DNA ligase-AMP adducts: identification of yeast DNA ligase polypeptides.

Yeast DNA ligase is radioactively labelled in vitro by incubating a crude cell extract with [alpha-32P]ATP. The product of this reaction is the stable covalent ligase-AMP adduct, which can be characterized by its reactivity with either pyrophosphate or nicked DNA and visualized by gel electrophoresis and autoradiography. The Saccharomyces cerevisiae DNA ligase was identified as an 89 kDa polypeptide by exploiting the fact that transformants with multiple copies of the plasmid-encoded DNA ligase (CDC9) gene overproduce the enzyme by two orders of magnitude. A similar strategy has been used to identify the Schizosaccharomyces pombe DNA ligase as an 87 kDa polypeptide. Both values agree well with the coding capacities of the respective cloned gene sequences. When the S. cerevisiae ligase is greatly overproduced with respect to wild-type levels, a second polypeptide of 78.5 kDa is also labelled and has the same properties as the 89 kDa adduct. We suggest that this polypeptide is generated by proteolysis.

An Arg/Lys-rich core peptide mimics TRBP binding to the HIV-1 TAR RNA upper-stem/loop.

TRBP is a cellular protein that binds to the HIV-1 leader RNA, TAR. Circular dichroism experiments have shown that a 24 amino acid peptide (TR1), located within a dsRNA binding domain (dsRBD) of TRBP, binds TAR with a 3:1 stoichiometry, eliciting a conformational change involving base unstacking. The binding characteristics of synthetic structural variants of TAR indicate that guanine residues play a key role in the TR1-RNA interaction and that binding sites exist in the upper-stem/loop and lower stem region of TAR. Deletion analysis of TR1 has led to the identification of a 15 amino acid subpeptide (TR13) which is necessary and sufficient to bind to the high affinity upper-stem/loop binding site of TAR. Alanine scanning of TR13 has revealed that mutations in either Lys or Arg residues result in altered TAR-binding, and molecular modelling/docking experiments have shown that the two Arg residues of TR13 can interact with two appropriately spaced guanine residues in the upper-stem/loop of TAR. The TR13 lysine residues appear to be essential for maintaining structural integrity and the correct positioning of the Arg side-chains. We propose that TRBP binds TAR by means of a "2-G hook" motif and that the binding specificity of this particular member of the family of double-stranded RNA-binding proteins lies within the highly conserved dsRBD core motif. Finally, our results also suggest that TRBP may function in vivo by modifying the tertiary structure of TAR RNA.

Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations.

Medicago truncatula, a diploid autogamous legume, is currently being developed as a model plant for the study of root endosymbiotic associations, including nodulation and mycorrhizal colonization. An important requirement for such a plant is the possibility of rapidly introducing and analyzing chimeric gene constructs in root tissues. For this reason, we developed and optimized a convenient protocol for Agrobacterium rhizogenes-mediated transformation of M. truncatula. This unusual protocol, which involves the inoculation of sectioned seedling radicles, results in rapid and efficient hairy root organogenesis and the subsequent development of vigorous "composite plants." In addition, we found that kanamycin can be used to select for the cotransformation of hairy roots directly with gene constructs of interest. M. truncatula composite plant hairy roots have a similar morphology to normal roots and can be nodulated successfully by their nitrogen-fixing symbiotic partner, Sinorhizobium meliloti. Furthermore, spatiotemporal expression of the Nod factor-responsive reporter pMtENOD11-gusA in hairy root epidermal tissues is indistinguishable from that observed in Agrobacterium tumefaciens-transformed lines. M. truncatula hairy root explants can be propagated in vitro, and we demonstrate that these clonal lines can be colonized by endomycorrhizal fungi such as Glomus intraradices with the formation of arbuscules within cortical cells. Our results suggest that M. truncatula hairy roots represent a particularly attractive system with which to study endosymbiotic associations in transgenically modified roots.

Medicago truncatula ENOD11: a novel RPRP-encoding early nodulin gene expressed during mycorrhization in arbuscule-containing cells.

Leguminous plants establish endosymbiotic associations with both rhizobia (nitrogen fixation) and arbuscular mycorrhizal fungi (phosphate uptake). These associations involve controlled entry of the soil microsymbiont into the root and the coordinated differentiation of the respective partners to generate the appropriate exchange interfaces. As part of a study to evaluate analogies at the molecular level between these two plant-microbe interactions, we focused on genes from Medicago truncatula encoding putative cell wall repetitive proline-rich proteins (RPRPs) expressed during the early stages of root nodulation. Here we report that a novel RPRP-encoding gene, MtENOD11, is transcribed during preinfection and infection stages of nodulation in root and nodule tissues. By means of reverse transcription-polymerase chain reaction and a promoter-reporter gene strategy, we demonstrate that this gene is also expressed during root colonization by endomycorrhizal fungi in inner cortical cells containing recently formed arbuscules. In contrast, no activation of MtENOD11 is observed during root colonization by a nonsymbiotic, biotrophic Rhizoctonia fungal species. Analysis of transgenic Medicago spp. plants expressing pMtENOD11-gusA also revealed that this gene is transcribed in a variety of nonsymbiotic specialized cell types in the root, shoot, and developing seed, either sharing high secretion/metabolite exchange activity or subject to regulated modifications in cell shape. The potential role of early nodulins with atypical RPRP structures such as ENOD11 and ENOD12 in symbiotic and nonsymbiotic cellular contexts is discussed.

