The gastrointestinal tract as the portal of entry for foreign macromolecules: fate of DNA and proteins

The gastrointestinal tract (GIT) of mammals is the main portal of entry for foreign DNA and proteins. We have documented the fate of orally administered DNA or protein in the GIT of the mouse. The gene for the Green Fluorescent Protein (GFP) (4.7 kb) and the genomes of bacteriophage M13 (7.25 kb) and adenovirus type 2 (Ad2; 35.9 kb) were used as test DNAs. Persistence of these DNAs in the GIT was monitored by Southern hybridization and fluorescent in situ hybridization (FISH) or by PCR. For studies on proteins, recombinant glutathione-S-transferase was fed to mice. Survival of the protein in the GIT was then assessed by Western blotting. Depending on feeding schedules and food regimens, but irrespective of mouse strain or DNA length, fragments of the GFP gene or other DNAs were detectable for up to 18 h after feeding by Southern blot analysis. The GFP DNA could be visualized by FISH in cecal epithelia. A high fiber diet reduced the time required for food to pass through the GIT, and foreign DNA was cleared more rapidly. A high fat diet or complexing of the foreign DNA with protamine or lipofectin did not extend DNA persistence times. Undegraded GST protein was detected only in foregut contents up to 30 min after feeding. At 15 and 30 min post feeding, trace amounts of GST were found in extracts of the kidney. The GIT is constantly exposed to highly recombinogenic fragments of foreign DNA and to intact foreign proteins. Our data have implications for studies on carcinogenesis and mutagenesis, and on the pathogenicity of infectious proteins such as prions.The first two authors contributed equally to this work     

On the fate of orally ingested foreign DNA in mice: chromosomal association and placental transmission to the fetus

We have previously shown that, when administered orally to mice, bacteriophage M13 DNA, as a paradigm foreign DNA without homology to the mouse genome, can persist in fragmented form in the gastrointestinal tract, penetrate the intestinal wall, and reach the nuclei of leukocytes, spleen and liver cells. Similar results were obtained when a plasmid containing the gene for the green fluorescent protein (pEGFP-C1) was fed to mice. In spleen, the foreign DNA was detected in covalent linkage to DNA with a high degree of homology to mouse genes, perhaps pseudogenes, or to authentic E. coli DNA. We have now extended these studies to the offspring of mice that were fed regularly during pregnancy with a daily dose of 50 g of M13 or pEGFP-C1 DNA. Using the polymerase chain reaction (PCR) or the fluorescent in situ hybridization (FISH) method, foreign DNA, orally ingested by pregnant mice, can be discovered in various organs of fetuses and of newborn animals. The M13 DNA fragments have a length of about 830 bp. In various organs of the mouse fetus, clusters of cells contain foreign DNA as revealed by FISH. The foreign DNA is invariably located in the nuclei. We have never found all cells of the fetus to be transgenic for the foreign DNA. This distribution pattern argues for a transplacental pathway rather than for germline transmission which might be expected only after long-time feeding regimens. In rare cells of three different fetuses, whose mothers have been fed with M13 DNA during gestation, the foreign DNA was detected by FISH in association with both chromatids. Is maternally ingested foreign DNA a potential mutagen for the developing fetus? Received: 15 April 1998 / Accepted: 15 June 1998     

Ingested foreign (phage M13) DNA survives transiently in the gastrointestinal tract and enters the bloodstream of mice

