Monohybrid and dihybrid segregations in the progenies of tobacco transformed for kanamycin resistance with a Ti-vector system

A chimeric DNA construction having nopaline synthase promoter, coding sequences of neomycin phosphotransferase gene conferring resistance to antibiotic kanamycin and OCS (octopine synthase) polyadenylation sequences bracketed by T-DNA ends was transferred to tobacco. Leaf discs were infected withA. tumefaciens containing disarmed, cointegrate plasmid pGV3850:: 1103 and allowed to form a callus in the presence of kanamycin. Shoots regenerated from infected leaf discs either through the callus or arising directly were further selected for their ability to root in kanamycin-containing media. Among the nine transgenic plants that were progeny tested, the transferred bacterial gene segregated as monohybrid ratio (3 Kan^R: 1 Kan^s) in seven. Segregation data of two plant progenies indicated the presence of two independent loci of Kan^R DNA insertion (15 Kan^R: 1 Kan ^s ). Back-cross segregation data were consistent with the monohybrid or independent assortment of duplicate factors. Thus in the two cases, a minimum independent integration of two copies of T-DNA each with a Kan^R marker is inferred.     

Genetic tools for selective labeling of proteins with α-15N-amino acids

Summary A collection of genetic tools that can be used to manipulate amino acid metabolism in Escherichia coli is described. The set comprises 21 strains of bacteria, each containing a different genetic defect that is closely linked to a selectable transposon marker. These tools can be used to construct strains of E. coli with ideal genotypes for residue-specific, selective labeling of proteins with nearly any ¹⁵N-amino acid. By using strains which have been modified to contain the appropriate genetic lesions to control amino acid biosynthesis, dilution of the isotope by endogenous amino acid biosynthesis and scrambling of the label to other types of residues can be avoided.Abbreviations ¹⁵N-amino acid -¹⁵N-amino acid - Cam^R chloramphenicol-resistant - DPA diaminopimelic acid - Hfr high-frequency recombinant - LB Luria broth - Kan^R kanamycin resistant - P1 bacteriophage P1 - pfu plaque-forming units - Str^R streptomycin-resistant - Tet^R tetracycline-resistant     

Intergeneric transfer of a partial genome and direct production of monosomic addition plants by microprotoplast fusion

Results are reported on the transfer of single, specific chromosomes carrying kanamycin resistance (Kan^R) and -glucuronidase (GUS) traits from a transformed donor line of potato (Solanum tuberosum) to a recipient line of the tomato species Lycopersicon peruvianum through microprotoplast fusion. Polyethylene glycol-induced mass fusion between donor potato microprotoplasts containing one or a few chromosomes and normal recipient diploid L. peruvianum protoplasts gave several Kan^R calli. A high frequency of plants regenerated from Kan^R calli expressed both Kan^R and GUS, and contained one or two copies of npt-II and a single copy of gus. Genomic in situ hybridization showed that several microprotoplast hybrid plants had one single potato donor chromosome carrying npt-II and gus genes and the complete chromosome complement of the recipient L. peruvianum (monosomic additions). Several monosomic-addition hybrid plants could be regenerated within the short time of 3 months and they were phenotypically normal, resembling the recipient line. These results suggest that the transfer of single chromosomes is tolerated better than is the transfer of the whole donor genome. The unique advantages of microprotoplast fusion are discussed: these include the direct production of monosomic addition lines for the transfer and introgression of economically important traits in sexually-incongruent species, the construction of chromosome-specific DNA libaries, high-resolution physical mapping and the identification of alien chromosome domains related to gene expression.     

