Activation of a cryptic splice site in the tax gene of HTLV-I by a single nucleotide change

We identified a T-to-C mutation 2 nucleotides (nt) upstream from the AG in a GT-AG intron between exons 2 and 3 in the human T-cell leukemia virus type I (HTLV-I) tax mRNA. This mutation resulted in the preferential usage of an alternative splice site, causing a 75-nt elongation of tax mRNA and reduced production of viral antigens. When the clone containing this T-to-C mutation was reverted to the wild-type (T) DNA sequence, normal splicing of tax mRNA ensued and viral production was restored. These results suggest that the nucleotide at the position 2nt upstream from the AG in a GT-AG intron is important for the proper splicing of the HTLV-I tax gene, although it is not considered important for splicing in eukaryotes.     

Activation of the cryptic DNA binding function of mutant forms of p53.

Wild type p53 assembles into a latent multiprotein complex which can be activated for sequence-specific DNA binding in vitro by proteins targeting the carboxy-terminal domain. Using an optimized system coupling the post-translational modification of wild type p53 to activation of sequence specific DNA binding, we examined the affects of common mutations on the cryptic DNA binding function of p53. Two mutant forms of p53 were shown to be efficiently converted from the latent state by PAb421 and DnaK, but were defective in activation by casein kinase II, indicating that mutant p53 may not be receptive to allosteric regulation by casein kinase II phosphorylation. A reactive sulfhydryl group is absolutely required for DNA binding by wild type and mutant forms of p53 once converted to the activated state. Together, these data show that some mutant forms of p53 harbour the wild-type machinery required to engage in sequence-specific DNA binding and define a signalling pathway whose inactivation may directly result in a loss of p53 function.     

Activation of cellular signaling by 8-oxoguanine DNA glycosylase-1-initiated DNA base excision repair

Accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) in the DNA results in genetic instability and mutagenesis, and is believed to contribute to carcinogenesis, aging processes and various aging-related diseases. 8-OxoG is removed from the DNA via DNA base excision repair (BER), initiated by 8-oxoguanine DNA glycosylase-1 (OGG1). Our recent studies have shown that OGG1 binds its repair product 8-oxoG base with high affinity at a site independent from its DNA lesion-recognizing catalytic site and the OGG1•8-oxoG complex physically interacts with canonical Ras family members. Furthermore, exogenously added 8-oxoG base enters the cells and activates Ras GTPases; however, a link has not yet been established between cell signaling and DNA BER, which is the endogenous source of the 8-oxoG base. In this study, we utilized KG-1 cells expressing a temperature-sensitive mutant OGG1, siRNA ablation of gene expression, and a variety of molecular biological assays to define a link between OGG1-BER and cellular signaling. The results show that due to activation of OGG1-BER, 8-oxoG base is released from the genome in sufficient quantities for activation of Ras GTPase and resulting in phosphorylation of the downstream Ras targets Raf1, MEK1,2 and ERK1,2. These results demonstrate a previously unrecognized mechanism for cellular responses to OGG1-initiated DNA BER.     

Activation of a cryptic streptomycin-resistance gene in the Bacteroides erm transposon, Tn4551

Bacteroides compound transposons encoding erm resistance are highly homologous but previous studies have shown some divergence of Tn4551. Results presented here describe a novel Tn4551 streptomycin-resistance gene, aadS, that was phenotypically silent in wild-type Bacteroides. However, aadS expression could be activated by a trans-acting chromosomal mutation. The aadS-encoded peptide displayed significant homology to Gram-positive streptomycin-dependent adenyltransferases, and enzymatic analysis confirmed the production of this activity. Examination of the nucleotide sequence showed that 200 bp upstream of aadS, the DNA base composition changed abruptly from 31% G+C to 48% G+C. These two regions were demarcated by a DNA sequence with homology to the recombination hot spots reported for Tn21 and the Bacteroides ermFU gene and to sequences at the ends of the chromosomal Bacteroides conjugal element, XBU4422.     

5-甲基胞嘧啶修饰对沉默抗生素基因的激活

对真核生物的表观遗传学研究表明,5-甲基胞嘧啶修饰参与了多种重要生理功能。虽然在原核生物中也存在5-甲基胞嘧啶修饰,但其具体功能尚未确定。大肠杆菌编码的Dcm甲基转移酶负责DNA的5-甲基胞嘧啶修饰[1],有研究报道显示,Dcm与细菌的限制修饰系统相关[2];也有研究报道dcm基因能影响大肠杆菌中核糖体基因的表达,从而影响初级代谢和次级代谢[3]。本期介绍了高婕、贺新义等发表的论文"大肠杆菌甲基转移酶dcm基因的表达对变铅青链霉菌的多效性影响"[4],作者巧妙地利用变铅青链霉菌的DNA无甲基化修饰这一特点,将大肠杆菌dcm基因导入变铅青链霉菌,研究了5-甲基胞嘧啶修饰在变铅青链霉菌中的功能。结果发现,DNA的5-甲基胞嘧啶修饰不仅可影响变铅青链霉菌的形态和生理分化,而且还能激活放线紫红素沉默基因的表达。论文作者以变铅青链霉菌为材料,拓展了对原核生物DNA5-甲基胞嘧啶修饰的生理功能的认识。以此为基础的深入研究,不仅有助于揭示5-甲基胞嘧啶修饰在原核生物中的功能,而且有可能为沉默抗生素基因的表达或抗生素产量的提高提供一个新的途径。     

