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Isolation of mtpim proves Tnt1 a useful reverse genetics tool in Medicago truncatula and uncovers new aspects of AP1-like functions in legumes.

Comparative studies help shed light on how the huge diversity in plant forms found in nature has been produced. We use legume species to study developmental differences in inflorescence architecture and flower ontogeny with classical models such as Arabidopsis thaliana or Antirrhinum majus. Whereas genetic control of these processes has been analyzed mostly in pea (Pisum sativum), Medicago truncatula is emerging as a promising alternative system for these studies due to the availability of a range of genetic tools. To assess the use of the retrotransposon Tnt1 for reverse genetics in M. truncatula, we screened a small Tnt1-mutagenized population using degenerate primers for MADS-box genes, known controllers of plant development. We describe here the characterization of mtpim, a new mutant caused by the insertion of Tnt1 in a homolog to the PROLIFERATING INFLORESCENCE MERISTEM (PIM)/APETALA1 (AP1)/SQUAMOSA genes. mtpim shows flower-to-inflorescence conversion and altered flowers with sepals transformed into leaves, indicating that MtPIM controls floral meristem identity and flower development. Although more extreme, this phenotype resembles the pea pim mutants, supporting the idea that M. truncatula could be used to complement analysis of reproductive development already initiated in pea. In fact, our study reveals aspects not shown by analysis of pea mutants: that the mutation in the AP1 homolog interferes with the specification of floral organs from common primordia and causes conversion of sepals into leaves, in addition to true conversion of flowers into inflorescences. The isolation of mtpim represents a proof of concept demonstrating that Tnt1 populations can be efficiently used in reverse genetics screenings in M. truncatula.

Amino Acid Sequence↗

Perspective: new genetic tools for studying retinal development and disease.

The use of knock-out and transgenic mice has been instrumental for advancing our understanding of retinal development and disease. In this perspective, we review existing genetic approaches to studying retinal development and present a series of new genetic tools that complement the use of standard knock-out and transgenic mice. Particular emphasis is placed on elucidating cell-autonomous and non-cell-autonomous roles of genes important for retinal development and disease in vivo. In addition, a series of gene-swapping vectors can be used to elucidate the function of proteins that regulate key processes in retinal development and a wide variety of retinopathies.

Animals↗

Genetic tools in pathogenic nocardioform actinomycetes.

Nocardioform actinomycetes are Gram-positive bacteria with high G+C content. Several species of Mycobacterium, Corynebacterium, Nocardia and Rhodococcus are important human or animal pathogens. Transposon mutagenesis and homologous recombination are powerful genetic tools used for the identification of pathogenicity determinants. Transposable elements with potential application to mutagenize pathogenic nocardioform actinomycetes are described. Homologous recombination experiments which have been recently achieved in mycobacteria and related actinomycetes are commented on.

Actinomycetales↗

A broad-host-range vector of incompatibility group Q can work as a plasmid vector in Neisseria meningitidis: a new genetical tool.

Plasmid pHT128, a derivative of the broad-host-range IncQ vector pGSS33, was successfully introduced into Neisseria meningitidis. Under optimal conditions, pHT128 was transferred from Escherichia coli to N. meningitidis by triparental conjugation at a frequency of 10(-5)-10(-6). The copy number of pHT128 in N. meningitidis was almost the same as in E. coli, in which the copy number of IncQ plasmids per chromosome is estimated to be 10. pHT128 was maintained as an episome in N. meningitidis in the presence of chloramphenicol, a marker of the plasmid. It was also shown that an opc or pilE1 gene cloned on pHT128 could be expressed in N. meningitidis under control of the tac promoter and could complement a mutation of opc or pilE1, respectively. In addition, the conjugational introduction of pHT128 into N. meningitidis was demonstrated to be independent of natural transformation competence. All the results indicate that pHT128 is a useful vector for N. meningitidis as a new genetical tool.

Conjugation, Genetic↗

Use of colicin-based genetic tools for studying bacterial protein transport.

Transport of proteins across the envelope of Gram-negative bacteria is a very challenging domain of investigation, which involves membrane-embedded proteinaceous complexes at which specific targeting occurs. These transporters (translocon or secreton) have been studied both with genetics and biochemistry. In this review we report recent developments that should help to identify novel interactions that exist within these complexes, and to decipher the signals that specifically direct transported proteins to the cognate system. These developments are exclusively based on the re-routing of colicins to these molecular machineries. The re-routing induces a lethal situation in the case of efficient or inefficient transport, depending on the system, thus creating a genetic tool for selection of mutations that correct or generate a transport default.

