In vitro and in vivo transfection and expression of plasmid-based non-viral vector for erythropoietin gene therapy

A non-viral gene therapy vector, pcDNA3-EPO, was constructed by subcloning erythropoietin (EPO) cDNA into plasmid pcDNA3. After liposome-mediated transfection of the NIH 3T3 cells in vitro, EPO expression in the culture medium was detected by ELISA and amounted to 1.25 ± 0.3 IU ml^–1. The biological activity of this EPO in the medium was detected after intramuscular injection of BALB/c mice. PCR of genomic DNA and RT-PCR of total RNA also confirmed that the plasmid pcDNA3-EPO had been transfected into the cells. A pool of pcDNA3-EPO transfectants, which stably expressed EPO, was obtained by G418 selection. When pcDNA3-EPO was combined into liposomes and intramuscularly injected into BALB/c mice, the reticulocyte ratio in the positive mice was three times higher than that in the control mice. In vivo expression was maintained in mice for at least one month.     

Small cryptic plasmids of Streptococcus pneumoniae belong to the pC194/pUB110 family of rolling circle plasmids

The DNA sequences of two related plasmids pPR1 and pPR3 described previously in Streptococcus pneumoniae isolates from Germany and Spain were now determined. Both plasmids belong to a family of rolling circle (RC) plasmids found in a variety of bacteria. Their GC content with 32% is lower than that of the S. pneumoniae chromosomal DNA. The plasmid pPR3 has a molecular size of 3160 bp with four putative open reading frames, whereas pPR1 contained a deletion of 313 bp that included the 5′-part of ORF2 and upstream regions and differed by three bp from pPR3. The predicted protein of ORF1 showed high similarity to replication proteins of RC plasmids with 74% identical amino acids to RepA of Streptococcus thermophilus plasmids. Sequences similar to the plus origin of replication of ssDNA plasmids were present in both plasmids. They also contained a 152-bp region with over 83% identity to the minus origin of replication of the Streptococcus agalacticae plasmid pMV158.     

接合转移质粒pUCP24T的构建与性能研究

目的 构建能在大肠埃希菌(E.coli)和铜绿假单胞菌(PA)间高效接合转移的质粒.方法 通过NCBI blastn程序分析质粒pCVD442接合反应起始序列oriT,PCR扩增oriT后插入pMD18-T,再将pMD1 8-oriT转化E.coli DH5α,经酶切、测序验证后用SmaI和HindIIII双酶切亚克隆至质粒pUCP24,获得质粒pUCP24T,将pUCP24T转化E.coli后研究pUCP24T从E.coli到PA的接合效率和质粒稳定性等.结果 成功构建pUCP24T重组质粒,在E.coli和PA的接合实验中,E.coli(pUCP24T)-PA共培养2h的接合效率平均为3.588×10-2,按12 h一代连续9代继代培养108 h,抗生素压力下质粒保存率为98.29％,无抗生素的培养基中质粒保存率为21.76％.结论 成功构建高接合效率的接合转移质粒pUCP24T.     

Interleukin-12 genetic administration suppressed metastatic liver tumor unsusceptible to CTL

A cytokine gene therapy approach was conducted against metastatic lesions of cytotoxic T lymphocyte (CTL)-unsusceptible tumor in mice. The EBV-based and conventional plasmid vectors that encode murine interleukin-12 (IL-12) gene (pGEG.mIL-12 and pG.mIL-12, respectively) were intravenously transfected into the mice that had received a subcutaneous inoculation of M5076 sarcoma cells. The pGEG.mIL-12 transfection drastically suppressed the subcutaneous as well as hepatic metastatic tumors, resulting in significant prolongation of survival period of the animals. Although single administration with pG.mIL-12 was not effective, repetitive transfection with the plasmid significantly prolonged the longevity of the mice-bearing the metastatic liver tumors. Multiple transfection with either pGEG.mIL-12 or pG.mIL-12 also suppressed peritoneal carcinomatosis in mice that had been injected with M5076 cells into the peritoneal cavity. It was suggested that a high level IL-12 production elicited by the intravenous delivery of the cytokine gene may be quite effective in inhibiting metastatic and CTL-unsusceptible neoplasms.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of plants by the pTF-FC2 plasmid is efficient and strictly dependent on the MobA protein

In the transformation of plants by Agrobacterium tumefaciens the VirD2 protein has been shown to pilot T-DNA during its transfer to the plant cell nucleus. Other studies have shown that the MobA protein of plasmid RSF1010 is capable of mediating its transfer from Agrobacterium cells to plant cells by a similar process. We have demonstrated previously that plasmid pTF-FC2, which has some similarity to RSF1010, is also able to transfer DNA efficiently. In this study, we performed a mutational analysis of the roles played by A. tumefaciens VirD2 and pTF-FC2 MobA in DNA transfer-mediated by A. tumefaciens carrying pTF-FC2. We show that MobA+/VirD2+ and MobA+/VirD2– strains were equally proficient in their ability to transfer a pTF-FC2-derived plasmid DNA to plants and to transform them. However, the MobA–/VirD2+ strain showed a DNA transfer efficiency of 0.03% compared with that of the other two strains. This sharply contrasts with our results that VirD2 can rather efficiently cleave the oriT sequence of pFT-FC2 in vitro. We therefore conclude that MobA plays a major VirD2-independent role in plant transformation by pTF-FC2.     

