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PubMed2003

Analysis of the Arabidopsis MADS AFFECTING FLOWERING gene family: MAF2 prevents vernalization by short periods of cold.

The Arabidopsis FLOWERING LOCUS C (FLC) gene is a key floral repressor in the maintenance of a vernalization response. In vernalization-sensitive genetic backgrounds, FLC levels are high, and they decline after exposure to long cold periods. Four FLC paralogs (MAF2 [MADS AFFECTING FLOWERING2] to MAF5) are arranged in a tandem array on the bottom of Arabidopsis chromosome V. We used a reverse genetics approach to analyze their functions. Loss-of-function and gain-of-function studies indicate that MAF2 acts as a floral repressor. In particular, maf2 mutant plants display a pronounced vernalization response when subjected to relatively short cold periods, which are insufficient to elicit a strong flowering response in the wild type, despite producing a large reduction in FLC levels. MAF2 expression is less sensitive to vernalization than that of FLC, and its repressor activity is exerted independently or downstream of FLC transcription. Thus, MAF2 can prevent premature vernalization in response to brief cold spells. Overexpression of MAF3 or MAF4 produces alterations in flowering time that suggest that these genes also act as floral repressors and might contribute to the maintenance of a vernalization requirement. However, the final gene in the cluster, MAF5, is upregulated by vernalization. Therefore, MAF5 could play an opposite role to FLC in the vernalization response.

Acclimatization↗

PubMed2004

Hepatitis C virus infection in human liver tissue engrafted in mice with an infectious molecular clone.

BACKGROUND/AIMS: Recent advances in molecular cloning of hepatitis C virus (HCV) have enabled us to apply some available HCV molecular clones to experimental studies. However, these investigations have been restricted to chimpanzee models or 'isolated hepatocytes' from tree shrews. In this study, we engrafted 'human liver tissue' into immunodeficient mice and investigated HCV infection using an infectious molecular clone. METHODS: Human liver tissues from normal (non-HCV-infected) liver were transplanted into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We then inoculated the mice with sera from HCV-infected patients or an infectious HCV molecular clone. HCV RNA was assessed using nested reverse-transcription polymerase chain reaction (PCR), real-time detection PCR and in situ PCR. RESULTS: Without any growth support, normal human liver tissues survived in NOD/SCID mice while maintaining the original viable hepatic architecture. HCV RNA was detected in the mice serum until the fourth week after the inoculation. In situ PCR and immunohistochemistry clearly demonstrated positive signals for HCV in the cytoplasm of infected hepatocytes, while the engrafted human liver tissues showed no apparent morphological changes indicative of infection. CONCLUSION: Engraftment of human liver tissues into NOD/SCID mice and infection with HCV molecular clones could offer a reverse genetic strategy for HCV infection.

Animals↗

PubMed1998

Yarrowia lipolytica: a model organism for protein secretion studies.

This paper reviews the advantages of the yeast Yarrowia lipolytica as a tool in the study of protein secretion. Work has been focused on the early steps leading the polypeptide, from the cytoplasmic ribosomes where it is synthesized, to the lumen of the endoplasmic reticulum. Using a thermosensitive allele of the 7SL RNA, the first in vivo evidence for a co-translational translocation was shown. Genetic screens allowed the identification of several new components of the translocation apparatus: Sls1p, an ER lumenal component involved in both translocation and lumenal transit; Tsr1p, involved in SRP-ribosome targeting; Tsr3p. Major translocation partners were also identified by reverse genetics (Sec61p, Sec62p, Kar2p, Srp54p, Sec65p).

Fungal Proteins↗

PubMed1990

Genetic methods for study of trans-acting genes involved in processing of precursors to yeast cytoplasmic transfer RNAs.

All of the strategies to identify mutants defective in pre-tRNA processing have proved to be useful. However, in some of these strategies (such as inability to derepress a nonsense suppressor, correction of processing-defective substrates, and reverse genetics) only a single mutant has been isolated. This is not because the methods have proved to be inefficient, but rather because the field is still in infancy. Further work is needed to saturate the types of mutants found by each procedure. Undoubtedly, new strategies will be designed.

Alleles↗

PubMed1995

Molecular and genetic control of chitin biosynthesis in fungi.

Chitin is the most important structural component of the cell walls of fungi. Its synthesis involves the transfer of N-acetylglucosaminyl residues from the universal donor UDPGlcNAC to the growing chain. This reaction is catalyzed by an ill-defined enzyme called chitin synthetase. By use of diverse techniques, including reverse genetics, it has been possible to isolate mutants affected in chitin biosynthesis in vitro and in vivo. These studies have permitted the identification of several genes that code for the catalytic components of the enzyme, and probably for ancillary reactions. In general, two types of genes have been identified in fungi. The so-called CHS genes, from which two families, and possibly three classes exist, code for chitin synthetases activated by proteases. All fungi thus far studied contain more than one CHS gene, which are normally dispensable. The second class are larger, essential genes coding for the catalytic polypeptide of chitin synthetases non-activated by proteolysis, and probably made of more than one polypeptide. These are labeled CSD2 or CAL1. It may be hoped that our knowledge of chitin synthetase will make them the most suitable targets for new strategies to control fungal infections.