Survival and multiplication ofRhizobium japonicum strains in silt loam

Summary Antibiotic resistant mutants 8-0 Str^R, 110 Tet^R and 138 Kan^R derived from wild typeRhizobium japonicum strains were inoculated into silt loam soil to cell concentrations greater than 2×10⁸/g of soil. Population changes were monitored using antibiotic media and strain identification was done using immunodiffusion assay on microcores of soil. Immunodiffusion bands formed by the mutant strains with homologous antisera essentially duplicated bands formed by the parent strain. Strains 110 Tet^R and 8-0 Str^R had cross reacting antigens whereas antigens of strain 138 Kan^R reacted only with the homologous antiserum. Populations ofR. japonicum strains introduced into sterile soil increased over a period of four weeks under both single and mixed culture inoculations. All populations decreased by the end of six weeks and thereafter remained constant. When theseR. japonicum strains were introduced into non-sterile soil, the population did not increase over the initial population added. Population decreased gradually for two weeks and then maintained thereafter. It was possible to recover very low populations of antibiotic resistantR. japonicum strains from both sterile and unsterile soils using media containing specific antibiotics. Detection ofR. japonicum strains by immunodiffusion was accomplished only when the population was 10⁹ cells/g of soil. The method using antibiotic resistant mutants permitted an evaluation of the interactions of variousR. japonicum strains in soil with respect to their survival and multiplication.     

Production of tyrosine from sucrose or glucose achieved by rapid genetic changes to phenylalanine-producing <Emphasis Type="Italic">Escherichia coli</Emphasis> strains

Escherichia coli K12 strains producing l-phenylalanine were converted to l-tyrosine-producing strains using a novel genetic method for gene replacement. We deleted a region of the E. coli K12 chromosome including the pheA gene encoding chorismate mutase/prephenate dehydratase, its leader peptide (pheL), and its promoter using a new polymerase chain reaction-based method that does not leave a chromosomal scar. For high level expression of tyrA, encoding chorismate mutase/prephenate dehydrogenase, its native promoter was replaced with the strong trc promoter. The linked ΔpheLA and Ptrc-tyrA::Kan^R genetic modifications were moved into l-phenylalanine producing strains by generalized transduction to convert l-phenylalanine-producing strains to l-tyrosine-producing strains. Moreover, introduction of a plasmid carrying genes responsible for sucrose degradation into these strains enabled l-tyrosine-production from sucrose.     

Large-Scale Reverse Genetics in Arabidopsis: Case Studies from the Chloroplast 2010 Project

