Inhibition of TnA translocation by TnA.

Plasmids already containing TnA showed decreased susceptibility to the translocation of a further TnA unit when compared with related plasmids that did not contain TnA. The translocation immunity imposed by TnA is exerted only on the plasmid of which it is part. It is suggested that this desensitization by a translocation unit is a general phenomenon that reduces the mutational effects of translocation.     

Rap1 prevents telomere fusions by nonhomologous end joining

Telomeres protect chromosomes from end-to-end fusions. In yeast Saccharomyces cerevisiae, the protein Rap1 directly binds telomeric DNA. Here, we use a new conditional allele of RAP1 and show that Rap1 loss results in frequent fusions between telomeres. Analysis of the fusion point with restriction enzymes indicates that fusions occur between telomeres of near wild-type length. Telomere fusions are not observed in cells lacking factors required for nonhomologous end joining (NHEJ), including Lig4 (ligase IV), KU and the Mre11 complex. SAE2 and TEL1 do not affect the frequency of fusions. Together, these results show that Rap1 is essential to block NHEJ between telomeres. Since the presence of Rap1 at telomeres has been conserved through evolution, the establishment of NHEJ suppression by Rap1 could be universal.     

Chromosome fusions following telomere loss are mediated by single-strand annealing

Progressive telomere shortening eventually results in chromosome fusions and genome instability as the cell's ability to distinguish chromosome ends from DNA double-strand breaks is compromised. In fission yeast, such events frequently produce stable survivors with all circular chromosomes. To shed light on the repair pathways that mediate chromosome end fusions and generate circular chromosomes, we have examined a diverse array of DNA repair factors. We show that telomere attrition-induced chromosome fusions are dependent on the fission yeast homologs of Rad52, the ERCC1/XPF endonuclease, the single-stranded DNA-binding protein RPA, and the Srs2 and Werner/Bloom helicases, but not Ku and ligase 4. Consistent with a recombinational mechanism of single-strand annealing, cloned junctions map to four of five homology regions in subtelomeric DNA. A comparison with telomere uncapping caused by the absence of the double-stranded telomere-binding protein Taz1 demonstrates that the circumstances and cause of telomere dysfunction profoundly affect which DNA repair pathway is engaged.     

High-level bacterial cellulase accumulation in chloroplast-transformed tobacco mediated by downstream box fusions

The Thermobifida fusca cel6A gene encoding an endoglucanase was fused to three different downstream box (DB) regions to generate cel6A genes with 14 amino acid fusions. The DB-Cel6A fusions were inserted into the tobacco (Nicotiana tabacum cv. Samsun) chloroplast genome for protein expression. Accumulation of Cel6A protein in transformed tobacco leaves varied over approximately two orders of magnitude, dependent on the identity of the DB region fused to the cel6A open reading frame (ORF). Additionally, the DB region fused to the cel6A ORF affected the accumulation of Cel6A protein in aging leaves, with the most effective DB regions allowing for high level accumulation of Cel6A protein in young, mature, and old leaves, while Cel6A protein accumulation decreased with leaf age when less effective DB regions were fused to the cel6A ORF. In the most highly expressed DB-Cel6A construct, enzymatically active Cel6A protein accumulated at up to 10.7% of total soluble leaf protein (%TSP). The strategy used for high-level endoglucanase expression may be useful for expression of other cellulolytic enzymes in chloroplasts, ultimately leading to cost-effective heterologous enzyme production for cellulosic ethanol using transplastomic plants.     

Plasmid mediated silver resistance in Acinetobacter baumannii

Acinetobacter baumannii BL88, an environmental isolate, was resistant to 13 metals and 10 antibiotics. Plumbagin cured resistance to silver, cadmium, antimony, streptomycin and ampicillin at varying frequencies. However, only silver resistance transferred (1 × 10^–6 recepient^–1) to Escherichia coli K12 during conjugation. Correspondingly there was transfer of a 54 kb plasmid (pUPI199) from A. baumannii BL88. The plasmid transformed E. coli DH5 cells at a frequency of 1 × 10^–8 recepient^–1. The growth rate of E. coli DH5; (pUPI199) was slower as compared with E. coli DH5. Plasmid pUPI199 was 76 and 9.6% stable in the host A. baumannii BL88 in the presence and absence of selection pressure, respectively. A. baumannii BL88 was found to accumulate and retain silver whereas E. coli DH5 (pUPI199) effluxed 63% of the accumulated silver ions.     

Plasmid mediated metal and antibiotic resistance in marinePseudomonas

Pseudomonas sp isolated from the Bay of Bengal (Madras coast) contained a single large plasmid (pMR1) of 146 kb. Plasmid curing was not successful with mitomycin C, sodium dodecyl sulfate, acridine orange, nalidixic acid or heat. Transfer of mercury resistance from marinePseudomonas toEscherichia coli occurred during mixed culture incubation in liquid broth at 10^–4 to 10^–5 ml^–1. However, transconjugants lacked the plasmid pMR1 and lost their ability to resist mercury. Transformation of pMR1 intoE. coli competent cells was successful; however, the efficiency of transformation (1.49×10² Hg^r transformants g^–1 pMR1 DNA) was low.E. coli transformants containing the plasmid pMR1 conferred inducible resistance to mercury, arsenic and cadmium compounds similar to the parental strain, but with increased expression. The mercury resistant transformants exhibited mercury volatilization activity. A correlation existed between metal and antibiotic resistance in the plasmid pMR1.     

