Discrimination between RNA circles, interlocked RNA circles and lariats using two-dimensional polyacrylamide gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis can be used to identify structural forms of RNA such as linear RNA, circular RNA, interlocked circles and lariats. The procedure is based upon the characteristic migration behaviour of the degradation products derived from the intact structures present already before the start of the experiment or formed during or after electrophoresis in the first dimension. After autoradiography to detect the positions of the radiolabeled RNA molecules, circles broken during electrophoresis of the first dimension give rise to horizontal lines touching the diagonal formed by linear RNAs at a point corresponding to the length of the RNA circle from which it was derived. Products derived from interlocked RNA circles by breakage after completion of the first dimension appear on a vertical line underneath the intact complex and consist of free RNA circles and their linear derivatives. Broken lariats give rise to two lines depending on the location of the break. Lariats with broken tails are present on a line to a position that corresponds to the length of their tail and that runs parallel to the diagonal formed by linear products. Lariats with a broken eye form a line running from the position of the intact product to the diagonal formed by the linear RNAs.     

Analyzing the mosaic structure of genes

Summary Some genes in prokaryotes consist of a mosaic of regions derived from different ancestors by horizontal gene transfer. A method is described for demonstrating the statistical significance of such mosaic structure and for locating the crossover points separating different regions.     

Transduction of certain genes by an autonomously replicating Bacillus thuringiensis phage

A derivative of Bacillus thuringiensis subsp. kurstaki HD1 (HD1-9) released transducing phage (TP21) from late exponential cultures. Three of seven markers tested were transduced into Bacillus cereus, but only two of these (cysC and trpB/F) were transduced at a frequency of more than 100 times the reversion rates. A limited transduction capacity was given further support in that few chromosomal markers were carried in the HD1-9 lysate, as demonstrated by Southern hybridization. Restriction fragments from the phage DNA and from total B. thuringiensis DNA hybridized to an insertion sequence (IS231-like) probe, which may provide a region of homology for transduction. All of the B. cereus transductants contained the phage as a 44-kb plasmid, and each could transduce both the cys and trp genes to other B. cereus auxotrophs, albeit at lower frequencies than those for the B. thuringiensis transducing phage. In some cases, especially for cys, the transduced gene was integrated into the chromosome of the recipient, whereas the trp gene in many cases appeared to be lost with curing of the 44-kb plasmid. In addition, some B. cereus transductants lost prototrophy but retained a 44-kb plasmid, consistent with the presence of TP21 helper phage. These phage may mediate the subsequent transduction from B. cereus phototrophs. TP21 replicates as a plasmid and, at least under the conditions studied, selectively transfers markers to B. cereus.     

Stability and expression of bacterial genes in replicating geminivirus vectors in plants.

Bacterial beta-glucuronidase (gus) and neomycin phosphotransferase (neo) genes were introduced into coat protein replacement vectors based on DNA A of tomato golden mosaic virus (TGMV). Recombinant gus and neo vectors up to 1.1 kbp larger than DNA A were shown to replicate stably in transgenic plants containing partial dimers (master copies) of the vectors integrated into their chromosomal DNA in the absence of DNA B. Beta-glucuronidase and neomycin phosphotransferase activities in independently transformed plants were proportional to the copy number of the double-stranded forms of the vector. Deletion analysis has shown that an essential part of the TGMV coat protein promoter, including a TATA box, lies within 76 nt upstream of the initiation codon of the gene. An increase in expression of a neo gene was obtained by replacing this 76 nt sequence by an 800 nt sequence containing a cauliflower mosaic virus 35S RNA promoter with no effect on the ability of the vector to replicate or on its stability in transgenic plants. Systemic infection of plants by agroinoculation with TGMV vectors larger than DNA A in the presence of DNA B resulted in deletions in the vector DNA in some, but not all, plants. Possible reasons for vector instability in systemically infected plants, and vector stability in transgenic plants containing master copies of the vector, are discussed.     

Transduction of certain genes by an autonomously replicating Bacillus thuringiensis phage.