Traditionally, phenotype-driven forward genetic plant mutant studies have been among the most successful approaches to revealing the roles of genes and their products and elucidating biochemical, developmental, and signaling pathways. A limitation is that it is time consuming, and sometimes technically challenging, to discover the gene responsible for a phenotype by map-based cloning or discovery of the insertion element. Reverse genetics is also an excellent way to associate genes with phenotypes, although an absence of detectable phenotypes often results when screening a small number of mutants with a limited range of phenotypic assays. The Arabidopsis Chloroplast 2010 Project (www.plastid.msu.edu) seeks synergy between forward and reverse genetics by screening thousands of sequence-indexed Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutants for a diverse set of phenotypes. Results from this project are discussed that highlight the strengths and limitations of the approach. We describe the discovery of altered fatty acid desaturation phenotypes associated with mutants of At1g10310, previously described as a pterin aldehyde reductase in folate metabolism. Data are presented to show that growth, fatty acid, and chlorophyll fluorescence defects previously associated with antisense inhibition of synthesis of the family of acyl carrier proteins can be attributed to a single gene insertion in Acyl Carrier Protein4 (At4g25050). A variety of cautionary examples associated with the use of sequence-indexed T-DNA mutants are described, including the need to genotype all lines chosen for analysis (even when they number in the thousands) and the presence of tagged and untagged secondary mutations that can lead to the observed phenotypes.Decoding of the Arabidopsis (Arabidopsis thaliana) genome sequence earlier this decade (Arabidopsis Genome Initiative, 2000) provided the opportunity to determine the functions of approximately 27,000 protein-coding genes. One or more functions of a small percentage of genes are currently experimentally determined, typically from mutant or transgenic analysis or through biochemistry. However, roles for the vast majority of plant genes are either more or less accurately predicted by DNA sequence homology or unpredictable based upon DNA sequence (Arabidopsis Genome Initiative, 2000; Cho and Walbot, 2001; Rhee et al., 2008; for recent specific examples, see Gao et al., 2009; Schilmiller et al., 2009). Because of the uncertainty associated with homology-based function assessment, high-throughput approaches to gene function identification are needed to expand the universe of genes with experimental annotation.In contrast to organisms amenable to targeted gene replacement, such as bacteria, yeast, and mouse (Wendland, 2003; Wu et al., 2007; Adams and van der Weyden, 2008), obtaining a gene knockout is not as efficient in flowering plants. In Arabidopsis, the conventional way of creating a gene knockout is by insertional mutagenesis via Agrobacterium tumefaciens-mediated transformation (Krysan et al., 1999). Using this technique, a large piece of T-DNA is inserted into the genome in an untargeted manner (Alonso et al., 2003). If it lands within a coding or regulatory region, the T-DNA can influence the expression of the corresponding gene. While the probability of any single insertion element causing a mutation in a gene of interest is low, sequencing of hundreds of thousands of independent insertion sites has led to a collection of mutants in the majority of genes (http://signal.SALK.edu/tabout.html; Alonso et al., 2003).T-DNA mutants can be a valuable tool for forward genetics, in which hundreds or thousands of mutants are subjected to phenotypic assays (Feldmann, 1991; Kuromori et al., 2006), but reverse genetics is the most common way in which these mutant collections are utilized. Typically, a small number of candidate genes are tested for a role in a particular biological process by reducing or increasing gene expression and assaying one or more phenotypes (for review, see Page and Grossniklaus, 2002; Alonso and Ecker, 2006). The availability of a gene-indexed T-DNA mutant collection allows researchers to rapidly obtain mutant lines for their genes of interest (http://signal.SALK.edu/cgi-bin/tdnaexpress). The availability of a large collection of indexed mutant or RNA interference lines in other model organisms has facilitated large-scale reverse genetics studies (Piano et al., 2000; Giaever et al., 2002; Ho et al., 2009).In the course of a large reverse genetics project (The Chloroplast 2010 Project; http://www.plastid.msu.edu/), more than 3,500 T-DNA lines harboring insertions in nuclear genes, most of which were computationally predicted to encode chloroplast-targeted proteins, were subjected to a diverse set of phenotypic screens (Lu et al., 2008). In total, 85 phenotypic observations ranging from quantitative metabolite measurements to qualitative phenotypic observations are collected for each mutant line, and the data are stored in a relational database (http://bioinfo.bch.msu.edu/2010_LIMS). This approach seeks to take advantage of the best features of forward and reverse genetics by screening a large number of lines with mutations in known genes. Unlike conventional genetics screens, where plants are assayed for one or a small number of traits, this project surveys varied phenotypes.In this study, a variety of phenotypic variants were analyzed. In some cases, independent mutants of the same gene were found to have similar phenotypes, revealing new information about those genes. In other examples, a single homozygous mutant allele was found to have a detectable phenotype. These run the gamut from cases where secondary mutations are strongly implicated in causing the phenotype, to an example where an analogous maize (Zea mays) mutant is known to have a similar phenotype, to other instances where the causative mutation is yet to be identified. In several examples of secondary mutations, the phenotype was not due to a T-DNA insertion, reinforcing the idea that these untagged alleles are a cause for concern in conducting large-scale reverse genetics screens (Vitha et al., 2003; Adham et al., 2005; Zolman et al., 2008), while providing opportunities for gene function discovery by map-based cloning or whole genome sequence analysis.     

Non-contiguous finished genome sequence and description of Halopiger goleamassiliensis sp. nov.