Amino Acid Sequence↗

PubMed2006

Genomic approaches to drug discovery.

Considerable progress has been made in exploiting the enormous amount of genomic and genetic information for the identification of potential targets for drug discovery and development. New tools that incorporate pathway information have been developed for gene expression data mining to reflect differences in pathways in normal and disease states. In addition, forward and reverse genetics used in a high-throughput mode with full-length cDNA and RNAi libraries enable the direct identification of components of signaling pathways. The discovery of the regulatory function of microRNAs highlights the importance of continuing the investigation of the genome with sophisticated tools. Furthermore, epigenetic information including DNA methylation and histone modifications that mediate important biological processes add to the possibilities to identify novel drug targets and patient populations that will benefit from new therapies.

Animals↗

PubMed2004

Bioterrorism and emerging infectious disease - antimicrobials, therapeutics and immune-modulators. SARS coronavirus.

The purpose of this meeting was to provide a forum for expert presentations and discussion about the threats of bioterrorism and emerging infectious diseases, and to address the issues relating to epidemics, prevention of infection and treatment of some of these emerging infectious diseases classified as potential agents of bioterror. Included in the talks were state-of-the-art presentations about infectious clone technology and recombinant viruses, pathogen and receptor interactions at the cellular and molecular level, genomic responses to infection, and new information on antiviral mechanisms of action. Severe acute respiratory syndrome (SARS) and progress toward understanding the epidemic was addressed, and other sessions were presented concerning immune therapy and immunopotentiation of disease, siRNA and gene silencing, host responses to pathogen infections, as well as the use of genetic engineering to circumvent and direct the immune response. Many discussions were held and data were presented about possible compounds and new drugs that may have antiviral properties, yet there were few discussions of any available new drugs. This report addresses reverse genetics of SARS virus, as well as its epidemiology, and a host of different recent approaches to developing antivirals effective against SARS, including some potential vaccine candidates. Also presented are hypotheses about the human immune response to SARS infection, as well as immune therapies against botulinum and anthrax toxins. This report also addresses antiviral approaches exploiting siRNAs, and different aspects of the host immune response to many of the different dangerous pathogens discussed at this meeting. Finally, approaches to circumventing and directing the immune response using genetic engineering will be reported.

Adjuvants, Immunologic↗

PubMed1991

[Cytomolecular aspects of colorectal carcinoma].

This report reviewed recent remarkable progresses on the cytomolecular mechanisms in colorectal carcinogenesis. Colorectal carcinoma is a good model for the study of multi-step progression, because we can obtain adenomatous polyps which are considered as a precancerous form. Furthermore, a familial syndrome, which is characterized by numerous adenomas of the colon, is available for linkage analysis. Recently, the p53 and DCC genes have been identified as candidate tumor suppressor genes on chromosome 17p and 18q respectively. In this paper, we present the multiple genetic alterations in colorectal carcinoma, including activation of K-ras gene and inactivation of tumor suppressor gene such as p53 and DCC genes as well as loss of heterozygosity and approach to the gene responsible for adenomatous polyposis coli by reverse genetics.

Adenomatous Polyposis Coli↗

PubMed2002

Molecular and genetic aspects of plant responses to osmotic stress.

Drought, high salinity and freezing impose osmotic stress on plants. Plants respond to the stress in part by modulating gene expression, which eventually leads to the restoration of cellular homeostasis, detoxification of toxins and recovery of growth. The signal transduction pathways mediating these adaptations can be dissected by combining forward and reverse genetic approaches with molecular, biochemical and physiological studies. Arabidopsis is a useful genetic model system for this purpose and its relatives including the halophyte Thellungiella halophila, can serve as valuable complementary genetic model systems.

Journal Article↗

PubMed1995

HpaII methyltransferase is mutagenic in Escherichia coli.

A genetic reversion assay to study C-to-T mutations within CG sites in DNA is described. It was used to demonstrate that the presence of HpaII methyltransferase (MTase) in Escherichia coli causes a substantial increase in C-to-T mutations at CG sites. This is similar to the known mutagenic effects of E. coli MTase Dcm within its own recognition sequence. With this genetic system, a homolog of an E. coli DNA repair gene in Haemophilus parainfluenzae was tested for antimutagenic activity. Unexpectedly, the homolog was found to have little effect on the reversion frequency. The system was also used to show that HpaII and SssI MTases can convert cytosine to uracil in vitro. These studies define 5-methylcytosine as an intrinsic mutagen and further elaborate the mutagenic potential of cytosine MTases.

