Pattern recognition in the computer analysis of nucleotide sequences

We have used an algorithm from the pattern recognition theory "generalized portrait" to find a distinguishing vector for Escherichia coli promoters. We have made an attempt to solve closely linked problems for choosing significant signs of that signal, multiple alignment and for calculation of the recognition vector (matrix). The promoters with known strength have been ranged with this vector. The analysis of the occurrence of predicted promoters has been carried out. The promoters search program for IBM-compatible computers is available from the authors.     

A single-copy promoter-cloning vector for use in Escherichia coli.

We have constructed and tested a single-copy-plasmid vector (pEU720) based on the IncFII-group plasmid, R100, that is useful for cloning promoters in front of lacZ. The vector is 15 kb long and contains a unique XhoI site in front of lacZ.     

Tissue-specific expression is conferred by a sequence from the 5'' end of the rat albumin gene.

We have constructed a transient expression vector containing 400 bp of rat albumin gene immediate 5'-flanking sequences inserted 5' to the bacterial enzyme chloramphenicol acetyl transferase (CAT). We have transfected various clones of rat hepatoma cells representing different states of expression of the liver phenotype with this vector (pALB-cat) and also with two control vectors containing viral promoters (pSVE-cat and pRSV-cat), and measured activity of the bacterial enzyme CAT in cellular extracts 48 h later. The albumin flanking sequences are able to direct highly efficient CAT expression, compared with the control vectors, only in cells which express their own albumin gene: the albumin-negative hepatoma cells are at least 100 times less efficient in expressing CAT after transfection with the pALB-cat plasmid than are the albumin-positive ones. An unexpected result of our study is the total inability of the rat albumin flanking sequences to direct expression in albumin-producing mouse hepatoma cells.     

p lambda Zd39: a new type of cDNA expression vector for low background, high efficiency directional cloning.

We have developed a new type of bacteriophage lambda vector which provides a strong biological selection against non-recombinants that is independent of the sequences immediately surrounding the cloning site. This system, which we call 'selective substitution', is ideally suited for cDNA expression vectors where it is necessary to flank the cDNA insert with sequence elements (promoters etc.) required to produce a biologically active mRNA in vivo. Selective substitution is a general method, which may be applied to many types of vectors. In this report, we have specifically applied selective substitution to the construction of a new mammalian retrovirus expression vector. The level of background obtained with this vector (that is, the number of plaques obtained when the vector is ligated in the absence of insert DNA) is 0.02% when compared to ligation with restriction fragments and 0.1% to 0.4% when compared to ligation with newly synthesized cDNA. These features have allowed us to easily and efficiently generate several large cDNA libraries using total and size selected cDNA.     

Using RNA interference to develop dengue virus resistance in genetically modified Aedes aegypti

Diseases caused by arthropod-borne viruses are significant public health problems, and novel methods are needed to control pathogen transmission. We hypothesize that genetic manipulation of Aedes aegypti mosquitoes can profoundly and permanently reduce vector competence and subsequent transmission of dengue viruses (DENV) to human hosts. We have identified RNA interference (RNAi) as a potential anti-viral, intracellular pathway in the vector that can be triggered by expression of virus-specific, double stranded RNAs (dsRNAs) to reduce vector competence to DENV. We identified DENV-derived RNA segments using recombinant Sindbis viruses to trigger RNAi, that when expressed in mosquitoes ablate homologous DENV replication and transmission. We also demonstrated that heritable expression of DENV-derived dsRNA in cultured mosquito cells can silence virus replication. We now have developed a number of transgenic mosquito lines that transcribe the effector dsRNA from constitutive promoters such as immediate early 1 (baculovirus) and polyubiquitin (Drosophila melanogaster). We have detected DENV-specific small interfering RNAs, the hallmark of RNAi, in at least one of these lines. Surprisingly, none of these lines expressed dsRNA in relevant tissues (e.g., midguts) that will ultimately affect transmission. A major challenge now is to express the effector dsRNA from tissue-specific promoters to allow RNAi to silence virus replication at critical sites in the vector such as midguts and salivary glands. If successful, this strategy has the advantage of harnessing a naturally occurring vector response to block DENV infection in a mosquito vector and profoundly affect virus transmission.     

pHYPER, a shRNA vector for high-efficiency RNA interference in embryonic stem cells

RNA interference (RNAi) is a powerful method to generate loss-of-function phenotypes. Plasmid vectors with RNA polymerase III promoters have been developed to express short hairpin RNAs (shRNAs) in mammalian cells. In order to optimize the efficiency of these vectors in embryonic stem (ES) cells, we have constructed and tested several plasmids, based on the H1 promoter; that direct the expression of shRNAs. The original pSUPER vector was used as a reference in this study. This vector drives the expression of shRNAs from a basic 0.2-kb H1 promoter; which exhibits a variable expression when integrated into the genome of ES cells. We used a 2.5-kb mouse genomic fragment containing the H1 promoter to construct a new H1 shRNA vector pHYPER. A comparison of this vector with the basic 0.2-kb H1 vector showed that pHYPER directs the synthesis of higher amounts of shRNAs. Using epifluorescence and fluorescent-activated cell sorting (FACS) analysis, we demonstrated that pHYPER is 4-fold more active than the 0.2-kb H1-based vector after integration into the genome of mouse ES cells. We provide a new, improved H1 shRNA vector that is optimized for both transient transfection studies and the generation of stable ES cell lines.     

A plasmid vehicle suitable for the molecular cloning and characterization of mammalian promoters.

