An computer information system for genetic engineering

The article describes the vectors data base and the software for its use (the VECTOR-PC system). At present the prototype versions of data base and VECTOR-PC exist and are in test exploitation. The original data base entry format contains 17 main fields for specific genetic engineering information. The VECTOR-PC system includes programs for data base search and support, and also the "genetic engineering designer", which allows the user to design his own hypothetic structures from the objects of data base and to receive detailed information about them. The system is destined for IBM PC or compatible computers.     

A model selection-based interval-mapping method for autopolyploids

While extensive progress has been made in quantitative trait locus (QTL) mapping for diploid species, similar progress in QTL mapping for polyploids has been limited due to the complex genetic architecture of polyploids. To date, QTL mapping in polyploids has focused mainly on tetraploids with dominant and/or codominant markers. Here, we extend this view to include any even ploidy level under a dominant marker system. Our approach first selects the most likely chromosomal marker configurations using a Bayesian selection criterion and then fits an interval-mapping model to each candidate. Profiles of the likelihood-ratio test statistic and the maximum-likelihood estimates (MLEs) of parameters including QTL effects are obtained via the EM algorithm. Putative QTL are then detected using a resampling-based significance threshold, and the corresponding parental configuration is identified to be the underlying parental configuration from which the data are observed. Although presented via pseudo-doubled backcross experiments, this approach can be readily extended to other breeding systems. Our method is applied to single-dose restriction fragment autotetraploid alfalfa data, and the performance is investigated through simulation studies.     

A novel screening system improves genetic correction by internal exon replacement

Trans-splicing is a powerful approach to reprogram the genome. It can be used to replace 5', 3' or internal exons. The latter approach has been characterized by low efficiency, as the requirements to promote internal trans-splicing are largely uncharacterized. The trans-splicing process is induced by engineered 'RNA trans-splicing molecules' (RTMs), which target a selected pre-mRNA to be reprogrammed via two complementary binding domains. To facilitate the development of more efficient RTMs for therapeutic applications we constructed a novel fluorescence based screening system. We incorporated exon 52 of the COL17A1 gene into a GFP-based cassette system as the target exon. This exon is mutated in many patients with the devastating skin blistering disease epidermolysis bullosa. In a double transfection assay we were able to rapidly identify optimal binding domains targeted to sequences in the surrounding introns 51 and 52. The ability to replace exon 52 was then evaluated in a more endogenous context using a target containing COL17A1 exon 51-intron 51-exon 52-intron 52-exon 53. Two selected RTMs produced significantly higher levels of GFP expression in up to 61% assayed cells. This novel approach allows for rapid identification of efficient RTMs for internal exon replacement.     

Inverse metabolic engineering: A strategy for directed genetic engineering of useful phenotypes

The classical method of metabolic engineering, identifying a rate-determining step in a pathway and alleviating the bottleneck by enzyme overexpression, has motivated much research but has enjoyed only limited practical success. Intervention of other limiting steps, of counterbalancing regulation, and of unknown coupled pathways often confounds this direct approach. Here the concept of inverse metabolic engineering is codified and its application is illustrated with several examples. Inverse metabolic engineering means the elucidation of a metabolic engineering strategy by: first, identifying, constructing, or calculating a desired phenotype; second, determining the genetic or the particular environmental factors conferring that phenotype; and third, endowing that phenotype on another strain or organism by directed genetic or environmental manipulation. This paradigm has been successfully applied in several contexts, including elimination of growth factor requirements in mammalian cell culture and increasing the energetic efficiency of microaerobic bacterial respiration. (c) 1996 John Wiley & Sons, Inc.     

A quorum-sensing signaling system essential for genetic competence in Streptococcus mutans is involved in biofilm formation

In a previous study, a quorum-sensing signaling system essential for genetic competence in Streptococcus mutans was identified, characterized, and found to function optimally in biofilms (Li et al., J. Bacteriol. 183:897-908, 2001). Here, we demonstrate that this system also plays a role in the ability of S. mutans to initiate biofilm formation. To test this hypothesis, S. mutans wild-type strain NG8 and its knockout mutants defective in comC, comD, comE, and comX, as well as a comCDE deletion mutant, were assayed for their ability to initiate biofilm formation. The spatial distribution and architecture of the biofilms were examined by scanning electron microscopy and confocal scanning laser microscopy. The results showed that inactivation of any of the individual genes under study resulted in the formation of an abnormal biofilm. The comC mutant, unable to produce or secrete a competence-stimulating peptide (CSP), formed biofilms with altered architecture, whereas the comD and comE mutants, which were defective in sensing and responding to the CSP, formed biofilms with reduced biomass. Exogenous addition of the CSP and complementation with a plasmid containing the wild-type comC gene into the cultures restored the wild-type biofilm architecture of comC mutants but showed no effect on the comD, comE, or comX mutant biofilms. The fact that biofilms formed by comC mutants differed from the comD, comE, and comX mutant biofilms suggested that multiple signal transduction pathways were affected by CSP. Addition of synthetic CSP into the culture medium or introduction of the wild-type comC gene on a shuttle vector into the comCDE deletion mutant partially restored the wild-type biofilm architecture and further supported this idea. We conclude that the quorum-sensing signaling system essential for genetic competence in S. mutans is important for the formation of biofilms by this gram-positive organism.     

