Inhibition of cytosine methylation allows efficient cloning of T-DNA tagged plant DNA ofArabidopsis thaliana by plasmid rescue

Summary Plasmid rescue can provide an efficient way of cloning T-DNA-tagged genomic DNA of plants. However, rescue has often been hampered by extensive rearrangements in the cloned DNA. We have demonstrated using a transgenic line ofArabidopsis thaliana that the plant DNA flanking the T-DNA tag was heavily cytosine methylated. This methylation could be completely inhibited by growing the plants in the presence of azacytidine. Rescue of the T-DNA tag together with the flanking plant genomic DNA sequences from nontreated control plants into an modified cytosine restriction (mcr) proficient strain ofEscherichia coli resulted in rearrangements of the majority of the rescued plasmids. These rearrangements could be avoided if the methylation was inhibited in the transgenic plants by azacytidine treatment or by cloning into anmcr-deficient strain ofE. coli. The results indicate that cytosine methylation of the DNA in the transgenic plants is the main cause of the DNA rearrangements observed during plasmid rescue and suggest efficient strategies to eliminate such artifacts.     

Transposable elements in the fungal plant pathogenFusarium oxysporum

The genome of the fungal plant pathogenFusarium oxysporum contains at least six different families of transposable elements. Representatives of both DNA transposons and retrotransposons have been identified, either by cloning of dispersed repetitive sequences (Foret andpalm) or by trapping in the nitrate reductase gene (Fot1, Fot2 Impala andHop).Fot1 andImpala elements are related to theTc1 andmariner class of transposons. These transposable elements can affect gene structure and function in several ways: inactivation of the target gene through insertion, diversification of the nucleotide sequence by imprecise excisions, and probably chromosomal rearrangements as suggested by the extensive karyotype variation observed among field isolates. Comparisons of the distribution of these elements inFusarium populations have improved our understanding of population structure and epidemiology and provided support for horizontal genetic transfer. Also they could be developed as genetic tools for tagging genes, a cloning strategy that is particularly promising in imperfect fungi.     

Identification of the<Emphasis Type="Italic">bphC</Emphasis> gene for<Emphasis Type="Italic">meta</Emphasis>-cleavage of aromatic pollutants from a metagenomic library derived from lake waters

Useful genes can be screened from various environments by construction of metagenomic DNA libraries. In this study, water samples were collected from several lakes in mid Korea, and analyzed by T-RFLP to examine diversities of the microbial communities. The crude DNAs were extracted by the SDS-based freezing-thawing method, and then further purified using an UltraClean^TM kit (MoBio, USA). The metagenomic libraries were constructed with the DNAs partially digested withEcoR I,BamH I, andSac II inEscherichia coli DH 10B using the pBACe3.6 vector. About 44.0 Mb of metagenomic libraries were obtained with average inserts 13∼15 kb in size. ThebphC genes responsible for degradation of aromatic hydrocarbons viameta-cleavage were identified from the metagenomic libraries by colony hybridization using thebphC specific sequence as a probe. The 2,3-dihydroxybiphenyl (2,3-DHBP) dioxygenase gene (bphC), capable of degradation of 2,3-DHBP, was cloned and its nucleotide sequences analyzed. The genes consisted of 966 and 897 base pairs with an ATG initiation codon and a TGA termination codon. The activity of the 2,3-DHBP dioxygenase was highly expressed to 2,3-DHBP and showed a broad substrate range to 2,3-DHBP, catechol, 3-methylcatechol and 4-methylcatechol. These results indicated that thebphC gene identified from the metagenomes derived from lake water might be useful in the development of a potent strain for degradation of aromatic pollutants.     

Umchs5,a Gene Coding for a Class IV Chitin Synthase inUstilago maydis

A fragment corresponding to a conserved region of a fifth gene coding for chitin synthase in the plant pathogenic fungusUstilago maydiswas amplified by means of the polymerase chain reaction (PCR). The amplified fragment was utilized as a probe for the identification of the whole gene in a genomic library of the fungus. The predicted gene product ofUmchs5has highest similarity with class IV chitin synthases encoded by theCHS3genes fromSaccharomyces cerevisiaeandCandida albicans, chs-4fromNeurospora crassa,andchsEfromAspergillus nidulans. Umchs5null mutants were constructed by substitution of most of the coding sequence with the hygromycin B resistance cassette. Mutants displayed significant reduction in growth rate, chitin content, and chitin synthase activity, specially in the mycelial form. Virulence to corn plantules was also reduced in the mutants. PCR was also used to obtain a fragment of a sixth chitin synthase,Umchs6.It is suggested that multigenic control of chitin synthesis inU. maydisoperates as a protection mechanism for fungal viability in which the loss of one activity is partially compensated by the remaining enzymes.     

Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

Recombination protein A gene,recA, fromSpirulina platensis IAM-M135

We report the cloning and sequencing of therecA gene fromSpirulina platensis. A genomic library ofSpirulina was constructed in pUC19 and screened by PCR using oligonucleotides corresponding to the conserved amino acid sequences ofAnabaena variabilis andSynechococcus RecA proteins. TheSpirulina recA gene consists of an open reading frame (ORF) of 1095 nucleotides encoding a protein (365 residues) which shares an identity of 79%, 70% and 57% with the RecA proteins ofAnabaena variabilis, Synechococcus andEscherichia coli respectively. TherecA gene is located close to one end of the clonedBglII fragment and has only 53 bp of 5 nucleotides. The isolation of this gene has implications for the development of gene transfer system(s) forSpirulina.     

ERECTA is required for protection against heat-stress in the AS1/ AS2 pathway to regulate adaxial-abaxial leaf polarity in Arabidopsis

In seed plants, formation of the adaxial–abaxial polarity is of primary importance in leaf patterning. Since Arabidopsis thaliana (L.) Heynh. genes ASYMMETRIC LEAVES1 (AS1) and ASYMMETRIC LEAVES2 (AS2) are key regulators in specifying adaxial leaf identity, and ERECTA is involved in the AS1/AS2 pathway for regulating adaxial–abaxial polarity [L. Xu et al. (2003) Development 130:4097–4107], we studied the physiological functions of the ERECTA protein in plant development. We analyzed the effects of different environmental conditions on a special leaf structure in the as1 and as2 mutants. This structure, called the lotus-leaf, reflects a severe loss of adaxial–abaxial polarity in leaves. Higher concentrations of salt or other osmotic substance and lower temperature severely affected plant growth both in the wild type and the mutants, but did not affect lotus-leaf frequency in the as1 and as2 mutants. as1 and as2 mutants exhibited a very low lotus-leaf frequency at 22°C, a temperature that favors Arabidopsis growth. The lotus-leaf frequency rose significantly with an increase in growth temperature, and only in plants that are in the erecta mutation background. These results suggest that ERECTA function is required for reducing plant sensitivity to heat stress during adaxial–abaxial polarity formation in leaves.Abbreviations AS1, AS2 ASYMMETRIC LEAVES1, 2 - ER ERECTA     

CENTRORADIALIS/TERMINAL FLOWER 1 gene homolog is conserved inN. tabacum, a determinate inflorescence plant

A mutation in theCENTRORADIALIS (CEN) gene ofAntirrhinum and in theTERMINAL FLOWER 1 (TFL1) gene ofArabidopsis causes their indeterminate inflorescence to determinate. We clonedCEN/TFL1 homologs fromNicotiana tabacum, the wild-type of which has a determinate inflorescence. TheCEN gene was expressed in the inflorescnece meristem and kept its inflorescence meristem identity, whereas the tobacco homolog (NCH) was expressed at a low level throughout the plant’s development. AlthoughCEN andNCH are highly homologous genes, they may have been recruited to different developmental functions during their evolution. TwoNCH genes are derived from amphidiploidN. tabacum, but both of them hybridized with its diploid parents,N. sylvestris andN. tomentosiformis. Southern blotting, and the genomic organization ofTFL1 inArabidopsis revealed that anotherCEN homolog exists in the genome ofArabidopsis. These results suggest that there are two copies of theCEN homolog per diploid plant. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology” These two authors contributed to this work equally.     

