The in-planta induced ecp2 gene of the tomato pathogen Cladosporium fulvum is not essential for pathogenicity

During the colonization of tomato leaves, the fungal pathogen Cladosporium fulvum excretes low-molecular-weight proteins in the intercellular spaces of the host tissue. These proteins are encoded by the ecp genes which are highly expressed in C. fulvum while growing in planta but are not, or are only weakly, expressed in C. fulvum grown in vitro. To investigate the function of the putative pathogenicity gene ecp2, encoding the 17-kDa protein ECP2, we performed two successive disruptions of the gene. In the first of these, the ecp2 gene was interrupted by a hygromycin B resistance gene cassette. In the second gene disruption, the ecp2 gene was completely deleted from the genome, and replaced by a phleomycin resistance gene cassette. Both disruption mutants were still pathogenic on tomato seedlings, indicating that the C. fulvum ecp2 gene is not essential for pathogenicity in tomato.     

Gene testings in relation with gene diagnosis and therapy

Recent advances in the molecular biology research have devoted greatly to the clinical applications of genetic informations. Gene diagnosis and therapy is a new medical field to deal with these applications. Nowadays diseases related with gene abnormalities have been shown to reach 8,450, and gene testings to cover all these should be very wide and variable. So that any one of the clinical laboratories in a hospital or even research institutes and laboratory centers should not be able to handle all the testings. A model system to promote gene testings effectively in Japan, which includes a special department in a hospital and limited specifications to the individual laboratories or centers, were presented. In the case of monogenic genetic disorders, gene testing results are useful not only to confirm clinical diagnosis but to predict future development of disorders preclinically and even during the prenatal or pregestational period. Accordingly, these may evoke critical ethical and social problems. Many of common diseases are polygenic in nature and gene testings in these disorders are not directed to the diagnosis but are quite useful to see the individual characteristics of the patients related to the special phenotypes or even the fate of disease process. Acquired malignancies are known to be resulted from a somatic gene mutation and progress by the associations of further abnormalities. Only limited gene testings are already now in use. Wide future development in this field is expected after getting detailed meanings of gene testings in the individual cancers. Gene testings for infectious diseases are known to be quite effective on the early and accurate diagnosis, and many of these are already covered by the health insurance. In conclusion, gene testings are quite important but variable and laboratory personnel should not be able to deal all of these properly. Considering future development of gene therapy, rapid establishment in a hospital of a special department, Department of Gene Diagnosis and Therapy, which is consisted from 3 divisions such as genetic counseling, gene testing and management, and monitoring and adviser of gene therapy, is highly recommended.     

From genes to gene medicines: recent advances in nonviral gene delivery

Over the last decade, research in somatic gene therapy has focused on selected approaches to deliver therapeutic genes to cells both ex vivo and in vivo. While most current gene therapy clinical trials are based on cell- and viral-mediated approaches, nonviral gene medicines are emerging as potentially safe and effective in the treatment of a wide variety of genetic and acquired diseases. Nonviral technologies consist of plasmid-based expression systems containing a gene encoding a therapeutic protein and synthetic gene delivery systems. In addition to the therapeutic gene, plasmid-based expression systems contain other genetic sequences to control the in vivo production and secretion of a protein. They may include elements that prolong extrachromosomal gene expression, cell-specific promoters and, optionally, gene switches for enabling drug-regulated gene therapy. Unique gene delivery systems will be required depending upon the biology and (patho)physiology of the target tissue. This review provides a critical view of gene therapy with a major focus on advanced nonviral technologies to control the in vivo location and function of administered genes.     