Bacterial Proteins↗

Tn5 as a molecular genetics tool: In vitro transposition and the coupling of in vitro technologies with in vivo transposition.

The development of in vitro transposition technologies have provided many powerful tools for the molecular genetics research laboratory. In this chapter we describe some of these tools with a focus on the Tn5 transposition system. Tn5 technologies are particularly useful because the Tn5 transposition system has simple requirements, is efficient, random in target recognition, and robust. In particular we will describe the use of in vitro Tn5 transposition in transposon tagging and in the generation of nested deletions. We will also describe a unique in vitro/in vivo technology in which Tn5 inserts can be generated in a wide spectrum of bacterial species through the electroporation of preformed tranposase-transposon DNA complexes.

Base Sequence↗

Inferring sex-biased dispersal from population genetic tools: a review.

Sex-biased dispersal, where individuals of one sex stay or return to their natal site (or group) to breed while individuals of the other sex are prone to disperse, is a wide-spread pattern in vertebrate organisms. In general, mammals exhibit male-biased dispersal whereas birds exhibit female-bias. Dispersal estimates are often difficult to obtain from direct field observations. Here we describe different methods for inferring sex-specific dispersal using population genetic tools and discuss the problems they can raise. We distinguish two types of methods: those based on bi-parental markers (eg comparison of male/female relatedness, F(st) and assignment probabilities) and those relying on the comparison between markers with different modes of inheritance (eg mtDNA markers and microsatellites). Finally, we discuss statistical problems that are encountered with these different methods (eg pseudoreplication, problems due to the comparison of distinct markers). While the genetic methods to detect sex-biased dispersal are now relatively well developed, their interpretation can prove problematic due to the confounding effects of factors such as the mating system of the species. Moreover, the relative power of these methods is not well known and requires further investigation.

Animals↗

Development of genetic tools for Lactobacillus sakei: disruption of the beta-galactosidase gene and use of lacZ as a reporter gene To study regulation of the putative copper ATPase, AtkB.

Downstream from the ptsHI operon of Lactobacillus sakei, the genes atkY and atkB, organized in an operon, were observed. The two putative proteins, AtkB and AtkY, show sequence similarity to the Enterococcus hirae copper P-type ATPase, responsible for copper efflux, and its negative regulator. Characterization of AtkB as a copper P-type ATPase could not be demonstrated since an atkB mutant did not show any phenotype. Thus, another strategy was followed in order to investigate the transcriptional regulation of the atkYB locus, leading to the development of new genetic tools for L. sakei. A plasmid was constructed, the use of which allowed gene replacement at the lacLM locus in L. sakei by two successive crossovers. A strain deleted of the lacLM operon encoding the beta-galactosidase of L. sakei was constructed by this method, and the Escherichia coli lacZ gene could then be used as a reporter gene to investigate the regulation of atkYB. Results show that the atkYB operon is induced by small concentrations of CuSO(4) (30 to 40 microM) but not when CuSO(4) is omitted or added at higher concentrations.

Adenosine Triphosphatases↗

A genomic-scale view of the cAMP response element-enhancer decoy: a tumor target-based genetic tool.

Enhancer DNA decoy oligodeoxynucleotides (ODNs) inhibit transcription by competing for transcription factors. A decoy ODN composed of the cAMP response element (CRE) inhibits CRE-directed gene transcription and tumor growth without affecting normal cell growth. Here, we use DNA microarrays to analyze the global effects of the CRE-decoy ODN in cancer cell lines and in tumors grown in nude mice. The CRE-decoy up-regulates the AP-2beta transcription factor gene in tumors but not in the livers of host animals. The up-regulated expression of AP-2beta is clustered with the up-regulation of other genes involved in development and cell differentiation. Concomitantly, another cluster of genes involved in cell proliferation and transformation is down-regulated. The observed alterations indicate that CRE-directed transcription favors tumor growth. The CRE-decoy ODN, therefore, may serve as a target-based genetic tool to treat cancer and other diseases in which CRE-directed transcription is abnormally used.

Adenocarcinoma↗

Properties of the maize transposable element Activator in transgenic tobacco plants: a versatile inter-species genetic tool.