Is the epithelial lining of the mammalian gastrointestinal (GI) tract a tight barrier against the uptake of ingested foreign DNA or can such foreign DNA penetrate into the organism? We approached this question by pipette-feeding circular or linearized double-stranded phage M13 DNA to mice or by adding M13 DNA to the food of mice whose fecal excretions had previously been shown to be devoid of this DNA. At various post-prandial times, the feces of the animals was tested for M 13 DNA sequences by Southern or dot blot hybridization or by the polymerase chain reaction (PCR). On Southern blot hybridization, the majority of M13 DNA fragments were found in the size range between < 200 and 400 by (base pairs). For the PCR analysis, synthetic oligodeoxyribonucleotide primers were spaced on the M13 DNA molecule such that the sizes of the persisting M13 DNA fragments could be determined. We also extracted DNA from whole blood or from sedimented blood cells of the animals at different times after feeding M t3 DNA and examined these DNA preparations for the presence of M13 DNA by dot blot hybridization or by PCR. M13 DNA fragments were found between 1 and 7 h postprandially in the feces of mice. By PCR analysis, fragments of 712, 976, and 1692 by in length were detected. In DNA from blood, M13 DNA fragments of up to 472 by were found by PCR between 2 and 6 h after feeding. Dot blot or Southern blot hybridization revealed M13 DNA at 2 and 4 h, but not at 1, 8 or 24 h after feeding. This DNA was shown to be DNase sensitive. M13 DNA was found both in blood cells and in the serum. A segment of about 400 by of the DNA amplified by PCR from feces or blood was analyzed for its nucleotide sequence which was found to be identical to that of authentic M13 DNA, except for a few deviations. M13 DNA could not be detected in the feces or in the blood of the animals prior to feeding or prior to 1 h and later than 7 h after feeding. These controls attest to the validity of the results and also argue against the possibility that the murine GI tract had been colonized by phage M13. Moreover, M13 DNA-positive bacterial colonies were never isolated from the feces of animals that had ingested M13 DNA. The results of reconstitution experiments suggested that 2 to 4% of the orally administered M13 DNA could be detected in the GI tract of mice. A proportion of about 0.01% to 0.1% of the M13 DNA fed could be retrieved from the blood.     

Detection of secondary predation by PCR analyses of the gut contents of invertebrate generalist predators

Predation by generalist predators is difficult to study in the field because of the complex effects of positive and negative interactions within and between predator species and guilds. Predation can be monitored by molecular means, through identification of prey DNA within predators. However, polymerase chain reaction (PCR) amplification of prey DNA from predators cannot discriminate between primary and secondary predation (hyperpredation), in which one predator feeds on another that has recently eaten the target prey. Here we quantify, for the first time, the potential error caused by detection of prey DNA following secondary predation, using an aphid-spider-carabid model. First, the aphid Sitobion avenae was fed to the spider Tenuiphantes tenuis and the carabid Pterostichus melanarius, and the postconsumption detection periods, for prey DNA within predators, were calculated. Aphids were then fed to spiders and the spiders to carabids. Aphid DNA was detected in the predators using primers that amplified 245- and 110-bp fragments of the mitochondrial cytochrome oxidase I gene. Fragment size and predator sex had no significant effect on detection periods. Secondary predation could be detected for up to 8 h, when carabids fed on spiders immediately after the latter had consumed aphids. Beetles tested positive up to 4 h after eating spiders that had digested their aphid prey for 4 h. Clearly, the extreme sensitivity of PCR makes detection of secondary predation more likely, and the only reliable answer in future may be to use PCR to identify, in parallel, instances of intraguild predation.     

应用xMAP液念芯片多重快速检测四种病原微生物的研究

目的:建立一种多重、快速、特异性好、灵敏度高的病原微生物检测方法。方法:根据GenBank数据库中的小肠结肠炎耶尔森氏菌、单核细胞增生性李斯特菌、产气荚膜梭菌、鼠疫耶尔森氏菌基因序列,分别针对ail、hly、cpe、3a基因设计4对引物和4条探针。通过重叠PCR扩增各目的基因并构建重组质粒,以该重组质粒DNA为模板,通过多重PCR同时扩增上述4个基因,建立xMAP液态芯片检测技术,在此基础上对标准菌株基因组DNA进行检测并验证该方法的特异性和敏感性。结果:xMAP液态芯片对质粒DNA和标准菌株基因组DNA的检测结果与多重PCR结果一致。该方法能在3.5 h内同时完成对4种病原菌的检测,特异性好,且敏感性要高于PCR方法,灵敏度最高可达200CFU/ml。结论:xMAP液态芯片技术是病原微生物的多重快速检测的新方法,具有很好的应用价值和前景。     