5-Methylcytosine↗

PubMed2001

The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus is a unique glycolytic enzyme that belongs to the cupin superfamily.

Pyrococcus furiosus uses a variant of the Embden-Meyerhof pathway during growth on sugars. All but one of the genes that encode the glycolytic enzymes of P. furiosus have previously been identified, either by homology searching of its genome or by reversed genetics. We here report the isolation of the missing link of the pyrococcal glycolysis, the phosphoglucose isomerase (PGI), which was purified to homogeneity from P. furiosus and biochemically characterized. The P. furiosus PGI, a dimer of identical 23.5-kDa subunits, catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate, with K(m) values of 1.99 and 0.63 mm, respectively. An optimum pH of 7.0 has been determined in both directions, and at its optimum temperature of 90 degrees C the enzyme has a half-life of 2.4 h. The N-terminal sequence was used for the identification of the pgiA gene in the P. furiosus genome. The pgiA transcription start site has been determined, and a monocistronic messenger was detected in P. furiosus during growth on maltose and pyruvate. The pgiA gene was functionally expressed in Escherichia coli BL21(DE3). The deduced amino acid sequence of this first archaeal PGI revealed that it is not related to its bacterial and eukaryal counterparts. In contrast, this archaeal PGI shares similarity with the cupin superfamily that consists of a variety of proteins that are generally involved in sugar metabolism in both prokaryotes and eukaryotes. As for the P. furiosus PGI, distinct phylogenetic origins have previously been reported for other enzymes from the pyrococcal glycolytic pathway. Apparently, convergent evolution by recruitment of several unique enzymes has resulted in the unique Pyrococcus glycolysis.

Amino Acid Sequence↗

PubMed2003

[Genetics for the non-geneticist. Semantics and basic facts].

This chapter is an introduction to a new language, that of genetics. The first part recalls and illustrates the elementary concepts of molecular genetics, with emphasis on the structure of DNA and the chromosomes, that of genes, the genetic code and the genetic factors of sexual differentiation. The second part explains medical molecular genetics which is based on a fundamental concept: polymorphism of DNA, a normal and physiological phenomenon which arises mostly from genetic exchanges which occur during meiosis. Monogenic diseases with mendelian transmission may be autosomal dominant or recessive or linked to the X-chromosome. The understanding of multigenic diseases requires the use of much more complex techniques based on so-called "reverse" genetics or on the "candidate gene" method.

Chromosomes↗

PubMed2005

Amazing grass: developmental genetics of maize domestication.

Crop plants were domesticated by prehistoric farmers through artificial selection to provide a means of feeding the human population. This article discusses the developmental genetics of crop domestication and improvement, including the historical framework and recent approaches in maize and other grasses. In many cases, selecting for a plant form that correlates with productivity involves controlling meristem activity. In the domestication of modern maize from its progenitor Zea mays ssp. parviglumis, QTL (quantitative trait loci) mapping, genetics and population genomics approaches have identified several genes that contain signatures of selection. Only a few genes involved in the derivation of the highly productive maize ear have been identified, including teosinte glume architecture1 and ramosa1. Future prospects hinge on forward and reverse genetics, as well as on other approaches from the developing discipline of evo-devo (evolutionary developmental biology).

Animals↗

PubMed2000

Disruption of the Arabidopsis photosystem I gene psaE1 affects photosynthesis and impairs growth.

The psae1-1 mutant of Arabidopsis was identified on the basis of a decrease in the effective quantum yield of photosystem II, among a collection of plants subjected to transposon tagging with the Enhancer element. The steady-state redox level and the rate of re-oxidation of P700 are significantly altered in psae1-1 mutants. The responsible mutation was localised to psaE1, one of two Arabidopsis genes that encode subunit E of photosystem I. An additional mutant allele, psae1-2, was identified by reverse genetics. In wild-type plants, the psaE1 transcript is expressed at a higher level than psaE2 mRNA. In the mutants, however, the E1 transcript was barely detectable, and was expressed only in small groups of wild-type cells resulting from somatic reversions. As a consequence, the amount of PsaE protein present in the mutant is significantly reduced. Concomitantly, the levels of other stromal photosystem I subunits (PsaC and PsaD) are also affected. Mutant plants showed a marked increase in light sensitivity and photoinhibition. Additional effects of the psae1 mutation include light green pigmentation, an increase in chlorophyll fluorescence and a decrease of approximately 50% in growth rate under greenhouse conditions.

Alleles↗

PubMed1994

Genetic approaches to study Legionella pneumophila pathogenicity.