Activation of a cryptic 5' splice site by U1 snRNA

In the course of analyzing 5' splice site mutations in the second intron of Schizosaccharomyces pombe cdc2, we identified a cryptic 5' junction containing a nonconsensus nucleotide at position +2. An even more unusual feature of this cryptic 5' junction was its pattern of activation. By analyzing the profile of splicing products for an extensive series of cdc2 mutants in the presence and absence of compensatory U1 alleles, we have obtained evidence that the natural 5' splice site participates in activation of the cryptic 5' splice site, and that it does so via base pairing to U1 snRNA. Furthermore, the results of follow-up experiments strongly suggest that base pairing between U1 snRNA and the cryptic 5' junction itself plays a dominant role in its activation. Most remarkably, a mutant U1 can activate the cryptic 5' splice site even in the presence of a wild-type sequence at the natural 5' junction, providing unambiguous evidence that this snRNA redirects splicing via base pairing. Although previous work has demonstrated that U5 and U6 snRNAs can activate cryptic 5' splice sites through base pairing interactions, this is the first example in which U1 snRNA has been implicated in the final selection of a cryptic 5' junction.     

Transformation of Escherichia coli by a specific DNA restriction fragment.

Summary Specific transformation of a rifampicin sensitive strain of Escherichia coli to rifampicin resistance has been performed by a single, defined DNA restriction fragment carrying the genetic information for the subunit of E. coli RNA polymerase. In this transformation the transforming genetic character has been substituted for the corresponding recipient gene locus by recombination. The value of the described transformation system for locating genetic markers on DNA restriction fragments is discussed in comparison to previously reported in vitro systems.     

隐性次级代谢产物生物合成基因簇的激活及天然产物定向发现

传统的"活性-化合物"天然药物发现方法导致大量已知化合物被重复分离,大大加剧了新药发现的难度。规模化基因组测序揭示了微生物基因组中存在大量的隐性(cryptic)次级代谢产物生物合成基因簇,如何激活这些隐性基因簇成为当今世界天然产物研究领域的难点与热点。本文从途径特异性和多效性两个角度综述了隐性生物合成基因簇激活策略;同时,对基因组信息指导下结构导向(structure-guided)的化合物定向分离技术进行了归纳。隐性基因簇的激活为定向发掘具有优良活性的新型天然产物提供了新的契机。     

Integration and partial excision of a cryptic plasmid in Pseudomonas syringae pv. phaseolicola.

A virulent strain of Pseudomonas syringae pv. phaseolicola, a pathogen of the common bean Phaseolus vulgaris (L.), was shown to harbor a 98-megadalton cryptic plasmid, pMC7105. After exposure of this strain to the plasmid-curing agent mitomycin C, a colony was isolated which had no detectable extrachromosomal DNA. Hybridization of labeled pMC7105 probe to nitrocellulose filters containing Southern-blotted BamHI cleavage products of cellular DNA revealed that pMC7105 was integrated into the chromosome rather than cured from this strain. Imprecise excision of pMC7105 resulted in the formation of three smaller plasmids of 34, 50, and 58 megadaltons. BamHI and EcoRI fingerprint analyses revealed that these plasmids were excised from a common region of pMC7105. The BamHI fragments of pMC7105 which were not present in the excision plasmids remained integrated and could be detected by hybridization of pMC7105 probe to Southern-blotted cellular DNA from these strains. Certain chromosomal fragments also had homology with the pMC7105 probe. The excision plasmids were stably maintained and neither integration nor excision altered the pathogenicity of these strains.     

Activation of a novel human transforming gene, ret, by DNA rearrangement

A novel transforming gene was detected by transfection of NIH 3T3 cells with human lymphoma DNA. The tumor DNA induced a single focus in primary transfections, whereas DNAs of transformed NIH cells induced transformation with high efficiencies in secondary and tertiary assays. Molecular clones spanning about 37 kb of human sequence were isolated from tertiary transformant DNA. Blot hybridization indicated that the transforming gene consisted of two segments that were unlinked in both normal human and primary lymphoma DNAs. The two segments of human DNA were cotranscribed in transformed NIH cells but not in any human cells examined. The transforming gene thus appeared to be activated by recombination between two unlinked human DNA segments, possibly by cointegration during transfection.     