应用xMAP液态芯片多重快速检测四种病原微生物的研究

目的：建立一种多重、快速、特异性好、灵敏度高的病原微生物检测方法。方法：根据GenBank数据库中的小肠结肠炎耶尔森氏菌、单核细胞增生性李斯特菌、产气荚膜梭菌、鼠疫耶尔森氏菌基因序列,分别针对ail、hly、cpe、3a基因设计4对引物和4条探针。通过重叠PCR扩增各目的基因并构建重组质粒,以该重组质粒DNA为模板,通过多重PCR同时扩增上述4个基因,建立xMAP液态芯片检测技术,在此基础上对标准菌株基因组DNA进行检测并验证该方法的特异性和敏感性。结果：xMAP液态芯片对质粒DNA和标准菌株基因组DNA的检测结果与多重PCR结果一致。该方法能在3.5 h内同时完成对4种病原菌的检测,特异性好,且敏感性要高于PCR方法,灵敏度最高可达200CFU/ml。结论：xMAP液态芯片技术是病原微生物的多重快速检测的新方法,具有很好的应用价值和前景。     

PCR assay of DNA damage and repair at the gene level in brain and spleen of gamma-irradiated young and old rats.

The PCR amplification of fragments of transcribed (beta-actin, p53) and nontranscribed (IgE, heavy chain) genes in brain and spleen DNA from gamma-irradiated and unirradiated 2- and 28-month-old rats was studied. The amplification levels of fragments of these genes in DNA from old rats were substantially lower than those from young rats, which suggested that these gene fragments in old-rat DNA contained lesions blocking thermostable polymerase in PCR. The beta-actin and IgE gene fragments of spleen DNA from old rats exhibited a significantly higher level of lesions inhibiting Tth polymerase compared to analogous fragments of brain DNA from the same animals. DNA from the tissues of gamma-irradiated rats showed the amount of damage inhibiting amplification to be dependent on animal age and the postirradiation time before DNA isolation. As judged from the changes in the amplification level of gene fragments, there was no preferential fast repair of lesions in the actively transcribed gene beta-actin compared to the nontranscribed gene IgE (heavy chain) in the brain and spleen of gamma-irradiated young and old rats. The amplification results suggest that equal amounts of DNA lesions were repaired in the brain of both old and young rats during the first 0.5 h of the postirradiation time (fast-repair phase), whereas in the subsequent postirradiation period over 5 h (slow-repair phase), the efficiency of damage elimination in the brain DNA of old rats was markedly lower. As for the spleen tissue, the elimination of lesions blocking Tth polymerase was much lower in old gamma-irradiated animals for both of the repair phases.     

Green fluorescent protein gene-transfected peafowl somatic cells participate in the development of chicken embryos

This study was performed to investigate whether the embryonic somatic cells are capable of reconstituting and participating in the embryonic development of chickens to produce chimeras. In order to track the migration behavior of the donor cells, a cell line, originally isolated from an Indian peafowl embryo, was fluorescent-labeled by transfection of the cells with enhanced Green Fluorescent Protein (GFP) and Neomycin resistant (Neo) genes prior to injection into the stage X blastoderm of White Leghorn chickens. The injection was performed with a medium in the presence of 1-5% polyethylene glycol. The development of putative chimeric embryos between the stages three and 24 was examined for GFP expression under fluorescent light. To trace the peafowl cells in the developing chicken embryos, both a species-specific genetic marker originating from the mitochondrial DNA cytochrome b (cyt b) gene and a DNA fragment of GFP gene were used. Of the 185 fertile eggs manipulated, 173 developed into embryos. Fifty-five of them showed positive GFP patches in extra-embryonic tissues, and 15 expressed GFP in intra-embryonic tissues such as those of the head, heart, and gonad. PCR analysis revealed that PCR fragments for the peafowl mitochondrial DNA cyt b and GFP genes were detected in the samples of the GFP positive extra- and intra-embryonic tissues of the chimeras. The present results provide evidence that fluorescent-labeled peafowl embryonic cells carrying GFP and Neo genes are able to participate in the development of chicken embryos to generate chimeras.     