The maize controlling element Activator (Ac) transposes autonomously from an integrated T-DNA vector to new sites in the genomes of tobacco and other heterologous plant species. Here we demonstrate that critical functions required for transposition of Ac in maize are conserved in tobacco and that Ac transposes at high frequency for at least five generations. Ac structure and terminal sequences are conserved upon transposition and a characteristic 8-bp duplication of target sequences is generated upon integration. Ac remains unmethylated, transcriptionally active, and capable to trans-activate transposition of the nonautonomous Dissociation (Ds) element throughout several generations. In tobacco, as in maize, Ac transposes adjacent to low copy or unique DNA, and transcriptional analysis of unique target DNA provides evidence that an Ac element transposed into a gene. In maize, increasing copies of Ac delay the timing and reduce the frequency of early transposition of Ac and Ds. In tobacco, increasing copies of Ac correlate with an increased frequency of Ds trans-activation. These data firmly establish that the Ac and Ds transposable elements are versatile genetic tools well suited for use as insertional mutagens and demonstrate that thorough investigation of mechanism and regulation of transposition is facilitated in heterologous settings.

Base Sequence↗

CRE-enhancer DNA decoy: a tumor target-based genetic tool.

Enhancer DNA decoy oligonucleotides (ODNs) inhibit transcription by competing for transcription factors. A decoy ODN composed of the cAMP response element (CRE) inhibits CRE-directed gene transcription and tumor growth without affecting normal cell growth. We used DNA microarrays to analyze the global gene expression in tumors exposed to the CRE-decoy ODN. The CRE decoy upregulated the AP-2beta transcription factor gene in tumors but not in the livers of host animals. The upregulated expression of AP-2beta was clustered with other upregulated genes involved in development and cell differentiation. Concomitantly, another cluster of genes involved in cell proliferation and transformation was downregulated. The observed alterations indicate that CRE-directed transcription favors tumor growth. Evidence presented here suggests that the CRE-decoy ODN may provide a target-based genetic tool for treating cancer, viral diseases, and other diseases in which CRE-directed transcription is abnormally used.

Animals↗

Development of a markerless genetic exchange method for Methanosarcina acetivorans C2A and its use in construction of new genetic tools for methanogenic archaea.

A new genetic technique for constructing mutants of Methanosarcina acetivorans C2A by using hpt as a counterselectable marker was developed. Mutants with lesions in the hpt gene, encoding hypoxanthine phosphoribosyltransferase, were shown to be >35-fold more resistant to the toxic base analog 8-aza-2,6-diaminopurine (8ADP) than was the wild type. Reintroduction of the hpt gene into a Delta hpt host restored 8ADP sensitivity and provided the basis for a two-step strategy involving plasmid integration and excision for recombination of mutant alleles onto the M. acetivorans chromosome. We have designated this method markerless exchange because, although selectable markers are used during the process, they are removed in the final mutants. Thus, the method can be repeated many times in the same cell line. The method was validated by construction of Delta proC Delta hpt mutants, which were recovered at a frequency of 22%. Additionally, a Methanosarcina-Escherichia shuttle vector, encoding the Escherichia coli proC gene as a new selectable marker, was constructed for use in proC hosts. Finally, the markerless exchange method was used to recombine a series of uidA reporter gene fusions into the M. acetivorans proC locus. In vitro assay of beta-glucuronidase activity in extracts of these recombinants demonstrated, for the first time, the utility of uidA as a reporter gene in Methanosarcina: A >5,000-fold range of promoter activities could be measured by using uidA: the methyl-coenzyme M reductase operon fusion displayed approximately 300-fold-higher activity than did the serC gene fusion, which in turn had 16-fold-higher activity than did a fusion to the unknown orf2 gene.

Alleles↗

Transposition of Tn916 to different sites in the chromosome of Neisseria meningitidis: a genetic tool for meningococcal mutagenesis.

The difficulty in obtaining mutants in pathogenic Neisseria has limited the ability to genetically define determinants responsible for virulence as well as the ability to generate a genetic map. We show that the 16.5kb conjugative transposon Tn916 can be introduced into Neisseria meningitidis on the suicide vectors pAM120 and pAM170. After introduction, Tn916 transposed to different sites in the chromosome of recipient meningococci, apparently at random, and was stably incorporated. Following its integration into the meningococcal chromosome, Tn916 did not appear to readily express its conjugative and transpositional functions. However, chromosomal DNA from Tn916-carrying meningococci could be used to transform other meningococcal strains to tetracycline resistance. These studies indicate that Tn916 may be an important tool for genetic analysis of N. meningitidis.

Chromosomes↗

Genetic Tools in the Nakaseomyces clade for Evolutionary Comparisons of Signal Transduction Pathways.