A derivative of Bacillus thuringiensis subsp. kurstaki HD1 (HD1-9) released transducing phage (TP21) from late exponential cultures. Three of seven markers tested were transduced into Bacillus cereus, but only two of these (cysC and trpB/F) were transduced at a frequency of more than 100 times the reversion rates. A limited transduction capacity was given further support in that few chromosomal markers were carried in the HD1-9 lysate, as demonstrated by Southern hybridization. Restriction fragments from the phage DNA and from total B. thuringiensis DNA hybridized to an insertion sequence (IS231-like) probe, which may provide a region of homology for transduction. All of the B. cereus transductants contained the phage as a 44-kb plasmid, and each could transduce both the cys and trp genes to other B. cereus auxotrophs, albeit at lower frequencies than those for the B. thuringiensis transducing phage. In some cases, especially for cys, the transduced gene was integrated into the chromosome of the recipient, whereas the trp gene in many cases appeared to be lost with curing of the 44-kb plasmid. In addition, some B. cereus transductants lost prototrophy but retained a 44-kb plasmid, consistent with the presence of TP21 helper phage. These phage may mediate the subsequent transduction from B. cereus phototrophs. TP21 replicates as a plasmid and, at least under the conditions studied, selectively transfers markers to B. cereus.     

DNA polymerase I and recBC enzyme support the covalent closing of hydrogen-bonded lambda DNA circles in extracts of Escherichia coli cells

The covalent closing of hydrogen-bonded lambda DNA circles in Escherichia coli extract was observed to require DNA polymerase I, recBC enzyme and ATP. This covalent closing activity was lost in strains harbouring a mutation in one of the genes responsible for production of the enzymes mentioned above, and was recovered by combining these mutant extracts. ATP could be replaced with dATP, but not appreciably with any of the other nucleoside triphosphates. High concentrations of ATP inhibited the closure. K+ or NH4+ (0.2M) was required for optimal activity and NMN was a strong inhibitor.     

Analyzing microarray data using CLANS

Analysis of microarray experiments is complicated by the huge amount of data involved. Searching for groups of co-expressed genes is akin to searching for protein families in a database as, in both cases, small subsets of genes with similar features are to be found within vast quantities of data. CLANS was originally developed to find protein families in large sets of amino acid sequences where the amount of data involved made phylogenetic approaches overly cumbersome. We present a number of improvements that greatly extend the previous version of CLANS and show its application to microarray data as well as its ability of incorporating additional information to facilitate interactive analysis. AVAILABILITY: The program is available for download from: http://bioinfoserver.rsbs.anu.edu.au/downloads/clans/     

Autonomously replicating episomes contain mdr1 genes in a multidrug-resistant human cell line.

Gene amplification in human tumor cells is frequently mediated by extrachromosomal elements (e.g., double minute chromosomes [DMs]). Recent experiments have shown that DMs can be formed from smaller, submicroscopic circular precursors referred to as episomes (S. M. Carroll, M. L. DeRose, P. Gaudray, C. M. Moore, D. R. Needham-Vandevanter, D. D. Von Hoff and G. M. Wahl, Mol. Biol. 8:1525-1533, 1988). To investigate whether episomes are generally involved as intermediates in gene amplification, we determined whether they mediate the amplification of the mdr1 gene, which when overexpressed engenders cross resistance to multiple lipophilic drugs. A variety of methods including electrophoresis of undigested DNAs in high-voltage gradients, NotI digestion, and production of double-strand breaks by gamma irradiation were used to distinguish between mdr1 sequences amplified on submicroscopic circular molecules and those amplified within DMs or chromosomal DNA. The gamma-irradiation procedure provides a new method for detecting and determining the size of circular molecules from 50 kilobases (kb) to greater than 1,000 kb. These methods revealed that some of the amplified mdr1 genes in vinblastine-resistant KB-V1 cells are contained in supercoiled circular molecules of approximately 600 and approximately 750 kb. Analysis of the replication of these molecules by a Meselson-Stahl density shift experiment demonstrated that they replicate approximately once in a cell cycle. The data lend further support to a model for gene amplification in which DMs are generally formed from smaller, autonomously replicating precursors.     

Transformation of Aspergillus parasiticus using autonomously replicating plasmids from Aspergillus nidulans

Abstract A genetic transformation system for the aflatoxin-producing fungus Aspergillus parasiticus using two autonomously replicating plasmids from A. nidulans (ARp1 and pDHG25) is reported. Transformation frequencies using the plasmid pDHG25 were from 5 × 10² to 2.5 × 10⁴ transformants per 10⁶ viable protoplasts and μg DNA. The stability of the plasmids in the transformants was also studied. This transformation system offers a new opportunity to clone genes related to aflatoxin production using appropriate aflatoxin-defective mutants.     