We have constructed a plasmid, pPSG4, that carries only the coding sequences for polyomavirus (Py) small, middle and truncated large T-antigens. A unique HindIII site allows the introduction of foreign promoters directly in front of viral coding sequences. The simian virus 40 (SV40) early and late, adenovirus-2 (Ad-2) major late and herpes simplex virus-1 (HSV-1) thymidine kinase (TK) promoters all confer on pPSG4 the ability to transform rat embryonic fibroblasts with high efficiency. Sequential deletion of the 72 bp repeats, the 21 bp repeats and the TATA box from the SV40 early region in pPSG4 produced a 50, then 30 and then a further 5 to 10-fold decrease in transformation efficiency, respectively. Thus pPSG4 is a convenient vector for the cloning and characterization of mammalian promoters.     

Construction of a low-temperature protein expression system using a cold-adapted bacterium, Shewanella sp. strain Ac10, as the host

A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4 degrees C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce beta-lactamase in Shewanella sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4 degrees C and 139 mg/liter of culture at 18 degrees C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BglA, from a psychrophilic bacterium, Desulfotalea psychrophila DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the Escherichia coli T7 promoter system.     

The Use of Heterologous Promoters for Adeno-Associated Virus (AAV) Protein Expression in AAV Vector Production

Although adeno-associated virus (AAV) vectors are potentially useful gene transfer vehicles for gene therapy, the vector production system is currently at the developmental stage. We constructed AAV helper plasmids (Rep and Cap expression plasmids) by replacing a native AAV promoter, p5, with various heterologous promoters to examine whether the efficiency of AAV vector production was influenced by modulating the AAV protein expression pattern. The helper plasmids containing heterologous promoters (EF, CMV, SV40, B19p6, and CAG promoters, respectively) expressed Rep78/68 more efficiently than a conventional helper plasmid (pIM45), but the expression of Rep52/40 and Cap decreased, resulting in a significant reduction in AAV vector production. Furthermore, the efficiency of vector production never fully recovered even if the Cap proteins were supplied by an additional expression plasmid. A large amount of Rep78/68 and/or a reduced level of Rep52/40 may have deleterious effects on AAV vector production. The present findings will aid in the development of a more efficient AAV vector production system.     

Cloning and characterisation of the Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase (GUT2) promoter.

The Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase (GUT2) promoter and part of the protein-coding region have been isolated on a 6.3-kb genomic DNA fragment. Nucleotide sequence analysis shows that the promoter has many structural features in common with yeast glycolytic enzyme promoters. Chromosomal mapping indicates that this genomic fragment is located on chromosome XII. The GUT2 promoter has been used to construct a recombinant human albumin (reHA) secretion vector; yeast transformed with this vector secrete reHA into the culture supernatant.     

Development of a murine prostatespecific 2-in-1 inducible bicistronic expression vector

Transgenic research often suffers from the lack of strong tissue specific promoters and the lack of suitable antibodies for transgene detection. We have now constructed a novel prostate specific 2-in-1 Tetracycline-off (Tet-off), bicistronic expression vector, designated PbTetOIE, for transgenic research. The vector allows potent induction as well as inducible suppression of transgene expression in the prostate epithelial cells, and also allows the detection of transgene expression at the cellular level through the detection of the internal enhanced green fluorescent protein (EGFP) downstream of the transgene.     

Vector for regulated expression of cloned genes in a wide range of gram-negative bacteria.

A pKT231-based broad-host-range plasmid vector was constructed which enabled regulation of expression of cloned genes in a wide range of gram-negative bacteria. This vector, pNM185, contained upstream of its EcoRI, SstI, and SstII cloning sites the positively activated pm twin promoters of the TOL plasmid and xylS, the gene of the positive regulator of these promoters. Expression of cloned genes was induced with micromolar quantities of benzoate or m-toluate, the inexpensive coinducers of the pm promoters. Expression of a test gene, xylE, which specifies catechol 2,3-dioxygenase, cloned in this vector was tested in representative strains of a variety of gram-negative bacteria. Regulated expression of xylE was observed in most strains examined, and induced levels of enzyme representing up to 5% of total cellular protein and ratios of induced:noninduced levels of enzyme up to a factor of 600 were observed. The level of xylE gene expression in different bacteria tended to be correlated with their phylogenetic distance from Pseudomonas putida.     

The Nicotiana chloroplast genome. IX. Identification of regions active as prokaryotic promoters in Escherichia coli

The galK-expression plasmid vector system pKO1 has been used to clone Nicotiana chloroplast (ct) promoters that function in Escherichia coli. The randomly cloned promoter-containing restriction fragments have been located on the ct genome and originate both from those regions encoding ribosomal and transfer RNAs and from locations elsewhere on the ct genome. The results provide the first demonstration that sequences which function as prokaryotic promoters exist in the ct genome.     

Both the <Emphasis Type="Italic">AOX1</Emphasis> promoter and the <Emphasis Type="Italic">FLD1</Emphasis> promoter work together in a <Emphasis Type="Italic">Pichia pastoris</Emphasis> expression vector

We constructed a Pichia pastoris expression vector with two strongly inducible promoters (an alcohol oxidase 1 promoter and a formaldehyde dehydrogenase 1 promoter) based on pPIC9 k. To test the function of these promoters, the vector was used to co-express two genes that encode for green fluorescent protein (GFP) and a portion of a gelatin gene (an intra- and extracellular protein). The gelatin gene was placed under the control of PAOX1, while the GFP was under the control of PFLD1. The two proteins were simultaneously expressed upon induction with 0.5% (v/v) methanol. The two promoters functioned effectively and their coexistence on one vector did not affect their efficiency in protein expression. Thus, it was possible to simultaneously induce the expression of at least two proteins from one vector, using two different promoters.