Activation of a cryptic crystal protein gene of Bacillus thuringiensis subspecies kurstaki by gene fusion and determination of the crystal protein insecticidal specificity

DNA hybridization with the insecticidal crystal protein gene cryllA (formerly cryBl) of Bacillus thuringiensis supspecies kurstaki has shown that subspecies kurstaki contains a cryllA-related sequence in addition to the cryllA gene (Donovan et al., 1988a). We have cloned the cryllA-related sequence and have determined that the sequence, which has been designated cryllB, is 89% identical to the cryllA gene. Recombinant B. thuringiensis cells harbouring the cloned cryllB gene produced very little CryllB protein. A high level of production of the CryllB protein was achieved by fusing the regulatory region of the crylllA crystal protein gene to the cryllB gene. The CryllB protein was found to be highly toxic to Lymantria dispar, Heliothis virescens and Trichoplusia ni, and was not toxic to Aedes aegypti.     

Improved gene correction efficiency with a tailed duplex DNA fragment

A 606-base single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, corrects a hygromycin resistance and enhanced green fluorescent protein (Hyg-EGFP) fusion gene more efficiently than a PCR fragment, which is the conventional type of DNA fragment used in gene correction. Here, a tailed duplex, obtained by annealing an oligonucleotide to the ss DNA fragment, was used in the correction. The tailed duplex may be a good substrate for the RAD51 protein, an important enzyme in homologous recombination, which could be the gene correction pathway. The annealing of the oligonucleotides enhanced the correction efficiency of the Hyg-EGFP gene, especially when annealed in the 3'-region of the ss DNA fragment. Both the length and backbone structure of the oligonucleotides affected the gene correction efficiency. This type of gene correction device was also effective for another target gene, the rpsL gene. The results obtained in this study indicate that tailed duplex DNA fragments are effective nucleic acids for gene correction.     

Stabilization of anE. coli plasmid by a mini-F fragment of DNA

Summary In anEscherichia coli K-12 strain (trpA trpE tnaA) cultured in LB broth without selective pressure, a pBR322 derivative containing the gene for tryptophan synthase (pBR322-trpBA) was found to be unstable. After 70 cell-number doublings, only 50% of the host cells retained the gene for ampicillin resistance (Ap^r). Insertion of the mini-F fragment of F factor DNA into this plasmid could effectively reduce the plasmid loss. Partial derepression of the tryptophan promotor-operator by 3-indopleacrylic acid further decreased the stability of the pBR322-trpBA but not that of the mini-F inserted plasmid (pBR322F-trpBA) The vector pBR322F-trpBA could be maintained at high copy number in the culture after 100 generations of growth; the culture was able to overproduce tryptophan synthase in the presence of 3-indoleacrylic acid.l-Tryptophan was produced from indole andl-serine using andE. coli host transformed with.pBR322F-trpBA DNA. After 8 h of incubation, the expression level was approximately 180 g/l.     

Sequence of a yeast DNA fragment containing a chromosomal replicator and a tRNA Glu 3 gene.

The sequence of a 1.9 kb Bam x Hind III fragment from yeast has been determined. This fragment is part of a yeast 6.7 kb Hind III segment cloned into pBR322 (pY20). The fragment carries a single gene for a glutamate tRNA which has no intron. According to genetic analyses [1] this fragment also contains a yeast chromosomal replicator. We have analyzed the sequence for potential open reading frames and for several structural features which are thought to be involved in the initiation of DNA replication. Hybridization studies have revealed that portions of this sequence are repeated within the yeast genome.     

Molecular cloning of the human DNA excision repair gene ERCC-6.

The UV-sensitive, nucleotide excision repair-deficient Chinese hamster mutant cell line UV61 was used to identify and clone a correcting human gene, ERCC-6. UV61, belonging to rodent complementation group 6, is only moderately UV sensitive in comparison with mutant lines in groups 1 to 5. It harbors a deficiency in the repair of UV-induced cyclobutane pyrimidine dimers but permits apparently normal repair of (6-4) photoproducts. Genomic (HeLa) DNA transfections of UV61 resulted, with a very low efficiency, in six primary and four secondary UV-resistant transformants having regained wild-type UV survival. Southern blot analysis revealed that five primary and only one secondary transformant retained human sequences. The latter line was used to clone the entire 115-kb human insert. Coinheritance analysis demonstrated that five of the other transformants harbored a 100-kb segment of the cloned human insert. Since it is extremely unlikely that six transformants all retain the same stretch of human DNA by coincidence, we conclude that the ERCC-6 gene resides within this region and probably covers most of it. The large size of the gene explains the extremely low transfection frequency and makes the gene one of the largest cloned by genomic DNA transfection. Four transformants did not retain the correcting ERCC-6 gene and presumably have reverted to the UV-resistant phenotype. One of these appeared to have amplified an endogenous, mutated CHO ERCC-6 allele, indicating that the UV61 mutation is leaky and can be overcome by gene amplification.