高GC含量DNA模板的PCR扩增

目的：探索高GC含量DNA的PCR扩增条件,为扩增达托霉素生物合成基因簇及拼接奠定基础。方法：在PCR扩增体系中,使用高保真的聚合酶及添加不同浓度的DMSO、7-deaza-dGTP等增强剂,并选择合适的PCR循环程序,优化富含GC的DNA的PCR扩增条件。结果：向反应体系中额外添加1%~4%的DMSO可以显著提高富含GC的DNA的PCR扩增产物量,但会降低其特异性;7-deaza-dGTP可以提高扩增产物的特异性及保真度,但产量会有所下降。应用touch down PCR并在体系中添加7-deaza-dGTP能够提高扩增产物的特异性和产率,增加扩增的保真度。结论：应用优化的PCR扩增条件将所有达托霉素生物合成基因簇分段扩增出来,并可扩增出长达6 kb的片段,且序列完全正确,可以进行后续拼接。     

Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library

Chemical synthesis of oligonucleotides is a widely used tool in the field of biochemistry. Several methods for gene synthesis have been introduced in the growing area of genomics. In this paper, a novel method of constructing dsDNA is proposed. Short (28-mer) oligo fragments from a library were assembled through successive annealing and ligation processes, followed by PCR. First, two oligo fragments annealed to form a dsDNA molecule. The double-stranded oligo was immobilized onto magnetic beads (solid support) via streptavidin-biotin binding. Next, single-stranded oligo fragments were added successively through ligation to form the complete DNA molecule. The synthesized DNA was amplified through PCR and gel electrophoresis was used to characterize the product. Sanger sequencing showed that more than 97% of the nucleotides matched the expected sequence. Extending the length of the DNA molecule by adding single-stranded oligonucleotides from a basis set (library) via ligation enables a more convenient and rapid mechanism for the design and synthesis of oligonucleotides on the go. Coupled with an automated dispensing system and libraries of short oligo fragments, this novel DNA synthesis method would offer an efficient and cost-effective method for producing dsDNA.     

Biodistribution and blood clearance of plasmid DNA administered in arginine peptide complexes

Background

Peptide/DNA complexes have great potential as non-viral methods for gene delivery. Despite promising results for peptide-mediated gene delivery technology, an effective systemic peptide-based gene delivery system has not yet been developed.

Methods

This study used pCMV-Luc as a model gene to investigate the biodistribution and the in vivo efficacy of arginine peptide-mediated gene delivery by polymerase chain reaction (PCR).

Results

Plasmid DNA was detected in all organs tested 1 h after intraperitoneal administration of arginine/DNA complexes, indicating that the arginine/DNA complexes disseminated widely through the body. The plasmid was primarily detected in the spleen, kidney, and diaphragm 24 h post administration. The mRNA expression of plasmid DNA was noted in the spleen, kidney, and diaphragm for up to 2 weeks, and in the other major organs, for at least 1 week. Blood clearance studies showed that injected DNA was found in the blood as long as 6 h after injection.

Conclusions

Taken together, our results demonstrated that arginine/DNA complexes are stable in blood and are effective for in vivo gene delivery. These findings suggest that intraperitoneal administration of arginine/DNA complexes is a promising tool in gene therapy.     

A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers

A set of mapping markers have been designed for Arabidopsis thaliana that correspond to DNA fragments amplifed by the polymerase chain reaction (PCR). The ecotype of origin of these amplified fragments can be determined by cleavage with a restriction endo-nuclease. Specifically, 18 sets of PCR primers were synthesized, each of which amplifies a single mapped DNA sequence from the Columbia and Landsberg erecta ecotypes. Also identifed was at least one restriction endonuclease for each of these PCR products that generates ecotype-specific digestion patterns. Using these co-dominant cleaved amplified polymorphic sequences (CAPS), an Arabidopsis gene can be unambiguously mapped to one of the 10 Arabidopsis chromosome arms in a single cross using a limited number of F₂ progeny.     