TheCBP2 gene fromSaccharomyces douglasii is a functional homologue of theSaccharomyces cerevisiae gene and is essential for respiratory growth in the presence of a wild-type (intron-containing) mitochondrial genome

InSaccharomyces cerevisiae the only known role of theCBP2 gene is the excision of the fifth intron of the mitochondrialcyt b gene (bI5). We have cloned theCBP2 gene fromSaccharomyces douglasii (a close relative ofS. cerevisiae). A comparison of theS. douglasii andS. cerevisiae sequences shows that there are 14% nucleotide substitutions in the coding region, with transitions being three times more frequent than transversions. At the protein level sequence identity is 87%. We have demonstrated that theS. douglasii CBP2 gene is essential for respiratory growth in the presence of a wild-typeS. douglasii mitochondrial genome, but not in the presence of an intronlessS. cerevisiae mitochondrial genome. Also theS. douglasii andS. cerevisiae CBP2 genes are completely interchangeable, even though the intron bI5 is absent from theS. douglasii mitochondrial genome.     

TheisfA mutation inhibits mutator activity and processing of UmuD protein inEscherichia coli recA730 strains

Further studies on theisfA mutation responsible for anti-SOS and antimutagenic activities inEscherichia coli are described. We have previously shown that theisfA mutation inhibits mutagenesis and other SOS-dependent phenomena, possibly by interfering with RecA coprotease activity. TheisfA mutation has now been demonstrated also to suppress mutator activity inE. coli recA730 andrecA730 lexA51(Def) strains that constitutively express RecA coprotease activity. We further show that the antimutator activity of theisfA mutation is related to inhibition of RecA coprotease-dependent processing of UmuD. Expression of UmuD' from plasmid pGW2122 efficiently restores UV-induced mutagenesis in therecA730 isfA strain and partially restores its mutator activity. On the other hand, overproduction of UmuD'C proteins from pGW2123 plasmid markedly enhances UV sensitivity with no restoration of mutability.     

Rapid amplification of genomic DNA ends bynla III partial digestion and polynucleotide tailing

We report a simple and efficient method, which combines restriction endonuclease digestion and deoxynucleotide tailing, for cloning unknown genomic sequences adjacent to a known sequence. Total genomic DNA is partially digested with the frequent-cutting restriction enzymeNla III. A homo-oligomeric cytosine tail is added by terminal transferase. The tailed DNA fragments are used as the template for cloning flanking regions from all sequences of interest. A first round PCR amplification is performed with a gene-specific primer and the selective (modified polyguanine) anchor primer complementary to the cytosine tail and theNla III recognition site, with a universal amplification primer sequence at its 5′ end. This is followed by another PCR amplification with a nested gene-specific primer and the universal amplification primer. Finally, the amplified products are fractionated, cloned, and sequenced. Using this method, we cloned the upstream region of a salt-induced gene based upon a partial cDNA clone (RSC5-U) obtained from sunflower (Helianthus annuus L.).     

Structure and expression of a nuclear gene for the PSI-D subunit of photosystem I inNicotiana sylvestris

The PSI-D subunit is the ferredoxin-binding site of photosystem I, and is encoded by the nuclear genepsaD. We isolated apsaD genomic clone fromNicotiana sylvestris, by screening a genomic library with apsaD cDNA which we previously cloned fromN. sylvestris (Yamamotoet al., Plant Mol Biol 17: 1251, 1991). Nucleotide sequence analysis revealed that this genomic clone contains apsaD gene, which does not correspond to thepsaD cDNA, so we designated these genespsaDb andpsaDa, respectively. ThepsaDb clone encodes a protein of 214 amino acids uninterrupted by introns. The N-terminal sequence determined for theN. sylvestris PSI-D protein encoded bypsaDb begins at the 49th residue. The products ofpsaDa andpsaDb share 82.7% and 79.5% identity at the amino acid and nucleotide levels, respectively. Genomic Southern analysis showed that two copies ofpsaD are present in theN. sylvestris genome. Ribonuclease protection assays and immunoblot analysis inN. sylvestris indicate that both genes are expressed in leaves, stems and flower buds, but neither is expressed in roots. During leaf development, the ratio ofpsaDb topsaDa mRNA increases from 0.12 in leaf buds to 0.36 in mature leaves. The relative abundance of the corresponding proteins decreased over the same developmental period. These results indicate that differential regulation mechanisms controlpsaDa andpsaDb expression at both the mRNA and protein levels during leaf development.     