Prolactin induction of the alpha 2-Macroglobulin gene in rat ovarian granulosa cells: stat 5 activation and binding to the interleukin-6 response element

Lantibiotics form a group of modified peptides with unique structures, containing post-translationally modified amino acids such as dehydrated and lanthionine residues. In the gram-positive bacteria that secrete these lantibiotics, the gene clusters flanking the structural genes for various linear (type A) lantibiotics have recently been characterized. The best studied representatives are those of nisin (nis), subtilin (spa), epidermin (epi), Pep5 (pep), cytolysin (cyl), lactocin S (las) and lacticin 481 (lct). Comparison of the lantibiotic gene clusters shows that they contain conserved genes that probably encode similar functions. The nis, spa, epi and pep clusters contain lanB and lanC genes that are presumed to code for two types of enzymes that have been implicated in the modification reactions characteristic of all lantibiotics, i.e. dehydration and thio-ether ring formation. The cyl, las and lct gene clusters have no homologue of the lanB gene, but they do contain a much larger lanM gene that is the lanC gene homologue. Most lantibiotic gene clusters contain a lanP gene encoding a serine protease that is presumably involved in the proteolytic processing of the prelantibiotics. All clusters contain a lanT gene encoding an ABC transporter likely to be involved in the export of (precursors of) the lantibiotics. The lanE, lanF and lanG genes in the nis, spa and epi clusters encode another transport system that is possibly involved in self-protection. In the nisin and subtilin gene clusters two tandem genes, lanR and lanK, have been located that code for a two-component regulatory system. Finally, non-homologous genes are found in some lantibiotic gene clusters. The nisI and spaI genes encode lipoproteins that are involved in immunity, the pepI gene encodes a membrane-located immunity protein, and epiD encodes an enzyme involved in a post-translational modification found only in the C-terminus of epidermin. Several genes of unknown function are also found in the las gene cluster. A database has been assembled for all putative gene products of type A lantibiotic gene clusters. Database searches, multiple sequence alignment and secondary structure prediction have been used to identify conserved sequence segments in the LanB, LanC, LanE, LanF, LanG, LanK, LanM, LanP, LanR and LanT gene products that may be essential for structure and function. This database allows for a rapid screening of newly determined sequences in lantibiotic gene clusters.     

Cancer gene therapy

Developments in molecular genetics, immunology, molecular and cellular biology, and tumor biology have given rise to the field of cancer gene therapy. Several gene delivery vehicles have been developed and are being examined in clinical trials. Most cancer gene therapy strategies involve introduction of genes to augment existing therapies. An overview is provided on gene delivery vehicles, gene therapy strategies, and cancer gene therapy clinical trials.     

Gene transfer by guanidinium-cholesterol cationic lipids into airway epithelial cells in vitro and in vivo

Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis. Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo. The results obtained indicate that the lipid bis(guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture. Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo. Transfected cells were detected both in the surface epithelium and in submucosal glands. In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system.     

Structure of the smallest salivary-gland secretory protein gene in Chironomus tentans

The salivary gland secretion in the dipteran Chironomus tentans is composed of approximately 15 different secretory proteins. The most well known of the corresponding genes are the four closely related Balbiani ring (BR) genes, in which the main part of each approximately 40-kb gene is composed of tandemly arranged repetitive units. Six of the seven additional secretory protein genes described share structural similarities with the BR genes and are members of the same BR multigene family. Here we report the identification of a new secretory protein gene, the sp12 gene, encoding the smallest component of the C. tentans salivary gland secretion. The gene has a corresponding mRNA length of approximately 0.7 kb and codes for a protein with a calculated molecular weight of 7,619 Da. The sp12 gene was characterized in seven Chironomus species. Based on a comparison of the orthologous gene sequences, we conclude that the sp12 gene has a repetitive structure consisting of diverged 21-bp-long repeats. The repeat structure and the codon composition are similar to the so-called SR regions of the BR genes and the sp12 gene may represent a diverged member of the BR multigene family.     

Assessment of cell death

The root-associated bacterium Azospirillum brasilense Sp7 produces the growth-stimulating phytohormone indole-3-acetic acid (= IAA) via the indole-3-pyruvate pathway. The DNA region containing ipdC, the structural gene for indole-3-pyruvate decarboxylase, was identified in a cosmid gene library of strain Sp7 by hybridization and has been sequenced. Upstream of the gene, two other ORF homologous to gltX and cysS were sequenced that are transcribed in the opposite direction. A functional analysis of the cloned ipdC region has been performed. To test the expression of the gene, a lacZ-Km cartridge was introduced into the gene. By this construct, tryptophan-dependent stimulation of gene expression in A. brasilense Sp7 was observed. Evidences for the existence of another copy of the ipdC gene in the Azospirillum genome are also reported.     