Sequence analysis of amplified DNA fragments containing the region encoding the putative lipase substrate-binding domain and genotyping of Aeromonas hydrophila

DNA fragments were amplified by PCR from all tested strains of Aeromonas hydrophila, A. caviae, and A. sobria with primers designed based on sequence alignment of all lipase, phospholipase C, and phospholipase A1 genes and the cytotonic enterotoxin gene, all of which have been reported to have the consensus region of the putative lipase substrate-binding domain. All strains showed lipase activity, and all amplified DNA fragments contained a nucleotide sequence corresponding to the substrate-binding domain. Thirty-five distinct nucleotide sequence patterns and 15 distinct deduced amino acid sequence patterns were found in the amplified DNA fragments from 59 A. hydrophila strains. The deduced amino acid sequences of the amplified DNA fragments from A. caviae and A. sobria strains had distinctive amino acids, suggesting a species-specific sequence in each organism. Furthermore, the amino acid sequence patterns appear to differ between clinical and environmental isolates among A. hydrophila strains. Some strains whose nucleotide sequences were identical to one another in the amplified region showed an identical DNA fingerprinting pattern by repetitive extragenic palindromic sequence-PCR genotyping. These results suggest that A. hydrophila, and also A. caviae and A. sobria strains, have a gene encoding a protein with lipase activity. Homologs of the gene appear to be widely distributed in Aeromonas strains, probably associating with the evolutionary genetic difference between clinical and environmental isolates of A. hydrophila. Additionally, the distinctive nucleotide sequences of the genes could be attributed to the genotype of each strain, suggesting that their analysis may be helpful in elucidating the genetic heterogeneity of Aeromonas.     

大黄鱼虹彩病毒PCR快速检测试剂盒的研制

针对近年来由虹彩病毒所引起的海水养殖鱼类疾病呈日趋严重的态势,该文在证实了引发我国大黄鱼大规模流行病的病原为一种虹彩病毒及测定病毒全基因组序列(111,760bp;GenBank accession numberl．AY779031)的基础上,通过与已报道虹彩病毒核酸序列进行分析比较,结合生物信息学手段,确定了以虹彩病毒ATPase基因保守区序列(295bp)作为扩增靶序列,设计合成了一对特异性引物,通过改进PCR模板的制备方法和优化扩增条件,建立了大黄鱼虹彩病毒PCR快速检测技术,并开发成简便、快速、实用的检测试剂盒,该试剂盒的检测灵敏度相当于30个病毒粒子,模板制备时间约30min、回收率为52％、半个工作日即可得到准确的结果,无非特异性扩增带,适用于大黄鱼虹彩病毒病的早期快速诊断、苗种的检疫及水质环境的监测,目前正在推广应用。     

利用PCR方法鉴定hiTAIL-PCR扩增产物中的质粒骨架片段

T-DNA突变体是研究基因功能的重要资源。高效热不对称交错PCR (hiTAIL-PCR)是克隆突变体中T-DNA插入位点侧翼序列的常用方法。然而我们发现, 利用hiTAIL-PCR克隆到的一些侧翼序列并不对应于宿主的染色体DNA序列, 而是质粒的骨架DNA片段。通过设置1组RB-S4/AC1或者LB-A4/AC1对照反应, 用PCR方法鉴定了hiTAIL-PCR扩增产物中位于T-DNA侧翼的质粒骨架片段。在后续分析中, 通过排除这些片段, 提高了利用hiTAIL-PCR获得宿主染色体DNA片段的效率。同时, 通过调整反应程序, 使得整个PCR的反应时间也大为缩短。在拟南芥(Arabidopsis thaliana) T-DNA突变体drf1侧翼序列的克隆实例中, 对照反应的引入将hiTAIL-PCR中需鉴定的22条扩增产物降至4条, 效率提高了81.8%。     

