SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
The genome of Mycobacterium smegmatis mc(2)155 contains a 56kb duplicated region. We isolated a mutant of mc(2)155 lacking this duplication (DeltaDRKIN). This mutation did not affect the growth rate, surface properties or transformation efficiency of the organism, confirming the potential utility of DeltaDRKIN for the study of genes contained within the duplicated region.
Rare earth elements have been emitted into the environment largely as fertilizer components. This has caused much fear about whether they would influence our environment, especially on the metabolism and genetics of microorganisms. In this article, the trivalent ion of a rare earth element, lanthanum, was studied for the effects on growth, transformation, and gene expression of Escherichia coli. The results showed that La3+ at concentrations from 50 to 150 microg/mL stimulated the endogenic metabolism and ectogenic metabolism, but had few effects on gene expression. La3+ at lower concentrations from 0.5 to 30 microg/mL inhibit intensively E. coli-absorbing external DNA, decreasing the transformation efficiency. It is also supported by observations using transmission electron microscopy. Our results are significant in understanding the function of rare earth elements to microorganisms and assessing the risk of application of rare earth compounds.
We report on the second phase of a programme to select a relative of Arabidopsis thaliana for use in large-scale molecular genetic studies of nickel (Ni) and zinc (Zn) hyperaccumulation. We also report on the relatedness among Thlaspi caerulescens accessions and the utility of using O-acetyl-L-serine as a marker for Ni and Zn hyperaccumulation potential. Twenty-seven new accessions of metal-accumulating species collected in the Czech Republic, France, Greece, Italy, Slovenia and the USA during Spring-Summer 2002 were evaluated. The criteria established for selection were hyperaccumulation of metals (Ni and Zn); compact growth habit; reasonable time to flowering; production of > or = 1000 seeds per plant; self-fertility; compact diploid genome; high sequence similarity to A. thaliana; > or = 0.1% transformation efficiency with easy selection. We conclude that the best candidate identified in the first phase was the best candidate overall: T. caerulescens accession St Félix de Pallières.
Petunia hybrida genetic transformation continues to be a valuable tool for genetic research into biochemical pathways and gene expression, as well as generating commercial products with varying floral colors. In this chapter, we describe a simple and reproducible genetic transformation protocol for generating transgenic petunia plants harboring a gene of interest and selectable marker. The system utilizes Agrobacterium tumefaciens for transgene integration with plant recovery via shoot organogenesis from leaf explant material. Selection for transgenic plants is achieved using the bar gene conferring resistance to glufosinate or nptII gene for resistance to kanamycin. Transformation efficiencies of around 10% are achievable with shoots being recovered about 8 wk after transgene insertion and rooted plants transferred to the greenhouse about twelve weeks after inoculation.
Advances in sequencing technology enabling rapid and inexpensive whole-genome sequencing highlight how few genes are functionally characterized. This problem is particularly acute in filamentous fungi, where even in the best studied organisms upward of half of genes are poorly characterized or unannotated. High-throughput tools to identify gene function exist for single-celled organisms, like yeast and bacteria. However, filamentous fungi present challenges to high-throughput gene characterization, including low transformation efficiency and multinucleate cells. Filamentous fungi are critical components of nutrient cycling in ecosystems, form symbioses with plants that improve nutrient uptake, and are devastating human, plant, and animal pathogens causing millions of deaths and substantial crop loss each year. Thus, it is critical to overcome challenges to rapid gene characterization in filamentous fungi. We generated a library of hundreds of millions of uniquely barcoded plasmids containing a broad host-range drug resistance marker for ectopic insertion into filamentous fungal genomes by Agrobacterium tumefaciens. We then optimized A. tumefaciens mediated transformation of the biocontrol agent Trichoderma atroviride and made an insertional mutagenesis library containing 83,311 barcoded insertions, disrupting 5,331 of 11,863 predicted genes. This library enables high-throughput screens to rapidly connect genotype to phenotype. Quantifying relative barcode abundance in the pooled library before and after exposure to experimental conditions identified candidate genes and recovered known pathway components in amino acid biosynthetic, fructose utilization, and xylose utilization pathways. This resource establishes a scalable platform for high-throughput functional genomics in filamentous fungi, enabling investigations of fungal biology to improve medical outcomes, biotechnology, and sustainable agriculture.