Plasmid mediated enhancement of uv resistance in Streptococcus faecalis

A 38.5-Mdal plasmid of Streptococcus faecalis subsp. zymogenes has been shown to enhance survival following uv irradiation. In addition, the presence of this plasmid increases the mutation frequencies following uv irradiation and enhances “W”-reactivation. The data presented indicate that S. faecalis has an inducible error-prone repair system and that the plasmid enhances these repair functions.     

Tetracycline resistance determined by pBR322 is mediated by one polypeptide

Only one polypeptide specified by plasmid pBR322 is necessary to determine tetracycline resistance. Small deletions in pBR322 constructed in vitro which result in the lack of ability to confer tetracycline resistance in vivo also result in the absence or alteration of this polypeptide in vivo. Other deletions define the extent of material necessary to encode this polypeptide. A correction to the DNA sequence of the tetracycline resistance cistron has been determined which confirms these observations.     

Highly efficient selection of phage antibodies mediated by display of antigen as Lpp-OmpA' fusions on live bacteria

Delayed infectivity panning (DIP) is a novel approach for the in vivo isolation of interacting protein pairs. DIP combines phage display and cell surface display of polypeptides as follows: an antigen is displayed in many copies on the surface of F(+) Escherichia coli cells by fusing it to a Lpp-OmpA' hybrid. To prevent premature, non-specific infection by phage, the cells are rendered functionally F(-) by growth at 16 degrees C. The antigen-displaying cells are used to capture antibody-displaying phage by virtue of the antibody-antigen interaction. Following removal of unbound phage, infection of the cells by bound phage is initiated by raising the temperature to 37 degrees C that facilitates F pilus expression. The phage then dissociate from the antigen and infect the bacteria through the F pilus. Using specific scFv antibodies and the human ErbB2 proto-oncogene and IL2-Ralpha chain as model antibody-antigen pairs, we demonstrate enrichment of those phage that display a specific antibody over phage that display an irrelevant antibody of over 1,000,000 in a single DIP cycle. We further show the successful isolation of anti-toxin, anti-receptor, anti-enzyme and anti-peptide antibodies from several immune phage libraries, a shuffled library and a large synthetic human library. The effectiveness of DIP makes it suitable for the isolation of rare clones present in large libraries.Since DIP can be applied for most of the phage libraries already existing, it could be a powerful tool for the rapid isolation and characterization of binders in numerous protein-protein interactions.     

Verprolin cytokinesis function mediated by the Hof one trap domain

In budding yeast, partitioning of the cytoplasm during cytokinesis can proceed via a pathway dependent on the contractile actomyosin ring, as in other eukaryotes, or alternatively via a septum deposition pathway dependent on an SH3 domain protein, Hof1/Cyk2 (the yeast PSTPIP1 ortholog). In dividing yeast cells, Hof1 forms a ring at the bud neck distinct from the actomyosin ring, and this zone is active in septum deposition. We previously showed the yeast Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) ortholog, verprolin/Vrp1/End5, interacts with Hof1 and facilitates Hof1 recruitment to the bud neck. A Vrp1 fragment unable to interact with yeast WASP (Las17/Bee1), localize to the actin cytoskeleton or function in polarization of the cortical actin cytoskeleton nevertheless retains function in Hof1 recruitment and cytokinesis. Here, we show the ability of this Vrp1 fragment to bind the Hof1 SH3 domain via its Hof one trap (HOT) domain is critical for cytokinesis. The Vrp1 HOT domain consists of three tandem proline-rich motifs flanked by serines. Unexpectedly, the Hof1 SH3 domain itself is not required for cytokinesis and indeed appears to negatively regulate cytokinesis. The Vrp1 HOT domain promotes cytokinesis by binding to the Hof1 SH3 domain and counteracting its inhibitory effect.     

Transposition of TnA does not generate deletions

Summary We have examined the incidence of loss of the TnA unit, Tn801, from RP1 under conditions where transposition of Tn801 to another replicon, R388, was readily detected. We found that the frequency of transposition of Tn801 from RP1 to R388 exceeded, by at least a factor of one hundred, the frequency at which it was deleted from RP1. We conclude that, in general, transposition of Tn801 does not generate derivatives of the donor plasmid which specifically lack Tn801. The relevance of these findings to the mechanism of transposition is discussed.     

Capping one end of an actin filament affects elongation at the other end

The rates of elongation at the free ends of actin filaments were compared to those of intact filaments, when the one end was masked with muscle beta-actinin or cytochalasin D, using fixed actoheavy meromyosin and Limulus acrosomal actin bundles as seeds. Experimental conditions were chosen so as to prevent spontaneous filament formation as far as possible. The rate of elongation at the barbed end of fixed actoheavy meromyosin was reduced to about one-fourth when the other pointed end was capped by beta-actinin, and that at the pointed end was reduced to one-third when the barbed end was blocked by cytochalasin D. Similar effects were also observed with the packed actin bundles of horseshoe crab sperm, although the decreases in elongation were less marked: 50-60% of the control both in the presence of beta-actinin and cytochalasin D. To explain the peculiar "end effect" described above, it is proposed that possible conformational changes at one end of an actin filament caused by the binding of a capping substance are transmitted successively to the other end so as to affect the elongation there.     