HBV preS/S基因克隆及其在酿酒酵母中的表达

目的:探索利用酿酒酵母系统表达乙型肝炎病毒(HBV)preS／S基因。方法:利用PCR技 术,以HBV病毒DNA为模板,体外扩增HBV preS／S基因。然后构建重组表达载体pESC-preS／S。 用LiAc法转化酿酒酵母YPH50,选取重组菌进行培养,并诱导表达外源蛋白。提取蛋白浓缩后 进行SDS-PAGE分析,并经Western blot分析鉴定。结果:实验结果表明重组菌能够表达HBV preS／S蛋白。结论:利用酿酒酵母系统可成功表达HBV preS／S基因,为制备新型预防性疫苗提供 条件。     

PCR analysis of x- and y-type genes present at the complex Glu-A1 locus in durum and bread wheat

Genes (x-type) corresponding to different high-molecular-weight glutenin subunits encoded at the Glu-A1 locus present in bread- and durum-wheat cultivars have been selectively amplified by the polymerase chain reaction (PCR). DNA fragments corresponding to an unexpressed x-type gene were also amplified. As unexpressed y-type genes may or may not contain an 8-kb transposon-like insertion, two different sets of primers were designed to obtain amplification of DNA fragments corresponding to these genes. Amplified DNA fragments were also digested with restriction enzymes. The digestion patterns of amplified fragments corresponding to unusual x-type subunits showed similarities with genes encoding the most common subunits 2^* and 1. The unexpressed amplified x-type gene showed a restriction pattern similar to the one obtained with the allelic gene encoding high-molecular-weight glutenin subunit 1; homologies were also found within the repetitive region of the linked y-type genes. On the basis of these observations it is postulated that an ancestral active x-type gene, most likely corresponding to subunit 1, was silenced following the insertion of the 8-kb transposon-like fragment into the linked y-type gene. Received: 8 April 1996 / Accepted: 30 August 1996     

PCR amplification of large genomic fragments from human and simian immunodeficiency virus infected cell lines.

Polymerase chain reaction (PCR) has been used to amplify the large fragments from viral genomic DNA of SIV from wild caught, asymptomatic Erythrocebus monkeys from Western Africa (Senegal) and also from HIV-2 infected cell lines. By using consensus primer sequences from highly conserved stretches of gag, pol and env genes, two halves of the viral genome of HIV-2 and SIV (isolated from west African Erythrocebus monkeys) have amplified by PCR. One half spans 5200 bp from within the U3 region of the 5' long terminal repeat (LTR) into pol gene and an overlapping fragment spans 3700 bp from the pol gene into U5 region of 3' LTR. Also fragments ranging from 1-2.3 kb from gag pol and env genes have been successfully amplified. Our data demonstrate that primers used to amplify large segments from viral DNA yield better results if they are derived from a consensus sequence of a highly conserved stretch of the viral genome.     

Microarrays for the detection of HBV and HDV

The increasing pace of development in molecular biology during the last decade has had a direct effect on mass testing and diagnostic applications, including blood screening. We report the model Microarray that has been developed for Hepatitis B virus (HBV) and Hepatitis D virus (HDV) detection. The specific primer pairs of PCR were designed using the Primer Premier 5.00 program according to the conserved regions of HBV and HDV. PCR fragments were purified and cloned into pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. The DNA microarray was prepared by robotically spotting PCR products onto the surface of glass slides. Sequences were aligned, and the results obtained showed that the products of PCR amplification were the required specific gene fragments of HBV, and HDV. Samples were labeled by Restriction Display PCR (RD-PCR). Gene chip hybridizing signals showed that the specificity and sensitivity required for HBV and HDV detection were satisfied. Using PCR amplified products to construct gene chips for the simultaneous clinical diagnosis of HBV and HDV resulted in a quick, simple, and effective method. We conclude that the DNA microarray assay system might be useful as a diagnostic technique in the clinical laboratory. Further applications of RD-PCR for the sample labeling could speed up microarray multi-virus detection.