Application of biotechnology to the cultivated mushroom, Agaricus bisporus, has been hampered thus far by the lack of a transformation system. Here, transformation of both a homo- and a heterokaryotic strain of A. bisporus to hygromycin B resistance is described. Transforming DNA was integrated into the A. bisporus genome and stably maintained throughout vegetative growth. Transformants of the heterokaryotic strain formed transgenic fruiting bodies. Promoters derived from the unrelated ascomycete Aspergillus nidulans and from A. bisporus itself, were able to drive expression of the hygromycin B resistance gene. Expression controlled by a fragment of 265 bp from the A. bisporus GPD promoter was sufficient to generate transformants. However, transformation efficiency was not enhanced by using this homologous promoter.
Some genital human papillomavirus (HPV) types, such as 16 and 18, are highly associated with malignant cervical tumors while others, such as HPV 6, are only rarely found in these malignancies. The E7 oncoproteins of HPV 6, 16 and 18 each have a 17 amino acid region with striking homology to adenovirus E1a and SV40 LT. E1a, LT and the E7 oncoprotein of HPV16 all bind the cellular Rb protein in vitro, and for E1a and LT this region of homology contains sequences essential for interaction with Rb. We have now found that in HPV 16 E7 this region (amino acids 21-37) contains two separate biochemical activities, each of which contributes to E7-mediated transformation. Rb binding was localized to the N terminus of this region, while the C terminus was shown to serve as a substrate for casein kinase (CK) II, which phosphorylated serine-31 and serine-32. Replacement of the two serines by non-phosphorylatable amino acids led to a reduction in transforming activity and abolished phosphorylation but did not affect Rb binding. Rb binding and CK II phosphorylation were also examined for the E7 proteins of HPV 6 and HPV 18. HPV 16 and 18 E7 bound similar amounts of Rb, but HPV 6 E7 consistently bound less. Phosphorylation rates also varied, with HPV 18 E7 being 2-fold faster than HPV 16 E7, which in turn was 2-fold faster than HPV 6 E7. We conclude that Rb binding and phosphorylation of E7 by CKII are independent activities which are required for efficient transformation by E7 and that these activities correlate directly with the relative oncogenic potential of these viruses.
A method for cloning mRNAs has been used which results in a high yield of recombinants containing complete 5'-terminal mRNA sequences. It is not dependent on self-priming to generate double-stranded DNA and therefore the S1 nuclease digestion step is not required. Instead, the cDNA is dCMP-tailed at its 3'-end with terminal deoxynucleotidyl transferase (TdT). The synthesis of the second strand is primed by oligo(dG) hybridized to the 3'-tail. Double-stranded cDNA is subsequently tailed with dCTP and annealed to dGMP-tailed vector DNA. This approach overcomes the loss of the 5'-terminal mRNA sequences and the problem of artifacts which may be introduced into cloned cDNA sequences. Chicken lysozyme cDNA was cloned into pBR322 by this procedure with a transformation efficiency of 5 x 10(3) recombinant clones per ng of ds-cDNA. Sequence analysis revealed that at least nine out of nineteen randomly isolated plasmids contained the entire 5'-untranslated mRNA sequence. The data strongly support the conclusion that the 5'-untranslated region of the lysozyme mRNA is heterogeneous in length.
The genome of Vibrio cholerae consists of two circular chromosomes of different sizes. Here, a comparative analysis of the replication origins of the large chromosomes (oriCIvc) of classical and El Torbio types of the pathogen is reported. Extensive nucleotide sequence analyses revealed that the oriCIvc region has six DnaA boxes instead of the five found in Escherichia coli oriC. The additional DnaA box, designated Rv, was unique in V. cholerae as well as in other members of the family Vibrionaceae. However, Rv was not found to be essential for the autonomous replication function of the 307-bp oriCIvc minimal region. In contrast to El Tor and the recently evolved V. cholerae 0139 strains, the oriCIvc region of the classical biotype showed only a single base transition (T-->G) in a highly conserved AT-rich 13-mer R repeat region. From the minichromosome copy number and its transformational efficiency analyses, it appears that the single base substitution in the oriCIvc of the classical biotype has a significant effect on its replication initiation.