Halopiger goleamassiliensis strain IIH3^T sp. nov. is a novel, extremely halophilic archaeon within the genus Halopiger. This strain was isolated from an evaporitic sediment in El Golea Lake, Ghardaïa region (Algeria). The type strain is strain IIH3^T. H. goleamassiliensis is moderately thermophilic, neutrophilic, non-motile and coccus-shaped. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,906,923 bp long genome contains 3,854 protein-encoding genes and 49 RNA genes (1 gene is 16S rRNA, 1 gene is 23S rRNA, 3 genes are 5S rRNA, and 44 are tRNA genes).     

Captured Segment Exchange: A Strategy for Custom Engineering Large Genomic Regions in Drosophila melanogaster

Site-specific recombinases (SSRs) are valuable tools for manipulating genomes. In Drosophila, thousands of transgenic insertions carrying SSR recognition sites have been distributed throughout the genome by several large-scale projects. Here we describe a method with the potential to use these insertions to make custom alterations to the Drosophila genome in vivo. Specifically, by employing recombineering techniques and a dual recombinase-mediated cassette exchange strategy based on the phiC31 integrase and FLP recombinase, we show that a large genomic segment that lies between two SSR recognition-site insertions can be “captured” as a target cassette and exchanged for a sequence that was engineered in bacterial cells. We demonstrate this approach by targeting a 50-kb segment spanning the tsh gene, replacing the existing segment with corresponding recombineered sequences through simple and efficient manipulations. Given the high density of SSR recognition-site insertions in Drosophila, our method affords a straightforward and highly efficient approach to explore gene function in situ for a substantial portion of the Drosophila genome.     

T-DNA mediated disruption of essential gametophytic genes in Arabidopsis is unexpectedly rare and cannot be inferred from segregation distortion alone

Many genes are thought to be expressed during the haploid phase in plants, however, very few haploid-specific genes have been isolated so far. T-DNA insertion mutagenesis is a powerful tool for generating mutations that affect gametophyte viability and function, as disruption of a gene essential for these processes should lead to a defect in the transmission of the gametes. Mutants can therefore be screened on the basis of segregation distortion for a reporter resistance gene contained in the T-DNA. We have screened the Versailles collection of Arabidopsis transformants for 1:1 Kan^R:Kan^S segregation after selfing, focussing on gametophyte mutations which show normal transmission through one gametophyte and cause lethality or dysfunction of the other. Only 1.3% (207) of the 16,000 lines screened were scored as good candidates. Thorough genetic analysis of 38 putative T-DNA transmission defect lines (Ttd) identified 8 defective gametophyte mutants, which all showed 0 to 1% T-DNA transmission through the pollen. During the screen, we observed a high background of low-penetrance mutations, often affecting the function of both gametophytes, and many lines which were likely to carry chromosomal rearrangements. The reasons for the small number of retained lines (all male gametophytic) are discussed, as well as the finding that, for most of them, residual T-DNA transmission is obtained through the affected gametophyte.     

T-DNA mediated disruption of essential gametophytic genes in Arabidopsis is unexpectedly rare and cannot be inferred from segregation distortion alone

Many genes are thought to be expressed during the haploid phase in plants, however, very few haploid-specific genes have been isolated so far. T-DNA insertion mutagenesis is a powerful tool for generating mutations that affect gametophyte viability and function, as disruption of a gene essential for these processes should lead to a defect in the transmission of the gametes. Mutants can therefore be screened on the basis of segregation distortion for a reporter resistance gene contained in the T-DNA. We have screened the Versailles collection of Arabidopsis transformants for 1:1 Kan^R:Kan^S segregation after selfing, focussing on gametophyte mutations which show normal transmission through one gametophyte and cause lethality or dysfunction of the other. Only 1.3% (207) of the 16,000 lines screened were scored as good candidates. Thorough genetic analysis of 38 putative T-DNA transmission defect lines (Ttd) identified 8 defective gametophyte mutants, which all showed 0 to 1% T-DNA transmission through the pollen. During the screen, we observed a high background of low-penetrance mutations, often affecting the function of both gametophytes, and many lines which were likely to carry chromosomal rearrangements. The reasons for the small number of retained lines (all male gametophytic) are discussed, as well as the finding that, for most of them, residual T-DNA transmission is obtained through the affected gametophyte. Received: 27 July 1998 / Accepted: 16 September 1998     