Identification of disease genes and somatic gene therapy: an overview and prospects for the aged

Positional cloning strategies for identification of disease genes include genetic linkage analysis in disease families and identification of individuals in whom the disease is associated with a specific chromosomal anomaly. Once a genomic region likely to contain the disease gene has been identified, overlapping genomic clones are isolated and the candidate gene sought. Somatic gene therapy entails introduction of the cloned gene into somatic cells to either replace genetically defective functions or alter pathological disease processes. Transfer of the gene may be accomplished by either DNA- or viral-mediated methods into a variety of tissue targets. Once the success and reliability of ongoing gene therapy trials for various human diseases is established, it may then be considered in the prevention and treatment of chronic, disabling diseases such as Alzheimer's disease, Parkinson's disease, arthritis, and diabetes as well as intervention of immunosenescence, when the relevant genes have been cloned. Ethical considerations for gene therapy for aging are similar to those for gene therapy in general. In addition, the ethics of gene therapy for treating diseases versus intervention of the normal aging process must be considered.     

Renal cellular transport, metabolism, and cytotoxicity of S-(6-purinyl)glutathione, a prodrug of 6-mercaptopurine, and analogues

Plasmids currently used for nonviral gene transfer have the disadvantage of carrying a bacterial origin of replication and an antibiotic resistance gene. There is, therefore, a risk of uncontrolled dissemination of the therapeutic gene and the antibiotic resistance gene. Minicircles are new DNA delivery vehicles which do not have such elements and are consequently safer as they exhibit a high level of biological containment. They are obtained in E. coli by att site-specific recombination mediated by the phage lambda integrase. The desired eukaryotic expression cassette, bounded by the lambda attP and attB sites was cloned on a recombinant plasmid. The expression cassette was excised in vivo after thermoinduction of the integrase gene leading to the formation of two supercoiled molecules the minicircle and the starting plasmid lacking the expression cassette. In various cell lines, purified minicircles exhibited a two- to 10-fold higher luciferase reporter gene activity than the unrecombined plasmid. This could be due to either the removal of unnecessary plasmid sequences, which could affect gene expression or the smaller size of mini-circle which may confer better extracellular and intracellular bioavailability and result in improved gene delivery properties.     

Liver-directed gene transfer vectors

The ultimate goal of liver-directed gene therapy for genetic diseases is the stable expression of a therapeutic transgene in a significant proportion of hepatocytes. This article considers the various liver-directed gene transfer procedures studied so far. Performances and limitations of currently available vector systems are discussed with respect to their clinical relevance. Although some improvements have been reported, naked DNA and nonviral gene transfer vectors induce transient expression in only a limited number of cells. Clinical applications of retrovirus-mediated gene transfer are hampered by the need to induce hepatocyte division. First-generation adenovirus vectors are highly efficient; however, they induce an immune response leading to the rapid rejection of transduced cells. Promising new vector systems have emerged, including gutless adenovirus vectors, adeno-associated vectors, and lentivirus vectors. However, these systems are still poorly documented and their relevance to liver-directed gene therapy must be confirmed.     