A Thermus thermophilus selector strain for production of thermostable and thermoactive alpha-galactosidase was constructed. For this purpose, the native alpha-galactosidase gene (agaT) of T. thermophilus TH125 was inactivated to prevent background activity. In our first attempt, insertional mutagenesis of agaT by using a cassette carrying a kanamycin resistance gene led to bacterial inability to utilize melibiose (alpha-galactoside) and galactose as sole carbohydrate sources due to a polar effect of the insertional inactivation. A Gal(+) phenotype was assumed to be essential for growth on melibiose. In a Gal(-) background, accumulation of galactose or its metabolite derivatives produced from melibiose hydrolysis could interfere with the growth of the host strain harboring recombinant alpha-galactosidase. Moreover, the AgaT(-) strain had to be Km(s) for establishment of the plasmids containing alpha-galactosidase genes and the kanamycin resistance marker. Therefore, a suitable selector strain (AgaT(-) Gal(+) Km(s)) was generated by applying integration mutagenesis in combination with phenotypic selection. To produce heterologous alpha-galactosidase in T. thermophilus, the isogenes agaA and agaB of Bacillus stearothermophilus KVE36 were cloned into an Escherichia coli-Thermus shuttle vector. The region containing the E. coli plasmid sequence (pUC-derived vector) was deleted before transformation of T. thermophilus with the recombinant plasmids. As a result, transformation efficiency and plasmid stability were improved. However, growth on minimal agar medium containing melibiose was achieved only following random selection of the clones carrying a plasmid-based mutation that had promoted a higher copy number and greater stability of the plasmid.
We describe the self-selection of replication origins of undescribed cryptic plasmids from Thermus aquaticus Y-VII-51B (ATCC 25105) and a Thermus sp. strain (ATCC 27737) by random insertion of a thermostable kanamycin adenyltransferase cartridge. Once selected, these autonomous replication origins were cloned into the Escherichia coli vector pUC9 or pUC19. The bifunctional plasmids were analyzed for their sizes, relationships, and properties as shuttle vectors for Thermus-Escherichia cloning. Seven different vectors with diverse kanamycin resistance levels, stabilities, transformation efficiencies, and copy numbers were obtained. As a general rule, those from T. aquaticus (pLU1 to pLU4) were more stable than those from the Thermus sp. (pMY1 to pMY3). To probe their usefulness, we used one of the plasmids (pMY1) to clone in E. coli a modified form of the cellulase gene (celA) from Clostridium thermocellum in which the native signal peptide was replaced in vitro by that from the S-layer gene of T. thermophilus HB8. The hybrid product was expressed and exported by E. coli. When the gene was transferred by transformation into T. thermophilus, the cellulase protein was also expressed and secreted at 70 degrees C.
A number of factors that are known to influence genetic transformation were evaluated to optimize Agrobacterium-mediated transformation of hypocotyl explants of cauliflower variety Pusa Snowball K-1. The binary vector p35SGUSINT mobilized into Agrobacterium strain GV2260 was used for transformation and transient GUS expression was used as the basis for identifying the most appropriate conditions for transformation. Explant age, preculture period, bacterial strain and density were found to be critical determinants of transformation efficiency. Using the optimized protocol, the synthetic cryIA(b) gene was mobilized into cauliflower. Molecular analyses of transgenics established the integration and expression of the transgene. Insect bioassays indicated the effectiveness of the transgene against infestation by diamondback moth (Plutella xylostella) larvae
The uptake of circular and linear plasmid RSF0885 deoxyribonucleic acids, (DNAs) obtained from Haemophilus parainfluenzae 14, in both homologous and heterologous recipients was studied and compared with that of chromosomal DNA. High concentrations of divalent cations stimulated the uptake of either circular or linear plasmid DNA in H. parainfluenzae 14 competent cells but did not affect the uptake of chromosomal DNA. The biological activity of linear plasmid DNA was similar to that of circular DNA, and the transforming efficiencies for ampicillin resistance of both molecular forms were stimulated by divalent ions. Plasmid DNA was taken up efficiently either with or without the addition of divalent ions but was not biologically active in the heterologous Haemophilus influenzae Rd recipient. Our results suggest that in H. parainfluenzae 14 some of the steps for chromosomal and plasmid DNA uptake are different.
We previously demonstrated that a point mutation in the last intron of the human H-ras oncogene causes a significant increase in its expression and transforming efficiency. Here we establish the basis of this phenomenon. Using gene reconstruction experiments, we have identified a negative-acting element in the intron that is completely inactivated by the mutation. The effects of other nucleotide alterations introduced into this region suggested that the negative element might constitute an alternative exon. Transcripts containing this putative exon were identified and S1 nuclease analysis confirmed that the mutation prevents their synthesis. The abundance of these transcripts is low, apparently due to message instability and/or defective processing. The predicted product of the alternative transcript is suggested to lack transforming potential. Our findings demonstrate that alternative splicing normally operates to suppress p21H-ras expression and that this negative control is abolished by a variety of mutations that interfere with this process.