Cloning of the cDNA and genomic clones for glutathione synthetase fromArabidopsis thaliana and complementation of agsh2 mutant in fission yeast

Glutathione is essential for protecting plants from a range of environmental stresses, including heavy metals where it acts as a precursor for the synthesis of phytochelatins. A 1658 bp cDNA clone for glutathione synthetase (gsh2) was isolated fromArabidopsis thaliana plants that were actively synthesizing glutathione upon exposure to cadmium. The sequence of the clone revealed a protein with an estimated molecular mass of 53858 Da that was very similar to the protein from higher eukaryotes, was less similar to the gene from the fission yeast,Schizosaccharomyces pombe, and shared only a small region of similarity with theEscherichia coli protein. A 4.3 kbSstI fragment containing the genomic clone for glutathione synthetase was also isolated and sequenced. A comparison of the cDNA and genomic sequences revealed that the gene was composed of twelve exons.When theArabidopsis cDNA cloned in a special shuttle vector was expressed in aS. pombe mutant deficient in glutathione synthetase activity, the plant cDNA was able to complement the yeast mutation. Glutathione synthetase activity was measurable in wild-type yeast cells, below detectable levels in thegsh2 ^- mutant, and restored to substantial levels by the expression of theArabidopsis cDNA. TheS. pombe mutant expressing the plant cDNA had near wild type levels of total cellular thiols,¹⁰⁹Cd²⁺ binding activity, and cadmium resistance. Since theArabidopsis cDNA was under control of a thiamine-repressible promoter, growth of the transformed yeast on thiamine-free medium increased expression of the cDNA resulting in increases in cadmium resistance.     

High-resolution genetic and physical mapping of the cauliflower high-β-carotene gene<Emphasis Type="Italic"> Or</Emphasis> (<Emphasis Type="Italic">Orange</Emphasis>)

Mutation in the cauliflower gene Or causes high levels of -carotene to accumulate in various tissues of the plant that are normally devoid of carotenoids. To decipher the molecular basis by which Or regulates carotenoid accumulation, we have undertaken the isolation of Or by a map-based cloning strategy. Two previously isolated, locus-specific, sequence-characterized amplified region (SCAR) markers that flank Or were employed for the analysis of a large segregating population consisting of 1632 F₂ individuals, and a high-resolution genetic linkage map of the Or locus region was developed. To facilitate positional cloning, we constructed a cauliflower genomic library in a bacterial artificial chromosome (BAC) vector, using high molecular weight DNA from Or homozygotes. The BAC library comprises 60,288 clones with an average insert size of 110 kb, and represents an estimated 10-fold coverage of the genome. A BAC contig encompassing the Or locus was established by screening the library with a marker that is closely linked to Or and by identifying overlapping BAC clones by chromosome walking. Physical mapping delimited the Or locus to a 50-kb DNA fragment within a single BAC clone, which corresponds to a genetic interval of 0.3 cM.Communicated by R. Hagemann     

Development of a new transformation-competent artificial chromosome (TAC) vector and construction of tomato and rice TAC libraries

Recent research has shown that BIBAC (binary bacterial artificial chromosome) and TAC (transformation-competent artificial chromosome) vector systems are very useful tools for map-based cloning of agronomically important genes in plant species. We have developed a new TAC vector that is suitable for both dicot and monocot transformation. Using this new TAC vector, we constructed large-insert genomic libraries of tomato and rice. The tomato library contains 96,996 clones (28.3-38.5 kb insert size) and has 3.18 haploid genome equivalents. The rice TAC library has 32.7 kb average insert size and has 9.24 haploid genome equivalents. The quality of these two libraries was tested using PCR to verify genome coverage. Individual clones were characterized to confirm insert integrity by Southern analysis, end sequencing and genetic mapping. To investigate the potential application of these TAC libraries in map-based cloning, TAC constructs containing a 45 kb fragment were introduced into the rice genome via Agrobacterium-mediated transformation. Molecular analysis indicates that the 45 kb fragment was successfully transferred into the rice genome. Although rearrangements of the introduced DNA were detected, 50% of regenerated plants contained at least one intact copy of the 45 kb clone and associated vector sequences. These libraries provide us with a valuable resource to rapidly isolate important genes in tomato and rice.     

