrane-bound genomic DNA templates were re-used at least 15 times for PCR with specific amplification of the desired gene each time. PCR amplifications of specific sequences of p53, p16, CYP1A1, CYP2D6, GSTM1 and GSTM3 were performed independently on the same strips of uncharged nylon membrane containing genomic DNA. PCR products were subjected to restriction fragment length polymorphism analysis, single-strand conformational polymorphism analysis and/or dideoxy sequencing to confirm PCR-amplified gene sequences. We found that PCR fragments obtained by amplification from bound genomic DNA as template were identical in sequence to those of PCR products obtained from free genomic DNA in solution. PCR was performed using as little as 5 ng genomic or 4 fg plasmid DNA bound to membrane. These results suggest that DNA covalently bound to membrane can be re-used for sample-specific PCR amplifications, providing a potentially unlimited source of DNA for PCR.     

Preliminary studies on DNA retardation by MutS applied to the detection of point mutations in clinical samples

MutS ability to bind DNA mismatches was applied to the detection of point mutations in PCR products. MutS recognized mismatches from single up to five nucleotides and retarded the electrophoretic migration of mismatched DNA. The electrophoretic detection of insertions/deletions above three nucleotides is also possible without MutS, thanks to the DNA mobility shift caused by the presence of large insertion/deletion loops in the heteroduplex DNA. Thus, the method enables the search for a broad range of mutations: from single up to several nucleotides. The mobility shift assays were carried out in polyacrylamide gels stained with SYBR-Gold. One assay required 50-200 ng of PCR product and 1-3 microg of Thermus thermophilus his6-MutS protein. The advantages of this approach are: the small amounts of DNA required for the examination, simple and fast staining, no demand for PCR product purification, no labelling and radioisotopes required. The method was tested in the detection of cancer predisposing mutations in RET, hMSH2, hMLH1, BRCA1, BRCA2 and NBS1 genes. The approach appears to be promising in screening for unknown point mutations.     

The new strategy for allele identification of the genes coding for pertussis toxin subunit S1 (ptx S1) and pertactin (prn) in Bordetella pertussis

Bordetella pertussis strains demonstrate polymorphism in toxin subunit S1 (PT S1) and pertactin (Prn), which belong to major protective antigens of the pathogen. Changes in the distribution of particular alleles of ptxS1 and prn genes in local B. pertussis populations have been proposed as possible factors influencing the vaccination effectiveness. We have developed a new methodology for the identification of the alleles, which eliminates the necessity of DNA sequencing. The approach is based on the evaluation of the number of sequence repeats and detection of specific nucleotides at polymorphic sites of the genes, and utilizes products of their full or partial PCR amplification. The approach is available for a laboratory with standard equipment. The total conformity of the strategy with the DNA sequencing-based approach was proved on the full set of reference strains and a group of Polish clinical isolates. The new methodology was used to investigate a collection of 120 Polish B. pertussis strains isolated from the 1960s to 2001. Similarly to findings from other countries and to earlier Polish data, the tendency to change the vaccine types of PT S1 and Prn by the antigenically different ones was observed.     

DNA聚合酶与引物/模板的相互作用对PCR效率的影响

PCR是体外酶促合成特异DNA片段的一种方法,引物的优劣直接关系到PCR的特异性与成功与否。传统的PCR引物设计软件基本上忽略了DNA聚合酶与引物/模板的亲和性对PCR效率的影响。为揭示DNA聚合酶与引物/模板的相互作用是否对PCR的效率有影响,通过构建Taq DNA 聚合酶与不同序列引物/模板DNA相互作用的三维结构模型,采用MM/GBSA方法计算复合物的结合自由能,以结合自由能为参数,为人血清白蛋白基因(Human Serum Albumin gene,HSA gene)和结核杆菌pyrF基因(Mycobacterium tuberculosis pyrF gene)设计了PCR引物。PCR实验结果表明,引物的PCR效率与结合自由能相关：引物与聚合酶的结合自由能越低,PCR实验的效率相对越高。这说明DNA聚合酶与引物/模板的相互作用对PCR效率有重要影响。因此,引物/模板DNA与聚合酶的结合自由能可以作为PCR引物设计的新参数。     

Isolation and characterization of nucleotide-binding site and C-terminal leucine-rich repeat-resistance gene candidates in bananas

Commercial banana varieties are highly susceptible to fungal pathogens, as well as bacterial pathogens, nematodes, viruses, and insect pests. The largest known family of plant resistance genes encodes proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for the isolation of candidate genes in banana that may be involved in plant defense. Six degenerate PCR primers were designed to target NBS and additional domains were tested on commercial banana species Musa acuminata subsp malaccensis and the Musa AAB Group propagated in vitro and plants maintained in a greenhouse. Total DNA was isolated by a modified CTAB extraction technique. Four resistance gene analogs were amplified and deposited in GenBank and assigned numbers HQ199833-HQ199836. The predicted amino acid sequences compared to the amino acid sequences of known resistance genes (MRGL1, MRGL2, MRGL3, and MRGL4) revealed significant sequence similarity. The presence of consensus domains, namely kinase-1a, kinase-2 and hydrophobic domain, provided evidence that the cloned sequences belong to the typical non-Toll/interleukin-1 receptor-like domain NBS-LRR gene family.     

Amplification of bacterial genomic DNA by the polymerase chain reaction and direct sequencing after asymmetric amplification: application to the study of periplasmic permeases.

The polymerase chain reaction (PCR) has been used to amplify DNA fragments by using eucaryotic genomic DNA as a template. We show that bacterial genomic DNA can be used as a template for PCR amplification. We demonstrate that DNA fragments at least as large as 4,400 base pairs can be amplified with fidelity and that the amplified DNA can be used as a substrate for most operations involving DNA. We discuss problems inherent in the direct sequencing of the amplified product, one of the important exploitations of this methodology. We have solved the problems by developing an "asymmetric amplification" method in which one of the oligonucleotide primers is used in limiting amounts, thus allowing the accumulation of single-stranded copies of only one of the DNA strands. As an illustration of the use of PCR in bacteria, we have amplified, sequenced, and subcloned several DNA fragments carrying mutations in genes of the histidine permease operon. These mutations are part of a preliminary approach to studying protein-protein interactions in transport, and their nature is discussed.     

Analysis of the methylation status of imprinted genes based on methylation-specific polymerase chain reaction combined with denaturing high-performance liquid chromatography

A procedure for the analysis of the methylation status of imprinted genes is described. The method offers a rapid and reliable alternative to conventional methods such as Southern blots and methylation-specific polymerase chain reaction (PCR) (i.e., allele-specific methylation-specific PCR). The efficient resolution of the differentially methylated alleles is demonstrated for three human imprinted genes: SNRPN, LIT1 (alias KCNQ1OT1), and H19. Abnormal imprinting of SNRPN is associated with the Angelman/Prader-Willi syndromes, and that of LIT1 and H19 with the Beckwith-Wiedemann syndrome. The method is based on methylation-specific PCR followed by denaturing high-performance liquid chromatography (MSP/DHPLC). Briefly, genomic DNA is initially subjected to an in vitro bisulfite treatment, whereby unmethylated cytosines are deaminated. Subsequent PCR amplifications, using primers specific for modified DNA, are aimed at DNA segments that show parent-of-origin-specific methylation. PCR conditions are chosen that allow an efficient amplification of both alleles. The PCR products representing the two alleles are identical in size; they differ, however, at a number of positions within the amplified DNA segment. The DHPLC analysis allows very efficient resolution of the two populations of PCR products, providing qualitative and quantitative results.