The function of herpes simplex virus genes: a primer for genetic engineering of novel vectors.

Herpes simplex virus vectors are being developed for delivery and expression of human genes to the central nervous system, selective destruction of cancer cells, and as carriers for genes encoding antigens that induce protective immunity against infectious agents. Vectors constructed to meet these objectives must differ from wild-type virus with respect to host range, reactivation from latency, and expression of viral genes. The vectors currently being developed are (i) helper free amplicons, (ii) replication defective viruses, and (iii) genetically engineered replication competent viruses with restricted host range. Whereas the former two types of vectors require stable, continuous cell lines expressing viral genes for their replication, the replication competent viruses will replicate on approved primary human cell strains.

Negative-strand RNA viruses: genetic engineering and applications.

The negative-strand RNA viruses are a broad group of animal viruses that comprise several important human pathogens, including influenza, measles, mumps, rabies, respiratory syncytial, Ebola, and hantaviruses. The development of new strategies to genetically manipulate the genomes of negative-strand RNA viruses has provided us with new tools to study the structure-function relationships of the viral components and their contributions to the pathogenicity of these viruses. It is also now possible to envision rational approaches--based on genetic engineering techniques--to design live attenuated vaccines against some of these viral agents. In addition, the use of different negative-strand RNA viruses as vectors to efficiently express foreign polypeptides has also become feasible, and these novel vectors have potential applications in disease prevention as well as in gene therapy.

Genetic engineering of carbohydrate biosynthetic pathways in transgenic mice demonstrates cell cycle-associated regulation of glycoconjugate production in small intestinal epithelial cells.

Proliferation, migration-associated differentiation, and cell death occur continuously and in a spatially well-organized fashion along the crypt-villus axis of the mouse small intestine, making it an attractive system for studying how these processes are regulated and interrelated. A pathway for producing glycoconjugates was engineered in adult FVB/N transgenic mice by expressing a human alpha 1,3/4-fucosyltransferase (alpha 1,3/4-FT; EC 2.4.1.65) along the length of this crypt-villus axis. The alpha 1,3/4-FT can use lacto-N-tetraose or lacto-neo-N-tetraose core chains to generate Lewis (Le) blood group antigens Le(a) or Le(x), respectively, and H type 1 or H type 2 core chains to produce Leb and Le(y). Single- and multilabel immunohistochemical studies revealed that expression of the alpha 1,3/4-FT results in production of Le(a) and Leb antigens in both undifferentiated proliferated crypt cells and in differentiated postmitotic villus-associated epithelial cells. In contrast, Le(x) antigens were restricted to crypt cells. Villus enterocytes can be induced to reenter the cell cycle by expression of simian virus 40 tumor antigen under the control of a promoter that only functions in differentiated members of this lineage. Bitransgenic animals, generated from a cross of FVB/N alpha 1,3/4-FT with FVB/N simian virus 40 tumor antigen mice, expand the range of Le(x) expression to include villus-associated enterocytes that have reentered the cell cycle. Thus, the fucosylations unveil a proliferation-dependent switch in oligosaccharide production, as defined by a monoclonal antibody specific for the Le(x) epitope. These findings show that genetic engineering of oligosaccharide biosynthetic pathways can be used to define markers for entry into, or progression through, the cell cycle and to identify changes in endogenous carbohydrate metabolism that occur when proliferative status is altered in a manner that is not deleterious to the system under study.

Genetic engineering of vein grafts resistant to atherosclerosis.

Previously, researchers have speculated that genetic engineering can improve the long-term function of vascular grafts which are prone to atherosclerosis and occlusion. In this study, we demonstrated that an intraoperative gene therapy approach using antisense oligodeoxynucleotide blockage of medial smooth muscle cell proliferation can prevent the accelerated atherosclerosis that is responsible for autologous vein graft failure. Selective blockade of the expression of genes for two cell cycle regulatory proteins, proliferating cell nuclear antigen and cell division cycle 2 kinase, was achieved in the smooth muscle cells of rabbit jugular veins grafted into the carotid arteries. This alteration of gene expression successfully redirected vein graft biology away from neointimal hyperplasia and toward medial hypertrophy, yielding conduits that more closely resembled normal arteries. More importantly, these genetically engineered grafts proved resistant to diet-induced atherosclerosis. These findings establish the feasibility of developing genetically engineered bioprostheses that are resistant to failure and better suited to the long-term treatment of occlusive vascular disease.

Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects

Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330- to 393-fold) tobacco budworm Heliothis virescens, cotton bollworm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5,000–10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field.

Plant genetic resources: What can they contribute toward increased crop productivity?

To feed a world population growing by up to 160 people per minute, with >90% of them in developing countries, will require an astonishing increase in food production. Forecasts call for wheat to become the most important cereal in the world, with maize close behind; together, these crops will account for ≈80% of developing countries’ cereal import requirements. Access to a range of genetic diversity is critical to the success of breeding programs. The global effort to assemble, document, and utilize these resources is enormous, and the genetic diversity in the collections is critical to the world’s fight against hunger. The introgression of genes that reduced plant height and increased disease and viral resistance in wheat provided the foundation for the “Green Revolution” and demonstrated the tremendous impact that genetic resources can have on production. Wheat hybrids and synthetics may provide the yield increases needed in the future. A wild relative of maize, Tripsacum, represents an untapped genetic resource for abiotic and biotic stress resistance and for apomixis, a trait that could provide developing world farmers access to hybrid technology. Ownership of genetic resources and genes must be resolved to ensure global access to these critical resources. The application of molecular and genetic engineering technologies enhances the use of genetic resources. The effective and complementary use of all of our technological tools and resources will be required for meeting the challenge posed by the world’s expanding demand for food.

Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (Lupinus angustifolius L.) expressing a sunflower seed albumin gene

With the aim of improving the nutritive value of an important grain legume crop, a chimeric gene specifying seed-specific expression of a sulfur-rich, sunflower seed albumin was stably transformed into narrow-leafed lupin (Lupinus angustifolius L.). Sunflower seed albumin accounted for 5% of extractable seed protein in a line containing a single tandem insertion of the transferred DNA. The transgenic seeds contained less sulfate and more total amino acid sulfur than the nontransgenic parent line. This was associated with a 94% increase in methionine content and a 12% reduction in cysteine content. There was no statistically significant change in other amino acids or in total nitrogen or total sulfur contents of the seeds. In feeding trials with rats, the transgenic seeds gave statistically significant increases in live weight gain, true protein digestibility, biological value, and net protein utilization, compared with wild-type seeds. These findings demonstrate the feasibility of using genetic engineering to improve the nutritive value of grain crops.

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690–2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.

High-resolution mapping and isolation of a yeast artificial chromosome contig containing fw2.2: A major fruit weight quantitative trait locus in tomato

A high-resolution physical and genetic map of a major fruit weight quantitative trait locus (QTL), fw2.2, has been constructed for a region of tomato chromosome 2. Using an F2 nearly isogenic line mapping population (3472 individuals) derived from Lycopersicon esculentum (domesticated tomato) × Lycopersicon pennellii (wild tomato), fw2.2 has been placed near TG91 and TG167, which have an interval distance of 0.13 ± 0.03 centimorgan. The physical distance between TG91 and TG167 was estimated to be ≤ 150 kb by pulsed-field gel electrophoresis of tomato DNA. A physical contig composed of six yeast artificial chromosomes (YACs) and encompassing fw2.2 was isolated. No rearrangements or chimerisms were detected within the YAC contig based on restriction fragment length polymorphism analysis using YAC-end sequences and anchored molecular markers from the high-resolution map. Based on genetic recombination events, fw2.2 could be narrowed down to a region less than 150 kb between molecular markers TG91 and HSF24 and included within two YACs: YAC264 (210 kb) and YAC355 (300 kb). This marks the first time, to our knowledge, that a QTL has been mapped with such precision and delimited to a segment of cloned DNA. The fact that the phenotypic effect of the fw2.2 QTL can be mapped to a small interval suggests that the action of this QTL is likely due to a single gene. The development of the high-resolution genetic map, in combination with the physical YAC contig, suggests that the gene responsible for this QTL and other QTLs in plants can be isolated using a positional cloning strategy. The cloning of fw2.2 will likely lead to a better understanding of the molecular biology of fruit development and to the genetic engineering of fruit size characteristics.