Cloning and characterization of the Schizosaccharomyces pombe DNA ligase gene CDC17.

The Schizosaccharomyces pombe CDC17 gene has been cloned by complementation of the cdc17 mutant coding for temperature-sensitive DNA ligase. An allele-specific suppressor active only in the presence of a high osmotic pressure was also isolated. The cloned CDC17 gene failed to complement the analogous DNA ligase mutation, cdc9, in Saccharomyces cerevisiae, although the reverse complementation was successful [Barker and Johnston, Eur. J. Biochem. 134 (1983) 315-319]. The CDC17 gene specifies a 2.8-kb transcript.

The tyrosyl-tRNA synthetase from Escherichia coli. Complete nucleotide sequence of the structural gene.

The structural component of the tyrS gene of Escherichia coli, comprising 1269 base pairs, has been fully sequenced by the combined M13/dideoxychain termination approach. The gene has a codon usage pattern which is typical of highly expressed proteins and similar to other Escherichia coli aminoacyl-tRNA synthetase genes. Peptide purification and sequencing has been used to locate the N-terminus and to provide confirmation of 95% of the translated protein sequence. This latter yields on Mr of 47,403 for the Escherichia coli tyrosyl-tRNA synthetase, and reveals considerable homology with the primary structure of the analogous enzyme isolated from Bacillus staerothermophilus.

Targeted inoculation of Medicago truncatula in vitro root cultures reveals MtENOD11 expression during early stages of infection by arbuscular mycorrhizal fungi.

• An in vitro targeted inoculation technique has been developed for studying the earliest stages of arbuscular endomycorrhizal (AM) infection of Medicago truncatula roots, and in particular the spatio-temporal expression of the early nodulin gene MtENOD11. • Agrobacterium rhizogenes transformed root explants were derived either from Myc + M. truncatula or from the infection-defective Myc - mutant TR26 ( dmi2-2 ), both expressing the pMtENOD11-gusA fusion. The normal positive geotropism of these roots, coupled with the negative geotropism of Gigaspora germ tubes allowed oriented growth of the two symbiotic partners, facilitating the identification of initial fungal/root contacts. • Early infection events at the stage of appressoria and/or internal hyphae could be observed for over 50% of the inoculated explants, revealing that MtENOD11 is expressed transiently in both epidermal and cortical cells at sites of hyphal penetration in Myc + roots, but not in epidermal cells in contact with appressoria in Myc - roots. • We propose that a direct link exists between MtENOD11 gene expression and cellular events required for fungal penetration, thereby extending analogies between rhizobial and AM host root infection processes.

Cloning and amplified expression of the tyrosyl-tRNA synthetase genes of Bacillus stearothermophilus and Escherichia coli.

Two fragments of DNA which carry the genes coding for the tyrosyl-tRNA synthetases of Escherichia coli and Bacillus stearothermophilus have been cloned into the plasmid pBR322 and were selected by complementation of an E. coli temperature-sensitive mutant. Transformation of this strain with either of the recombinant plasmids results in a 100-fold increase in tyrosyl-tRNA synthetase activity measured in vitro and the protein products co-migrate with the corresponding purified enzymes on polyacrylamide gels.

Factor analysis of body hair distributions.

Using observations on the presence or absence of terminal hair in 13 locations on 700 white adult males, descriptive statistics were tabulated, phi coefficients of intercorrelation were computed, principal axis factor loadings were extracted, and varimax rotation of factors to simple structure was performed. The resulting rotated factors were interpreted as five independent components of body hair distribution: dorsal (less sacral), pubic extension, thoracic, sacral, and abdominal.

Characterisation of an autonomously replicating sequence from the fission yeast Schizosaccharomyces pombe.

A DNA sequence has been isolated from Schizosaccharomyces pombe which promotes high frequency transformation of plasmids in the same organism. It is closely linked to the DNA ligase gene CDC17 and has therefore been named ARS17 although in structure it differs substantially from ARS elements in Saccharomyces cerevisiae. ARS17 spans some 1.8 kb of DNA and deletion of any part of this region affects activity. Moreover, there does not appear to be any short sequence which is, by itself, sufficient for high frequency transformation. ARS17 lies between and partly overlaps two divergently transcribed genes and it is extremely AT rich. It lacks the consensus sequence found in S. cerevisiae ARSs and it has no ARS activity in S. cerevisiae.

Saccharomyces cerevisiae cdc9, a structural gene for yeast DNA ligase which complements Schizosaccharomyces pombe cdc17.