The genus Nakaseomyces provides four species that are closely related but have different characteristics. For example, N. glabratus (formerly known as Candida glabrata) is a common human pathogen, whereas N. bracarensis and N. nivariensis have been isolated in clinical settings but are not common human pathogens. N. delphensis was isolated from fruit and there is no evidence it is pathogenic. Given the differences, we developed the clade as a molecular genetic system where we could introduce plasmids and assess transcriptional output from cloned promoters. We engineered a CRISPR/Cas9 plasmid that allows for rapid Gibson cloning of gRNAs, generated auxotrophic strains for amino acids and nucleotides, and introduced plasmids into each species. We used promoter-YFP plasmids to determine that while there are differences between the species, each species likely has intact thiamine and phosphate (THI and PHO) signal transduction pathways, and that gene expression in N. glabratus and N. bracarensis is more similar to one another than to the other two species. Finally, we determine that N. glabratus, N. bracarensis, and N. nivariensis persist in a murine macrophage for 24 h, whereas N. delphensis does not. This work describes new molecular tools for genetic manipulation in the Nakaseomyces clade and allows for evolutionary questions to be explored.

Signal Transduction↗

Genetic tools for pseudomonads, rhizobia, and other gram-negative bacteria.

Gram-negative bacteria are extraordinarily diverse microorganisms that present a wide variety of characteristics worthy of genetic investigation. For historical reasons, the application of recombinant DNA technology to gram-negative bacteria in general has always lagged behind that of E. coli and its close relatives. However, the past 10 years have seen dramatic advances in the development of new tools and vectors for genetic analysis in non-E. coli hosts. Applications include various kinds of genetic manipulation, conjugation, transposition, site-specific recombination, protein secretion, protein purification, cell suicide, microbial ecology, biodegradation, and plant and animal pathogenicity.

Cloning, Molecular↗

Genetic tools, Kuhnean theoretical shift and the geneticization process.

The growing use of genetic tests in medical practice has a strong influence on some widespread notions of health and unhealth. Two consequences of this phenomenon are: (i) important changes in the meaning of these current notions and, therefore, (ii) the arrival of a new taxonomy or rearrangement for the so-called "health-concepts". This paper attempts to demonstrate that both facts fuel a theoretical change that might be considered a model of scientific Kuhnean change in a fundamental aspect. On the other hand, this theoretical shift process offers the basis for approaching a present-day controversy, i.e. whether Western society is going through a period of geneticization or not, from a new perspective.

Attitude to Health↗

Quantitative genetic tools for insecticide resistance risk assessment: estimating the heritability of resistance.

Quantitative genetic studies of resistance can provide estimates of genetic parameters not available with other types of genetic analyses. Three methods are discussed for estimating the amount of additive genetic variation in resistance to individual insecticides and subsequent estimation of the heritability (h2) of resistance. Sibling analysis and offspring-parent regression permit direct estimates of h2 by comparing the resistance phenotypes of individuals of known relatedness. Threshold trait analyses, performed on data from selection experiments, provide estimates of realized heritability. Procedures are outlined for predicting changes in resistance to insecticides based on h2 estimates. Quantitative genetic theory is examined as it relates to resistance and resistance as a quantitative trait; quantitative genetic methods also are unique in providing estimates of genetic correlations between traits. Comments are included on estimates of genetic correlation between resistance and phenotypic traits (e.g., development time) and how they may be used to predict changes in the genetic aspects of phenology that result from insecticide applications (i.e., to predict how the reproductive capacity of future generations will differ from that of the treated generation).

Analysis of Variance↗

Genetic tools in the study of drug self-administration.

Recent studies have shown that large genetic differences exist in the extent to which orally delivered ethanol will come to serve as a positive reinforcer under operantly defined conditions. In addition, these studies suggest that a significant correlation exists between results from two-bottle choice studies of ethanol drinking and operant self-administration studies of ethanol functioning as a reinforcer. The present paper reports further genetic influences on ethanol self-administration which were found using Long Sleep and Short Sleep mice, bred selectively for high and low duration of loss of the righting reflex in responses to ethanol, respectively. It was possible to establish ethanol as a reinforcer in Long Sleep mice but not in Short Sleep mice. These results indicate that neurosensitivity to ethanol may determine the absolute amount of ethanol consumption but is not highly related to the ability of ethanol to serve as a positive reinforcer. In addition, this paper presents genetic correlations which indicate that (a) ethanol preference and self-administration are highly correlated across genotype; (b) sensitivity to ethanol and self-administration of this drug are not highly genetically correlated; (c) ethanol is not self-administered in operant studies solely for its caloric value; and (d) there exist important genetic determinants of drug reinforced behavior.

Alcohol Drinking↗