变形杆菌的耐药性及接合性R质粒的检测

本文报告了从烧伤病人感染创面分离、鉴定的35株变形杆菌的药敏试验及接合性R质粒的检测结果。35株变形杆菌双测试的6种抗菌药物都具有不同程度的耐药性。其中,对6种药物同时耐受的有26株(74.3％)。对其中的27株做了接合试验,接合性R质粒的检出率为70.4%。     

Comparison of the nucleotide sequences of the meta-cleavage pathway genes of TOL plasmid pWW0 from Pseudomonas putida with other meta-cleavage genes suggests that both single and multiple nucleotide substitutions contribute to enzyme evolution

TOL plasmid pWW0 from Pseudomonas putida mt-2 encodes catabolic enzymes required for the oxidation of toluene and xylenes. The structural genes for these catabolic enzymes are clustered into two operons, the xylCMABN operon, which encodes a set of enzymes required for the transformation of toluene/xylenes to benzoate/toluates, and the xylXYZLTEGFJQKIH operon, which encodes a set of enzymes required for the transformation of benzoate/toluates to Krebs cycle intermediates. The latter operon can be divided physically and functionally into two parts, the xylXYZL cluster, which is involved in the transformation of benzoate/toluates to (methyl)catechols, and the xylTEGFJQKIH cluster, which is involved in the transformation of (methyl)catechols to Krebs cycle intermediates. Genes isofunctional to xylXYZL are present in Acinetobacter calcoaceticus, and constitute a benzoate-degradative pathway, while xylTEGFJQKIH homologous encoding enzymes of a methylphenol-degradative pathway and a naphthalene-degradative pathway are present on plasmid pVI150 from P. putida CF600, and on plasmid NAH7 from P. putida PpG7, respectively. Comparison of the nucleotide sequences of the xylXYZLTEGFJQKIH genes with other isofunctional genes suggested that the xylTEGFJQKIH genes on the TOL plasmid diverged from these homologues 20 to 50 million years ago, while the xylXYZL genes diverged from the A. calcoaceticus homologues 100 to 200 million years ago. In codons where amino acids are not conserved, the substitution rate in the third base was higher than that in synonymous codons. This result was interpreted as indicating that both single and multiple nucleotide substitutions contributed to the amino acid-substituting mutations, and hence to enzyme evolution. This observation seems to be general because mammalian globin genes exhibit the same tendency.     

Enigmatic double-stranded RNA in Japonica rice

We have found a linear, 16 kb, double-stranded RNA (dsRNA) in symptomless Japonica rice (Oryza sativa L.) that is not found in Indica rice (Oryza sativa L.). The dsRNA was detected in every tissue and at every developmental stage, and its copy number was approximately constant (about 20 copies/cell). Double-stranded RNA was also detected in two strains of Oryza rufipogon (an ancestor of O. sativa). Hybridization experiments indicated that the dsRNA of O. rufipogon was homologous but not identical to that of O. sativa. The sequence of about 13.2 kb of the dsRNA was determined and two open reading frames (ORFs) were found. The larger ORF (ORF B) was more than 12 351 nucleotides long and encoded more than 4 117 amino acid residues.     

Mutations in a Saccharomyces cerevisme host showing increased holding stability of the heterologous plasmid pSRI

Summary We have isolated Saccharomyces cerevisiae mutants, smp, showing stable maintenance of plasmid pSRI, a Zygosaccharomyces rouxii plasmid. The smp mutants were recessive and were classified into at least three different complementation groups. The three mutants also showed increased stability of YRp plasmids and the mutations are additive for plasmid stability. One mutation, smp1, confers a respiration-deficient (rho ⁰) phenotype and several Rho^– mutants independently isolated by ethidium bromide treatment of the same yeast strain also showed increased stabilities of pSR1 and YRp plasmids. The wild-type S. cerevisiae cells showed a strongly biased distribution of pSR1 molecules as well as YRp plasmids to the mother cells at mitosis, while the smpf mutant did not show this bias. Another mutation, smp3, at a locus linked to ade2 on chromosome XV, confers temperature-sensitive growth. The SMP3 gene encodes a 59.9 kDa hydrophobic protein and disruption of the gene is lethal.     

用PCR法直接快速筛查重组阳性克隆

应用ＰＣＲ法快速筛查插入有苯丙氨酸脱氨酶ｃＤＮＡ重组阳性克隆。方法：用于ＰＣＲ扩增的引物是位于载体ｐＥＴ２３ｂ启动子处的Ｔ７启动子引物和位于目的基因ＰＡＬｃＤＮＡ３’端终止密码ＴＡＡ处的引物。以灭菌吸头挑一单菌落加入ＰＣＲ体系扩增。结果：在筛查的３个克隆中,有２个阳性克降,并且插入方向正确,经ＤＮＡ序列测定得到进一步证实。结论：以ＰＣＲ方法筛查重组阳性克隆,可以简便快速鉴定插入片段的大小和方面,不