A new technique for genetic engineering of Agrobacterium Ti plasmid

A new technique is described that allows easy introduction of foreign genetic elements into specific regions of Agrobacterium tumefaciens DNA. It uses plasmids that (1) can be introduced, but not maintained in A. tumefaciens, (2) have a region homologous to the genome of the recipient, and (3) have an appropriate marker. Selection for the marker will yield transconjugants in which the introduced plasmid has recombined with the host genome. Applications of the technique are described.     

GENESIS, a knowledge-based genetic engineering simulation system for representation of genetic data and experiment planning.

We have built a knowledge-based genetic engineering simulation system-- GENESIS-- capable of representing both domain-specific and general knowledge. Information is stored within a hierarchically-organized framework composed of structures called units. A series of sophisticated editors enables no-computer specialist molecular geneticists to construct a knowledge base through direct interaction with the computer. Three types of knowledge specific to the domain of molecular genetics, MAPS, sequences and RULES are discussed in detail with examples.     

A genetic locus essential for formate-dependent growth of Bradyrhizobium japonicum.

A genetic locus essential for the formate-dependent growth of Bradyrhizobium japonicum was isolated by complementation of ethyl methanesulfonate-induced mutants with a cosmid gene library of B. japonicum DNA. Three related cosmids containing 18.7 kilobase pairs of B. japonicum DNA in common were identified as being able to restore formate-dependent growth capability to mutants lacking either ribulosebisphosphate carboxylase or both ribulosebisphosphate carboxylase and phosphoribulokinase activities. To further localize the complementing gene(s), a series of four deletions spanning a total of 16.1 kilobase pairs were introduced into the B. japonicum chromosome. Each resulting deletion mutant lacked formate dehydrogenase activity and lacked ribulosebisphosphate carboxylase activity and immunologically detectable protein. Three of the four also lacked phosphoribulokinase activity. Two other mutants in which the deletion-bearing recombinant plasmid had integrated into the chromosome also lacked ribulosebisphosphate carboxylase activity and protein and phosphoribulokinase activities. The genetic locus defined by these mutants could contain the structural genes for these enzymes or a regulatory gene(s) controlling their expression or both.     

Improvement of downstream processing of recombinant proteins by means of genetic engineering methods

The rapid advancement of genetic engineering has allowed to produce an impressive number of proteins on a scale which would not have been achieved by classical biotechnology. At the beginning of this development research was focussed on elucidating the mechanisms of protein overexpression. The appearance of inclusion bodies may illustrate the success. In the meantime, genetic engineering is not only expected to achieve overexpression, but to improve the whole process of protein production. For downstream processing of recombinant proteins, the synthesis of fusion proteins is of primary importance. Fusion with certain proteins or peptides may protect the target protein from proteolytic degradation and may alter its solubility. Intracellular proteins may be translocated by means of fusions with signal peptides. Affinity tags as fusion complements may render protein separation and purification highly selective. These methods as well as similar ones for improving the downstream processing of proteins will be discussed on the basis of recent literature.     

Transposon-free insertions for insect genetic engineering

Methods involving the release of transgenic insects in the field hold great promise for controlling vector-borne diseases and agricultural pests. Insect transformation depends on nonautonomous transposable elements as gene vectors. The resulting insertions are stable in the absence of suitable transposase, however, such absence cannot always be guaranteed. We describe a method for post-integration elimination of all transposon sequences in the pest insect Medfly, Ceratitis capitata. The resulting insertions lack transposon sequences and are therefore impervious to transposase activity.     

A genetic interaction between the core and NS3 proteins of hepatitis C virus is essential for production of infectious virus

By analogy to other members of the Flaviviridae family, the hepatitis C virus (HCV) core protein is presumed to oligomerize to form the viral nucleocapsid, which encloses the single-stranded RNA genome. Core protein is directed to lipid droplets (LDs) by domain 2 (D2) of the protein, and this process is critical for virus production. Domain 1 (D1) of core is also important for infectious particle morphogenesis, although its precise contribution to this process is poorly understood. In this study, we mutated amino acids 64 to 75 within D1 of core and examined the ability of these mutants to produce infectious virus. We found that residues 64 to 66 are critical for generation of infectious progeny, whereas 67 to 75 were dispensable for this process. Further investigation of the defective 64 to 66 mutant (termed JFH1(T)-64-66) revealed it to be incapable of producing infectious intracellular virions, suggesting a fault during HCV assembly. Furthermore, isopycnic gradient analyses revealed that JFH1(T)-64-66 assembled dense intracellular species of core, presumably representing nucleocapsids. Thus, amino acids 64 to 66 are seemingly not involved in core oligomerization/nucleocapsid assembly. Passaging of JFH1(T)-64-66 led to the emergence of a single compensatory mutation (K1302R) within the helicase domain of NS3 that completely rescued its ability to produce infectious virus. Importantly, the same NS3 mutation abrogated virus production in the context of wild-type core protein. Together, our results suggest that residues 64 to 66 of core D1 form a highly specific interaction with the NS3 helicase that is essential for the generation of infectious HCV particles at a stage downstream of nucleocapsid assembly.     

