Synthetic Polylinker Research Articles

Modeling Oculopharyngeal Muscular Dystrophy in Myotube Cultures Reveals Reduced Accumulation of Soluble Mutant PABPN1 Protein

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by an alanine tract expansion mutation in poly(A) binding protein nuclear 1 (expPABPN1). To model OPMD in a myogenic and physiological context, we generated mouse myoblast cell clones stably expressing either human wild type (WT) or expPABPN1 at low levels. Transgene expression is induced on myotube differentiation and results in formation of insoluble nuclear PABPN1 aggregates that are similar to those observed in patients with OPMD. Quantitative analysis of PABPN1 in myotube cultures revealed that expPABPN1 accumulation and aggregation is greater than that of the WT protein. We found that aggregation of expPABPN1 is more affected than WT PABPN1 by inhibition of proteasome activity. Consistent with this, in myotube cultures expressing expPABPN1, deregulation of the proteasome was identified as the most significantly perturbed pathway. Differences in the accumulation of soluble WT and expPABPN1 were consistent with differences in ubiquitination and rate of protein turnover. This study demonstrates, for the first time to our knowledge, that, in myotubes, the ratio of soluble/insoluble expPABPN1 is significantly lower compared with that of the WT protein. We suggest that this difference can contribute to muscle weakness in OPMD.

Impaired Ca2+ Store Functions in Skeletal and Cardiac Muscle Cells from Sarcalumenin-deficient Mice

Sarcalumenin (SAR), specifically expressed in striated muscle cells, is a Ca2+-binding protein localized in the sarcoplasmic reticulum (SR) of the intracellular Ca2+ store. By generating SAR-deficient mice, we herein examined its physiological role. The mutant mice were apparently normal in growth, health, and reproduction, indicating that SAR is not essential for fundamental muscle functions. SAR-deficient skeletal muscle carrying irregular SR ultrastructures retained normal force generation but showed slow relaxation phases after contractions. A weakened Ca2+ uptake activity was detected in the SR prepared from mutant muscle, indicating that SAR contributes to Ca2+ buffering in the SR lumen and also to the maintenance of Ca2+ pump proteins. Cardiac myocytes from SAR-deficient mice showed slow contraction and relaxation accompanied by impaired Ca2+ transients, and the mutant mice exhibited a number of impairments in cardiac performance as determined in electrocardiography, ventricular catheterization, and echocardiography. The results obtained demonstrate that SAR plays important roles in improving the Ca2+ handling functions of the SR in striated muscle.

Antibiotic resistance gene cassettes derived from the omega interposon for use in E. coli and Streptomyces.

Three antibiotic resistance gene cassettes, derived from the omega interposon (Prentki and Krisch (1984) Gene 29, 303-313) were constructed. These cassettes carry different antibiotic resistance genes, conferring resistance to geneticin, hygromycin or viomycin, flanked by short inverted repeats containing transcription and translation termination signals and synthetic polylinkers. These cassettes were designated omega aac, omega hyg and omega vph. Resistance phenotypes conferred by these constructions are selectable in E. coli and Streptomyces. These cassettes can be used for insertional mutagenesis or for vector construction.

Maturation-specific translational enhancement mediated by the 5'-UTR of a late pollen transcript.

The tomato lat52 gene encodes an abundant protein specifically expressed in the vegetative cell of the pollen grain during pollen maturation which is essential for normal pollen tube growth. Multiple upstream cis-regulatory elements controlling the level and specificity of lat52 transcription have previously been identified. This research investigated the role of the 5'-untranslated region (5'-UTR) in controlling lat52 expression. In transient expression assays, gene fusion constructs containing the lat52 5'-UTR were expressed in pollen at levels 13- to 60-fold above those in which synthetic polylinker sequences replaced the lat52 5'-UTR. This enhancement was shown to be independent of both the promoter sequences, the linked reporter gene and the 3'-UTR. Analysis of RNA and protein levels in transgenic plants containing such gene fusions demonstrated that the lat52 5'-UTR conferred a dramatically increased translational yield to heterologous transcripts in a pollen-specific and strictly developmentally regulated manner during the final stages of pollen maturation. These results represent a novel example of translational enhancement in plants in that translational yield is regulated developmentally in a cell-specific manner via sequences located within the 5'-UTR.

Cloning vectors for lactococci based on a plasmid encoding resistance to cadmium.