Genomic characterization of the Neurofibromatosis Type 1 gene of Fugu rubripes

The genomic structure of the Neurofibromatosis Type1 (NF1) gene of Fugu rubripes was investigated by sequence analysis of two overlapping cosmids. The Fugu NF1 gene spans 27 kb and is 13 times smaller than the human counterpart owing primarily to reduced intron size. The predicted amino acid sequence is highly related to that of human neurofibromin, exhibiting an overall similarity of 91.5%. Nearly all exons described for the human NF1 gene could be identified, except exon 12b and the alternatively spliced exons 9br and 48a. With the exception of the splice acceptor site in front of exon 16, all splice sites are in identical positions to those found in the human gene. Intron 1, which is 100-140 kb long in humans, spans 2575 bp in the Fugu NF1 gene. Another large intron of the human NF1 gene, intron 27b (45-50 kb), is 3942 bp of size in Fugu. Sequences related to the OMgp gene (Oligodendrocyte-Myelin-glycoprotein) or the EVI2A gene (ecotropic viral integration site), which are inserted into human NF1 intron 27b, were not detected in the corresponding Fugu intron. However, a single exon gene with similarity to the human EVI2B gene has been found on the reverse strand of Fugu intron 27b. This suggests that the human EVI2B gene and the Fugu gene in intron 27b have a common ancestor. We found the expression of this inserted gene in liver and kidney, but not in brain tissue of Fugu rubripes.     

Gene therapy of single-gene disorders: preface to the special section

Gene therapy was introduced into clinical practice with great excitement, much publicity and considerable optimism in the early 1990s. Scientific evaluation of the early clinical trials has, however, greatly reduced the initial optimism. Follow-up studies have revealed that many early gene therapy trials mainly represented gene transfer into patients, possibly with short-term effects, but not true gene therapy where the course of the disease is permanently affected. This has lead to critical re-evaluation of the approaches taken. Clearly, more basic understanding is needed of the molecular mechanisms of the diseases treated. For this purpose, better animal models for human diseases are necessary. One of the biggest obstacles for gene therapy has been the lack of adequate vector systems. Development of new vectors for efficient and targeted delivery and uptake of therapeutic genes is a crucial area where progress needs to be made. The rationale for gene therapy depends largely on the type of disease to be treated. Recessively inherited single-gene disorders represent diseases where the concept of gene therapy--addition of a therapeutic gene to restore the lost function of two mutant alleles--is easily understood and rarely questioned. However, most gene therapy protocols are focused on multifactorial diseases such as malignancies where the therapeutic approach is quite different. While gene transfer technologies are being developed into truly effective gene therapy, the fight against inherited single-gene disorders also continues at population level by carrier screening and prenatal diagnostics where rapid methodological developments are taking place.     

Evolution of the mitochondrial ATPase 6 gene in Drosophila: unusually high level of polymorphism in D. melanogaster

We have determined 1990 bp mitochondrial DNA sequence which extends from 3' end of the cytochrome oxidase subunit I (COI) gene to 5' end of the COIII gene from two sibling species of Drosophila, D. simulans and D. mauritiana. Analyses of the sequences and part of the NADH dehydrogenase subunit 2 gene and the COI gene together with those from D. melanogaster and D. yakuba revealed that amino-acid substitution rate of the ATPase 6 gene seems to be higher in some strains of D. melanogaster than in the other species. High level of amino-acid polymorphism in this gene was observed in D. melanogaster. Synonymous substitution rate is relatively constant in all the genes examined, suggesting that mutation rate is not higher in the ATPase 6 gene of D. melanogaster. The amino-acid substitutions found specifically in D. melanogaster are at the sites which are not conserved among mammals, yeast and E. coli. These sites of the ATPase 6 gene might lose the selective constraint in D. melanogaster, and the amino-acid substitutions can be explained by neutral mutations and random genetic drift.     

Transgenetic investigations of prion diseases of humans and animals

Prions cause transmissible and genetic neurodegenerative diseases. Infectious prion particles are composed largely, if not entirely, of an abnormal isoform of the prion protein (PrPSc), which is encoded by a chromosomal gene. Although the PrP gene is single copy, transgenic mice with both alleles of the PrP gene ablated develop normally. A post-translational process, as yet unidentified, converts the cellular prion protein (PrPC) into PrPSc. Scrapie incubation times, neuropathology and prion synthesis in transgenic mice are controlled by the PrP gene. Mutations in the PrP gene are genetically linked to development of neurodegeneration. Transgenic mice expressing mutant PrP spontaneously develop neurological dysfunction and spongiform neuropathology. Investigations of prion diseases using transgenesis promise to yield much new information about these once enigmatic disorders.