Techniques for genetic engineering in mycobacteria

The study of mycobacterial genetics has experienced quick technical developments in the past ten years, despite a relatively slow start, caused by difficulties in accessing these recalcitrant species. The study of mycobacterial pathogenesis is important in the development of new ways of treating tuberculosis and leprosy, now that the emergence of antibiotic-resistant strains has reduced the effectiveness of current therapies. The tuberculosis vaccine strain M. bovis BCG might be used as a vector for multivalent vaccination. Also, non-pathogenic mycobacterial strains have many possible biotechnological applications. After giving a historical overview of methods and techniques, we will discuss recent developments in the search for alternative host strains and DNA transfer systems. Special attention will be given to the development of vectors and techniques for stabilizing foreign DNA in mycobacteria.     

Heat shock proteins: essential proteins for apoptosis regulation

Many different external and intrinsic apoptotic stimuli induce the accumulation in the cells of a set of proteins known as stress or heat shock proteins (HSPs). HSPs are conserved proteins present in both prokaryotes and eukaryotes. These proteins play an essential role as molecular chaperones by assisting the correct folding of nascent and stress-accumulated misfolded proteins, and by preventing their aggregation. HSPs have a protective function, that is they allow the cells to survive to otherwise lethal conditions. Various mechanisms have been proposed to account for the cytoprotective functions of HSPs. Several of these proteins have demonstrated to directly interact with components of the cell signalling pathways, for example those of the tightly regulated caspase-dependent programmed cell death machinery, upstream, downstream and at the mitochondrial level. HSPs can also affect caspase-independent apoptosis-like process by interacting with apoptogenic factors such as apoptosis-inducing factor (AIF) or by acting at the lysosome level. This review will describe the different key apoptotic proteins interacting with HSPs and the consequences of these interactions in cell survival, proliferation and apoptotic processes. Our purpose will be illustrated by emerging strategies in targeting these protective proteins to treat haematological malignancies.     

A fluorescent cassette-based strategy for engineering multiple domain fusion proteins

Background

The engineering of fusion proteins has become increasingly important and most recently has formed the basis of many biosensors, protein purification systems, and classes of new drugs. Currently, most fusion proteins consist of three or fewer domains, however, more sophisticated designs could easily involve three or more domains. Using traditional subcloning strategies, this requires micromanagement of restriction enzymes sites that results in complex workaround solutions, if any at all.

Results

Therefore, to aid in the efficient construction of fusion proteins involving multiple domains, we have created a new expression vector that allows us to rapidly generate a library of cassettes. Cassettes have a standard vector structure based on four specific restriction endonuclease sites and using a subtle property of blunt or compatible cohesive end restriction enzymes, they can be fused in any order and number of times. Furthermore, the insertion of PCR products into our expression vector or the recombination of cassettes can be dramatically simplified by screening for the presence or absence of fluorescence.

Conclusions

Finally, the utility of this new strategy was demonstrated by the creation of basic cassettes for protein targeting to subcellular organelles and for protein purification using multiple affinity tags.

     

New plasmid vector and host system for genetic engineering in Bacillus sphaericus.

A new plasmid, pNQ116, was constructed in Bacillus sphaericus by cloning a promoter fragment from B. sphaericus Ts-1 into pNQ112. The plasmid (CmrKmr, 5.23 kb) contains a restriction endonuclease polylinker used for cloning foreign genes, and its cat-86 gene is expressed at high levels from the Ts-1 promoter. This plasmid vector has been transformed into B. sphaericus AS 1.270, AS 1.465, AS 1.469, and 2362, at frequencies of 10(2)-10(3) transformants per microgram of DNA, and is maintained stably under nonselective conditions in these host strains. The presence of pNQ116 in B. sphaericus 2362 does ot interfere with the mosquito larvicidal activity of the organism.     

Combined use of extraction and genetic engineering for protein purification: recovery of beta-galactosidase fused proteins.

Partitioning of beta-galactosidase in aqueous two-phase systems of poly(ethylene glycol) and potassium phosphate is reviewed. The affinity of Escherichia coli beta-galactosidase for the PEG-rich phase dominates also in beta-galactosidase fusion proteins and the concept of using beta-galactosidase as an affinity handle for extraction of other proteins, after fusion, is discussed. A hypothesis is presented, assuming that tryptophan residues at the surface of beta-galactosidase is responsible for its partitioning to the PEG rich phase, and the concept of poly-tryptophan handles fused to the target protein for extraction is introduced.     

An electrofocusing system for the analysis of proteins and their genetic variants.

The isoelectric points of proteins can be determined in acrylamide gels containing urea and neutral detergents. We report here a method which permits sereening of putative genetic variants for isoelectric point differences. The method requires only small quantities of material. The analyses are performed without prior fractionation of the starting material.