Establishment of genomic library technology mediated by non-homologous end joining mechanism in Yarrowia lipolytica

Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits. The non-homologous end joining (NHEJ), which enables foreign DNA fragments to be randomly integrated into different chromosomal sites, shows prominent capability in genomic libraries construction. In this study, we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process, employment of defective Ura marker and optimization of iterative transformations, which enhanced genes integration efficiency by 4.67, 22.74 and 1.87 times, respectively. We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of CrtE, CrtB and CrtI, with 23.8 times higher production than rDNA integration through homologous recombination (HR). The NHEJ-mediated genomic library technology also achieved random and scattered integration of loxP and vox sites, with the copy number up to 65 and 53, respectively, creating potential for further application of recombinase mediated genome rearrangement in Y. lipolytica. This work provides a high-efficient NHEJ-mediated genomic library technology, which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h. This novel technology also lays an excellent foundation for the development of other genetic technologies in Y. lipolytica.

     

Plasmid mediated mutagenesis of a cellular gene in transfected eukaryotic cells.

NIH3T3 cells are widely used in transformation assays and readily take up transfected DNA. A system has been devised using NIH3T3 cells to measure the mutagenic effect of transfected DNA on recipient cell genes. NIH3T3 cells can be mutated to 6-thioguanine resistance at a frequency which suggests that at least a portion of the cells have only one functional copy of the HGPRT gene. They have a low spontaneous background mutation frequency (approximately 1 X 10(-7)). Transfection of three different plasmids into NIH3T3 cells induced 6-thioguanine resistant mutants at frequencies ranging from 3 to 11 fold above background. The mutant phenotype is stable and reversion frequencies of several mutants are less than or equal to 1 X 10(-7). Southern blot analysis of the HGPRT gene in several mutants showed that 4 of 26 mutants (15.4%) had detectable alterations in the structure of the HGPRT gene. Interestingly 3 of the 4 mutants showing rearrangements were obtained by transfection of the HSV-2 morphological transforming region.     

Transposition of lambda placMu is mediated by the A protein altered at its carboxy-terminal end

Lambda placMu phages are derivatives of bacteriophage lambda that use the transposition machinery of phage Mu to insert into chromosomal and cloned genes. When inserted in the proper fashion, these phages yield stable fusions to the Escherichia coli lac operon in a single step. We have determined the amount of DNA from the c end of phage Mu present in one of these phages, lambda placMu3, and have shown that this phage carries a 3137-bp fragment of Mu DNA. This DNA segment carries the Mu c-end attachment site and encodes the Mu genes cts62, ner+, and gene A lacking 179 bp at its 3' end (A'). The product of this truncated gene A' retains transposase activity and is sufficient for the transposition of lambda placMu. This was demonstrated by showing that lambda placMu derivatives carrying the A am1093 mutation in the A' gene are unable to transpose by themselves in a Su- strain, but their transposition can be triggered by coinfection with lambda pMu507(A+ B+). We have constructed several new lambda placMu phages that carry the A' am1093 gene and the kan gene, which confers resistance to kanamycin. Chromosomal insertions of these new phages are even more stable than those of the previously reported lambda placMu phages, which makes them useful tools for genetic analysis.     

Effect of x-irradiation of one partner on hybrid frequency in fusions between Chinese hamster cells

Sublines derived from V79 and B150 Chinese hamster cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and thymidine kinase (TK) respectively. When these strains are cultivated together without virus treatment in HAT medium, hybrid colonies with normal enzymic patterns arise in high frequency. The ratio of proliferating hybrid colonies to the minority cell inoculum in mixed cultures is approximately 1/1000. This observation has been used to examine the effect of x-irradiation pretreatment of one cell type on the frequency of hybrid formation in mixed cultures. B150 cells (TK-) were irradiated in monolayer cultures at dose levels of 200–4000r (180 KV, 15 ma). After trypsinization, the irradiated cells were mixed with unirradiated V79 cells (subline 129, HGPRT-) and cultivated in HAT medium. The per cent decline in hybrid frequency as a function of x-irradiation was compared to the relative decline in viability of x-irradiated B150 cells when assayed in cultures alone. These curves diverged markedly, with the frequency of proliferating hybrids decreasing much less rapidly with radiation dose than survival of B150 cells. By contrast, when B150 cells were subjected to acute heat shock for graded intervals and subsequently mixed with untreated V79–129 cells, both viability of B150 cells and hybrid formation declined at the same rate. Our results suggest that lethally irradiated cells can still fuse with normal cells, and may contribute functional chromosomes to the hybrid karyotype. At limiting doses of x-irradiation this technique may be useful as a means for induced segregation of marker chromosomes.