Pressure-mediated oligonucleotide transfection of rat and human cardiovascular tissues

The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but their in vivo application is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability of ex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibit target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipultation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.

Aldehyde dehydrogenase class 3 expression: Identification of a cornea-preferred gene promoter in transgenic mice

Aldehyde dehydrogenase class 3 (ALDH3) constitutes 20–40% of the total water-soluble proteins in the mammalian cornea. Here, we show by Northern blot analysis that ALDH3 expression in the mouse is at least 500-fold higher in the cornea than in any other tissue examined, with very low levels of expression detected in the stomach, urinary bladder, ocular lens, and lung. Histochemical localization reveals that this exceptional level of expression in the mouse cornea occurs in the anterior epithelial cells and that little ALDH3 is present in the keratocytes or corneal endothelial cells. A 13-kbp mouse ALDH3 promoter fragment containing >12 kbp of the 5′ flanking sequence, the 40-bp untranslated first exon, and 29 bp of intron 1 directed cat reporter gene expression to tissues that express the endogenous ALDH3 gene, except that transgene promoter activity was higher in the stomach and bladder than in the cornea. By contrast, when driven by a 4.4-kbp mouse ALDH3 promoter fragment [1,050-bp 5′ flanking region, exon 1, intron 1 (3.4 kbp), and 7 bp of exon 2] expression of the cat reporter gene was confined to the corneal epithelial cells, except for very low levels in the liver, effectively reproducing the corneal expression pattern of the endogenous ALDH3 gene. These results indicate that tissue-specific expression of ALDH3 is determined by positive and negative elements in the 5′ flanking region of the gene and suggests putative silencers located in intron 1. We demonstrate regulatory sequences capable of directing cornea-specific gene expression, affording the opportunity for genetic engineering in this transparent tissue.

Plant biology in the future

In the beginning of modern plant biology, plant biologists followed a simple model for their science. This model included important branches of plant biology known then. Of course, plants had to be identified and classified first. Thus, there was much work on taxonomy, genetics, and physiology. Ecology and evolution were approached implicitly, rather than explicitly, through paleobotany, taxonomy, morphology, and historical geography. However, the burgeoning explosion of knowledge and great advances in molecular biology, e.g., to the extent that genes for specific traits can be added (or deleted) at will, have created a revolution in the study of plants. Genomics in agriculture has made it possible to address many important issues in crop production by the identification and manipulation of genes in crop plants. The current model of plant study differs from the previous one in that it places greater emphasis on developmental controls and on evolution by differential fitness. In a rapidly changing environment, the current model also explicitly considers the phenotypic variation among individuals on which selection operates. These are calls for the unity of science. In fact, the proponents of “Complexity Theory” think there are common algorithms describing all levels of organization, from atoms all the way to the structure of the universe, and that when these are discovered, the issue of scaling will be greatly simplified! Plant biology must seriously contribute to, among other things, meeting the nutritional needs of the human population. This challenge constitutes a key part of the backdrop against which future evolution will occur. Genetic engineering technologies are and will continue to be an important component of agriculture; however, we must consider the evolutionary implications of these new technologies. Meeting these demands requires drastic changes in the undergraduate curriculum. Students of biology should be trained in molecular, cellular, organismal, and ecosystem biology, including all living organisms.

Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance

The recently cloned NPR1 gene of Arabidopsis thaliana is a key regulator of acquired resistance responses. Upon induction, NPR1 expression is elevated and the NPR1 protein is activated, in turn inducing expression of a battery of downstream pathogenesis-related genes. In this study, we found that NPR1 confers resistance to the pathogens Pseudomonas syringae and Peronospora parasitica in a dosage-dependent fashion. Overexpression of NPR1 leads to enhanced resistance with no obvious detrimental effect on the plants. Thus, for the first time, a single gene is shown to be a workable target for genetic engineering of nonspecific resistance in plants.