An 8.8-kb plasmid (pND302) was identified in Lactococcus lacti spp lactis M71 which encodes cadmium resistance (CdR). Most of the commercial lactococcal strains tested were sensitive to cadmium. Therefore, CdR should provide a useful selectable marker for constructing cloning vectors in lactococci. pND302 was mapped with a number of restriction enzymes and found to contain a unique EcoRI site suitable for cloning. Two E. coli/L lactis shuttle cloning vectors, pND304 and pND624, were constructed by subcloning of the E. coli plasmids pBR322 and pGEM-7Zf(+) containing a 1.6-kb gene encoding nisin resistance (NisR) of lactococcal origin into the EcoRI site of pND302, separately. The E. coli DNA component of pND624 was removed and the resulting plasmid, pND625, consisted of only lactococcal DNA, expressing NisR and CdR, with two synthetic polylinkers that contain multiple restriction sites for versatile cloning. Both pND302 and pND625 can be transformed by electroporation into L. lactis LMO230 at 10(3)/micrograms DNA and maintained stably in LMO230. The results indicated that pND302 and pND625 are potential food-grade cloning vectors for lactococci.

Isolation of Lactococcus lactis nonsense suppressors and construction of a food‐grade cloning vector

Nonsense suppressor strains of Lactococcus lactis were isolated using plasmids containing nonsense mutations or as revertants of a nonsense auxotrophic mutant. The nonsense suppressor gene was cloned from two suppressor strains and the DNA sequence determined. One suppressor is an ochre suppressor with an altered tRNA(gln) and the other an amber suppressor with an altered tRNA(ser). The nonsense suppressors allowed isolation of nonsense mutants of a lytic bacteriophage and suppressible auxotrophic mutants of L. lactis MG1363. A food-grade cloning vector based totally on DNA from Lactococcus and a synthetic polylinker with 11 unique restriction sites was constructed using the ochre suppressor as a selectable marker. Selection, following electroporation of a suppressible purine auxotroph, can be done on purine-free medium. The pepN gene from L. lactis Wg2 was subcloned resulting in a food-grade plasmid giving a four- to fivefold increase in lysine aminopeptidase activity.

Efficient Transformation of the Cephamycin C Producer Nocardia lactamdurans and Development of Shuttle and Promoter-Probe Cloning Vectors

A high transformation efficiency (1 x 10 to 7 x 10 transformants per mug of DNA) of Nocardia lactamdurans LC411 was obtained by direct treatment of mycelium with polyethylene glycol 1000 and cesium chloride. A variety of vectors from Streptomyces lividans, Brevibacterium lactofermentum, Rhodococcus fascians, and a Nocardia (Amycolatopsis) sp. were tested; transformants could be obtained only with vectors derived from an endogenous plasmid of the Amycolatopsis sp. strain DSM 43387. Vectors carrying the kanamycin resistance gene (kan) as a selective marker were constructed. The transformation procedure has been optimized by using one of these vectors (pULVK1) and studying the influence of the age of the culture, concentrations of cesium chloride and polyethylene glycol, amount of plasmid DNA, and nutrient supplementations of the growth medium. Versatile shuttle cloning vectors (pULVK2 and pULVK3) have been developed by subcloning the pBluescript KS(+) multiple cloning site or a synthetic polylinker containing several unique restriction sites (EcoRV, DraI, BamHI, SstI, EcoRI, and HindIII). A second marker, the apramycin resistance gene (amr) has been added to the vectors (pULVK2A), allowing insertional inactivation of one of the markers while using the second one for selection. An alternative marker, the amy gene of Streptomyces griseus (pULAM2), which is easily detected by the release of extracellular amylase in transformants of N. lactamdurans carrying this vector, has been added. Two promoter-probe plasmids, pULVK4 and pULVK5, have been constructed, with the promoterless xylE gene as a reporter, for utilization in N. lactamdurans.

Expression of parasite antigens in Escherichia coli

Immunological detection of cloned parasite antigens in E. coli offers an efficient means of identifying parasite antigens which may be relevant to immunity. Moreover, this technique enables the sequence of an antigen to be determined and also enables purification of the recombinant protein from bacterial lysates. The variety of different vectors available means that specific vectors are available for most purposes. Useful vectors are available which have not yet been employed to study parasites. Lambda phage has been extensively used while other phage vector systems have not, even though they could offer advantages over existing systems. Cloning in the M13 vector for example, allows direct sequencing of cloned parasite genes or gene fragments from expressing clones (Messing, 1983). A series of M13 vectors has been constructed with a Hind 111 cloning site in all three reading frames specifically for this purpose (Lathe et al., 1984). Some plasmid vectors which could be used rely on expression of hybrid fusion proteins. The pUR vectors 278, 279, 289, 290, 291 and 292 (Ruther & Muller-Hill, 1983) have a full length lac Z gene with synthetic polylinkers at the 5' end in all three phases so that a hybrid β-1 fusion protein can be produced under the control of the lac UV5 promotor. The pEX 1–3 vectors (Stanley et al., 1984) have lambda Pl promotor and N-terminus linked to β-galactosidase and contain a 5' synthetic polylinker in three translational phases. This vector was designed for the cloning of short regions of foreign DNA which can be probed with antibodies to define an epitope. This approach to dissecting antigens has great potential for the analysis of complex antigens like those of P. falciparum The interest in expressing foreign genes in bacteria ensures that host-vector systems will continue to improve. Although prokaryotic expression systems have obviously worked successfully for the expression of many foreign genes, problems still exist where carbohydrate addition or other eukaryotic post-translational modifications are important in the generation of epitopes. One approach to solving these problems associated with heterologous expression of proteins, is to use eukaryotic host-vector systems such as yeast or vaccinia virus infected mammalian cells (Mackett et al., 1984). The vaccinia virus system has been used to express the P. knowlesi sporozoite antigen (Smith et al., 1984) and a hybrid form of the normally secreted P. falciparum S antigen has been expressed on the surface of vaccinia virus infected cells, anchored in the cell membrane (Langford et al., 1986). These live, infectious viruses can be used as live vaccines. Special problems exist with these systems and it is still not possible to generate the large libraries possible using E. coil. Consequently, it is not possible to routinely detect rare sequences in eukaryotic libraries as it is in E. coli. The ability to generate large amounts of parasite fusion proteins will allow production of reagents for detecting infections. Furthermore, it should soon be possible to study parasitic diseases that hitherto could not be studied and to isolate defined elements that can be used to vaccinate against parasitic disease.

Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of Gram-negative bacteria

We have constructed a series of derivatives of the Ω interposon [Prentki and Krisch, Gene 29 (1984) 303–313 ] that can be used for in vitro insertional mutagenesis. Each of these DNA fragments carries a different antibiotic or Hg 2+ resistance gene (Ap R, Cm R, Tc R, Km R or Hg R) which is flanked, in inverted orientation, by transcription and translation termination signals and by synthetic polylinkers. The DNA of these interposons can be easily purified and then inserted, by in vitro ligation, into a plasmid linearized either at random by DNase I or at specific sites by restriction enzymes. Plasmid molecules which contain an interposon insertion can be identified by expression of its drug resistance. The position of the interposon can be precisely mapped by the restriction sites in the flanking polylinker. To verify their properties we have used these Ω derivatives to mutagenize a broad host range plasmid which contains the entire meta-cleavage pathway of the toluene degradation plasmid pWW0 of Pseudomonas putida. Insertion of these interposons in the plasmid between the promoter and the catechol 2,3-dioxygenase (C23O) gene dramatically reduced the expression of this enzyme in Escherichia coli. We also show that when a plasmid containing an ω interposon is transferred by conjugal mobilization from E. coli to P. putida, Agrobacterium tumefaciens, Erwinia chrysanthemi, Paracoccus denitrificans or Rhizobium leguminosarum, the appropriate interposon drug resistance is usually expressed and, compared to the non-mutated plasmid, much reduced levels of C23O activity are detected. Thus, the selection and/or characterization of Ω insertional mutations can be carried out in these bacterial species.

Ω mutagenesis in gram-negative bacteria: a selectable interposon which is strongly polar in a wide range of bacterial species

We have used the 2.0-kb DNA fragment Ω [Prentki and Krisch, Gene 29 (1984) 303–313] to mutagenize in vitro a broad-host-range plasmid carrying the entire meta-cleavage pathway of the Pseudomonas putida TOL plasmid pWW0. The mutant plasmids were subsequently introduced by conjugal mobilization into a variety of Gram-negative bacteria. The Ω fragment carries a selectable marker ( aadA +; Spc R /Sm R), which is expressed in all species tested, as well as flanking transcription and translation termination signals and synthetic Polylinkers. Expression of the plasmid-bome catechol 2,3-dioxygenase (C230) gene, situated downstream from the site of Ω insertion, was substantially reduced in all strains tested. The transcription terminators originally cloned from bacteriophage T4 gene 32, are apparently functional in a wide range of hosts. Insertional mutagenesis with the Ω ‘interposon’ can thus be used in a wide variety of species, with the advantages of a positive selection for the presence of the fragment, the termination of RNA and protein synthesis beyond the site of insertion, and genetic stability of the resulting mutation.

In vitro insertional mutagenesis with a selectable DNA fragment

A new method for in vitro insertional mutagenesis of genes cloned in Escherichia coli is presented. This simple procedure combines the advantages of in vitro DNA linker mutagenesis with those of in vivo transposition mutagenesis. It makes use of the Ω fragment, a 2.0-kb DNA segment consisting of an antibiotic resistance gene (the Sm r/Spc r gene of the R100.1 plasmid) flanked by short inverted repeats carrying transcription and translation termination signals and synthetic polylinkers. The Ω fragment is inserted into a linearized plasmid by in vitro ligation, and the recombinant DNA molecules are selected by their resistance to streptomycin and spectinomycin. The Ω fragment terminates RNA and protein synthesis prematurely, thus allowing the definition and mapping of both transcription and translation units. Because of the symmetrical structure of Ω, the same effect is obtained with insertions in either orientation. The antibiotic resistance gene can be subsequently excised from the mutated molecules, leaving behind its flanking restriction site(s).