Kidney cells of an Indian muntjak were transformed with simian virus 40 (SV40). The transformation efficiency of the tertiary cultures was very high when estimated by the agar suspension culture method. The efficiency was about 0.015% when infected at an input multiplicity of 0.4 p.f.u./cell. Clonal cell lines were established from the colonies in soft agar medium. Most of the cell lines and their subclones produced a small amount of infectious SV40. The SV40 virion antigen-positive cells in a clone increased from 0.2% to about 40% by the treatment with mitomycin C. More than 70% of the cells in two cell lines were normal in G- and C-banded karyotypes, indicating that chromosomal change is not a necessary step in the process of transformation of the Indian muntjak cells with SV40.
Replication of the bovine papillomavirus (BPV-1) DNA requires both the viral E1 and E2 gene products. The minimal origin of replication, which resides in a 60-basepair fragment centered on the unique HpaI site of the BPV-1 genome, can be bound by the E1 protein and is flanked by E2 binding sites. The integrity of the region surrounding the unique HpaI restriction enzyme site is important for E1 binding and DNA replication, but the E2 binding sites are not required. The ability of E1 to complex with E2 and the requirement for both factors in DNA replication have led to the hypothesis that the E1/E2 complex may have a direct role in replication. Therefore we have studied mutations in the BPV-1 origin region for E1, E2, and E1/E2 complex binding and for their effect on transformation and replication in the context of the complete viral genome. Transformation and DNA replication were observed with some mutated viral genomes dramatically reduced for E1 binding. These mutated origins could bind significant amounts of E1 in the presence of E2, suggesting that binding of the E1/E2 complex could compensate for the loss of E1 binding in DNA replication. One mutation, with an eight-nucleotide insertion at the HpaI site, was still bound by E1 and the E1/E2 complex, yet did not replicate or transform efficiently. Thus although the binding of E1 or the E1/E2 complex to the origin appears to be necessary for replication, it is not sufficient.
The gram-negative bacterial pathogen Helicobacter pylori, an important aetiological agent of gastroduodenal disease in humans, belongs to a group of bacterial species displaying competence for genetic transformation. Here, we describe the comB gene locus of H. pylori involved in DNA transformation competence. It consists of a cluster of four tandemly arranged genes with partially overlapping open reading frames, orf2, comB1, comB2 and comB3, constituting a single transcriptional unit. Orf2 encodes a 37-amino-acid peptide carrying a signal sequence, whereas comB1, comB2 and comB3 produce 29 kDa, 38 kDa and 42 kDa proteins, respectively, as demonstrated by immunoblotting with specific antisera. For Orf2 and ComB1, no homologous proteins were identified in the database. For ComB3, the best homologies were found with TraS/TraB from the Pseudomonas aeruginosa conjugative plasmid RP1 and TrbI of plasmid RP4, VirB10 from the Ti plasmid of Agrobacterium tumefaciens and PtlG, a protein involved in secretion of pertussis toxin of Bordetella pertussis. Defined transposon knock-out mutants in individual comB genes resulted in transformation-defective phenotypes ranging from a 90% reduction to a complete loss of the natural transformation efficiency. The comB2 and comB3 genes show homology to HP0528 and HP0527, respectively, located on the cagII pathogenicity island of H. pylori strain 26695.
Expression of antisense c-src RNAs in rat and mouse fibroblasts had a dramatic effect on the function of polyoma virus middle T (mT). Antisense c-src RNA decreased the amount of mT:pp60c-src complexes in de novo virus-infected cells and prevented expression of the transformed phenotype in rat F111 cells. Expression of antisense c-src RNA in infected NIH3T3 cells also reduced the formation of mT:pp60c-src complexes but did not affect the ability of polyoma virus to carry out a productive infection. Further analysis of the effects of antisense c-src RNA in uninfected cells revealed that pp60c-src is required for cell growth. When pp60c-src synthesis was reduced, F111 cells stopped proliferating and showed decreased S6 phosphorylation in response to serum. However, F111 cells expressing reduced pp60c-src could be efficiently transformed by v-rasHa, even in the presence of low serum. Thus, pp60c-src appears to function as a component of a signal transduction pathway which regulates cell proliferation in response to serum.