The Saccharomyces cerevisiae cdc9 gene has been cloned in the vector YRp12 by complementation of the temperature-sensitive lesion in vivo. The gene is contained within a 3300-base-pair fragment of DNA, which maps to the chromosomal locus of cdc9 and which is able to complement a DNA-ligase-deficient mutant of the fission yeast Schizosaccharomyces pombe.

Electron microscopic studies of condensed mitotic chromosomes in the fission yeast Schizosaccharomyces pombe.

By blocking cells in mitosis with the anti-fungal drug thiabendazole, it has been possible to carry out ultrastructural studies on the condensed chromosomes of the fission yeast, Schizosaccharomyces pombe. It is estimated that the DNA in these chromosomes is compacted approximately 1000-fold, and that the nucleoprotein density is similar to that of higher eukaryotic metaphase chromosomes. A basic structural component of the condensed chromosomes appears to be a 50-60 nm fibre, which is often visible in a loop configuration on the periphery of the chromatids. This is reminiscent of the 50-60 nm fibre loops which are frequently seen in preparations of metaphase chromosomes, and suggests that mechanisms of nucleoprotein folding may be similar in both lower and higher eukaryotes.

Molecular characterisation of the DNA ligase gene, CDC17, from the fission yeast Schizosaccharomyces pombe.

We have sequenced a 4200-base-pair fragment of Schizosaccharomyces pombe DNA which encompasses the entire DNA ligase gene, CDC17. S1 mapping has enabled us to identify two small introns (40 and 62 nucleotides) at the 5' end of the coding region of the gene and their 3' internal conserved sequences match the CTRAY consensus found in other S. pombe introns. The major transcription initiation and 3' polyadenylation sites have been mapped and are preceded by higher eukaryotic-like TATA and AATAAA sequences respectively. Furthermore, the CDC17 mRNA carries a poly(A) tail whose length (approximately 250 nucleotides) is typical of that found in higher eukaryotic mRNAs, and is in contrast to the much shorter polyadenylated sequences found for the mRNAs of the budding yeast, Saccharomyces cerevisiae. The deduced amino acid sequence of the S. pombe DNA ligase predicts a protein of 86182 daltons, and an overall 53% homology with the same enzyme from S. cerevisiae. In particular, a stretch of 24 amino acids with 100% sequence homology spans the putative ATP-binding region which is also conserved in T4 and T7 bacteriophage DNA ligases.

The expression in meiosis of genes which are transcribed periodically in the mitotic cell cycle of budding yeast.

The mitotic cell cycle genes CDC 8, 9 and 21 in Saccharomyces cerevisiae, together with the histone H2A gene, are transcribed discontinuously in meiosis. Message from all four genes initially declines in amount, then increases abruptly to reach maximal levels during premeiotic DNA synthesis before again declining. This response occurs only in meiotic cells; in asporogenous diploids the transcript simply declines in amount. In contrast, message from four genes with no known specific meiotic function (including the actin gene) shows the same profile in both sporogenous and asporogenous diploids. In mitotic cells the three CDC genes appear to be transcribed at the same time in the cell cycle, whereas in meiosis their transcripts accumulate with different kinetics, suggesting either that they have different turnover rates in meiotic cells or that the timing of their transcription is different.

Expression of polyoma virus middle-T antigen in Saccharomyces cerevisiae.

The polyoma middle-T gene, lacking its intron, was inserted into a yeast expression plasmid containing the phosphoglycerate kinase promoter. Such plasmids transformed yeast at low frequency and these transformants expressed middle-T antigen at a level of approximately 0.1% cell protein. Furthermore, expression of this protein was frequently lost during growth in liquid culture and this loss of middle-T was accompanied by a twofold increase in the rate of growth. The spontaneous production of a truncated middle-T antigen, lacking the C terminus, was also observed; the expression of this protein did not inhibit the growth rate of the cells. Recovery and analysis of the expression plasmids encoding the truncated molecule showed that a single C X G base pair had been deleted from a run of nine consecutive C X G base pairs (Pyr nucleotide 1239--1247) within the middle-T coding region. This frame-shift mutation results in premature termination of the protein and loss of the strongly hydrophobic region of the molecule believed to be responsible for the membrane association of middle-T antigen.

Efficient transformation of Medicago truncatula cv. Jemalong using the hypervirulent Agrobacterium tumefaciens strain AGL1.

The efficiency of Agrobacterium tumefaciens transformation of the model legume Medicago truncatula cv. Jemalong (genotype 2HA) was evaluated for strains LBA 4404, C58pMP90, C58pGV2260 and AGL1. Binary vectors carrying promoter- gus/ gfp reporter gene fusions and the nptII gene as selectable marker were used for plant in vitro transformation/regeneration. The highest transformation efficiency was obtained with the disarmed hypervirulent strain AGL1 (Ti plasmid TiBo542), for which the percentage of explants forming kanamycin (Km)-resistant calli was double that obtained with each of the other three strains. In addition, we were able to reduce the time necessary for plant regeneration using AGL1, with 24% of the explants generating Km-resistant transgenic plantlets within only 4-5 months of culture. Transgene expression in planta was analysed and found to be conserved in the T(1) descendents.