Evolution of the<Emphasis Type="Italic"> Drosophila broad</Emphasis> locus: the<Emphasis Type="Italic"> Manduca sexta broad</Emphasis> Z4 isoform has biological activity in<Emphasis Type="Italic"> Drosophila</Emphasis>

The Drosophila melanogaster broad locus is essential for normal metamorphic development. Broad encodes three genetically distinct functions (rbp, br, and 2Bc) and a family of four zinc-finger DNA-binding proteins (Z1-Z4). The Z1, Z2, and Z3 protein isoforms are primarily associated with the rbp, br, and 2Bc genetic functions respectively. The Z4 protein isoform also provides some rbp genetic function, however an essential function for the Z4 isoform in metamorphosis has not been identified. To determine the degree of conservation of Z4 function between the tobacco hornworm Manduca sexta and Drosophila we generated transgenic Drosophila expressing the Manduca broad Z4 isoform and used this transgene to rescue rbp mutant lethality during Drosophila metamorphosis. We find that the Manduca Z4 protein has significant biological activity in Drosophila with respect to rescue of rbp-associated lethality. There was also some overlap in effects on cuticle gene expression between the Manduca Z4 and Drosophila Z1 isoforms that was not shared with the Drosophila Z4 isoform. Our findings show that Z4 function has been conserved over the 260-million-year period since the divergence of Diptera and Lepidoptera, and are consistent with the hypothesis that the Drosophila Z4 and Manduca Z4 isoforms have essential roles in metamorphosis.Edited by M. Akam     

TREGED: A new strategy for inducing deletions in plant genomes

We have created a DNA construct, TREGED (transposon-and recombinase-mediated genome deletion), that will automatically induce deletions in plant genomes. TREGED contains the maizeAc/Ds transposon, the yeast R-RS site-specific recombination system, the bacterialtetR repression systems, a novel artificial superintron, and the marker genesGUS andLc. The novelty of TREGED is that only one cross is required to trigger a sequence of events leading to deletion and, simultaneously, to a color assay to detect the deletion. Crossing is done to introduceAc transposase which activatesDs transposition from TREGED to a nearby chromosome region.Ds transposition, in turn, activates recombination between an engineeredRS site on TREGED and anRS site on the transposedDs fragment, thus deleting the genome segment between TREGED andDs. The recombination event also deletesLc orGUS and part oftetR, which triggers expression ofGUS orLc color genes for an upstream or downstream deletion respectively. Each TREGED insertion site will produce multiple kinds of deletions identifiable by inspecting a single F₁ plant and its progeny for colored tissue. The color markers can also be used to differentiate between deletion and other more rare events such as translocation and inversion. We anticipate TREGED will greatly simplify the steps required to obtain useful deletions—eventually allowing the creation of detailed deletion libraries. Such libraries will be particularly useful for anlaysis of gene and chromatin function in plant species with large genomes.     

Influence of the<Emphasis Type="Italic">SMT2</Emphasis> knock-out on hypocotyl elongation in<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Inhibitors are very important in the study of hormone function. Brasinazole (Brz) is a specific inhibitor of brassinosteroids (BRs) biosynthesis. To expand our knowledge of the molecular mechanisms of plant steroid signaling, we performed genetic screening using medium containing Brz under dark conditions. Mutants insensitive to Brz developlonger hypocotyls than their wild type counterparts. We isolatedabz453 as a Brz insensitive mutant. TAIL-PCR and the segregation ratio of T2 plants indicated a single T-DNA insertion at the 24-Sterol C-methyltransferase (SMT2) gene in theabz453 mutant. Recapitulation for putative FCP serine phosphatase (FSP), the gene neighboringSMT2, indicated no significant phenotypes, but theSMT2 anti-sense (SMT2-AS) line developed longer hypocotyls than the wild type in medium containing Brz. Additionally, theSMT2-AS line displayed similar phenotypes to theabz453 line in soil including enhanced growth and smaller silique. Theabz453 andSMT2-AS mutants showed phenotypes similar to those of wild type in medium containing benzylaminopurine, pacrobutrazol and ACC (precursor for ethylene) under dark conditions. However, when brassinolide (BL) dose response was observed, theabz453 andSMT2-AS lines showed higher sensitivity than wild type. Theabz453/det2 andabz453/bri1-119 double mutants showed enhanced growth compared to thedet2 andbri1-119 line under both dark and light conditions. Specially, in dark conditions double mutants displayed nearly 2- and 1.5-fold longer hypocotyls thandet2 andbri1-119 plants. Brz insensitivity to theSMT2 knock-out mutant and phenotypes of double mutants indicate that not only do BRI1 and DET2 influence the BRs response, as evidenced by hypocotyl elongation, but another sterol derived signals may also be affected in mutants, suggesting that another pathway is involved in hypocotyl elongation due to SMT2.