Non-contiguous finished genome sequence and description of Sulfurimonas hongkongensis sp. nov., a strictly anaerobic denitrifying,hydrogen- and sulfur-oxidizing chemolithoautotroph isolated from marine sediment

Here, we report a type strain AST-10 representing a novel species Sulfurimonas hongkongensis within Epsilonproteobacteria, which is involved in marine sedimentary sulfur oxidation and denitrification. Strain AST-10^T (= DSM 22096^T = JCM 18418^T) was isolated from the coastal sediment at the Kai Tak Approach Channel connected to Victoria Harbour in Hong Kong. It grew chemolithoautotrophically using thiosulfate, sulfide or hydrogen as the sole electron donor and nitrate as the electron acceptor under anoxic conditions. It was rod-shaped and grew at 15-35°C (optimum at 30°C), pH 6.5-8.5 (optimum at 7.0-7.5), and 10-60 g L^-1 NaCl (optimum at 30 g L^-1). Genome sequencing and annotation of strain AST-10^T showed a 2,302,023 bp genome size, with 34.9% GC content, 2,290 protein-coding genes, and 42 RNA genes, including 3 rRNA genes.     

Identification of Genes That Promote or Inhibit Olfactory Memory Formation in Drosophila

Genetic screens in Drosophila melanogaster and other organisms have been pursued to filter the genome for genetic functions important for memory formation. Such screens have employed primarily chemical or transposon-mediated mutagenesis and have identified numerous mutants including classical memory mutants, dunce and rutabaga. Here, we report the results of a large screen using panneuronal RNAi expression to identify additional genes critical for memory formation. We identified >500 genes that compromise memory when inhibited (low hits), either by disrupting the development and normal function of the adult animal or by participating in the neurophysiological mechanisms underlying memory formation. We also identified >40 genes that enhance memory when inhibited (high hits). The dunce gene was identified as one of the low hits and further experiments were performed to map the effects of the dunce RNAi to the α/β and γ mushroom body neurons. Additional behavioral experiments suggest that dunce knockdown in the mushroom body neurons impairs memory without significantly affecting acquisition. We also characterized one high hit, sickie, to show that RNAi knockdown of this gene enhances memory through effects in dopaminergic neurons without apparent effects on acquisition. These studies further our understanding of two genes involved in memory formation, provide a valuable list of genes that impair memory that may be important for understanding the neurophysiology of memory or neurodevelopmental disorders, and offer a new resource of memory suppressor genes that will aid in understanding restraint mechanisms employed by the brain to optimize resources.     

Genomic analysis of Skermanella stibiiresistens type strain SB22T

Members of genus Skermanella were described as Gram-negative, motile, aerobic, rod-shaped, obligate-heterotrophic bacteria and unable to fix nitrogen. In this study, the genome sequence of Skermanella stibiiresistens SB22^T is reported. Phylogenetic analysis using core proteins confirmed the phylogenetic assignment based on 16S rRNA gene sequences. Strain SB22^T has all the proteins for complete glycolysis, tricarboxylic acid cycle and pentose phosphate pathway. The RuBisCO encoding genes cbbL1S1 and nitrogenase delta subunit gene anfG are absent, consistent with its inability to fix carbon and nitrogen, respectively. In addition, the genome possesses a series of flagellar assembly and chemotaxis genes to ensure its motility.     