异染色质及基因关闭

异染色质普遍存在于真核生物的染色质中,和细胞分裂、生存竞争等有密切关系,尤其在调节基因的活性上有重要作用。组蛋白尾的修饰,决定着异染色质的形成和解聚,从而控制基因的启闭,这一机制被称为组蛋白密码。本文以裂殖酵母的交配型区为例介绍了异染色质的的形成及维持机理。组蛋白密码可能是DNA遗传密码外生命的又一调节机制,而对异染色质形成和结构功能的研究,将成为破译组蛋白密码的钥匙。     

Site-directed mutagenesis by complementary-strand synthesis using a closing oligonucleotide and double-stranded DNA templates

An approach for generating structures capable of directing full-length complementary-strand synthesis for double-stranded plasmid DNA is described. The structures are formed following heat denaturation and cooling of linearized plasmid DNA molecules in the presence of what is referred to as a "closing" oligonucleotide. Consisting of a sequence complementary to the free ends of one of the two plasmid strands, the closing oligonucleotide functions as an agent for recircularization of a DNA strand and generation of a primer-circular template structure suitable for polymerase-dependent full-length complementary-strand synthesis and ligation into a covalently closed heteroduplex molecule. When combined with a mutagenic oligonucleotide and uracil-substituted DNA templates, this approach allows site-directed mutagenesis to be performed directly on double-stranded DNA with a mutant formation efficiency of about 50%, a level amenable to rapid screening by DNA sequencing.     

Model-driven approaches for in vitro combination therapy using ONYX-015 replicating oncolytic adenovirus

Replicating genetically modified adenoviruses have shown promise as a new treatment approach against cancer. Recombinant adenoviruses replicate only in cancer cells which contain certain mutations, such as the loss of functional p53, as is the case in the virus ONYX-015. The successful entry of the viral particle into target cells is strongly dependent on the presence of the main receptor for adenovirus, the coxsackie- and adenovirus receptor (CAR). This receptor is frequently down-regulated in highly malignant cells, rendering this population less vulnerable to viral attack. It has been shown that the use of MEK inhibitors can up-regulate CAR expression, resulting in enhanced adenovirus entry into the cells. However, inhibition of MEK results in G1 cell cycle arrest, rendering infected cells temporarily unable to produce virus. This forces a tradeoff. While drug mediated up-regulation of CAR enhances virus entry into cancer cells, the consequent cell cycle arrest inhibits production of new virus particles and the replication of the virus. Optimal control-based schedules of MEK inhibitor application should increase the efficacy of this treatment, maximizing the overall tumor toxicity by exploiting the dynamics of CAR expression and viral production. We introduce a mathematical model of these dynamics and show simple optimal control based strategies which motivate this approach.     

Effects of mutations in DNA repair genes on formation of ribosomal DNA circles and life span in Saccharomyces cerevisiae

A cause of aging in Saccharomyces cerevisiae is the accumulation of extrachromosomal ribosomal DNA circles (ERCs). Introduction of an ERC into young mother cells shortens life span and accelerates the onset of age-associated sterility. It is important to understand the process by which ERCs are generated. Here, we demonstrate that homologous recombination is necessary for ERC formation. rad52 mutant cells, defective in DNA repair through homologous recombination, do not accumulate ERCs with age, and mutations in other genes of the RAD52 class have varying effects on ERC formation. rad52 mutation leads to a progressive delocalization of Sir3p from telomeres to other nuclear sites with age and, surprisingly, shortens life span. We speculate that spontaneous DNA damage, perhaps double-strand breaks, causes lethality in mutants of the RAD52 class and may be an initial step of aging in wild-type cells.     

Analyzing time-series microarray data reveals key genes in spinal cord injury

Although many scholars have utilized high-throughput microarrays to delineate gene expression patterns after spinal cord injury (SCI), no study has evaluated gene changes in raphe magnus (RM) and somatomotor cortex (SMTC), two areas in brain primarily affected by SCI. In present study, we aimed to analyze the differentially expressed genes (DEGs) of RM and SMTC between SCI model and sham injured control at 4, 24 h, 7, 14, 28 days, and 3 months using microarray dataset GSE2270 downloaded from gene expression omnibus and unpaired significance analysis of microarray method. Protein–protein interaction (PPI) network was constructed for DEGs at crucial time points and significant biological functions were enriched using DAVID. The results indicated that more DEGs were identified at 14 days in RM and at 4 h/3 months in SMTC after SCI. In the PPI network for DEGs at 14 days in RM, interleukin 6, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), FBJ murine osteosarcoma viral oncogene homolog (FOS), tumor necrosis factor, and nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) were the top 5 hub genes; In the PPI network for DEGs at 3 months in SMTC, the top 5 hub genes were ubiquitin B, Ras‐related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1), FOS, Janus kinase 2 and vascular endothelial growth factor A. Hedgehog and Wnt signaling pathways were the top 2 significant pathways in RM. These hub DEGs and pathways may be underlying therapeutic targets for SCI.