Legionella pneumophila is an intracellular pathogen replicating in human macrophages during the course of infection of the lungs. Infection by legionellae often leads to severe pneumonia, termed Legionnaires' disease. Genetic approaches to identify the factors responsible for L. pneumophila pathogenicity started with the construction of genomic libraries in Escherichia coli. Various L. pneumophila-specific genes were cloned in E. coli K-12 by identification using functional assays, antibody screening and hybridization ('reverse genetics'). By disrupting the genes via allelic exchange, mutants have been created to assess the influence of the factors on pathogenicity. Among the cloned genes, only for the gene product of the mip gene, encoding a 24-kDa surface-associated protein (macrophage infectivity potentiator) unequivocal evidence for its contribution to pathogenicity could be provided. Two hemolytic factors that have been cloned do not seem to play a role in L. pneumophila pathogenicity. Genetic systems for transposon mutagenesis of the L. pneumophila genome (Tn5, Tn903dIIlacZ, MudphoA), including Tn phoA shuttle mutagenesis, have been established and specifically adapted to identify mutants which displayed an impaired capability to multiply inside macrophages and with a reduced in vivo virulence. Furthermore, by complementation of avirulent mutants, genetic loci could be identified which restored the virulence.

Gene Expression Regulation, Bacterial↗

PubMed1992

Correlation of genetic and physical structure in the region surrounding the I2 Fusarium oxysporum resistance locus in tomato.

The dominant gene I2 confers on tomato (Lycopersicon esculentum) resistance against the fungus Fusarium oxysporum f. sp. lycopersici race 2. A restriction fragment length polymorphism (RFLP) marker, TG105, has recently been found to be tightly linked to I2. The potential for cloning this gene by a reverse genetics approach prompted us to describe in both genetic and physical detail the region surrounding the I2 locus on chromosome 11. We have analyzed patterns of segregation of RFLP markers on chromosome 11 and Fusarium resistance in 140 F2 plants from a cross between Fusarium-resistant and susceptible parental lines. Marker TG105 mapped 0.4 centiMorgan (CM) from I2. Physical analysis of TG105 and its flanking RFLP markers, TG26 and TG36, by pulsed field gradient gel electrophoresis (PFGE) yielded a restriction map for this region encompassing at least 620 kb of the tomato genome. TG105 and TG26 hybridized to the same 175 kb MluI-NruI restriction fragment. We have therefore linked two genetically distinct RFLP markers. Based on the 4.1 cM distance between them, we have assigned a mean value of 43 kb for each cM recombination distance in the vicinity of I2. This local ratio between physical and genetic distances is more than 10-fold below the average for the tomato genome. It should therefore be possible to clone I2 by chromosome walking from TG105.

Fusarium↗

PubMed1993

Genetics of epilepsy: an overview.

Studies of the genetics of epilepsy have, until recently, involved epidemiologic or segregation analyses of phenotypic characteristics of a number of seizure disorders. Technical advances in molecular biology involving gene mapping and gene identification have made it possible to examine the heritability of various epilepsy syndromes. Using "reverse genetics" or positional cloning, it is possible to identify an abnormal protein through gene isolation and cloning. Genes are localized through analysis of linkage to phenotypic markers (proteins) or DNA markers such as restriction fragment length polymorphisms, variable number of tandem repeats, and dinucleotides. Methods used to obtain DNA of interest involve digestion of genomic DNA with specific restriction endonucleases or amplification of DNA by polymerase chain reaction technology. Gel electrophoresis is the basis for the separation of different sized DNA. Inherited disorders for which a gene has been cloned or localized have highly penetrant, well-defined clinical phenotypes with no remissions and abundant clinical material. Genetic epilepsies, however, are variably penetrant age-dependent disorders with heterogeneous clinical phenotypes. Despite these difficulties, three genetic epilepsies have been mapped to specific chromosomes: benign familial neonatal convulsions to 20q, juvenile myoclonic epilepsy to 6p, and Baltic progressive myoclonus epilepsy to 21q. Further progress in understanding genetic epilepsies will depend on better definition of syndrome phenotypes, isolation of the epilepsy gene(s), and identification of the abnormal protein(s).

Animals↗

PubMed2006

An overview of C. elegans biology.

The establishment of Caenorhabditis elegans as a "model organism" began with the efforts of Sydney Brenner in the early 1960s. Brenner's focus was to find a suitable animal model in which the tools of genetic analysis could be used to define molecular mechanisms of development and nervous system function. C. elegans provides numerous experimental advantages for such studies. These advantages include a short life cycle, production of large numbers of offspring, easy and inexpensive laboratory culture, forward and reverse genetic tractability, and a relatively simple anatomy. This chapter will provide a brief overview of C. elegans biology.

Animals↗