Non-random distribution of transduction termini in transductants from the integrated R plasmid, R100-1

Summary Tra ⁺and tra ^–derivatives of drug resistance plasmid, R100-1, were isolated by phage P1 from an Hfr donor with integrated R100-1 and then analyzed by complementation tests with tra ^–point mutants of Flac. Tra ⁺derivatives of R100-1 carrying tetracycline resistance alone and those carrying all six drug-resistance genes could support transfer of tra ^–point mutants of Flac except Flac traJ, whereas all of tra ^–derivatives of R100-1 failed to complement any one of tra ^–point mutants of Flac. This suggests that these tra ^–derivatives of R100-1 carrying tetracycline resistance gene are deleted for all the transfer genes impaired in the Flac point mutants tested. We assume a hot point, probably a specific base sequence similar to an IS element, at the left of the tetracycline gene (Fig. 1) becomes a transduction terminus in transduction of the integrated R100-1 by phage P1. Complementation analysis of tra ^–derivatives carrying five resistance genes except the tetracycline gene led us to a supposition that a gene(s), probably analogous to traJ of the F plasmid, is located on R100-1 near the tetracycline gene which plays an important regulatory role for self-transfer as well as for the complementation of tra ^–Flac mutants.     

Complete genome sequence of Kosakonia sacchari type strain SP1T

Kosakonia sacchari sp. nov. is a new species within the new genus Kosakonia, which was included in the genus Enterobacter. K sacchari is a nitrogen-fixing bacterium named for its association with sugarcane (Saccharum officinarum L.). K sacchari bacteria are Gram-negative, aerobic, non-spore-forming, motile rods. Strain SP1^T (=CGMCC1.12102^T=LMG 26783^T) is the type strain of the K sacchari sp. nov and is able to colonize and fix N₂ in association with sugarcane plants, thus promoting plant growth. Here we summarize the features of strain SP1^T and describe its complete genome sequence. The genome contains a single chromosome and no plasmids, 4,902,024 nucleotides with 53.7% GC content, 4,460 protein-coding genes and 105 RNA genes including 22 rRNA genes, 82 tRNA genes, and 1 ncRNA gene.Key words : endophyte, Enterobacter, Kosakonia, nitrogen fixation, plant growth-promoting bacteria, sugarcane     

MicroRNA-218 Is Deleted and Downregulated in Lung Squamous Cell Carcinoma

MicroRNAs (miRNAs) are a family of small, non-coding RNA species functioning as negative regulators of multiple target genes including tumour suppressor genes and oncogenes. Many miRNA gene loci are located within cancer-associated genomic regions. To identify potential new amplified oncogenic and/or deleted tumour suppressing miRNAs in lung cancer, we inferred miRNA gene dosage from high dimensional arrayCGH data. From miRBase v9.0 (http://microrna.sanger.ac.uk), 474 human miRNA genes were physically mapped to regions of chromosomal loss or gain identified from a high-resolution genome-wide arrayCGH study of 132 primary non-small cell lung cancers (NSCLCs) (a training set of 60 squamous cell carcinomas and 72 adenocarcinomas). MiRNAs were selected as candidates if their immediately flanking probes or host gene were deleted or amplified in at least 25% of primary tumours using both Analysis of Copy Errors algorithm and fold change (≥±1.2) analyses. Using these criteria, 97 miRNAs mapped to regions of aberrant copy number. Analysis of three independent published lung cancer arrayCGH datasets confirmed that 22 of these miRNA loci showed directionally concordant copy number variation. MiR-218, encoded on 4p15.31 and 5q35.1 within two host genes (SLIT2 and SLIT3), in a region of copy number loss, was selected as a priority candidate for follow-up as it is reported as underexpressed in lung cancer. We confirmed decreased expression of mature miR-218 and its host genes by qRT-PCR in 39 NSCLCs relative to normal lung tissue. This downregulation of miR-218 was found to be associated with a history of cigarette smoking, but not human papilloma virus. Thus, we show for the first time that putative lung cancer-associated miRNAs can be identified from genome-wide arrayCGH datasets using a bioinformatics mapping approach, and report that miR-218 is a strong candidate tumour suppressing miRNA potentially involved in lung cancer.